Dr. Michael T. Longaker

All Publications

• The DERMIS Study: Methodologies, Results, and Implications for the Future. Journal of diabetes science and technology Hirsch, I. B., Khakpour, D., Joseph, J., Shinohara, M. M., Wang, R. K., Klueh, U., Kreutzner, D., Riveline, J. P., Jacquemier, P., Maier, L., Longaker, M. T., Parkin, C. G., Pieber, T., Kalus, A. 2024: 19322968241298005

  Abstract

  Ongoing innovation in diabetes technologies has led to the development of advanced tools such as automated insulin delivery (AID) systems that adjust insulin delivery in response to current and predicted glucose levels, residual insulin action, and other inputs (eg, meal and exercise announcements). However, infusion sets continue to be the "Achilles heel" of accurate and precise insulin delivery and continued device use. A recent study by Kalus et al (DERMIS Study) revealed higher vessel density and signals of inflammation by optical coherence tomography (OCT), in addition to increased inflammation, fat necrosis, fibrosis, and eosinophilic infiltration by histopathology. Although the study provided a comprehensive description of what was happening, the results raise important questions that require additional research. On February 29, 2024, the Leona M. and Harry B. Helmsley Charitable Trust sponsored a conference to begin addressing these issues. This article summarizes the DERMIS study findings and testing methodologies discussed at the conference and proposes the next steps for developing insulin infusion sets that reduce the variability in insulin delivery and extend wear.

  View details for DOI 10.1177/19322968241298005

  View details for PubMedID 39633523

• Characterizing Fibroblast Heterogeneity in Diabetic Wounds Through Single-Cell RNA-Sequencing. Biomedicines Wang, H. H., Korah, M., Jing, S. L., Berry, C. E., Griffin, M. F., Longaker, M. T., Januszyk, M. 2024; 12 (11)

  Abstract

  Diabetes mellitus is an increasingly prevalent chronic metabolic disorder characterized by physiologic hyperglycemia that, when left uncontrolled, can lead to significant complications in multiple organs. Diabetic wounds are common in the general population, yet the underlying mechanism of impaired healing in such wounds remains unclear. Single-cell RNA-sequencing (scRNAseq) has recently emerged as a tool to study the gene expression of heterogeneous cell populations in skin wounds. Herein, we review the history of scRNAseq and its application to the study of diabetic wound healing, focusing on how innovations in single-cell sequencing have transformed strategies for fibroblast analysis. We summarize recent research on the role of fibroblasts in diabetic wound healing and describe the functional and cellular heterogeneity of skin fibroblasts. Moreover, we highlight future opportunities in diabetic wound fibroblast research, with a focus on characterizing distinct fibroblast subpopulations and their lineages. Leveraging single-cell technologies to explore fibroblast heterogeneity and the complex biology of diabetic wounds may reveal new therapeutic targets for improving wound healing and ultimately alleviate the clinical burden of chronic wounds.

  View details for DOI 10.3390/biomedicines12112538

  View details for PubMedID 39595104

• Charles "Chuck" K.F. Chan (1975-2024). Cell stem cell Ambrosi, T. H., Longaker, M. T. 2024; 31 (10): 1391-1392

  View details for DOI 10.1016/j.stem.2024.09.005

  View details for PubMedID 39366359

• Clinical, mechanistic, and therapeutic landscape of cutaneous fibrosis. Science translational medicine Li, D. J., Berry, C. E., Wan, D. C., Longaker, M. T. 2024; 16 (766): eadn7871

  Abstract

  When dysregulated, skin fibrosis can lead to a multitude of pathologies. We provide a framework for understanding the wide clinical spectrum, mechanisms, and management of cutaneous fibrosis encompassing a variety of matrix disorders, fibrohistiocytic neoplasms, injury-induced scarring, and autoimmune scleroses. Underlying such entities are common mechanistic pathways that leverage morphogenic signaling, immune activation, and mechanotransduction to modulate fibroblast function. In light of the limited array of available treatments for cutaneous fibrosis, scientific insights have opened new therapeutic and investigative avenues for conditions that still lack effective interventions.

  View details for DOI 10.1126/scitranslmed.adn7871

  View details for PubMedID 39321265

• Author Correction: Sexually dimorphic estrogen sensing in skeletal stem cells controls skeletal regeneration. Nature communications Andrew, T. W., Koepke, L. S., Wang, Y., Lopez, M., Steininger, H., Struck, D., Boyko, T., Ambrosi, T. H., Tong, X., Sun, Y., Gulati, G. S., Murphy, M. P., Marecic, O., Tevlin, R., Schallmoser, K., Strunk, D., Seita, J., Goodman, S. B., Yang, F., Longaker, M. T., Yang, G. P., Chan, C. K. 2024; 15 (1): 8030

  View details for DOI 10.1038/s41467-024-51829-1

  View details for PubMedID 39271692

• Editorial for Special Issue on Artificial Intelligence in Tissue Engineering and Biology. Tissue engineering. Part A Guo, J. L., Januszyk, M., Longaker, M. T. 2024

  Abstract

  N/A.

  View details for DOI 10.1089/ten.TEA.2024.0240

  View details for PubMedID 39162812

• Marvels of spiny mouse regeneration: cellular players and their interactions in restoring tissue architecture in mammals. Current opinion in genetics & development Tomasso, A., Disela, V., Longaker, M. T., Bartscherer, K. 2024; 87: 102228

  Abstract

  Understanding the cellular and molecular determinants of mammalian tissue regeneration and repair is crucial for developing effective therapies that restore tissue architecture and function. In this review, we focus on the cell types involved in scarless wound response and regeneration of spiny mice (Acomys). Comparative -omics approaches with scar-prone mammals have revealed species-specific peculiarities in cellular behavior during the divergent healing trajectories. We discuss the developing views on which cell types engage in restoring the architecture of spiny mouse tissues through a co-ordinated spatiotemporal response to injury. While yet at the beginning of understanding how cells interact in these fascinating animals to regenerate tissues, spiny mice hold great promise for scar prevention and anti-fibrotic treatments.

  View details for DOI 10.1016/j.gde.2024.102228

  View details for PubMedID 39047585

• Understanding the Foreign Body Response via Single-Cell Meta-Analysis. Biology Liang, N. E., Parker, J. B., Lu, J. M., Januszyk, M., Wan, D. C., Griffin, M., Longaker, M. T. 2024; 13 (7)

  Abstract

  Foreign body response (FBR) is a universal reaction to implanted biomaterial that can affect the function and longevity of the implant. A few studies have attempted to identify targets for treating FBR through the use of single-cell RNA sequencing (scRNA-seq), though the generalizability of these findings from an individual study may be limited. In our study, we perform a meta-analysis of scRNA-seq data from all available FBR mouse studies and integrate these data to identify gene signatures specific to FBR across different models and anatomic locations. We identify subclusters of fibroblasts and macrophages that emerge in response to foreign bodies and characterize their signaling pathways, gene ontology terms, and downstream mediators. The fibroblast subpopulations enriched in the setting of FBR demonstrated significant signaling interactions in the transforming growth factor-beta (TGF-beta) signaling pathway, with known pro-fibrotic mediators identified as top expressed genes in these FBR-derived fibroblasts. In contrast, FBR-enriched macrophage subclusters highly expressed pro-fibrotic and pro-inflammatory mediators downstream of tumor necrosis factor (TNF) signaling. Cell-cell interactions were additionally interrogated using CellChat, with identification of key signaling interactions enriched between fibroblasts and macrophages in FBR. By combining multiple FBR datasets, our meta-analysis study identifies common cell-specific gene signatures enriched in foreign body reactions, providing potential therapeutic targets for patients requiring medical implants across a myriad of devices and indications.

  View details for DOI 10.3390/biology13070540

  View details for PubMedID 39056733

• Role of ferroptosis in radiation-induced soft tissue injury. Cell death discovery Berry, C. E., Kendig, C. B., An, N., Fazilat, A. Z., Churukian, A. A., Griffin, M., Pan, P. M., Longaker, M. T., Dixon, S. J., Wan, D. C. 2024; 10 (1): 313

  Abstract

  Ionizing radiation has been pivotal in cancer therapy since its discovery. Despite its therapeutic benefits, IR causes significant acute and chronic complications due to DNA damage and the generation of reactive oxygen species, which harm nucleic acids, lipids, and proteins. While cancer cells are more vulnerable to ionizing radiation due to their inefficiency in repairing damage, healthy cells in the irradiated area also suffer. Various types of cell death occur, including apoptosis, necrosis, pyroptosis, autophagy-dependent cell death, immunogenic cell death, and ferroptosis. Ferroptosis, driven by iron-dependent lipid peroxide accumulation, has been recognized as crucial in radiation therapy's therapeutic effects and complications, with extensive research across various tissues. This review aims to summarize the pathways involved in radiation-related ferroptosis, findings in different organs, and drugs targeting ferroptosis to mitigate its harmful effects.

  View details for DOI 10.1038/s41420-024-02003-5

  View details for PubMedID 38969638

  View details for PubMedCentralID PMC11226648

• Single-cell transcriptional analysis of irradiated skin reveals changes in fibroblast subpopulations and variability in caveolin expression. Radiation oncology (London, England) Kameni, L. E., Griffin, M., Berry, C. E., Shariatzadeh, S., Downer, M. A., Valencia, C., Fazilat, A. Z., Nazerali, R., Momeni, A., Januszyk, M., Longaker, M. T., Wan, D. C. 2024; 19 (1): 82

  Abstract

  Radiation-induced fibrosis (RIF) is an important late complication of radiation therapy, and the resulting damaging effects of RIF can significantly impact reconstructive outcomes. There is currently a paucity of effective treatment options available, likely due to the continuing knowledge gap surrounding the cellular mechanisms involved. In this study, detailed analyses of irradiated and non-irradiated human skin samples were performed incorporating histological and single-cell transcriptional analysis to identify novel features guiding development of skin fibrosis following radiation injury.Paired irradiated and contralateral non-irradiated skin samples were obtained from six female patients undergoing post-oncologic breast reconstruction. Skin samples underwent histological evaluation, immunohistochemistry, and biomechanical testing. Single-cell RNA sequencing was performed using the 10X single cell platform. Cells were separated into clusters using Seurat in R. The SingleR classifier was applied to ascribe cell type identities to each cluster. Differentially expressed genes characteristic to each cluster were then determined using non-parametric testing.Comparing irradiated and non-irradiated skin, epidermal atrophy, dermal thickening, and evidence of thick, disorganized collagen deposition within the extracellular matrix of irradiated skin were readily appreciated on histology. These histologic features were associated with stiffness that was higher in irradiated skin. Single-cell RNA sequencing revealed six predominant cell types. Focusing on fibroblasts/stromal lineage cells, five distinct transcriptional clusters (Clusters 0-4) were identified. Interestingly, while all clusters were noted to express Cav1, Cluster 2 was the only one to also express Cav2. Immunohistochemistry demonstrated increased expression of Cav2 in irradiated skin, whereas Cav1 was more readily identified in non-irradiated skin, suggesting Cav1 and Cav2 may act antagonistically to modulate fibrotic cellular responses.In response to radiation therapy, specific changes to fibroblast subpopulations and enhanced Cav2 expression may contribute to fibrosis. Altogether, this study introduces a novel pathway of caveolin involvement which may contribute to fibrotic development following radiation injury.

  View details for DOI 10.1186/s13014-024-02472-z

  View details for PubMedID 38926892

  View details for PubMedCentralID 6329848

• Hematoxylin and Eosin Architecture Uncovers Clinically Divergent Niches in Pancreatic Cancer. Tissue engineering. Part A Guo, J. L., Lopez, D. M., Mascharak, S., Foster, D. S., Khan, A., Davitt, M. F., Nguyen, A. T., Burcham, A. R., Chinta, M. S., Guardino, N. J., Griffin, M., Miller, E., Januszyk, M., Raghavan, S. S., Longacre, T. A., Delitto, D. J., Norton, J. A., Longaker, M. T. 2024

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) represents one of the only cancers with an increasing incidence rate and is often associated with intra- and peri-tumoral scarring, referred to as desmoplasia. This scarring is highly heterogeneous in extracellular matrix (ECM) architecture and plays complex roles in both tumor biology and clinical outcomes that are not yet fully understood. Using hematoxylin and eosin (H&E), a routine histological stain utilized in existing clinical workflows, we quantified ECM architecture in 85 patient samples to assess relationships between desmoplastic architecture and clinical outcomes such as survival time and disease recurrence. By utilizing unsupervised machine learning (ML) to summarize a latent space across 147 local (e.g. fiber length, solidity) and global (e.g. fiber branching, porosity) H&E-based features, we identified a continuum of histological architectures that were associated with differences in both survival and recurrence. Further, we mapped H&E architectures to a CO-Detection by indEXing (CODEX) reference atlas, revealing localized cell- and protein-based niches associated with outcome-positive vs. outcome-negative scarring in the tumor microenvironment. Overall, our study utilizes standard H&E staining to uncover clinically relevant associations between desmoplastic organization and PDAC outcomes, offering a translatable pipeline to support prognostic decision-making and a blueprint of spatial-biological factors for modeling by tissue engineering methods.

  View details for DOI 10.1089/ten.TEA.2024.0039

  View details for PubMedID 38874979

• Ferroptosis Inhibition with Deferoxamine Alleviates Radiation-Induced Fibrosis. Research square Berry, C. E., Kendig, C., Bs, T. L., Brenac, C., Griffin, M., Guo, J., Kameni, L., Dixon, S. J., Longaker, M. T., Wan, D. 2024

  Abstract

  Radiation-induced fibrosis (RIF) is a debilitating sequelae of radiation therapy that has been shown to improve with topical treatment with the iron chelator deferoxamine (DFO). We investigated whether DFO exerts this effect through attenuation of ferroptosis, a recently described iron-dependent pathway of cell death.Adult C57BL/6J mice were treated with topical DFO or ferrostastin-1 (Fer-1) and irradiated with 30 Grays of ionizing radiation to the dorsal skin to promote development of chronic RIF. Immunofluorescent staining with 4-hydroxynonenal (4-HNE) antibody was carried out directly following irradiation to assess ferroptosis activity. Perfusion testing with laser Doppler was performed throughout the healing interval. Eight weeks following radiation, dorsal skin was harvested and analyzed histologically and biomechanically.Immunohistochemical staining demonstrated lower presence of 4-HNE in non-irradiated skin, DFO-treated skin, and Fer-1-treated skin compared to irradiated, untreated skin. DFO resulted in histological measurements (dermal thickness and collagen content) that resembled normal skin, while Fer-1 treatment yielded less significant improvements. These results were mirrored by analysis of extracellular matrix ultrastructure and biomechanical testing, which recapitulated the ability of topical DFO treatment to alleviate RIF across these parameters while Fer-1 resulted in less notable improvement. Finally, perfusion levels in DFO treated irradiated skin were similar to measurements in normal skin, while Fer-1 treatment did not impact this feature.Ferroptosis contributes to the development of RIF and attenuation of this process leads to reduced skin injury. DFO further improves RIF through additional enhancement of perfusion not seen with Fer-1.

  View details for DOI 10.21203/rs.3.rs-4314380/v1

  View details for PubMedID 38853919

  View details for PubMedCentralID PMC11160928

• Understanding Tendon Fibroblast Biology and Heterogeneity. Biomedicines DiIorio, S. E., Young, B., Parker, J. B., Griffin, M. F., Longaker, M. T. 2024; 12 (4)

  Abstract

  Tendon regeneration has emerged as an area of interest due to the challenging healing process of avascular tendon tissue. During tendon healing after injury, the formation of a fibrous scar can limit tendon strength and lead to subsequent complications. The specific biological mechanisms that cause fibrosis across different cellular subtypes within the tendon and across different tendons in the body continue to remain unknown. Herein, we review the current understanding of tendon healing, fibrosis mechanisms, and future directions for treatments. We summarize recent research on the role of fibroblasts throughout tendon healing and describe the functional and cellular heterogeneity of fibroblasts and tendons. The review notes gaps in tendon fibrosis research, with a focus on characterizing distinct fibroblast subpopulations in the tendon. We highlight new techniques in the field that can be used to enhance our understanding of complex tendon pathologies such as fibrosis. Finally, we explore bioengineering tools for tendon regeneration and discuss future areas for innovation. Exploring the heterogeneity of tendon fibroblasts on the cellular level can inform therapeutic strategies for addressing tendon fibrosis and ultimately reduce its clinical burden.

  View details for DOI 10.3390/biomedicines12040859

  View details for PubMedID 38672213

  View details for PubMedCentralID PMC11048404

• Deferoxamine topical cream superior to patch in rescuing radiation-induced fibrosis of unwounded and wounded skin. Journal of cellular and molecular medicine Berry, C. E., Abbas, D. B., Griffin, M., Lintel, H., Guo, J., Kameni, L., Churukian, A. A., Fazilat, A. Z., Chen, K., Gurtner, G. C., Longaker, M. T., Momeni, A., Wan, D. C. 2024; 28 (8): e18306

  Abstract

  Topical patch delivery of deferoxamine (DFO) has been studied as a treatment for this fibrotic transformation in irradiated tissue. Efficacy of a novel cream formulation of DFO was studied as a RIF therapeutic in unwounded and excisionally wounded irradiated skin. C57BL/6J mice underwent 30 Gy of radiation to the dorsum followed by 4 weeks of recovery. In a first experiment, mice were separated into six conditions: DFO 50 mg cream (D50), DFO 100 mg cream (D100), soluble DFO injections (DI), DFO 1 mg patch (DP), control cream (Vehicle), and irradiated untreated skin (IR). In a second experiment, excisional wounds were created on the irradiated dorsum of mice and then divided into four treatment groups: DFO 100 mg Cream (W-D100), DFO 1 mg patch (W-DP), control cream (W-Vehicle), and irradiated untreated wounds (W-IR). Laser Doppler perfusion scans, biomechanical testing, and histological analysis were performed. In irradiated skin, D100 improved perfusion compared to D50 or DP. Both D100 and DP enhanced dermal characteristics, including thickness, collagen density and 8-isoprostane staining compared to untreated irradiated skin. D100 outperformed DP in CD31 staining, indicating higher vascular density. Extracellular matrix features of D100 and DP resembled normal skin more closely than DI or control. In radiated excisional wounds, D100 facilitated faster wound healing and increased perfusion compared to DP. The 100 mg DFO cream formulation rescued RIF of unwounded irradiated skin and improved excisional wound healing in murine skin relative to patch delivery of DFO.

  View details for DOI 10.1111/jcmm.18306

  View details for PubMedID 38613357

• Modelling and targeting mechanical forces in organ fibrosis. Nature reviews bioengineering Mascharak, S., Guo, J. L., Griffin, M., Berry, C. E., Wan, D. C., Longaker, M. T. 2024; 2 (4): 305-323

  Abstract

  Few efficacious therapies exist for the treatment of fibrotic diseases, such as skin scarring, liver cirrhosis and pulmonary fibrosis, which is related to our limited understanding of the fundamental causes and mechanisms of fibrosis. Mechanical forces from cell-matrix interactions, cell-cell contact, fluid flow and other physical stimuli may play a central role in the initiation and propagation of fibrosis. In this Review, we highlight the mechanotransduction mechanisms by which various sources of physical force drive fibrotic disease processes, with an emphasis on central pathways that may be therapeutically targeted to prevent and reverse fibrosis. We then discuss engineered models of mechanotransduction in fibrosis, as well as molecular and biomaterials-based therapeutic approaches for limiting fibrosis and promoting regenerative healing phenotypes in various organs. Finally, we discuss challenges within fibrosis research that remain to be addressed and that may greatly benefit from next-generation bioengineered model systems.

  View details for DOI 10.1038/s44222-023-00144-3

  View details for PubMedID 39552705

• Understanding wound healing in obesity. World journal of experimental medicine Cotterell, A., Griffin, M., Downer, M. A., Parker, J. B., Wan, D., Longaker, M. T. 2024; 14 (1): 86898

  Abstract

  Obesity has become more prevalent in the global population. It is associated with the development of several diseases including diabetes mellitus, coronary heart disease, and metabolic syndrome. There are a multitude of factors impacted by obesity that may contribute to poor wound healing outcomes. With millions worldwide classified as obese, it is imperative to understand wound healing in these patients. Despite advances in the understanding of wound healing in both healthy and diabetic populations, much is unknown about wound healing in obese patients. This review examines the impact of obesity on wound healing and several animal models that may be used to broaden our understanding in this area. As a growing portion of the population identifies as obese, understanding the underlying mechanisms and how to overcome poor wound healing is of the utmost importance.

  View details for DOI 10.5493/wjem.v14.i1.86898

  View details for PubMedID 38590299

  View details for PubMedCentralID PMC10999071

• Successful topical treatment of human biofilms using multiple antibiotic elution from a collagen-rich hydrogel. Scientific reports Sharma, A. D., Jarman, E. H., Kuppalli, K., Murphy, M. J., Longaker, M. T., Gurtner, G., Fox, P. M. 2024; 14 (1): 5621

  Abstract

  Chronic non-healing wounds significantly strain modern healthcare systems, affecting 1-2% of the population in developed countries with costs ranging between $28.1 and $96.8 billion annually. Additionally, it has been established that chronic wounds resulting from comorbidities, such as peripheral vascular disease and diabetes mellitus, tend to be polymicrobial in nature. Treatment of polymicrobial chronic wounds with oral and IV antibiotics can result in antimicrobial resistance, leading to more difficult-to-treat wounds. Ideally, chronic ulcers would be topically treated with antibiotic combinations tailored to the microbiome of a patient's wound. We have previously shown that a topical collagen-rich hydrogel (cHG) can elute single antibiotics to inhibit bacterial growth in a manner that is nontoxic to mammalian cells. Here, we analyzed the microbiology of cultures taken from human patients diagnosed with diabetes mellitus suffering from chronic wounds present for more than 6 weeks. Additionally, we examined the safety of the elution of multiple antibiotics from collagen-rich hydrogel in mammalian cells in vivo. Finally, we aimed to create tailored combinations of antibiotics impregnated into cHG to successfully target and treat infections and eradicate biofilms cultured from human chronic diabetic wound tissue. We found that the majority of human chronic wounds in our study were polymicrobial in nature. The elution of multiple antibiotics from cHG was well-tolerated in mammalian cells, making it a potential topical treatment of the polymicrobial chronic wound. Finally, combinations of antibiotics tailored to each patient's microbiome eluted from a collagen-rich hydrogel successfully treated bacterial cultures isolated from patient samples via an in vitro assay.

  View details for DOI 10.1038/s41598-024-54477-z

  View details for PubMedID 38454046

• A tension offloading patch mitigates dermal fibrosis induced by pro-fibrotic skin injections. Research square Talbott, H. E., Griffin, M. F., Mascharak, S., Parker, J. B., Kuhnert, M. M., Guo, J. L., Diaz Deleon, N. M., Lavin, C., Abbas, D., Guardino, N., Morgan, A., Valencia, C., Cotterell, A., Longaker, M. T., Wan, D. C. 2024

  Abstract

  Skin fibrosis is a clinical problem with devastating impacts but limited treatment options. In the setting of diabetes, insulin administration often causes local dermal fibrosis, leading to a range of clinical sequelae including impeded insulin absorption. Mechanical forces are important drivers of fibrosis and, clinically, physical tension offloading at the skin level using an elastomeric patch significantly reduces wound scarring. However, it is not known whether tension offloading could similarly prevent skin fibrosis in the setting of pro-fibrotic injections. Here, we develop a porcine model using repeated local injections of bleomycin to recapitulate key features of insulin-induced skin fibrosis. Using histologic, tissue ultrastructural, and biomechanical analyses, we show that application of a tension-offloading patch both prevents and rescues existing skin fibrosis from bleomycin injections. By applying single-cell transcriptomic analysis, we find that the fibrotic response to bleomycin involves shifts in myeloid cell dynamics from favoring putatively pro-regenerative to pro-fibrotic myeloid subtypes; in a mechanomodulatory in vitro platform, we show that these shifts are mechanically driven and reversed by exogenous IL4. Finally, using a human foreskin xenograft model, we show that IL4 treatment mitigates bleomycin-induced dermal fibrosis. Overall, this study highlights that skin tension offloading, using an FDA cleared, commercially available patch, could have significant potential clinical benefit for the millions of patients dependent on insulin.

  View details for DOI 10.21203/rs.3.rs-3915097/v1

  View details for PubMedID 38464040

• Single cell pharmacogenic pipeline identifies novel opportunities in uterine leiomyosarcoma Daniel, S. K., Foster, D., Nosrati, F., Korah, M., Fallah, M., Sun, B. J., Loftus, T., Hu, D., Dua, M., Visser, B., Poultsides, G., Kirane, A., Longaker, M., Ganjoo, K., Lee, B., Delitto, D. SPRINGER. 2024: S42

  View details for Web of Science ID 001185577500085

• IL-1RA and IL-12 Decrease Pancreatic Ductal Adenocarcinoma Tumor Mass and Prolong Survival by Inhibiting Mechanoresponsive Cafs Morgan, A., Griffin, M., Downer, M., Lindsay, H., Parker, J. B., Guo, J. L., Longaker, M. T., Norton, J. A. LIPPINCOTT WILLIAMS & WILKINS. 2023: S455-S456

  View details for Web of Science ID 001094086301392

• Single-Cell RNA-Seq Analysis Reveals Similar Fibroblasts in Irradiated Skin of Humans, Mice, and Pigs Kameni, L., Berry, C., Griffin, M., Januszyk, M., Downer, M., Huber, J. L., Parker, J., Momeni, A., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2023: S389

  View details for Web of Science ID 001094086301247

• Single-Cell RNA-Sequencing Identifies Modulator of Foreign Body Response with Use of Acellular Dermal Matrix in Breast Reconstruction Liang, N., Tevlin, R., Griffin, M., Parker, J. B., Henn, D., Navarro, R. S., Dung Nguyen, Momeni, A., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2023: S389-S390

  View details for Web of Science ID 001094086301248

• Healing of Chronically Irradiated Excisional Wounds Improved by Topical Deferoxamine Treatment Berry, C., Griffin, M., Kameni, L., Abbas, D., Downer, M., Parker, J. B., Liang, N., Januszyk, M., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2023: S382-S383

  View details for Web of Science ID 001094086301234

• Tissue Microenvironment a Key Driver in Fibrotic Capsules Formed During Foreign Body Response Parker, J. B., Griffin, M., Downer, M., Morgan, A., Guo, J. L., Liang, N., Berry, C., Diiorio, S., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2023: S393

  View details for Web of Science ID 001094086301255

• Fat Grafting Treatment for Radiation-Induced Fibrosis Results in Downregulation of Inflammatory and Fibrotic Signaling Pathways Berry, C., Abbas, D., Kameni, L., Griffin, M., Downer, M., Parker, J. B., Guo, J. L., Januszyk, M., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2023: S381-S382

  View details for Web of Science ID 001094086301232

• Investigating Dysmotility and the Expansion of Glial Cells and Interstitial Cells of Cajal in Crohn's Disease Strictures Using a Novel Surgical Mouse Model Kim, A., Bauer-rowe, K. E., Griffin, M., Foster, D., Longaker, M. T., Hyun, J. S. LIPPINCOTT WILLIAMS & WILKINS. 2023: S355

  View details for Web of Science ID 001094086301169

• Analysis of Collagen Extracellular Matrix Ultrastructure in Mouse Long Bone Distraction Osteogenesis Diiorio, S., Guo, J. L., Griffin, M., Huber, J. L., Downer, M., Berry, C., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2023: S378-S379

  View details for Web of Science ID 001094086301225

• Grafting of Human Foreskin onto Murine Dorsum Provides a Novel Model of Chronic Radiation-Induced Fibrosis Berry, C., Abbas, D., Griffin, M., Kameni, L., Downer, M., Parker, J. B., Liang, N., Dilorio, S., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2023: S398

  View details for Web of Science ID 001094086301270

• Meta-Analysis of Single-Cell Transcriptomics Data of Cardiac Fibroblasts Reveals Temporal Heterogeneity of Cardiac Fibroblast Response after MI Lu, J., Januszyk, M., Griffin, M., Guo, J. L., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2023: S68

  View details for Web of Science ID 001094086300124

• Novel Deferoxamine Cream Formulation Improves Perfusion, Elasticity, and Tissue Architecture in Chronically Irradiated Murine Skin Compared to Transdermal Patch Berry, C., Abbas, D., Kameni, L., Griffin, M., Downer, M., Parker, J. B., Liang, N., Guo, J. L., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2023: S386

  View details for Web of Science ID 001094086301241

• Pioglitazone Decreases Adipogenisis Leading to Melanoma Skin Tumor Suppression Downer, M., Griffin, M., Morgan, A., Parker, J. B., Berry, C., Li, D. J., Liang, N., Kameni, L., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2023: S387-S388

  View details for Web of Science ID 001094086301244

• Ferropotosis Levels Decrease in Response to Deferoxamine Treatment in Irradiated Murine Skin Berry, C., Kameni, L., Griffin, M., Downer, M., Parker, J. B., Guo, J. L., Liang, N., Januszyk, M., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2023: S382

  View details for Web of Science ID 001094086301233

• Where There Is Fat, There Is Fibrosis: Elucidating the Mechanisms of Creeping Fat-Driven Stricture Formation Bauer-Rowe, K. E., Kim, A., Griffin, M., Liang, N., Foster, D., Guo, J. L., Norton, J. A., Longaker, M. T., Hyun, J. S. LIPPINCOTT WILLIAMS & WILKINS. 2023: S365-S366

  View details for Web of Science ID 001094086301190

• A Review of Radiation-Induced Vascular Injury and Clinical Impact. Annals of plastic surgery Kameni, L. E., Januszyk, M., Berry, C. E., Downer, M. A., Parker, J. B., Morgan, A. G., Valencia, C., Griffin, M., Li, D. J., Liang, N. E., Momeni, A., Longaker, M. T., Wan, D. C. 2023

  Abstract

  ABSTRACT: The number of cancer survivors continues to increase because of advances in therapeutic modalities. Along with surgery and chemotherapy, radiotherapy is a commonly used treatment modality in roughly half of all cancer patients. It is particularly helpful in the oncologic treatment of patients with breast, head and neck, and prostate malignancies. Unfortunately, among patients receiving radiation therapy, long-term sequalae are often unavoidable, and there is accumulating clinical evidence suggesting significant radiation-related damage to the vascular endothelium. Ionizing radiation has been known to cause obliterative fibrosis and increased wall thickness in irradiated blood vessels. Clinically, these vascular changes induced by ionizing radiation can pose unique surgical challenges when operating in radiated fields. Here, we review the relevant literature on radiation-induced vascular damage focusing on mechanisms and signaling pathways involved and highlight microsurgical anastomotic outcomes after radiotherapy. In addition, we briefly comment on potential therapeutic strategies, which may have the ability to mitigate radiation injury to the vascular endothelium.

  View details for DOI 10.1097/SAP.0000000000003723

  View details for PubMedID 37962260

• Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing. Journal of visualized experiments : JoVE Foster, D. S., Griffin, M., Januszyk, M., Delitto, D., Norton, J. A., Longaker, M. T. 2023

  Abstract

  Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

  View details for DOI 10.3791/65831

  View details for PubMedID 37902368

• Desmoplastic stromal signatures predict patient outcomes in pancreatic ductal adenocarcinoma. Cell reports. Medicine Mascharak, S., Guo, J. L., Foster, D. S., Khan, A., Davitt, M. F., Nguyen, A. T., Burcham, A. R., Chinta, M. S., Guardino, N. J., Griffin, M., Lopez, D. M., Miller, E., Januszyk, M., Raghavan, S. S., Longacre, T. A., Delitto, D. J., Norton, J. A., Longaker, M. T. 2023: 101248

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related death. Hallmarks include desmoplasia with variable extracellular matrix (ECM) architecture and a complex microenvironment with spatially defined tumor, stromal, and immune populations. Nevertheless, the role of desmoplastic spatial organization in patient/tumor variability remains underexplored, which we elucidate using two technologies. First, we quantify ECM patterning in 437 patients, revealing architectures associated with disease-free and overall survival. Second, we spatially profile the cellular milieu of 78 specimens using codetection by indexing, identifying an axis of pro-inflammatory cell interactions predictive of poorer outcomes. We discover that clinical characteristics, including neoadjuvant chemotherapy status, tumor stage, and ECM architecture, correlate with differential stromal-immune organization, including fibroblast subtypes with distinct niches. Lastly, we define unified signatures that predict survival with areas under the receiver operating characteristic curve (AUCs) of 0.872-0.903, differentiating survivorship by 655 days. Overall, our findings establish matrix ultrastructural and cellular organizations of fibrosis linked to poorer outcomes.

  View details for DOI 10.1016/j.xcrm.2023.101248

  View details for PubMedID 37865092

• Allele-specific expression reveals genetic drivers of tissue regeneration in mice. Cell stem cell Mack, K. L., Talbott, H. E., Griffin, M. F., Parker, J. B., Guardino, N. J., Spielman, A. F., Davitt, M. F., Mascharak, S., Downer, M., Morgan, A., Valencia, C., Akras, D., Berger, M. J., Wan, D. C., Fraser, H. B., Longaker, M. T. 2023

  Abstract

  In adult mammals, skin wounds typically heal by scarring rather than through regeneration. In contrast, "super-healer" Murphy Roths Large (MRL) mice have the unusual ability to regenerate ear punch wounds; however, the molecular basis for this regeneration remains elusive. Here, in hybrid crosses between MRL and non-regenerating mice, we used allele-specific gene expression to identify cis-regulatory variation associated with ear regeneration. Analyzing three major cell populations (immune, fibroblast, and endothelial), we found that genes with cis-regulatory differences specifically in fibroblasts were associated with wound-healing pathways and also co-localized with quantitative trait loci for ear wound-healing. Ectopic treatment with one of these proteins, complement factor H (CFH), accelerated wound repair and induced regeneration in typically fibrotic wounds. Through single-cell RNA sequencing (RNA-seq), we observed that CFH treatment dramatically reduced immune cell recruitment to wounds, suggesting a potential mechanism for CFH's effect. Overall, our results provide insights into the molecular drivers of regeneration with potential clinical implications.

  View details for DOI 10.1016/j.stem.2023.08.010

  View details for PubMedID 37714154

• Commentary on: Postoperative Mechanomodulation Decreases T-Junction Dehiscence After Reduction Mammaplasty: Early Scar Analysis From a Randomized Controlled Trial. Aesthetic surgery journal Berry, C. E., Longaker, M. T., Wan, D. C. 2023

  View details for DOI 10.1093/asj/sjad281

  View details for PubMedID 37625787

• Understanding Fibroblast Heterogeneity in Form and Function. Biomedicines Parker, J. B., Valencia, C., Akras, D., DiIorio, S. E., Griffin, M. F., Longaker, M. T., Wan, D. C. 2023; 11 (8)

  Abstract

  Historically believed to be a homogeneous cell type that is often overlooked, fibroblasts are more and more understood to be heterogeneous in nature. Though the mechanisms behind how fibroblasts participate in homeostasis and pathology are just beginning to be understood, these cells are believed to be highly dynamic and play key roles in fibrosis and remodeling. Focusing primarily on fibroblasts within the skin and during wound healing, we describe the field's current understanding of fibroblast heterogeneity in form and function. From differences due to embryonic origins to anatomical variations, we explore the diverse contributions that fibroblasts have in fibrosis and plasticity. Following this, we describe molecular techniques used in the field to provide deeper insights into subpopulations of fibroblasts and their varied roles in complex processes such as wound healing. Limitations to current work are also discussed, with a focus on future directions that investigators are recommended to take in order to gain a deeper understanding of fibroblast biology and to develop potential targets for translational applications in a clinical setting.

  View details for DOI 10.3390/biomedicines11082264

  View details for PubMedID 37626760

• Understanding the Role of Adipocytes and Fibroblasts in Cancer. Annals of plastic surgery Downer, M. A., Griffin, M. F., Morgan, A. G., Parker, J. B., Li, D. J., Berry, C. E., Liang, N. E., Kameni, L., Cotterell, A. C., Akras, D., Valencia, C., Longaker, M. T., Wan, D. C. 2023

  Abstract

  Cancer is currently the second leading cause of death in the United States. There is increasing evidence that the tumor microenvironment (TME) is pivotal for tumorigenesis and metastasis. Recently, adipocytes and cancer-associated fibroblasts (CAFs) in the TME have been shown to play a major role in tumorigenesis of different cancers, specifically melanoma. Animal studies have shown that CAFs and adipocytes within the TME help tumors evade the immune system, for example, by releasing chemokines to blunt the effectiveness of the host defense. Although studies have identified that adipocytes and CAFs play a role in tumorigenesis, adipocyte transition to fibroblast within the TME is fairly unknown. This review intends to elucidate the potential that adipocytes may have to transition to fibroblasts and, as part of the TME, a critical role that CAFs may play in affecting the growth and invasion of tumor cells. Future studies that illuminate the function of adipocytes and CAFs in the TME may pave way for new antitumor therapies.

  View details for DOI 10.1097/SAP.0000000000003658

  View details for PubMedID 37553786

• Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing. Nature communications Henn, D., Zhao, D., Sivaraj, D., Trotsyuk, A., Bonham, C. A., Fischer, K. S., Kehl, T., Fehlmann, T., Greco, A. H., Kussie, H. C., Moortgat Illouz, S. E., Padmanabhan, J., Barrera, J. A., Kneser, U., Lenhof, H., Januszyk, M., Levi, B., Keller, A., Longaker, M. T., Chen, K., Qi, L. S., Gurtner, G. C. 2023; 14 (1): 4729

  Abstract

  Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.

  View details for DOI 10.1038/s41467-023-40519-z

  View details for PubMedID 37550295

• Del1 Is a Growth Factor for Skeletal Progenitor Cells in the Fracture Callus. Biomolecules Sun, Y., Boyko, T., Marecic, O., Struck, D., Mann, R. K., Andrew, T. W., Lopez, M., Tong, X., Goodman, S. B., Yang, F., Longaker, M. T., Chan, C. K., Yang, G. P. 2023; 13 (8)

  Abstract

  Failure to properly form bone or integrate surgical implants can lead to morbidity and additional surgical interventions in a significant proportion of orthopedic surgeries. While the role of skeletal stem cells (SSCs) in bone formation and repair is well-established, very little is known about the factors that regulate the downstream Bone, Cartilage, Stromal, Progenitors (BCSPs). BCSPs, as transit amplifying progenitor cells, undergo multiple mitotic divisions to expand the pool of lineage committed progenitors allowing stem cells to preserve their self-renewal and stemness. Del1 is a protein widely expressed in the skeletal system, but its deletion led to minimal phenotype changes in the uninjured mouse. In this paper, we demonstrate that Del1 is a key regulator of BCSP expansion following injury. In Del1 knockout mice, there is a significant reduction in the number of BCSPs which leads to a smaller callus and decreased bone formation compared with wildtype (WT) littermates. Del1 serves to promote BCSP proliferation and prevent apoptosis in vivo and in vitro. Moreover, exogenous Del1 promotes proliferation of aged human BCSPs. Our results highlight the potential of Del1 as a therapeutic target for improving bone formation and implant success. Del1 injections may improve the success of orthopedic surgeries and fracture healing by enhancing the proliferation and survival of BCSPs, which are crucial for generating new bone tissue during the process of bone formation and repair.

  View details for DOI 10.3390/biom13081214

  View details for PubMedID 37627279

• Medical Biology of Cancer-Associated Fibroblasts in Pancreatic Cancer. Biology Morgan, A., Griffin, M., Kameni, L., Wan, D. C., Longaker, M. T., Norton, J. A. 2023; 12 (8)

  Abstract

  Pancreatic cancer is one of the deadliest forms of cancer with one of the lowest 5-year survival rates of all cancer types. A defining characteristic of pancreatic cancer is the existence of dense desmoplastic stroma that, when exposed to stimuli such as cytokines, growth factors, and chemokines, generate a tumor-promoting environment. Cancer-associated fibroblasts (CAFs) are activated during the progression of pancreatic cancer and are a crucial component of the tumor microenvironment (TME). CAFs are primarily pro-tumorigenic in their activated state and function as promoters of cancer invasion, proliferation, metastasis, and immune modulation. Aided by many signaling pathways, cytokines, and chemokines in the tumor microenvironment, CAFs can originate from many cell types including resident fibroblasts, mesenchymal stem cells, pancreatic stellate cells, adipocytes, epithelial cells, endothelial cells, and other cell types. CAFs are a highly heterogeneous cell type expressing a variety of surface markers and performing a wide range of tumor promoting and inhibiting functions. Single-cell transcriptomic analyses have revealed a high degree of specialization among CAFs. Some examples of CAF subpopulations include myofibrotic CAFs (myCAFs), which exhibit a matrix-producing contractile phenotype; inflammatory CAFs (iCAF) that are classified by their immunomodulating, secretory phenotype; and antigen-presenting CAFs (apCAFs), which have antigen-presenting capabilities and express Major Histocompatibility Complex II (MHC II). Over the last several years, various attempts have been undertaken to describe the mechanisms of CAF-tumor cell interaction, as well as CAF-immune cell interaction, that contribute to tumor proliferation, invasion, and metastasis. Although our understanding of CAF biology in cancer has steadily increased, the extent of CAFs heterogeneity and their role in the pathobiology of pancreatic cancer remains elusive. In this regard, it becomes increasingly evident that further research on CAFs in pancreatic cancer is necessary.

  View details for DOI 10.3390/biology12081044

  View details for PubMedID 37626931

• Call for Special Issue Papers: Artificial Intelligence in Tissue Engineering and Biology. Tissue engineering. Part A Guo, J. L., Januszyk, M., Longaker, M. T. 2023

  View details for DOI 10.1089/ten.tea.2023.29049.cfp

  View details for PubMedID 37466469

• Call for Special Issue Papers: Artificial Intelligence in Tissue Engineering and Biology. Tissue engineering. Part B, Reviews Guo, J. L., Januszyk, M., Longaker, M. T. 2023

  View details for DOI 10.1089/ten.teb.2023.29019.cfp

  View details for PubMedID 37466464

• Call for Special Issue Papers: Artificial Intelligence in Tissue Engineering and Biology. Tissue engineering. Part C, Methods Guo, J. L., Januszyk, M., Longaker, M. T. 2023

  View details for DOI 10.1089/ten.tec.2023.29040.cfp

  View details for PubMedID 37466465

• Thumb Osteoarthritis: Stem Cell Activation, Niche Augmentation and Tissue Regeneration Murphy, M., Takematsu, E., Koepke, L., Tong, X., Butler, G., Ambrosi, T., Hoover, M., Wang, Y., Zhao, L., Wong, J., Reid, A., Longaker, M., Chan, C. MARY ANN LIEBERT, INC. 2023

  View details for Web of Science ID 001126807000330

• Thumb Osteoarthritis: Stem Cell Activation, Niche Augmentation and Tissue Regeneration Murphy, M., Takematsu, E., Koepke, L., Tong, X., Butler, G., Ambrosi, T., Hoover, M., Wang, Y., Zhao, L., Wong, J., Reid, A., Longaker, M., Chant, C. MARY ANN LIEBERT, INC. 2023

  View details for Web of Science ID 001057609600337

• Reduction of Tendon Fibrosis Using Galectin-3 Inhibitors. Plastic and reconstructive surgery Spielman, A. F., Griffin, M. F., Titan, A. L., Guardino, N., Cotterell, A. C., Akras, D., Wan, D. C., Longaker, M. T. 2023

  Abstract

  BACKGROUND: Fibrosis is a complication of both tendon injuries and repairs. We aim to develop a mouse model to assess tendon fibrosis and to identify an antifibrotic agent capable of overcoming tendon fibrosis.METHODS: Adult C57Bl/6 mice underwent a skin incision to expose the Achilles tendon, followed by 50% tendon injury and abrasion with sandpaper. Sham surgeries were conducted on contralateral hindlimbs. Histology and immunofluorescent staining for fibrotic markers (Col1, alpha-SMA) were used to confirm that the model induced tendon fibrosis. A second experiment was conducted to further examine the role of alpha-SMA in adhesion formation using alpha-SMA.mTmG mice (6-8 weeks old) (n=3) with the same injury model. The control group (tendon injury) was compared to the sham group, using the contralateral limb with skin incision only. A second experiment was conducted to further examine the role of alpha-SMA in adhesion formation using alpha-SMA.mTmG mice (6-8 weeks old) (n=3) with the same injury model. The control group (tendon injury) was compared to the sham group, using the contralateral limb with skin incision only. Lastly, alpha-SMA.mTmG mice were randomized to either condition 1. Tendon injury (control group) or 2. Tendon injury with Galectin-3 inhibitor (Gal3i) treatment at time of injury (treatment group).RESULTS: Histological analyses confirmed tendon thickening and collagen deposition after tendon injury and abrasion compared to control. Immunofluorescence showed higher levels of Col1 and alpha-SMA protein expression after injury compared to sham (*p<0.05). RT-qPCR also demonstrated increased gene expression of Col1 and alpha-SMA after injury compared to sham (*p<0.05). Gal3 protein expression also increased after injury and co-localized with alpha-SMA positive fibroblasts surrounding the fibrotic tendon. Gal3i treatment decreased collagen deposition and scarring observed in the treatment group (*p<0.05). Flow cytometry analysis further showed reduced numbers of profibrotic fibroblasts (CD26+) in the treatment compared to the control group (*p<0.05).CONCLUSIONS: Our study provides a reproducible and reliable model to investigate tendon fibrosis. Findings suggest the potential of Gal3i to overcome fibrosis resulting from tendon injuries.

  View details for DOI 10.1097/PRS.0000000000010880

  View details for PubMedID 37344932

• Purification and functional characterization of novel human skeletal stem cell lineages. Nature protocols Hoover, M. Y., Ambrosi, T. H., Steininger, H. M., Koepke, L. S., Wang, Y., Zhao, L., Murphy, M. P., Alam, A. A., Arouge, E. J., Butler, M. G., Takematsu, E., Stavitsky, S. P., Hu, S., Sahoo, D., Sinha, R., Morri, M., Neff, N., Bishop, J., Gardner, M., Goodman, S., Longaker, M., Chan, C. K. 2023

  Abstract

  Human skeletal stem cells (hSSCs) hold tremendous therapeutic potential for developing new clinical strategies to effectively combat congenital and age-related musculoskeletal disorders. Unfortunately, refined methodologies for the proper isolation of bona fide hSSCs and the development of functional assays that accurately recapitulate their physiology within the skeleton have been lacking. Bone marrow-derived mesenchymal stromal cells (BMSCs), commonly used to describe the source of precursors for osteoblasts, chondrocytes, adipocytes and stroma, have held great promise as the basis of various approaches for cell therapy. However, the reproducibility and clinical efficacy of these attempts have been obscured by the heterogeneous nature of BMSCs due to their isolation by plastic adherence techniques. To address these limitations, our group has refined the purity of individual progenitor populations that are encompassed by BMSCs by identifying defined populations of bona fide hSSCs and their downstream progenitors that strictly give rise to skeletally restricted cell lineages. Here, we describe an advanced flow cytometric approach that utilizes an extensive panel of eight cell surface markers to define hSSCs; bone, cartilage and stromal progenitors; and more differentiated unipotent subtypes, including an osteogenic subset and three chondroprogenitors. We provide detailed instructions for the FACS-based isolation of hSSCs from various tissue sources, in vitro and in vivo skeletogenic functional assays, human xenograft mouse models and single-cell RNA sequencing analysis. This application of hSSC isolation can be performed by any researcher with basic skills in biology and flow cytometry within 1-2 days. The downstream functional assays can be performed within a range of 1-2 months.

  View details for DOI 10.1038/s41596-023-00836-5

  View details for PubMedID 37316563

  View details for PubMedCentralID 6568007

• Emerging frontiers in regenerative medicine. Science (New York, N.Y.) McKinley, K. L., Longaker, M. T., Naik, S. 2023; 380 (6647): 796-798

  Abstract

  Bridging knowledge gaps could enable regenerative therapy.

  View details for DOI 10.1126/science.add6492

  View details for PubMedID 37228215

• Circulating and extracellular vesicle-derived microRNAs as biomarkers in bone-related diseases. Frontiers in endocrinology Huber, J., Longaker, M. T., Quarto, N. 2023; 14: 1168898

  Abstract

  MicroRNAs (miRNA) are small non-coding RNA molecules that regulate posttranscriptional gene expression by repressing messengerRNA-targets. MiRNAs are abundant in many cell types and are secreted into extracellular fluids, protected from degradation by packaging in extracellular vesicles. These circulating miRNAs are easily accessible, disease-specific and sensitive to small changes, which makes them ideal biomarkers for diagnostic, prognostic, predictive or monitoring purposes. Specific miRNA signatures can be reflective of disease status and development or indicators of poor treatment response. This is especially important in malignant diseases, as the ease of accessibility of circulating miRNAs circumvents the need for invasive tissue biopsy. In osteogenesis, miRNAs can act either osteo-enhancing or osteo-repressing by targeting key transcription factors and signaling pathways. This review highlights the role of circulating and extracellular vesicle-derived miRNAs as biomarkers in bone-related diseases, with a specific focus on osteoporosis and osteosarcoma. To this end, a comprehensive literature search has been performed. The first part of the review discusses the history and biology of miRNAs, followed by a description of different types of biomarkers and an update of the current knowledge of miRNAs as biomarkers in bone related diseases. Finally, limitations of miRNAs biomarker research and future perspectives will be presented.

  View details for DOI 10.3389/fendo.2023.1168898

  View details for PubMedID 37293498

  View details for PubMedCentralID PMC10244776

• Attenuating Chronic Fibrosis: Decreasing Foreign Body Response with Acellular Dermal Matrix. Tissue engineering. Part B, Reviews Liang, N. E., Griffin, M., Berry, C. E., Parker, J. B., Downer, M. A., Wan, D. C., Longaker, M. T. 2023

  Abstract

  Surgical implants are increasingly used across multiple medical disciplines, with applications ranging from tissue reconstruction to improving compromised organ and limb function. Despite their significant potential for improving health and quality of life, biomaterial implant function is severely limited by the body's immune response to its presence: this is known as the foreign body response and is characterized by chronic inflammation and fibrotic capsule formation. This response can result in life-threatening sequelae such as implant malfunction, superimposed infection and associated vessel thrombosis, in addition to soft tissue disfigurement. Patients may require frequent medical visits, as well as repeated invasive procedures, increasing the burden on an already strained healthcare system. Currently, the foreign body response and the cells and molecular mechanisms that mediate it are poorly understood. With applications across a wide array of surgical specialties, acellular dermal matrix has emerged as a potential solution to the fibrotic reaction seen with FBR. Though the mechanisms by which acellular dermal matrix decreases chronic fibrosis remain to be clearly characterized, animal studies across diverse surgical models point to its biomimetic properties that facilitate decreased periprosthetic inflammation and improved host cell incorporation.

  View details for DOI 10.1089/ten.TEB.2023.0060

  View details for PubMedID 37212342

• Craniofacial Dysmorphology and Impaired Suture Patterning in a Mouse Model of Frank-Ter-Haar Syndrome Huber, J., Menon, S., Lopez-Torres, M., Guo, J. L., Longaker, M. T., Quarto, N. LIPPINCOTT WILLIAMS & WILKINS. 2023: S89

  View details for Web of Science ID 000989943300229

• Topical Vanadate Improves Excisional Wound Healing in Murine Model Lintel, H., Abbas, D., Mackay, D. D., Griffin, M., Spielman, A., Guardino, N., Churukian, A., Guo, J. L., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2023: S91

  View details for Web of Science ID 000989943300234

• Lineage Tracing Reveals Adipocytes Are Responsible for Muscle Fibrosis Following Nerve Injury Spielman, A., Griffin, M., Cotterell, A. C., Erich, K., Guo, B. L., Abbas, D., Parker, J. L., Lintel, H., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2023: S92

  View details for Web of Science ID 000989943300237

• Developing a Mouse Model to Evaluate Tibial Distraction Osteogenesis DiIorio, S., Tevlin, R., Shah, H. N., Salhotra, A., Griffin, M., Januszyk, M., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2023: S90

  View details for Web of Science ID 000989943300230

• Radiation Injury Genetically Alters Fibroblast Subpopulations to Induce Fibrosis Abbas, D., Griffin, M., Guo, J. L., Lavin, C. V., Fahy, E. J., Guardino, N. J., Lintel, H., Januszyk, M., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2023: S94

  View details for Web of Science ID 000989943300243

• Radiation Injury Therapy with Transdermal Deferoxamine Patch is Dose Dependent Abbas, D., Griffin, M., Lavin, C. V., Fahy, E. J., Lintel, H., Guardino, N. J., Guo, J. L., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2023: S91

  View details for Web of Science ID 000989943300233

• Quantitative Analysis of the Collagen Matrix Ultrastructure in Mouse Hearts after Myocardial Infarction Lu, J., Griffin, M., Guo, J. L., Mascharak, S., Guardino, N. J., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2023: S13-S14

  View details for Web of Science ID 000989943300035

• Overcoming Radiation Induced Oral Fibrosis through the Down Regulation of Wnt Signaling Using Bmp-7 Inhibitors Cotterell, A. C., Griffin, M., Spielman, A., Guardino, N., Lintel, H., Abbas, D., Guo, J. L., Parker, J., Wan, D., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2023: S90

  View details for Web of Science ID 000989943300232

• The effects of mechanical force on fibroblast behavior in cutaneous injury. Frontiers in surgery Berry, C. E., Downer, M., Morgan, A. G., Griffin, M., Liang, N. E., Kameni, L., Laufey Parker, J. B., Guo, J., Longaker, M. T., Wan, D. C. 2023; 10: 1167067

  Abstract

  Wound healing results in the formation of scar tissue which can be associated with functional impairment, psychological stress, and significant socioeconomic cost which exceeds 20 billion dollars annually in the United States alone. Pathologic scarring is often associated with exaggerated action of fibroblasts and subsequent excessive accumulation of extracellular matrix proteins which results in fibrotic thickening of the dermis. In skin wounds, fibroblasts transition to myofibroblasts which contract the wound and contribute to remodeling of the extracellular matrix. Mechanical stress on wounds has long been clinically observed to result in increased pathologic scar formation, and studies over the past decade have begun to uncover the cellular mechanisms that underly this phenomenon. In this article, we will review the investigations which have identified proteins involved in mechano-sensing, such as focal adhesion kinase, as well as other important pathway components that relay the transcriptional effects of mechanical forces, such as RhoA/ROCK, the hippo pathway, YAP/TAZ, and Piezo1. Additionally, we will discuss findings in animal models which show the inhibition of these pathways to promote wound healing, reduce contracture, mitigate scar formation, and restore normal extracellular matrix architecture. Recent advances in single cell RNA sequencing and spatial transcriptomics and the resulting ability to further characterize mechanoresponsive fibroblast subpopulations and the genes that define them will be summarized. Given the importance of mechanical signaling in scar formation, several clinical treatments focused on reducing tension on the wound have been developed and are described here. Finally, we will look toward future research which may reveal novel cellular pathways and deepen our understanding of the pathogenesis of pathologic scarring. The past decade of scientific inquiry has drawn many lines connecting these cellular mechanisms that may lead to a map for the development of transitional treatments for patients on the path to scarless healing.

  View details for DOI 10.3389/fsurg.2023.1167067

  View details for PubMedID 37143767

  View details for PubMedCentralID PMC10151708

• Combination of Distinct Vascular Stem/Progenitor Cells for Neovascularization and Ischemic Rescue. Arteriosclerosis, thrombosis, and vascular biology Zhao, L., Lee, A. S., Sasagawa, K., Sokol, J., Wang, Y., Ransom, R. C., Zhao, X., Ma, C., Steininger, H. M., Koepke, L. S., Borrelli, M. R., Brewer, R. E., Lee, L. L., Huang, X., Ambrosi, T. H., Sinha, R., Hoover, M. Y., Seita, J., Weissman, I. L., Wu, J. C., Wan, D. C., Xiao, J., Longaker, M. T., Nguyen, P. K., Chan, C. K. 2023

  Abstract

  Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia.The ActinCreER/R26VT2/GK3 Rainbow reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA-sequencing was used to characterize distinct vessel-forming populations and their interactions.Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells: VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105highSca1low, which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105lowSca1high and forms stunted vessels and fat. Interestingly, cotransplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice.These findings suggest that autologous cotransplantation of synergistic VSPCs from nonessential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease.

  View details for DOI 10.1161/ATVBAHA.122.317943

  View details for PubMedID 37051932

• Establishing a xenograft model with CD-1 nude mice to study human skin wound repair. Plastic and reconstructive surgery Abbas, D. B., Griffin, M., Fahy, E. J., Spielman, A. F., Guardino, N. J., Pu, A., Lintel, H., Lorenz, H. P., Longaker, M. T., Wan, D. C. 2023

  Abstract

  A significant gap exists in the translatability of small animal models to human subjects. One important factor is poor laboratory models involving human tissue. Thus, we have created a viable postnatal human skin xenograft model using athymic mice.Discarded human foreskins were collected following circumcision. All subcutaneous tissue was removed from these samples sterilely. Host CD-1 nude mice were then anesthetized, and dorsal skin was sterilized. A 1.2cm diameter, full-thickness section of dorsal skin was excised. The foreskin sample was then placed into the full-thickness defect in the host mice and sutured into place. Xenografts underwent dermal wounding using a 4 mm punch biopsy after engraftment. Xenografts were monitored for 14 days after wounding and then harvested.At 14 days postoperatively, all mice survived the procedure. Grossly, the xenograft wounds showed formation of a human scar at POD-14. H&E and Masson Trichome staining confirmed scar formation in the wounded human skin. Using a novel Artificial Intelligence (AI) algorithm using Picrosirius-Red staining, scar formation was confirmed in human wounded skin compared to the unwounded skin. Histologically, CD31 + immunostaining confirmed vascularization of the xenograft. The xenograft exclusively showed human collagen I, CD26 +, and human nuclear antigen in the human scar without any staining of these human markers in the murine skin.The proposed model demonstrates wound healing to be a local response from tissue resident human fibroblasts and allows for reproducible evaluation of human skin wound repair in a preclinical model.

  View details for DOI 10.1097/PRS.0000000000010465

  View details for PubMedID 36988644

• Mechanoresponsive Pancreatic Ductal Adenocarcinoma Cancer Associated Fibroblasts Shows an FAK-Dependent Subtype Divergent from Canonical Fibrotic TGFB-Pathway Dependence Foster, D., Delitto, D., Januszyk, M., Yost, K., Griffin, M., Guo, J., Guardino, N., Delitto, A., Chinta, M., Burcham, A., Nguyen, A., Bauer-, K., Berry, C., Kim, A., Nosrati, F., Wapnir, I., Chang, H., Norton, J. A., Longaker, M. SPRINGER. 2023: S30-S31

  View details for Web of Science ID 001046841200059

• Denervation during mandibular distraction osteogenesis results in impaired bone formation. Scientific reports Tevlin, R., Griffin, M., Chen, K., Januszyk, M., Guardino, N., Spielman, A., Walters, S., Gold, G. E., Chan, C. K., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2023; 13 (1): 2097

  Abstract

  Mandibular distraction osteogenesis (DO) is mediated by skeletal stem cells (SSCs) in mice, which enact bone regeneration via neural crest re-activation. As peripheral nerves are essential to progenitor function during development and in response to injury, we questioned if denervation impairs mandibular DO. C57Bl6 mice were divided into two groups: DO with a segmental defect in the inferior alveolar nerve (IAN) at the time of mandibular osteotomy ("DO Den") and DO with IAN intact ("DO Inn"). DO Den demonstrated significantly reduced histological and radiological osteogenesis relative to DO Inn. Denervation preceding DO results in reduced SSC amplification and osteogenic potential in mice. Single cell RNA sequencing analysis revealed that there was a predominance of innervated SSCs in clusters dominated by pathways related to bone formation. A rare human patient specimen was also analyzed and suggested that histological, radiological, and transcriptional alterations seen in mouse DO may be conserved in the setting of denervated human mandible distraction. Fibromodulin (FMOD) transcriptional and protein expression were reduced in denervated relative to innervated mouse and human mandible regenerate. Finally, when exogenous FMOD was added to DO-Den and DO-Inn SSCs undergoing in vitro osteogenic differentiation, the osteogenic potential of DO-Den SSCs was increased in comparison to control untreated DO-Den SSCs, modeling the superior osteogenic potential of DO-Inn SSCs.

  View details for DOI 10.1038/s41598-023-27921-9

  View details for PubMedID 36747028

• An Inexpensive 3D Printed Mouse Model of Successful, Complication-free Long Bone Distraction Osteogenesis. Plastic and reconstructive surgery. Global open Tevlin, R., Shah, H. N., Salhotra, A., Di Iorio, S. E., Griffin, M., Januszyk, M., Wan, D. C., Longaker, M. T. 2023; 11 (2): e4674

  Abstract

  Distraction osteogenesis (DO) is used for skeletal defects; however, up to 50% of cases exhibit complications. Previous mouse models of long bone DO have been anecdotally hampered by postoperative complications, expense, and availability. To improve clinical techniques, cost-effective, reliable animal models are needed. Our focus was to develop a new mouse tibial distractor, hypothesized to result in successful, complication-free DO.A lightweight tibial distractor was developed using CAD and 3D printing. The device was fixed to the tibia of C57Bl/6J mice prior to osteotomy. Postoperatively, mice underwent 5 days latency, 10 days distraction (0.15 mm every 12 hours), and 28 days consolidation. Bone regeneration was examined on postoperative day 43 using micro-computed tomography (μCT) and Movat's modified pentachrome staining on histology (mineralized volume fraction and pixels, respectively). Costs were recorded. We compared cohorts of 11 mice undergoing sham, DO, or acute lengthening (distractor acutely lengthened 3.0 mm).The histological bone regenerate was significantly increased in DO (1,879,257 ± 155,415 pixels) compared to acute lengthening (32847 ± 1589 pixels) (P < 0.0001). The mineralized volume fraction (bone/total tissue volume) of the regenerate was significantly increased in DO (0.9 ± 0.1) compared to acute lengthening (0.7 ± 0.1) (P < 0.001). There was no significant difference in bone regenerate between DO and sham. The distractor was relatively low cost ($11), with no complications.Histology and µCT analysis confirmed that the proposed tibial DO model resulted in successful bone formation. Our model is cost-effective and reproducible, enabling implementation in genetically dissectible transgenic mice.

  View details for DOI 10.1097/GOX.0000000000004674

  View details for PubMedID 36798717

  View details for PubMedCentralID PMC9925097

• Chelating the valley of death: Deferoxamine's path from bench to wound clinic. Frontiers in medicine Parker, J. B., Griffin, M. F., Downer, M. A., Akras, D., Berry, C. E., Cotterell, A. C., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2023; 10: 1015711

  Abstract

  There is undisputable benefit in translating basic science research concretely into clinical practice, and yet, the vast majority of therapies and treatments fail to achieve approval. The rift between basic research and approved treatment continues to grow, and in cases where a drug is granted approval, the average time from initiation of human trials to regulatory marketing authorization spans almost a decade. Albeit with these hurdles, recent research with deferoxamine (DFO) bodes significant promise as a potential treatment for chronic, radiation-induced soft tissue injury. DFO was originally approved by the Food and Drug Administration (FDA) in 1968 for the treatment of iron overload. However, investigators more recently have posited that its angiogenic and antioxidant properties could be beneficial in treating the hypovascular and reactive-oxygen species-rich tissues seen in chronic wounds and radiation-induced fibrosis (RIF). Small animal experiments of various chronic wound and RIF models confirmed that treatment with DFO improved blood flow and collagen ultrastructure. With a well-established safety profile, and now a strong foundation of basic scientific research that supports its potential use in chronic wounds and RIF, we believe that the next steps required for DFO to achieve FDA marketing approval will include large animal studies and, if those prove successful, human clinical trials. Though these milestones remain, the extensive research thus far leaves hope for DFO to bridge the gap between bench and wound clinic in the near future.

  View details for DOI 10.3389/fmed.2023.1015711

  View details for PubMedID 36873870

• Bioprinted Hydrogels for Fibrosis and Wound Healing: Treatment and Modeling. Gels (Basel, Switzerland) Guo, J. L., Longaker, M. T. 2022; 9 (1)

  Abstract

  Three-dimensional (3D) printing has been used to fabricate biomaterial scaffolds with finely controlled physical architecture and user-defined patterning of biological ligands. Excitingly, recent advances in bioprinting have enabled the development of highly biomimetic hydrogels for the treatment of fibrosis and the promotion of wound healing. Bioprinted hydrogels offer more accurate spatial recapitulation of the biochemical and biophysical cues that inhibit fibrosis and promote tissue regeneration, augmenting the therapeutic potential of hydrogel-based therapies. Accordingly, bioprinted hydrogels have been used for the treatment of fibrosis in a diverse array of tissues and organs, including the skin, heart, and endometrium. Furthermore, bioprinted hydrogels have been utilized for the healing of both acute and chronic wounds, which present unique biological microenvironments. In addition to these therapeutic applications, hydrogel bioprinting has been used to generate in vitro models of fibrosis in a variety of soft tissues such as the skin, heart, and liver, enabling high-throughput drug screening and tissue analysis at relatively low cost. As biological research begins to uncover the spatial biological features that underlie fibrosis and wound healing, bioprinting offers a powerful toolkit to recapitulate spatially defined pro-regenerative and anti-fibrotic cues for an array of translational applications.

  View details for DOI 10.3390/gels9010019

  View details for PubMedID 36661787

• Multiplexed evaluation of mouse wound tissue using oligonucleotide barcoding with single-cell RNA sequencing. STAR protocols Januszyk, M., Griffin, M., Mascharak, S., Talbott, H. E., Chen, K., Henn, D., Spielman, A. F., Parker, J. B., Liang, N. E., Cotterell, A., Guardino, N., Foster, D. S., Wagh, D., Coller, J., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2022; 4 (1): 101946

  Abstract

  Despite its rapidly increased availability for the study of complex tissue, single-cell RNA sequencing remains prohibitively expensive for large studies. Here, we present a protocol using oligonucleotide barcoding for the tagging and pooling of multiple samples from healing wounds, which are among the most challenging tissue types for this application. We describe steps to generate skin wounds in mice, followed by tissue harvest and oligonucleotide barcoding. This protocol is also applicable to other species including rats, pigs, and humans. For complete details on the use and execution of this protocol, please refer to Stoeckius etal. (2018),1 Galiano etal. (2004),2 and Mascharak etal. (2022).3.

  View details for DOI 10.1016/j.xpro.2022.101946

  View details for PubMedID 36525348

• Topical vanadate improves tensile strength and alters collagen organization of excisional wounds in a mouse model. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society Lintel, H., Abbas, D. B., Mackay, D. J., Griffin, M., Lavin, C. V., Berry, C. E., Guardino, N. J., Guo, J. L., Momeni, A., Mackay, D. R., Longaker, M. T., Wan, D. C. 2022

  Abstract

  Wound dehiscence, oftentimes a result of the poor tensile strength of early healing wounds, is a significant threat to the postoperative patient, potentially causing life-threatening complications. Vanadate, a protein tyrosine phosphatase inhibitor, has been shown to alter the organization of deposited collagen in healing wounds and significantly improve the tensile strength of incisional wounds in rats. In this study, we sought to explore the effects of locally administered vanadate on tensile strength and collagen organization in both the early and remodeling phases of excisional wound healing in a murine model. Wild-type mice underwent stented excisional wounding on their dorsal skin and were divided equally into three treatment conditions: vanadate injection, saline injection control, and an untreated control. Tensile strength testing, in vivo suction Cutometer analysis, gross wound measurements, and histologic analysis were performed during healing, immediately upon wound closure, and after four weeks of remodeling. We found that vanadate treatment significantly increased the tensile strength of wounds and their stiffness relative to control wounds, both immediately upon healing and into the remodeling phase. Histologic analysis revealed that these biomechanical changes were likely the result of increased collagen deposition and an altered collagen organization composed of thicker and distinctly organized collagen bundles. Given the risk that dehiscence poses to all operative patients, vanadate presents an interesting therapeutic avenue to improve the strength of post-operative wounds and unstable chronic wounds in order to reduce the risk of dehiscence.

  View details for DOI 10.1111/wrr.13062

  View details for PubMedID 36484112

• Macrophage inflammatory and regenerative response periodicity is programmed by cell cycle and chromatin state. Molecular cell Daniel, B., Belk, J. A., Meier, S. L., Chen, A. Y., Sandor, K., Czimmerer, Z., Varga, Z., Bene, K., Buquicchio, F. A., Qi, Y., Kitano, H., Wheeler, J. R., Foster, D. S., Januszyk, M., Longaker, M. T., Chang, H. Y., Satpathy, A. T. 2022

  Abstract

  Cell cycle (CC) facilitates cell division via robust, cyclical gene expression. Protective immunity requires the expansion of pathogen-responsive cell types, but whether CC confers unique gene expression programs that direct the subsequent immunological response remains unclear. Here, we demonstrate that single macrophages (MFs) adopt different plasticity states in CC, which leads to heterogeneous cytokine-induced polarization, priming, and repolarization programs. Specifically, MF plasticity to interferon gamma (IFNG) is substantially reduced during S-G2/M, whereas interleukin 4 (IL-4) induces S-G2/M-biased gene expression, mediated by CC-biased enhancers. Additionally, IL-4 polarization shifts the CC-phase distribution of MFs toward the G2/M phase, providing a subpopulation-specific mechanism for IL-4-induced, dampened IFNG responsiveness. Finally, we demonstrate CC-dependent MF responses in murine and human disease settings invivo, including Th2-driven airway inflammation and pulmonary fibrosis, where MFs express an S-G2/M-biased tissue remodeling gene program. Therefore, MF inflammatory and regenerative responses are gated by CC in a cyclical, phase-dependent manner.

  View details for DOI 10.1016/j.molcel.2022.11.017

  View details for PubMedID 36521490

• Standardized Quantitative Sensory Testing to assess insufficient recovery of touch discrimination in free flap surgery in extremity reconstruction. Plastic and reconstructive surgery Huber, J., Scharberth, A., Maier, C., Wallner, C., Wagner, J. M., Dadras, M., Longaker, M. T., Lehnhardt, M., Behr, B. 2022

  Abstract

  BACKGROUND: With major advances in microsurgical techniques, free tissue transfer has become a widely adopted approach to treat complex soft tissue defects. However, sensory recovery is poor leaving the anaesthetic skin prone to injuries.METHODS: 28 patients with 22 anterior lateral thigh flaps and 6 latissimus dorsi flaps on their extremities participated in the study. Quantitative sensory testing and 2 points discrimination was performed in three test areas and one control on the contralateral unaffected extremity. Physical disability, mental health, quality of life and characteristics of pain were assessed by the painDetect, DASH, LEFS and sf12 questionnaires, respectively.RESULTS: Somatosensory profiles of all flaps were characterized by an overall loss of nerve function. Small-fibre function was mostly recovered while large-fibre function and thus touch discrimination was severely impaired. Mechanical detection thresholds improved over time and from periphery to the centre. Reported pain was mild to moderate and correlated with decreased physical function.CONCLUSION: Standardized Quantitative sensory testing provides a useful tool kit to assess the sensory regeneration after surgical treatment of soft tissue defects. After free tissue transfer small-fibre function recovers with nerve ingrowth in a centripetal direction from the flap margins to the centre, likely via collateral axonal sprouting from the undamaged nerves surrounding the flap. Myelinated fibres recover slowly and inefficiently.

  View details for DOI 10.1097/PRS.0000000000009860

  View details for PubMedID 36374559

• Adipose-Derived Stromal Cell-based Therapies for Radiation-Induced Fibrosis. Advances in wound care Berry, C., Abbas, D. B., Lintel, H., Churukian, A., Griffin, M., Guo, J., Cotterell, A. C., Parker, J., Downer, M. A., Longaker, M. T., Wan, D. C. 2022

  Abstract

  SIGNIFICANCE: Half of all cancer patients receive radiation therapy as a component of their treatment regimen, and the most common resulting complication is radiation-induced fibrosis of the skin and soft tissue. This thickening of the dermis paired with decreased vascularity results in functional limitations, aesthetic concerns, and poses unique challenges when considering surgical exploration or reconstruction. Existing therapeutic options for radiation-induced fibrosis of the skin are limited both in scope and efficacy. Cell-based therapies have emerged as a promising means of utilizing regenerative cell populations to improve both functional and aesthetic outcomes, and even as prophylaxis for radiation-induced fibrosis.RECENT ADVANCES: As one of the leading areas of cell-based therapy research, adipose-derived stromal cells (ADSCs) demonstrate significant therapeutic potential in the treatment of radiation-induced fibrosis (RIF). The introduction of the ADSC-augmented fat graft has shown clinical utility. Recent research dedicated to characterizing specific ADSC subpopulations points toward further granularity in understanding of the mechanisms driving the well-established clinical outcomes seen with fat grafting therapy.CRITICAL ISSUES: Various animal models of radiation-induced fibrosis demonstrated improved clinical outcomes following treatment with cell-based therapies, but the cellular and molecular basis underlying these effects remains poorly understood.FUTURE DIRECTIONS: Recent literature has focused on improving the efficacy of cell-based therapies, most notably through 1) augmentation of fat grafts with platelet-rich plasma and 2) the modification of expressed RNA through epitranscriptomics. For the latter, new and promising gene targets continue to be identified which have the potential to reverse the effects of fibrosis by increasing angiogenesis, decreasing inflammation, and promoting adipogenesis.

  View details for DOI 10.1089/wound.2022.0103

  View details for PubMedID 36345216

• Machine Learning-Based Desmoplastic Signatures Predict Patient Outcomes in Pancreatic Ductal Adenocarcinoma Guo, J. L., Mascharak, S., Foster, D. S., Guardino, N. J., Griffin, M., Miller, E., Raghavan, S., Longacre, T. A., Norton, J. A., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2022: S53-S54

  View details for Web of Science ID 000867877000135

• Inhibition of Yes-Associated Protein Promotes Skin Wound Regeneration in Large Animals Januszyk, M., Talbott, H. E., Griffin, M., Guardino, N., Spielman, A., Guo, J. L., Mascharak, S., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2022: S196

  View details for DOI 10.1097/01.XCS.0000894508.92389.a2

  View details for Web of Science ID 000867889300379

• Denervation During Mouse and Human Mandibular Distraction Osteogenesis Results in Impaired Osteogenesis Tevlin, R., Griffin, M., Chen, K., Januszyk, M., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2022: S202

  View details for DOI 10.1097/01.XCS.0000894560.64479.80

  View details for Web of Science ID 000867889300393

• Adipocytes the Forgotten Culprit in Skin Fibrosis: Exploring the Mechanism of Fat Driven Skin Fibrosis Griffin, M., Guardino, N., Spielman, A. F., Mascharak, S., Parker, J. L., Guo, J. L., Abbas, D., Wan, D. C., Bauer-Rowe, K. E., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2022: S199

  View details for DOI 10.1097/01.XCS.0000894536.38023.90

  View details for Web of Science ID 000867889300387

• Adipocyte Progenitor Cells Embedded in Collagen Gels Accelerate Bone Formation in a Murine Calvarial Critical Defect Model Cotterell, A. C., Griffin, M., Guardino, N., Spielman, A. F., Lintel, H., Abbas, D., Guo, J. L., Parker, J. L., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2022: S198-S199

  View details for DOI 10.1097/01.XCS.0000894532.90979.c9

  View details for Web of Science ID 000867889300386

• Transdermal Deferoxamine Improves Acute Wound Healing in Chronic Irradiated Skin in a Mouse Model Lintel, H., Abbas, D., Lavin, C., Griffin, M., Guo, J. L., Guardino, N., Churukian, A., Chen, K., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2022: S211-S212

  View details for DOI 10.1097/01.XCS.0000894656.51739.b7

  View details for Web of Science ID 000867889300416

• Fibroblast Subpopulations Are Modified with Fat Grafting to Treat Radiation-Induced Fibrosis Abbas, D. B., Guo, J. L., Griffin, M., Lintel, H., Guardino, N. J., Berry, C. E., Pu, A., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2022: S202-S203

  View details for DOI 10.1097/01.XCS.0000894568.35895.12

  View details for Web of Science ID 000867889300395

• Semantics Matter: Cheiloschisis Web-Based Information Differs from Cleft Lip Abbas, D. B., Lintel, H., Guardino, N. J., Griffin, M., Guo, J. L., Cotterell, A. C., Spielman, A. F., Parker, J. L., Longaker, M. T., Wan, D. C. LIPPINCOTT WILLIAMS & WILKINS. 2022: S209

  View details for DOI 10.1097/01.XCS.0000894628.90579.08

  View details for Web of Science ID 000867889300410

• Where There Is Fat There Is Fibrosis: Elucidating the Mechanisms of Creeping Fat-Driven Stricture Formation Bauer-Rowe, K. E., Kim, A., Griffin, M., Foster, D., Guardino, N., Guo, J. L., Talbott, H. E., Norton, J. A., Hyun, J. S., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2022: S59-S60

  View details for DOI 10.1097/01.XCS.0000893384.03562.27

  View details for Web of Science ID 000867889300098

• Multi-Modal Analysis of Cell Populations and Architectural States Mediating the Progression and Resolution of Pulmonary Fibrosis Guo, J. L., Griffin, M., Guardino, N. J., Abbas, D., Lu, J., Spielman, A., Lintel, H., Cotterell, A. C., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2022: S82

  View details for Web of Science ID 000867877000204

• Engrailed-Positive Fibroblasts: The Primary Cell Type Present in Fibrotic Capsules During Foreign Body Response Parker, J. B., Griffin, M., Mascharak, S., Spielman, A., Cotterell, A. C., Abbas, D., Lintel, H., Januszyk, M., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2022: S68

  View details for Web of Science ID 000867877000171

• Reversal of Senescence in Skin-derived Fibroblasts Using Exogenous Mechanical Stimulation Guo, J. L., Griffin, M., Guardino, N. J., Chen, K., Cotterell, A. C., Abbas, D., Spielman, A., Lintel, H., Wan, D. C., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2022: S70

  View details for Web of Science ID 000867877000176

• Sexually dimorphic estrogen sensing in skeletal stem cells controls skeletal regeneration. Nature communications Andrew, T. W., Koepke, L. S., Wang, Y., Lopez, M., Steininger, H., Struck, D., Boyko, T., Ambrosi, T. H., Tong, X., Sun, Y., Gulati, G. S., Murphy, M. P., Marecic, O., Telvin, R., Schallmoser, K., Strunk, D., Seita, J., Goodman, S. B., Yang, F., Longaker, M. T., Yang, G. P., Chan, C. K. 2022; 13 (1): 6491

  Abstract

  Sexually dimorphic tissues are formed by cells that are regulated by sex hormones. While a number of systemic hormones and transcription factors are known to regulate proliferation and differentiation of osteoblasts and osteoclasts, the mechanisms that determine sexually dimorphic differences in bone regeneration are unclear. To explore how sex hormones regulate bone regeneration, we compared bone fracture repair between adult male and female mice. We found that skeletal stem cell (SSC) mediated regeneration in female mice is dependent on estrogen signaling but SSCs from male mice do not exhibit similar estrogen responsiveness. Mechanistically, we found that estrogen acts directly on the SSC lineage in mice and humans by up-regulating multiple skeletogenic pathways and is necessary for the stem cell's ability to self- renew and differentiate. Our results also suggest a clinically applicable strategy to accelerate bone healing using localized estrogen hormone therapy.

  View details for DOI 10.1038/s41467-022-34063-5

  View details for PubMedID 36310174

• Multiomic analysis reveals conservation of cancer-associated fibroblast phenotypes across species and tissue of origin. Cancer cell Foster, D. S., Januszyk, M., Delitto, D., Yost, K. E., Griffin, M., Guo, J., Guardino, N., Delitto, A. E., Chinta, M., Burcham, A. R., Nguyen, A. T., Bauer-Rowe, K. E., Titan, A. L., Salhotra, A., Jones, R. E., da Silva, O., Lindsay, H. G., Berry, C. E., Chen, K., Henn, D., Mascharak, S., Talbott, H. E., Kim, A., Nosrati, F., Sivaraj, D., Ransom, R. C., Matthews, M., Khan, A., Wagh, D., Coller, J., Gurtner, G. C., Wan, D. C., Wapnir, I. L., Chang, H. Y., Norton, J. A., Longaker, M. T. 2022

  Abstract

  Cancer-associated fibroblasts (CAFs) are integral to the solid tumor microenvironment. CAFs were once thought to be a relatively uniform population of matrix-producing cells, but single-cell RNA sequencing has revealed diverse CAF phenotypes. Here, we further probed CAF heterogeneity with a comprehensive multiomics approach. Using paired, same-cell chromatin accessibility and transcriptome analysis, we provided an integrated analysis of CAF subpopulations over a complex spatial transcriptomic and proteomic landscape to identify three superclusters: steady state-like (SSL), mechanoresponsive (MR), and immunomodulatory (IM) CAFs. These superclusters are recapitulated across multiple tissue types and species. Selective disruption of underlying mechanical force or immune checkpoint inhibition therapy results in shifts in CAF subpopulation distributions and affected tumor growth. As such, the balance among CAF superclusters may have considerable translational implications. Collectively, this research expands our understanding of CAF biology, identifying regulatory pathways in CAF differentiation and elucidating therapeutic targets in a species- and tumor-agnostic manner.

  View details for DOI 10.1016/j.ccell.2022.09.015

  View details for PubMedID 36270275

• Characterization of Mechanoresponsive Inflammatory Cells during Wound Healing Chen, K., Griffin, M., Henn, D., Bonham, C. A., Fischer, K., Padmanabhan, J., Trotsyuk, A. A., Sivaraj, D., Leeolou, M., Kussie, H. C., Huskins, S., Steele, S., Perrault, D., Longaker, M. T., Gurtner, G. C. WILEY. 2022: A22

  View details for Web of Science ID 000847524300054

• Machine Learning in Tissue Engineering. Tissue engineering. Part A Guo, J. L., Januszyk, M., Longaker, M. T. 2022

  Abstract

  Machine learning (ML) and artificial intelligence have accelerated scientific discovery, augmented clinical practice, and deepened fundamental understanding of many biological phenomena. ML technologies have now been applied to diverse areas of tissue engineering research, including biomaterial design, scaffold fabrication, and cell/tissue modeling. Emerging ML-empowered strategies include machine-optimized polymer synthesis, predictive modeling of scaffold fabrication processes, complex analyses of structure-function relationships, and deep learning of spatialized cell phenotypes and tissue composition. The emergence of ML in tissue engineering, while relatively recent, has already enabled increasingly complex and multivariate analyses of the relationships between biological, chemical, and physical factors in driving tissue regenerative outcomes. This review highlights the novel methodologies, emerging strategies, and areas of potential growth within this rapidly evolving area of research.

  View details for DOI 10.1089/ten.TEA.2022.0128

  View details for PubMedID 35943870

• Wound healing, fibroblast heterogeneity, and fibrosis. Cell stem cell Talbott, H. E., Mascharak, S., Griffin, M., Wan, D. C., Longaker, M. T. 2022; 29 (8): 1161-1180

  Abstract

  Fibroblasts are highly dynamic cells that play a central role in tissue repair and fibrosis. However, the mechanisms by which they contribute to both physiologic and pathologic states of extracellular matrix deposition and remodeling are just starting to be understood. In this review article, we discuss the current state of knowledge in fibroblast biology and heterogeneity, with a primary focus on the role of fibroblasts in skin wound repair. We also consider emerging techniques in the field, which enable an increasingly nuanced and contextualized understanding of these complex systems, and evaluate limitations of existing methodologies and knowledge. Collectively, this review spotlights a diverse body of research examining an often-overlooked cell type-the fibroblast-and its critical functions in wound repair and beyond.

  View details for DOI 10.1016/j.stem.2022.07.006

  View details for PubMedID 35931028

• Exploring the Overlooked Roles and Mechanisms of Fibroblasts in the Foreign Body Response. Advances in wound care Parker, J., Griffin, M., Spielman, A. F., Wan, D. C., Longaker, M. T. 2022

  Abstract

  Significance Foreign body response (FBR), wherein a fibrotic capsule forms around an implanted structure, is a common surgical complication that often leads to pain, discomfort, and eventual revision surgeries. Though believed to have some mechanistic overlap with normal wound healing, much remains to be discovered about the specific mechanism by which this occurs. Recent Advances Current understanding of FBR has focused on the roles of the immune system and the biomaterial, both major contributors to FBR. However, another key player, the fibroblast, is often overlooked. This review summarizes key contributors of FBR, focusing on the roles of fibroblasts. As much remains to be discovered about fibroblasts' specific roles in FBR, we draw on current knowledge of fibroblast subpopulations and functions during wound healing. We also provide an overview on candidate biomaterials and signalling pathways involved in FBR. Critical Issues and Future Directions While the global implantable medical devices market is considerable and continues to appreciate in value, FBR remains one of the most common surgical implant complications. In parallel with the continued development of candidate biomaterials, further exploration of potential fibroblast subpopulations at a transcriptional level would provide key insights into further understanding the underlying mechanisms by which fibrous encapsulation occurs, and unveil novel directions for anti-fibrotic and regenerative therapies in the future.

  View details for DOI 10.1089/wound.2022.0066

  View details for PubMedID 35819293

• Transdermal deferoxamine administration improves excisional wound healing in chronically irradiated murine skin. Journal of translational medicine Lintel, H., Abbas, D. B., Lavin, C. V., Griffin, M., Guo, J. L., Guardino, N., Churukian, A., Gurtner, G. C., Momeni, A., Longaker, M. T., Wan, D. C. 2022; 20 (1): 274

  Abstract

  BACKGROUND: Radiation-induced skin injury is a well-known risk factor for impaired wound healing. Over time, the deleterious effects of radiation on skin produce a fibrotic, hypovascular dermis poorly suited to wound healing. Despite increasing understanding of the underlying pathophysiology, therapeutic options remain elusive. Deferoxamine (DFO), an iron-chelating drug, has been shown in prior murine studies to ameliorate radiation-induced skin injury as well as improve wound healing outcomes in various pathologic conditions when administered transdermally. In this preclinical study, we evaluated the effects of deferoxamine on wound healing outcomes in chronically irradiated murine skin.METHODS: Wild-type mice received 30Gy of irradiation to their dorsal skin and were left to develop chronic fibrosis. Stented excisional wounds were created on their dorsal skin. Wound healing outcomes were compared across 4 experimental conditions: DFO patch treatment, vehicle-only patch treatment, untreated irradiated wound, and untreated nonirradiated wounds. Gross closure rate, wound perfusion, scar elasticity, histology, and nitric oxide assays were compared across the conditions.RESULTS: Relative to vehicle and untreated irradiated wounds, DFO accelerated wound closure and reduced the frequency of healing failure in irradiated wounds. DFO augmented wound perfusion throughout healing and upregulated angiogenesis to levels observed in nonirradiated wounds. Histology revealed DFO increased wound thickness, collagen density, and improved collagen fiber organization to more closely resemble nonirradiated wounds, likely contributing to the observed improved scar elasticity. Lastly, DFO upregulated inducible nitric oxide synthase and increased nitric oxide production in early healing wounds.CONCLUSION: Deferoxamine treatment presents a potential therapeutic avenue through which to target impaired wound healing in patients following radiotherapy.

  View details for DOI 10.1186/s12967-022-03479-4

  View details for PubMedID 35715816

• Profibrotic Signaling Pathways and Surface Markers Are Upregulated in Fibroblasts of Human Striae Distensae and in a Mouse Model System. Plastic and reconstructive surgery Borrelli, M. R., Griffin, M., Chen, K., Diaz, N. M., Adem, S., Mascharak, S., Shen, A. H., Ngaage, L. M., Lewis, N., Longaker, M. T., Gurtner, G., Wan, D. C., Lorenz, H. P. 2022

  Abstract

  INTRODUCTION: Striae distensae (SD) are common disfiguring cutaneous lesions but lack effective treatments due to an incomplete understanding of their pathophysiology. Dermal fibroblasts likely play an important role. We investigate the cellular-molecular features distinguishing fibroblasts from human SD and normal skin (NS). We also develop a mouse model of SD.METHODS: Human SD and NS samples were compared for tensile strength and histological structure. Fibroblasts from SD and NS were isolated by fluorescence-activated cell sorting (FACS) for gene expression analysis. Immunofluorescence staining and FACS were used to confirm gene expression data at the protein level. A mouse model of SD formation was created by administering corticosteroids and mechanically loading the dorsal skin.RESULTS: Human SD exhibited reduced tensile strength, more disordered collagen fibers, and epidermal atrophy compared to human NS. There were 296 upregulated genes in SD fibroblasts, including the profibrotic lineage and surface marker CD26. Upregulated genes were involved in profibrotic and mechanoresponsive signaling pathways (TGFbeta and FAK-PI3-AKT-signaling). In contrast, 571 genes were downregulated, including CD74 and genes of the AMPK pathway. Increased CD26 and decreased CD74 expression was confirmed by FACS and immunofluorescence. Similar cutaneous histological and gene expression changes were induced in hypercortisolemic mice by mechanically loading the dorsal skin.CONCLUSIONS: Fibroblasts from human SD exhibit increased profibrotic and decreased antifibrotic signaling. CD26 and CD74 are promising surface markers that may be targeted therapeutically. Our mouse model of SD can be used as a platform to test the efficacy of potential therapeutic agents.

  View details for DOI 10.1097/PRS.0000000000009363

  View details for PubMedID 35666152

• Beyond the Scar: A Basic Science Review of Wound Remodeling. Advances in wound care Spielman, A. F., Griffin, M., Parker, J., Cotterell, A. C., Wan, D. C., Longaker, M. T. 2022

  Abstract

  SIGNIFICANCE: Increasing development of experimental animal models has allowed for the study of scar formation. However, many pathophysiological unknowns remain in the longest stage of healing, the remodeling stage, which may continue for a year or more. The wound healing process results in different types of scarring classified as normal or pathological depending on failures at each stage. Failures can also occur during wound remodeling, but the molecular mechanisms driving the wound remodeling process have yet to be investigated.RECENT ADVANCES: While current understanding of wound repair is based on investigations of acute healing, these experimental models have informed knowledge of key components of remodeling. This review examines the components that contribute to collagen organization and the final scar, including cell types, their regulation, and signaling pathways. Dysregulation in any one of these components causes pathologic healing.CRITICAL ISSUES AND FUTURE DIRECTIONS: As wounds continue to remodel months to years after re-epithelization, new models to better understand long-term remodeling will be critical for improving healing outcomes. Further investigation of the contributions of fibroblasts and cell signaling pathways involved during remodeling as well as their potential failures may inform new approaches in promoting regenerative healing beyond re-epithelization.

  View details for DOI 10.1089/wound.2022.0049

  View details for PubMedID 35658581

• Partial Tendon Injury at the Tendon-to-Bone Enthesis Activates Skeletal Stem Cells. Stem cells translational medicine Titan, A. L., Davitt, M., Foster, D., Salhotra, A., Menon, S., Chen, K., Fahy, E., Lopez, M., Jones, R. E., Baiu, I., Burcham, A., Januszyk, M., Gurtner, G., Fox, P., Chan, C., Quarto, N., Longaker, M. 2022

  Abstract

  The tendon enthesis plays a critical role in facilitating movement and reducing stress within joints. Partial enthesis injuries heal in a mechanically inferior manner and never achieve healthy tissue function. The cells responsible for tendon-to-bone healing remain incompletely characterized and their origin is unknown. Here, we evaluated the putative role of mouse skeletal stem cells (mSSCs) in the enthesis after partial-injury. We found that mSSCs were present at elevated levels within the enthesis following injury and that these cells downregulated TGFβ signaling pathway elements at both the RNA and protein levels. Exogenous application of TGFβ post-injury led to a reduced mSSC response and impaired healing, whereas treatment with a TGFβ inhibitor (SB43154) resulted in a more robust mSSC response. Collectively, these data suggest that mSSCs may augment tendon-to-bone healing by dampening the effects of TGFβ signaling within the mSSC niche.

  View details for DOI 10.1093/stcltm/szac027

  View details for PubMedID 35640155

• Disrupting mechanotransduction decreases fibrosis and contracture in split-thickness skin grafting. Science translational medicine Chen, K., Henn, D., Januszyk, M., Barrera, J. A., Noishiki, C., Bonham, C. A., Griffin, M., Tevlin, R., Carlomagno, T., Shannon, T., Fehlmann, T., Trotsyuk, A. A., Padmanabhan, J., Sivaraj, D., Perrault, D. P., Zamaleeva, A. I., Mays, C. J., Greco, A. H., Kwon, S. H., Leeolou, M. C., Huskins, S. L., Steele, S. R., Fischer, K. S., Kussie, H. C., Mittal, S., Mermin-Bunnell, A. M., Diaz Deleon, N. M., Lavin, C., Keller, A., Longaker, M. T., Gurtner, G. C. 2022; 14 (645): eabj9152

  Abstract

  Burns and other traumatic injuries represent a substantial biomedical burden. The current standard of care for deep injuries is autologous split-thickness skin grafting (STSG), which frequently results in contractures, abnormal pigmentation, and loss of biomechanical function. Currently, there are no effective therapies that can prevent fibrosis and contracture after STSG. Here, we have developed a clinically relevant porcine model of STSG and comprehensively characterized porcine cell populations involved in healing with single-cell resolution. We identified an up-regulation of proinflammatory and mechanotransduction signaling pathways in standard STSGs. Blocking mechanotransduction with a small-molecule focal adhesion kinase (FAK) inhibitor promoted healing, reduced contracture, mitigated scar formation, restored collagen architecture, and ultimately improved graft biomechanical properties. Acute mechanotransduction blockade up-regulated myeloid CXCL10-mediated anti-inflammation with decreased CXCL14-mediated myeloid and fibroblast recruitment. At later time points, mechanical signaling shifted fibroblasts toward profibrotic differentiation fates, and disruption of mechanotransduction modulated mesenchymal fibroblast differentiation states to block those responses, instead driving fibroblasts toward proregenerative, adipogenic states similar to unwounded skin. We then confirmed these two diverging fibroblast transcriptional trajectories in human skin, human scar, and a three-dimensional organotypic model of human skin. Together, pharmacological blockade of mechanotransduction markedly improved large animal healing after STSG by promoting both early, anti-inflammatory and late, regenerative transcriptional programs, resulting in healed tissue similar to unwounded skin. FAK inhibition could therefore supplement the current standard of care for traumatic and burn injuries.

  View details for DOI 10.1126/scitranslmed.abj9152

  View details for PubMedID 35584231

• Pullulan-Collagen Hydrogel Wound Dressing Promotes Dermal Remodeling and Wound Healing Compared to Commercially Available Collagen Dressings. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society Chen, K., Sivaraj, D., Davitt, M., Leeolou, M. C., Henn, D., Steele, S. R., Huskins, S. L., Trotsyuk, A. A., Kussie, H. C., Greco, A., Padmanabhan, J., Perrault, D. P., Zamaleeva, A. I., Longaker, M. T., Gurtner, G. C. 2022

  Abstract

  Biological scaffolds such as hydrogels provide an ideal, physio-mimetic of native ECM that can improve wound healing outcomes after cutaneous injury. While most studies have focused on the benefits of hydrogels in accelerating wound healing, there is minimal data directly comparing different hydrogel material compositions. In this study, we utilized a splinted excisional wound model that recapitulates human-like wound healing in mice and treated wounds with three different collagen hydrogel dressings. We assessed the feasibility of applying each dressing and performed histologic and histopathologic analysis on the explanted scar tissues to assess variations in collagen architecture and alignment, as well as tissue response. Our data indicate that the material properties of hydrogel dressings can significantly influence healing time, cellular response, and resulting architecture of healed scars. Specifically, our pullulan-collagen hydrogel dressing accelerated wound closure and promoted healed tissue with less dense, more randomly aligned, and shorter collagen fibers. Further understanding of how hydrogel properties affect the healing and resulting scar architecture of wounds may lead to novel insights and further optimization of the material properties of wound dressings. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1111/wrr.13012

  View details for PubMedID 35384131

• REGENERATION OF CARTILAGE THOUGH ACTIVATION OF TISSUE RESIDENT SKELETAL STEM CELLS AND AUGMENTATION OF THE NICHE Murphy, M. P., Koepke, L. S., Lopez, M. T., Tong, X., Ambrosi, T. H., Gulati, G., Marecic, O., Wang, Y., Ransom, R. C., Hoover, M., Longaker, M. T., Chan, C. F. MARY ANN LIEBERT, INC. 2022: S375

  View details for Web of Science ID 000821187301490

• Fat Grafts Augmented With Vitamin E Improve Volume Retention and Radiation-Induced Fibrosis. Aesthetic surgery journal Abbas, D. B., Lavin, C. V., Fahy, E. J., Griffin, M., Guardino, N. J., Nazerali, R. S., Nguyen, D. H., Momeni, A., Longaker, M. T., Wan, D. C. 2022

  Abstract

  Treatments for radiation-induced fibrosis range from vitamin E and pentoxifylline systemically to deferoxamine and fat grafting locally. Regarding fat grafting, volume retention hinders its long-term functionality and is affected by two factors: inflammation and necrosis secondary to hypovascularity.We aimed to simultaneously improve fat graft retention and radiation-induced fibrosis by integrating vitamin E and pentoxifylline into fat grafts locally.Forty adult CD-1 nude male mice at 6 weeks of age underwent scalp irradiation and recovered for four weeks to allow for the development of fibrosis. Mice received 200μL of donor human fat graft to the scalp. Mice were separated into 4 conditions: no grafting, fat graft without treatment, graft treated with pentoxifylline, and graft treated with vitamin E. Fat graft volume retention was monitored in-vivo using microCT scans at weeks 0, 1, 2, 4, 6, and 8 after grafting. Histological and cytokine analysis of the scalp skin and fat grafts were also performed.Vitamin E (VE) treated grafts had significant improvement in dermal thickness and collagen density of overlying skin compared to all other groups. VE decreased 8-isoprostane and increased CD31 + staining compared to the other grafted groups. Cytokine analysis revealed decreased inflammatory and increased angiogenic markers in both the fat graft and overlying skin of the vitamin E group. Fat graft volume retention was significantly improved in the vitamin E group starting at 1 week post grafting.Radiation-induced fibrosis and fat graft volume retention are both simultaneously improved with local administration of vitamin E.

  View details for DOI 10.1093/asj/sjac066

  View details for PubMedID 35350074

• Mechanical Stimulation Reverses Pro-Fibrotic Transcriptional States in Senescent Fibroblasts Guo, J. L., Griffin, M., Guardino, N., Chen, K., Gurtner, G. C., Longaker, M. T. WILEY. 2022: A33-A34

  View details for Web of Science ID 000763583000075

• Characterization of Mechanoresponsive Inflammatory Cells during Wound Healing Chen, K., Griffin, M., Henn, D., Bonham, C. A., Fischer, K., Padmanabhan, J., Trotsyuk, A. A., Sivaraj, D., Leeolou, M. C., Kussie, H. C., Huskins, S. L., Steele, S., Perrault, D., Longaker, M. T., Gurtner, G. C. WILEY. 2022: A5

  View details for Web of Science ID 000763583000021

• Overcoming Radiation Induced Oral Fibrosis Through The Down Regulation Of WNT Signaling Using BMP-7 Inhibitors Guardino, N., Griffin, M., Spielman, A. F., Abbas, D. B., King, M., Parker, J., Wan, D., Longaker, M. T. WILEY. 2022: A5

  View details for Web of Science ID 000763583000022

• Adipocytes Transition To Pro-Fibrotic Fibroblasts And Contribute To Muscle Fibrosis Following Nerve Injury Spielman, A. F., Griffin, M., Guardino, N., desJardins-Park, H. E., Bauer-Rowe, K. E., Guo, J. L., Parker, J., Abbas, D. B., Wan, D., Longaker, M. T. WILEY. 2022: A3-A4

  View details for Web of Science ID 000763583000018

• Vitamin E Treated Fat Grafts Demonstrate Improved Volume Retention And Decreased Radiation-Induced Fibrosis Abbas, D. B., Lavin, C. V., Fahy, E. J., Lintel, H., Griffin, M., Nazerali, R., Dung Nguyen, Momeni, A., Longaker, M. T., Wan, D. WILEY. 2022: A16

  View details for Web of Science ID 000763583000040

• Mechanical Stimulation Reverses Pro-Fibrotic Transcriptional States in Senescent Fibroblasts Guo, J. L., Griffin, M., Guardino, N., Chen, K., Gurtner, G. C., Longaker, M. T. WILEY. 2022: A14-A16

  View details for Web of Science ID 000763583000039

• Interactions Of Fibroblasts Versus Macrophages In An In Vitro Model Of Scar Formation And Wound Healing Huskins, S. L., Griffin, M., Steele, S., Thomas, B., Kussie, H. C., Sivaraj, D., Leeolou, M. C., Trotsyuk, A. A., Padmanabhan, J., Longaker, M. T., Gurtner, G. C., Chen, K. WILEY. 2022: A53-A54

  View details for Web of Science ID 000763583000115

• Pullulan-Collagen Hydrogel Wound Dressing Promotes Dermal Remodeling and Healing in an Excisional Wound Model Leeolou, M. C., Sivaraj, D., Davitt, M., Henn, D., Steele, S., Huskins, S. L., Trotsyuk, A. A., Kussie, H. C., Greco, A., Perrault, D., Padmanabhan, J., Longaker, M. T., Chen, K., Gurtner, G. C. WILEY. 2022: A24

  View details for Web of Science ID 000763583000056

• The Oral Mucosa Hosts Distinct Fibroblast Subpopulations to Facilitate Regenerative Wound Repair Griffin, M., Cook, J., Bofelli, D., Guardino, N., Spielman, A. F., Januszyk, M., Chen, K., Abbas, D. B., Zwick, R., Klein, O., Longaker, M. T. WILEY. 2022: A12-A13

  View details for Web of Science ID 000763583000035

• Transdermal Deferoxamine Enhances Wound Healing In Chronically Irradiated Skin In Mice Lintel, H., Abbas, D. B., Lavin, C. V., Griffin, M., Guardino, N., Guo, J. L., Spielman, A. F., Gurtner, G. C., Longaker, M. T., Wan, D. WILEY. 2022: A29-A30

  View details for Web of Science ID 000763583000068

• Characterization of Mechanoresponsive Inflammatory Cells during Wound Healing Chen, K., Griffin, M., Henn, D., Bonham, C. A., Fischer, K., Padmanabhan, J., Trotsyuk, A. A., Sivaraj, D., Leeolou, M. C., Kussie, H. C., Huskins, S. L., Steele, S., Perrault, D., Longaker, M. T., Gurtner, G. C. WILEY. 2022: A31-A32

  View details for Web of Science ID 000763583000072

• Discussion: Beyond the Scalpel: Attracting and Nurturing Surgeon-Scientists in Plastic Surgery. Plastic and reconstructive surgery Liao, E. C., Longaker, M. T. 1800; 149 (2): 517-518

  View details for DOI 10.1097/PRS.0000000000008787

  View details for PubMedID 35077431

• Tractable Human Skeletal Stem Cell Diversity Shapes Bone Development and Regeneration Ambrosi, T., Taheri, S., Sinha, R., Goodnough, L., Steininger, H., Weissman, I., Longaker, M., Sahoo, D., Chan, C. WILEY. 2022: 266-267

  View details for Web of Science ID 000778074501234

• Multi-omic analysis reveals divergent molecular events in scarring and regenerative wound healing. Cell stem cell Mascharak, S., Talbott, H. E., Januszyk, M., Griffin, M., Chen, K., Davitt, M. F., Demeter, J., Henn, D., Bonham, C. A., Foster, D. S., Mooney, N., Cheng, R., Jackson, P. K., Wan, D. C., Gurtner, G. C., Longaker, M. T. 1800

  Abstract

  Regeneration is the holy grail of tissue repair, but skin injury typically yields fibrotic, non-functional scars. Developing pro-regenerative therapies requires rigorous understanding of the molecular progression from injury to fibrosis or regeneration. Here, we report the divergent molecular events driving skin wound cells toward scarring or regenerative fates. We profile scarring versus YAP-inhibition-induced wound regeneration at the transcriptional (single-cell RNA sequencing), protein (timsTOF proteomics), and tissue (extracellular matrix ultrastructural analysis) levels. Using cell-surface barcoding, we integrate these data to reveal fibrotic and regenerative "molecular trajectories" of healing. We show that disrupting YAP mechanotransduction yields regenerative repair by fibroblasts with activated Trps1 and Wnt signaling. Finally, via invivo gene knockdown and overexpression in wounds, we identify Trps1 as a key regulatory gene that is necessary and partially sufficient for wound regeneration. Our findings serve as a multi-omic map of wound regeneration and could have therapeutic implications for pathologic fibroses.

  View details for DOI 10.1016/j.stem.2021.12.011

  View details for PubMedID 35077667

• Harnessing a Feasible and Versatile ex vivo Calvarial Suture 2-D Culture System to Study Suture Biology. Frontiers in physiology Quarto, N., Menon, S., Griffin, M., Huber, J., Longaker, M. T. 2022; 13: 823661

  Abstract

  As a basic science, craniofacial research embraces multiple facets spanning from molecular regulation of craniofacial development, cell biology/signaling and ultimately translational craniofacial biology. Calvarial sutures coordinate development of the skull, and the premature fusion of one or more, leads to craniosynostosis. Animal models provide significant contributions toward craniofacial biology and clinical/surgical treatments of patients with craniofacial disorders. Studies employing mouse models are costly and time consuming for housing/breeding. Herein, we present the establishment of a calvarial suture explant 2-D culture method that has been proven to be a reliable system showing fidelity with the in vivo harvesting procedure to isolate high yields of skeletal stem/progenitor cells from small number of mice. Moreover, this method allows the opportunity to phenocopying models of craniosynostosis and in vitro tamoxifen-induction of ActincreERT2;R26Rainbow suture explants to trace clonal expansion. This versatile method tackles needs of large number of mice to perform calvarial suture research.

  View details for DOI 10.3389/fphys.2022.823661

  View details for PubMedID 35222087

• So You Want to Be an Innovator? Plastic and reconstructive surgery Rohrich, R. J., Rosen, J., Longaker, M. T. 2021; 148 (5S): 55S-57S

  View details for DOI 10.1097/01.prs.0000794856.25019.d0

  View details for PubMedID 34699492

• Osteoskeletal Regenerative Ability of Exosomes Derived From Adipose-derived Stem Cells Upon Inhibition of Transforming Growth Factor-beta-signaling Huber, J. L., Menon, S., Longaker, M. T., Quarto, N. ELSEVIER SCIENCE INC. 2021: E194

  View details for Web of Science ID 000718306700472

• Transdermal Deferoxamine in a Porcine Model Is a Safe Treatment to Improve Elasticity Secondary to Radiation-induced Fibrosis Abbas, D. B., Fahy, E. J., Lavin, C. V., Griffin, M., Deleon, N., King, M. E., Gurtner, G. C., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: E164

  View details for Web of Science ID 000718306700398

• CD34+CD146+Adipose-derived Stromal Cells (ASCs) Enrichment of Fat Grafts Enhance Regeneration of Irradiated Skin and Graft Retention Deleon, N., Abbas, D. B., Borrelli, M. R., Adem, S., Lavin, C. V., Griffin, M., King, M. E., Lee, D., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: E198

  View details for Web of Science ID 000718306700482

• Dermal Iron Chelation Reduces Indirect Radiation Injury Lavin, C. V., Fahy, E. J., Abbas, D. B., Griffin, M., Lee, D. K., Deleon, N., Guardino, N., Gurtner, G. C., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: E155

  View details for Web of Science ID 000718306700375

• Dorsal Skin Fibrosis Secondary to Radiation Is Mitigated by Fat Grafting in Engrailed-1 Mice Abbas, D. B., Fahy, E. J., Griffin, M., Lavin, C. V., Deleon, N., King, M. E., Lee, D., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: E159-E160

  View details for Web of Science ID 000718306700387

• Suprazygomatic Maxillary Nerve Blocking Reduces Postoperative Pain and Opioid Use Following Bimaxillary Osteotomy Lavin, C. V., Lee, D. K., Fahy, E. J., Abbas, D. B., Griffin, M., King, M. E., Momeni, A., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: E157

  View details for Web of Science ID 000718306700380

• Optimizing Cutometer Mpa 580 Calculated Parameters to Determine In-vivo Elasticity of Human Skin Abbas, D. B., Lavin, C. V., Fahy, E., Griffin, M., Guardino, N., Lee, D., Gurtner, G. C., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: E162

  View details for Web of Science ID 000718306700394

• Temperature Influence on Scanning Laser Doppler Flowmetry in Anesthetized Mice Fahy, E. J., Lavin, C. V., Abbas, D., Griffin, M., King, M. E., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: E157

  View details for Web of Science ID 000718306700381

• Acellular Dermal Matrix Grafts Decrease Radiation-Induced Contracture and Dermal Thickening Deleon, N., Lavin, C. D., Abbas, D., Griffin, M., King, M. E., Fahy, E. J., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: E197

  View details for Web of Science ID 000718306700480

• Adipose-Derived Stromal Cell (ASC) Subpopulation with Adipogenic Capabilities Increase Fat Graft Quality in Irradiated Tissue Deleon, N., Adem, S., Borrelli, M. R., Abbas, D. B., Lavin, C. V., Griffin, M., King, M. E., Lee, D., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: E197-E198

  View details for Web of Science ID 000718306700481

• Acellular Dermal Matrix Modulation of the Peri-Prosthetic Breast Microenvironment During Breast Reconstruction Tevlin, R., Januszyk, M., Griffin, M., Shefren, K., Chan, C. F., Momeni, A., Wan, D. C., Longaker, M. T. ELSEVIER SCIENCE INC. 2021: S195-S196

  View details for Web of Science ID 000718303100367

• Fibroblast Sub-Populations Dynamically Change Composition to Heal Dorsal Skin Radiation Wounds in Wild-Type Mice Abbas, D. B., Griffin, M., Fahy, E. J., Lavin, C., Lee, D., Mascharak, S., King, M., Januszyk, M., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: S207-S208

  View details for Web of Science ID 000718303100389

• Adipose Precursor Cell-Embedded Collagen Gels Attenuate Inflammation and Improve Tissue Perfusion in Cutaneous Wounds Fahy, E. J., Griffin, M., Abbas, D., Lavin, C. V., King, M. E., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: S196

  View details for Web of Science ID 000718303100368

• Denervation During Mandibular Distraction Osteogenesis Results in Impaired Osteogenesis Tevlin, R., Januszyk, M., Griffin, M., Salhotra, A., Wan, D. C., Chan, C. F., Longaker, M. T. ELSEVIER SCIENCE INC. 2021: S196-S197

  View details for Web of Science ID 000718303100369

• Topical Deferoxamine Patch Is Superior to Direct Injection for the Treatment of Radiation-Induced Skin Fibrosis Lavin, C. V., Abbas, D. B., Fahy, E. J., Lee, D. K., Griffin, M., Deleon, N., Mascharak, S., Gurtner, G. C., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: S202-S203

  View details for Web of Science ID 000718303100381

• Effects of beta(3) Adrenergic Receptor Agonist Treatment in Murine Full Thickness Dorsal Cutaneous Wounds Fahy, E. J., Griffin, M., Abbas, D., Lavin, C. V., King, M. E., Lee, D., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: S197-S198

  View details for Web of Science ID 000718303100371

• Cancer-Associated Fibroblasts Share Highly Conserved Phenotypes and Functions Across Tumor Types and Species Foster, D. S., Januszyk, M., Yost, K. E., Chinta, M., Titan, A. L., Wapnir, I. L., Gurtner, G. C., Chang, H. Y., Norton, J. A., Longaker, M. T. ELSEVIER SCIENCE INC. 2021: S243-S244

  View details for Web of Science ID 000718303100463

• Where There Is Fat There Is Fibrosis: Elucidating the Mechanisms of Creeping Fat-Driven Stricture Formation Bauer-Rowe, K. E., Griffin, M., Foster, D., desJardins-Park, H. E., Mascharak, S., Norton, J. A., Hyun, J. S., Longaker, M. T. ELSEVIER SCIENCE INC. 2021: S65

  View details for Web of Science ID 000718303100105

• Single-Cell RNA Sequencing Reveals Fibroblast Heterogeneity Across Mouse and Human Embryonic Origins Griffin, M., King, M. W., Guardino, N., Tevlin, R., Fahy, E. J., Mascharak, S., Abbas, D., Lavin, C. V., Wan, D., Longaker, M. ELSEVIER SCIENCE INC. 2021: S201-S202

  View details for Web of Science ID 000718303100378

• Local Vitamin E Administration Improves Fat Graft Retention and Radiation-Induced Fibrosis in a Mouse Model Abbas, D. B., Lavin, C. V., Fahy, E. J., Griffin, M., King, M. E., Lee, D., Dung Nguyen, Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2021: S199

  View details for Web of Science ID 000718303100374

• Integrated spatial multiomics reveals fibroblast fate during tissue repair. Proceedings of the National Academy of Sciences of the United States of America Foster, D. S., Januszyk, M., Yost, K. E., Chinta, M. S., Gulati, G. S., Nguyen, A. T., Burcham, A. R., Salhotra, A., Ransom, R. C., Henn, D., Chen, K., Mascharak, S., Tolentino, K., Titan, A. L., Jones, R. E., da Silva, O., Leavitt, W. T., Marshall, C. D., des Jardins-Park, H. E., Hu, M. S., Wan, D. C., Wernig, G., Wagh, D., Coller, J., Norton, J. A., Gurtner, G. C., Newman, A. M., Chang, H. Y., Longaker, M. T. 2021; 118 (41)

  Abstract

  In the skin, tissue injury results in fibrosis in the form of scars composed of dense extracellular matrix deposited by fibroblasts. The therapeutic goal of regenerative wound healing has remained elusive, in part because principles of fibroblast programming and adaptive response to injury remain incompletely understood. Here, we present a multimodal -omics platform for the comprehensive study of cell populations in complex tissue, which has allowed us to characterize the cells involved in wound healing across both time and space. We employ a stented wound model that recapitulates human tissue repair kinetics and multiple Rainbow transgenic lines to precisely track fibroblast fate during the physiologic response to skin injury. Through integrated analysis of single cell chromatin landscapes and gene expression states, coupled with spatial transcriptomic profiling, we are able to impute fibroblast epigenomes with temporospatial resolution. This has allowed us to reveal potential mechanisms controlling fibroblast fate during migration, proliferation, and differentiation following skin injury, and thereby reexamine the canonical phases of wound healing. These findings have broad implications for the study of tissue repair in complex organ systems.

  View details for DOI 10.1073/pnas.2110025118

  View details for PubMedID 34620713

• A comparative analysis of deferoxamine treatment modalities for dermal radiation-induced fibrosis. Journal of cellular and molecular medicine Lavin, C. V., Abbas, D. B., Fahy, E. J., Lee, D. K., Griffin, M., Diaz Deleon, N. M., Mascharak, S., Chen, K., Momeni, A., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2021

  Abstract

  The iron chelator, deferoxamine (DFO), has been shown to potentially improve dermal radiation-induced fibrosis (RIF) in mice through increased angiogenesis and reduced oxidative damage. This preclinical study evaluated the efficacy of two DFO administration modalities, transdermal delivery and direct injection, as well as temporal treatment strategies in relation to radiation therapy to address collateral soft tissue fibrosis. The dorsum of CD-1 nude mice received 30Gy radiation, and DFO (3mg) was administered daily via patch or injection. Treatment regimens were prophylactic, during acute recovery, post-recovery, or continuously throughout the experiment (n=5 per condition). Measures included ROS-detection, histology, biomechanics and vascularity changes. Compared with irradiated control skin, DFO treatment decreased oxidative damage, dermal thickness and collagen content, and increased skin elasticity and vascularity. Metrics of improvement in irradiated skin were most pronounced with continuous transdermal delivery of DFO. In summary, DFO administration reduces dermal fibrosis induced by radiation. Although both treatment modalities were efficacious, the transdermal delivery showed greater effect than injection for each temporal treatment strategy. Interestingly, the continuous patch group was more similar to normal skin than to irradiated control skin by most measures, highlighting a promising approach to address detrimental collateral soft tissue injury following radiation therapy.

  View details for DOI 10.1111/jcmm.16913

  View details for PubMedID 34612609

• Inhibiting Fibroblast Mechanotransduction Modulates Severity of Idiopathic Pulmonary Fibrosis. Advances in wound care Trotsyuk, A. A., Chen, K., Kwon, S. H., Ma, K. C., Henn, D., Mermin-Bunnell, A. M., Mittal, S., Padmanabhan, J., Larson, M. R., Steele, S. R., Sivaraj, D., Bonham, C. A., Noishiki, C., Rodrigues, M., Jiang, Y., Jing, S., Niu, S., Chattopadhyay, A., Perrault, D. P., Leeolou, M. C., Fischer, K., Gurusankar, G., Choi Kussie, H., Wan, D. C., Januszyk, M., Longaker, M. T., Gurtner, G. C. 2021

  Abstract

  OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that affects 63 in every 100,000 Americans. Its etiology remains unknown, although inflammatory pathways appear to be important. Given the dynamic environment of the lung, we examined the significance of mechanotransduction on both inflammatory and fibrotic signaling during IPF.INNOVATION: Mechanotransduction pathways have not been thoroughly examined in the context of lung disease and pharmacologic approaches for IPF do not currently target these pathways. The interplay between mechanical strain and inflammation in pulmonary fibrosis remain incompletely understood.APPROACH: In this study, we used conditional KO mice to block mechanotransduction by knocking out FAK (Focal Adhesion Kinase) expression in fibroblasts, followed by induction of pulmonary fibrosis using bleomycin. We examined both normal human and human IPF fibroblasts and used immunohistochemistry, qRT-PCR, and Western Blot to evaluate the effects of FAK inhibition (FAKI) on modulating fibrotic and inflammatory genes.RESULTS: Our data indicate that deletion of FAK in mice reduces expression of fibrotic and inflammatory genes in lungs. Similarly, mechanical straining in normal human lung fibroblasts activates inflammatory and fibrotic pathways. FAK inhibition decreases these signals but has less effect on IPF fibroblasts as compared to normal human fibroblasts.CONCLUSION: Administering FAKI at early stages of fibrosis may attenuate the FAK-mediated fibrotic response pathway in IPF, potentially mediating disease progression.

  View details for DOI 10.1089/wound.2021.0077

  View details for PubMedID 34544267

• From Chronic Wounds to Scarring: The Growing Healthcare Burden of Under- and Over-healing Wounds. Advances in wound care desJardins-Park, H. E., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2021

  Abstract

  SIGNIFICANCE: Wound healing is the largest medical market without an existing small molecule/drug treatment. Both "under-healing" (chronic wounds) and "over-healing" (scarring) cause a substantial biomedical burden and lifelong consequences for patients. These problems cost tens of billions of dollars per year in the United States alone, a number expected to grow as the population ages and the prevalence of common comorbidities (e.g., diabetes) rises. However, no therapies currently exist to produce the "ideal" healing outcome: efficient wound repair via regeneration of normal tissue. Recent Advances: Ongoing research continues to illuminate possible therapeutic avenues for wound healing. By identifying underlying mechanisms of wound repair - for instance, tissue mechanics' role in fibrosis, or cell populations that modulate wound healing and scarring - novel molecular targets may be defined. This Advances in Wound Care Forum issue includes reviews of scientific literature and original research from the Hagey Laboratory for Pediatric Regenerative Medicine at Stanford and its alumni, including developing approaches for encouraging wound healing, minimizing fibrosis, and coaxing regeneration.CRITICAL ISSUES: Wound healing problems reflect an enormous and rapidly expanding clinical burden. The issues of both under- and over-healing wound outcomes will continue to expand as their underlying causes (e.g., diabetes) grow. Targeted treatments are needed to enable wound repair with functional tissue restoration and decreased scarring.FUTURE DIRECTIONS: Basic scientists will continue to refine understanding of factors driving undesirable wound outcomes. These discoveries are beginning to be translated and, in the coming years, will hopefully form the foundation for anti-scarring drugs and other wound therapeutics.

  View details for DOI 10.1089/wound.2021.0039

  View details for PubMedID 34521257

• A Novel Xenograft Model Demonstrates Human Fibroblast Behavior During Skin Wound Repair and Fibrosis. Advances in wound care Borrelli, M., Shen, A. H., Griffin, M., Mascharak, S., Adem, S., Diaz Deleon, N. M., Ngaage, L. M., Longaker, M. T., Wan, D. C., Lorenz, H. P. 2021

  Abstract

  OBJECTIVE: Xenografts of human skin on immunodeficient mice provide a means of assessing human skin physiology and its response to wounding.APPROACH: We describe a novel xenograft model using full-thickness human neonatal foreskin to examine human skin wound repair and fibroblast heterogeneity. Full-thickness 8-mm human neonatal foreskin biopsies were sutured into the dorsum of NSG (NOD.Cg-Prkdc scidIl2rgtm1Wjl/SzJ) pups as subcutaneous grafts and exposed to cutaneous grafts at the time of weaning (postnatal day 21). To model fibrosis, xenografts were wounded with 5-mm linear incisions and monitored until post-wound day (PWD) 14. To explore whether our model can be used to test the efficacy of topical therapies, wounded xenografts were injected with fibroblast growth factor-2 (FGF2) for the first four consecutive PWDs. Xenografts were harvested for analysis by histology and fluorescence-activated cell sorting (FACS).RESULTS: Xenografts successfully engrafted with evidence of mouse-human anastomoses and resembled native neonatal foreskin at the gross and microscopic level. Wounded xenografted skin scarred with human collagen and an expansion of CD26-positive human fibroblasts. Collagen scar was quantitated by neural network analysis, which revealed distinct clustering of collagen fiber networks from unwounded skin and wounded skin at PWD7 and PWD14. Collagen fiber networks within FGF2-treated wounds at PWD14 resembled those in untreated wounded xenografts at PWD7, suggesting that FGF2 treatment at time of wounding can reduce fibrosis. Innovation and Conclusion: This novel xenograft model can be used to investigate acute fibrosis, fibroblast heterogeneity, and the efficacy of antifibrotic agents during wound repair in human skin.

  View details for DOI 10.1089/wound.2020.1392

  View details for PubMedID 34521222

• Understanding Scarring in the Oral Mucosa. Advances in wound care Griffin, M., Fahy, E., King, M., Guardino, N., Chen, K., Abbas, D. B., Lavin, C., Diaz Deleon, N. M., Lorenz, H. P., Longaker, M. T., Wan, D. C. 2021

  Abstract

  SIGNIFICANCE: Skin inevitably heals with the formation of a fibrotic scar. Patients affected by skin fibrosis suffer from long-term psychological and physical burdens. Recent Advances: Since the discovery of fetal scarless skin-wound healing, research has hoped to identify and mimic scarless healing for adult skin. Oral mucosa healing in adults provides the closest example to fetal scarless healing. Injuries to the oral mucosa heal with very minimal scarring. Understanding the mechanisms through which this process occurs may bring us closer to achieving scarless healing in adults.CRITICAL ISSUES: In this review, we summarize the current evidence that illustrates distinct mechanisms involved in oral mucosal healing. We discuss the role of the oral niche in contributing to wound repair. The intrinsic properties of immune cells, fibroblasts, and keratinocytes within the oral mucosa that support regenerative repair are provided. We highlight the contribution of cytokines, growth factors, and chemokine secretion in permitting a scarless mucosal environment. Furthermore, we discuss the role of stem cell-like progenitor populations in the mucosa that may contribute to wound healing. We also provide suggestions for future studies that are needed to achieve scarless healing in adults.FUTURE DIRECTIONS: Many characteristics of the oral mucosa have been shown to contribute to decreased scarring, but the specific mechanism(s) is unclear. Advancing our understanding of oral healing may yield therapeutic therapies that can be used to overcome dermal fibrosis and scarring.

  View details for DOI 10.1089/wound.2021.0038

  View details for PubMedID 34470520

• Standardizing dimensionless cutometer parameters to determine in-vivo elasticity of human skin. Advances in wound care Abbas, D. B., Lavin, C., Fahy, E., Griffin, M., Guardino, N., King, M., Chen, K., Lorenz, H. P., Gurtner, G. C., Longaker, M. T., Momeni, A., Wan, D. C. 2021

  Abstract

  OBJECTIVE: Skin fibrosis places an enormous burden on patients and society, but disagreement exists over methods to quantify severity of skin scarring. A suction cutometer measures skin elasticity in-vivo, but it has not been widely adopted due to inconsistency in data produced. We investigated variability of several dimensionless parameters generated by the cutometer to improve their precision and accuracy.APPROACH: Twenty adult human subjects underwent suction cutometer measurement of normal skin and fibrotic scars. Using Mode 1, each subject underwent 5 trials with each trial containing 4 curves. R0/2/5/6/7 and Q1/2/3 data were collected. Analyses were performed on these calculated parameters.RESULTS: R0/2/5/6/7 and Q1/2 parameters from curves 1-4 demonstrated significant differences, while these same parameters were not significantly different when only using curves 2-4. Individual analysis of all parameters between curve 1 and every subsequent curve was statistically significant for R0, R2, R5, R6, R7, Q1, and Q2. No differences were appreciated for parameter Q3. Comparison between normal skin and fibrotic scars were significantly different for parameters R5, Q1, and Q3.INNOVATION: Our study is the first demonstration of accurate comparison between normal skin and fibrotic scars using the dimensionless parameters of a suction cutometer.CONCLUSION: Measured parameters from the first curve of each trial were significantly different from subsequent curves for both normal skin and fibrotic scars. Precision and reproducibility of data from dimensionless parameters can therefore be improved by removing the first curve. R5, Q1, and Q3 parameters differentiated normal skin as more elastic than fibrotic scars.

  View details for DOI 10.1089/wound.2021.0082

  View details for PubMedID 34470542

• JUN promotes hypertrophic skin scarring via CD36 in preclinical in vitro and in vivo models. Science translational medicine Griffin, M. F., Borrelli, M. R., Garcia, J. T., Januszyk, M., King, M., Lerbs, T., Cui, L., Moore, A. L., Shen, A. H., Mascharak, S., Diaz Deleon, N. M., Adem, S., Taylor, W. L., desJardins-Park, H. E., Gastou, M., Patel, R. A., Duoto, B. A., Sokol, J., Wei, Y., Foster, D., Chen, K., Wan, D. C., Gurtner, G. C., Lorenz, H. P., Chang, H. Y., Wernig, G., Longaker, M. T. 2021; 13 (609): eabb3312

  Abstract

  [Figure: see text].

  View details for DOI 10.1126/scitranslmed.abb3312

  View details for PubMedID 34516825

• Modulating cellular responses to mechanical forces to promote wound regeneration. Advances in wound care Mascharak, S., desJardins-Park, H. E., Davitt, M. F., Guardino, N. J., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2021

  Abstract

  SIGNIFICANCE: Skin scarring poses a major biomedical burden for hundreds of millions of patients annually. However, this burden could be mitigated by therapies that promote wound regeneration, with full recovery of skin's normal adnexa, matrix ultrastructure, and mechanical strength. Recent Advances: The observation of wound regeneration in several mouse models suggests a retained capacity for postnatal mammalian skin to regenerate under the right conditions. Mechanical forces are a major contributor to skin fibrosis and a prime target for devices and therapeutics that could promote skin regeneration.CRITICAL ISSUES: Wound induced hair neogenesis, Acomys "spiny" mice, Murphy Roths Large (MRL) mice, and mice treated with mechanotransduction inhibitors all show various degrees of wound regeneration. Comparison of regenerating wounds in these models against scarring wounds reveals differences in ECM interactions and in mechanosensitive activation of key signaling pathways, including Wnt, Sonic hedgehog, Focal Adhesion Kinase, and Yes-associated protein. The advent of single cell "omics" technologies has deepened this understanding and revealed that regeneration may recapitulate development in certain contexts, though it is unknown whether these mechanisms are relevant to healing in tight-skinned animals such as humans.FUTURE DIRECTIONS: While early findings in mice are promising, comparison across model systems is needed to resolve conflicting mechanisms and to identify conserved master regulators of skin regeneration. There also remains a dire need for studies on mechanomodulation of wounds in large, tight-skinned animals such as red Duroc pigs, which better approximate human wound healing.

  View details for DOI 10.1089/wound.2021.0040

  View details for PubMedID 34465219

• The role of Wnt signaling in skin fibrosis. Medicinal research reviews Griffin, M. F., Huber, J., Evan, F. J., Quarto, N., Longaker, M. T. 2021

  Abstract

  Skin fibrosis is the excessive deposition of extracellular matrix in the dermis. Cutaneous fibrosis can occur following tissue injury, including burns, trauma, and surgery, resulting in scars that are disfiguring, limit movement and cause significant psychological distress for patients. Many molecular pathways have been implicated in the development of skin fibrosis, yet effective treatments to prevent or reverse scarring are unknown. The Wnt signaling pathways are known to play an important role in skin homeostasis, skin injury, and in the development of fibrotic skin diseases. This review provides a detailed overview of the role of the canonical Wnt signaling pathways in regulating skin scarring. We also discuss how Wnt signaling interacts with other known fibrotic molecular pathways to cause skin fibrosis. We further provide a summary of the different Wnt inhibitor types available for treating skin scarring. Understanding the role of the Wnt pathway in cutaneous fibrosis will accelerate the development of effective Wnt modulators for the treatment of skin fibrosis.

  View details for DOI 10.1002/med.21853

  View details for PubMedID 34431110

• Decellularized Adipose Matrices can Alleviate Radiation-induced Skin Fibrosis. Advances in wound care Adem, S., Abbas, D. B., Lavin, C., Fahy, E., Griffin, M., Diaz Deleon, N. M., Borrelli, M. R., Mascharak, S., Shen, A. H., Patel, R. A., Longaker, M. T., Nazerali, R. S., Wan, D. C. 2021

  Abstract

  OBJECTIVE: Radiation therapy is commonplace for cancer treatment but often results in fibrosis and atrophy of surrounding soft tissue. Decellularized adipose matrices (DAMs) have been reported to improve these soft tissue defects through the promotion of adipogenesis. These matrices are decellularized by a combination of physical, chemical, and enzymatic methods to minimize their immunologic effects while promoting their regenerative effects. In this study, we aimed to explore the regenerative ability of a DAM (Renuva, MTF Biologics, New Jersey, USA) in radiation-induced soft tissue injury.APPROACH: Fresh human lipoaspirate or DAM was injected into the irradiated scalp of CD-1 nude mice, and volume retention was monitored radiographically over 8 weeks. Explanted grafts were histologically assessed, and overlying skin was examined histologically and biomechanically. Irradiated human skin was also evaluated from patients following fat grafting or DAM injection. However, integrating data between murine and human skin in all cohorts is limited given the genetic variability between the two species.RESULTS: Volume retention was found to be greater with fat grafts, though DAM retention was nonetheless appreciated at irradiated sites. Improvement in both mouse and human irradiated skin overlying fat and DAM grafts was observed in terms of biomechanical stiffness, dermal thickness, collagen density, collagen fiber networks, and skin vascularity.INNOVATION: This is the first demonstration of the use of DAMs for augmenting the regenerative potential of irradiated mouse and human skin.CONCLUSIONS: These findings support use of DAMs to address soft tissue atrophy following radiation therapy. Morphological characteristics of the irradiated skin can also be improved with DAM grafting.

  View details for DOI 10.1089/wound.2021.0008

  View details for PubMedID 34346243

• Skeletal stem and progenitor cells maintain cranial suture patency and prevent craniosynostosis. Nature communications Menon, S., Salhotra, A., Shailendra, S., Tevlin, R., Ransom, R. C., Januszyk, M., Chan, C. K., Behr, B., Wan, D. C., Longaker, M. T., Quarto, N. 2021; 12 (1): 4640

  Abstract

  Cranial sutures are major growth centers for the calvarial vault, and their premature fusion leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cellsderived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. Crossing Axin2LacZ/+ mouse, endowing enhanced Wnt activation, to a Twist1+/- mouse model of coronal craniosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co-transplantation of these cells with Wnt3a prevents resynostosis following suturectomy in Twist1+/- mice. Our study reveals that decrease and/or imbalance of skeletal stem/progenitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis.

  View details for DOI 10.1038/s41467-021-24801-6

  View details for PubMedID 34330896

• Distinct skeletal stem cell types orchestrate long bone skeletogenesis. eLife Ambrosi, T. H., Sinha, R., Steininger, H. M., Hoover, M. Y., Murphy, M. P., Koepke, L. S., Wang, Y., Lu, W., Morri, M., Neff, N. F., Weissman, I. L., Longaker, M. T., Chan, C. K. 2021; 10

  Abstract

  Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research.

  View details for DOI 10.7554/eLife.66063

  View details for PubMedID 34280086

• Mechanical Strain Drives Myeloid Cell Differentiation Toward Pro-Inflammatory Subpopulations. Advances in wound care Chen, K., Henn, D., Sivaraj, D., Bonham, C. A., Griffin, M., Choi Kussie, H., Padmanabhan, J., Trotsyuk, A. A., Wan, D. C., Januszyk, M., Longaker, M. T., Gurtner, G. C. 2021

  Abstract

  OBJECTIVE: After injury, humans and other mammals heal by forming fibrotic scar tissue with diminished function, and this healing process involves the dynamic interplay between resident cells within the skin and cells recruited from the circulation. Recent studies have provided mounting evidence that external mechanical forces stimulate intracellular signaling pathways to drive fibrotic processes.INNOVATION: While most studies have focused on studying mechanotransduction in fibroblasts, recent data suggest that mechanical stimulation may also shape the behavior of immune cells, referred to as "mechano-immunomodulation". However, the effect of mechanical strain on myeloid cell recruitment and differentiation remains poorly understood and has never been investigated at the single cell level.APPROACH: In this study, we utilized a three-dimensional (3D) in vitro culture system that permits the precise manipulation of mechanical strain applied to cells. We cultured myeloid cells and used single cell RNA-sequencing to interrogate the effects of strain on myeloid differentiation and transcriptional programming.RESULTS: Our data indicate that myeloid cells are indeed mechanoresponsive, with mechanical stress influencing myeloid differentiation. Mechanical strain also upregulated a cascade of inflammatory chemokines, most notably from the Ccl family.CONCLUSION: Further understanding of how mechanical stress affects myeloid cells in conjunction with other cell types in the complicated, multicellular milieu of wound healing may lead to novel insights and therapies for the treatment of fibrosis.

  View details for DOI 10.1089/wound.2021.0036

  View details for PubMedID 34278820

• Deferoxamine to minimize fibrosis during radiation therapy. Advances in wound care Tevlin, R., Longaker, M. T., Wan, D. 2021

  Abstract

  Significance By 2030, there will be over 4 million radiation-treated cancer survivors living in the US. Irradiation triggers inflammation, fibroblast activation, and extracellular matrix deposition in addition to reactive oxygen species (ROS) generation, leading to a chronic inflammatory response. Radiation-induced fibrosis (RIF) is a progressive pathology resulting in skin pigmentation, reduced elasticity, ulceration and dermal thickening, cosmetic deformity, pain, and the need for reconstructive surgery. Recent Advances Deferoxamine (DFO) is an FDA-approved iron chelator for blood dyscrasia management, which has been found to be pro-angiogenic, to decrease free radical formation, and reduce cell death. DFO has shown great promise in the treatment and prophylaxis of RIF in preclinical studies. Critical Issues Systemic DFO has a short half-life and is cumbersome to deliver to patients intravenously. Transdermal DFO delivery is complicated by its high atomic mass and hydrophilicity, preventing stratum corneum penetration. A transdermal drug delivery system was developed to address these challenges, in addition to a strategy for topical administration. Future Directions DFO has great potential to translate from bench to bedside. An important step in translation of DFO for RIF prophylaxis is to ensure that DFO treatment does not affect the efficacy of radiation therapy. Furthermore, following an initial plethora of studies reporting DFO treatment by intravenous and subcutaneous routes, a significant advantage of recent studies is the success of transdermal and topical delivery. Given the strong foundation of basic scientific research supporting the use of DFO treatment on RIF, clinicians will be closely following the results of the ongoing human studies.

  View details for DOI 10.1089/wound.2021.0021

  View details for PubMedID 34074152

• Disrupting Mechanotransduction Reduces Scar Formation And Restores Cellular Subpopulations In A Large Animal Model Of Skin Grafting Chen, K., Henn, D., Bonham, C. A., Noishiki, C., Barrera, J. A., Carlomagno, T. C., Shannon, T., Mays, C. J., Trotsyuk, A. A., Padmanabhan, J., Longaker, M. T., Januszyk, M., Gurtner, G. C. WILEY. 2021: A12-A13

  View details for Web of Science ID 000650720500039

• Single Cell RNA Sequencing Reveals Fibroblast Heterogeneity Across Embryonic Origins Of Skin Griffin, M., King, M., Chen, K., desJardins-Park, H., Mascharak, S., Fahy, E., Guardino, N., Lavin, C., Abbas, D., Januszyk, M., Wan, D., Longaker, M. WILEY. 2021: A11-A12

  View details for Web of Science ID 000650720500037

• Novel Genetic Analysis Of MRL Mice Reveals That Complement Inhibition By Factor H Reduces Scarring desJardins-Park, H. E., Mack, K. L., Guardino, N., Griffin, M., Davitt, M. F., Mascharak, S., Wan, D. C., Fraser, H. B., Longaker, M. T. WILEY. 2021: A13

  View details for Web of Science ID 000650720500040

• Adipocytes In Dermal Wounds Undergo Conversion To Pro-fibrotic Fibroblasts That Contribute To Scar Formation Guardino, N., desJardins-Park, H. E., Griffin, M., Bauer-Rowe, K. E., King, M. E., King, M. E., Mascharak, S., Longaker, M. T. WILEY. 2021: A31

  View details for Web of Science ID 000650720500078

• Transgenic Inhibition Of Engrailed-1 Results In Endogenous Postnatal Skin Regeneration Mascharak, S., desJardins-Park, H. E., Davitt, M. F., Chen, K., Griffin, M., Guardino, N., Lorenz, H., Wan, D. C., Gurtner, G. C., Longaker, M. T. WILEY. 2021: A14-A15

  View details for Web of Science ID 000650720500043

• Wnt-active Engrailed-1 Lineage-negative Fibroblasts Mediate Postnatal Skin Regeneration Mascharak, S., desJardins-Park, H. E., Januszyk, M., Chen, K., Davitt, M. F., Demeter, J., Henn, D., Griffin, M., Bonham, C. A., Mooney, N., Cheng, R., Jackson, P. K., Wan, D. C., Gurtner, G. C., Longaker, M. T. WILEY. 2021: A30

  View details for Web of Science ID 000650720500076

• Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring. Science (New York, N.Y.) Mascharak, S., desJardins-Park, H. E., Davitt, M. F., Griffin, M., Borrelli, M. R., Moore, A. L., Chen, K., Duoto, B., Chinta, M., Foster, D. S., Shen, A. H., Januszyk, M., Kwon, S. H., Wernig, G., Wan, D. C., Lorenz, H. P., Gurtner, G. C., Longaker, M. T. 2021; 372 (6540)

  Abstract

  Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. Engrailed-1 lineage-positive fibroblasts (EPFs) are known to function in scarring, but Engrailed-1 lineage-negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating Engrailed-1 expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives Engrailed-1 activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents Engrailed-1 activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).

  View details for DOI 10.1126/science.aba2374

  View details for PubMedID 33888614

• Discussion: Overcoming the Patent Gap: A Guide to Patenting for Plastic Surgeons. Plastic and reconstructive surgery Januszyk, M., desJardins-Park, H. E., Gurtner, G. C., Longaker, M. T. 2021; 148 (4): 918-919

  View details for DOI 10.1097/PRS.0000000000008394

  View details for PubMedID 34550949

• An Evolutionary Conserved Signaling Network Between Mouse and Human Underlies the Differential Osteoskeletal Potential of Frontal and Parietal Calvarial Bones. Frontiers in physiology Menon, S., Huber, J., Duldulao, C., Longaker, M. T., Quarto, N. 2021; 12: 747091

  Abstract

  The mammalian calvarial vault is an ancient and highly conserved structure among species, however, the mechanisms governing osteogenesis of the calvarial vault and how they might be conserved across mammalian species remain unclear. The aim of this study was to determine if regional differences in osteogenic potential of the calvarial vault, first described in mice, extend to humans. We derived human frontal and parietal osteoblasts from fetal calvarial tissue, demonstrating enhanced osteogenic potential both in vitro and in vivo of human frontal derived osteoblasts compared to parietal derived osteoblasts. Furthermore, we found shared differential signaling patterns in the canonical WNT, TGF-beta, BMP, and FGF pathways previously described in the mouse to govern these regional differences in osteogenic potential. Taken together, our findings unveil evolutionary conserved similarities both at functional and molecular level between the mouse and human calvarial bones, providing further support that studies employing mouse models, are suitable for translational studies to human.

  View details for DOI 10.3389/fphys.2021.747091

  View details for PubMedID 34744787

• Striae Distensae: Scars without Wounds. Plastic and reconstructive surgery Borrelli, M. R., Griffin, M., Ngaage, L. M., Longaker, M. T., Lorenz, H. P. 2021; 148 (1): 77-87

  Abstract

  Striae distensae, or stretch marks, are common linear lesions of atrophic skin characterized histologically by epidermal atrophy, absent rete ridges, and alterations in connective tissue architecture. Hormonal excess, mechanical stress, and genetic predisposition are all associated with striae distensae, but their exact pathogenesis remains unknown. Despite a multitude of options, no single treatment has yet proven effective. In this article, the authors describe an up-to-date overview of striae distensae in terms of their etiology, pathophysiology, and therapeutic options. Further research is required to better elucidate their pathophysiology and to develop targeted effective treatments.

  View details for DOI 10.1097/PRS.0000000000008065

  View details for PubMedID 34181606

• Aged skeletal stem cells generate an inflammatory degenerative niche. Nature Ambrosi, T. H., Marecic, O., McArdle, A., Sinha, R., Gulati, G. S., Tong, X., Wang, Y., Steininger, H. M., Hoover, M. Y., Koepke, L. S., Murphy, M. P., Sokol, J., Seo, E. Y., Tevlin, R., Lopez, M., Brewer, R. E., Mascharak, S., Lu, L., Ajanaku, O., Conley, S. D., Seita, J., Morri, M., Neff, N. F., Sahoo, D., Yang, F., Weissman, I. L., Longaker, M. T., Chan, C. K. 2021

  Abstract

  Loss of skeletal integrity during ageing and disease is associated with an imbalance in the opposing actions of osteoblasts and osteoclasts1. Here we show that intrinsic ageing of skeletal stem cells (SSCs)2 in mice alters signalling in the bone marrow niche and skews the differentiation of bone and blood lineages, leading to fragile bones that regenerate poorly. Functionally, aged SSCs have a decreased bone- and cartilage-forming potential but produce more stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell RNA-sequencing studies link the functional loss to a diminished transcriptomic diversity of SSCs in aged mice, which thereby contributes to the transformation of the bone marrow niche. Exposure to a youthful circulation through heterochronic parabiosis or systemic reconstitution with young haematopoietic stem cells did not reverse the diminished osteochondrogenic activity of aged SSCs, or improve bone mass or skeletal healing parameters in aged mice. Conversely, the aged SSC lineage promoted osteoclastic activity and myeloid skewing by haematopoietic stem and progenitor cells, suggesting that the ageing of SSCs is a driver of haematopoietic ageing. Deficient bone regeneration in aged mice could only be returned to youthful levels by applying a combinatorial treatment of BMP2 and a CSF1 antagonist locally to fractures, which reactivated aged SSCs and simultaneously ablated the inflammatory, pro-osteoclastic milieu. Our findings provide mechanistic insights into the complex, multifactorial mechanisms that underlie skeletal ageing and offer prospects for rejuvenating the aged skeletal system.

  View details for DOI 10.1038/s41586-021-03795-7

  View details for PubMedID 34381212

• Disrupting biological sensors of force promotes tissue regeneration in large organisms. Nature communications Chen, K., Kwon, S. H., Henn, D., Kuehlmann, B. A., Tevlin, R., Bonham, C. A., Griffin, M., Trotsyuk, A. A., Borrelli, M. R., Noishiki, C., Padmanabhan, J., Barrera, J. A., Maan, Z. N., Dohi, T., Mays, C. J., Greco, A. H., Sivaraj, D., Lin, J. Q., Fehlmann, T., Mermin-Bunnell, A. M., Mittal, S., Hu, M. S., Zamaleeva, A. I., Keller, A., Rajadas, J., Longaker, M. T., Januszyk, M., Gurtner, G. C. 2021; 12 (1): 5256

  Abstract

  Tissue repair and healing remain among the most complicated processes that occur during postnatal life. Humans and other large organisms heal by forming fibrotic scar tissue with diminished function, while smaller organisms respond with scarless tissue regeneration and functional restoration. Well-established scaling principles reveal that organism size exponentially correlates with peak tissue forces during movement, and evolutionary responses have compensated by strengthening organ-level mechanical properties. How these adaptations may affect tissue injury has not been previously examined in large animals and humans. Here, we show that blocking mechanotransduction signaling through the focal adhesion kinase pathway in large animals significantly accelerates wound healing and enhances regeneration of skin with secondary structures such as hair follicles. In human cells, we demonstrate that mechanical forces shift fibroblasts toward pro-fibrotic phenotypes driven by ERK-YAP activation, leading to myofibroblast differentiation and excessive collagen production. Disruption of mechanical signaling specifically abrogates these responses and instead promotes regenerative fibroblast clusters characterized by AKT-EGR1.

  View details for DOI 10.1038/s41467-021-25410-z

  View details for PubMedID 34489407

• Xenogeneic skin transplantation promotes angiogenesis and tissue regeneration through activated Trem2+ macrophages. Science advances Henn, D., Chen, K., Fehlmann, T., Trotsyuk, A. A., Sivaraj, D., Maan, Z. N., Bonham, C. A., Barrera, J. A., Mays, C. J., Greco, A. H., Moortgat Illouz, S. E., Lin, J. Q., Steele, S. R., Foster, D. S., Padmanabhan, J., Momeni, A., Nguyen, D., Wan, D. C., Kneser, U., Januszyk, M., Keller, A., Longaker, M. T., Gurtner, G. C. 2021; 7 (49): eabi4528

  Abstract

  [Figure: see text].

  View details for DOI 10.1126/sciadv.abi4528

  View details for PubMedID 34851663

• Angiogenic CD34+CD146+ adipose-derived stromal cells augment recovery of soft tissue after radiotherapy. Journal of tissue engineering and regenerative medicine Diaz Deleon, N. M., Adem, S., Lavin, C. V., Abbas, D. B., Griffin, M., King, M. E., Borrelli, M. R., Patel, R. A., Fahy, E. J., Lee, D., Shen, A. H., Momeni, A., Longaker, M. T., Wan, D. C. 2021

  Abstract

  Radiation therapy is effective for cancer treatment but may also result in collateral soft tissue contracture, contour deformities, and non-healing wounds. Autologous fat transfer has been described to improve tissue architecture and function of radiation-induced fibrosis and these effects may be augmented by enrichment with specific adipose-derived stromal cells (ASCs) with enhanced angiogenic potential. CD34+CD146+, CD34+CD146-, or CD34+ unfractionated (UF) human ASCs were isolated by flow cytometry and used to supplement human lipoaspirate placed beneath the scalp of irradiated mice. Volume retention was followed radiographically and fat grafts as well as overlying soft tissue were harvested after eight weeks for histologic and biomechanical analyses. Radiographic evaluation revealed the highest volume retention in fat grafts supplemented with CD34+CD146+ ASCs, and these grafts were also found to have greater histologic integrity than other groups. Irradiated skin overlying CD34+CD146+ ASC-enriched grafts was significantly more vascularized than other treatment groups, had significantly less dermal thickness and collagen deposition, and the greatest improvement in fibrillin staining and return of elasticity. Radiation therapy obliterates vascularity and contributes to scarring and loss of tissue function. ASC-enrichment of fat grafts with CD34+CD146+ ASCs not only enhances fat graft vascularization and retention, but also significantly promotes improvement in overlying radiation-injured soft tissue. This regenerative effect on skin is highly promising for patients with impaired wound healing and deformities following radiotherapy. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/term.3253

  View details for PubMedID 34582109

• Epidermal-Derived Hedgehog Signaling Drives Mesenchymal Proliferation during Digit Tip Regeneration. Journal of clinical medicine Maan, Z. N., Rinkevich, Y., Barrera, J., Chen, K., Henn, D., Foster, D., Bonham, C. A., Padmanabhan, J., Sivaraj, D., Duscher, D., Hu, M., Yan, K., Januszyk, M., Longaker, M. T., Weissman, I. L., Gurtner, G. C. 2021; 10 (18)

  Abstract

  Hand injuries often result in significant functional impairments and are rarely completely restored. The spontaneous regeneration of injured appendages, which occurs in salamanders and newts, for example, has been reported in human fingertips after distal amputation, but this type of regeneration is rare in mammals and is incompletely understood. Here, we study fingertip regeneration by amputating murine digit tips, either distally to initiate regeneration, or proximally, causing fibrosis. Using an unbiased microarray analysis, we found that digit tip regeneration is significantly associated with hair follicle differentiation, Wnt, and sonic hedgehog (SHH) signaling pathways. Viral over-expression and genetic knockouts showed the functional significance of these pathways during regeneration. Using transgenic reporter mice, we demonstrated that, while both canonical Wnt and HH signaling were limited to epidermal tissues, downstream hedgehog signaling (through Gli) occurred in mesenchymal tissues. These findings reveal a mechanism for epidermal/mesenchyme interactions, governed by canonical hedgehog signaling, during digit regeneration. Further research into these pathways could lead to improved therapeutic outcomes after hand injuries in humans.

  View details for DOI 10.3390/jcm10184261

  View details for PubMedID 34575372

• The Adrenergic System in Plastic and Reconstructive Surgery: Physiology and Clinical Considerations. Annals of plastic surgery Fahy, E. J., Griffin, M. n., Lavin, C. n., Abbas, D. n., Longaker, M. T., Wan, D. n. 2021

  Abstract

  The primary organ systems and tissues concerning plastic and reconstructive surgery include the integument, vasculature, subcutis, and peripheral nerves, because these may individually or collectively be injured requiring reconstruction, or indeed be used in reconstruction themselves through grafts, flaps, or anastomoses. Adrenergic receptors are present throughout these anatomic components on the vasculature, adipose, platelets, immune cells, keratinocytes, melanocytes, fibroblasts, peripheral nerves, and tendons. Herein, the influence of adrenergic signaling on the physiology of anatomic components related to plastic surgery is discussed, along with clinical considerations of this systems involvement in procedures, such as free flap reconstruction, skin grafting, fat grafting, and other areas relevant to plastic and reconstructive surgery. Current evidence as well as potential for further investigation is discussed.

  View details for DOI 10.1097/SAP.0000000000002706

  View details for PubMedID 33833152

• Proceed with Caution: Mouse Deep Digit Flexor Tendon Injury Model. Plastic and reconstructive surgery. Global open Titan, A. L., Fahy, E. n., Chen, K. n., Foster, D. S., Bennett-Kennett, R. n., Dauskardt, R. H., Gurtner, G. C., Chang, J. n., Fox, P. M., Longaker, M. T. 2021; 9 (1): e3359

  Abstract

  The purpose of this study was to determine the feasibility of using mouse models for translational study of flexor tendon repair and reconstruction.Quantitative data detailing the gross anatomy, biomechanical characteristics, and microscopic structure of the deep digit flexor tendon (DDF) of the mouse hindpaw were obtained. Histological characterization of the DDF and the anatomy of the digit in the mouse hindpaw are detailed. Biomechanical testing determined the load-to-failure, stress, elastic modulus, and the site of tendon failure.In gross anatomy, the origins and insertions of the mouse deep digit flexor tendon are similar to those of the human digit, surrounded by a synovial sheath that is only 1- to 2-cells thick. A neurovascular network runs on each side of the digit outside the synovial sheath, but does not clearly penetrate it. The thickness of the DDF is 0.14 ± 0.03 mm and the width is 0.3 ± 0.03 mm. The thickness of the DDF is less than that of 9-0 nylon needle. The mean failure force of the deep flexor tendon was 2.79 ± 0.53N.The gross anatomy of the mouse hindpaw digit is similar to that of the human digit except for key differences seen in the synovial sheath and vascular supply. The dimensions of the mouse DDF make it challenging to create a clinically translatable repair model using currently available surgical techniques. Despite the similarities between the human and mouse anatomy, and the powerful basic science tools available in murine models, mice are an unreliable model for assessing flexor tendon injury and repair.

  View details for DOI 10.1097/GOX.0000000000003359

  View details for PubMedID 33552814

  View details for PubMedCentralID PMC7859083

• Craniofacial and Long Bone Development in the Context of Distraction Osteogenesis. Plastic and reconstructive surgery Shah, H. N., Jones, R. E., Borrelli, M. R., Robertson, K. n., Salhotra, A. n., Wan, D. C., Longaker, M. T. 2021; 147 (1): 54e–65e

  Abstract

  Bone retains regenerative potential into adulthood, and surgeons harness this plasticity during distraction osteogenesis. The underlying biology governing bone development, repair, and regeneration is divergent between the craniofacial and appendicular skeleton. Each type of bone formation is characterized by unique molecular signaling and cellular behavior. Recent discoveries have elucidated the cellular and genetic processes underlying skeletal development and regeneration, providing an opportunity to couple biological and clinical knowledge to improve patient care.A comprehensive literature review of basic and clinical literature regarding craniofacial and long bone development, regeneration, and distraction osteogenesis was performed.The current understanding in craniofacial and long bone development and regeneration is discussed, and clinical considerations for the respective distraction osteogenesis procedures are presented.Distraction osteogenesis is a powerful tool to regenerate bone and thus address a number of craniofacial and appendicular skeletal deficiencies. The molecular mechanisms underlying bone regeneration, however, remain elusive. Recent work has determined that embryologic morphogen gradients constitute important signals during regeneration. In addition, striking discoveries have illuminated the cellular processes underlying mandibular regeneration during distraction osteogenesis, showing that skeletal stem cells reactivate embryologic neural crest transcriptomic processes to carry out bone formation during regeneration. Furthermore, innovative adjuvant therapies to complement distraction osteogenesis use biological processes active in embryogenesis and regeneration. Additional research is needed to further characterize the underlying cellular mechanisms responsible for improved bone formation through adjuvant therapies and the role skeletal stem cells play during regeneration.

  View details for DOI 10.1097/PRS.0000000000007451

  View details for PubMedID 33370054

• Wounds Inhibit Tumor Growth In Vivo ANNALS OF SURGERY Hu, M. S., Maan, Z. N., Leavitt, T., Hong, W., Rennert, R. C., Marshall, C. D., Borrelli, M. R., Zhu, T. N., Esquivel, M., Zimmermann, A., McArdle, A., Chung, M. T., Foster, D. S., Jones, R., Gurtner, G. C., Giaccia, A. J., Lorenz, H., Weissman, I. L., Longaker, M. T. 2021; 273 (1): 173–80

  View details for DOI 10.1097/SLA.0000000000003255

  View details for Web of Science ID 000613348700039

• Endogenous Mechanisms of Craniomaxillofacial Repair: Toward Novel Regenerative Therapies. Frontiers in oral health desJardins-Park, H. E., Mascharak, S., Longaker, M. T., Wan, D. C. 1800; 2: 676258

  Abstract

  In the fields of oral and craniomaxillofacial surgery, regeneration of multiple tissue types-including bone, skin, teeth, and mucosal soft tissue-is often a desired outcome. However, limited endogenous capacity for regeneration, as well as predisposition of many tissues to fibrotic healing, may prevent recovery of normal form and function for patients. Recent basic science research has advanced our understanding of molecular and cellular pathways of repair in the oral/craniofacial region and how these are influenced by local microenvironment and embryonic origin. Here, we review the current state of knowledge in oral and craniomaxillofacial tissue repair/regeneration in four key areas: bone (in the context of calvarial defects and mandibular regeneration during distraction osteogenesis); skin (in the context of cleft lip/palate surgery); oral mucosa (in the context of minimally scarring repair of mucosal injuries); and teeth (in the context of dental disease/decay). These represent four distinct healing processes and outcomes. We will discuss both divergent and conserved pathways of repair in these contexts, with an eye toward fundamental mechanisms of regeneration vs. fibrosis as well as translational research directions. Ultimately, this knowledge can be leveraged to develop new cell-based and molecular treatment strategies to encourage bone and soft tissue regeneration in oral and craniomaxillofacial surgery.

  View details for DOI 10.3389/froh.2021.676258

  View details for PubMedID 35048022

• Exosomes - a tool for bone tissue engineering. Tissue engineering. Part B, Reviews Huber, J. L., Griffin, M., Longaker, M. T., Quarto, N. 2020

  Abstract

  Mesenchymal stem cells (MSC) have been repeatedly shown to be a valuable source for cell-based therapy in regenerative medicine, including bony tissue repair. However, engraftment at the injury site is poor. Recently, it has been suggested that MSCs and other cells act via a paracrine signaling mechanism. Exosomes are nanostructures that have been implicated in this process. They carry DNA, RNA, proteins and lipids and play an important role in cell-to-cell communication directly modulating their target cell at a transcriptional level. In a bone microenvironment, they have been shown to increase osteogenesis and osteogenic differentiation in vivo and in vitro. In the following review, we will discuss the most advanced and significant knowledge of biological functions of exosomes in bone regeneration and their clinical applications in osseous diseases.

  View details for DOI 10.1089/ten.TEB.2020.0246

  View details for PubMedID 33297857

• Prrx1 Fibroblasts Represent a Pro-fibrotic Lineage in the Mouse Ventral Dermis. Cell reports Leavitt, T., Hu, M. S., Borrelli, M. R., Januszyk, M., Garcia, J. T., Ransom, R. C., Mascharak, S., desJardins-Park, H. E., Litzenburger, U. M., Walmsley, G. G., Marshall, C. D., Moore, A. L., Duoto, B., Adem, S., Foster, D. S., Salhotra, A., Shen, A. H., Griffin, M., Shen, E. Z., Barnes, L. A., Zielins, E. R., Maan, Z. N., Wei, Y., Chan, C. K., Wan, D. C., Lorenz, H. P., Chang, H. Y., Gurtner, G. C., Longaker, M. T. 2020; 33 (6): 108356

  Abstract

  Fibroblast heterogeneity has been shown within the unwounded mouse dorsal dermis, with fibroblast subpopulations being identified according to anatomical location and embryonic lineage. Using lineage tracing, we demonstrate that paired related homeobox 1 (Prrx1)-expressing fibroblasts are responsible for acute and chronic fibroses in the ventral dermis. Single-cell transcriptomics further corroborated the inherent fibrotic characteristics of Prrx1 fibroblasts during wound repair. In summary, we identify and characterize a fibroblast subpopulation in the mouse ventral dermis with intrinsic scar-forming potential.

  View details for DOI 10.1016/j.celrep.2020.108356

  View details for PubMedID 33176144

• Skeletal Stem Cells-A Paradigm Shift in the Field of Craniofacial Bone Tissue Engineering. Frontiers in dental medicine Tevlin, R., Longaker, M. T., Wan, D. C. 2020; 1

  Abstract

  Defects of the craniofacial skeleton arise as a direct result of trauma, diseases, oncological resection, or congenital anomalies. Current treatment options are limited, highlighting the importance for developing new strategies to restore form, function, and aesthetics of missing or damaged bone in the face and the cranium. For optimal reconstruction, the goal is to replace "like with like." With the inherent challenges of existing options, there is a clear need to develop alternative strategies to reconstruct the craniofacial skeleton. The success of mesenchymal stem cell-based approaches has been hampered by high heterogeneity of transplanted cell populations with inconsistent preclinical and clinical trial outcomes. Here, we discuss the novel characterization and isolation of mouse skeletal stem cell (SSC) populations and their response to injury, systemic disease, and how their re-activation in vivo can contribute to tissue regeneration. These studies led to the characterization of human SSCs which are able to self-renew, give rise to increasingly fate restricted progenitors, and differentiate into bone, cartilage, and bone marrow stroma, all on the clonal level in vivo without prior in vitro culture. SSCs hold great potential for implementation in craniofacial bone tissue engineering and regenerative medicine. As we begin to better understand the diversity and the nature of skeletal stem and progenitor cells, there is a tangible future whereby a subset of human adult SSCs can be readily purified from bone or activated in situ with broad potential applications in craniofacial tissue engineering.

  View details for DOI 10.3389/fdmed.2020.596706

  View details for PubMedID 35664558

  View details for PubMedCentralID PMC9161996

• Peripheral Motor Neuron Activity Influences over Local Sarcoma Progression Davitt, M., Foster, D., Mascharak, S., desJardins-Park, H., Norton, J., Longaker, M. T. ELSEVIER SCIENCE INC. 2020: S230–S231

  View details for Web of Science ID 000582792300421

• Radiation-Induced Soft Tissue Atrophy Impaired by Enhancement of Fat Grafts with CD146+Subpopulation of Adipose-Derived Stromal Cells Deleon, N., Borrelli, M. R., Adem, S., Shen, A., Abbas, D., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2020: E186

  View details for Web of Science ID 000582798100421

• Fat Grafting Depletes Profibrotic Prrx1-Positive Fibroblasts in Irradiated Skin and Mitigates Radiation-Induced Groin Contracture Borrelli, M. R., Adem, S., Deleon, N., Abbas, D., Chen, K., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2020: S225–S226

  View details for Web of Science ID 000582792300411

• Transdermal Deferoxamine Reduces Radiation-Induced Damage in Porcine Skin Adem, S., Deleon, N., Chen, K., Borrelli, M. R., Shen, A. H., Noishiki, C., Patel, R. A., Gurtner, G. C., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2020: E46–E47

  View details for Web of Science ID 000582798100088

• Transdermal Deferoxamine Treatment Mitigates Fibrosis in Irradiated Skin Shen, A. H., Borrelli, M. R., Deleon, N., Adem, S., Mascharak, S., Salhotra, A., Shah, H., Longaker, M. T., Gurtner, G. C., Wan, D. C. ELSEVIER SCIENCE INC. 2020: S235

  View details for Web of Science ID 000582792300431

• Wounds Heal by Tissue-Resident Fibroblast Progenitors that Proliferate Polyclonally and Mechanoresponsively Foster, D. S., Chinta, M., Salhotra, A., Nguyen, A. T., Burcham, A., Mascharak, S., Januszyk, M., Gurtner, G. C., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2020: S236–S237

  View details for Web of Science ID 000582792300433

• Ectoderm-Derived Wnt and Hedgehog Signaling Drive Digit Tip Regeneration Barrera, J., Maan, Z. N., Foster, D., Henn, D., Chen, K., Bonham, C., Januszyk, M., Longaker, M. T., Weissman, I., Gurtner, G. C. ELSEVIER SCIENCE INC. 2020: S186

  View details for Web of Science ID 000582792300339

• Understanding Long Bone Regeneration through the Development of a Novel Murine Distraction Device Shah, H. N., Salhotra, A., Wan, D. C., Longaker, M. T. ELSEVIER SCIENCE INC. 2020: E173

  View details for Web of Science ID 000582798100387

• Detection, Scoring, and Classification of Solid Organ Fibroses with Machine Learning Analysis Mascharak, S., desJardins-Park, H. E., Davitt, M., Foster, D. S., Chinta, M., Wan, D. C., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2020: S222

  View details for Web of Science ID 000582792300403

• Adipose-Derived Stromal Cells within Transplanted Fat Hone to Blood Vessels and Assume a Pericyte Structure Borrelli, M. R., Adem, S., Deleon, N., Abbas, D., Momeni, A., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2020: E183

  View details for Web of Science ID 000582798100412

• A Surgical Model for Investigating the Role of Creeping Fat in Intestinal Fibrosis Bauer-Rowe, K. E., Foster, D., Titan, A., Chinta, M., desJardins-Park, H., Griffin, M., Longaker, M. T. ELSEVIER SCIENCE INC. 2020: S50–S51

  View details for Web of Science ID 000582792300070

• Grafted Fat Depletes the Profibrotic Engrailed-1-Positive Fibroblast Subpopulation and Ameliorates Radiation-Induced Scalp Fibrosis Borrelli, M. R., Deleon, N., Adem, S., Abbas, D., Momeni, A., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2020: E186

  View details for Web of Science ID 000582798100420

• Elucidating Molecular Drivers of Wound Regeneration in MRL Mice Via Novel Transcriptomic Analyses desJardins-Park, H. E., Mack, K. L., Davitt, M. F., Griffin, M., Mascharak, S., Fraser, H. B., Longaker, M. T. ELSEVIER SCIENCE INC. 2020: S225

  View details for Web of Science ID 000582792300410

• Mechanisms of bone development and repair. Nature reviews. Molecular cell biology Salhotra, A., Shah, H. N., Levi, B., Longaker, M. T. 2020

  Abstract

  Bone development occurs through a series of synchronous events that result in the formation of the body scaffold. The repair potential of bone and its surrounding microenvironment - including inflammatory, endothelial and Schwann cells - persists throughout adulthood, enabling restoration of tissue to its homeostatic functional state. The isolation of a single skeletal stem cell population through cell surface markers and the development of single-cell technologies are enabling precise elucidation of cellular activity and fate during bone repair by providing key insights into the mechanisms that maintain and regenerate bone during homeostasis and repair. Increased understanding of bone development, as well as normal and aberrant bone repair, has important therapeutic implications for the treatment of bone disease and ageing-related degeneration.

  View details for DOI 10.1038/s41580-020-00279-w

  View details for PubMedID 32901139

• Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing. Micromachines Januszyk, M., Chen, K., Henn, D., Foster, D. S., Borrelli, M. R., Bonham, C. A., Sivaraj, D., Wagh, D., Longaker, M. T., Wan, D. C., Gurtner, G. C. 2020; 11 (9)

  Abstract

  Background: Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of the most difficult target specimens, due to the significant amount of fibrinous debris, extracellular matrix components, and non-viable cells inherent in tissue routinely obtained from debridement. Methods: Here, we examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. Results: 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/L, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells. Conclusions: scRNA-seq of clinical wound samples can be achieved using minor modifications to standard processing protocols and data analysis methods. This simple approach can capture widespread transcriptional differences between diabetic and non-diabetic tissue obtained from matched wound locations.

  View details for DOI 10.3390/mi11090815

  View details for PubMedID 32872278

• Articular cartilage regeneration by activated skeletal stem cells. Nature medicine Murphy, M. P., Koepke, L. S., Lopez, M. T., Tong, X., Ambrosi, T. H., Gulati, G. S., Marecic, O., Wang, Y., Ransom, R. C., Hoover, M. Y., Steininger, H., Zhao, L., Walkiewicz, M. P., Quarto, N., Levi, B., Wan, D. C., Weissman, I. L., Goodman, S. B., Yang, F., Longaker, M. T., Chan, C. K. 2020

  Abstract

  Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage1. The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation2-4. Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis5-7. We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.

  View details for DOI 10.1038/s41591-020-1013-2

  View details for PubMedID 32807933

• Fibroblast Heterogeneity in Wound Healing: Hurdles to Clinical Translation. Trends in molecular medicine Mascharak, S., desJardins-Park, H. E., Longaker, M. T. 2020

  Abstract

  Recent work has revealed that fibroblasts are remarkably heterogeneous cells, but the appropriate lens through which to study this variation (lineage, phenotype, and plasticity) and its relevance to human biology remain unclear. In this opinion article, we comment on recent breakthroughs in our understanding of fibroblast heterogeneity during skin wound healing, and on open questions that must be addressed to clinically translate these findings in order to minimize scarring in patients. We emphasize the need for experimental models of wound healing that better approximate human biology, as well as comparison of scarring and regenerative phenotypes to uncover master regulators of fibrosis.

  View details for DOI 10.1016/j.molmed.2020.07.008

  View details for PubMedID 32800679

• Rewriting the Future: Promises and Limits of Germline Gene Editing in Craniofacial Surgery. The Journal of craniofacial surgery Davitt, M., Mascharak, S., desJardins-Park, H., Chinta, M., Wan, D. C., Longaker, M. T. 2020

  View details for DOI 10.1097/SCS.0000000000006602

  View details for PubMedID 32796298

• Prophylactic treatment with transdermal deferoxamine mitigates radiation-induced skin fibrosis. Scientific reports Shen, A. H., Borrelli, M. R., Adem, S., Deleon, N. M., Patel, R. A., Mascharak, S., Yen, S. J., Sun, B. Y., Taylor, W. L., Januszyk, M., Nguyen, D. H., Momeni, A., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2020; 10 (1): 12346

  Abstract

  Radiation therapy can result in pathological fibrosis of healthy soft tissue. The iron chelator deferoxamine (DFO) has been shown to improve skin vascularization when injected into radiated tissue prior to fat grafting. Here, we evaluated whether topical DFO administration using a transdermal drug delivery system prior to and immediately following irradiation (IR) can mitigate the chronic effects of radiation damage to the skin. CD-1 nude immunodeficient mice were split into four experimental groups: (1) IR alone (IR only), (2) DFO treatment for two weeks after recovery from IR (DFO post-IR), (3) DFO prophylaxis with treatment through and post-IR (DFO ppx), or (4) no irradiation or DFO (No IR). Immediately following IR, reactive oxygen species and apoptotic markers were significantly decreased and laser doppler analysis revealed significantly improved skin perfusion in mice receiving prophylactic DFO. Six weeks following IR, mice in the DFO post-IR and DFO ppx groups had improved skin perfusion and increased vascularization. DFO-treated groups also had evidence of reduced dermal thickness and collagen fiber network organization akin to non-irradiated skin. Thus, transdermal delivery of DFO improves tissue perfusion and mitigates chronic radiation-induced skin fibrosis, highlighting a potential role for DFO in the treatment of oncological patients.

  View details for DOI 10.1038/s41598-020-69293-4

  View details for PubMedID 32704071

• Doxycycline Reduces Scar Thickness and Improves Collagen Architecture ANNALS OF SURGERY Moore, A. L., desJardins-Park, H. E., Duoto, B. A., Mascharak, S., Murphy, M. P., Irizarry, D. M., Foster, D. S., Jones, R. E., Barnes, L. A., Marshall, C. D., Ransom, R. C., Wernig, G., Longaker, M. T. 2020; 272 (1): 183–93

  View details for DOI 10.1097/SLA.0000000000003172

  View details for Web of Science ID 000568895500044

• Radiation-induced skin fibrosis is reversed by transdermal delivery of deferoxamine Borrelli, M. R., Adem, S., Diaz, N., Mascharak, S., Sen, A., Januszyk, M., Nguyen, D., Momeni, A., Gurtner, G. C., Longaker, M. T., Wan, D. C. WILEY. 2020: S51–S52

  View details for Web of Science ID 000548418300116

• Reactive oxygen species and apoptotic proteins in irradiated murine skin decrease with deferoxamine treatment Shen, A. H., Deleon, N., Adem, S., Borrelli, M. R., Salhotra, A., Shah, H., Gurtner, G. C., Longaker, M. T., Wan, D. C. WILEY. 2020: S32–S33

  View details for Web of Science ID 000548418300070

• Activation of skeletal stems cells in response to long bone distraction osteogenesis Salhotra, A., Shah, H. N., Lopez, M. T., Wan, D. C., Longaker, M. T. WILEY. 2020: S25–S26

  View details for Web of Science ID 000548418300053

• Stretch marks are abundant in CD26-positive human dermal fibroblasts and exhibit increased profibrotic mechanosensitive signaling Borrelli, M. R., Griffin, M., Ngaage, L. M., Mascharak, S., Lewis, N., Januszyk, M., Wan, D. C., Longaker, M. T., Lorenz, H. P. WILEY. 2020: S32

  View details for Web of Science ID 000548418300069

• Inhibiting mechanotransduction signaling changes fibroblast heterogeneity and promotes tissue regeneration in healing wounds Chen, K., Kwon, S., Henn, D., Kuehlmann, B. A., Bonham, C. A., Padmanabhan, J., Noishiki, C., Barrera, J., Longaker, M. T., Januszyk, M., Gurtner, G. C. WILEY. 2020: S13–S14

  View details for Web of Science ID 000548418300026

• Inhibiting mechanotransduction signaling changes fibroblast heterogeneity and promotes tissue regeneration in healing wounds Chen, K., Kwon, S., Henn, D., Kuehlmann, B. A., Bonham, C. A., Padmanabhan, J., Noishiki, C., Barrera, J., Longaker, M. T., Januszyk, M., Gurtner, G. C. WILEY. 2020: S12–S13

  View details for Web of Science ID 000548418300023

• A novel murine distraction device investigating long bone regeneration Shah, H. N., Salhotra, A., Lopez, M. T., Wan, D. C., Longaker, M. T. WILEY. 2020: S16

  View details for Web of Science ID 000548418300032

• Radiation-induced fibrosis in porcine skin improves with transdermal deferoxamine treatment Adem, S., Borrelli, M. R., Deleon, N., Shen, A. H., Chen, K., Noishiki, C., Gurtner, G. C., Longaker, M. T., Wan, D. C. WILEY. 2020: S31–S32

  View details for Web of Science ID 000548418300068

• Harnessing novel gene expression analyses to identify drivers of regenerative ear wound healing in MRL mice desJardins-Park, H. E., Mack, K. L., Davitt, M. F., Griffin, M., Fraser, H. B., Longaker, M. T. WILEY. 2020: S25

  View details for Web of Science ID 000548418300052

• Fibroblast Heterogeneity in and Its Implications for Plastic and Reconstructive Surgery: A Basic Science Review PLASTIC AND RECONSTRUCTIVE SURGERY-GLOBAL OPEN desJardins-Park, H. E., Chinta, M. S., Foster, D. S., Borrelli, M. R., Shen, A. H., Wan, D. C., Longaker, M. T. 2020; 8 (6)

  View details for DOI 10.1097/GOX.0000000000002927

  View details for Web of Science ID 000552760800046

• Fibroblast Heterogeneity in and Its Implications for Plastic and Reconstructive Surgery: A Basic Science Review. Plastic and reconstructive surgery. Global open desJardins-Park, H. E., Chinta, M. S., Foster, D. S., Borrelli, M. R., Shen, A. H., Wan, D. C., Longaker, M. T. 2020; 8 (6): e2927

  Abstract

  Fibroblasts' integral role in tissue development, maintenance, and disease represents a fast-growing field of basic science research. Although fibroblasts were long thought to be a homogeneous cell population, recent research has illuminated the unforeseen complexity of these cells, giving rise to the rapidly expanding research field of "fibroblast heterogeneity." Fibroblasts play a critical role in states of tissue fibrosis such as skin scarring, which affects hundreds of millions of patients annually and causes severe aesthetic, developmental, and functional morbidity. Beyond scarring, major organ fibrosis is an enormous public health concern responsible for nearly half of all deaths in the United States. Because fibrosis is a conserved response to tissue damage in all organs, the study of fibroblasts throughout the body may help us to understand their role in the conditions most relevant to plastic and reconstructive surgery-for instance, skin scarring (eg, from burns, traumatic lacerations, or surgical incisions), "pathological" scarring (hypertrophic scars, keloids), and capsular contracture. Here, we present a basic science review of fibroblast heterogeneity in wound healing, cancer, organ fibrosis, and human dermal architecture. The field of fibroblast heterogeneity is young, and many of the insights discussed have yet to be translated clinically. However, plastic surgeons stand in a unique position to bridge these discoveries into clinical realities. We hope this information can spur readers to consider both what questions in plastic surgery can be studied from the lens of fibroblast heterogeneity, and how these preclinical insights can be translated to improving care of our patients.

  View details for DOI 10.1097/GOX.0000000000002927

  View details for PubMedID 32766071

  View details for PubMedCentralID PMC7339369

• Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation. eLife Carter, A. C., Xu, J., Nakamoto, M. Y., Wei, Y., Zarnegar, B. J., Shi, Q., Broughton, J. P., Ransom, R. C., Salhotra, A., Nagaraja, S. D., Li, R., Dou, D. R., Yost, K. E., Cho, S., Mistry, A., Longaker, M. T., Khavari, P. A., Batey, R. T., Wuttke, D. S., Chang, H. Y. 2020; 9

  Abstract

  The Xist lncRNA mediates X chromosome inactivation (XCI)1,2. Here we show that Spen, an Xist-binding repressor protein essential for XCI3-9, binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen inactivation activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing10-11. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.

  View details for DOI 10.7554/eLife.54508

  View details for PubMedID 32379046

• Pro-Fibrotic CD26-Positive Fibroblasts Are Present in Greater Abundance in Breast Capsule Tissue of Irradiated Breasts AESTHETIC SURGERY JOURNAL Borrelli, M. R., Irizzary, D., Patel, R. A., Dung Nguyen, Momeni, A., Longaker, M. T., Wan, D. C. 2020; 40 (4): 369–79

  View details for DOI 10.1093/asj/sjz109

  View details for Web of Science ID 000558971600024

• Pancreatic Cancer Associated Fibroblasts (CAF): Under-Explored Target for Pancreatic Cancer Treatment. Cancers Norton, J. n., Foster, D. n., Chinta, M. n., Titan, A. n., Longaker, M. n. 2020; 12 (5)

  Abstract

  Pancreatic cancer is the 4th leading cause of cancer deaths in the United States. The pancreatic cancer phenotype is primarily a consequence of oncogenes disturbing the resident pancreas parenchymal cell repair program. Many solid tumor types including pancreatic cancer have severe tumor fibrosis called desmoplasia. Desmoplastic stroma is coopted by the tumor as a support structure and CAFs aid in tumor growth, invasion, and metastases. This stroma is caused by cancer associated fibroblasts (CAFs), which lay down extensive connective tissue in and around the tumor cells. CAFs represent a heterogeneous population of cells that produce various paracrine molecules such as transforming growth factor-beta (TGF-beta) and platelet derived growth factors (PDGFs) that aid tumor growth, local invasion, and development of metastases. The hard, fibrotic shell of desmoplasia serves as a barrier to the infiltration of both chemo- and immunotherapy drugs and host immune cells to the tumor. Although there have been recent improvements in chemotherapy and surgical techniques for management of pancreatic cancer, the majority of patients will die from this disease. Therefore, new treatment strategies are clearly needed. CAFs represent an under-explored potential therapeutic target. This paper discusses what we know about the role of CAFs in pancreatic cancer cell growth, invasion, and metastases. Additionally, we present different strategies that are being and could be explored as anti-CAF treatments for pancreatic cancer.

  View details for DOI 10.3390/cancers12051347

  View details for PubMedID 32466266

• CD34+CD146+ adipose-derived stromal cells enhance engraftment of transplanted fat. Stem cells translational medicine Borrelli, M. R., Patel, R. A., Blackshear, C. n., Vistnes, S. n., Diaz Deleon, N. M., Adem, S. n., Shen, A. H., Sokol, J. n., Momeni, A. n., Nguyen, D. n., Longaker, M. T., Wan, D. C. 2020

  Abstract

  Fat grafting is a surgical technique able to reconstruct and regenerate soft tissue. The adipose-derived stromal cells (ASCs) within the stromal vascular fraction are believed to drive these beneficial effects. ASCs are increasingly recognized to be a heterogeneous group, comprised of multiple stem and progenitor subpopulations with distinct functions. We hypothesized the existence of an ASC subpopulation with enhanced angiogenic potential. Human ASCs that were CD34+CD146+, CD34+CD146-, or CD34+ unfractionated (UF) were isolated by flow cytometry for comparison of expression of proangiogenic factors and endothelial tube-forming potential. Next, lipoaspirate was enriched with either CD34+CD146+, CD34+CD146-, CD34+ UF ASCs, or was not enriched, and grafted beneath the scalp skin of immunodeficient CD-1 Nude mice (10 000 cells/200 μL/graft). Fat retention was monitored radiographically more than 8 weeks and fat grafts were harvested for histological assessment of quality and vascularization. The CD34+CD146+ subpopulation comprised ~30% of ASCs, and exhibited increased expression of vascular endothelial growth factor and angiopoietin-1 compared to CD34+CD146- and CD34+ UF ASCs, and increased expression of fibroblast growth factor-2 compared to CD34+CD146- ASCs. The CD34+CD146+ subpopulation exhibited enhanced induction of tube-formation compared to CD34+CD146- ASCs. Upon transplantation, fat enriched CD34+CD146+ ASCs underwent less resorption and had improved histologic quality and vascularization. We have identified a subpopulation of CD34+ ASCs with enhanced angiogenic effects in vitro and in vivo, likely mediated by increased expression of potent proangiogenic factors. These findings suggest that enriching lipoaspirate with CD34+CD146+ ASCs may enhance fat graft vascularization and retention in the clinical setting.

  View details for DOI 10.1002/sctm.19-0195

  View details for PubMedID 32543083

• Tuning Macrophage Phenotype to Mitigate Skeletal Muscle Fibrosis. Journal of immunology (Baltimore, Md. : 1950) Stepien, D. M., Hwang, C. n., Marini, S. n., Pagani, C. A., Sorkin, M. n., Visser, N. D., Huber, A. K., Edwards, N. J., Loder, S. J., Vasquez, K. n., Aguilar, C. A., Kumar, R. n., Mascharak, S. n., Longaker, M. T., Li, J. n., Levi, B. n. 2020

  Abstract

  Myeloid cells are critical to the development of fibrosis following muscle injury; however, the mechanism of their role in fibrosis formation remains unclear. In this study, we demonstrate that myeloid cell-derived TGF-β1 signaling is increased in a profibrotic ischemia reperfusion and cardiotoxin muscle injury model. We found that myeloid-specific deletion of Tgfb1 abrogates the fibrotic response in this injury model and reduces fibro/adipogenic progenitor cell proliferation while simultaneously enhancing muscle regeneration, which is abrogated by adaptive transfer of normal macrophages. Similarly, a murine TGFBRII-Fc ligand trap administered after injury significantly reduced muscle fibrosis and improved muscle regeneration. This study ultimately demonstrates that infiltrating myeloid cell TGF-β1 is responsible for the development of traumatic muscle fibrosis, and its blockade offers a promising therapeutic target for preventing muscle fibrosis after ischemic injury.

  View details for DOI 10.4049/jimmunol.1900814

  View details for PubMedID 32161098

• Macrophage Subpopulation Dynamics Shift following Intravenous Infusion of Mesenchymal Stromal Cells. Molecular therapy : the journal of the American Society of Gene Therapy Kosaric, N. n., Srifa, W. n., Bonham, C. A., Kiwanuka, H. n., Chen, K. n., Kuehlmann, B. A., Maan, Z. N., Noishiki, C. n., Porteus, M. H., Longaker, M. T., Gurtner, G. C. 2020

  Abstract

  Intravenous infusion of mesenchymal stromal cells (MSCs) is thought to be a viable treatment for numerous disorders. Although the intrinsic immunosuppressive ability of MSCs has been credited for this therapeutic effect, their exact impact on endogenous tissue-resident cells following delivery has not been clearly characterized. Moreover, multiple studies have reported pulmonary sequestration of MSCs upon intravenous delivery. Despite substantial efforts to improve MSC homing, it remains unclear whether MSC migration to the site of injury is necessary to achieve a therapeutic effect. Using a murine excisional wound healing model, we offer an explanation of how sequestered MSCs improve healing through their systemic impact on macrophage subpopulations. We demonstrate that infusion of MSCs leads to pulmonary entrapment followed by rapid clearance, but also significantly accelerates wound closure. Using single-cell RNA sequencing of the wound, we show that following MSC delivery, innate immune cells, particularly macrophages, exhibit distinctive transcriptional changes. We identify the appearance of a pro-angiogenic CD9+ macrophage subpopulation, whose induction is mediated by several proteins secreted by MSCs, including COL6A1, PRG4, and TGFB3. Our findings suggest that MSCs do not need to act locally to induce broad changes in the immune system and ultimately treat disease.

  View details for DOI 10.1016/j.ymthe.2020.05.022

  View details for PubMedID 32531238

• Elucidating the fundamental fibrotic processes driving abdominal adhesion formation. Nature communications Foster, D. S., Marshall, C. D., Gulati, G. S., Chinta, M. S., Nguyen, A. n., Salhotra, A. n., Jones, R. E., Burcham, A. n., Lerbs, T. n., Cui, L. n., King, M. E., Titan, A. L., Ransom, R. C., Manjunath, A. n., Hu, M. S., Blackshear, C. P., Mascharak, S. n., Moore, A. L., Norton, J. A., Kin, C. J., Shelton, A. A., Januszyk, M. n., Gurtner, G. C., Wernig, G. n., Longaker, M. T. 2020; 11 (1): 4061

  Abstract

  Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.

  View details for DOI 10.1038/s41467-020-17883-1

  View details for PubMedID 32792541

• Evaluation of Outcomes Following Surgery for Locally Advanced Pancreatic Neuroendocrine Tumors. JAMA network open Titan, A. L., Norton, J. A., Fisher, A. T., Foster, D. S., Harris, E. J., Worhunsky, D. J., Worth, P. J., Dua, M. M., Visser, B. C., Poultsides, G. A., Longaker, M. T., Jensen, R. T. 2020; 3 (11): e2024318

  Abstract

  Although outcome of surgical resection of liver metastases from pancreatic neuroendocrine tumors (PNETs) has been extensively studied, little is known about surgery for locally advanced PNETs; it was listed recently by the European neuroendocrine tumor society as a major unmet need.To evaluate the outcome of patients who underwent surgery for locally aggressive PNETs.This retrospective single-center case series reviewed consecutive patients who underwent resection of T3/T4 PNETs at a single academic institution. Data collection occurred from 2003 to 2018. Data analysis was performed in August 2019.Disease-free survival (primary outcome) and overall mortality (secondary outcome) were assessed with Kaplan-Meier analysis. Recurrence risk (secondary outcome, defined as identification of tumor recurrence on imaging) was assessed with Cox proportional hazard models adjusting for covariates.In this case series, 99 patients with locally advanced nondistant metastatic PNET (56 men [57%]) with a mean (SEM) age of 57.0 (1.4) years and a mean (SEM) follow-up of 5.3 (0.1) years underwent surgically aggressive resections. Of those, 4 patients (4%) underwent preoperative neoadjuvant treatment (including peptide receptor radionuclide therapy and chemotherapy); 18 patients (18%) underwent pancreaticoduodenectomy, 68 patients (69%) had distal or subtotal pancreatic resection, 10 patients (10%) had total resection, and 3 patients (3%) had other pancreatic procedures. Additional organ resection was required in 86 patients (87%): spleen (71 patients [71%]), major blood vessel (17 patients [17%]), bowel (2 patients [2%]), stomach (4 patients [4%]), and kidney (2 patients [2%]). Five-year disease-free survival was 61% (61 patients) and 5-year overall survival was 91% (91 patients). Of those living, 75 patients (76%) had an Eastern Cooperative Oncology Group score of less than or equal to 1 at last followup. Lymph node involvement (HR, 7.66; 95% CI, 2.78-21.12; P < .001), additional organ resected (HR, 6.15; 95% CI, 1.61-23.55; P = .008), and male sex (HR, 3.77; 95% CI, 1.68-8.97; P = .003) were associated with increased risk of recurrence. Functional tumors had a lower risk of recurrence (HR, 0.23; CI, 0.06-0.89; P = .03). Required resection of blood vessels was not associated with a significant increase recurrence risk.In this case series, positive lymph node involvement and resection of organs with tumor involvement were associated with an increased recurrence risk. These subgroups may require adjuvant systemic treatment. These findings suggest that patients with locally advanced PNETs who undergo surgical resection have excellent disease-free and overall survival.

  View details for DOI 10.1001/jamanetworkopen.2020.24318

  View details for PubMedID 33146734

• The antifibrotic adipose-derived stromal cell: Grafted fat enriched with CD74+ adipose-derived stromal cells reduces chronic radiation-induced skin fibrosis. Stem cells translational medicine Borrelli, M. R., Patel, R. A., Adem, S. n., Diaz Deleon, N. M., Shen, A. H., Sokol, J. n., Yen, S. n., Chang, E. Y., Nazerali, R. n., Nguyen, D. n., Momeni, A. n., Wang, K. C., Longaker, M. T., Wan, D. C. 2020

  Abstract

  Fat grafting can reduce radiation-induced fibrosis. Improved outcomes are found when fat grafts are enriched with adipose-derived stromal cells (ASCs), implicating ASCs as key drivers of soft tissue regeneration. We have identified a subpopulation of ASCs positive for CD74 with enhanced antifibrotic effects. Compared to CD74- and unsorted (US) ASCs, CD74+ ASCs have increased expression of hepatocyte growth factor, fibroblast growth factor 2, and transforming growth factor β3 (TGF-β3) and decreased levels of TGF-β1. Dermal fibroblasts incubated with conditioned media from CD74+ ASCs produced less collagen upon stimulation, compared to fibroblasts incubated with media from CD74- or US ASCs. Upon transplantation, fat grafts enriched with CD74+ ASCs reduced the stiffness, dermal thickness, and collagen content of overlying skin, and decreased the relative proportions of more fibrotic dermal fibroblasts. Improvements in several extracellular matrix components were also appreciated on immunofluorescent staining. Together these findings indicate CD74+ ASCs have antifibrotic qualities and may play an important role in future strategies to address fibrotic remodeling following radiation-induced fibrosis.

  View details for DOI 10.1002/sctm.19-0317

  View details for PubMedID 32563212

• Immobilization after injury alters extracellular matrix and stem cell fate. The Journal of clinical investigation Huber, A. K., Patel, N. n., Pagani, C. A., Marini, S. n., Padmanabhan, K. n., Matera, D. L., Said, M. n., Hwang, C. n., Hsu, G. C., Poli, A. A., Strong, A. L., Visser, N. D., Greenstein, J. A., Nelson, R. n., Li, S. n., Longaker, M. T., Tang, Y. n., Weiss, S. J., Baker, B. M., James, A. W., Levi, B. n. 2020

  Abstract

  Cells sense extracellular environment and mechanical stimuli and translate these signals into intracellular responses through mechanotransduction and alters cell maintenance, proliferation, and differentiation. Here we use a mouse model of trauma induced heterotopic ossification (HO) to examine how cell-extrinsic forces impact MPC fate. After injury, single cell (sc) RNA sequencing of the injury site reveals an early increase in MPC genes associated with pathways of cell adhesion and ECM-receptor interactions, and MPC trajectories to cartilage and bone. Immunostaining uncovers active mechanotransduction after injury with increased focal adhesion kinase signaling and nuclear translocation of transcriptional co-activator TAZ, inhibition of which mitigates HO. Similarly, joint immobilization decreases mechanotransductive signaling, and completely inhibits HO. Joint immobilization decreases collagen alignment and increases adipogenesis. Further, scRNA sequencing of the HO site after injury with or without immobilization identifies gene signatures in mobile MPCs correlating with osteogenesis, while signatures from immobile MPCs with adipogenesis. scATAC-seq in these same MPCs confirm that in mobile MPCs, chromatin regions around osteogenic genes are open, while in immobile MPCs, regions around adipogenic genes are open. Together these data suggest that joint immobilization after injury results in decreased ECM alignment, altered MPC mechanotransduction, and changes in genomic architecture favoring adipogenesis over osteogenesis, resulting in decreased formation of HO.

  View details for DOI 10.1172/JCI136142

  View details for PubMedID 32673290

• Mechanisms of bone development and repair Nature reviews. Molecular cell biology Shah, H. N., Salhotra, A., Levi, B., Longaker, M. T. 2020

  Abstract

  Bone development occurs through a series of synchronous events that result in the formation of the body scaffold. The repair potential of bone and its surrounding microenvironment - including inflammatory, endothelial and Schwann cells - persists throughout adulthood, enabling restoration of tissue to its homeostatic functional state. The isolation of a single skeletal stem cell population through cell surface markers and the development of single-cell technologies are enabling precise elucidation of cellular activity and fate during bone repair by providing key insights into the mechanisms that maintain and regenerate bone during homeostasis and repair. Increased understanding of bone development, as well as normal and aberrant bone repair, has important therapeutic implications for the treatment of bone disease and ageing-related degeneration.

  View details for DOI 10.1038/s41580-020-00279-w

• Understanding the impact of fibroblast heterogeneity on skin fibrosis. Disease models & mechanisms Griffin, M. F., desJardins-Park, H. E., Mascharak, S. n., Borrelli, M. R., Longaker, M. T. 2020; 13 (6)

  Abstract

  Tissue fibrosis is the deposition of excessive extracellular matrix and can occur as part of the body's natural wound healing process upon injury, or as a consequence of diseases such as systemic sclerosis. Skin fibrosis contributes to significant morbidity due to the prevalence of injuries resulting from trauma and burn. Fibroblasts, the principal cells of the dermis, synthesize extracellular matrix to maintain the skin during homeostasis and also play a pivotal role in all stages of wound healing. Although it was previously believed that fibroblasts are homogeneous and mostly quiescent cells, it has become increasingly recognized that numerous fibroblast subtypes with unique functions and morphologies exist. This Review provides an overview of fibroblast heterogeneity in the mammalian dermis. We explain how fibroblast identity relates to their developmental origin, anatomical site and precise location within the skin tissue architecture in both human and mouse dermis. We discuss current evidence for the varied functionality of fibroblasts within the dermis and the relationships between fibroblast subtypes, and explain the current understanding of how fibroblast subpopulations may be controlled through transcriptional regulatory networks and paracrine communications. We consider how fibroblast heterogeneity can influence wound healing and fibrosis, and how insight into fibroblast heterogeneity could lead to novel therapeutic developments and targets for skin fibrosis. Finally, we contemplate how future studies should be shaped to implement knowledge of fibroblast heterogeneity into clinical practice in order to lessen the burden of skin fibrosis.

  View details for DOI 10.1242/dmm.044164

  View details for PubMedID 32541065

• "Tissues in a Dish": A Review of Organoids in Plastic Surgery. Plastic and reconstructive surgery. Global open Chinta, M. S., desJardins-Park, H. E., Wan, D. C., Longaker, M. T. 2020; 8 (4): e2787

  Abstract

  Organoids are in vitro miniaturized organ models-or, colloquially, "organs in a dish." These 3-dimensional, multicellular structures are classically derived from pluripotent or multipotent stem cells. When guided by tissue-specific molecular factors, these cells exhibit self-organizing abilities that allow them to accurately recapitulate the architecture and function of the organ of interest. Organoid technology is a rapidly expanding field that endows researchers with an unprecedented ability to recreate, study, and manipulate complex biologic processes in vitro. When compared with standard 2- and 3-dimensional culture systems, which rely on co-culturing pre-established cell types, organoids provide a more biomimetic model with which to study the intercellular interactions necessary for in vivo organ function and architecture. Organoids have the potential to impact all avenues of medicine, including those fields most relevant to plastic and reconstructive surgery such as wound healing, oncology, craniofacial reconstruction, and burn care. In addition to their ability to serve as a novel tool for studying human-specific disease, organoids may be used for tissue engineering with the goal of developing biomimetic soft-tissue substitutes, which would be especially valuable to the plastic surgeon. Although organoids hold great promise for the field of plastic surgery, technical challenges in creating vascularized, multilineage organoids must be overcome to allow for the integration of this technology in clinical practice. This review provides a brief history of the organoid, highlights its potential clinical applications, discusses certain limitations, and examines the impact that this technology may have on the field of plastic and reconstructive surgery.

  View details for DOI 10.1097/GOX.0000000000002787

  View details for PubMedID 32440447

  View details for PubMedCentralID PMC7209840

• Fat Grafting Rescues Radiation-Induced Joint Contracture. Stem cells (Dayton, Ohio) Borrelli, M. R., Diaz Deleon, N. M., Adem, S., Patel, R. A., Mascharak, S., Shen, A. H., Irizarry, D., Nguyen, D., Momeni, A., Longaker, M. T., Wan, D. C. 2019

  Abstract

  The aim of this study was to explore the therapeutic effects of fat grafting on radiation-induced hind limb contracture. Radiation therapy (RT) is used to palliate and/or cure a range of malignancies but causes inevitable and progressive fibrosis of surrounding soft tissue. Pathological fibrosis may lead to painful contractures which limit movement and negatively impact quality of life. Fat grafting is able to reduce and/or reverse radiation-induced soft tissue fibrosis. We explored whether fat grafting could improve extensibility in irradiated and contracted hind limbs of mice. Right hind limbs of female 60-day-old CD-1 nude mice were irradiated. Chronic skin fibrosis and limb contracture developed. After 4weeks, irradiated hind limbs were then injected with (a) fat enriched with stromal vascular cells (SVCs); (b) fat only; (c) saline; or (d) nothing (n = 10/group). Limb extension was measured at baseline and every 2weeks for 12weeks. Hind limb skin then underwent histological analysis and biomechanical strength testing. Irradiation significantly reduced limb extension but was progressively rescued by fat grafting. Fat grafting also reduced skin stiffness and reversed the radiation-induced histological changes in the skin. The greatest benefits were found in mice injected with fat enriched with SVCs. Hind limb radiation induces contracture in our mouse model which can be improved with fat grafting. Enriching fat with SVCs enhances these beneficial effects. These results underscore an attractive approach to address challenging soft tissue fibrosis in patients following RT.

  View details for DOI 10.1002/stem.3115

  View details for PubMedID 31793745

• A fine balance in tendon healing. Nature cell biology Titan, A. L., Longaker, M. T. 2019

  View details for DOI 10.1038/s41556-019-0432-0

  View details for PubMedID 31768049

• Macrophage Transplantation Fails to Improve Repair of Critical-Sized Calvarial Defects. The Journal of craniofacial surgery Borrelli, M. R., Hu, M. S., Hong, W. X., Oliver, J. D., Duscher, D., Longaker, M. T., Lorenz, H. P. 2019

  Abstract

  INTRODUCTION: Over 500,000 bone grafting procedures are performed every year in the United States for neoplastic and traumatic lesions of the craniofacial skeleton, costing $585 million in medical care. Current bone grafting procedures are limited, and full-thickness critical-sized defects (CSDs) of the adult human skull thus pose a substantial reconstructive challenge for the craniofacial surgeon. Cell-based strategies have been shown to safely and efficaciously accelerate the rate of bone formation in CSDs in animals. The authors recently demonstrated that supraphysiological transplantation of macrophages seeded in pullalan-collagen composite hydrogels significantly accelerated wound healing in wild type and diabetic mice, an effect mediated in part by enhancing angiogenesis. In this study, the authors investigated the bone healing effects of macrophage transplantation into CSDs of mice.METHODS: CD1 athymic nude mice (60 days of age) were anesthetized, and unilateral full-thickness critical-sized (4 mm in diameter) cranial defects were created in the right parietal bone, avoiding cranial sutures. Macrophages were isolated from FVB-L2G mice and seeded onto hydroxyapatite-poly (lactic-co-glycolic acid) (HA-PLGA) scaffolds (1.0 * 10 cells per CSD). Scaffolds were incubated for 24 hours before they were placed into the CSDs. Macrophage survival was assessed using three-dimensional in vivo imaging system (3D IVIS)/micro-CT. Micro-CT at 0, 2, 4, 6, and 8 weeks was performed to evaluate gross bone formation, which was quantified using Adobe Photoshop. Microscopic evidence of bone regeneration was assessed at 8 weeks by histology. Bone formation and macrophage survival were compared at each time point using independent samples t tests.RESULTS: Transplantation of macrophages at supraphysiological concentration had no effect on the formation of bones in CSDs as assessed by either micro-CT data at any time point analyzed (all P > 0.05). These results were corroborated by histology. 3D IVIS/micro-CT demonstrated survival of macrophages through 8 weeks.CONCLUSION: Supraphysiologic delivery of macrophages to CSDs of mice had no effect on bone formation despite survival of transplanted macrophages through to 8 weeks posttransplantation. Further research into the physiological effects of macrophages on bone regeneration is needed to assess whether recapitulation of these conditions in macrophage-based therapy can promote the healing of large cranial defects.

  View details for DOI 10.1097/SCS.0000000000005797

  View details for PubMedID 31609958

• Outcomes of Fat Grafting in Irradiated Tissue Are Improved by Pre-Treatment with Transdermal Deferoxamine Borrelli, M. R., Patel, R. A., Sokol, J., Momeni, A., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2019: E216

  View details for Web of Science ID 000492749600518

• Role of the Skeletal Stem Cell in Achilles Tendon to Bone Interface Healing Titan, A. L., Jones, R., Salhotra, A., Robertson, K. S., Foster, D., Menon, S., Murphy, M., Lucero, G. V., Chan, C. K., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S228–S229

  View details for Web of Science ID 000492740900438

• Effect of Mechanical Loading on Clonality of Injured Flexor Tendons after Repair Titan, A. L., Foster, D., Jones, R., Salhotra, A., Nguyen, A. T., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S221

  View details for Web of Science ID 000492740900423

• JUN Drives Pathologic Scarring by Activating Key Fibroproliferative Pathways in Fibroblast Subpopulations Borrelli, M. R., Garcia, J. T., Moore, A. L., Patel, R. A., Mascharak, S., Duoto, B., Cui, L., Wan, D. C., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: E215–E216

  View details for Web of Science ID 000492749600516

• CD146(+) Adipose-Derived Stromal Cells Have Proangiogenic Qualities and Enhance the Regenerative Potential of Grafted Fat Borrelli, M. R., Patel, R. A., Blackshear, C., Vistnes, S., Deleon, N., Nazerali, R., Momeni, A., Dung Nguyen, Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2019: S284

  View details for Web of Science ID 000492740900554

• Cancer-Associated Fibroblasts Persist but Show Decreased Fibroblast Activation Protein Expression after Neoadjuvant Chemotherapy in Human Pancreatic Ductal Adenocarcinoma Foster, D. S., Nguyen, A. T., Chinta, M., Titan, A. L., Salhotra, A., Jones, R., Mascharak, S., Norton, J., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S257–S258

  View details for Web of Science ID 000492740900501

• Tumors Co-Opt Fibroblast Wound Healing Capacity Foster, D. S., Mascharak, S., Nguyen, A. T., Chinta, M., Salhotra, A., Titan, A. L., Jones, R., da Silva, O., Norton, J. A., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S231–S232

  View details for Web of Science ID 000492740900444

• Endogenous Breast Cancer Shows Clonal Proliferation of Cancer Associated Fibroblasts at Primary Tumor and Metastatic Sites Foster, D. S., Chinta, M., Nguyen, A. T., Salhotra, A., Ransom, R., Jones, R., Titan, A. L., Mascharak, S., Norton, J. A., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S262

  View details for Web of Science ID 000492740900510

• Much-Needed Clarification and Guidance on Cell-Based Therapies for Musculoskeletal Disorders - Secondary Publication. Journal of orthopaedic research : official publication of the Orthopaedic Research Society Murphy, M. P., Wan, D. C., Longaker, M. T. 2019

  View details for DOI 10.1002/jor.24486

  View details for PubMedID 31573106

• Fibroblast Proliferation in Wound Healing Is Clonal and Focal Adhesion Kinase-Dependent Chinta, M., Foster, D., Nguyen, A. T., Salhotra, A., Ransom, R. C., Jones, R., Titan, A. L., Marshall, C., Mascharak, S., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S223

  View details for Web of Science ID 000492740900427

• CD74+Adipose-Derived Stromal Cells Have Anti-Fibrotic Effects in Grafted Fat in the Irradiated and Non-Irradiated Setting Borrelli, M. R., Patel, R. A., Sokol, J., Dung Nguyen, Momeni, A., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2019: E214

  View details for Web of Science ID 000492749600512

• Regenerative Skin Healing Through Targeted Modulation of Engrailed1-Negative Fibroblasts Mascharak, S., desJardins-Park, H. E., Moore, A. L., Borrelli, M. R., Chinta, M., Foster, D., Lorenz, H., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S228

  View details for Web of Science ID 000492740900437

• CD26(+) Fibroblasts Increase in Abundance in Breast Capsule Tissue Surrounding Irradiated Breasts Borrelli, M. R., Patel, R. A., Dung Nguyen, Momeni, A., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2019: S220

  View details for Web of Science ID 000492740900421

• Intrinsic Chromatin State and Extrinsic Wound-Related Cues Can Coordinate to Activate Fibroblasts for Scarring desJardins-Park, H. E., Moore, A. L., Litzenburger, U., Mascharak, S., Chinta, M., Ransom, R. C., Hu, M. S., Lorenz, H. P., Chang, H. Y., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S223–S224

  View details for Web of Science ID 000492740900428

• A Revised Perspective of Skeletal Stem Cell Biology. Frontiers in cell and developmental biology Ambrosi, T. H., Longaker, M. T., Chan, C. K. 2019; 7: 189

  Abstract

  Bone-related maladies are a major health burden on modern society. Loss of skeletal integrity and regeneration capacity through aging, obesity, and disease follows from a detrimental shift in bone formation and resorption dynamics. Targeting tissue-resident adult stem cells offers a potentially innovative paradigm in the development of therapeutic strategies against organ dysfunction. While the essential role of skeletal stem cells (SSCs) for development, growth, and maintenance of the skeleton has been generally established, a common consensus on the exact identity and definition of a pure bona fide SSC population remains elusive. The controversies stem from conflicting results between different approaches and criteria for isolation, detection, and functional evaluation; along with the interchangeable usage of the terms SSC and "mesenchymal stromal/stem cell (MSC)". A great number of prospective bone-forming stem cell populations have been reported with various characteristic markers, often describing overlapping cell populations with widely unexplored heterogeneity, species specificity, and distribution at distinct skeletal sites, bone regions, and microenvironments, thereby creating confusion that may complicate future advances in the field. In this review, we examine the state-of-the-art knowledge of SSC biology and try to establish a common ground for the definition and terminology of specific bone-resident stem cells. We also discuss recent advances in the identification of highly purified SSCs, which will allow detailed interrogation of SSC diversity and regulation at the single-cell level.

  View details for DOI 10.3389/fcell.2019.00189

  View details for PubMedID 31572721

  View details for PubMedCentralID PMC6753172

• A Revised Perspective of Skeletal Stem Cell Biology FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY Ambrosi, T. H., Longaker, M. T., Chan, C. F. 2019; 7

  View details for DOI 10.3389/fcell.2019.00189

  View details for Web of Science ID 000485330300001

• Coordinating Tissue Regeneration Through Transforming Growth Factor-beta Activated Kinase 1 Inactivation and Reactivation STEM CELLS Hsieh, H., Agarwal, S., Cholok, D. J., Loder, S. J., Kaneko, K., Huber, A., Chung, M. T., Ranganathan, K., Habbouche, J., Li, J., Butts, J., Reimer, J., Kaura, A., Drake, J., Breuler, C., Priest, C. R., Nguyen, J., Brownley, C., Peterson, J., Ozgurel, S., Niknafs, Y. S., Li, S., Inagaki, M., Scott, G., Krebsbach, P. H., Longaker, M. T., Westover, K., Gray, N., Ninomiya-Tsuji, J., Mishina, Y., Levi, B. 2019; 37 (6): 766–78

  View details for DOI 10.1002/stem.2991

  View details for Web of Science ID 000474038500008

• TOPICAL FOCAL ADHESION KINASE INHIBITOR PROMOTES SKIN REGENERATION AND SCAR PREVENTION IN A PRECLINICAL PORCINE MODEL Kwon, S., Kuehlmann, B., Dohi, T., Trotsyuk, A. A., Hu, M. S., Inayathullah, M., Rajadas, J., Longaker, M. T., Gurtner, G. C. WILEY. 2019: A11–A12

  View details for Web of Science ID 000463117000043

• Pro-Fibrotic CD26-Positive Fibroblasts are Present in Greater Abundance in Breast Capsule Tissue of Irradiated Breasts. Aesthetic surgery journal Borrelli, M. R., Irizzary, D., Patel, R. A., Nguyen, D., Momeni, A., Longaker, M. T., Wan, D. C. 2019

  Abstract

  BACKGROUND: Breast capsular contracture is a major problem following implant-based breast reconstruction, particularly in the setting of radiation therapy. Recent work has identified a fibrogenic fibroblast subpopulation characterized by CD26 surface marker expression.OBJECTIVE: This work aimed to investigate the role of CD26-positive fibroblasts in the formation of breast implant capsules following radiation therapy.METHODS: Breast capsule specimens were obtained from irradiated and non-irradiated breasts of 10 patients following bilateral mastectomy and unilateral irradiation at the time of expander-implant exchange, under institutional review board approval. Specimens were processed for Hematoxylin and Eosin staining, as well as for immunohistochemistry and fluorescence activated cell sorting (FACS) for CD26-positive fibroblasts. Expression of fibrotic genes and production of collagen was compared between CD26-positive, CD26-negative, and unsorted fibroblasts.RESULTS: Capsule specimens from irradiated breast tissue were thicker and had greater CD26-postive cells on immunofluorescence imaging and on FACS analysis, than did capsule specimens from the non-irradiated breast. Compared to CD26-negative fibroblasts, CD26-positive fibroblasts produced more collagen and had increased expression of the profibrotic genes IL8, TGF-beta1, COL1A1, and TIMP4.CONCLUSIONS: CD26-positive fibroblasts were found in a significantly greater abundance in capsules of irradiated compared to non-irradiated breasts and demonstrated greater fibrotic potential. This fibrogenic fibroblast subpopulation may play an important role in the development of capsular contracture following irradiation, and its targeted depletion or moderation may represent a potential therapeutic option.

  View details for PubMedID 30972420

• Fat Grafting into Younger Recipients Improves Volume Retention in an Animal Model PLASTIC AND RECONSTRUCTIVE SURGERY Chung, N. N., Ransom, R. C., Blackshear, C. P., Irizarry, D. M., Yen, D., Momeni, A., Lee, G. K., Nguyen, D. H., Longaker, M. T., Wan, D. C. 2019; 143 (4): 1067–75

  View details for DOI 10.1097/PRS.0000000000005483

  View details for Web of Science ID 000474582600052

• The Spectrum of Scarring in Craniofacial Wound Repair. Frontiers in physiology desJardins-Park, H. E., Mascharak, S., Chinta, M. S., Wan, D. C., Longaker, M. T. 2019; 10: 322

  Abstract

  Fibrosis is intimately linked to wound healing and is one of the largest causes of wound-related morbidity. While scar formation is the normal and inevitable outcome of adult mammalian cutaneous wound healing, scarring varies widely between different anatomical sites. The spectrum of craniofacial wound healing spans a particularly diverse range of outcomes. While most craniofacial wounds heal by scarring, which can be functionally and aesthetically devastating, healing of the oral mucosa represents a rare example of nearly scarless postnatal healing in humans. In this review, we describe the typical wound healing process in both skin and the oral cavity. We present clinical correlates and current therapies and discuss the current state of research into mechanisms of scarless healing, toward the ultimate goal of achieving scarless adult skin healing.

  View details for DOI 10.3389/fphys.2019.00322

  View details for PubMedID 30984020

  View details for PubMedCentralID PMC6450464

• The Spectrum of Scarring in Craniofacial Wound Repair FRONTIERS IN PHYSIOLOGY desJardins-Park, H. E., Mascharak, S., Chinta, M. S., Wan, D. C., Longaker, M. T. 2019; 10

  View details for DOI 10.3389/fphys.2019.00322

  View details for Web of Science ID 000462815300002

• Small molecule inhibition of dipeptidyl peptidase-4 enhances bone marrow progenitor cell function and angiogenesis in diabetic wounds TRANSLATIONAL RESEARCH Whittam, A. J., Maan, Z. N., Duscher, D., Barrera, J. A., Hu, M. S., Fischer, L. H., Khong, S., Kwon, S., Wong, V. W., Walmsley, G. G., Giacco, F., Januszyk, M., Brownlee, M., Longaker, M. T., Gurtner, G. C. 2019; 205: 51–63

  View details for DOI 10.1016/j.trsl.2018.10.006

  View details for Web of Science ID 000459843700005

• Wounds Inhibit Tumor Growth In Vivo. Annals of surgery Hu, M. S., Maan, Z. N., Leavitt, T., Hong, W. X., Rennert, R. C., Marshall, C. D., Borrelli, M. R., Zhu, T. N., Esquivel, M., Zimmermann, A., McArdle, A., Chung, M. T., Foster, D. S., Jones, R. E., Gurtner, G. C., Giaccia, A. J., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2019

  Abstract

  OBJECTIVE: The aim of this study was to determine the interaction of full thickness excisional wounds and tumors in vivo.SUMMARY OF BACKGROUND DATA: Tumors have been described as wounds that do not heal due to similarities in stromal composition. On the basis of observations of slowed tumor growth after ulceration, we hypothesized that full thickness excisional wounds would inhibit tumor progression in vivo.METHODS: To determine the interaction of tumors and wounds, we developed a tumor xenograft/allograft (human head and neck squamous cell carcinoma SAS/mouse breast carcinoma 4T1) wound mouse model. We examined tumor growth with varying temporospatial placement of tumors and wounds or ischemic flap. In addition, we developed a tumor/wound parabiosis model to understand the ability of tumors and wounds to recruit circulating progenitor cells.RESULTS: Tumor growth inhibition by full thickness excisional wounds was dose-dependent, maintained by sequential wounding, and relative to distance. This effect was recapitulated by placement of an ischemic flap directly adjacent to a xenograft tumor. Using a parabiosis model, we demonstrated that a healing wound was able to recruit significantly more circulating progenitor cells than a growing tumor. Tumor inhibition by wound was unaffected by presence of an immune response in an immunocompetent model using a mammary carcinoma. Utilizing functional proteomics, we identified 100 proteins differentially expressed in tumors and wounds.CONCLUSION: Full thickness excisional wounds have the ability to inhibit tumor growth in vivo. Further research may provide an exact mechanism for this remarkable finding and new advances in wound healing and tumor biology.

  View details for PubMedID 30829705

• Coordinating Tissue Regeneration through TGF-beta Activated Kinase 1 (TAK1) In-activation and Re-activation. Stem cells (Dayton, Ohio) Sung Hsieh, H. H., Agarwal, S., Cholok, D. J., Loder, S. J., Kaneko, K., Huber, A., Chung, M. T., Ranganathan, K., Habbouche, J., Li, J., Butts, J., Reimer, J., Kaura, A., Drake, J., Breuler, C., Priest, C. R., Nguyen, J., Brownley, C., Peterson, J., Ozgurel, S. U., Niknafs, Y. S., Li, S., Inagaki, M., Scott, G., Krebsbach, P., Longaker, M. T., Westover, K., Gray, N., Ninomiya-Tsuji, J., Mishina, Y., Levi, B. 2019

  Abstract

  Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of TGF-beta activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by re-activation to elicit differentiation and extracellular matrix (ECM) production. While the current regenerative medicine paradigm is centered on the effects of drug treatment ("drug on"), the impact of drug withdrawal ("drug off") implicit in these regimens are unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment ("drug on") and subsequent withdrawal ("drug off") through genetic modification, was used here to inactivate and re-activate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the "drug on" (Cre-mediated inactivation) and "drug off" (Flp-mediated re-activation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. SIGNIFICANCE STATEMENT: We target the TAK1 pathway to reduce heterotopic ossification, a pathologic condition in which bone develops within muscle or soft tissues. We show that Tak1 knockout leads to cellular proliferation; this can be harnessed to increase the number of cells present at the injury site. Using a mouse model, we inactivate and reactivate the Tak1 gene. We show that inactivation and reactivation of Tak1 can improve bony healing through the coordination of increased proliferation (inactivation) followed by differentiation (reactivation). This approach elucidates a new paradigm in regenerative medicine in which coordination between treatment and withdrawal of treatment can augment healing. © AlphaMed Press 2019.

  View details for PubMedID 30786091

• Fat Chance: The Rejuvenation of Irradiated Skin PLASTIC AND RECONSTRUCTIVE SURGERY-GLOBAL OPEN Borrelli, M. R., Patel, R. A., Sokol, J., Dung Nguyen, Momeni, A., Longaker, M. T., Wan, D. C. 2019; 7 (2): e2092

  Abstract

  Radiotherapy (RT) helps cure and palliate thousands of patients with a range of malignant diseases. A major drawback, however, is the collateral damage done to tissues surrounding the tumor in the radiation field. The skin and subcutaneous tissue are among the most severely affected regions. Immediately following RT, the skin may be inflamed, hyperemic, and can form ulcers. With time, the dermis becomes progressively indurated. These acute and chronic changes cause substantial patient morbidity, yet there are few effective treatment modalities able to reduce radiodermatitis. Fat grafting is increasingly recognized as a tool able to reverse the fibrotic skin changes and rejuvenate the irradiated skin. This review outlines the current progress toward describing and understanding the cellular and molecular effects of fat grafting in irradiated skin. Identification of the key factors involved in the pathophysiology of fibrosis following RT will inform therapeutic interventions to enhance its beneficial effects.

  View details for PubMedID 30881833

• Wound healing and fibrosis: current stem cell therapies. Transfusion Jones, R. E., Foster, D. S., Hu, M. S., Longaker, M. T. 2019; 59 (S1): 884-892

  Abstract

  Scarring is a result of the wound healing response and causes tissue dysfunction after injury. This process is readily evident in the skin, but also occurs internally across organ systems in the form of fibrosis. Stem cells are crucial to the innate tissue healing response and, as such, present a possible modality to therapeutically promote regenerative healing while minimizing scaring. In this review, the cellular basis of scaring and fibrosis is examined. Current stem cell therapies under exploration for skin wound healing and internal organ fibrosis are discussed. While most therapeutic approaches rely on the direct application of progenitor-type cells to injured tissue to promote healing, novel strategies to manipulate the scarring response are also presented. As our understanding of developmental and stem cell biology continues to increase, therapies to encourage regeneration of healthy functional tissue after damage secondary to injury or disease will continue to expand.

  View details for DOI 10.1111/trf.14836

  View details for PubMedID 30737822

• Fat Grafting into Younger Recipients Improves Volume Retention in an Animal Model. Plastic and reconstructive surgery Chung, N. N., Ransom, R. C., Blackshear, C. P., Irizarry, D. M., Yen, D., Momeni, A., Lee, G. K., Nguyen, D. H., Longaker, M. T., Wan, D. C. 2019

  Abstract

  BACKGROUND: Soft tissue deficits associated with various craniofacial anomalies can be addressed by fat grafting, although outcomes remain unpredictable. Furthermore, consensus does not exist for timing of these procedures. While some advocate approaching soft tissue reconstruction after the underlying skeletal foundation has been corrected, other studies have suggested earlier grafting may exploit a younger recipient niche that is more conducive for fat graft survival. As there is a dearth of research investigating effects of recipient age on fat graft volume retention, this study compared the effectiveness of fat grafting in younger versus older animals through a longitudinal, in vivo analysis.METHODS: Human lipoaspirate from three healthy female donors was grafted subcutaneously over the calvarium of immunocompromised mice. Volume retention over 8 weeks was evaluated using micro-computed tomography in three experimental ages - 3-weeks old, 6-months old, and 1-year old. Histology was performed on explanted grafts to evaluate graft health and vascularity. Recipient site vascularity was also evaluated by confocal microscopy.RESULTS: Greatest retention of fat graft volume was noted in the youngest group compared to both older groups (*p < 0.05) at 6 and 8 weeks following grafting. Histological and immunohistochemical analyses revealed that improved retention in younger groups was associated with greater fat graft integrity and more robust vascularization.CONCLUSION: Our study provides evidence that grafting fat into a younger recipient site correlates with improved volume retention over time, suggesting that beginning soft tissue reconstruction with fat grafting in patients at an earlier age may be preferable to late correction.

  View details for PubMedID 30730498

• Heterogeneity in old fibroblasts is linked to variability in reprogramming and wound healing. Nature Mahmoudi, S. n., Mancini, E. n., Xu, L. n., Moore, A. n., Jahanbani, F. n., Hebestreit, K. n., Srinivasan, R. n., Li, X. n., Devarajan, K. n., Prélot, L. n., Ang, C. E., Shibuya, Y. n., Benayoun, B. A., Chang, A. L., Wernig, M. n., Wysocka, J. n., Longaker, M. T., Snyder, M. P., Brunet, A. n. 2019; 574 (7779): 553–58

  Abstract

  Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5-8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain 'activated fibroblasts' that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals.

  View details for DOI 10.1038/s41586-019-1658-5

  View details for PubMedID 31645721

• Induction of the Fetal Scarless Phenotype in Adult Wounds: Impossible? REGENERATIVE MEDICINE AND PLASTIC SURGERY: SKIN AND SOFT TISSUE, BONE, CARTILAGE, MUSCLE, TENDON AND NERVES Hu, M. S., Borrelli, M. R., Longaker, M. T., Lorenz, H., Duscher, D., Shiffman, M. A. 2019: 3-17

  View details for DOI 10.1007/978-3-030-19962-3_1

  View details for Web of Science ID 000652015400003

• In Vitro and In Vivo Osteogenic Differentiation of Human Adipose-Derived Stromal Cells BONE MORPHOGENETIC PROTEINS Marshall, C. D., Brett, E. A., Moore, A. L., Wan, D. C., Longaker, M. T., Rogers, M. B. 2019; 1891: 9-18

  View details for DOI 10.1007/978-1-4939-8904-1_2

  View details for Web of Science ID 000693352500003

• Skeletal Stem Cell-Schwann Cell Circuitry in Mandibular Repair. Cell reports Jones, R. E., Salhotra, A. n., Robertson, K. S., Ransom, R. C., Foster, D. S., Shah, H. N., Quarto, N. n., Wan, D. C., Longaker, M. T. 2019; 28 (11): 2757–66.e5

  Abstract

  Regenerative paradigms exhibit nerve dependency, including regeneration of the mouse digit tip and salamander limb. Denervation impairs regeneration and produces morphological aberrancy in these contexts, but the direct effect of innervation on the stem and progenitor cells enacting these processes is unknown. We devised a model to examine nerve dependency of the mouse skeletal stem cell (mSSC), the progenitor responsible for skeletal development and repair. We show that after inferior alveolar denervation, mandibular bone repair is compromised because of functional defects in mSSCs. We present mSSC reliance on paracrine factors secreted by Schwann cells as the underlying mechanism, with partial rescue of the denervated phenotype by Schwann cell transplantation and by Schwann-derived growth factors. This work sheds light on the nerve dependency of mSSCs and has implications for clinical treatment of mandibular defects.

  View details for DOI 10.1016/j.celrep.2019.08.021

  View details for PubMedID 31509739

• Radiation-Induced Skin Fibrosis: Pathogenesis, Current Treatment Options, and Emerging Therapeutics. Annals of plastic surgery Borrelli, M. R., Shen, A. H., Lee, G. K., Momeni, A. n., Longaker, M. T., Wan, D. C. 2019; 83 (4S Suppl 1): S59–S64

  Abstract

  Radiotherapy (RT) has become an indispensable part of oncologic treatment protocols for a range of malignancies. However, a serious adverse effect of RT is radiodermatitis; almost 95% of patients develop moderate to severe skin reactions following radiation treatment. In the acute setting, these can be erythema, desquamation, ulceration, and pain. Chronically, soft tissue atrophy, alopecia, and stiffness can be noted. Radiodermatitis can delay oncologic treatment protocols and significantly impair quality of life. There is currently a paucity of effective treatment options and prevention strategies for radiodermatitis. Importantly, recent preclinical and clinical studies have suggested that fat grafting may be of therapeutic benefit, reversing detrimental changes to soft tissue following RT. This review outlines the damaging effects of RT on the skin and soft tissue as well as discusses available treatment options for radiodermatitis. Emerging strategies to mitigate detrimental, chronic radiation-induced changes are also presented.

  View details for DOI 10.1097/SAP.0000000000002098

  View details for PubMedID 31513068

• Pressure Injury. Annals of surgery Hajhosseini, B. n., Longaker, M. T., Gurtner, G. C. 2019

  Abstract

  Pressure injury is seen across all healthcare settings and affects people of any age and health condition. It imposes a significant burden, with annual costs of up to $17.8 billion in the United States alone. Despite considerable resources it exhausts, the disease remains very prevalent, and the incidence is on the rise. This is in part due to aging population, growing number of nursing home residents, poorly understood biology, and dismal track record of clinical research in this field.In our Review Article, we discuss the disease pathophysiology, clinical manifestation, evidence based recommendations for risk assessment, prevention and timely management, existing challenges, and directions to improve research on the field. This article encompasses dedicated sections on the full spectrum of the pressure related pathologies including "conventional pressure ulcers", "medical device related pressure injuries", "pressure injuries in mucosal membranes", "pressure injuries in pediatric population", "pressure injury at end of life", and the "role of pressure in pathogenesis of diabetic foot ulcers".

  View details for DOI 10.1097/SLA.0000000000003567

  View details for PubMedID 31460882

• Discussion: Adipose-Derived Stem Cells and Ceiling Culture-Derived Preadipocytes Cultured from Subcutaneous Fat Tissue Differ in Their Epigenetic Characteristics and Osteogenic Potential. Plastic and reconstructive surgery Borrelli, M. R., Longaker, M. T., Wan, D. C. 2019; 144 (3): 656–57

  View details for DOI 10.1097/PRS.0000000000005914

  View details for PubMedID 31461021

• Flexor Tendon: Development, Healing, Adhesion Formation, and Contributing Growth Factors. Plastic and reconstructive surgery Titan, A. L., Foster, D. S., Chang, J. n., Longaker, M. T. 2019; 144 (4): 639e–647e

  Abstract

  Management of flexor tendon injuries of the hand remains a major clinical problem. Even with intricate repair, adhesion formation remains a common complication. Significant progress has been made to better understand the mechanisms of healing and adhesion formation. However, there has been slow progress in the clinical prevention and reversal of flexor tendon adhesions. The goal of this article is to discuss recent literature relating to tendon development, tendon healing, and adhesion formation to identify areas in need of further research. Additional research is needed to understand and compare the molecular, cellular, and genetic mechanisms involved in flexor tendon morphogenesis, postoperative healing, and mechanical loading. Such knowledge is critical to determine how to improve repair outcomes and identify new therapeutic strategies to promote tissue regeneration and prevent adhesion formation.

  View details for DOI 10.1097/PRS.0000000000006048

  View details for PubMedID 31568303

• A Clearing Technique to Enhance Endogenous Fluorophores in Skin and Soft Tissue. Scientific reports Foster, D. S., Nguyen, A. T., Chinta, M. n., Salhotra, A. n., Jones, R. E., Mascharak, S. n., Titan, A. L., Ransom, R. C., da Silva, O. L., Foley, E. n., Briger, E. n., Longaker, M. T. 2019; 9 (1): 15791

  Abstract

  Fluorescent proteins are used extensively in transgenic animal models to label and study specific cell and tissue types. Expression of these proteins can be imaged and analyzed using fluorescent and confocal microscopy. Conventional confocal microscopes cannot penetrate through tissue more than 4-6 μm thick. Tissue clearing procedures overcome this challenge by rendering thick specimens into translucent tissue. However, most tissue clearing techniques do not satisfactorily preserve expression of endogenous fluorophores. Using simple adjustments to the BABB (Benzoic Acid Benzyl Benzoate) clearing methodology, preservation of fluorophore expression can be maintained. Modified BABB tissue clearing is a reliable technique to clear skin and soft tissue specimens for the study of dermal biology, wound healing and fibrotic pathologies.

  View details for DOI 10.1038/s41598-019-50359-x

  View details for PubMedID 31673001

• Much-Needed Clarification and Guidance on Cell-Based Therapies for Musculoskeletal Disorders. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research Murphy, M. P., Wan, D. C., Longaker, M. T. 2019

  View details for DOI 10.1002/jbmr.3841

  View details for PubMedID 31545871

• In Vitro and In Vivo Osteogenic Differentiation of Human Adipose-Derived Stromal Cells. Methods in molecular biology (Clifton, N.J.) Marshall, C. D., Brett, E. A., Moore, A. L., Wan, D. C., Longaker, M. T. 2019; 1891: 9–18

  Abstract

  Adipose-derived stromal cells (ASCs) are a promising population of cells that may be useful for the regeneration of human tissue defects. ASCs are capable of forming bone tissue in vitro and in vivo. Further work is required to determine the optimal conditions that will allow human ASCs to regenerate tissue in clinically significant tissue defects. Here we present three experimental protocols that are indispensable for the study of ASC osteogenic activity.

  View details for PubMedID 30414122

• Tissue Engineering and Regenerative Medicine in Craniofacial Reconstruction and Facial Aesthetics. The Journal of craniofacial surgery Borrelli, M. R., Hu, M. S., Longaker, M. T., Lorenz, H. P. 2019

  Abstract

  The craniofacial region is anatomically complex and is of critical functional and cosmetic importance, making reconstruction challenging. The limitations of current surgical options highlight the importance of developing new strategies to restore the form, function, and esthetics of missing or damaged soft tissue and skeletal tissue in the face and cranium. Regenerative medicine (RM) is an expanding field which combines the principles of tissue engineering (TE) and self-healing in the regeneration of cells, tissues, and organs, to restore their impaired function. RM offers many advantages over current treatments as tissue can be engineered for specific defects, using an unlimited supply of bioengineered resources, and does not require immunosuppression. In the craniofacial region, TE and RM are being increasingly used in preclinical and clinical studies to reconstruct bone, cartilage, soft tissue, nerves, and blood vessels. This review outlines the current progress that has been made toward the engineering of these tissues for craniofacial reconstruction and facial esthetics.

  View details for DOI 10.1097/SCS.0000000000005840

  View details for PubMedID 31369496

• Doxycycline Reduces Scar Thickness and Improves Collagen Architecture. Annals of surgery Moore, A. L., desJardins-Park, H. E., Duoto, B. A., Mascharak, S., Murphy, M. P., Irizarry, D. M., Foster, D. S., Jones, R. E., Barnes, L. A., Marshall, C. D., Ransom, R. C., Wernig, G., Longaker, M. T. 2018

  Abstract

  OBJECTIVE: To investigate the effects of local doxycycline administration on skin scarring.BACKGROUND: Skin scarring represents a major source of morbidity for surgical patients. Doxycycline, a tetracycline antibiotic with off-target effects on the extracellular matrix, has demonstrated antifibrotic effects in multiple organs. However, doxycycline's potential effects on skin scarring have not been explored in vivo.METHODS: Female C57BL/6J mice underwent dorsal wounding following an established splinted excisional skin wounding model. Doxycycline was administered by local injection into the wound base following injury. Wounds were harvested upon complete wound closure (postoperative day 15) for histological examination and biomechanical testing of scar tissue.RESULTS: A one-time dose of 3.90 mM doxycycline (2 mg/mL) within 12 hours of injury was found to significantly reduce scar thickness by 24.8% (P < 0.0001) without compromising tensile strength. The same effect could not be achieved by oral dosing. In doxycycline-treated scar matrices, collagen I content was significantly reduced (P = 0.0317) and fibers were favorably arranged with significantly increased fiber randomness (P = 0.0115). Common culprits of altered wound healing mechanics, including angiogenesis and inflammation, were not impacted by doxycycline treatment. However, engrailed1 profibrotic fibroblasts, responsible for scar extracellular matrix deposition, were significantly reduced with doxycycline treatment (P = 0.0005).CONCLUSIONS: Due to the substantial improvement in skin scarring and well-established clinical safety profile, locally administered doxycycline represents a promising vulnerary agent. As such, we favor rapid translation to human patients as an antiscarring therapy.

  View details for PubMedID 30585822

• Mechanoresponsive stem cells acquire neural crest fate in jaw regeneration NATURE Ransom, R. C., Carter, A. C., Salhotra, A., Leavitt, T., Marecic, O., Murphy, M. P., Lopez, M. L., Wei, Y., Marshall, C. D., Shen, E. Z., Jones, R., Sharir, A., Klein, O. D., Chan, C. F., Wan, D. C., Chang, H. Y., Longaker, M. T. 2018; 563 (7732): 514-+

  View details for DOI 10.1038/s41586-018-0650-9

  View details for Web of Science ID 000450960000045

• Small molecule inhibition of dipeptidyl peptidase-4 enhances bone marrow progenitor cell function and angiogenesis in diabetic wounds. Translational research : the journal of laboratory and clinical medicine Whittam, A. J., Maan, Z. N., Duscher, D., Barrera, J. A., Hu, M. S., Fischer, L. H., Khong, S., Kwon, S. H., Wong, V. W., Walmsley, G. G., Giacco, F., Januszyk, M., Brownlee, M., Longaker, M. T., Gurtner, G. C. 2018

  Abstract

  In diabetes, stromal cell-derived factor-1 (SDF-1) expression and progenitor cell recruitment are reduced. Dipeptidyl peptidase-4 (DPP-4) inhibits SDF-1 expression and progenitor cell recruitment. Here we examined the impact of the DPP-4 inhibitor, MK0626, on progenitor cell kinetics in the context of wound healing. Wildtype (WT) murine fibroblasts cultured under high-glucose to reproduce a diabetic microenvironment were exposed to MK0626, glipizide, or no treatment, and SDF-1 expression was measured with ELISA. Diabetic mice received MK0626, glipizide, or no treatment for 6 weeks and then were wounded. Immunohistochemistry was used to quantify neovascularization and SDF-1 expression. Gene expression was measured at the RNA and protein level using quantitative polymerase chain reaction and ELISA, respectively. Flow cytometry was used to characterize bone marrow-derived mesenchymal progenitor cell (BM-MPC) population recruitment to wounds. BM-MPC gene expression was assayed using microfluidic single cell analysis. WT murine fibroblasts exposed to MK0626 demonstrated increased SDF-1 expression. MK0626 treatment significantly accelerated wound healing and increased wound vascularity, SDF-1 expression, and dermal thickness in diabetic wounds. MK0626 treatment increased the number of BM-MPCs present in bone marrow and in diabetic wounds. MK0626 had no effect on BM-MPC population dynamics. BM-MPCs harvested from MK0626-treated mice exhibited increased chemotaxis in response to SDF-1 when compared to diabetic controls. Treatment with a DPP-4 inhibitor significantly improved wound healing, angiogenesis, and endogenous progenitor cell recruitment in the setting of diabetes.

  View details for PubMedID 30452888

• DEL1 protects against chondrocyte apoptosis through integrin binding. The Journal of surgical research Wang, Z., Boyko, T., Tran, M. C., LaRussa, M., Bhatia, N., Rashidi, V., Longaker, M. T., Yang, G. P. 2018; 231: 1–9

  Abstract

  BACKGROUND: Osteoarthritis (OA) is a debilitating disease process, affecting mobility and overall health of millions. Current treatment is for symptomatic relief and discovery of approaches to halt or reverse damage is imperative. Deletion of developmental endothelial locus-1 (Del1) has been shown to increase severity of OA in knockout mice. We examined the intracellular pathways involved in the ability of DEL1 to protect chondrocytes from apoptosis and anoikis and hypothesized that it functioned via integrin signaling.MATERIALS AND METHODS: Primary human chondrocytes were treated with various inducers of apoptosis, including anoikis, in the presence of added DEL1 or bovine serum albumin as control. Various inhibitors of integrin binding were examined for their effect on DEL1 activity. Downstream signaling pathway components were detected by immunoblotting.RESULTS: The addition of DEL1 protected chondrocytes from multiple inducers of apoptosis as measured by cell survival, terminal deoxynucleotidyl transferase dUTP nick end labeling and caspase 3/7 assays (P<0.05). The effect of DEL1 was blocked by RGD peptides and by antibodies directed to integrin alphaVbeta3, but not by controls or antibody to integrin alpha1 (P<0.05). Treatment with DEL1 promoted ERK and AKT activation when cells were attached, but only AKT activation under conditions of anoikis.CONCLUSIONS: DEL1 protected chondrocytes from apoptosis in response to activators of either the intrinsic or extrinsic pathways, and to anoikis. This effect was mediated primarily through integrin alphaVbeta3. This represents a therapeutic target for therapies to prevent cartilage degeneration in OA.

  View details for PubMedID 30278915

• Discussion: CRISPR Craft: DNA Editing the Reconstructive Ladder PLASTIC AND RECONSTRUCTIVE SURGERY Hu, M. S., Longaker, M. T., Wan, D. C. 2018; 142 (5): 1365–66

  View details for DOI 10.1097/PRS.0000000000004949

  View details for Web of Science ID 000448330200083

• DEL1 protects against chondrocyte apoptosis through integrin binding JOURNAL OF SURGICAL RESEARCH Wang, Z., Boyko, T., Tran, M. C., LaRussa, M., Bhatia, N., Rashidi, V., Longaker, M. T., Yang, G. P. 2018; 231: 1–9

  View details for DOI 10.1016/j.jss.2018.04.066

  View details for Web of Science ID 000445911700001

• Discussion: CRISPR Craft: DNA Editing the Reconstructive Ladder. Plastic and reconstructive surgery Hu, M. S., Longaker, M. T., Wan, D. C. 2018; 142 (5): 1365-1366

  View details for DOI 10.1097/PRS.0000000000004949

  View details for PubMedID 30511993

• Mechanoresponsive stem cells acquire neural crest fate in jaw regeneration. Nature Ransom, R. C., Carter, A. C., Salhotra, A., Leavitt, T., Marecic, O., Murphy, M. P., Lopez, M. L., Wei, Y., Marshall, C. D., Shen, E. Z., Jones, R. E., Sharir, A., Klein, O. D., Chan, C. K., Wan, D. C., Chang, H. Y., Longaker, M. T. 2018

  Abstract

  During both embryonic development and adult tissue regeneration, changes in chromatin structure driven by master transcription factors lead to stimulus-responsive transcriptional programs. A thorough understanding of how stem cells in the skeleton interpret mechanical stimuli and enact regeneration would shed light on how forces are transduced to the nucleus in regenerative processes. Here we develop a genetically dissectible mouse model of mandibular distraction osteogenesis-which isa process that is used in humans to correct an undersized lower jawthat involves surgically separating the jaw bone, whichelicits new bone growth in the gap. We use this model to show that regions of newly formed bone are clonally derived from stem cells that reside in the skeleton. Using chromatin and transcriptional profiling, we show that these stem-cell populations gain activity within the focal adhesion kinase (FAK) signalling pathway, and that inhibiting FAK abolishes new bone formation. Mechanotransduction via FAK in skeletal stem cells during distraction activates a gene-regulatory program and retrotransposons that are normally active in primitive neural crest cells, from which skeletal stem cells arise during development. This reversion to a developmental state underlies the robust tissue growth that facilitates stem-cell-based regeneration of adult skeletal tissue.

  View details for PubMedID 30356216

• Author Correction: Genetic dissection of clonal lineage relationships with hydroxytamoxifen liposomes. Nature communications Ransom, R. C., Foster, D. S., Salhotra, A., Jones, R. E., Marshall, C. D., Leavitt, T., Murphy, M. P., Moore, A. L., Blackshear, C. P., Brett, E. A., Wan, D. C., Longaker, M. T. 2018; 9 (1): 4411

  Abstract

  In the original version of this Article, the authors inadvertently omitted Elizabeth A. Brett, who contributed to the generation of the histology figures, from the author list.This has now been corrected in both the PDF and HTML versions of the Article.

  View details for PubMedID 30341306

• Genetic dissection of clonal lineage relationships with hydroxytamoxifen liposomes (vol 9, 2971, 2018) NATURE COMMUNICATIONS Ransom, R. C., Foster, D. S., Salhotra, A., Jones, R., Marshall, C. D., Leavitt, T., Murphy, M. P., Moore, A. L., Blackshear, C. P., Brett, E. A., Wan, D. C., Longaker, M. T. 2018; 9

  View details for DOI 10.1038/s41467-018-06927-2

  View details for Web of Science ID 000447698700001

• Twist1-Haploinsufficiency Selectively Enhances the Osteoskeletal Capacity of Mesoderm-Derived Parietal Bone Through Downregulation of Fgf23. Frontiers in physiology Quarto, N., Shailendra, S., Meyer, N. P., Menon, S., Renda, A., Longaker, M. T. 2018; 9: 1426

  Abstract

  Craniofacial development is a program exquisitely orchestrated by tissue contributions and regulation of genes expression. The basic helix-loop-helix (bHLH) transcription factor Twist1 expressed in the skeletal mesenchyme is a key regulator of craniofacial development playing an important role during osteoskeletogenesis. This study investigates the postnatal impact of Twist1 haploinsufficiency on the osteoskeletal ability and regeneration on two calvarial bones arising from tissues of different embryonic origin: the neural crest-derived frontal and the mesoderm-derived parietal bones. We show that Twist1 haplonsufficiency as well Twist1-sh-mediated silencing selectively enhanced osteogenic and tissue regeneration ability of mesoderm-derived bones. Transcriptomic profiling, gain-and loss-of-function experiments revealed that Twist1 haplonsufficiency triggers its selective activity on mesoderm-derived bone through a sharp downregulation of the bone-derived hormone Fgf23 that is upregulated exclusively in wild-type parietal bone.

  View details for DOI 10.3389/fphys.2018.01426

  View details for PubMedID 30374308

  View details for PubMedCentralID PMC6196243

• Twist1-Haploinsufficiency Selectively Enhances the Osteoskeletal Capacity of Mesoderm-Derived Parietal Bone Through Downregulation of Fgf23 FRONTIERS IN PHYSIOLOGY Quarto, N., Shailendra, S., Meyer, N. P., Menon, S., Renda, A., Longaker, M. T. 2018; 9

  View details for DOI 10.3389/fphys.2018.01426

  View details for Web of Science ID 000447266900001

• Management of Chronic Wounds-2018. JAMA Jones, R. E., Foster, D. S., Longaker, M. T. 2018; 320 (14): 1481-1482

  View details for DOI 10.1001/jama.2018.12426

  View details for PubMedID 30326512

• Iron Chelation with Transdermal Deferoxamine Accelerates Healing of Murine Sickle Cell Ulcers. Advances in wound care Rodrigues, M., Bonham, C. A., Minniti, C. P., Gupta, K., Longaker, M. T., Gurtner, G. C. 2018; 7 (10): 323-332

  Abstract

  Objective: Sickle cell ulcers (SCUs) are a devastating comorbidity affecting patients with sickle cell disease (SCD). SCUs form over the medial or lateral malleoli of the lower extremity, are slow to heal, and prone to recidivism. Some SCUs may never heal, leading to chronic pain and foot deformities. There is no specific and effective therapy for SCUs. Systemic deferoxamine (DFO) has been demonstrated to prevent some of the sequelae of SCD by chelating iron. In this study, we tested the ability of DFO delivered via a transdermal delivery system (DFO-TDDS) to accelerate healing in a murine model of SCU. Approach: Excisional wounds were created in a transgenic murine model of SCD expressing >99% human sickle hemoglobin, and healing rates were compared with wounds in wild-type mice. Next, excisional wounds in SCD mice were treated with DFO-TDDS, DFO injection, or left untreated. Wound closure rates, histology, and iron in the healed wounds were analyzed. Results: Wounds in SCD mice healed significantly slower than wild-type mice (***p < 0.001). DFO-TDDS-treated wounds demonstrated significantly accelerated time to closure, reduced size, and improved wound remodeling compared with untreated wounds (***p < 0.001) and DFO injection treatment (*p < 0.05). DFO released from the TDDS into wounds resulted in chelation of excessive dermal-free iron. Innovation: DFO-TDDS is a novel therapeutic that is effective in healing wounds in sickle cell mice. Conclusion: DFO-TDDS significantly accelerates healing of murine SCUs by chelation of excessive free iron and is currently manufactured in an FDA-compliant facility to be translated for treating human SCUs.

  View details for DOI 10.1089/wound.2018.0789

  View details for PubMedID 30374417

  View details for PubMedCentralID PMC6203233

• Utilizing Confocal Microscopy to Characterize Human and Mouse Adipose Tissue TISSUE ENGINEERING PART C-METHODS Blackshear, C. P., Borrelli, M. R., Shen, E. Z., Ransom, R., Chung, N. N., Vistnes, S. M., Irizarry, D., Nazerali, R., Momeni, A., Longaker, M. T., Wan, D. C. 2018; 24 (10): 566–77

  View details for DOI 10.1089/ten.tec.2018.0154

  View details for Web of Science ID 000447725200002

• Engrailed1-Positive Fibroblasts May Modulate Transcription of the TGF-beta Pathway in the Transition from Scarless Healing to Scarring Phenotype Moore, A. L., Marshall, C. D., Des Jardins-Park, H. E., Duoto, B. A., Mascharak, S., Barnes, A., Ransom, R. C., Hu, M. S., Lorenz, H., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: E221–E222

  View details for DOI 10.1016/j.jamcollsurg.2018.08.599

  View details for Web of Science ID 000447772500535

• Mouse Model with cJUN Over-Expression Eludes to Deep Dermal Fibroblast Expansion and Immune Cell Recruitment as the Biologic Mechanism of Hypertrophic Scarring Moore, A. L., Duoto, B. A., Des Jardins-Park, H. E., Mascharak, S., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: S208

  View details for DOI 10.1016/j.jamcollsurg.2018.07.456

  View details for Web of Science ID 000447760600409

• Translational Approach Using Trimodal Manipulation of Resident Skeletal Stem Cells for Articular Cartilage Repair Murphy, M. P., Koepke, L. S., Lopez, M., Ransom, R. C., Brewer, R. E., Borrelli, M. R., Marecic, O., Chan, C. F., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: S213–S214

  View details for DOI 10.1016/j.jamcollsurg.2018.07.457

  View details for Web of Science ID 000447760600420

• Nerve-Dependent Mandibular Regeneration by Skeletal Stem Cells in Fracture Repair Jones, R., Ransom, R. C., Salhotra, A., Foster, D. S., Wan, D. C., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: S197

  View details for DOI 10.1016/j.jamcollsurg.2018.07.434

  View details for Web of Science ID 000447760600388

• Acta2, Tnc, and Col24a1 Expression Are Associated with Abdominal Adhesion Formation Marshall, C. D., Foster, D. S., Ransom, R. C., Manjunath, A., Gulati, G., Hu, M. S., Moore, A. L., Barnes, L. A., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: E128

  View details for DOI 10.1016/j.jamcollsurg.2018.08.347

  View details for Web of Science ID 000447772500304

• Reduced Scar Thickness Achieved by Topical Doxycycline Is Mediated by Specific Skin Fibroblast Populations and Not Immune Cell Infiltrate Moore, A. L., Murphy, M. P., Irizarry, D. M., Des Jardins-Park, H. E., Duoto, B. A., Mascharak, S., Foster, D. S., Jones, R., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: S210–S211

  View details for DOI 10.1016/j.jamcollsurg.2018.07.462

  View details for Web of Science ID 000447760600413

• Clonal Analysis of Local Fibroblasts in Wound Healing and Tumor Stroma Foster, D. S., Ransom, R. C., Nguyen, A. T., Salhotra, A., Jones, R. E., Hu, M. S., Norton, J. A., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: S236

  View details for DOI 10.1016/j.jamcollsurg.2018.07.623

  View details for Web of Science ID 000447760600466

• Automated Quantification of Vessel Structure: A Novel Method for Analysis of Angiogenesis in Wound Healing Jardins-Park, H., Mascharak, S., Moore, A. L., Duoto, B. A., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: E196

  View details for Web of Science ID 000447772500469

• Method of Isolating and Transplanting the Hematopoietic Stem Cell with Its Microenvironment Which Improves Functional Hematopoietic Engraftment Borrelli, M. R., Lopez, M., Gulati, G., Murphy, M. P., Sinha, R., Longaker, M. T., Weissman, I. L., Newman, A. M., Chan, C. K., Sokol, J. ELSEVIER SCIENCE INC. 2018: E224

  View details for DOI 10.1016/j.jamcollsurg.2018.08.605

  View details for Web of Science ID 000447772500541

• Identification of the Human Skeletal Stem Cell. Cell Chan, C. K., Gulati, G. S., Sinha, R., Tompkins, J. V., Lopez, M., Carter, A. C., Ransom, R. C., Reinisch, A., Wearda, T., Murphy, M., Brewer, R. E., Koepke, L. S., Marecic, O., Manjunath, A., Seo, E. Y., Leavitt, T., Lu, W., Nguyen, A., Conley, S. D., Salhotra, A., Ambrosi, T. H., Borrelli, M. R., Siebel, T., Chan, K., Schallmoser, K., Seita, J., Sahoo, D., Goodnough, H., Bishop, J., Gardner, M., Majeti, R., Wan, D. C., Goodman, S., Weissman, I. L., Chang, H. Y., Longaker, M. T. 2018; 175 (1): 43

  Abstract

  Stem cell regulation and hierarchical organization ofhuman skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.

  View details for PubMedID 30241615

• Identification of the Human Skeletal Stem Cell CELL Chan, C. F., Gulati, G. S., Sinha, R., Tompkins, J., Lopez, M., Carter, A. C., Ransom, R. C., Reinisch, A., Wearda, T., Murphy, M., Brewer, R. E., Koepke, L. S., Marecic, O., Manjunath, A., Seo, E., Leavitt, T., Lu, W., Allison Nguyen, Conley, S. D., Salhotra, A., Ambrosi, T. H., Borrelli, M. R., Siebel, T., Chan, K., Schallmoser, K., Seita, J., Sahoo, D., Goodnough, H., Bishop, J., Gardner, M., Majeti, R., Wan, D. C., Goodman, S., Weissman, I. L., Chang, H. Y., Longaker, M. T. 2018; 175 (1): 43-+

  View details for DOI 10.1016/j.cell.2018.07.029

  View details for Web of Science ID 000445120000013

• The evolving relationship of wound healing and tumor stroma. JCI insight Foster, D. S., Jones, R. E., Ransom, R. C., Longaker, M. T., Norton, J. A. 2018; 3 (18)

  Abstract

  The stroma in solid tumors contains a variety of cellular phenotypes and signaling pathways associated with wound healing, leading to the concept that a tumor behaves as a wound that does not heal. Similarities between tumors and healing wounds include fibroblast recruitment and activation, extracellular matrix (ECM) component deposition, infiltration of immune cells, neovascularization, and cellular lineage plasticity. However, unlike a wound that heals, the edges of a tumor are constantly expanding. Cell migration occurs both inward and outward as the tumor proliferates and invades adjacent tissues, often disregarding organ boundaries. The focus of our review is cancer associated fibroblast (CAF) cellular heterogeneity and plasticity and the acellular matrix components that accompany these cells. We explore how similarities and differences between healing wounds and tumor stroma continue to evolve as research progresses, shedding light on possible therapeutic targets that can result in innovative stromal-based treatments for cancer.

  View details for DOI 10.1172/jci.insight.99911

  View details for PubMedID 30232274

• Utilizing Confocal Microscopy to Characterize Human and Mouse Adipose Tissue. Tissue engineering. Part C, Methods Blackshear, C., Borrelli, M. R., Shen, E. Z., Ransom, R. C., Chung, N. N., Vistnes, S., Irizarry, D., Nazerali, R., Momeni, A., Longaker, M. T., Wan, D. C. 2018

  Abstract

  Significant advances in our understanding of human obesity, endocrinology, and metabolism have been made possible by murine comparative models, in which anatomically analogous fat depots are utilized; however, current research has questioned how truly analogous these depots are. In this study, we assess the validity of the analogy from the perspective of cellular architecture. Whole tissue mounting, confocal microscopy, and image reconstruction software were employed to characterize the three-dimensional structure of the inguinal fat pad in mice, gluteofemoral fat in humans, and subcutaneous adipose tissue of the human abdominal wall. Abdominal and gluteofemoral adipose tissue specimens from 12 human patients and bilateral inguinal fat pads from 12 mice were stained for adipocytes, blood vessels, and a putative marker for adipose-derived multipotent progenitor cells, CD34. Samples were whole-mounted and imaged with laser scanning confocal microscopy. Expectedly, human adipocytes were larger and demonstrated greater size heterogeneity. Mouse fat displayed significantly higher vascular density compared to human fat when normalized to adipocyte count. There was no significant difference in the concentration of CD34+ stromal cells from either species. However, the mean distance between CD34+ stromal cells and blood vessels was significantly greater in human fat. Finally, mouse inguinal fat contained larger numbers of brown adipocytes than did human gluteofemoral or human abdominal fat. Overall, the basic architecture of human adipose tissue differs significantly from that of mice. Insofar as human gluteofemoral fat differs from human abdominal adipose tissue, it was closer to mouse inguinal fat, being its comparative developmental analogue. These differences likely confer variance in functional properties between the two sources, and thus must be considered when designing murine models of human disease.

  View details for PubMedID 30215305

• Pathway Analysis of Gene Expression in Murine Fetal and Adult Wounds. Advances in wound care Hu, M. S., Hong, W. X., Januszyk, M., Walmsley, G. G., Luan, A., Maan, Z. N., Moshrefi, S., Tevlin, R., Wan, D. C., Gurtner, G. C., Longaker, M. T., Lorenz, H. P. 2018; 7 (8): 262-275

  Abstract

  Objective: In early gestation, fetal wounds heal without fibrosis in a process resembling regeneration. Elucidating this remarkable mechanism can result in tremendous benefits to prevent scarring. Fetal mouse cutaneous wounds before embryonic day (E)18 heal without scar. Herein, we analyze expression profiles of fetal and postnatal wounds utilizing updated gene annotations and pathway analysis to further delineate between repair and regeneration. Approach: Dorsal wounds from time-dated pregnant BALB/c mouse fetuses and adult mice at various time points were collected. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was utilized to select genes with >2-fold expression differences with a false discovery rate of <2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways. Results: Our analysis identified 471 differentially expressed genes in fetal versus adult wounds following injury. Utilizing enrichment analysis of significant genes, we identified the top 20 signaling pathways that were upregulated and downregulated at 1 and 12 h after injury. At 24 h after injury, we discovered 18 signaling pathways upregulated in adult wounds and 11 pathways upregulated in fetal wounds. Innovation: These novel target genes and pathways may reveal repair mechanisms of the early fetus that promote regeneration over fibrosis. Conclusion: Our microarray analysis recognizes hundreds of possible genes as candidates for regulators of scarless versus scarring wound repair. Enrichment analysis reveals 109 signaling pathways related to fetal scarless wound healing.

  View details for DOI 10.1089/wound.2017.0779

  View details for PubMedID 30087802

  View details for PubMedCentralID PMC6080120

• Wound Healing Research at the Hagey Laboratory for Pediatric Regenerative Medicine at Stanford University School of Medicine. Advances in wound care Hu, M. S., Longaker, M. T. 2018; 7 (8): 257-261

  View details for DOI 10.1089/wound.2018.0787

  View details for PubMedID 30087801

  View details for PubMedCentralID PMC6080091

• Fibroblasts and wound healing: an update. Regenerative medicine des Jardins-Park, H. E., Foster, D. S., Longaker, M. T. 2018

  View details for PubMedID 30062921

• Genetic dissection of clonal lineage relationships with hydroxytamoxifen liposomes. Nature communications Ransom, R. C., Foster, D. S., Salhotra, A., Jones, R. E., Marshall, C. D., Leavitt, T., Murphy, M. P., Moore, A. L., Blackshear, C. P., Wan, D. C., Longaker, M. T. 2018; 9 (1): 2971

  Abstract

  Targeted genetic dissection of tissues to identify precise cell populations has vast biological and therapeutic applications. Here we develop an approach, through thepackaging and delivery of 4-hydroxytamoxifen liposomes (LiTMX), that enables localized induction of CreERT2 recombinase in mice. Our method permits precise, in vivo, tissue-specific clonal analysis with both spatial and temporal control. This technology is effective using mice with both specific and ubiquitous Cre drivers in a variety of tissue types, under conditions of homeostasis and post-injury repair, and is highly efficient for lineage tracing and genetic analysis. This methodology is directly and immediately applicable to the developmental biology, stem cell biology and regenerative medicine, and cancer biology fields.

  View details for PubMedID 30061668

• Genetic dissection of clonal lineage relationships with hydroxytamoxifen liposomes NATURE COMMUNICATIONS Ransom, R. C., Foster, D. S., Salhotra, A., Jones, R., Marshall, C. D., Leavitt, T., Murphy, M. P., Moore, A. L., Blackshear, C. P., Wan, D. C., Longaker, M. T. 2018; 9

  View details for DOI 10.1038/s41467-018-05436-6

  View details for Web of Science ID 000440149300004

• Fibroblasts and wound healing: an update REGENERATIVE MEDICINE desJardins-Park, H. E., Foster, D. S., Longaker, M. T. 2018; 13 (5): 491–95

  View details for DOI 10.2217/rme-2018-0073

  View details for Web of Science ID 000449034700001

• Iron Chelation with Transdermal Deferoxamine Accelerates Healing of Murine Sickle Cell Ulcers ADVANCES IN WOUND CARE Rodrigues, M., Bonham, C. A., Minniti, C. P., Gupta, K., Longaker, M. T., Gurtner, G. C. 2018

  View details for DOI 10.1089/wound.2018.0789

  View details for Web of Science ID 000435552200001

• Isolation and functional assessment of mouse skeletal stem cell lineage NATURE PROTOCOLS Gulati, G. S., Murphy, M. P., Marecic, O., Lopez, M., Brewer, R. E., Koepke, L. S., Manjunath, A., Ransom, R. C., Salhotra, A., Weissman, I. L., Longaker, M. T., Chan, C. F. 2018; 13 (6): 1294–1309

  Abstract

  There are limited methods available to study skeletal stem, progenitor, and progeny cell activity in normal and diseased contexts. Most protocols for skeletal stem cell isolation are based on the extent to which cells adhere to plastic or whether they express a limited repertoire of surface markers. Here, we describe a flow cytometry-based approach that does not require in vitro selection and that uses eight surface markers to distinguish and isolate mouse skeletal stem cells (mSSCs); bone, cartilage, and stromal progenitors (mBCSPs); and five downstream differentiated subtypes, including chondroprogenitors, two types of osteoprogenitors, and two types of hematopoiesis-supportive stroma. We provide instructions for the optimal mechanical and chemical digestion of bone and bone marrow, as well as the subsequent flow-cytometry-activated cell sorting (FACS) gating schemes required to maximally yield viable skeletal-lineage cells. We also describe a methodology for renal subcapsular transplantation and in vitro colony-formation assays on the isolated mSSCs. The isolation of mSSCs can be completed in 9 h, with at least 1 h more required for transplantation. Experience with flow cytometry and mouse surgical procedures is recommended before attempting the protocol. Our system has wide applications and has already been used to study skeletal response to fracture, diabetes, and osteoarthritis, as well as hematopoietic stem cell-niche interactions in the bone marrow.

  View details for PubMedID 29748647

• Wound Healing Research at the Hagey Laboratory for Pediatric Regenerative Medicine at Stanford University School of Medicine ADVANCES IN WOUND CARE Hu, M. S., Longaker, M. T. 2018: 1–5

  View details for DOI 10.1089/wound.2018.0787

  View details for Web of Science ID 000429094900001

• A Fibroblast Is Not a Fibroblast Is Not a Fibroblast JOURNAL OF INVESTIGATIVE DERMATOLOGY Hu, M. S., Moore, A. L., Longaker, M. T. 2018; 138 (4): 729–30

  Abstract

  Fibrosis after injury is a huge public health concern, leading to morbidity, mortality, and expenditure of billions of health care dollars. Recent mouse studies have shown that dermal fibroblasts are heterogeneous. New research using single-cell RNA sequencing to identify major fibroblast populations in humans is paving the way to a better understanding of fibroblast heterogeneity and fibrosis.

  View details for PubMedID 29579454

• Pathway Analysis of Gene Expression in Murine Fetal and Adult Wounds ADVANCES IN WOUND CARE Hu, M. S., Hong, W., Januszyk, M., Walmsley, G. G., Luan, A., Maan, Z. N., Moshrefi, S., Tevlin, R., Wan, D. C., Gurtner, G. C., Longaker, M. T., Lorenz, H. 2018

  View details for DOI 10.1089/wound.2017.0779

  View details for Web of Science ID 000429548900001

• Three-Dimensional Ultrasound Versus Computerized Tomography in Fat Graft Volumetric Analysis ANNALS OF PLASTIC SURGERY Blackshear, C., Rector, M., Chung, N., Irizarry, D., Flacco, J., Brett, E., Momeni, A., Lee, G., Longaker, M. T., Wan, D. C. 2018; 80 (3): 293–96

  Abstract

  Studies evaluating fat grafting in mice have frequently used micro-computed tomography (micro-CT) as an accurate radiographic tool to measure longitudinal volume retention without killing the animal. Over the past decade, however, microultrasonography has emerged as an equally powerful preclinical imaging tool. Given their respective strengths in 3-dimensional reconstruction, there is no study to our knowledge that directly compares micro-CT with microultrasound in volumetric analysis. In this study, we compared the performance of micro-CT with microultrasound in the evaluation of adipose tissue graft volume in a murine model. Fifteen immunodeficient mice were given 200 μL of adipose tissue grafts. In vivo volumetric analysis of the grafts by micro-CT and microultrasound was conducted at discrete time points up to postoperative day 105. Three mice were killed at multiple time points, and explanted grafts were reimaged by CT and ultrasound, as mentioned previously. Analysis revealed that in vivo graft volumes measured by micro-CT do not differ significantly from those of microultrasound. Furthermore, both micro-CT and microultrasound were capable of accurately measuring fat grafts as in vivo volumes closely correlated with explanted volumes. Finally, ultrasound was found to yield improved soft tissue contrast compared with micro-CT. Therefore, either modality may be used, depending on experimental needs.

  View details for DOI 10.1097/SAP.0000000000001183

  View details for Web of Science ID 000425352000021

  View details for PubMedID 28678028

  View details for PubMedCentralID PMC5752634

• beta-Catenin-Dependent Wnt Signaling: A Pathway in Acute Cutaneous Wounding PLASTIC AND RECONSTRUCTIVE SURGERY Carre, A. L., Hu, M. S., James, A. W., Kawai, K., Galvez, M. G., Longaker, M. T., Lorenz, H. 2018; 141 (3): 669–78

  Abstract

  Acute wound healing is a dynamic process that results in the formation of scar tissue. The mechanisms of this process are not well understood; numerous signaling pathways are thought to play a major role. Here, the authors have identified β-catenin-dependent Wnt signaling as an early acute-phase reactant in acute wound healing and scar formation.The authors created 6-mm full-thickness excisional cutaneous wounds on adult β-catenin-dependent Wnt signal (BAT-gal) reporter mice. The expression of canonical Wnt after wounding was analyzed using X-gal staining and quantitative real-time polymerase chain reaction. Next, recombinant mouse Wnt3a (rmWnt3a) was injected subcutaneously to the wound edge, daily. The mice were killed at stratified time points, up to 15 days after injury. Histologic analysis, quantitative real-time polymerase chain reaction, and Western blot were performed.Numerous individual Wnt ligands increased in expression after wounding, including Wnt3a, Wnt4, Wnt10a, and Wnt11. A specific pattern of Wnt activity was observed, localized to the hair follicle and epidermis. Mice injected with rmWnt3a exhibited faster wound closure, increased scar size, and greater expression of fibroblast growth factor receptor-2 and type I collagen.The authors' data suggest that β-catenin-dependent Wnt signaling expression increases shortly after cutaneous wounding, and exogenous rmWnt3a accelerates reepithelialization, wound matrix maturation, and scar formation. Future experiments will focus on the intersection of Wnt signaling and other known profibrotic cytokines.

  View details for PubMedID 29481398

• Deferoxamine Preconditioning of Irradiated Tissue Improves Perfusion and Fat Graft Retention PLASTIC AND RECONSTRUCTIVE SURGERY Flacco, J., Chung, N., Blackshear, C. P., Irizarry, D., Momeni, A., Lee, G. K., Dung Nguyen, Gurtner, G. C., Longaker, M. T., Wan, D. C. 2018; 141 (3): 655–65

  Abstract

  Radiation therapy is a mainstay in the treatment of many malignancies, but collateral damage to surrounding tissue, with resultant hypovascularity, fibrosis, and atrophy, can be difficult to reconstruct. Fat grafting has been shown to improve the quality of irradiated skin, but volume retention of the graft is significantly decreased. Deferoxamine is a U.S. Food and Drug Administration-approved iron-chelating medication for acute iron intoxication and chronic iron overload that has also been shown to increase angiogenesis. The present study evaluates the effects of deferoxamine treatment on irradiated skin and subsequent fat graft volume retention.Mice underwent irradiation to the scalp followed by treatment with deferoxamine or saline and perfusion and were analyzed using laser Doppler analysis. Human fat grafts were then placed beneath the scalp and retention was also followed up to 8 weeks radiographically. Finally, histologic evaluation of overlying skin was performed to evaluate the effects of deferoxamine preconditioning.Treatment with deferoxamine resulted in significantly increased perfusion, as demonstrated by laser Doppler analysis and CD31 immunofluorescent staining (p < 0.05). Increased dermal thickness and collagen content secondary to irradiation, however, were not affected by deferoxamine (p > 0.05). Importantly, fat graft volume retention was significantly increased when the irradiated recipient site was preconditioned with deferoxamine (p < 0.05).The authors' results demonstrated increased perfusion with deferoxamine treatment, which was also associated with improved fat graft volume retention. Preconditioning with deferoxamine may thus enhance fat graft outcomes for soft-tissue reconstruction following radiation therapy.

  View details for PubMedID 29135894

  View details for PubMedCentralID PMC5826842

• Embryonic skin development and repair. Organogenesis Hu, M. S., Borrelli, M. R., Hong, W. X., Malhotra, S., Cheung, A. T., Ransom, R. C., Rennert, R. C., Morrison, S. D., Lorenz, H. P., Longaker, M. T. 2018: 1–18

  Abstract

  Fetal cutaneous wounds have the unique ability to completely regenerate wounded skin and heal without scarring. However, adult cutaneous wounds heal via a fibroproliferative response which results in the formation of a scar. Understanding the mechanism(s) of scarless wound healing leads to enormous clinical potential in facilitating an environment conducive to scarless healing in adult cutaneous wounds. This article reviews the embryonic development of the skin and outlines the structural and functional differences in adult and fetal wound healing phenotypes. A review of current developments made towards applying this clinical knowledge to promote scarless healing in adult wounds is addressed.

  View details for PubMedID 29420124

• Review of the Current Management of Pressure Ulcers ADVANCES IN WOUND CARE Boyko, T. V., Longaker, M. T., Yang, G. P. 2018; 7 (2): 57–67

  Abstract

  Significance: The incidence of pressure ulcers is increasing due to our aging population and the increase in the elderly living with disability. Learning how to manage pressure ulcers appropriately is increasingly important for all professionals in wound care. Recent Advances: Many new dressings and treatment modalities have been developed over the recent years and the goal of this review is to highlight their benefits and drawbacks to help providers choose their tools appropriately. Critical Issues: Despite an increased number of therapies available on the market, none has demonstrated any clear benefit over the others and pressure ulcer treatment remains frustrating and time-consuming. Future Directions: Additional research is needed to develop products more effective in prevention and treatment of pressure ulcers.

  View details for PubMedID 29392094

  View details for PubMedCentralID PMC5792240

• Cutaneous Scarring: Basic Science, Current Treatments, and Future Directions ADVANCES IN WOUND CARE Marshall, C. D., Hu, M. S., Leavitt, T., Barnes, L. A., Lorenz, H., Longaker, M. T. 2018; 7 (2): 29–45

  Abstract

  Significance: Scarring of the skin from burns, surgery, and injury constitutes a major burden on the healthcare system. Patients affected by major scars, particularly children, suffer from long-term functional and psychological problems. Recent Advances: Scarring in humans is the end result of the wound healing process, which has evolved to rapidly repair injuries. Wound healing and scar formation are well described on the cellular and molecular levels, but truly effective molecular or cell-based antiscarring treatments still do not exist. Recent discoveries have clarified the role of skin stem cells and fibroblasts in the regeneration of injuries and formation of scar. Critical Issues: It will be important to show that new advances in the stem cell and fibroblast biology of scarring can be translated into therapies that prevent and reduce scarring in humans without major side effects. Future Directions: Novel therapies involving the use of purified human cells as well as agents that target specific cells and modulate the immune response to injury are currently undergoing testing. In the basic science realm, researchers continue to refine our understanding of the role that particular cell types play in the development of scar.

  View details for PubMedID 29392092

  View details for PubMedCentralID PMC5792238

• Mechanical Forces in Cutaneous Wound Healing: Emerging Therapies to Minimize Scar Formation ADVANCES IN WOUND CARE Barnes, L. A., Marshall, C. D., Leavitt, T., Hu, M. S., Moore, A. L., Gonzalez, J. G., Longaker, M. T., Gurtner, G. C. 2018; 7 (2): 47–56

  Abstract

  Significance: Excessive scarring is major clinical and financial burden in the United States. Improved therapies are necessary to reduce scarring, especially in patients affected by hypertrophic and keloid scars. Recent Advances: Advances in our understanding of mechanical forces in the wound environment enable us to target mechanical forces to minimize scar formation. Fetal wounds experience much lower resting stress when compared with adult wounds, and they heal without scars. Therapies that modulate mechanical forces in the wound environment are able to reduce scar size. Critical Issues: Increased mechanical stresses in the wound environment induce hypertrophic scarring via activation of mechanotransduction pathways. Mechanical stimulation modulates integrin, Wingless-type, protein kinase B, and focal adhesion kinase, resulting in cell proliferation and, ultimately, fibrosis. Therefore, the development of therapies that reduce mechanical forces in the wound environment would decrease the risk of developing excessive scars. Future Directions: The development of novel mechanotherapies is necessary to minimize scar formation and advance adult wound healing toward the scarless ideal. Mechanotransduction pathways are potential targets to reduce excessive scar formation, and thus, continued studies on therapies that utilize mechanical offloading and mechanomodulation are needed.

  View details for PubMedID 29392093

  View details for PubMedCentralID PMC5792236

• Mesenchymal Stromal Cells and Cutaneous Wound Healing: A Comprehensive Review of the Background, Role, and Therapeutic Potential STEM CELLS INTERNATIONAL Hu, M. S., Borrelli, M. R., Lorenz, H., Longaker, M. T., Wan, D. C. 2018: 6901983

  Abstract

  Cutaneous wound repair is a highly coordinated cascade of cellular responses to injury which restores the epidermal integrity and its barrier functions. Even under optimal healing conditions, normal wound repair of adult human skin is imperfect and delayed healing and scarring are frequent occurrences. Dysregulated wound healing is a major concern for global healthcare, and, given the rise in diabetic and aging populations, this medicoeconomic disease burden will continue to rise. Therapies to reliably improve nonhealing wounds and reduce scarring are currently unavailable. Mesenchymal stromal cells (MSCs) have emerged as a powerful technique to improve skin wound healing. Their differentiation potential, ease of harvest, low immunogenicity, and integral role in native wound healing physiology make MSCs an attractive therapeutic remedy. MSCs promote cell migration, angiogenesis, epithelialization, and granulation tissue formation, which result in accelerated wound closure. MSCs encourage a regenerative, rather than fibrotic, wound healing microenvironment. Recent translational research efforts using modern bioengineering approaches have made progress in creating novel techniques for stromal cell delivery into healing wounds. This paper discusses experimental applications of various stromal cells to promote wound healing and discusses the novel methods used to increase MSC delivery and efficacy.

  View details for PubMedID 29887893

  View details for PubMedCentralID PMC5985130

• Embryonic skin development and repair ORGANOGENESIS Hu, M. S., Borrelli, M. R., Hong, W., Malhotra, S., Cheung, A. M., Ransom, R. C., Rennert, R. C., Morrison, S. D., Lorenz, H., Longaker, M. T. 2018; 14 (1): 46–63

  View details for DOI 10.1080/15476278.2017.1421882

  View details for Web of Science ID 000435701100005

• Management of Chronic Wounds—2018 JAMA Jones, R., Foster, D. S., Longaker, M. T. 2018

  View details for DOI 10.1001/jama.2018.12426

• Wound Healing and Fibrosis: Current Stem Cell Therapies Transfusion Jones, R., Foster, D. S., Hu, M., Longaker, M. T. 2018

  View details for DOI 10.1111/trf.14836

• Skin development and regeneration, and the control of fibrosis REGENERATIVE ENGINEERING AND DEVELOPMENTAL BIOLOGY: PRINCIPLES AND APPLICATIONS Hu, M. S., Lorenz, H., Longaker, M. T., Gardiner, D. M. 2018: 543–53

  View details for Web of Science ID 000461747900026

• The evolving relationship of wound healing and tumor stroma JCI Insight Foster, D. S., Jones, R., Ransom, R., Longaker, M. T., Norton, J. A. 2018

  Abstract

  The stroma in solid tumors contains a variety of cellular phenotypes and signaling pathways associated with wound healing, leading to the concept that a tumor behaves as a wound that does not heal. Similarities between tumors and healing wounds include fibroblast recruitment and activation, extracellular matrix (ECM) component deposition, infiltration of immune cells, neovascularization, and cellular lineage plasticity. However, unlike a wound that heals, the edges of a tumor are constantly expanding. Cell migration occurs both inward and outward as the tumor proliferates and invades adjacent tissues, often disregarding organ boundaries. The focus of our review is cancer associated fibroblast (CAF) cellular heterogeneity and plasticity and the acellular matrix components that accompany these cells. We explore how similarities and differences between healing wounds and tumor stroma continue to evolve as research progresses, shedding light on possible therapeutic targets that can result in innovative stromal-based treatments for cancer.

  View details for DOI 10.1172/jci.insight.99911

• Scarless wound healing: Transitioning from fetal research to regenerative healing. Wiley interdisciplinary reviews. Developmental biology Moore, A. L., Marshall, C. D., Barnes, L. A., Murphy, M. P., Ransom, R. C., Longaker, M. T. 2018; 7 (2)

  Abstract

  Since the discovery of scarless fetal skin wound healing, research in the field has expanded significantly with the hopes of advancing the finding to adult human patients. There are several differences between fetal and adult skin that have been exploited to facilitate scarless healing in adults including growth factors, cytokines, and extracellular matrix substitutes. However, no one therapy, pathway, or cell subtype is sufficient to support scarless wound healing in adult skin. More recently, products that contain or mimic fetal and adult uninjured dermis were introduced to the wound healing market with promising clinical outcomes. Through our review of the major experimental targets of fetal wound healing, we hope to encourage research in areas that may have a significant clinical impact. Additionally, we will investigate therapies currently in clinical use and evaluate whether they represent a legitimate advance in regenerative medicine or a vulnerary agent. WIREs Dev Biol 2018, 7:e309. doi: 10.1002/wdev.309 This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration Plant Development > Cell Growth and Differentiation Adult Stem Cells, Tissue Renewal, and Regeneration > Environmental Control of Stem Cells.

  View details for PubMedID 29316315

• Pathway Analysis of Gene Expression of E14 Versus E18 Fetal Fibroblasts ADVANCES IN WOUND CARE Hu, M. S., Borrelli, M. R., Januszyk, M., Luan, A., Malhotra, S., Walmsley, G. G., Hong, W., Tevlin, R., Gurtner, G. C., Longaker, M. T., Lorenz, H. P. 2018; 7 (1): 1–10

  Abstract

  Objective: Fetuses early in gestation heal skin wounds without forming scars. The biological mechanisms behind this process are largely unknown. Fibroblasts, however, are cells known to be intimately involved in wound healing and scar formation. We examined fibroblasts in different stages of development to characterize differences in gene expression that may result in the switch from regenerative wound repair to repair with scarring. Approach: Fibroblasts were isolated and cultured from the back skin of BALB/c wild-type mouse fetuses at embryonic day (E)14 and E18 (n = 10). The fibroblast total RNA was extracted, and microarray analysis was conducted using chips containing 42,000 genes. Significance analysis of microarrays was performed to identify genes with greater than twofold expression difference and a false discovery rate of less than two. Identified genes subsequently underwent enrichment analysis to detect differentially expressed pathways. Results: Two hundred seventy-five genes were differentially expressed between E14 and E18 in fetal fibroblasts. Thirty genes were significantly downregulated and 245 genes were significantly upregulated at E18 compared with E14. Ingenuity pathway analysis identified the top 20 signaling pathways differentially activated in fetal fibroblasts between the E18 and E14 time points. Innovation: To our knowledge, this work represents the first instance where differentially expressed genes and signaling pathways between fetal fibroblasts at E14 and E18 have been studied. Conclusion: The genes and pathways identified here potentially underlie the mechanism behind the transition from fetal wound healing via regeneration to wound healing by repair, and may prove to be key targets for future therapeutics.

  View details for PubMedID 29344429

• An Improved Humanized Mouse Model for Excisional Wound Healing Using Double Transgenic Mice ADVANCES IN WOUND CARE Hu, M. S., Cheng, J., Borrelli, M. R., Leavitt, T., Walmsley, G. G., Zielins, E. R., Hong, W., Cheung, A. M., Duscher, D., Maan, Z. N., Irizarry, D. M., Stephan, B., Parsa, F., Wan, D. C., Gurtner, G. C., Lorenz, H., Longaker, M. T. 2018; 7 (1): 11–17

  Abstract

  Objective: Splinting full-thickness cutaneous wounds in mice has allowed for a humanized model of wound healing. Delineating the epithelial edge and assessing time to closure of these healing wounds via macroscopic visualization have remained a challenge. Approach: Double transgenic mice were created by crossbreeding K14-Cre and ROSAmT/mG reporter mice. Full-thickness excisional wounds were created in K14-Cre/ROSAmT/mG mice (n = 5) and imaged using both normal and fluorescent light on the day of surgery, and every other postoperative day (POD) until wound healing was complete. Ten blinded observers analyzed a series of images from a single representative healing wound, taken using normal or fluorescent light, to decide the POD when healing was complete. K14-Cre/ROSAmT/mG mice (n = 4) were subsequently sacrificed at the four potential days of rated wound closure to accurately determine the histological point of wound closure using microscopic fluorescence imaging. Results: Average time to wound closure was rated significantly longer in the wound series images taken using normal light, compared with fluorescent light (mean POD 13.6 vs. 11.6, *p = 0.008). Fluorescence imaging of histological samples indicated that reepithelialization was complete at 12 days postwounding. Innovation: We describe a novel technique, using double transgenic mice K14-Cre/ROSAmT/mG and fluorescence imaging, to more accurately determine the healing time of wounds in mice upon macroscopic evaluation. Conclusion: The accuracy by which wound healing can be macroscopically determined in vivo in mouse models of wound healing is significantly enhanced using K14-Cre/ROSAmT/mG double transgenic mice and fluorescence imaging.

  View details for PubMedID 29344430

• Transdermal Deferoxamine Significantly Enhances Healing of Sickle Cell Ulcers Rodrigues, M., Bonham, C. A., Inayathullah, M., Rajadas, J., Yang, G. P., Caterina, M. P., Gupta, K., Longaker, M. T., Gurtner, G. C. WILEY. 2018: A11

  View details for Web of Science ID 000430308600039

• Inhibition of IRE1 results in decreased scar formation. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society Boyko, T. V., Bam, R. n., Jiang, D. n., Wang, Z. n., Bhatia, N. n., Tran, M. C., Longaker, M. T., Koong, A. C., Yang, G. P. 2018

  Abstract

  Wound healing is characterized by the production of large amounts of protein necessary to replace lost cellular mass and extracellular matrix. The unfolded protein response (UPR) is an important adaptive cellular response to increased protein synthesis. One of the main components of the UPR is IRE1, an endoplasmic reticulum transmembrane protein with endonuclease activity that produces the activated form of the transcription factor XBP1. Using luciferase reporter mice for Xbp1 splicing, we showed that IRE1 was up-regulated during excisional wound healing at the time in wound healing consistent with that of the proliferative phase, when the majority of protein synthesis for cellular proliferation and matrix deposition occurs. Furthermore, using a small molecule inhibitor of IRE1 we demonstrated that inhibition of IRE1 led to decreased scar formation in treated mice. Results were recapitulated in a hypertrophic scar mouse model. These data help provide a cellular pathway to target in the treatment of hypertrophic scarring and keloid disorders.

  View details for PubMedID 29316036

• PHD-2 Suppression in Mesenchymal Stromal Cells Enhances Wound Healing PLASTIC AND RECONSTRUCTIVE SURGERY Ko, S., Nauta, A. C., Morrison, S. D., Hu, M. S., Zimmermann, A. S., Chung, M. T., Glotzbach, J. P., Wong, V. W., Walmsley, G. G., Lorenz, H., Chan, D. A., Gurtner, G. C., Giaccia, A. J., Longaker, M. T. 2018; 141 (1): 55E–67E

  Abstract

  Cell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing.Mesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics.PHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p < 0.05). Human umbilical vein endothelial cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p < 0.05). Wounds treated with PHD-2 knockdown mesenchymal stromal cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p < 0.05). Histologic studies revealed increased blood vessel density and increased cellularity in the wounds treated with PHD-2 knockdown mesenchymal stromal cells (p < 0.05).Silencing PHD-2 in mesenchymal stromal cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family transcription factors and up-regulation of multiple downstream angiogenic factors.

  View details for PubMedID 29280872

  View details for PubMedCentralID PMC5747314

• Prrx1 Labels the Fibrogenic Fibroblast in the Ventral Dermis Hu, M., Leavitt, T., Garcia, J., Ransom, R., Litzenburger, U., Walmsley, G., Marshall, C., Moore, A., Mascharak, S., Chan, C., Wan, D., Lorenz, P., Chang, H., Longaker, M. WILEY. 2018: A4

  View details for Web of Science ID 000430308600009

• Noncoding RNAs in Wound Healing: A New and Vast Frontier ADVANCES IN WOUND CARE Luan, A., Hu, M. S., Leavitt, T., Brett, E. A., Wang, K. C., Longaker, M. T., Wan, D. C. 2018; 7 (1): 19–27

  Abstract

  Significance: Wound healing requires a highly orchestrated coordination of processes that are not yet fully understood. Therefore, available clinical therapies are thus far limited in their efficacy in preventing and treating both chronic wounds and scars. Current gene-based therapeutics is largely based on our understanding of the protein-coding genome and proteins involved in known wound healing pathways. Recent Advances: Noncoding RNAs such as microRNAs and long noncoding RNAs have recently been found to be significant modulators of gene expression in diverse cellular pathways. Research has now implicated noncoding RNAs in nearly every stage of the wound healing process, suggesting that they may serve as clinical therapeutic targets. Noncoding RNAs are critical regulators in processes such as angiogenesis and cutaneous cell migration and proliferation, including classically described biological pathways previously attributed to mostly protein constituents. Critical Issues: The complexity and diversity of the interactions of noncoding RNAs with their targets and other binding partners require thorough characterization and understanding of their functions before they may be altered to modulate human wound healing pathways. Future Directions: Research in the area of noncoding RNAs continues to rapidly expand our understanding of their potential roles in physiological and pathological wound healing. Coupled with improving technologies to enhance or suppress target noncoding RNA in vivo, these advances hold great promise in the development of new therapies for wound healing.

  View details for PubMedID 29344431

  View details for PubMedCentralID PMC5770091

• Ultrasound-assisted liposuction provides a source for functional adipose-derived stromal cells CYTOTHERAPY Duscher, D., Maan, Z. N., Luan, A., Aitzetmueller, M. M., Brett, E. A., Atashroo, D., Whittam, A. J., Hu, M. S., Walmsley, G. G., Houschyar, K. S., Schilling, A. F., Machens, H., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2017; 19 (12): 1491–1500

  Abstract

  Regenerative medicine employs human mesenchymal stromal cells (MSCs) for their multi-lineage plasticity and their pro-regenerative cytokine secretome. Adipose-derived mesenchymal stromal cells (ASCs) are concentrated in fat tissue, and the ease of harvest via liposuction makes them a particularly interesting cell source. However, there are various liposuction methods, and few have been assessed regarding their impact on ASC functionality. Here we study the impact of the two most popular ultrasound-assisted liposuction (UAL) devices currently in clinical use, VASER (Solta Medical) and Lysonix 3000 (Mentor) on ASCs.After lipoaspirate harvest and processing, we sorted for ASCs using fluorescent-assisted cell sorting based on an established surface marker profile (CD34+CD31-CD45-). ASC yield, viability, osteogenic and adipogenic differentiation capacity and in vivo regenerative performance were assessed.Both UAL samples demonstrated equivalent ASC yield and viability. VASER UAL ASCs showed higher osteogenic and adipogenic marker expression, but a comparable differentiation capacity was observed. Soft tissue healing and neovascularization were significantly enhanced via both UAL-derived ASCs in vivo, and there was no significant difference between the cell therapy groups.Taken together, our data suggest that UAL allows safe and efficient harvesting of the mesenchymal stromal cellular fraction of adipose tissue and that cells harvested via this approach are suitable for cell therapy and tissue engineering applications.

  View details for PubMedID 28917626

  View details for PubMedCentralID PMC5723208

• Cell-Based Soft Tissue Reconstruction in a Hydrogel Scaffold ANNALS OF PLASTIC SURGERY Blackshear, C. P., Flacco, J. S., Vistnes, S. M., Chung, N. N., Irizarry, D., Brett, E. A., Yen, D. J., Momeni, A., Longaker, M. T., Wan, D. C. 2017; 79 (6): 618–22

  Abstract

  Renevia is a hyaluronin-gelatin crosslinked matrix scaffold that has been studied as an alternative to adipose transfer in soft tissue reconstruction. It is designed to emulate the native extracellular matrix environment by supporting stromal vascular fraction (SVF) cell attachment, survival, and proliferation, thus promoting cell-based volume restoration. However, the concentration of incorporated cells for a clinically relevant result has yet to be determined.Five experimental groups of seven CD-1 nude immunodeficient mice were given 250 μL grafts of the following composition: 1 million human SVF cells per mL of Renevia scaffold, 6 million human SVF cells per mL scaffold, 12 million human SVF cells per mL scaffold, Renevia scaffold-alone or human adipose tissue-alone. Volumetric analysis was conducted at discrete time points over 16 weeks using 3-dimensional ultrasound, after which time the grafts were explanted for histologic analysis.At the conclusion of the study at week 16, the Renevia scaffold group incorporating the highest concentration of human SVF cells (12 million cells per mL scaffold) had significantly greater volume retention compared with the 2 lower concentrations, scaffold-alone and fat-alone groups. Histology of the 12 million scaffold group revealed abundant adipocyte formation within the scaffold, exceeding that observed in the 6 million, 1 million, and scaffold-alone groups. The 12 million group also demonstrated significantly increased vascularity per CD31 staining.Stromal vascular fraction cells coupled with Renevia hydrogel scaffold can enhance soft tissue volume reconstruction. In this study, we observed the greatest effect with 12 million cells per mL. From the perspective of volume retention, incorporation of higher concentrations of SVF cells with Renevia may be an alternative to conventional adipose tissue grafting.

  View details for PubMedID 28671889

  View details for PubMedCentralID PMC5677558

• A MUSE for Skin Regeneration JOURNAL OF INVESTIGATIVE DERMATOLOGY Hu, M. S., Longaker, M. T. 2017; 137 (12): 2471–72

  Abstract

  With a rise in the prevalence of chronic wounds and other soft tissue defects, there is an urgent need to regenerate skin. Multilineage-differentiating stress-enduring cells were identified as distinct pluripotent stem cells in mesenchymal cell populations in humans. New research demonstrates the ability to effectively differentiate multilineage-differentiating stress-enduring cells into fibroblasts and keratinocytes for skin reconstitution.

  View details for PubMedID 29169463

• Calvarial Defects: Cell-Based Reconstructive Strategies in the Murine Model TISSUE ENGINEERING PART C-METHODS Murphy, M. P., Quarto, N., Longaker, M. T., Wan, D. C. 2017; 23 (12): 971–81

  Abstract

  Calvarial defects pose a continued clinical dilemma for reconstruction. Advancements within the fields of stem cell biology and tissue engineering have enabled researchers to develop reconstructive strategies using animal models. We review the utility of various animal models and focus on the mouse, which has aided investigators in understanding cranial development and calvarial bone healing. The murine model has also been used to study regenerative approaches to critical-sized calvarial defects, and we discuss the application of stem cells such as bone marrow-derived mesenchymal stromal cells, adipose-derived stromal cells, muscle-derived stem cells, and pluripotent stem cells to address deficient bone in this animal. Finally, we highlight strategies to manipulate stem cells using various growth factors and inhibitors and ultimately how these factors may prove crucial in future advancements within calvarial reconstruction using native skeletal stem cells.

  View details for PubMedID 28825366

  View details for PubMedCentralID PMC5734144

• Epigenetic Analysis of Scar Forming Fibroblasts Reveals Key Differences in Genes Associated with Fibrosis Moore, A. L., Marshall, C. D., Litzenburger, U., Barnes, L., Ransom, R., Hu, M., Leavitt, T., Chang, H. Y., Longaker, M. T. ELSEVIER SCIENCE INC. 2017: S200–S201

  View details for DOI 10.1016/j.jamcollsurg.2017.07.460

  View details for Web of Science ID 000413315300436

• Fat Graft Retention Decreases with Recipient Age Montenegro, C. F., Flacco, J., Chung, N. N., Irizarry, D. M., Brett, E. A., Blackshear, C. P., Nguyen, D., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2017: E144–E145

  View details for DOI 10.1016/j.jamcollsurg.2017.07.917

  View details for Web of Science ID 000413319300353

• Shifting Skeletal Stem Cell Dynamics Underlie Skeletal Aging in Mice Marecic, O., McArdle, A., Seo, E., Tevlin, R., Gulati, G. S., Murphy, M. P., Lopez, M., Weissman, I. L., Chan, C. K., Longaker, M. T. ELSEVIER SCIENCE INC. 2017: S166

  View details for DOI 10.1016/j.jamcollsurg.2017.07.374

  View details for Web of Science ID 000413315300353

• Activation of the Mouse Resident Skeletal Stem Cell for Articular Cartilage Repair Murphy, M. P., Lopez, M., Ransom, R. C., Marecic, O., Gulati, G. S., Chan, C. K., Longaker, M. T. ELSEVIER SCIENCE INC. 2017: S160

  View details for DOI 10.1016/j.jamcollsurg.2017.07.360

  View details for Web of Science ID 000413315300339

• Rescue of Del1 Knock Out Phenotype in Bone Fracture Healing in Mice Boyko, T. V., Marecic, O., Lopez, M., Seo, E., Tong, X., Chan, C., Longaker, M. T., Yang, G. P. ELSEVIER SCIENCE INC. 2017: S89–S90

  View details for DOI 10.1016/j.jamcollsurg.2017.07.192

  View details for Web of Science ID 000413315300183

• Doxycyline Improves Wound Healing via Nonantibiotic Associated Mechanisms Moore, A. L., Murphy, M. P., Irizarry, D. M., Brett, E. A., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2017: S162–S163

  View details for DOI 10.1016/j.jamcollsurg.2017.07.366

  View details for Web of Science ID 000413315300345

• Cellular Mechanisms Underlying Regeneration in Mandibular Distraction Osteogenesis Ransom, R. C., Leavitt, T., Murphy, M. P., Marecic, O., Lopez, M., Marshall, C. D., Barnes, L. A., Wan, D. C., Chan, C. K., Longaker, M. T. ELSEVIER SCIENCE INC. 2017: E143–E144

  View details for DOI 10.1016/j.jamcollsurg.2017.07.914

  View details for Web of Science ID 000413319300350

• Invited Commentary on: Role of Notch Signaling in the Physiological Patterning of Posterofrontal and Sagittal Cranial Sutures JOURNAL OF CRANIOFACIAL SURGERY Quarto, N., Longaker, M. T. 2017; 28 (6): 1626–27

  View details for PubMedID 28796102

• Strategic Targeting of Multiple BMP Receptors Prevents Trauma-Induced Heterotopic Ossification MOLECULAR THERAPY Agarwal, S., Loder, S. J., Breuler, C., Li, J., Cholok, D., Brownley, C., Peterson, J., Hsieh, H. H., Drake, J., Ranganathan, K., Niknafs, Y. S., Xiao, W., Li, S., Kumar, R., Tompkins, R., Longaker, M. T., Davis, T. A., Yu, P. B., Mishina, Y., Levi, B. 2017; 25 (8): 1974–87

  Abstract

  Trauma-induced heterotopic ossification (tHO) is a condition of pathologic wound healing, defined by the progressive formation of ectopic bone in soft tissue following severe burns or trauma. Because previous studies have shown that genetic variants of HO, such as fibrodysplasia ossificans progressiva (FOP), are caused by hyperactivating mutations of the type I bone morphogenetic protein receptor (T1-BMPR) ACVR1/ALK2, studies evaluating therapies for HO have been directed primarily toward drugs for this specific receptor. However, patients with tHO do not carry known T1-BMPR mutations. Here we show that, although BMP signaling is required for tHO, no single T1-BMPR (ACVR1/ALK2, BMPR1a/ALK3, or BMPR1b/ALK6) alone is necessary for this disease, suggesting that these receptors have functional redundancy in the setting of tHO. By utilizing two different classes of BMP signaling inhibitors, we developed a translational approach to treatment, integrating treatment choice with existing diagnostic options. Our treatment paradigm balances either immediate therapy with reduced risk for adverse effects (Alk3-Fc) or delayed therapy with improved patient selection but greater risk for adverse effects (LDN-212854).

  View details for PubMedID 28716575

  View details for PubMedCentralID PMC5542633

• Isotretinoin and Timing of Procedural Interventions A Systematic Review With Consensus Recommendations JAMA DERMATOLOGY Spring, L. K., Krakowski, A. C., Alam, M., Bhatia, A., Brauer, J., Cohen, J., Del Rosso, J. Q., Diaz, L., Dover, J., Eichenfield, L. F., Gurtner, G. C., Hanke, C., Jahnke, M. N., Kelly, K. M., Khetarpal, S., Kinney, M. A., Levy, M. L., Leyden, J., Longaker, M. T., Munavalli, G. S., Ozog, D. M., Prather, H., Shumaker, P. R., Tanzi, E., Torres, A., Velez, M., Waldman, A. B., Yan, A. C., Zaenglein, A. L. 2017; 153 (8): 802–9

  Abstract

  The notion that systemic isotretinoin taken within 6 to 12 months of cutaneous surgery contributes to abnormal scarring or delayed wound healing is widely taught and practiced; however, it is based on 3 small case series from the mid-1980s.To evaluate the body of literature to provide evidence-based recommendations regarding the safety of procedural interventions performed either concurrently with, or immediately following the cessation of systemic isotretinoin therapy.A panel of national experts in pediatric dermatology, procedural/cosmetic dermatology, plastic surgery, scars, wound healing, acne, and isotretinoin was convened. A systematic PubMed review of English-language articles published from 1982 to 2017 was performed using the following search terms: isotretinoin, 13-cis-retinoic acid, Accutane, retinoids, acitretin, surgery, surgical, laser, ablative laser, nonablative laser, laser hair removal, chemical peel, dermabrasion, wound healing, safety, scarring, hypertrophic scar, and keloid. Evidence was graded, and expert consensus was obtained.Thirty-two relevant publications reported 1485 procedures. There was insufficient evidence to support delaying manual dermabrasion, superficial chemical peels, cutaneous surgery, laser hair removal, and fractional ablative and nonablative laser procedures for patients currently receiving or having recently completed isotretinoin therapy. Based on the available literature, mechanical dermabrasion and fully ablative laser are not recommended in the setting of systemic isotretinoin treatment.Physicians and patients may have an evidence-based discussion regarding the known risk of cutaneous surgical procedures in the setting of systemic isotretinoin therapy. For some patients and some conditions, an informed decision may lead to earlier and potentially more effective interventions.

  View details for PubMedID 28658462

• Discussion: Combining Smoothened Agonist and NEL-Like Protein-1 Enhances Bone Healing. Plastic and reconstructive surgery Ransom, R. C., Longaker, M. T. 2017; 139 (6): 1397-1398

  View details for DOI 10.1097/PRS.0000000000003368

  View details for PubMedID 28538564

• Dynamic Rheology for the Prediction of Surgical Outcomes in Autologous Fat Grafting. Plastic and reconstructive surgery Luan, A., Zielins, E. R., Wearda, T., Atashroo, D. A., Blackshear, C. P., Raphel, J., Brett, E. A., Flacco, J., Alyono, M. C., Momeni, A., Heilshorn, S., Longaker, M. T., Wan, D. C. 2017

  Abstract

  Due to the abundance and biocompatibility of fat, lipotransfer has become an attractive method for treating soft tissue deficits. However, it is limited by unpredictable graft survival and retention. Currently, little is known about the viscoelastic properties of fat after various injection methods. Here, we assess the effects of cannula diameter, length, and shape on the viscoelastic properties, structure, and retention of fat.Human lipoaspirate was harvested using suction-assisted liposuction and prepared for grafting. A syringe pump was used to inject fat at a controlled flow rate through cannulas of varying gauge, length, and shape. Processed samples were tested in triplicate on an oscillatory rheometer to measure their viscoelastic properties. Fat grafts from each group were placed into the scalps of immunocompromised mice. After 8 weeks, graft retention was measured using micro-CT and grafts were explanted for histological analysis.Lipoaspirate injected through narrower, longer, and bent cannulas exhibited more shear thinning with diminished quality. The storage modulus (G') of fat processed with 18-gauge cannulas was significantly lower than when processed with 14-gauge or larger cannulas, which also corresponded with inferior in vivo histological structure. Similarly, the longer cannula group had a significantly lower G' than the shorter cannula, and was associated with decreased graft retention.Discrete modifications in the methods used for fat placement can have a significant impact on immediate graft integrity, and ultimately on graft survival and quality. Respecting these biomechanical influences during the placement phase of lipotransfer may allow surgeons to optimize outcomes.

  View details for DOI 10.1097/PRS.0000000000003578

  View details for PubMedID 28574947

• Protein-Nanoparticle Hydrogels That Self-assemble in Response to Peptide-Based Molecular Recognition. ACS biomaterials science & engineering Parisi-Amon, A., Lo, D. D., Montoro, D. T., Dewi, R. E., Longaker, M. T., Heilshorn, S. C. 2017; 3 (5): 750-756

  Abstract

  Recently, supramolecular hydrogels assembled through nonspecific interactions between polymers and nanoparticles (termed PNP systems) were reported to have rapid shear-thinning and self-healing properties amenable for cell-delivery applications in regenerative medicine. Here, we introduce protein engineering concepts into the design of a new family of PNP hydrogels to enable direct control over the polymer-nanoparticle interactions using peptide-based molecular recognition motifs. Specifically, we have designed a bifunctional peptide that induces supramolecular hydrogel assembly between hydroxy apatite nanoparticles and an engineered, recombinant protein. We demonstrate that this supramolecular assembly critically requires molecular recognition, as no assembly is observed in the presence of control peptides with a scrambled amino acid sequence. Titration of the bifunctional peptide enables direct control over the number of physical cross-links within the system and hence the resulting hydrogel mechanical properties. As with previous PNP systems, these materials are rapidly shear-thinning and self-healing. As proof-of-concept, we demonstrate that these materials are suitable for therapeutic cell delivery applications in a preclinical murine calvarial defect model.

  View details for DOI 10.1021/acsbiomaterials.6b00286

  View details for PubMedID 33440497

• Isolation of CD248-expressing stromal vascular fraction for targeted improvement of wound healing. Wound repair and regeneration Brett, E., Zielins, E. R., Chin, M., Januszyk, M., Blackshear, C. P., Findlay, M., Momeni, A., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2017

  Abstract

  Wound healing remains a global issue of disability, cost, and health. Addition of cells from the stromal vascular fraction (SVF) of adipose tissue has been shown to increase the rate of full thickness wound closure. This study aimed to investigate the angiogenic mechanisms of CD248+ SVF cells in the context of full thickness excisional wounds. Single cell transcriptional analysis was used to identify and cluster angiogenic gene-expressing cells, which was then correlated with surface marker expression. SVF cells isolated from human lipoaspirate were FACS sorted based on the presence of CD248. Cells were analyzed for angiogenic gene expression and ability to promote microvascular tubule formation in vitro. Following this, 6mm full thickness dermal wounds were created on the dorsa of immunocompromised mice and then treated with CD248+, CD248-, or unsorted SVF cells delivered in a pullalan-collagen hydrogel or the hydrogel alone. Wounds were measured every other day photometrically until closure. Wounds were also evaluated histologically at 7 and 14 days post-wounding and when fully healed to assess for reepithelialization and development of neovasculature. Wounds treated with CD248+ cells healed significantly faster than other treatment groups, and at 7 days, had quantitatively more reepithelialization. Concurrently, immunohistochemistry of CD31 revealed a much higher presence of vascularity in the CD248+ SVF cells treated group at the time of healing and at 14 days post-op, consistent with a pro-angiogenic effect of CD248+ cells in vivo. Therefore, using CD248+ pro-angiogenic cells obtained from SVF presents a viable strategy in wound healing by promoting increased vessel growth in the wound.

  View details for DOI 10.1111/wrr.12542

  View details for PubMedID 28464475

• Protein-Nanoparticle Hydrogels That Self-assemble in Response to Peptide-Based Molecular Recognition ACS BIOMATERIALS SCIENCE & ENGINEERING Parisi-Amon, A., Lo, D. D., Montoro, D. T., Dew, R. E., Longaker, M. T., Heilshorn, S. C. 2017; 3 (5): 750-756

  View details for DOI 10.1021/acsbiomaterials.6b00286

  View details for Web of Science ID 000401053800009

• A Review of Cell-Based Strategies for Soft Tissue Reconstruction. Tissue engineering. Part B, Reviews Brett, E., Chung, N., Leavitt, W. T., Momeni, A., Longaker, M. T., Wan, D. C. 2017

  Abstract

  Soft tissue reconstruction to restore volume to damaged or deficient tissue beneath the skin remains a challenging endeavor. Current techniques are centered around autologous fat transfer, or the use of synthetic substitutes, however, a great deal of scientific inquiry has been made into both the molecular mechanisms involved in, and limitations of, de novo adipogenesis, that is, the formation of new adipose tissue from precursor cells. To best comprehend these mechanisms, an understanding of defined markers for adipogenic differentiation, and knowledge of both commercially available and primary cell lines that enable in vitro and in vivo studies is necessary. We review the growth factors, proteins, cytokines, drugs, and molecular pathways that have shown promise in enhancing adipogenesis and vasculogenesis, in addition to the multitude of scaffolds that act as delivery vehicles to support these processes. While progress continues on these fronts, equally important is how researchers are optimizing clinically employed strategies such as autologous fat transfer through cell-based intervention, and the potential to augment this approach through isolation of preferentially adipogenic or angiogenic precursor subpopulations, which exists on the horizon. This review will highlight the novel molecular and synthetic modifications currently being studied for inducing adipose tissue regeneration on a cellular level, which will expand our arsenal of techniques for approaching soft tissue reconstruction.

  View details for DOI 10.1089/ten.TEB.2016.0455

  View details for PubMedID 28372485

• Purified Adipose-Derived Stromal Cells Provide Superior Fat Graft Retention Compared with Unenriched Stromal Vascular Fraction PLASTIC AND RECONSTRUCTIVE SURGERY Zielins, E. R., Brett, E. A., Blackshear, C. P., Flacco, J., Ransom, R. C., Longaker, M. T., Wan, D. C. 2017; 139 (4): 911–14

  Abstract

  Cell-assisted lipotransfer has shown much promise as a technique to improve fat graft retention in both mouse and human studies. However, the literature varies as to whether fresh stromal vascular fraction or culture-expanded adipose-derived stromal cells are used to augment volume retention. The authors' study sought to determine whether there was a significant advantage to using adipose-derived stromal cells over unpurified stromal vascular fraction cells in a mouse model of cell-assisted lipotransfer.

  View details for PubMedID 28350672

  View details for PubMedCentralID PMC5398091

• Discussion: Regeneration of Vascularized Corticocancellous Bone and Diploic Space Using Muscle- Derived Stem Cells: A Translational Biologic Alternative for Healing Critical Bone Defects PLASTIC AND RECONSTRUCTIVE SURGERY Murphy, M. P., Chan, C. K., Longaker, M. T. 2017; 139 (4): 906-907

  View details for DOI 10.1097/PRS.0000000000003210

  View details for PubMedID 28350669

• Discussion: Towards Microsurgical Correction of Cleft Lip Ex Utero via Restoration of Craniofacial Developmental Programs. Plastic and reconstructive surgery Ransom, R. C., Lorenz, H. P., Longaker, M. T. 2017

  View details for DOI 10.1097/PRS.0000000000003424

  View details for PubMedID 28338583

• Comparison of the Hydroxylase Inhibitor Dimethyloxalylglycine and the Iron Chelator Deferoxamine in Diabetic and Aged Wound Healing. Plastic and reconstructive surgery Duscher, D., Januszyk, M., Maan, Z. N., Whittam, A. J., Hu, M. S., Walmsley, G. G., Dong, Y., Khong, S. M., Longaker, M. T., Gurtner, G. C. 2017; 139 (3): 695e-706e

  Abstract

  A hallmark of diabetes mellitus is the breakdown of almost every reparative process in the human body, leading to critical impairments of wound healing. Stabilization and activity of the transcription factor hypoxia-inducible factor (HIF)-1α is impaired in diabetes, leading to deficits in new blood vessel formation in response to injury. In this article, the authors compare the effectiveness of two promising small-molecule therapeutics, the hydroxylase inhibitor dimethyloxalylglycine and the iron chelator deferoxamine, for attenuating diabetes-associated deficits in cutaneous wound healing by enhancing HIF-1α activation.HIF-1α stabilization, phosphorylation, and transactivation were measured in murine fibroblasts cultured under normoxic or hypoxic and low-glucose or high-glucose conditions following treatment with deferoxamine or dimethyloxalylglycine. In addition, diabetic wound healing and neovascularization were evaluated in db/db mice treated with topical solutions of either deferoxamine or dimethyloxalylglycine, and the efficacy of these molecules was also compared in aged mice.The authors show that deferoxamine stabilizes HIF-1α expression and improves HIF-1α transactivity in hypoxic and hyperglycemic states in vitro, whereas the effects of dimethyloxalylglycine are significantly blunted under hyperglycemic hypoxic conditions. In vivo, both dimethyloxalylglycine and deferoxamine enhance wound healing and vascularity in aged mice, but only deferoxamine universally augmented wound healing and neovascularization in the setting of both advanced age and diabetes.This first direct comparison of deferoxamine and dimethyloxalylglycine in the treatment of impaired wound healing suggests significant therapeutic potential for topical deferoxamine treatment in ischemic and diabetic disease.

  View details for DOI 10.1097/PRS.0000000000003072

  View details for PubMedID 28234841

  View details for PubMedCentralID PMC5327844

• Minimizing Skin Scarring through Biomaterial Design JOURNAL OF FUNCTIONAL BIOMATERIALS Moore, A. L., Marshall, C. D., Longaker, M. T. 2017; 8 (1)

  View details for DOI 10.3390/jfb8010003

  View details for Web of Science ID 000446638700002

• Laboratory Models for the Study of Normal and Pathologic Wound Healing. Plastic and reconstructive surgery Boyko, T. V., Longaker, M. T., Yang, G. P. 2017; 139 (3): 654-662

  Abstract

  Current knowledge of wound healing is based on studies using various in vitro and in vivo wound models. In vitro models allow for biological examination of specific cell types involved in wound healing. In vivo models generally provide the full spectrum of biological responses required for wound healing, including inflammation and angiogenesis, and provide cell-cell interactions not seen in vitro. In this review, the authors aim to delineate the most relevant wound healing models currently available and to discuss their strengths and limitations in their approximation of the human wound healing processes to aid scientists in choosing the most appropriate wound healing models for designing, testing, and validating their experiments.

  View details for DOI 10.1097/PRS.0000000000003077

  View details for PubMedID 28234843

• Fibroblasts become fat to reduce scarring. Science Chan, C. K., Longaker, M. T. 2017; 355 (6326): 693-694

  View details for DOI 10.1126/science.aam6748

  View details for PubMedID 28209860

• Excess Dermal Tissue Remodeling In Vivo: Does It Settle? Plastic and reconstructive surgery Leavitt, T., Hu, M. S., Zielins, E. R., Barnes, L. A., Marshall, C. D., Wan, D. C., Lorenz, H. P., Gurtner, G. C., Longaker, M. T. 2017; 139 (2): 415e-424e

  Abstract

  Surgical manipulation of skin may result in undesired puckering of excess tissue, which is generally assumed to settle over time. In this article, the authors address the novel question of how this excess tissue remodels.Purse-string sutures (6-0 nylon) were placed at the midline dorsum of 22 wild-type BALB/c mice in a circular pattern marked with tattoo ink. Sutures were cinched and tied under tension in the treatment group, creating an excess tissue deformity, whereas control group sutures were tied without tension. After 2 or 4 weeks, sutures were removed. The area of tattooed skin was measured up to 56 days after suture removal. Histologic analysis was performed on samples harvested 14 days after suture removal.The majority of excess tissue deformities flattened within 2 days after suture removal. However, the sutured skin in the treatment group decreased in area by an average of 18 percent from baseline (n = 9), compared to a 1 percent increase in the control group (n = 10) at 14 days after suture removal (p < 0.05). This was similarly observed at 28 days (treatment, -11.7 percent; control, 4.5 percent; n = 5; p = 0.0243). Despite flattening, deformation with purse-string suture correlated with increased collagen content of skin, in addition to increased numbers of myofibroblasts. Change in area did not correlate with duration of suture placement.Excess dermal tissue deformities demonstrate the ability to remodel with gross flattening of the skin, increased collagen deposition, and incomplete reexpansion to baseline area. Further studies will reveal whether our findings in this mouse model translate to humans.

  View details for DOI 10.1097/PRS.0000000000003026

  View details for PubMedID 28121870

• Rapid Isolation of BMPR-IB plus Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Marshall, C. D., Zielins, E. R., Brett, E. A., Blackshear, C. P., Hu, M. S., Leavitt, T., Barnes, L. A., Lorenz, H. P., Longaker, M. T., Wan, D. C. 2017

  Abstract

  Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

  View details for DOI 10.3791/55120

  View details for Web of Science ID 000397847700048

  View details for PubMedID 28287559

• Rapid Isolation of BMPR-IB plus Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Marshall, C. D., Zielins, E. R., Brett, E. A., Blackshear, C. P., Hu, M. S., Leavitt, T., Barnes, L. A., Lorenz, H. P., Longaker, M. T., Wan, D. C. 2017

  Abstract

  Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

  View details for DOI 10.3791/55120

  View details for Web of Science ID 000397847700048

  View details for PubMedID 28287559

• Sanativo Wound Healing Product Does Not Accelerate Reepithelialization in a Mouse Cutaneous Wound Healing Model. Plastic and reconstructive surgery Marshall, C. D., Hu, M. S., Leavitt, T., Barnes, L. A., Cheung, A. T., Malhotra, S., Lorenz, H. P., Delp, S. L., Quake, S. R., Longaker, M. T. 2017; 139 (2): 343-352

  Abstract

  Sanativo is an over-the-counter Brazilian product derived from Amazon rainforest plant extract that is purported to improve the healing of skin wounds. Two experimental studies have shown accelerated closure of nonsplinted excisional wounds in rat models. However, these models allow for significant contraction of the wound and do not approximate healing in the tight skin of humans.Full-thickness excisional wounds were created on the dorsal skin of mice and were splinted with silicone rings, a model that forces the wound to heal by granulation and reepithelialization. Sanativo or a control solution was applied either daily or every other day to the wounds. Photographs were taken every other day, and the degree of reepithelialization of the wounds was determined.With both daily and every-other-day applications, Sanativo delayed reepithelialization of the wounds. Average time to complete healing was faster with control solution versus Sanativo in the daily application group (9.4 versus 15.2 days; p < 0.0001) and the every-other-day application group (11 versus 13 days; p = 0.017). The size of visible scar at the last time point of the study was not significantly different between the groups, and no differences were found on histologic examination.Sanativo wound healing compound delayed wound reepithelialization in a mouse splinted excisional wound model that approximates human wound healing. The size of visible scar after complete healing was not improved with the application of Sanativo. These results should cast doubt on claims that this product can improve wound healing in humans.

  View details for DOI 10.1097/PRS.0000000000003013

  View details for PubMedID 28121865

• Detection of Stem Cell Transplant Rejection with Ferumoxytol MR Imaging: Correlation of MR Imaging Findings with Those at Intravital Microscopy. Radiology Daldrup-Link, H. E., Chan, C., Lenkov, O., Taghavigarmestani, S., Nazekati, T., Nejadnik, H., Chapelin, F., Khurana, A., Tong, X., Yang, F., Pisani, L., Longaker, M., Gambhir, S. S. 2017: 161139-?

  Abstract

  Purpose To determine whether endogenous labeling of macrophages with clinically applicable nanoparticles enables noninvasive detection of innate immune responses to stem cell transplants with magnetic resonance (MR) imaging. Materials and Methods Work with human stem cells was approved by the institutional review board and the stem cell research oversight committee, and animal experiments were approved by the administrative panel on laboratory animal care. Nine immunocompetent Sprague-Dawley rats received intravenous injection of ferumoxytol, and 18 Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice received rhodamine-conjugated ferumoxytol. Then, 48 hours later, immune-matched or mismatched stem cells were implanted into osteochondral defects of the knee joints of experimental rats and calvarial defects of Jax mice. All animals underwent serial MR imaging and intravital microscopy (IVM) up to 4 weeks after surgery. Macrophages of Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice express enhanced green fluorescent protein (GFP), which enables in vivo correlation of ferumoxytol enhancement at MR imaging with macrophage quantities at IVM. All quantitative data were compared between experimental groups by using a mixed linear model and t tests. Results Immune-mismatched stem cell implants demonstrated stronger ferumoxytol enhancement than did matched stem cell implants. At 4 weeks, T2 values of mismatched implants were significantly lower than those of matched implants in osteochondral defects of female rats (mean, 10.72 msec for human stem cells and 11.55 msec for male rat stem cells vs 15.45 msec for sex-matched rat stem cells; P = .02 and P = .04, respectively) and calvarial defects of recipient mice (mean, 21.7 msec vs 27.1 msec, respectively; P = .0444). This corresponded to increased recruitment of enhanced GFP- and rhodamine-ferumoxytol-positive macrophages into stem cell transplants, as visualized with IVM and histopathologic examination. Conclusion Endogenous labeling of macrophages with ferumoxytol enables noninvasive detection of innate immune responses to stem cell transplants with MR imaging. (©) RSNA, 2017 Online supplemental material is available for this article.

  View details for DOI 10.1148/radiol.2017161139

  View details for PubMedID 28128708

• Minimizing Skin Scarring through Biomaterial Design. Journal of functional biomaterials Moore, A. L., Marshall, C. D., Longaker, M. T. 2017; 8 (1)

  Abstract

  Wound healing continues to be a major burden to patients, though research in the field has expanded significantly. Due to an aging population and increasing comorbid conditions, the cost of chronic wounds is expected to increase for patients and the U.S. healthcare system alike. With this knowledge, the number of engineered products to facilitate wound healing has also increased dramatically, with some already in clinical use. In this review, the major biomaterials used to facilitate skin wound healing will be examined, with particular attention allocated to the science behind their development. Experimental therapies will also be evaluated.

  View details for DOI 10.3390/jfb8010003

  View details for PubMedID 28117733

• Pharmacological rescue of diabetic skeletal stem cell niches. Science translational medicine Tevlin, R., Seo, E. Y., Marecic, O., McArdle, A., Tong, X., Zimdahl, B., Malkovskiy, A., Sinha, R., Gulati, G., Li, X., Wearda, T., Morganti, R., Lopez, M., Ransom, R. C., Duldulao, C. R., Rodrigues, M., Nguyen, A., Januszyk, M., Maan, Z., Paik, K., Yapa, K., Rajadas, J., Wan, D. C., Gurtner, G. C., Snyder, M., Beachy, P. A., Yang, F., Goodman, S. B., Weissman, I. L., Chan, C. K., Longaker, M. T. 2017; 9 (372)

  Abstract

  Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing. Despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques, we analyzed intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function to identify specific mSSC niche-related abnormalities that could impair skeletal repair in diabetic (Db) mice. We discovered that high serum concentrations of tumor necrosis factor-α directly repressed the expression of Indian hedgehog (Ihh) in mSSCs and in their downstream skeletogenic progenitors in Db mice. When hedgehog signaling was inhibited during fracture repair, injury-induced mSSC expansion was suppressed, resulting in impaired healing. We reversed this deficiency by precise delivery of purified Ihh to the fracture site via a specially formulated, slow-release hydrogel. In the presence of exogenous Ihh, the injury-induced expansion and osteogenic potential of mSSCs were restored, culminating in the rescue of Db bone healing. Our results present a feasible strategy for precise treatment of molecular aberrations in stem and progenitor cell populations to correct skeletal manifestations of systemic disease.

  View details for DOI 10.1126/scitranslmed.aag2809

  View details for PubMedID 28077677

• Pharmacological rescue of diabetic skeletal stem cell niches SCIENCE TRANSLATIONAL MEDICINE Tevlin, R., Seo, E., Marecic, O., McArdle, A., Tong, X., Zimdahl, B., Malkovskiy, A., Sinha, R., Gulati, G., Li, X., Wearda, T., Morganti, R., Lopez, M., Ransom, R. C., Duldulao, C. R., Rodrigues, M., Nguyen, A., Januszyk, M., Maan, Z., Paik, K., Yapa, K., Rajadas, J., Wan, D. C., Gurtner, G. C., Snyder, M., Beachy, P. A., Yang, F., Goodman, S. B., Weissman, I. L., Chan, C. F., Longaker, M. T. 2017; 9 (372)

  View details for DOI 10.1126/scitranslmed.aag2809

  View details for Web of Science ID 000394445700005

• Hyaluronic acid synthesis is required for zebrafish tail fin regeneration. PloS one Ouyang, X., Panetta, N. J., Talbott, M. D., Payumo, A. Y., Halluin, C., Longaker, M. T., Chen, J. K. 2017; 12 (2)

  Abstract

  Using genome-wide transcriptional profiling and whole-mount expression analyses of zebrafish larvae, we have identified hyaluronan synthase 3 (has3) as an upregulated gene during caudal fin regeneration. has3 expression is induced in the wound epithelium within hours after tail amputation, and its onset and maintenance requires fibroblast growth factor, phosphoinositide 3-kinase, and transforming growth factor-ß signaling. Inhibition of hyaluronic acid (HA) synthesis by the small molecule 4-methylumbelliferone (4-MU) impairs tail regeneration in zebrafish larvae by preventing injury-induced cell proliferation. In addition, 4-MU reduces the expression of genes associated with wound epithelium and blastema function. Treatment with glycogen synthase kinase 3 inhibitors rescues 4-MU-induced defects in cell proliferation and tail regeneration, while restoring a subset of wound epithelium and blastema markers. Our findings demonstrate a role for HA biosynthesis in zebrafish tail regeneration and delineate its epistatic relationships with other regenerative processes.

  View details for DOI 10.1371/journal.pone.0171898

  View details for PubMedID 28207787

  View details for PubMedCentralID PMC5313160

• The Role of Skeletal Stem Cells in the Reconstruction of Bone Defects. The Journal of craniofacial surgery Murphy, M. P., Irizarry, D. n., Lopez, M. n., Moore, A. L., Ransom, R. C., Longaker, M. T., Wan, D. C., Chan, C. K. 2017; 28 (5): 1136–41

  Abstract

  Craniofacial surgery, since its inauguration, has been the culmination of collaborative efforts to solve complex congenital, dysplastic, oncological, and traumatic cranial bone defects. Now, 50 years on from the first craniofacial meeting, the collaborative efforts between surgeons, scientists, and bioengineers are further advancing craniofacial surgery with new discoveries in tissue regeneration. Recent advances in regenerative medicine and stem cell biology have transformed the authors' understanding of bone healing, the role of stem cells governing bone healing, and the effects of the niche environment and extracellular matrix on stem cell fate. This review aims at summarizing the advances within each of these fields.

  View details for PubMedID 28665863

• Human Adipose-Derived Stromal Cell Isolation Methods and Use in Osteogenic and Adipogenic In Vivo Applications. Current protocols in stem cell biology Brett, E., Tevlin, R., McArdle, A., Seo, E. Y., Chan, C. K., Wan, D. C., Longaker, M. T. 2017; 43

  Abstract

  Adipose tissue represents an abundant and easily accessible source of multipotent cells, which may serve as excellent building blocks for tissue engineering. This article presents a newly described protocol for isolating adipose-derived stromal cells (ASCs) from human lipoaspirate, compared to the standard protocol for harvesting ASCs established in 2001. Human ASC isolation is performed using two methods, and resultant cells are compared through cell yield, cell viability, cell proliferation and regenerative potential. The osteogenic and adipogenic potential of ASCs isolated using both protocols are assessed invitro and gene expression analysis is performed. The focus of this series of protocols is the regenerative potential of both cell populations in vivo. As such, the two in vivo animal models described are fat graft retention (soft tissue reconstruction) and calvarial defect healing (bone regeneration). The techniques described comprise fat grafting with cell assisted lipotransfer, and calvarial defect creation healed with cell-seeded scaffolds. © 2017 by John Wiley & Sons, Inc.

  View details for PubMedID 29140567

• Isolation of Live Fibroblasts by Fluorescence-Activated Cell Sorting FIBROSIS: METHODS AND PROTOCOLS Leavitt, T., Hu, M. S., Longaker, M. T., Rittie, L. 2017; 1627: 205–12

  Abstract

  Flow cytometry is a powerful tool in cell biology in that it allows real-time characterization of cellular phenotypes. Additionally, through the process of fluorescence-activated cell sorting (FACS), living cells can be isolated for future in vitro experiments, including single cell analysis. Here we describe the isolation of live fibroblasts from the dermis of the skin in mice using FACS. This method circumvents the need for ex vivo expansion in cell culture, which can alter phenotypic and functional characteristics of cells.

  View details for PubMedID 28836203

• Delivery of monocyte lineage cells in a biomimetic scaffold enhances tissue repair. JCI insight Hu, M. S., Walmsley, G. G., Barnes, L. A., Weiskopf, K. n., Rennert, R. C., Duscher, D. n., Januszyk, M. n., Maan, Z. N., Hong, W. X., Cheung, A. T., Leavitt, T. n., Marshall, C. D., Ransom, R. C., Malhotra, S. n., Moore, A. L., Rajadas, J. n., Lorenz, H. P., Weissman, I. L., Gurtner, G. C., Longaker, M. T. 2017; 2 (19)

  Abstract

  The monocyte lineage is essential to normal wound healing. Macrophage inhibition or knockout in mice results in impaired wound healing through reduced neovascularization, granulation tissue formation, and reepithelialization. Numerous studies have either depleted macrophages or reduced their activity in the context of wound healing. Here, we demonstrate that by increasing the number of macrophages or monocytes in the wound site above physiologic levels via pullulan-collagen composite dermal hydrogel scaffold delivery, the rate of wound healing can be significantly accelerated in both wild-type and diabetic mice, with no adverse effect on the quality of repair. Macrophages transplanted onto wounds differentiate into M1 and M2 phenotypes of different proportions at various time points, ultimately increasing angiogenesis. Given that monocytes can be readily isolated from peripheral blood without in vitro manipulation, these findings hold promise for translational medicine aimed at accelerating wound healing across a broad spectrum of diseases.

  View details for PubMedID 28978794

• Magnetic Nanoparticle-Based Upregulation of B-Cell Lymphoma 2 Enhances Bone Regeneration. Stem cells translational medicine Brett, E., Zielins, E. R., Luan, A., Ooi, C. C., Shailendra, S., Atashroo, D., Menon, S., Blackshear, C., Flacco, J., Quarto, N., Wang, S. X., Longaker, M. T., Wan, D. C. 2017; 6 (1): 151-160

  Abstract

  Clinical translation of cell-based strategies for tissue regeneration remains challenging because survival of implanted cells within hostile, hypoxic wound environments is uncertain. Overexpression of B-cell lymphoma 2 (Bcl-2) has been shown to inhibit apoptosis in implanted cells. The present study describes an "off the shelf" prefabricated scaffold integrated with magnetic nanoparticles (MNPs) used to upregulate Bcl-2 expression in implanted adipose-derived stromal cells for bone regeneration. Iron oxide cores were sequentially coated with branched polyethyleneimine, minicircle plasmid encoding green fluorescent protein and Bcl-2, and poly-β-amino ester. Through in vitro assays, increased osteogenic potential and biological resilience were demonstrated in the magnetofected group over control and nucleofected groups. Similarly, our in vivo calvarial defect study showed that magnetofection had an efficiency rate of 30%, which in turn resulted in significantly more healing compared with control group and nucleofected group. Our novel, prefabricated MNP-integrated scaffold allows for in situ postimplant temporospatial control of cell transfection to augment bone regeneration. Stem Cells Translational Medicine 2017;6:151-160.

  View details for DOI 10.5966/sctm.2016-0051

  View details for PubMedID 28170185

• Biomimetics of Bone Implants: The Regenerative Road. BioResearch open access Brett, E., Flacco, J., Blackshear, C., Longaker, M. T., Wan, D. C. 2017; 6 (1): 1-6

  Abstract

  The current strategies for healing bone defects are numerous and varied. At the core of each bone healing therapy is a biomimetic mechanism, which works to enhance bone growth. These range from porous scaffolds, bone mineral usage, collagen, and glycosaminoglycan substitutes to transplanted cell populations. Bone defects face a range of difficulty in their healing, given the composite of dense outer compact bone and blood-rich inner trabecular bone. As such, the tissue possesses a number of inherent characteristics, which may be clinically harnessed as promoters of bone healing. These include mechanical characteristics, mineral composition, native collagen content, and cellular fraction of bone. This review charts multiple biomimetic strategies to help heal bony defects in large and small osseous injury sites, with a special focus on cell transplantation.

  View details for DOI 10.1089/biores.2016.0044

  View details for PubMedID 28163982

• Commentary on: Adipose Stem Cell Function Maintained with Age: An Intra-Subject Study of Long-Term Cryopreserved Cells. Aesthetic surgery journal Irizarry, D., Longaker, M. T., Wan, D. C. 2016

  View details for DOI 10.1093/asj/sjw224

  View details for PubMedID 28039122

• Lectins bring benefits to bones ELIFE Chan, C. F., Ransom, R. C., Longaker, M. T. 2016; 5

  Abstract

  The discovery that proteins called c-type lectins promote bone growth could lead to new treatments for age-related bone disorders.

  View details for PubMedID 27960074

• A Novel Method of Human Adipose-Derived Stem Cell Isolation with Resultant Increased Cell Yield PLASTIC AND RECONSTRUCTIVE SURGERY Tevlin, R., McArdle, A., Brett, E., Chung, M. T., Paik, K., Seo, E. Y., Walmsley, G. G., Duldulao, C. R., Atashroo, D., Zielins, E., Vistnes, S., Chan, C. K., Wan, D. C., Longaker, M. T. 2016; 138 (6): 983E-996E

  Abstract

  The authors have developed a novel protocol for isolating adipose-derived stem cells from human lipoaspirate. In this study, they compare their new method to a previously published standard protocol.Human adipose-derived stem cell isolation was performed using two methods to compare cell yield, cell viability, cell proliferation, and regenerative potential. The new and conventional isolation methods differ in two key areas: the collagenase digestion buffer constituents and the use of an orbital shaker. The osteogenic and adipogenic potential of adipose-derived stem cells isolated using both protocols was assessed in vitro, and gene expression analysis was performed. To assess the ability of the isolated cells to generate bone in vivo, the authors created critical-size calvarial defects in mice, which were treated with adipose-derived stem cells loaded onto hydroxyapatite-coated poly(lactic-co-glycolic acid) scaffolds. To test the ability of the isolated cells to enhance adipogenesis, the cells were added to lipoaspirate and placed beneath the scalp of immunocompromised mice. Fat graft volume retention was subsequently assessed by serial computed tomographic volumetric scanning.The new method resulted in a 10-fold increased yield of adipose-derived stem cells compared with the conventional method. Cells harvested using the new method demonstrated significantly increased cell viability and proliferation in vitro (p < 0.05). New method cells also demonstrated significantly enhanced osteogenic and adipogenic differentiation capacity in vitro (p < 0.05) in comparison with the conventional method cells. Both cell groups demonstrated equivalent osteogenic and adipogenic regenerative potential in mice.The authors have developed a protocol that maximizes the yield of adipose-derived stem cells derived from lipoaspirate. The new method cells have increased osteogenic and adipogenic potential in vitro and are not inferior to conventional method cells in terms of their ability to generate bone and fat in vivo.Therapeutic, V.

  View details for DOI 10.1097/PRS.0000000000002790

  View details for PubMedID 27537222

• Commentary on: The Effects of Fat Harvesting and Preparation, Air Exposure, Obesity, and Stem Cell Enrichment on Adipocyte Viability Prior to Graft Transplantation. Aesthetic surgery journal Blackshear, C. P., Longaker, M. T., Wan, D. C. 2016; 36 (10): 1174-1175

  View details for PubMedID 27474768

• Dipeptidyl Peptidase-4, Wound Healing, Scarring, and Fibrosis. Plastic and reconstructive surgery Hu, M. S., Longaker, M. T. 2016; 138 (5): 1026-1031

  Abstract

  Scarring and fibrosis are an enormous public health concern, resulting in excessive morbidity and mortality in addition to countless lost health care dollars. Recent advances in cell and developmental biology promise a better understanding of scarring and fibrosis and may translate to new clinical therapies.

  View details for PubMedID 27782998

• Stem Cells in Bone Regeneration STEM CELL REVIEWS AND REPORTS Walmsley, G. G., Ransom, R. C., Zielins, E. R., Leavitt, T., Flacco, J. S., Hu, M. S., Lee, A. S., Longaker, M. T., Wan, D. C. 2016; 12 (5): 524-529

  Abstract

  Bone has the capacity to regenerate and repair itself. However, this capacity may be impaired or lost depending on the size of the defect or the presence of certain disease states. In this review, we discuss the key principles underlying bone healing, efforts to characterize bone stem and progenitor cell populations, and the current status of translational and clinical studies in cell-based bone tissue engineering. Though barriers to clinical implementation still exist, the application of stem and progenitor cell populations to bone engineering strategies has the potential to profoundly impact regenerative medicine.

  View details for DOI 10.1007/s12015-016-9665-5

  View details for Web of Science ID 000385138500003

  View details for PubMedID 27250635

  View details for PubMedCentralID PMC5053855

• Intestinal Smooth Muscle Cell Migration May Contribute to Abdominal Adhesion Formation Marshall, C. D., Hu, M. S., Leavitt, T., Ransom, R. C., Barnes, L. A., Lorenz, H., Longaker, M. T. ELSEVIER SCIENCE INC. 2016: E106–E107

  View details for DOI 10.1016/j.jamcollsurg.2016.08.269

  View details for Web of Science ID 000395825100229

• Inhibition of Unfolded Protein Response Decreases Scar Formation Boyko, T. V., Jiang, D., Cao, H., Koong, A. C., Longaker, M. T., Yang, G. P. ELSEVIER SCIENCE INC. 2016: S98

  View details for DOI 10.1016/j.jamcollsurg.2016.06.198

  View details for Web of Science ID 000393077500168

• Scarless wound healing: finding the right cells and signals. Cell and tissue research Leavitt, T., Hu, M. S., Marshall, C. D., Barnes, L. A., Lorenz, H. P., Longaker, M. T. 2016; 365 (3): 483-493

  Abstract

  From the moment we are born, every injury to the skin has the potential to form a scar, many of which can impair form and/or function. As such, scar management constitutes a billion-dollar industry. However, effectively promoting scarless wound healing remains an elusive goal. The complex interactions of wound healing contribute to our inability to recapitulate scarless wound repair as it occurs in nature, such as in fetal skin and the oral mucosa. However, many new advances have occurred in recent years, some of which have translated scientific findings from bench to bedside. In vivo lineage tracing has helped establish a variety of novel cellular culprits that may act as key drivers of the fibrotic response. These newly characterized cell populations present further targets for therapeutic intervention, some of which have previously demonstrated promising results in animal models. Here, we discuss several recent studies that identify exciting approaches for diminishing scar formation. Particular attention will also be paid to the canonical Wnt/β-catenin signaling pathway, which plays an important role in both embryogenesis and tissue repair. New insights into the differential effects of Wnt signaling on heterogeneous fibroblast and keratinocyte populations within the skin further demonstrate methods by which wound healing can be re-directed to a more fetal scarless phenotype. Graphical abstract Recent approaches to reducing scar formation. Representation showing novel scientific approaches for decreasing scar formation, including the targeting of pro-fibrotic cell populations based on surface molecule expression (e.g. DPP4(+) fibroblasts, ADAM12(+) pericytes). Modulation of cellular mechanotransduction pathways are another means to reduce scar formation, both at the molecular level or, macroscopically with dressings designed to offload tension, at cutaneous wound sites (ADAM12 a disintegrin and metalloprotease 12, DPP4 dipeptidyl peptidase-4, FAK focal adhesion kinase).

  View details for DOI 10.1007/s00441-016-2424-8

  View details for PubMedID 27256396

  View details for PubMedCentralID PMC5010960

• Mechanical Stimulation Increases Knee Meniscus Gene RNA-level Expression in Adipose-derived Stromal Cells PLASTIC AND RECONSTRUCTIVE SURGERY-GLOBAL OPEN Meier, E. M., Wu, B., Siddiqui, A., Tepper, D. G., Longaker, M. T., Lam, M. T. 2016; 4 (9): e864

  Abstract

  Efforts have been made to engineer knee meniscus tissue for injury repair, yet most attempts have been unsuccessful. Creating a cell source that resembles the complex, heterogeneous phenotype of the meniscus cell remains difficult. Stem cell differentiation has been investigated, mainly using bone marrow mesenchymal cells and biochemical means for differentiation, resulting in no solution. Mechanical stimulation has been investigated to an extent with no conclusion. Here, we explore the potential for and effectiveness of mechanical stimulation to induce the meniscal phenotype in adipose-derived stromal cells.Human adipose-derived stromal cells were chosen for their fibrogenic nature and conduciveness for chondrogenesis. Biochemical and mechanical stimulation were investigated. Biochemical stimulation included fibrogenic and chondrogenic media. For mechanical stimulation, a custom-built device was used to apply constant, cyclical, uniaxial strain for up to 6 hours. Strain and frequency varied.Under biochemical stimulation, both fibrogenic (collagen I, versican) and chondrogenic (collagen II, Sox9, aggrecan) genes were expressed by cells exposed to either fibrogenic or chondrogenic biochemical factors. Mechanical strain was found to preferentially promote fibrogenesis over chondrogenesis, confirming that tensile strain is an effective fibrogenic cue. Three hours at 10% strain and 1 Hz in chondrogenic media resulted in the highest expression of fibrochondrogenic genes. Although mechanical stimulation did not seem to affect protein level expression, biochemical means did affect protein level presence of collagen fibers.Mechanical stimulation can be a useful differentiation tool for mechanoresponsive cell types as long as biochemical factors are also integrated.

  View details for PubMedID 27757329

• Magnetic Nanoparticle-Based Upregulation of B-Cell Lymphoma 2 Enhances Bone Regeneration. Stem cells translational medicine Brett, E., Zielins, E. R., Luan, A., Ooi, C. C., Shailendra, S., Atashroo, D., Menon, S., Blackshear, C., Flacco, J., Quarto, N., Wang, S. X., Longaker, M. T., Wan, D. C. 2016

  Abstract

  : Clinical translation of cell-based strategies for tissue regeneration remains challenging because survival of implanted cells within hostile, hypoxic wound environments is uncertain. Overexpression of B-cell lymphoma 2 (Bcl-2) has been shown to inhibit apoptosis in implanted cells. The present study describes an "off the shelf" prefabricated scaffold integrated with magnetic nanoparticles (MNPs) used to upregulate Bcl-2 expression in implanted adipose-derived stromal cells for bone regeneration. Iron oxide cores were sequentially coated with branched polyethyleneimine, minicircle plasmid encoding green fluorescent protein and Bcl-2, and poly-β-amino ester. Through in vitro assays, increased osteogenic potential and biological resilience were demonstrated in the magnetofected group over control and nucleofected groups. Similarly, our in vivo calvarial defect study showed that magnetofection had an efficiency rate of 30%, which in turn resulted in significantly more healing compared with control group and nucleofected group. Our novel, prefabricated MNP-integrated scaffold allows for in situ postimplant temporospatial control of cell transfection to augment bone regeneration.The use of adipose-derived stem cells as transplanted cells in wounded areas is desirable for their regenerative potential, but they are difficult to use owing to their fragility. Enhancing their survival in the context of a calvarial defect can be achieved by stimulating antiapoptotic protein expression in the cells themselves, through a plasmid expression vector. The present study used a nonintegrating minicircle plasmid encoding B-cell lymphoma 2 attached to a magnetic nanoparticle to facilitate in vivo transfection with temporospatial control (external magnetic field). This in situ system stimulates cell survival through gene expression and knock-on bone regeneration through cell survival.

  View details for PubMedID 27484867

• Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Keeney, M., Chung, M. T., Zielins, E. R., Paik, K. J., McArdle, A., Morrison, S. D., Ransom, R. C., Barbhaiya, N., Atashroo, D., Jacobson, G., Zare, R. N., Longaker, M. T., Wan, D. C., Yang, F. 2016; 104 (8): 2099-2107

  Abstract

  Scaffold-mediated gene delivery holds great promise for tissue regeneration. However, previous attempts to induce bone regeneration using scaffold-mediated non-viral gene delivery rarely resulted in satisfactory healing. We report a novel platform with sustained release of minicircle DNA (MC) from PLGA scaffolds to accelerate bone repair. MC was encapsulated inside PLGA scaffolds using supercritical CO2 , which showed prolonged release of MC. Skull-derived osteoblasts transfected with BMP-2 MC in vitro result in higher osteocalcin gene expression and mineralized bone formation. When implanted in a critical-size mouse calvarial defect, scaffolds containing luciferase MC lead to robust in situ protein production up to at least 60 days. Scaffold-mediated BMP-2 MC delivery leads to substantially accelerated bone repair as early as two weeks, which continues to progress over 12 weeks. This platform represents an efficient, long-term nonviral gene delivery system, and may be applicable for enhancing repair of a broad range of tissues types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2099-2107, 2016.

  View details for DOI 10.1002/jbm.a.35735

  View details for Web of Science ID 000379736500025

  View details for PubMedID 27059085

• Creation of Abdominal Adhesions in Mice JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Marshall, C. D., Hu, M. S., Leavitt, T., Barnes, L. A., Cheung, A. T., Malhotra, S., Lorenz, H. P., Longaker, M. T. 2016

  Abstract

  Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

  View details for DOI 10.3791/54450

  View details for Web of Science ID 000391742700091

  View details for PubMedID 27685681

• Winner of the Young Investigator Award of the Society for Biomaterials at the 10th World Biomaterials Congress, May 17-22, 2016, Montreal QC, Canada: Microribbon-based hydrogels accelerate stem cell-based bone regeneration in a mouse critical-size cranial defect model JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Han, L., Conrad, B., Chung, M. T., Deveza, L., Jiang, X., Wang, A., Butte, M. J., Longaker, M. T., Wan, D., Yang, F. 2016; 104 (6): 1321-1331

  Abstract

  Stem cell-based therapies hold great promise for enhancing tissue regeneration. However, the majority of cells die shortly after transplantation, which greatly diminishes the efficacy of stem cell-based therapies. Poor cell engraftment and survival remain a major bottleneck to fully exploiting the power of stem cells for regenerative medicine. Biomaterials such as hydrogels can serve as artificial matrices to protect cells during delivery and guide desirable cell fates. However, conventional hydrogels often lack macroporosity, which restricts cell proliferation and delays matrix deposition. Here we report the use of injectable, macroporous microribbon (μRB) hydrogels as stem cell carriers for bone repair, which supports direct cell encapsulation into a macroporous scaffold with rapid spreading. When transplanted in a critical-sized, mouse cranial defect model, μRB-based hydrogels significantly enhanced the survival of transplanted adipose-derived stromal cells (ADSCs) (81%) and enabled up to three-fold cell proliferation after 7 days. In contrast, conventional hydrogels only led to 27% cell survival, which continued to decrease over time. MicroCT imaging showed μRBs enhanced and accelerated mineralized bone repair compared to hydrogels (61% vs. 34% by week 6), and stem cells were required for bone repair to occur. These results suggest that paracrine signaling of transplanted stem cells are responsible for the observed bone repair, and enhancing cell survival and proliferation using μRBs further promoted the paracrine-signaling effects of ADSCs for stimulating endogenous bone repair. We envision μRB-based scaffolds can be broadly useful as a novel scaffold for enhancing stem cell survival and regeneration of other tissue types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1321-1331, 2016.

  View details for DOI 10.1002/jbm.a.35715

  View details for Web of Science ID 000375117200001

  View details for PubMedID 26991141

  View details for PubMedCentralID PMC5142823

• Expansion and Hepatic Differentiation of Adult Blood-Derived CD34(+) Progenitor Cells and Promotion of Liver Regeneration After Acute Injury STEM CELLS TRANSLATIONAL MEDICINE Hu, M., Li, S., Menon, S., Liu, B., Hu, M. S., Longaker, M. T., Lorenz, H. P. 2016; 5 (6): 723-732

  Abstract

  The low availability of functional hepatocytes has been an unmet demand for basic scientific research, new drug development, and cell-based clinical applications for decades. Because of the inability to expand hepatocytes in vitro, alternative sources of hepatocytes are a focus of liver regenerative medicine. We report a new group of blood-derived CD34(+) progenitor cells (BDPCs) that have the ability to expand and differentiate into functional hepatocyte-like cells and promote liver regeneration. BDPCs were obtained from the peripheral blood of an adult mouse with expression of surface markers CD34, CD45, Sca-1, c-kit, and Thy1.1. BDPCs can proliferate in vitro and differentiate into hepatocyte-like cells expressing hepatocyte markers, including CK8, CK18, CK19, α-fetoprotein, integrin-β1, and A6. The differentiated BDPCs (dBDPCs) also display liver-specific functional activities, such as glycogen storage, urea production, and albumin secretion. dBDPCs have cytochrome P450 activity and express specific hepatic transcription factors, such as hepatic nuclear factor 1α. To demonstrate liver regenerative activity, dBDPCs were injected into mice with severe acute liver damage caused by a high-dose injection of carbon tetrachloride (CCl4). dBDPC treatment rescued the mice from severe acute liver injury, increased survival, and induced liver regeneration. Because of their ease of access and application through peripheral blood and their capability of rapid expansion and hepatic differentiation, BDPCs have great potential as a cell-based therapy for liver disease.Hematopoietic stem/progenitor cell expansion and tissue-specific differentiation in vitro are challenges in regenerative medicine, although stem cell therapy has raised hope for the treatment of liver diseases by overcoming the scarcity of hepatocytes. This study identified and characterized a group of blood-derived progenitor cells (BDPCs) from the peripheral blood of an adult mouse. The CD34(+) progenitor-dominant BDPCs were rapidly expanded and hepatically differentiated into functional hepatocyte-like cells with our established coculture system. BDPC treatment increased animal survival and produced full regeneration in a severe liver injury mouse model caused by CCl4. BDPCs could have potential for liver cell therapies.

  View details for DOI 10.5966/sctm.2015-0268

  View details for PubMedID 27075766

  View details for PubMedCentralID PMC4878335

• Suction assisted liposuction does not impair the regenerative potential of adipose derived stem cells JOURNAL OF TRANSLATIONAL MEDICINE Duscher, D., Luan, A., Rennert, R. C., Atashroo, D., Maan, Z. N., Brett, E. A., Whittam, A. J., Ho, N., Lin, M., Hu, M. S., Walmsley, G. G., Wenny, R., Schmidt, M., Schilling, A. F., Machens, H., Huemer, G. M., Wan, D. C., Longaker, M. T., Gurtner, G. C. 2016; 14

  Abstract

  Adipose-derived stem cells (ASCs) have been identified as a population of multipotent cells with promising applications in tissue engineering and regenerative medicine. ASCs are abundant in fat tissue, which can be safely harvested through the minimally invasive procedure of liposuction. However, there exist a variety of different harvesting methods, with unclear impact on ASC regenerative potential. The aim of this study was thus to compare the functionality of ASCs derived from the common technique of suction-assisted lipoaspiration (SAL) versus resection.Human adipose tissue was obtained from paired abdominoplasty and SAL samples from three female donors, and was processed to isolate the stromal vascular fraction. Fluorescence-activated cell sorting was used to determine ASC yield, and cell viability was assayed. Adipogenic and osteogenic differentiation capacity were assessed in vitro using phenotypic staining and quantification of gene expression. Finally, ASCs were applied in an in vivo model of tissue repair to evaluate their regenerative potential.SAL specimens provided significantly fewer ASCs when compared to excised fat tissue, however, with equivalent viability. SAL-derived ASCs demonstrated greater expression of the adipogenic markers FABP-4 and LPL, although this did not result in a difference in adipogenic differentiation. There were no differences detected in osteogenic differentiation capacity as measured by alkaline phosphatase, mineralization or osteogenic gene expression. Both SAL- and resection-derived ASCs enhanced significantly cutaneous healing and vascularization in vivo, with no significant difference between the two groups.SAL provides viable ASCs with full capacity for multi-lineage differentiation and tissue regeneration, and is an effective method of obtaining ASCs for cell-based therapies.

  View details for DOI 10.1186/s12967-016-0881-1

  View details for Web of Science ID 000375475200004

  View details for PubMedID 27153799

  View details for PubMedCentralID PMC4859988

• Small Molecule Inhibition of Transforming Growth Factor Beta Signaling Enables the Endogenous Regenerative Potential of the Mammalian Calvarium TISSUE ENGINEERING PART A Senarath-Yapa, K., Li, S., Walmsley, G. G., Zielins, E., Paik, K., Britto, J. A., Grigoriadis, A. E., Wan, D. C., Liu, K. J., Longaker, M. T., Quarto, N. 2016; 22 (9-10): 707-720

  Abstract

  Current approaches for the treatment of skeletal defects are suboptimal, principally because the ability of bone to repair and regenerate is poor. Although the promise of effective cellular therapies for skeletal repair is encouraging, these approaches are limited by the risks of infection, cellular contamination, and tumorigenicity. Development of a pharmacological approach would therefore help avoid some of these potential risks. This study identifies transforming growth factor beta (TGFβ) signaling as a potential pathway for pharmacological modulation in vivo. We demonstrate that inhibition of TGFβ signaling by the small molecule SB431542 potentiates calvarial skeletal repair through activation of bone morphogenetic protein (BMP) signaling on osteoblasts and dura mater cells participating in healing of calvarial defects. Cells respond to inhibition of TGFβ signaling by producing higher levels of BMP2 that upregulates inhibitory Smad6 expression, thus providing a negative feedback loop to contain excessive BMP signaling. Importantly, study on human osteoblasts indicates that molecular mechanism(s) triggered by SB431542 are conserved. Collectively, these data provide insights into the use of small molecules to modulate key signaling pathways for repairing skeletal defects.

  View details for DOI 10.1089/ten.tea.2015.0527

  View details for Web of Science ID 000377380600001

  View details for PubMedID 27036931

  View details for PubMedCentralID PMC4876548

• Stem and progenitor cells: advancing bone tissue engineering. Drug delivery and translational research Tevlin, R., Walmsley, G. G., Marecic, O., Hu, M. S., Wan, D. C., Longaker, M. T. 2016; 6 (2): 159-173

  Abstract

  Unlike many other postnatal tissues, bone can regenerate and repair itself; nevertheless, this capacity can be overcome. Traditionally, surgical reconstructive strategies have implemented autologous, allogeneic, and prosthetic materials. Autologous bone-the best option-is limited in supply and also mandates an additional surgical procedure. In regenerative tissue engineering, there are myriad issues to consider in the creation of a functional, implantable replacement tissue. Importantly, there must exist an easily accessible, abundant cell source with the capacity to express the phenotype of the desired tissue, and a biocompatible scaffold to deliver the cells to the damaged region. A literature review was performed using PubMed; peer-reviewed publications were screened for relevance in order to identify key advances in stem and progenitor cell contribution to the field of bone tissue engineering. In this review, we briefly introduce various adult stem cells implemented in bone tissue engineering such as mesenchymal stem cells (including bone marrow- and adipose-derived stem cells), endothelial progenitor cells, and induced pluripotent stem cells. We then discuss numerous advances associated with their application and subsequently focus on technological advances in the field, before addressing key regenerative strategies currently used in clinical practice. Stem and progenitor cell implementation in bone tissue engineering strategies have the ability to make a major impact on regenerative medicine and reduce patient morbidity. As the field of regenerative medicine endeavors to harness the body's own cells for treatment, scientific innovation has led to great advances in stem cell-based therapies in the past decade.

  View details for DOI 10.1007/s13346-015-0235-1

  View details for PubMedID 25990836

  View details for PubMedCentralID PMC4654714

• Discussion: In Vitro Validation of a Closed Device Enabling the Purification of the Fluid Portion of Liposuction Aspirates. Plastic and reconstructive surgery Zielins, E. R., Longaker, M. T., Wan, D. C. 2016; 137 (4): 1168-1170

  View details for DOI 10.1097/PRS.0000000000002035

  View details for PubMedID 27018671

• Autologous Fat Grafting: The Science Behind the Surgery. Aesthetic surgery journal Zielins, E. R., Brett, E. A., Longaker, M. T., Wan, D. C. 2016; 36 (4): 488-496

  Abstract

  An invaluable part of the plastic surgeon's technical arsenal for soft tissue contouring, fat grafting continues to be plagued by unpredictable outcomes, resulting in either reoperation and/or patient dissatisfaction. Thus, extensive research has been conducted into the effects of adipose tissue procurement, processing, and placement on fat graft quality at both the cellular level and in terms of overall volume retention. Herein, we present an overview of the vast body of literature in these areas, with additional discussion of cell-assisted lipotransfer as a therapy to improve volume retention, and on the controversial use of autologous fat in the setting of prior irradiation.

  View details for DOI 10.1093/asj/sjw004

  View details for PubMedID 26961989

• Cell-Assisted Lipotransfer Improves Volume Retention in Irradiated Recipient Sites and Rescues Radiation-Induced Skin Changes STEM CELLS Luan, A., Duscher, D., Whittam, A. J., Paik, K. J., Zielins, E. R., Brett, E. A., Atashroo, D. A., Hu, M. S., Lee, G. K., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2016; 34 (3): 668-673

  Abstract

  Radiation therapy is not only a mainstay in the treatment of many malignancies but also results in collateral obliteration of microvasculature and dermal/subcutaneous fibrosis. Soft tissue reconstruction of hypovascular, irradiated recipient sites through fat grafting remains challenging; however, a coincident improvement in surrounding skin quality has been noted. Cell-assisted lipotransfer (CAL), the enrichment of fat with additional adipose-derived stem cells (ASCs) from the stromal vascular fraction, has been shown to improve fat volume retention, and enhanced outcomes may also be achieved with CAL at irradiated sites. Supplementing fat grafts with additional ASCs may also augment the regenerative effect on radiation-damaged skin. In this study, we demonstrate the ability for CAL to enhance fat graft volume retention when placed beneath the irradiated scalps of immunocompromised mice. Histologic metrics of fat graft survival were also appreciated, with improved structural qualities and vascularity. Finally, rehabilitation of radiation-induced soft tissue changes were also noted, as enhanced amelioration of dermal thickness, collagen content, skin vascularity, and biomechanical measures were all observed with CAL compared to unsupplemented fat grafts. Supplementation of fat grafts with ASCs therefore shows promise for reconstruction of complex soft tissue defects following adjuvant radiotherapy. Stem Cells 2016;34:668-673.

  View details for DOI 10.1002/stem.2256

  View details for Web of Science ID 000372552600013

• Cell-Assisted Lipotransfer Improves Volume Retention in Irradiated Recipient Sites and Rescues Radiation-Induced Skin Changes. Stem cells Luan, A., Duscher, D., Whittam, A. J., Paik, K. J., Zielins, E. R., Brett, E. A., Atashroo, D. A., Hu, M. S., Lee, G. K., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2016; 34 (3): 668-673

  Abstract

  Radiation therapy is not only a mainstay in the treatment of many malignancies but also results in collateral obliteration of microvasculature and dermal/subcutaneous fibrosis. Soft tissue reconstruction of hypovascular, irradiated recipient sites through fat grafting remains challenging; however, a coincident improvement in surrounding skin quality has been noted. Cell-assisted lipotransfer (CAL), the enrichment of fat with additional adipose-derived stem cells (ASCs) from the stromal vascular fraction, has been shown to improve fat volume retention, and enhanced outcomes may also be achieved with CAL at irradiated sites. Supplementing fat grafts with additional ASCs may also augment the regenerative effect on radiation-damaged skin. In this study, we demonstrate the ability for CAL to enhance fat graft volume retention when placed beneath the irradiated scalps of immunocompromised mice. Histologic metrics of fat graft survival were also appreciated, with improved structural qualities and vascularity. Finally, rehabilitation of radiation-induced soft tissue changes were also noted, as enhanced amelioration of dermal thickness, collagen content, skin vascularity, and biomechanical measures were all observed with CAL compared to unsupplemented fat grafts. Supplementation of fat grafts with ASCs therefore shows promise for reconstruction of complex soft tissue defects following adjuvant radiotherapy. Stem Cells 2016;34:668-673.

  View details for DOI 10.1002/stem.2256

  View details for PubMedID 26661694

• Enrichment of Adipose-Derived Stromal Cells for BMPR1A Facilitates Enhanced Adipogenesis TISSUE ENGINEERING PART A Zielins, E. R., Paik, K., Ransom, R. C., Brett, E. A., Blackshear, C. P., Luan, A., Walmsley, G. G., Atashroo, D. A., Senarath-Yapa, K., Momeni, A., Rennert, R., Sorkin, M., Seo, E. Y., Chan, C. K., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2016; 22 (3-4): 214-221

  Abstract

  Reconstruction of soft tissue defects has traditionally relied on the use of grafts and flaps, which may be associated with variable resorption and/or significant donor site morbidity. Cell-based strategies employing adipose-derived stromal cells (ASCs), found within the stromal vascular fraction (SVF) of adipose tissue, may offer an alternative strategy for soft tissue reconstruction. In this study, we investigated the potential of a bone morphogenetic protein receptor type 1A (BMPR1A)(+) subpopulation of ASCs to enhance de novo adipogenesis.Human lipoaspirate was enzymatically digested to isolate SVF and magnetic-activated cell separation was utilized to obtain BMPR1A(+) and BMPR1A(-) cells. These cells, along with unenriched cells, were expanded in culture and evaluated for adipogenic gene expression and in vitro adipocyte formation. Cells from each group were also labeled with a green fluorescent protein (GFP) lentivirus and transplanted into the inguinal fat pads, an adipogenic niche, of immunocompromised mice to determine their potential for de novo adipogenesis. Confocal microscopy along with staining of lipid droplets and vasculature was performed to evaluate the formation of mature adipocytes by transplanted cells.In comparison to BMPR1A(-) and unenriched ASCs, BMPR1A(+) cells demonstrated significantly enhanced adipogenesis when cultured in an adipogenic differentiation medium, as evidenced by increased staining with Oil Red O and increased expression of peroxisome proliferator-activating receptor gamma (PPAR-γ) and fatty acid-binding protein 4 (FABP4). BMPR1A(+) cells also formed significantly more adipocytes in vivo, as demonstrated by quantification of GFP+ adipocytes. Minimal formation of mature adipocytes was appreciated by BMPR1A(-) cells.BMPR1A(+) ASCs show an enhanced ability for adipogenesis in vitro, as shown by gene expression and histological staining. Furthermore, within an adipogenic niche, BMPR1A(+) cells possessed an increased capacity to generate de novo fat compared to BMPR1A(-) and unenriched cells. This suggests utility for the BMPR1A(+) subpopulation in cell-based strategies for soft tissue reconstruction.

  View details for DOI 10.1089/ten.tea.2015.0278

  View details for PubMedID 26585335

• Discussion: Transplantation of an LGR6(+) Epithelial Stem Cell-Enriched Scaffold for Repair of Full-Thickness Soft-Tissue Defects: The In Vitro Development of Polarized Hair-Bearing Skin PLASTIC AND RECONSTRUCTIVE SURGERY Hu, M. S., Longaker, M. T., Lorenz, H. 2016; 137 (2): 508–9

  View details for PubMedID 26818285

• Ultrasound-Assisted Liposuction Does Not Compromise the Regenerative Potential of Adipose-Derived Stem Cells. Stem cells translational medicine Duscher, D., Atashroo, D., Maan, Z. N., Luan, A., Brett, E. A., Barrera, J., Khong, S. M., Zielins, E. R., Whittam, A. J., Hu, M. S., Walmsley, G. G., Pollhammer, M. S., Schmidt, M., Schilling, A. F., Machens, H., Huemer, G. M., Wan, D. C., Longaker, M. T., Gurtner, G. C. 2016; 5 (2): 248-257

  Abstract

  Human mesenchymal stem cells (MSCs) have recently become a focus of regenerative medicine, both for their multilineage differentiation capacity and their excretion of proregenerative cytokines. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest because of their abundance in fat tissue and the ease of harvest via liposuction. However, little is known about the impact of different liposuction methods on the functionality of ASCs. Here we evaluate the regenerative abilities of ASCs harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration (SAL). Lipoaspirates were sorted using fluorescent assisted cell sorting based on an established surface-marker profile (CD34+/CD31-/CD45-), to obtain viable ASCs. Yield and viability were compared and the differentiation capacities of the ASCs were assessed. Finally, the regenerative potential of ASCs was examined using an in vivo model of tissue regeneration. UAL- and SAL-derived samples demonstrated equivalent ASC yield and viability, and UAL ASCs were not impaired in their osteogenic, adipogenic, or chondrogenic differentiation capacity. Equally, quantitative real-time polymerase chain reaction showed comparable expression of most osteogenic, adipogenic, and key regenerative genes between both ASC groups. Cutaneous regeneration and neovascularization were significantly enhanced in mice treated with ASCs obtained by either UAL or SAL compared with controls, but there were no significant differences in healing between cell-therapy groups. We conclude that UAL is a successful method of obtaining fully functional ASCs for regenerative medicine purposes. Cells harvested with this alternative approach to liposuction are suitable for cell therapy and tissue engineering applications. Significance: Adipose-derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency, diverse cytokine profile, and ease of harvest via liposuction. Alternative approaches to classical suction-assisted liposuction are gaining popularity; however, little evidence exists regarding the impact of different liposuction methods on the regenerative functionality of ASCs. Human ASC characteristics and regenerative capacity were assessed when harvested via ultrasound-assisted (UAL) versus standard suction-assisted liposuction. ASCs obtained via UAL were of equal quality when directly compared with the current gold standard harvest method. UAL is an adjunctive source of fully functional mesenchymal stem cells for applications in basic research and clinical therapy.

  View details for DOI 10.5966/sctm.2015-0064

  View details for PubMedID 26702129

  View details for PubMedCentralID PMC4729547

• Murine Dermal Fibroblast Isolation by FACS. Journal of visualized experiments : JoVE Walmsley, G. G., Maan, Z. N., Hu, M. S., Atashroo, D. A., Whittam, A. J., Duscher, D., Tevlin, R., Marecic, O., Lorenz, H. P., Gurtner, G. C., Longaker, M. T. 2016

  Abstract

  Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin(-)) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

  View details for DOI 10.3791/53430

  View details for PubMedID 26780559

  View details for PubMedCentralID PMC4781205

• Murine Dermal Fibroblast Isolation by FACS JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Walmsley, G. G., Maan, Z. N., Hu, M. S., Atashroo, D. A., Whittam, A. J., Duscher, D., Tevlin, R., Marecic, O., Lorenz, H. P., Gurtner, G. C., Longaker, M. T. 2016

  Abstract

  Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin(-)) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

  View details for DOI 10.3791/53430

  View details for Web of Science ID 000368577400023

  View details for PubMedCentralID PMC4781205

• Stem cells and chronic wound healing: state of the art CHRONIC WOUND CARE MANAGEMENT AND RESEARCH Leavitt, T., Hu, M. S., Marshall, C. D., Barnes, L. A., Longaker, M. T., Lorenz, H. 2016; 3: 7–27

  View details for DOI 10.2147/CWCMR.S84369

  View details for Web of Science ID 000382881200001

• Surveillance of Stem Cell Fate and Function: A System for Assessing Cell Survival and Collagen Expression In Situ. Tissue engineering. Part A Walmsley, G. G., Senarath-Yapa, K., Wearda, T. L., Menon, S., Hu, M. S., Duscher, D., Maan, Z. N., Tsai, J. M., Zielins, E. R., Weissman, I. L., Gurtner, G. C., Lorenz, H. P., Longaker, M. T. 2016; 22 (1-2): 31-40

  Abstract

  Cell-based therapy is an emerging paradigm in skeletal regenerative medicine. However, the primary means by which transplanted cells contribute to bone repair and regeneration remain controversial. To gain an insight into the mechanisms of how both transplanted and endogenous cells mediate skeletal healing, we used a transgenic mouse strain expressing both the topaz variant of green fluorescent protein under the control of the collagen, type I, alpha 1 promoter/enhancer sequence (Col1a1(GFP)) and membrane-bound tomato red fluorescent protein constitutively in all cell types (R26(mTmG)). A comparison of healing in parietal versus frontal calvarial defects in these mice revealed that frontal osteoblasts express Col1a1 to a greater degree than parietal osteoblasts. Furthermore, the scaffold-based application of adipose-derived stromal cells (ASCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), and osteoblasts derived from these mice to critical-sized calvarial defects allowed for investigation of cell survival and function following transplantation. We found that ASCs led to significantly faster rates of bone healing in comparison to BM-MSCs and osteoblasts. ASCs displayed both increased survival and increased Col1a1 expression compared to BM-MSCs and osteoblasts following calvarial defect transplantation, which may explain their superior regenerative capacity in the context of bone healing. Using this novel reporter system, we were able to elucidate how cell-based therapies impact bone healing and identify ASCs as an attractive candidate for cell-based skeletal regenerative therapy. These insights potentially influence stem cell selection in translational clinical trials evaluating cell-based therapeutics for osseous repair and regeneration.

  View details for DOI 10.1089/ten.TEA.2015.0221

  View details for PubMedID 26486617

  View details for PubMedCentralID PMC4741228

• Local and Circulating Endothelial Cells Undergo Endothelial to Mesenchymal Transition (EndMT) in Response to Musculoskeletal Injury. Scientific reports Agarwal, S., Loder, S., Cholok, D., Peterson, J., Li, J., Fireman, D., Breuler, C., Hsieh, H. S., Ranganathan, K., Hwang, C., Drake, J., Li, S., Chan, C. K., Longaker, M. T., Levi, B. 2016; 6: 32514-?

  Abstract

  Endothelial-to-mesenchymal transition (EndMT) has been implicated in a variety of aberrant wound healing conditions. However, unambiguous evidence of EndMT has been elusive due to limitations of in vitro experimental designs and animal models. In vitro experiments cannot account for the myriad ligands and cells which regulate differentiation, and in vivo tissue injury models may induce lineage-independent endothelial marker expression in mesenchymal cells. By using an inducible Cre model to mark mesenchymal cells (Scx-creERT/tdTomato + ) prior to injury, we demonstrate that musculoskeletal injury induces expression of CD31, VeCadherin, or Tie2 in mesenchymal cells. VeCadherin and Tie2 were expressed in non-endothelial cells (CD31-) present in marrow from uninjured adult mice, thereby limiting the specificity of these markers in inducible models (e.g. VeCadherin- or Tie2-creERT). However, cell transplantation assays confirmed that endothelial cells (ΔVeCadherin/CD31+/CD45-) isolated from uninjured hindlimb muscle tissue undergo in vivo EndMT when transplanted directly into the wound without intervening cell culture using PDGFRα, Osterix (OSX), SOX9, and Aggrecan (ACAN) as mesenchymal markers. These in vivo findings support EndMT in the presence of myriad ligands and cell types, using cell transplantation assays which can be applied for other pathologies implicated in EndMT including tissue fibrosis and atherosclerosis. Additionally, endothelial cell recruitment and trafficking are potential therapeutic targets to prevent EndMT.

  View details for DOI 10.1038/srep32514

  View details for PubMedID 27616463

• The role of stem cells in limb regeneration ORGANOGENESIS Zielins, E. R., Ransom, R. C., Leavitt, T. E., Longaker, M. T., Wan, D. C. 2016; 12 (1): 16-27

  Abstract

  Limb regeneration is a complex yet fascinating process observed to some extent in many animal species, though seen in its entirety in urodele amphibians. Accomplished by formation of a morphologically uniform intermediate, the blastema, scientists have long attempted to define the cellular constituents that enable regrowth of a functional appendage. Today, we know that the blastema consists of a variety of multipotent progenitor cells originating from a variety of tissues, and which contribute to limb tissue regeneration in a lineage-restricted manner. By continuing to dissect the role of stem cells in limb regeneration, we can hope to one day modulate the human response to limb amputation and facilitate regrowth of a working replacement.

  View details for DOI 10.1080/15476278.2016.1163463

  View details for Web of Science ID 000377930900002

  View details for PubMedID 27008101

  View details for PubMedCentralID PMC4882123

• Getting nervous about regeneration. Stem cell investigation Montoro, D. T., Muhonen, E. G., Longaker, M. T. 2016; 3: 71

  View details for PubMedID 27868053

• Del1 Knockout Mice Developed More Severe Osteoarthritis Associated with Increased Susceptibility of Chondrocytes to Apoptosis. PloS one Wang, Z., Tran, M. C., Bhatia, N. J., Hsing, A. W., Chen, C., LaRussa, M. F., Fattakhov, E., Rashidi, V., Jang, K. Y., Choo, K. J., Nie, X., Mathy, J. A., Longaker, M. T., Dauskardt, R. H., Helms, J. A., Yang, G. P. 2016; 11 (8)

  Abstract

  We identified significant expression of the matricellular protein, DEL1, in hypertrophic and mature cartilage during development. We hypothesized that this tissue-specific expression indicated a biological role for DEL1 in cartilage biology.Del1 KO and WT mice had cartilage thickness evaluated by histomorphometry. Additional mice underwent medial meniscectomy to induce osteoarthritis, and were assayed at 1 week for apoptosis by TUNEL staining and at 8 weeks for histology and OA scoring. In vitro proliferation and apoptosis assays were performed on primary chondrocytes.Deletion of the Del1 gene led to decreased amounts of cartilage in the ears and knee joints in mice with otherwise normal skeletal morphology. Destabilization of the knee led to more severe OA compared to controls. In vitro, DEL1 blocked apoptosis in chondrocytes.Osteoarthritis is among the most prevalent diseases worldwide and increasing in incidence as our population ages. Initiation begins with an injury resulting in the release of inflammatory mediators. Excessive production of inflammatory mediators results in apoptosis of chondrocytes. Because of the limited ability of chondrocytes to regenerate, articular cartilage deteriorates leading to the clinical symptoms including severe pain and decreased mobility. No treatments effectively block the progression of OA. We propose that direct modulation of chondrocyte apoptosis is a key variable in the etiology of OA, and therapies aimed at preventing this important step represent a new class of regenerative medicine targets.

  View details for DOI 10.1371/journal.pone.0160684

  View details for PubMedID 27505251

• Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs. PloS one Paik, K. J., Maan, Z. N., Zielins, E. R., Duscher, D., Whittam, A. J., Morrison, S. D., Brett, E. A., Ransom, R. C., Hu, M. S., Wu, J. C., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2016; 11 (3)

  Abstract

  The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

  View details for DOI 10.1371/journal.pone.0150927

  View details for PubMedID 26967994

  View details for PubMedCentralID PMC4788284

• An Overview of Direct Somatic Reprogramming: The Ins and Outs of iPSCs. International journal of molecular sciences Menon, S., Shailendra, S., Renda, A., Longaker, M., Quarto, N. 2016; 17 (1)

  Abstract

  Stem cells are classified into embryonic stem cells and adult stem cells. An evolving alternative to conventional stem cell therapies is induced pluripotent stem cells (iPSCs), which have a multi-lineage potential comparable to conventionally acquired embryonic stem cells with the additional benefits of being less immunoreactive and avoiding many of the ethical concerns raised with the use of embryonic material. The ability to generate iPSCs from somatic cells provides tremendous promise for regenerative medicine. The breakthrough of iPSCs has raised the possibility that patient-specific iPSCs can provide autologous cells for cell therapy without the concern for immune rejection. iPSCs are also relevant tools for modeling human diseases and drugs screening. However, there are still several hurdles to overcome before iPSCs can be used for translational purposes. Here, we review the recent advances in somatic reprogramming and the challenges that must be overcome to move this strategy closer to clinical application.

  View details for DOI 10.3390/ijms17010141

  View details for PubMedID 26805822

  View details for PubMedCentralID PMC4730380

• Mesenchymal Stromal Cells as Cell-Based Therapeutics for Wound Healing STEM CELLS INTERNATIONAL Malhotra, S., Hu, M. S., Marshall, C. D., Leavitt, T., Cheung, A. T., Gonzalez, J. G., Kaur, H., Lorenz, H. P., Longaker, M. T. 2016

  Abstract

  Chronic wounds are a source of substantial morbidity for patients and are a major financial burden for the healthcare system. There are no current therapies that reliably improve nonhealing wounds or reverse pathological scarring. Mesenchymal stromal cells (MSCs) are a promising source of novel cell-based therapies due to the ease of their harvest and their integral role in the native wound repair process. Recent work has addressed the problems of loss of plasticity and off-target delivery through use of modern bioengineering techniques. Here we describe the applications of MSCs harvested from different sources to the wound healing process and recent advances in delivery of MSCs to targeted sites of injury.

  View details for DOI 10.1155/2016/4157934

  View details for Web of Science ID 000373501200001

  View details for PubMedID 26966438

  View details for PubMedCentralID PMC4757746

• Surveillance of Stem Cell Fate and Function: A System for Assessing Cell Survival and Collagen Expression In Situ TISSUE ENGINEERING PART A Walmsley, G. G., Senarath-Yapa, K., Wearda, T. L., Menon, S., Hu, M. S., Duscher, D., Maan, Z. N., Tsai, J. M., Zielins, E. R., Weissman, I. L., Gurtner, G. C., Lorenz, H. P., Longaker, M. T. 2016; 22 (1-2): 31-40

  Abstract

  Cell-based therapy is an emerging paradigm in skeletal regenerative medicine. However, the primary means by which transplanted cells contribute to bone repair and regeneration remain controversial. To gain an insight into the mechanisms of how both transplanted and endogenous cells mediate skeletal healing, we used a transgenic mouse strain expressing both the topaz variant of green fluorescent protein under the control of the collagen, type I, alpha 1 promoter/enhancer sequence (Col1a1(GFP)) and membrane-bound tomato red fluorescent protein constitutively in all cell types (R26(mTmG)). A comparison of healing in parietal versus frontal calvarial defects in these mice revealed that frontal osteoblasts express Col1a1 to a greater degree than parietal osteoblasts. Furthermore, the scaffold-based application of adipose-derived stromal cells (ASCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), and osteoblasts derived from these mice to critical-sized calvarial defects allowed for investigation of cell survival and function following transplantation. We found that ASCs led to significantly faster rates of bone healing in comparison to BM-MSCs and osteoblasts. ASCs displayed both increased survival and increased Col1a1 expression compared to BM-MSCs and osteoblasts following calvarial defect transplantation, which may explain their superior regenerative capacity in the context of bone healing. Using this novel reporter system, we were able to elucidate how cell-based therapies impact bone healing and identify ASCs as an attractive candidate for cell-based skeletal regenerative therapy. These insights potentially influence stem cell selection in translational clinical trials evaluating cell-based therapeutics for osseous repair and regeneration.

  View details for DOI 10.1089/ten.tea.2015.0221

  View details for Web of Science ID 000368520300005

  View details for PubMedCentralID PMC4741228

• Reply: Studies in Fat Grafting: Part V. Cell-Assisted Lipotransfer to Enhance Fat Graft Retention Is Dose Dependent PLASTIC AND RECONSTRUCTIVE SURGERY Zielins, E. R., Longaker, M. T., Wan, D. C. 2015; 136 (6): 850E–851E

  View details for PubMedID 26322809

• Enhanced Activation of Canonical Wnt Signaling Confers Mesoderm-Derived Parietal Bone with Similar Osteogenic and Skeletal Healing Capacity to Neural Crest-Derived Frontal Bone PLOS ONE Li, S., Quarto, N., Senarath-Yapa, K., Grey, N., Bai, X., Longaker, M. T. 2015; 10 (10)

  Abstract

  Bone formation and skeletal repair are dynamic processes involving a fine-tuned balance between osteoblast proliferation and differentiation orchestrated by multiple signaling pathways. Canonical Wnt (cWnt) signaling is known to playing a key role in these processes. In the current study, using a transgenic mouse model with targeted disruption of axin2, a negative regulator of cWnt signaling, we investigated the impact of enhanced activation of cWnt signaling on the osteogenic capacity and skeletal repair. Specifically, we looked at two calvarial bones of different embryonic tissue origin: the neural crest-derived frontal bone and the mesoderm-derived parietal bone, and we investigated the proliferation and apoptotic activity of frontal and parietal bones and derived osteoblasts. We found dramatic differences in cell proliferation and apoptotic activity between Axin2-/- and wild type calvarial bones, with Axin2-/- showing increased proliferative activity and reduced levels of apoptosis. Furthermore, we compared osteoblast differentiation and bone regeneration in Axin2-/- and wild type neural crest-derived frontal and mesoderm-derived parietal bones, respectively. Our results demonstrate a significant increase either in osteoblast differentiation or bone regeneration in Axin2-/- mice as compared to wild type, with Axin2-/- parietal bone and derived osteoblasts displaying a "neural crest-derived frontal bone-like" profile, which is typically characterized by higher osteogenic capacity and skeletal repair than parietal bone. Taken together, our results strongly suggest that enhanced activation of cWnt signaling increases the skeletal potential of a calvarial bone of mesoderm origin, such as the parietial bone to a degree similar to that of a neural crest origin bone, like the frontal bone. Thus, providing further evidence for the central role played by the cWnt signaling in osteogenesis and skeletal-bone regeneration.

  View details for DOI 10.1371/journal.pone.0138059

  View details for Web of Science ID 000362178700009

  View details for PubMedID 26431534

  View details for PubMedCentralID PMC4592195

• RNA Sequencing for Identification of Differentially Expressed Noncoding Transcripts during Adipogenic Differentiation of Adipose-Derived Stromal Cells. Plastic and reconstructive surgery Luan, A., Paik, K. J., Li, J., Zielins, E. R., Atashroo, D. A., Spencley, A., Momeni, A., Longaker, M. T., Wang, K. C., Wan, D. C. 2015; 136 (4): 752-763

  Abstract

  Adipose-derived stromal cells represent a relatively abundant source of multipotent cells, with many potential applications in regenerative medicine. The present study sought to demonstrate the use of RNA sequencing in identifying differentially expressed transcripts, particularly long noncoding RNAs, associated with adipogenic differentiation to gain a clearer picture of the mechanisms responsible for directing adipose-derived stromal cell fate toward the adipogenic lineage.Human adipose-derived stromal cells were cultured in adipogenic differentiation media, and RNA was harvested at days 0, 1, 3, 5, and 7. Directional RNA sequencing libraries were prepared and sequenced. Paired-end reads were mapped to the human genome reference sequence hg19. Transcriptome assembly was performed and significantly differentially expressed transcripts were identified. Gene ontology term analysis was then performed to identify coding and noncoding transcripts of interest. Differential expression was verified by quantitative real-time polymerase chain reaction.Of 2868 significantly differentially expressed transcripts identified, 207 were noncoding. Enriched gene ontology terms among up-regulated coding transcripts notably reflected differentiation toward the adipogenic lineage. Enriched gene ontology terms among down-regulated coding transcripts reflected growth arrest. Guilt-by-association analysis revealed noncoding RNA candidates with potential roles in the process of adipogenic differentiation.The precise mechanisms that guide lineage-specific differentiation in multipotent cells are not yet fully understood. Defining long noncoding RNAs associated with adipogenic differentiation allows for potential manipulation of regulatory pathways in novel ways. The authors present RNA sequencing as a powerful tool for expanding the understanding of adipose-derived stromal cells and developing novel applications within regenerative medicine.

  View details for DOI 10.1097/PRS.0000000000001582

  View details for PubMedID 26090763

• Delivery of Macrophages in a Biomimetic Scaffold Accelerates Diabetic Wound Healing Through Enhanced Angiogenesis Walmsley, G. G., Hu, M. S., Duscher, D., Januszyk, M., Maan, Z. N., Senarath-Yapa, K., Tevlin, R., Zielins, E. R., Gurtner, G. C., Longaker, M. T. ELSEVIER SCIENCE INC. 2015: S113–S114

  View details for DOI 10.1016/j.jamcollsurg.2015.07.264

  View details for Web of Science ID 000361119700227

• Impairment in Fracture Healing in a Mouse Model of Type 2 Diabetes Is Driven by Skeletal Stem Cell Niche Dysregulation Tevlin, R., Seo, E., Mc Ardle, A., Tong, X., Januszyk, M., Yang, F., Gurtner, G. C., Chan, C. F., Weissman, I. L., Longaker, M. T. ELSEVIER SCIENCE INC. 2015: S115

  View details for DOI 10.1016/j.jamcollsurg.2015.07.268

  View details for Web of Science ID 000361119700230

• Microfluidic single cell transcriptional analysis reveals subpopulations of adipose derived stromal cells with enhanced angiogenic potential Zielins, E. R., Januszyk, M., Luan, A., Brett, E. A., Paik, K., Walmsley, G. G., Gurtner, G. C., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2015: E26

  View details for DOI 10.1016/j.jamcollsurg.2015.08.366

  View details for Web of Science ID 000386899000059

• Melanoma Progression Depends on CXCL12 Expression by Host Endothelium Maan, Z. N., Hu, M. S., Whittam, A., Fischer, L. H., Duscher, D., Walmsley, G. G., Januszyk, M., Whitmore, A. J., Longaker, M. T., Gurtner, G. C. ELSEVIER SCIENCE INC. 2015: S116

  View details for DOI 10.1016/j.jamcollsurg.2015.07.272

  View details for Web of Science ID 000361119700234

• Wounds outcompete tumors for neovascularization Hu, M. S., Maan, Z. N., Hong, W., Walmsley, G. G., Rennert, R. C., Atashroo, D., Gurtner, G. C., Giaccia, A. J., Lorenz, H., Longaker, M. T. ELSEVIER SCIENCE INC. 2015: E124

  View details for DOI 10.1016/j.jamcollsurg.2015.08.230

  View details for Web of Science ID 000386899000304

• Adipose-Derived Stem Cells Improve Engraftment of Full-Thickness Skin Grafts by Increasing Angiogenesis Hu, M. S., Hong, W., Maan, Z. N., Hu, M., Zimmermann, A. S., Walmsley, G. G., Chung, M., Lorenz, H., Longaker, M. T. ELSEVIER SCIENCE INC. 2015: S112

  View details for DOI 10.1016/j.jamcollsurg.2015.07.260

  View details for Web of Science ID 000361119700223

• Progenitor Cell Dysfunctions Underlie Some Diabetic Complications AMERICAN JOURNAL OF PATHOLOGY Rodrigues, M., Wong, V. W., Rennert, R. C., Davis, C. R., Longaker, M. T., Gurtner, G. C. 2015; 185 (10): 2607-2618

  Abstract

  Stem cells and progenitor cells are integral to tissue homeostasis and repair. They contribute to health through their ability to self-renew and commit to specialized effector cells. Recently, defects in a variety of progenitor cell populations have been described in both preclinical and human diabetes. These deficits affect multiple aspects of stem cell biology, including quiescence, renewal, and differentiation, as well as homing, cytokine production, and neovascularization, through mechanisms that are still unclear. More important, stem cell aberrations resulting from diabetes have direct implications on tissue function and seem to persist even after return to normoglycemia. Understanding how diabetes alters stem cell signaling and homeostasis is critical for understanding the complex pathophysiology of many diabetic complications. Moreover, the success of cell-based therapies will depend on a more comprehensive understanding of these deficiencies. This review has three goals: to analyze stem cell pathways dysregulated during diabetes, to highlight the effects of hyperglycemic memory on stem cells, and to define ways of using stem cell therapy to overcome diabetic complications.

  View details for DOI 10.1016/j.ajpath.2015.05.003

  View details for Web of Science ID 000362698300003

  View details for PubMedID 26079815

  View details for PubMedCentralID PMC4607762

• Skeletal Stem Cell Niche Aberrancies Underlie Impaired Fracture Healing in a Mouse Model of Type 2 Diabetes. Plastic and reconstructive surgery Tevlin, R., Young Seo, E., Marecic, O., Wearda, T., Mc Ardle, A., Januszyk, M., Gulati, G., Maan, Z., Hu, M. S., Walmsley, G. G., Gurtner, G. C., Chan, C. K., Weissman, I. L., Longaker, M. T. 2015; 136 (4): 73-?

  View details for DOI 10.1097/01.prs.0000472372.96995.3e

  View details for PubMedID 26397581

• My Journey as a Surgeon-Scientist Ten Years after Receiving the Inaugural Jacobson Promising Investigator Award JOURNAL OF THE AMERICAN COLLEGE OF SURGEONS Longaker, M. T. 2015; 221 (4): 880-882

  Abstract

  The First Joan L and Julius H Jacobson Promising Investigator Awardee, Michael T Longaker MD, FACS In 2005, the research committee of the American College of Surgeons was tasked with selecting the recipient of a newly established award, "The Joan L and Julius H Jacobson Promising Investigator Award." According to the Jacobsons, the $30,000 award funded by Dr Jacobson should be given at least once every 2 years to a surgeon investigator at "the tipping point," who can demonstrate that his/her research shows the promise of leading to a significant contribution to the practice of surgery and patient safety. Every year, the research committee receives many excellent nominations and has the difficult task of selecting 1 awardee. In 2005, the awardee was a young promising investigator, Michael T Longaker, MD, FACS. Ten years later, Dr Longaker, a prominent researcher in the field of "scar formation," presents his journey in research and the impact of the Jacobson award on his career. Dr Longaker is now a national and international figure in the field of wound healing, tissue regeneration, and stem cell research. Kamal MF Itani, MD, FACS and Gail Besner, MD, FACS, on behalf of the Research Committee of the American College of Surgeons.

  View details for DOI 10.1016/j.jamcollsurg.2015.06.018

  View details for Web of Science ID 000361067200015

  View details for PubMedID 26304185

• Identification and characterization of an injury-induced skeletal progenitor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Marecic, O., Tevlin, R., McArdle, A., Seo, E. Y., Wearda, T., Duldulao, C., Walmsley, G. G., Nguyen, A., Weissman, I. L., Chan, C. K., Longaker, M. T. 2015; 112 (32): 9920-9925

  Abstract

  The postnatal skeleton undergoes growth, remodeling, and repair. We hypothesized that skeletal progenitor cells active during these disparate phases are genetically and phenotypically distinct. We identified a highly potent regenerative cell type that we term the fracture-induced bone, cartilage, stromal progenitor (f-BCSP) in the fracture callus of adult mice. The f-BCSP possesses significantly enhanced skeletogenic potential compared with BCSPs harvested from uninjured bone. It also recapitulates many gene expression patterns involved in perinatal skeletogenesis. Our results indicate that the skeletal progenitor population is functionally stratified, containing distinct subsets responsible for growth, regeneration, and repair. Furthermore, our findings suggest that injury-induced changes to the skeletal stem and progenitor microenvironments could activate these cells and enhance their regenerative potential.

  View details for DOI 10.1073/pnas.1513066112

  View details for PubMedID 26216955

• High-Throughput Screening of Surface Marker Expression on Undifferentiated and Differentiated Human Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A Walmsley, G. G., Atashroo, D. A., Maan, Z. N., Hu, M. S., Zielins, E. R., Tsai, J. M., Duscher, D., Paik, K., Tevlin, R., Marecic, O., Wan, D. C., Gurtner, G. C., Longaker, M. T. 2015; 21 (15-16): 2281-2291

  Abstract

  Adipose tissue contains an abundant source of multipotent mesenchymal cells termed "adipose-derived stromal cells" (ASCs) that hold potential for regenerative medicine. However, the heterogeneity inherent to ASCs harvested using standard methodologies remains largely undefined, particularly in regards to differences across donors. Identifying the subpopulations of ASCs predisposed toward differentiation along distinct lineages holds value for improving graft survival, predictability, and efficiency. Human ASCs (hASCs) from three different donors were independently isolated by density-based centrifugation from adipose tissue and maintained in culture or differentiated along either adipogenic or osteogenic lineages using differentiation media. Undifferentiated and differentiated hASCs were then analyzed for the presence of 242 human surface markers by flow cytometry analysis. By comprehensively characterizing the surface marker profile of undifferentiated hASCs using flow cytometry, we gained novel insights into the heterogeneity underlying protein expression on the surface of cultured undifferentiated hASCs across different donors. Comparison of the surface marker profile of undifferentiated hASCs with hASCs that have undergone osteogenic or adipogenic differentiation allowed for the identification of surface markers that were upregulated and downregulated by osteogenic or adipogenic differentiation. Osteogenic differentiation induced upregulation of CD164 and downregulation of CD49a, CD49b, CD49c, CD49d, CD55, CD58, CD105, and CD166 while adipogenic differentiation induced upregulation of CD36, CD40, CD146, CD164, and CD271 and downregulation of CD49b, CD49c, CD49d, CD71, CD105, and CD166. These results lend support to the notion that hASCs isolated using standard methodologies represent a heterogeneous population and serve as a foundation for future studies seeking to maximize their regenerative potential through fluorescence-activated cell sorting-based selection before therapy.

  View details for DOI 10.1089/ten.tea.2015.0039

  View details for Web of Science ID 000359812700014

  View details for PubMedID 26020286

  View details for PubMedCentralID PMC4529076

• En1 fibroblasts and melanoma. Melanoma management Walmsley, G. G., Hu, M. S., Maan, Z. N., Rinkevich, Y., Weissman, I. L., Longaker, M. T. 2015; 2 (3): 191-192

  View details for DOI 10.2217/mmt.15.23

  View details for PubMedID 30190847

  View details for PubMedCentralID PMC6094637

• What Makes a Plastic Surgery Residency Program Attractive? An Applicant's Perspective PLASTIC AND RECONSTRUCTIVE SURGERY Atashroo, D. A., Luan, A., Vyas, K. S., Zielins, E. R., Maan, Z., Duscher, D., Walmsley, G. G., Lynch, M. P., Davenport, D. L., Wan, D. C., Longaker, M. T., Vasconez, H. C. 2015; 136 (1): 189-196

  Abstract

  Plastic surgery is among the most competitive specialties in medicine, but little is known about the attributes of programs that are most attractive to successful applicants. This study aimed to understand and provide insights regarding program characteristics that are most influential to students when ranking plastic surgery programs.An anonymous online survey was conducted with newly matched plastic surgery residents for the integrated and combined Match in 2012 and 2013. Subjects were queried regarding their demographics, qualifications, application experiences, and motivations for residency program selection.A total of 92 of 245 matched plastic surgery residents (38 percent) responded to the survey. The perception of resident happiness was the most positive factor influencing program ranking, followed by high operative volume, faculty mentorship, and strong research infrastructure. Perception of a program as "malignant" was the most negative attribute. Applicants with Step 1 scores greater than 245 received significantly more interviews (p =0.001) and considered resident benefits less important (p < 0.05), but geographic location more important (p =0.005). Applicants who published more than two articles also received more interviews (p =0.001) and ranked a strong research infrastructure and program reputation as significantly more important (p < 0.05). Forty-two percent of applicants completed an away rotation at the program with which they matched, and these applicants were more likely to match at their number one ranked program (p = 0.001).Plastic surgery applicants have differing preferences regarding the ideal training program, but some attributes resonate. These trends can guide programs for improvement in attracting the best applicants.

  View details for DOI 10.1097/PRS.0000000000001365

  View details for Web of Science ID 000357097900001

  View details for PubMedID 26111321

• Nanotechnology in bone tissue engineering NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE Walmsley, G. G., McArdle, A., Tevlin, R., Momeni, A., Atashroo, D., Hu, M. S., Feroze, A. H., Wong, V. W., Lorenz, P. H., Longaker, M. T., Wan, D. C. 2015; 11 (5): 1253-1263

  Abstract

  Nanotechnology represents a major frontier with potential to significantly advance the field of bone tissue engineering. Current limitations in regenerative strategies include impaired cellular proliferation and differentiation, insufficient mechanical strength of scaffolds, and inadequate production of extrinsic factors necessary for efficient osteogenesis. Here we review several major areas of research in nanotechnology with potential implications in bone regeneration: 1) nanoparticle-based methods for delivery of bioactive molecules, growth factors, and genetic material, 2) nanoparticle-mediated cell labeling and targeting, and 3) nano-based scaffold construction and modification to enhance physicochemical interactions, biocompatibility, mechanical stability, and cellular attachment/survival. As these technologies continue to evolve, ultimate translation to the clinical environment may allow for improved therapeutic outcomes in patients with large bone deficits and osteodegenerative diseases.Traditionally, the reconstruction of bony defects has relied on the use of bone grafts. With advances in nanotechnology, there has been significant development of synthetic biomaterials. In this article, the authors provided a comprehensive review on current research in nanoparticle-based therapies for bone tissue engineering, which should be useful reading for clinicians as well as researchers in this field.

  View details for DOI 10.1016/j.nano.2015.02.013

  View details for Web of Science ID 000363967100022

  View details for PubMedID 25791811

  View details for PubMedCentralID PMC4476906

• Studies in Fat Grafting: Part V. Cell-Assisted Lipotransfer to Enhance Fat Graft Retention Is Dose Dependent PLASTIC AND RECONSTRUCTIVE SURGERY Paik, K. J., Zielins, E. R., Atashroo, D. A., Maan, Z. N., Duscher, D., Luan, A., Walmsley, G. G., Momeni, A., Vistnes, S., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2015; 136 (1): 67-75

  Abstract

  Cell-assisted lipotransfer has shown much promise as a technique for improving fat graft take. However, the concentration of stromal vascular fraction cells required to optimally enhance fat graft retention remains unknown.Human lipoaspirate was processed for both fat transfer and harvest of stromal vascular fraction cells. Cells were then mixed back with fat at varying concentrations ranging from 10,000 to 10 million cells per 200 μl of fat. Fat graft volume retention was assessed by means of computed tomographic scanning over 8 weeks, and then fat grafts were explanted and compared histologically for overall architecture and vascularity.Maximum fat graft retention was seen at a concentration of 10,000 cells per 200 μl of fat. The addition of higher number of cells negatively impacted fat graft retention, with supplementation of 10 million cells producing the lowest final volumes, lower than fat alone. Interestingly, fat grafts supplemented with 10,000 cells showed significantly increased vascularity and decreased inflammation, whereas fat grafts supplemented with 10 million cells showed significant lipodegeneration compared with fat alone: The authors' study demonstrates dose dependence in the number of stromal vascular fraction cells that can be added to a fat graft to enhance retention. Although cell-assisted lipotransfer may help promote graft survival, this effect may need to be balanced with the increased metabolic load of added cells that may compete with adipocytes for nutrients during the postgraft period.

  View details for DOI 10.1097/PRS.0000000000001367

  View details for Web of Science ID 000357096300002

  View details for PubMedID 25829158

  View details for PubMedCentralID PMC4483157

• Emerging drugs for the treatment of wound healing EXPERT OPINION ON EMERGING DRUGS Zielins, E. R., Brett, E. A., Luan, A., Hu, M. S., Walmsley, G. G., Paik, K., Senarath-Yapa, K., Atashroo, D. A., Wearda, T., Lorenz, H. P., Wan, D. C., Longaker, M. T. 2015; 20 (2): 235-246

  Abstract

  Wound healing can be characterized as underhealing, as in the setting of chronic wounds, or overhealing, occurring with hypertrophic scar formation after burn injury. Topical therapies targeting specific biochemical and molecular pathways represent a promising avenue for improving and, in some cases normalizing, the healing process.A brief overview of both normal and pathological wound healing has been provided, along with a review of the current clinical guidelines and treatment modalities for chronic wounds, burn wounds and scar formation. Next, the major avenues for wound healing drugs, along with drugs currently in development, are discussed. Finally, potential challenges to further drug development, and future research directions are discussed.The large body of research concerning wound healing pathophysiology has provided multiple targets for topical therapies. Growth factor therapies with the ability to be targeted for localized release in the wound microenvironment are most promising, particularly when they modulate processes in the proliferative phase of wound healing.

  View details for DOI 10.1517/14728214.2015.1018176

  View details for Web of Science ID 000356118400007

  View details for PubMedID 25704608

• The Foreign Body Response: At the Interface of Surgery and Bioengineering PLASTIC AND RECONSTRUCTIVE SURGERY Major, M. R., Wong, V. W., Nelson, E. R., Longaker, M. T., Gurtner, G. C. 2015; 135 (5): 1489-1498

  Abstract

  The surgical implantation of materials and devices has dramatically increased over the past decade. This trend is expected to continue with the broadening application of biomaterials and rapid expansion of aging populations. One major factor that limits the potential of implantable materials and devices is the foreign body response, an immunologic reaction characterized by chronic inflammation, foreign body giant cell formation, and fibrotic capsule formation.The English literature on the foreign body response to implanted materials and devices is reviewed.Fibrotic encapsulation can cause device malfunction and dramatically limit the function of an implanted medical device or material. Basic science studies suggest a role for immune and inflammatory pathways at the implant-host interface that drive the foreign body response. Current strategies that aim to modulate the host response and improve construct biocompatibility appear promising.This review article summarizes recent basic science, preclinical, and clinicopathologic studies examining the mechanisms driving the foreign body response, with particular focus on breast implants and synthetic meshes. Understanding these molecular and cellular mechanisms will be critical for achieving the full potential of implanted biomaterials to restore human tissues and organs.

  View details for DOI 10.1097/PRS.0000000000001193

  View details for Web of Science ID 000353647100060

  View details for PubMedID 25919260

• Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential. Science Rinkevich, Y., Walmsley, G. G., Hu, M. S., Maan, Z. N., Newman, A. M., Drukker, M., Januszyk, M., Krampitz, G. W., Gurtner, G. C., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2015; 348 (6232)

  Abstract

  Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.

  View details for DOI 10.1126/science.aaa2151

  View details for PubMedID 25883361

  View details for PubMedCentralID PMC5088503

• Identification and isolation of a dermal lineage with intrinsic fibrogenic potential SCIENCE Rinkevich, Y., Walmsley, G. G., Hu, M. S., Maan, Z. N., Newman, A. M., Drukker, M., Januszyk, M., Krampitz, G. W., Gurtner, G. C., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2015; 348 (6232): 302-?

  Abstract

  Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.

  View details for DOI 10.1126/science.aaa2151

  View details for Web of Science ID 000352999000034

  View details for PubMedCentralID PMC5088503

• Peripheral blood-derived mesenchymal stem cells: candidate cells responsible for healing critical-sized calvarial bone defects. Stem cells translational medicine Li, S., Huang, K., Wu, J., Hu, M. S., Sanyal, M., Hu, M., Longaker, M. T., Lorenz, H. P. 2015; 4 (4): 359-368

  Abstract

  Postnatal tissue-specific stem/progenitor cells hold great promise to enhance repair of damaged tissues. Many of these cells are retrieved from bone marrow or adipose tissue via invasive procedures. Peripheral blood is an ideal alternative source for the stem/progenitor cells because of its ease of retrieval. We present a coculture system that routinely produces a group of cells from adult peripheral blood. Treatment with these cells enhanced healing of critical-size bone defects in the mouse calvarium, a proof of principle that peripheral blood-derived cells can be used to heal bone defects. From these cells, we isolated a subset of CD45(-) cells with a fibroblastic morphology. The CD45(-) cells were responsible for most of the differentiation-induced calcification activity and were most likely responsible for the enhanced healing process. These CD45(-) fibroblastic cells are plastic-adherent and exhibit a surface marker profile negative for CD34, CD19, CD11b, lineage, and c-kit and positive for stem cell antigen 1, CD73, CD44, CD90.1, CD29, CD105, CD106, and CD140α. Furthermore, these cells exhibited osteogenesis, chondrogenesis, and adipogenesis capabilities. The CD45(-) fibroblastic cells are the first peripheral blood-derived cells that fulfill the criteria of mesenchymal stem cells as defined by the International Society for Cellular Therapy. We have named these cells "blood-derived mesenchymal stem cells."

  View details for DOI 10.5966/sctm.2014-0150

  View details for PubMedID 25742693

  View details for PubMedCentralID PMC4367504

• Therapeutic applications of human adipose-derived stromal cells for soft tissue reconstruction. Discovery medicine Zielins, E. R., Luan, A., Brett, E. A., Longaker, M. T., Wan, D. C. 2015; 19 (105): 245-253

  Abstract

  Adipose derived stromal cells (ASCs) are a multipotent cell population derived from the stromal vascular fraction of lipoaspirate. Given their relatively broad differentiation potential and paracrine capabilities, ASCs represent a readily accessible, endogenous resource for novel reconstructive strategies. In particular, augmentation of autologous fat grafts with ASCs has already been employed clinically for restoration of soft tissue defects. While fat grafting alone remains highly unpredictable, enrichment of fat with supplemental ASCs, also known as cell-assisted lipotransfer (CAL), has been shown to significantly enhance volume retention. How addition of these cells to fat grafts results in improved outcomes, however, remains poorly understood. Furthermore, the safety of CAL in the setting of prior malignancy and post-radiation wound beds has yet to be fully determined, an important consideration for its use in cancer reconstruction. Thus, further studies to determine the how and why behind the efficacy of CAL are necessary before it can be widely adopted as a safe and reliable surgical technique.

  View details for PubMedID 25977187

• Therapeutic Applications of Human Adipose-Derived Stromal Cells for Soft Tissue Reconstruction DISCOVERY MEDICINE Zielins, E. R., Luan, A., Brett, E. A., Longaker, M. T., Wan, D. C. 2015; 105: 245-253

  Abstract

  Adipose derived stromal cells (ASCs) are a multipotent cell population derived from the stromal vascular fraction of lipoaspirate. Given their relatively broad differentiation potential and paracrine capabilities, ASCs represent a readily accessible, endogenous resource for novel reconstructive strategies. In particular, augmentation of autologous fat grafts with ASCs has already been employed clinically for restoration of soft tissue defects. While fat grafting alone remains highly unpredictable, enrichment of fat with supplemental ASCs, also known as cell-assisted lipotransfer (CAL), has been shown to significantly enhance volume retention. How addition of these cells to fat grafts results in improved outcomes, however, remains poorly understood. Furthermore, the safety of CAL in the setting of prior malignancy and post-radiation wound beds has yet to be fully determined, an important consideration for its use in cancer reconstruction. Thus, further studies to determine the how and why behind the efficacy of CAL are necessary before it can be widely adopted as a safe and reliable surgical technique.

  View details for Web of Science ID 000356922200001

• Studies in Fat Grafting: Part IV. Adipose-Derived Stromal Cell Gene Expression in Cell-Assisted Lipotransfer PLASTIC AND RECONSTRUCTIVE SURGERY Garza, R. M., Rennert, R. C., Paik, K. J., Atashroo, D., Chung, M. T., Duscher, D., Januszyk, M., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2015; 135 (4): 1045-1055

  Abstract

  Fat graft volume retention remains highly unpredictable, but addition of adipose-derived stromal cells to fat grafts has been shown to improve retention. The present study aimed to investigate the mechanisms involved in adipose-derived stromal cell enhancement of fat grafting.Adipose-derived stromal cells isolated from human lipoaspirate were labeled with green fluorescent protein and luciferase. Fat grafts enhanced with adipose-derived stromal cells were injected into the scalp and bioluminescent imaging was performed to follow retention of adipose-derived stromal cells within the fat graft. Fat grafts were also explanted at days 1, 5, and 10 after grafting for adipose-derived stromal cell extraction and single-cell gene analysis. Finally, CD31 immunohistochemical staining was performed on fat grafts enriched with adipose-derived stromal cells.Bioluminescent imaging demonstrated significant reduction in luciferase-positive adipose-derived stromal cells within fat grafts at 5 days after grafting. A similar reduction in viable green fluorescent protein-positive adipose-derived stromal cells retrieved from explanted grafts was also noted. Single-cell analysis revealed expression of multiple genes/markers related to cell survival and angiogenesis, including BMPR2, CD90, CD105, FGF2, CD248, TGFß1, and VEGFA. Genes involved in adipogenesis were not expressed by adipose-derived stromal cells. Finally, CD31 staining revealed significantly higher vascular density in fat grafts explanted at day 10 after grafting.Although adipose-derived stromal cell survival in the hypoxic graft environment decreases significantly over time, these cells provide multiple angiogenic growth factors. Therefore, improved fat graft volume retention with adipose-derived stromal cell enrichment may be attributable to improved graft vascularization.

  View details for DOI 10.1097/PRS.0000000000001104

  View details for Web of Science ID 000351910200043

  View details for PubMedID 25502860

  View details for PubMedCentralID PMC4376612

• TWIST1 Silencing Enhances In Vitro and In Vivo Osteogenic Differentiation of Human Adipose-Derived Stem Cells by Triggering Activation of BMP-ERK/FGF Signaling and TAZ Upregulation. Stem cells Quarto, N., Senarath-Yapa, K., Renda, A., Longaker, M. T. 2015; 33 (3): 833-847

  Abstract

  Mesenchymal stem cells (MSCs) show promise for cellular therapy and regenerative medicine. Human adipose tissue-derived stem cells (hASCs) represent an attractive source of seed cells in bone regeneration. How to effectively improve osteogenic differentiation of hASCs in the bone tissue engineering has become a very important question with profound translational implications. Numerous regulatory pathways dominate osteogenic differentiation of hASCs involving transcriptional factors and signaling molecules. However, how these factors combine with each other to regulate hASCs osteogenic differentiation still remains to be illustrated. The highly conserved developmental proteins TWIST play key roles for transcriptional regulation in mesenchymal cell lineages. This study investigates TWIST1 function in hASCs osteogenesis. Our results show that TWIST1 shRNA silencing increased the osteogenic potential of hASCs in vitro and their skeletal regenerative ability when applied in vivo. We demonstrate that the increased osteogenic capacity observed with TWIST1 knockdown in hASCs is mediated through endogenous activation of BMP and ERK/FGF signaling leading, in turn, to upregulation of TAZ, a transcriptional modulator of MSCs differentiation along the osteoblast lineage. Inhibition either of BMP or ERK/FGF signaling suppressed TAZ upregulation and the enhanced osteogenesis in shTWIST1 hASCs. Cosilencing of both TWIST1 and TAZ abrogated the effect elicited by TWIST1 knockdown thus, identifying TAZ as a downstream mediator through which TWIST1 knockdown enhanced osteogenic differentiation in hASCs. Our functional study contributes to a better knowledge of molecular mechanisms governing the osteogenic ability of hASCs, and highlights TWIST1 as a potential target to facilitate in vivo bone healing. Stem Cells 2015;33:833-847.

  View details for DOI 10.1002/stem.1907

  View details for PubMedID 25446627

• Live Fibroblast Harvest Reveals Surface Marker Shift In Vitro TISSUE ENGINEERING PART C-METHODS Walmsley, G. G., Rinkevich, Y., Hu, M. S., Montoro, D. T., Lo, D. D., McArdle, A., Maan, Z. N., Morrison, S. D., Duscher, D., Whittam, A. J., Wong, V. W., Weissman, I. L., Gurtner, G. C., Longaker, M. T. 2015; 21 (3): 314-321

  Abstract

  Current methods for the isolation of fibroblasts require extended ex vivo manipulation in cell culture. As a consequence, prior studies investigating fibroblast biology may fail to adequately represent cellular phenotypes in vivo. To overcome this problem, we describe a detailed protocol for the isolation of fibroblasts from the dorsal dermis of adult mice that bypasses the need for cell culture, thereby preserving the physiological, transcriptional, and proteomic profiles of each cell. Using the described protocol we characterized the transcriptional programs and the surface expression of 176 CD markers in cultured versus uncultured fibroblasts. The differential expression patterns we observed highlight the importance of a live harvest for investigations of fibroblast biology.

  View details for DOI 10.1089/ten.tec.2014.0118

  View details for Web of Science ID 000350043400009

  View details for PubMedID 25275778

  View details for PubMedCentralID PMC4346232

• The role and regulation of osteoclasts in normal bone homeostasis and in response to injury. Plastic and reconstructive surgery McArdle, A., Marecic, O., Tevlin, R., Walmsley, G. G., Chan, C. K., Longaker, M. T., Wan, D. C. 2015; 135 (3): 808-816

  Abstract

  Bone is a dynamic tissue, with a range of diverse functions, including locomotion, protection of internal organs, and hematopoiesis. Optimum treatment of fractures and/or bone defects requires knowledge of the complex cellular interactions involved with bone healing and remodeling. Emerging data have underscored the importance of osteoclasts in this process, playing a key role both in normal bone turnover and in facilitating bone regeneration. In this review, the authors discuss the basic principles of osteoclast biology, including its cellular origins, its function, and key regulatory mechanisms, in addition to conditions that arise when osteoclast function is altered.

  View details for DOI 10.1097/PRS.0000000000000963

  View details for PubMedID 25719699

• Scarless wound healing: chasing the holy grail. Plastic and reconstructive surgery Walmsley, G. G., Maan, Z. N., Wong, V. W., Duscher, D., Hu, M. S., Zielins, E. R., Wearda, T., Muhonen, E., McArdle, A., Tevlin, R., Atashroo, D. A., Senarath-Yapa, K., Lorenz, H. P., Gurtner, G. C., Longaker, M. T. 2015; 135 (3): 907-917

  Abstract

  Over 100 million patients acquire scars in the industrialized world each year, primarily as a result of elective operations. Although undefined, the global incidence of scarring is even larger, extending to significant numbers of burn and other trauma-related wounds. Scars have the potential to exert a profound psychological and physical impact on the individual. Beyond aesthetic considerations and potential disfigurement, scarring can result in restriction of movement and reduced quality of life. The formation of a scar following skin injury is a consequence of wound healing occurring through reparative rather than regenerative mechanisms. In this article, the authors review the basic stages of wound healing; differences between adult and fetal wound healing; various mechanical, genetic, and pharmacologic strategies to reduce scarring; and the biology of skin stem/progenitor cells that may hold the key to scarless regeneration.

  View details for DOI 10.1097/PRS.0000000000000972

  View details for PubMedID 25719706

• Exercise induces stromal cell-derived factor-1a-mediated release of endothelial progenitor cells with increased vasculogenic function. Plastic and reconstructive surgery Chang, E., Paterno, J., Duscher, D., Maan, Z. N., Chen, J. S., Januszyk, M., Rodrigues, M., Rennert, R. C., Bishop, S., Whitmore, A. J., Whittam, A. J., Longaker, M. T., Gurtner, G. C. 2015; 135 (2): 340e-50e

  Abstract

  Endothelial progenitor cells have been shown to traffic to and incorporate into ischemic tissues, where they participate in new blood vessel formation, a process termed vasculogenesis. Previous investigation has demonstrated that endothelial progenitor cells appear to mobilize from bone marrow to the peripheral circulation after exercise. In this study, the authors investigate potential etiologic factors driving this mobilization and investigate whether the mobilized endothelial progenitor cells are the same as those present at baseline.Healthy volunteers (n = 5) performed a monitored 30-minute run to maintain a heart rate greater than 140 beats/min. Venous blood samples were collected before, 10 minutes after, and 24 hours after exercise. Endothelial progenitor cells were isolated and evaluated.Plasma levels of stromal cell-derived factor-1α significantly increased nearly two-fold immediately after exercise, with a nearly four-fold increase in circulating endothelial progenitor cells 24 hours later. The endothelial progenitor cells isolated following exercise demonstrated increased colony formation, proliferation, differentiation, and secretion of angiogenic cytokines. Postexercise endothelial progenitor cells also exhibited a more robust response to hypoxic stimulation.Exercise appears to mobilize endothelial progenitor cells and augment their function by means of stromal cell-derived factor 1α-dependent signaling. The population of endothelial progenitor cells mobilized following exercise is primed for vasculogenesis with increased capacity for proliferation, differentiation, secretion of cytokines, and responsiveness to hypoxia. Given the evidence demonstrating positive regenerative effects of exercise, this may be one possible mechanism for its benefits.

  View details for DOI 10.1097/PRS.0000000000000917

  View details for PubMedID 25626819

• Reply: Studies in fat grafting: part I. Effects of injection technique on in vitro fat viability and in vivo volume retention; and studies in fat grafting: part II. Effects of injection mechanics on material properties of fat. Plastic and reconstructive surgery Wan, D. C., Gurtner, G. C., Longaker, M. T. 2015; 135 (2): 448e-9e

  View details for DOI 10.1097/PRS.0000000000000978

  View details for PubMedID 25626833

• Identification and specification of the mouse skeletal stem cell. Cell Chan, C. K., Seo, E. Y., Chen, J. Y., Lo, D., McArdle, A., Sinha, R., Tevlin, R., Seita, J., Vincent-Tompkins, J., Wearda, T., Lu, W., Senarath-Yapa, K., Chung, M. T., Marecic, O., Tran, M., Yan, K. S., Upton, R., Walmsley, G. G., Lee, A. S., Sahoo, D., Kuo, C. J., Weissman, I. L., Longaker, M. T. 2015; 160 (1-2): 285-298

  Abstract

  How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

  View details for DOI 10.1016/j.cell.2014.12.002

  View details for PubMedID 25594184

• Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation. Blood Reinisch, A., Etchart, N., Thomas, D., Hofmann, N. A., Fruehwirth, M., Sinha, S., Chan, C. K., Senarath-Yapa, K., Seo, E., Wearda, T., Hartwig, U. F., Beham-Schmid, C., Trajanoski, S., Lin, Q., Wagner, W., Dullin, C., Alves, F., Andreeff, M., Weissman, I. L., Longaker, M. T., Schallmoser, K., Majeti, R., Strunk, D. 2015; 125 (2): 249-260

  Abstract

  In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a bone marrow cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13 and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs) as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.

  View details for DOI 10.1182/blood-2014-04-572255

  View details for PubMedID 25406351

• Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis. Journal of visualized experiments : JoVE Zielins, E. R., Tevlin, R., Hu, M. S., Chung, M. T., McArdle, A., Paik, K. J., Atashroo, D., Duldulao, C. R., Luan, A., Senarath-Yapa, K., Walmsley, G. G., Wearda, T., Longaker, M. T., Wan, D. C. 2015

  View details for DOI 10.3791/52181

  View details for PubMedID 25650785

• Differences in Foetal, Adult Skin and Mucosal Repair STEM CELL BIOLOGY AND TISSUE ENGINEERING IN DENTAL SCIENCES Lo, D. D., Hu, M. S., Zimmermann, A. S., Longaker, M. T., Lorenz, H., Vishwakarma, A., Sharpe, P., Shi, S., Ramalingam, M. 2015: 691–702

  View details for DOI 10.1016/B978-0-12-397157-9.00055-2

  View details for Web of Science ID 000444643800052

• Stem Cell-Based Therapeutics to Improve Wound Healing. Plastic surgery international Hu, M. S., Leavitt, T., Malhotra, S., Duscher, D., Pollhammer, M. S., Walmsley, G. G., Maan, Z. N., Cheung, A. T., Schmidt, M., Huemer, G. M., Longaker, M. T., Lorenz, H. P. 2015; 2015: 383581-?

  Abstract

  Issues surrounding wound healing have garnered deep scientific interest as well as booming financial markets invested in novel wound therapies. Much progress has been made in the field, but it is unsurprising to find that recent successes reveal new challenges to be addressed. With regard to wound healing, large tissue deficits, recalcitrant wounds, and pathological scar formation remain but a few of our most pressing challenges. Stem cell-based therapies have been heralded as a promising means by which to surpass current limitations in wound management. The wide differentiation potential of stem cells allows for the possibility of restoring lost or damaged tissue, while their ability to immunomodulate the wound bed from afar suggests that their clinical applications need not be restricted to direct tissue formation. The clinical utility of stem cells has been demonstrated across dozens of clinical trials in chronic wound therapy, but there is hope that other aspects of wound care will inherit similar benefit. Scientific inquiry into stem cell-based wound therapy abounds in research labs around the world. While their clinical applications remain in their infancy, the heavy investment in their potential makes it a worthwhile subject to review for plastic surgeons, in terms of both their current and future applications.

  View details for DOI 10.1155/2015/383581

  View details for PubMedID 26649195

  View details for PubMedCentralID PMC4663003

• Assessment of viability of human fat injection into nude mice with micro-computed tomography. Journal of visualized experiments : JoVE Atashroo, D. A., Paik, K. J., Chung, M. T., McArdle, A., Senarath-Yapa, K., Zielins, E. R., Tevlin, R., Duldulao, C. R., Walmsley, G. G., Wearda, T., Marecic, O., Longaker, M. T., Wan, D. C. 2015

  View details for DOI 10.3791/52217

  View details for PubMedID 25590561

• A mouse fetal skin model of scarless wound repair. Journal of visualized experiments : JoVE Walmsley, G. G., Hu, M. S., Hong, W. X., Maan, Z. N., Lorenz, H. P., Longaker, M. T. 2015

  View details for DOI 10.3791/52297

  View details for PubMedID 25650841

• Impact of surgical innovation on tissue repair in the surgical patient. British journal of surgery Tevlin, R., Atashroo, D., Duscher, D., Mc Ardle, A., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2015; 102 (2): e41-55

  View details for DOI 10.1002/bjs.9672

  View details for PubMedID 25627135

• A mouse fetal skin model of scarless wound repair. Journal of visualized experiments : JoVE Walmsley, G. G., Hu, M. S., Hong, W. X., Maan, Z. N., Lorenz, H. P., Longaker, M. T. 2015: 52297-?

  Abstract

  Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

  View details for DOI 10.3791/52297

  View details for PubMedID 25650841

• Isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis. Journal of visualized experiments : JoVE Zielins, E. R., Tevlin, R., Hu, M. S., Chung, M. T., McArdle, A., Paik, K. J., Atashroo, D., Duldulao, C. R., Luan, A., Senarath-Yapa, K., Walmsley, G. G., Wearda, T., Longaker, M. T., Wan, D. C. 2015: 52181-?

  Abstract

  Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

  View details for DOI 10.3791/52181

  View details for PubMedID 25650785

• Assessment of viability of human fat injection into nude mice with micro-computed tomography. Journal of visualized experiments : JoVE Atashroo, D. A., Paik, K. J., Chung, M. T., McArdle, A., Senarath-Yapa, K., Zielins, E. R., Tevlin, R., Duldulao, C. R., Walmsley, G. G., Wearda, T., Marecic, O., Longaker, M. T., Wan, D. C. 2015

  Abstract

  Lipotransfer is a vital tool in the surgeon's armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.(1) However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. (1-3) To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 µl of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes. Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

  View details for DOI 10.3791/52217

  View details for PubMedID 25590561

• Impact of surgical innovation on tissue repair in the surgical patient. British journal of surgery Tevlin, R., Atashroo, D., Duscher, D., Mc Ardle, A., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2015; 102 (2): e41-55

  Abstract

  Throughout history, surgeons have been prolific innovators, which is hardly surprising as most surgeons innovate daily, tailoring their intervention to the intrinsic uniqueness of each operation, each patient and each disease. Innovation can be defined as the application of better solutions that meet new requirements, unarticulated needs or existing market needs. In the past two decades, surgical innovation has significantly improved patient outcomes, complication rates and length of hospital stay. There is one key area that has great potential to change the face of surgical practice and which is still in its infancy: the realm of regenerative medicine and tissue engineering.A literature review was performed using PubMed; peer-reviewed publications were screened for relevance in order to identify key surgical innovations influencing regenerative medicine, with a focus on osseous, cutaneous and soft tissue reconstruction.This review describes recent advances in regenerative medicine, documenting key innovations in osseous, cutaneous and soft tissue regeneration that have brought regenerative medicine to the forefront of the surgical imagination.Surgical innovation in the emerging field of regenerative medicine has the ability to make a major impact on surgery on a daily basis.

  View details for DOI 10.1002/bjs.9672

  View details for PubMedID 25627135

• Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation. Proceedings of the National Academy of Sciences of the United States of America Tan, S. H., Senarath-Yapa, K., Chung, M. T., Longaker, M. T., Wu, J. Y., Nusse, R. 2014; 111 (49): E5262-71

  Abstract

  Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated-to our knowledge for the first time-that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation.

  View details for DOI 10.1073/pnas.1420463111

  View details for PubMedID 25422448

• Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tan, S. H., Senarath-Yapa, K., Chung, M. T., Longaker, M. T., Wu, J. Y., Nusse, R. 2014; 111 (49): E5262-E5271

  Abstract

  Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated-to our knowledge for the first time-that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation.

  View details for DOI 10.1073/pnas.1420463111

  View details for Web of Science ID 000345921500004

  View details for PubMedID 25422448

• Biomaterials for Craniofacial Bone Engineering JOURNAL OF DENTAL RESEARCH Tevlin, R., Mcardle, A., Atashroo, D., Walmsley, G. G., Senarath-Yapa, K., Zielins, E. R., Paik, K. J., Longaker, M. T., Wan, D. C. 2014; 93 (12): 1187-1195

  View details for DOI 10.1177/0022034514547271

  View details for Web of Science ID 000345340400002

• Loss of keratinocyte focal adhesion kinase stimulates dermal proteolysis through upregulation of MMP9 in wound healing. Annals of surgery Wong, V. W., Garg, R. K., Sorkin, M., Rustad, K. C., Akaishi, S., Levi, K., Nelson, E. R., Tran, M., Rennert, R., Liu, W., Longaker, M. T., Dauskardt, R. H., Gurtner, G. C. 2014; 260 (6): 1138-1146

  Abstract

  To investigate how epithelial mechanotransduction pathways impact wound repair.Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9.A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds.Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses.Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

  View details for DOI 10.1097/SLA.0000000000000219

  View details for PubMedID 25389925

• Loss of Keratinocyte Focal Adhesion Kinase Stimulates Dermal Proteolysis Through Upregulation of MMP9 in Wound Healing ANNALS OF SURGERY Wong, V. W., Garg, R. K., Sorkin, M., Rustad, K. C., Akaishi, S., Levi, K., Nelson, E. R., Tran, M., Rennert, R., Liu, W., Longaker, M. T., Dauskardt, R. H., Gurtner, G. C. 2014; 260 (6): 1138-1146

  Abstract

  To investigate how epithelial mechanotransduction pathways impact wound repair.Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9.A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds.Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses.Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

  View details for DOI 10.1097/SLA.0000000000000219

  View details for Web of Science ID 000345217200030

• Positive Selection for Bone Morphogenetic Protein Receptor Type-IB Promotes Differentiation and Specification of Human Adipose-Derived Stromal Cells Toward an Osteogenic Lineage TISSUE ENGINEERING PART A McArdle, A., Chung, M. T., Paik, K. J., Duldulao, C., Chan, C., Rennert, R., Walmsley, G. G., Senarath-Yapa, K., Hu, M., Seo, E., Lee, M., Wan, D. C., Longaker, M. T. 2014; 20 (21-22): 3031-3040

  Abstract

  Adipose tissue represents an abundant and easily accessible source of multipotent cells that may serve as an excellent building block for tissue engineering. However, adipose-derived stromal cells (ASCs) are a heterogeneous group and subpopulations may be identified with enhanced osteogenic potential.Human ASC subpopulations were prospectively isolated based on expression of bone morphogenetic protein receptor type-IB (BMPR-IB). Unsorted, BMPR-IB(+), and BMPR-IB(-) cells were analyzed for their osteogenic capacity through histological staining and gene expression. To evaluate their in vivo osteogenic potential, critical-sized calvarial defects were created in immunocompromised mice and treated with unsorted, BMPR-IB(+), or BMPR-IB(-) cells. Healing was assessed using microcomputed tomography and pentachrome staining of specimens at 8 weeks.Increased osteogenic differentiation was noted in the BMPR-IB(+) subpopulation, as demonstrated by alkaline phosphatase staining at day 7 and extracellular matrix mineralization with Alizarin red staining at day 14. This was also associated with increased expression for osteocalcin, a late marker of osteogenesis. Radiographic analysis demonstrated significantly enhanced healing of critical-sized calvarial defects treated with BMPR-IB(+) ASCs compared with unsorted or BMPR-IB(-) cells. This was confirmed through pentachrome staining, which revealed more robust bone regeneration in the BMPR-IB(+) group.BMPR-IB(+) human ASCs have an enhanced ability to form bone both in vitro and in vivo. These data suggest that positive selection for BMPR-IB(+) and manipulation of the BMP pathway in these cells may yield a highly osteogenic subpopulation of cells for bone tissue engineering.

  View details for DOI 10.1089/ten.tea.2014.0101

  View details for Web of Science ID 000344592600021

  View details for PubMedCentralID PMC4229710

• Positive selection for bone morphogenetic protein receptor type-IB promotes differentiation and specification of human adipose-derived stromal cells toward an osteogenic lineage. Tissue engineering. Part A McArdle, A., Chung, M. T., Paik, K. J., Duldulao, C., Chan, C., Rennert, R., Walmsley, G. G., Senarath-Yapa, K., Hu, M., Seo, E., Lee, M., Wan, D. C., Longaker, M. T. 2014; 20 (21-22): 3031-3040

  Abstract

  Adipose tissue represents an abundant and easily accessible source of multipotent cells that may serve as an excellent building block for tissue engineering. However, adipose-derived stromal cells (ASCs) are a heterogeneous group and subpopulations may be identified with enhanced osteogenic potential.Human ASC subpopulations were prospectively isolated based on expression of bone morphogenetic protein receptor type-IB (BMPR-IB). Unsorted, BMPR-IB(+), and BMPR-IB(-) cells were analyzed for their osteogenic capacity through histological staining and gene expression. To evaluate their in vivo osteogenic potential, critical-sized calvarial defects were created in immunocompromised mice and treated with unsorted, BMPR-IB(+), or BMPR-IB(-) cells. Healing was assessed using microcomputed tomography and pentachrome staining of specimens at 8 weeks.Increased osteogenic differentiation was noted in the BMPR-IB(+) subpopulation, as demonstrated by alkaline phosphatase staining at day 7 and extracellular matrix mineralization with Alizarin red staining at day 14. This was also associated with increased expression for osteocalcin, a late marker of osteogenesis. Radiographic analysis demonstrated significantly enhanced healing of critical-sized calvarial defects treated with BMPR-IB(+) ASCs compared with unsorted or BMPR-IB(-) cells. This was confirmed through pentachrome staining, which revealed more robust bone regeneration in the BMPR-IB(+) group.BMPR-IB(+) human ASCs have an enhanced ability to form bone both in vitro and in vivo. These data suggest that positive selection for BMPR-IB(+) and manipulation of the BMP pathway in these cells may yield a highly osteogenic subpopulation of cells for bone tissue engineering.

  View details for DOI 10.1089/ten.TEA.2014.0101

  View details for PubMedID 24854876

• Reply: tension shielding with the embrace device: does it really improve scars? Plastic and reconstructive surgery Gurtner, G. C., Longaker, M. T. 2014; 134 (4): 664e-6e

  View details for DOI 10.1097/PRS.0000000000000560

  View details for PubMedID 25357068

• Diabetes irreversibly depletes bone marrow-derived mesenchymal progenitor cell subpopulations. Diabetes Januszyk, M., Sorkin, M., Glotzbach, J. P., Vial, I. N., Maan, Z. N., Rennert, R. C., Duscher, D., Thangarajah, H., Longaker, M. T., Butte, A. J., Gurtner, G. C. 2014; 63 (9): 3047-3056

  Abstract

  Diabetic vascular pathology is largely attributable to impairments in tissue recovery from hypoxia. Circulating progenitor cells have been postulated to play a role in ischemic recovery and deficiencies in these cells have been well described in diabetic patients. Here, we examine bone marrow-derived mesenchymal progenitor cells (BM-MPCs) that have previously been shown to be important for new blood vessel formation, and demonstrate significant deficits in the context of diabetes. Further, we determine that this dysfunction is attributable to intrinsic defects in diabetic BM-MPCs that are not correctable by restoring glucose homeostasis. We identify two transcriptionally distinct subpopulations that are selectively depleted by both type 1 and type 2 diabetes, and these subpopulations have pro-vasculogenic expression profiles, suggesting that they are vascular progenitor cells. These results suggest that the clinically observed deficits in progenitor cells may be attributable to selective and irreversible depletion of progenitor cell subsets in patients with diabetes.

  View details for DOI 10.2337/db13-1366

  View details for PubMedID 24740572

• Reduced Regenerative Capacity of Aged Adipose Derived Stem Cells is Caused by Alterations of Cell Subpopulation Dynamics Duscher, D., Rennert, R. C., Januszyk, M., Maan, Z. N., Whittam, A. J., Hu, M. S., Walmsley, G. G., Atashroo, D., Longaker, M. T., Gurtner, G. C. ELSEVIER SCIENCE INC. 2014: S136

  View details for DOI 10.1016/j.jamcollsurg.2014.07.326

  View details for Web of Science ID 000342420900287

• The Optimal Fat Graft: Evaluating the Biomechanical Effects of Diameter, Length, and Flow Rate during Fat Placement Atashroo, D., Wearda, T., Raphel, J., Paik, K., Zielins, E. R., Walmsley, G. G., Tevlin, R., Wan, D. C., Heilshorn, S., Longaker, M. T. ELSEVIER SCIENCE INC. 2014: S90

  View details for DOI 10.1016/j.jamcollsurg.2014.07.215

  View details for Web of Science ID 000342420900186

• Diminished Recruitment of Resident Skeletal Progenitor Cells in Diabetic Fracture Healing Tevlin, R., Seo, E., McArdle, A., Marecic, O., Wearda, T., Senarath-Yapa, K., Zielins, E. R., Chan, C. K., Weissman, I. L., Longaker, M. T. ELSEVIER SCIENCE INC. 2014: S82

  View details for DOI 10.1016/j.jamcollsurg.2014.07.193

  View details for Web of Science ID 000342420900165

• Heterochronic Parabiosis Rejuvenates Aged Bones McArdle, A., Tevlin, R., Marecic, O., Wearda, T., Senarath-Yapa, K., Walmsley, G. G., Snyder, M. P., Weissman, I. L., Chan, C. K., Longaker, M. T. ELSEVIER SCIENCE INC. 2014: S82–S83

  View details for DOI 10.1016/j.jamcollsurg.2014.07.195

  View details for Web of Science ID 000342420900167

• Response of Skeletal Progenitor Cells to Fracture Injury in a Mouse Model Marecic, O., McArdle, A., Seo, E., Tevlin, R., Duldulao, C., Wearda, T., Chan, C., Weissman, I. L., Longaker, M. T. ELSEVIER SCIENCE INC. 2014: S86

  View details for DOI 10.1016/j.jamcollsurg.2014.07.204

  View details for Web of Science ID 000342420900176

• Capillary force seeding of hydrogels for adipose-derived stem cell delivery in wounds. Stem cells translational medicine Garg, R. K., Rennert, R. C., Duscher, D., Sorkin, M., Kosaraju, R., Auerbach, L. J., Lennon, J., Chung, M. T., Paik, K., Nimpf, J., Rajadas, J., Longaker, M. T., Gurtner, G. C. 2014; 3 (9): 1079-1089

  Abstract

  Effective skin regeneration therapies require a successful interface between progenitor cells and biocompatible delivery systems. We previously demonstrated the efficiency of a biomimetic pullulan-collagen hydrogel scaffold for improving bone marrow-derived mesenchymal stem cell survival within ischemic skin wounds by creating a "stem cell niche" that enhances regenerative cytokine secretion. Adipose-derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility, and in this study we explored the utility of ASCs for hydrogel-based therapies. To optimize hydrogel cell seeding, a rapid, capillary force-based approach was developed and compared with previously established cell seeding methods. ASC viability and functionality following capillary hydrogel seeding were then analyzed in vitro and in vivo. In these experiments, ASCs were seeded more efficiently by capillary force than by traditional methods and remained viable and functional in this niche for up to 14 days. Additionally, hydrogel seeding of ASCs resulted in the enhanced expression of multiple stemness and angiogenesis-related genes, including Oct4, Vegf, Mcp-1, and Sdf-1. Moving in vivo, hydrogel delivery improved ASC survival, and application of both murine and human ASC-seeded hydrogels to splinted murine wounds resulted in accelerated wound closure and increased vascularity when compared with control wounds treated with unseeded hydrogels. In conclusion, capillary seeding of ASCs within a pullulan-collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC-seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting.

  View details for DOI 10.5966/sctm.2014-0007

  View details for PubMedID 25038246

  View details for PubMedCentralID PMC4149299

• Noncontact, low-frequency ultrasound therapy enhances neovascularization and wound healing in diabetic mice. Plastic and reconstructive surgery Maan, Z. N., Januszyk, M., Rennert, R. C., Duscher, D., Rodrigues, M., Fujiwara, T., Ho, N., Whitmore, A., Hu, M. S., Longaker, M. T., Gurtner, G. C. 2014; 134 (3): 402e-11e

  Abstract

  Chronic wounds are a major source of morbidity for patients and represent a significant health burden. Implementing noninvasive techniques that accelerate healing of these wounds would provide great benefit. Ultrasound appears to be an effective modality for the treatment of chronic wounds in humans. MIST Therapy is a noncontact, low-frequency ultrasound treatment delivered through a saline mist. A variety of mechanisms have been proposed to explain the efficacy of ultrasound therapy, but the underlying molecular and cellular pathways impacted by this technique remain unclear. The in vivo effect of noncontact, low-frequency ultrasound was therefore examined in a humanized excisional wound model.The treatment group received noncontact, low-frequency ultrasound therapy three times per week, whereas the control group received a standard dressing change. Wounds were photographed at regular intervals to calculate healing kinetics. Wound tissue was harvested and processed for histology, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay.The MIST group demonstrated significantly accelerated wound healing, with 17.3 days to wound closure compared with 24 days in the controls (p < 0.05). This improvement became evident by day 9, with healing evidenced by significantly decreased mean wound area relative to original size (68 percent versus 80 percent; p < 0.01). Expression of markers of neovascularization (stromal cell-derived factor 1, vascular endothelial growth factor, and CD31) was also increased in the wound beds of noncontact, low-frequency ultrasound-treated mice compared with controls.Noncontact, low-frequency ultrasound treatment improves neovascularization and wound closure rates in excisional wounds for diabetic mice, likely because of the stimulated release of angiogenic factors.

  View details for DOI 10.1097/PRS.0000000000000467

  View details for PubMedID 25158717

• A Randomized Controlled Trial of the embrace Advanced Scar Therapy Device to Reduce Incisional Scar Formation. Plastic and reconstructive surgery Longaker, M. T., Rohrich, R. J., Greenberg, L., Furnas, H., Wald, R., Bansal, V., Seify, H., Tran, A., Weston, J., Korman, J. M., Chan, R., Kaufman, D., Dev, V. R., Mele, J. A., Januszyk, M., Cowley, C., McLaughlin, P., Beasley, B., Gurtner, G. C. 2014; 134 (3): 536-546

  Abstract

  Scarring represents a significant biomedical burden in clinical medicine. Mechanomodulation has been linked to scarring through inflammation, but until now a systematic approach to attenuate mechanical force and reduce scarring has not been possible.The authors conducted a 12-month, prospective, open-label, randomized, multicenter clinical trial to evaluate abdominoplasty scar appearance following postoperative treatment with the embrace Advanced Scar Therapy device to reduce mechanical forces on healing surgical incisions. Incisions from 65 healthy adult subjects were randomized to receive embrace treatment on one half of an abdominoplasty incision and control treatment (surgeon's optimal care methods) on the other half. The primary endpoint for this study was the difference between assessments of scar appearance for the treated and control sides using the visual analogue scale scar score.Final 12-month study photographs were obtained from 36 subjects who completed at least 5 weeks of dressing application. The mean visual analogue scale score for embrace-treated scars (2.90) was significantly improved compared with control-treated scars (3.29) at 12 months (difference, 0.39; 95 percent confidence interval, 0.14 to 0.66; p = 0.027). Both subjects and investigators found that embrace-treated scars demonstrated significant improvements in overall appearance at 12 months using the Patient and Observer Scar Assessment Scale evaluation (p = 0.02 and p < 0.001, respectively). No serious adverse events were reported.These results demonstrate that the embrace device significantly reduces scarring following abdominoplasty surgery. To the authors' knowledge, this represents the first level I evidence for postoperative scar reduction.Therapeutic, II.

  View details for DOI 10.1097/PRS.0000000000000417

  View details for PubMedID 24804638

• The role of stem cells in aesthetic surgery: fact or fiction? Plastic and reconstructive surgery McArdle, A., Senarath-Yapa, K., Walmsley, G. G., Hu, M., Atashroo, D. A., Tevlin, R., Zielins, E., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2014; 134 (2): 193-200

  Abstract

  Stem cells are attractive candidates for the development of novel therapies, targeting indications that involve functional restoration of defective tissue. Although most stem cell therapies are new and highly experimental, there are clinics around the world that exploit vulnerable patients with the hope of offering supposed stem cell therapies, many of which operate without credible scientific merit, oversight, or other patient protection.We review the potential, as well as drawbacks, for incorporation of stem cells in cosmetic procedures. A review of FDA-approved indications and ongoing clinical trials with adipose stem cells is provided. Furthermore, a "snapshot" analysis of websites using the search terms "stem cell therapy" or "stem cell treatment" or "stem cell facelift" was performed.Despite the protective net cast by regulatory agencies such as the FDA and professional societies such as the American Society of Plastic Surgeons, we are witnessing worrying advertisements for procedures such as stem cell facelifts, stem cell breast augmentations, and even stem cell vaginal rejuvenation. The marketing and promotion of stem cell procedures in aesthetic surgery is not adequately supported by clinical evidence in the majority of cases.Stem cells offer tremendous potential, but the marketplace is saturated with unsubstantiated and sometimes fraudulent claims that may place patients at risk. With plastic surgeons at the forefront of stem cell-based regenerative medicine, it is critically important that we provide an example of a rigorous approach to research, data collection, and advertising of stem cell therapies.

  View details for DOI 10.1097/PRS.0000000000000404

  View details for PubMedID 24732654

• Studies in Fat Grafting: Part III. Fat Grafting Irradiated Tissue-Improved Skin Quality and Decreased Fat Graft Retention. Plastic and reconstructive surgery Garza, R. M., Paik, K. J., Chung, M. T., Duscher, D., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2014; 134 (2): 249-257

  Abstract

  Following radiation therapy, skin becomes fibrotic and can present a difficult problem for reconstructive surgeons. There is an increasing belief that fat grafting under irradiated skin can reverse the damage caused by radiation. The present study evaluated the effect of fat grafting on irradiated skin, along with fat graft quality and retention rates in irradiated tissue.Nine adult Crl:NU-Foxn1 CD-1 mice underwent 30-Gy external beam irradiation of the scalp. Four weeks after irradiation, scalp skin from irradiated and nonirradiated mice was harvested and compared histologically for dermal thickness, collagen content, and vascular density. Human fat grafts were then injected in the subcutaneous plane of the scalp. Skin assessment was performed in the irradiated group at 2 and 8 weeks after grafting, and fat graft retention was measured at baseline and every 2 weeks up to 8 weeks after grafting using micro-computed tomography. Finally, fat graft samples were explanted at 8 weeks, and quality scoring was performed.Fat grafting resulted in decreased dermal thickness, decreased collagen content, and increased vascular density in irradiated skin. Computed tomographic analysis revealed significantly decreased fat graft survival in the irradiated group compared with the nonirradiated group. Histologic scoring of explanted fat grafts demonstrated no difference in quality between the irradiated and nonirradiated groups.Fat grafting attenuates dermal collagen deposition and vessel depletion characteristic of radiation fibrosis. Although fat graft retention rates are significantly lower in irradiated than in nonirradiated tissue, the quality of retained fat between the groups is similar.

  View details for DOI 10.1097/PRS.0000000000000326

  View details for PubMedID 25068325

  View details for PubMedCentralID PMC4116637

• Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rinkevich, Y., Montoro, D. T., Muhonen, E., Walmsley, G. G., Lo, D., Hasegawa, M., Januszyk, M., Connolly, A. J., Weissman, I. L., Longaker, M. T. 2014; 111 (27): 9846-9851

  Abstract

  The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the digit tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated digit tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.

  View details for DOI 10.1073/pnas.1410097111

  View details for Web of Science ID 000338514800040

  View details for PubMedCentralID PMC4103362

• Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration. Proceedings of the National Academy of Sciences of the United States of America Rinkevich, Y., Montoro, D. T., Muhonen, E., Walmsley, G. G., Lo, D., Hasegawa, M., Januszyk, M., Connolly, A. J., Weissman, I. L., Longaker, M. T. 2014; 111 (27): 9846-9851

  Abstract

  The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the digit tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated digit tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.

  View details for DOI 10.1073/pnas.1410097111

  View details for PubMedID 24958860

• Studies in Fat Grafting: Part II. Effects of Injection Mechanics on Material Properties of Fat PLASTIC AND RECONSTRUCTIVE SURGERY Atashroo, D., Raphel, J., Chung, M. T., Paik, K. J., Parisi-Amon, A., McArdle, A., Senarath-Yapa, K., Zielins, E. R., Tevlin, R., Duldulao, C., Walmsley, G. G., Hu, M. S., Momeni, A., Domecus, B., Rimsa, J. R., Greenberg, L., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2014; 134 (1): 39-46

  Abstract

  Although fat grafting can address many soft-tissue deficits, results remain inconsistent. In this study, the authors compared physical properties of fat following injection using an automated, low-shear device or the modified Coleman technique.Lipoaspirate was obtained from nine patients and processed for injection using either a modified Coleman technique or an automated, low-shear device. Fat was passed through a 2-mm cannula and compared with minimally processed fat. A rheometer was used to measure the storage modulus and shear rate at which tissues began to lose their solid-like properties. Viscosity was also measured, and gross properties of treatment groups were evaluated qualitatively with a glass slide test.Fat injected through an automated, low-shear device closely matched physical properties of minimally processed fat. The storage modulus (G') of fat for the device group was greater than for the modified Coleman group, and the onset of breakdown was delayed. Similarly, viscosity measurement of fat from the automated device closely matched minimally processed fat and was greater than that of othe modified Coleman group.The physical properties of lipoaspirate processed using an automated, low-shear device with a 2-mm cannula preserved the intactness of fat more than the modified Coleman technique. The authors' rheologic data demonstrate less damage using an automated device compared with the modified Coleman technique and potentially support its use for improved fat graft integrity.

  View details for DOI 10.1097/PRS.0000000000000289

  View details for Web of Science ID 000338116400043

  View details for PubMedCentralID PMC4101917

• Studies in fat grafting: Part I. Effects of injection technique on in vitro fat viability and in vivo volume retention. Plastic and reconstructive surgery Chung, M. T., Paik, K. J., Atashroo, D. A., Hyun, J. S., McArdle, A., Senarath-Yapa, K., Zielins, E. R., Tevlin, R., Duldulao, C., Hu, M. S., Walmsley, G. G., Parisi-Amon, A., Momeni, A., Rimsa, J. R., Commons, G. W., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2014; 134 (1): 29-38

  Abstract

  Fat grafting has become increasingly popular for the correction of soft tissue deficits at many sites throughout the body. Long-term outcomes, however, depend on delivery of fat in the least traumatic fashion to optimize viability of the transplanted tissue. In this study, we compare the biologic properties of fat following injection using two methods.Lipoaspiration samples were obtained from five female donors and cellular viability, proliferation, and lipolysis were evaluated following injection using either a modified Coleman technique or an automated, low shear device. Comparisons were made to minimally processed, uninjected fat. Volume retention was also measured over twelve weeks following injection of fat under the scalp of immunodeficient mice using either the modified Coleman technique or the Adipose Tissue Injector. Finally, fat grafts were analyzed histologically.Fat viability and cellular proliferation were both significantly greater with the Adipose Tissue Injector relative to injection with the modified Coleman technique. In contrast, significantly less lipolysis was noted using the automated device. In vivo fat volume retention was significantly greater than with the modified Coleman technique at 4, 6, 8, and 12 week time points. This corresponded with significantly greater histological scores for healthy fat and lower scores for injury following injection with the device.Biological properties of injected tissues reflect how disruptive and harmful techniques for placement of fat may be, and our in vitro and in vivo data both support the use of the automated, low shear devices compared to the modified Coleman technique.

  View details for DOI 10.1097/PRS.0000000000000290

  View details for PubMedID 24622574

• Studies in fat grafting: Part II. Effects of injection mechanics on material properties of fat. Plastic and reconstructive surgery Atashroo, D., Raphel, J., Chung, M. T., Paik, K. J., Parisi-Amon, A., McArdle, A., Senarath-Yapa, K., Zielins, E. R., Tevlin, R., Duldulao, C., Walmsley, G. G., Hu, M. S., Momeni, A., Domecus, B., Rimsa, J. R., Greenberg, L., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2014; 134 (1): 39-46

  Abstract

  Although fat grafting can address many soft-tissue deficits, results remain inconsistent. In this study, the authors compared physical properties of fat following injection using an automated, low-shear device or the modified Coleman technique.Lipoaspirate was obtained from nine patients and processed for injection using either a modified Coleman technique or an automated, low-shear device. Fat was passed through a 2-mm cannula and compared with minimally processed fat. A rheometer was used to measure the storage modulus and shear rate at which tissues began to lose their solid-like properties. Viscosity was also measured, and gross properties of treatment groups were evaluated qualitatively with a glass slide test.Fat injected through an automated, low-shear device closely matched physical properties of minimally processed fat. The storage modulus (G') of fat for the device group was greater than for the modified Coleman group, and the onset of breakdown was delayed. Similarly, viscosity measurement of fat from the automated device closely matched minimally processed fat and was greater than that of othe modified Coleman group.The physical properties of lipoaspirate processed using an automated, low-shear device with a 2-mm cannula preserved the intactness of fat more than the modified Coleman technique. The authors' rheologic data demonstrate less damage using an automated device compared with the modified Coleman technique and potentially support its use for improved fat graft integrity.

  View details for DOI 10.1097/PRS.0000000000000289

  View details for PubMedID 25028817

• Tissue engineering and regenerative repair in wound healing. Annals of biomedical engineering Hu, M. S., Maan, Z. N., Wu, J., Rennert, R. C., Hong, W. X., Lai, T. S., Cheung, A. T., Walmsley, G. G., Chung, M. T., McArdle, A., Longaker, M. T., Lorenz, H. P. 2014; 42 (7): 1494-1507

  Abstract

  Wound healing is a highly evolved defense mechanism against infection and further injury. It is a complex process involving multiple cell types and biological pathways. Mammalian adult cutaneous wound healing is mediated by a fibroproliferative response leading to scar formation. In contrast, early to mid-gestational fetal cutaneous wound healing is more akin to regeneration and occurs without scar formation. This early observation has led to extensive research seeking to unlock the mechanism underlying fetal scarless regenerative repair. Building upon recent advances in biomaterials and stem cell applications, tissue engineering approaches are working towards a recapitulation of this phenomenon. In this review, we describe the elements that distinguish fetal scarless and adult scarring wound healing, and discuss current trends in tissue engineering aimed at achieving scarless tissue regeneration.

  View details for DOI 10.1007/s10439-014-1010-z

  View details for PubMedID 24788648

• Gene expression in fetal murine keratinocytes and fibroblasts JOURNAL OF SURGICAL RESEARCH Hu, M. S., Januszyk, M., Hong, W. X., Walmsley, G. G., Zielins, E. R., Atashroo, D. A., Maan, Z. N., McArdle, A., Takanishi, D. M., Gurtner, G. C., Longaker, M. T., Lorenz, H. P. 2014; 190 (1): 344-357

  Abstract

  Early fetuses heal wounds without the formation of a scar. Many studies have attempted to explain this remarkable phenomenon. However, the exact mechanism remains unknown. Herein, we examine the predominant cell types of the epidermis and dermis-the keratinocyte and fibroblast-during different stages of fetal development to better understand the changes that lead to scarring wound repair versus regeneration.Keratinocytes and fibroblasts were harvested and cultured from the dorsal skin of time-dated BALB/c fetuses. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was used to select genes with >2-fold expression differences with a false discovery rate <2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways.By comparing the gene expression profile of keratinocytes from E16 versus E18 fetuses, we identified 24 genes that were downregulated at E16. Analysis of E16 and E18 fibroblasts revealed 522 differentially expressed genes. Enrichment analysis showed the top 20 signaling pathways that were downregulated in E16 keratinocytes and upregulated or downregulated in E16 fibroblasts.Our data reveal 546 differentially expressed genes in keratinocytes and fibroblasts between the scarless and scarring transition. In addition, a total of 60 signaling pathways have been identified to be either upregulated or downregulated in these cell types. The genes and pathways recognized by our study may prove to be essential targets that may discriminate between fetal wound regeneration and adult wound repair.

  View details for DOI 10.1016/j.jss.2014.02.030

  View details for Web of Science ID 000338444700051

• Studies in Fat Grafting: Part I. Effects of Injection Technique on In Vitro Fat Viability and In Vivo Volume Retention PLASTIC AND RECONSTRUCTIVE SURGERY Chung, M. T., Paik, K. J., Atashroo, D. A., Hyun, J. S., McArdle, A., Senarath-Yapa, K., Zielins, E. R., Tevlin, R., Duldulao, C., Hu, M. S., Walmsley, G. G., Parisi-Amon, A., Momeni, A., Rimsa, J. R., Commons, G. W., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2014; 134 (1): 29-38

  Abstract

  Fat grafting has become increasingly popular for the correction of soft tissue deficits at many sites throughout the body. Long-term outcomes, however, depend on delivery of fat in the least traumatic fashion to optimize viability of the transplanted tissue. In this study, we compare the biologic properties of fat following injection using two methods.Lipoaspiration samples were obtained from five female donors and cellular viability, proliferation, and lipolysis were evaluated following injection using either a modified Coleman technique or an automated, low shear device. Comparisons were made to minimally processed, uninjected fat. Volume retention was also measured over twelve weeks following injection of fat under the scalp of immunodeficient mice using either the modified Coleman technique or the Adipose Tissue Injector. Finally, fat grafts were analyzed histologically.Fat viability and cellular proliferation were both significantly greater with the Adipose Tissue Injector relative to injection with the modified Coleman technique. In contrast, significantly less lipolysis was noted using the automated device. In vivo fat volume retention was significantly greater than with the modified Coleman technique at 4, 6, 8, and 12 week time points. This corresponded with significantly greater histological scores for healthy fat and lower scores for injury following injection with the device.Biological properties of injected tissues reflect how disruptive and harmful techniques for placement of fat may be, and our in vitro and in vivo data both support the use of the automated, low shear devices compared to the modified Coleman technique.

  View details for DOI 10.1097/PRS.0000000000000290

  View details for Web of Science ID 000338116400042

• Mechanotransduction and fibrosis JOURNAL OF BIOMECHANICS Duscher, D., Maan, Z. N., Wong, V. W., Rennert, R. C., Januszyk, M., Rodrigues, M., Hu, M., Whitmore, A. J., Whittam, A. J., Longaker, M. T., Gurtner, G. C. 2014; 47 (9): 1997-2005

  Abstract

  Scarring and tissue fibrosis represent a significant source of morbidity in the United States. Despite considerable research focused on elucidating the mechanisms underlying cutaneous scar formation, effective clinical therapies are still in the early stages of development. A thorough understanding of the various signaling pathways involved is essential to formulate strategies to combat fibrosis and scarring. While initial efforts focused primarily on the biochemical mechanisms involved in scar formation, more recent research has revealed a central role for mechanical forces in modulating these pathways. Mechanotransduction, which refers to the mechanisms by which mechanical forces are converted to biochemical stimuli, has been closely linked to inflammation and fibrosis and is believed to play a critical role in scarring. This review provides an overview of our current understanding of the mechanisms underlying scar formation, with an emphasis on the relationship between mechanotransduction pathways and their therapeutic implications.

  View details for DOI 10.1016/j.jbiomech.2014.03.031

  View details for Web of Science ID 000338621900009

• Fat or fiction: origins matter. Cell metabolism Wan, D. C., Longaker, M. T. 2014; 19 (6): 900-901

  Abstract

  Cell-lineage tracing has revealed a complex heterogeneity present in postnatal tissue and adult progenitors. Chau et al. (2014) and Long et al. (2014) provide further evidence for this among adipocytes, and their findings underscore the importance of cellular ontogeny not just for development but also for potential treatment of disease.

  View details for DOI 10.1016/j.cmet.2014.05.007

  View details for PubMedID 24896537

• Adipose-Derived Stem Cells: A Review of Signaling Networks Governing Cell Fate and Regenerative Potential in the Context of Craniofacial and Long Bone Skeletal Repair INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Senarath-Yapa, K., McArdle, A., Renda, A., Longaker, M. T., Quarto, N. 2014; 15 (6): 9314-9330

  Abstract

  Improvements in medical care, nutrition and social care are resulting in a commendable change in world population demographics with an ever increasing skew towards an aging population. As the proportion of the world's population that is considered elderly increases, so does the incidence of osteodegenerative disease and the resultant burden on healthcare. The increasing demand coupled with the limitations of contemporary approaches, have provided the impetus to develop novel tissue regeneration therapies. The use of stem cells, with their potential for self-renewal and differentiation, is one potential solution. Adipose-derived stem cells (ASCs), which are relatively easy to harvest and readily available have emerged as an ideal candidate. In this review, we explore the potential for ASCs to provide tangible therapies for craniofacial and long bone skeletal defects, outline key signaling pathways that direct these cells and describe how the developmental signaling program may provide clues on how to guide these cells in vivo. This review also provides an overview of the importance of establishing an osteogenic microniche using appropriately customized scaffolds and delineates some of the key challenges that still need to be overcome for adult stem cell skeletal regenerative therapy to become a clinical reality.

  View details for DOI 10.3390/ijms15069314

  View details for Web of Science ID 000338639000009

• Moisturizing different racial skin types. The Journal of clinical and aesthetic dermatology Wan, D. C., Wong, V. W., Longaker, M. T., Yang, G. P., Wei, F. 2014; 7 (6): 25-32

  Abstract

  The skin is a complex organ involved in thermoregulation, gas exchange, protection against pathogens, and barrier function to maintain proper hydration. When dry, the ability for skin to execute these tasks becomes impaired. Dry skin affects almost everyone as we age, but it is also dependent on external factors, such as dry climate, colder temperatures, and repeated washing. In addition, increasing evidence has shown racial variability in the physiological properties of skin, which directly impacts water content of the stratum corneum and sensitivity to exogenously applied agents. A multitude of products have been developed to treat dry skin, and as a group, moisturizers have been designed to either impart or restore hydration in the stratum corneum. Given the large number of moisturizers presently available, depending on individual components, several different mechanisms may be employed to promote skin hydration. As there exists dramatic racial variability in skin properties, certain moisturizers may thus be more effective in some and less effective in others to treat the common condition of dry skin.

  View details for PubMedID 25013536

• Adipose-derived stem cells: a review of signaling networks governing cell fate and regenerative potential in the context of craniofacial and long bone skeletal repair. International journal of molecular sciences Senarath-Yapa, K., McArdle, A., Renda, A., Longaker, M. T., Quarto, N. 2014; 15 (6): 9314-30

  Abstract

  Improvements in medical care, nutrition and social care are resulting in a commendable change in world population demographics with an ever increasing skew towards an aging population. As the proportion of the world's population that is considered elderly increases, so does the incidence of osteodegenerative disease and the resultant burden on healthcare. The increasing demand coupled with the limitations of contemporary approaches, have provided the impetus to develop novel tissue regeneration therapies. The use of stem cells, with their potential for self-renewal and differentiation, is one potential solution. Adipose-derived stem cells (ASCs), which are relatively easy to harvest and readily available have emerged as an ideal candidate. In this review, we explore the potential for ASCs to provide tangible therapies for craniofacial and long bone skeletal defects, outline key signaling pathways that direct these cells and describe how the developmental signaling program may provide clues on how to guide these cells in vivo. This review also provides an overview of the importance of establishing an osteogenic microniche using appropriately customized scaffolds and delineates some of the key challenges that still need to be overcome for adult stem cell skeletal regenerative therapy to become a clinical reality.

  View details for DOI 10.3390/ijms15069314

  View details for PubMedID 24865492

• In Vivo clonal analysis reveals lineage-restricted progenitor characteristics in Mammalian kidney development, maintenance, and regeneration. Cell reports Rinkevich, Y., Montoro, D. T., Contreras-Trujillo, H., Harari-Steinberg, O., Newman, A. M., Tsai, J. M., Lim, X., Van-Amerongen, R., Bowman, A., Januszyk, M., Pleniceanu, O., Nusse, R., Longaker, M. T., Weissman, I. L., Dekel, B. 2014; 7 (4): 1270-1283

  Abstract

  The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs) into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.

  View details for DOI 10.1016/j.celrep.2014.04.018

  View details for PubMedID 24835991

  View details for PubMedCentralID PMC4425291

• Tracking the elusive fibrocyte: identification and characterization of collagen-producing hematopoietic lineage cells during murine wound healing. Stem cells Suga, H., Rennert, R. C., Rodrigues, M., Sorkin, M., Glotzbach, J. P., Januszyk, M., Fujiwara, T., Longaker, M. T., Gurtner, G. C. 2014; 32 (5): 1347-1360

  Abstract

  Fibrocytes are a unique population of circulating cells reported to exhibit characteristics of both hematopoietic and mesenchymal cells, and play an important role in wound healing. However, putative fibrocytes have been found to lose expression of hematopoietic surface markers such as CD45 during differentiation, making it difficult to track these cells in vivo with conventional methodologies. In this study, to distinguish hematopoietic and nonhematopoietic cells without surface markers, we took advantage of the gene vav 1, which is expressed solely on hematopoietic cells but not on other cell types, and established a novel transgenic mouse, in which hematopoietic cells are irreversibly labeled with green fluorescent protein and nonhematopoietic cells with red fluorescent protein. Use of single-cell transcriptional analysis in this mouse model revealed two discrete types of collagen I (Col I) expressing cells of hematopoietic lineage recruited into excisional skin wounds. We confirmed this finding on a protein level, with one subset of these Col I synthesizing cells being CD45+ and CD11b+, consistent with the traditional definition of a fibrocyte, while another was CD45- and Cd11b-, representing a previously unidentified population. Both cell types were found to initially peak, then reduce posthealing, consistent with a disappearance from the wound site and not a loss of identifying surface marker expression. Taken together, we have unambiguously identified two cells of hematopoietic origin that are recruited to the wound site and deposit collagen, definitively confirming the existence and natural time course of fibrocytes in cutaneous healing. Stem Cells 2014;32:1347-1360.

  View details for DOI 10.1002/stem.1648

  View details for PubMedID 24446236

• The Role of Hypoxia-Inducible Factor in Wound Healing ADVANCES IN WOUND CARE Hong, W., Hu, M. S., Esquivel, M., Liang, G. Y., Rennert, R. C., McArdle, A., Paik, K. J., Duscher, D., Gurtner, G. C., Lorenz, H., Longaker, M. T. 2014; 3 (5): 390–99

  View details for DOI 10.1089/wound.2013.0520

  View details for Web of Science ID 000214959300004

• The Role of Hypoxia-Inducible Factor in Wound Healing. Advances in wound care Hong, W. X., Hu, M. S., Esquivel, M., Liang, G. Y., Rennert, R. C., McArdle, A., Paik, K. J., Duscher, D., Gurtner, G. C., Lorenz, H. P., Longaker, M. T. 2014; 3 (5): 390-399

  Abstract

  Significance: Poor wound healing remains a significant health issue for a large number of patients in the United States. The physiologic response to local wound hypoxia plays a critical role in determining the success of the normal healing process. Hypoxia-inducible factor-1 (HIF-1), as the master regulator of oxygen homeostasis, is an important determinant of healing outcomes. HIF-1 contributes to all stages of wound healing through its role in cell migration, cell survival under hypoxic conditions, cell division, growth factor release, and matrix synthesis throughout the healing process. Recent Advances: Positive regulators of HIF-1, such as prolyl-4-hydroxylase inhibitors, have been shown to be beneficial in enhancing diabetic ischemic wound closure and are currently undergoing clinical trials for treatment of several human-ischemia-based conditions. Critical Issues: HIF-1 deficiency and subsequent failure to respond to hypoxic stimuli leads to chronic hypoxia, which has been shown to contribute to the formation of nonhealing ulcers. In contrast, overexpression of HIF-1 has been implicated in fibrotic disease through its role in increasing myofibroblast differentiation leading to excessive matrix production and deposition. Both positive and negative regulators of HIF-1 therefore provide important therapeutic targets that can be used to manipulate HIF-1 expression where an excess or deficiency in HIF-1 is known to correlate with pathogenesis. Future Directions: Targeting HIF-1 during wound healing has many important clinical implications for tissue repair. Counteracting the detrimental effects of excessive or deficient HIF-1 signaling by modulating HIF-1 expression may improve future management of poorly healing wounds.

  View details for PubMedID 24804159

• Fat Transfer in 2014: What We Do Not Know PLASTIC AND RECONSTRUCTIVE SURGERY Longaker, M. T., Aston, S. J., Baker, D. C., Rohrich, R. J. 2014; 133 (5): 1305–7

  View details for PubMedID 24776560

• Mechanical offloading of incisional wounds is associated with transcriptional downregulation of inflammatory pathways in a large animal model ORGANOGENESIS Januszyk, M., Wong, V. W., Bhatt, K. A., Vial, I. N., Paterno, J., Longaker, M. T., Gurtner, G. C. 2014; 10 (2): 186-193

  Abstract

  Cutaneous scarring is a major source of morbidity and current therapies to mitigate scar formation remain ineffective. Although wound fibrosis and inflammation are highly linked, only recently have mechanical forces been implicated in these pathways. Our group has developed a topical polymer device that significantly reduces post-injury scar formation via the manipulation of mechanical forces. Here we extend these studies to examine the genomewide transcriptional effects of mechanomodulation during scar formation using a validated large animal model, the red Duroc pig. We demonstrate that mechanical loading of incisional wounds upregulates expression of genes associated with inflammatory and fibrotic pathways, and that device-mediated offloading of these wounds reverses these effects. Validation studies are needed to clarify the clinical significance of these findings.

  View details for DOI 10.4161/org.28818

  View details for Web of Science ID 000341807300005

  View details for PubMedID 24739276

  View details for PubMedCentralID PMC4154952

• LOW DOSE RADIATION FROM CARDIAC COMPUTED TOMOGRAPHY IS ASSOCIATED WITH DNA DAMAGE AND CELLULAR DEATH Hong, W., Lee, W., Liang, G., Chan, C., Sanchez-Freire, V., Hu, S., Longaker, M. T., Wu, J., Nguyen, P. ELSEVIER SCIENCE INC. 2014: A1047

  View details for DOI 10.1016/S0735-1097(14)61047-0

  View details for Web of Science ID 000359579101705

• The Role of Stem Cells During Scarless Skin Wound Healing. Advances in wound care Hu, M. S., Rennert, R. C., McArdle, A., Chung, M. T., Walmsley, G. G., Longaker, M. T., Lorenz, H. P. 2014; 3 (4): 304-314

  Abstract

  Significance: In early gestation, fetal skin wounds undergo regeneration and healing without a scar. This phenomenon is intrinsic to early fetal skin but disappears during late gestation. Adult wounds undergo repair via a fibroproliferative response that leads to incomplete regeneration of the original tissue and a resultant scar. This outcome can have devastating effects for patients and is a significant financial burden to the healthcare system. Recent Advances: Studies have demonstrated the possible role of several stem cells in wound healing. In particular, epidermal stem cells and mesenchymal stem cells have been implicated in wound repair and regeneration. Recently, stem cells with adult epidermal stem cell markers have been found in fetal skin dermis. These cells are thought to play a role in scarless fetal wound healing. Critical Issues: Despite numerous studies on scarless fetal wound healing, the exact mechanism is still largely unknown. Although inflammation is greatly reduced, the stem cell profile of regenerating fetal skin wounds remains unknown. Without a detailed understanding of stem cell differences between fetal and adult wounds, the ability to prevent or treat both normal and pathologic excessive scarring, in the form of keloids and hypertrophic scars, is limited. Future Directions: Further studies on differences between fetal and adult skin-specific stem cells may elucidate the mechanism of scarless wound healing in the early fetus. With this knowledge, the potential to reduce scarring in adult wounds may be achieved.

  View details for PubMedID 24761362

• The Role of Stem Cells During Scarless Skin Wound Healing ADVANCES IN WOUND CARE Hu, M., Rennert, R. C., McArdle, A., Chung, M. T., Walmsley, G. G., Longaker, M. T., Lorenz, H. 2014; 3 (4): 304–14

  View details for DOI 10.1089/wound.2013.0471

  View details for Web of Science ID 000214959200002

• Mechanotransduction and fibrosis. Journal of biomechanics Duscher, D., Maan, Z. N., Wong, V. W., Rennert, R. C., Januszyk, M., Rodrigues, M., Hu, M., Whitmore, A. J., Whittam, A. J., Longaker, M. T., Gurtner, G. C. 2014

  Abstract

  Scarring and tissue fibrosis represent a significant source of morbidity in the United States. Despite considerable research focused on elucidating the mechanisms underlying cutaneous scar formation, effective clinical therapies are still in the early stages of development. A thorough understanding of the various signaling pathways involved is essential to formulate strategies to combat fibrosis and scarring. While initial efforts focused primarily on the biochemical mechanisms involved in scar formation, more recent research has revealed a central role for mechanical forces in modulating these pathways. Mechanotransduction, which refers to the mechanisms by which mechanical forces are converted to biochemical stimuli, has been closely linked to inflammation and fibrosis and is believed to play a critical role in scarring. This review provides an overview of our current understanding of the mechanisms underlying scar formation, with an emphasis on the relationship between mechanotransduction pathways and their therapeutic implications.

  View details for DOI 10.1016/j.jbiomech.2014.03.031

  View details for PubMedID 24709567

• Abstract 33: Stress Offloading through Mechanomodulation is Associated with Down-Regulation of Inflammatory Pathways in a Large Animal Model. Plastic and reconstructive surgery Januszyk, M., Wong, V. W., Bhatt, K., Vial, I. N., Dauskardt, R., Longaker, M. T., Gurtner, G. C. 2014; 133 (3): 44-?

  View details for DOI 10.1097/01.prs.0000445066.09978.3b

  View details for PubMedID 25942144

• Abstract 8: SDF-1 Regulates Adipose Niche Homeostasis and Adipose Derived Stromal Cell Function. Plastic and reconstructive surgery Maan, Z. N., Rennert, R. C., Duscher, D., Januszyk, M., Paik, K., Chung, M. T., Paik, K., Fujiwara, T., Rodrigues, M., Ho, N., Baker, H., Perez, M., Hu, M., Sorkin, M., Longaker, M. T., Gurtner, G. C. 2014; 133 (3): 15-16

  View details for DOI 10.1097/01.prs.0000444941.23852.ce

  View details for PubMedID 25942119

• Abstract 10: Global and Endothelial Cell Specific Deletion of SDF-1 Results in Delayed Wound Healing. Plastic and reconstructive surgery Maan, Z. N., Ho, N., Rennert, R. C., Duscher, D., Sorkin, M., Rodrigues, M., Chen, J., Vial, I. N., Januszyk, M., Findlay, M., Hu, M., Walmsley, G., Longaker, M. T., Gurtner, G. C. 2014; 133 (3): 20-?

  View details for DOI 10.1097/01.prs.0000444963.66915.ba

  View details for PubMedID 25942121

• Abstract 135: improved engraftment of autologous skin grafts in diabetic mice with adipose-derived stem cells. Plastic and reconstructive surgery Hu, M., Hong, W. X., Senarath-Yapa, K., Zimmermann, A., Chung, M., Esquivel, M., McArdle, A., Walmsley, G., Maan, Z., Garza, R., Lorenz, H. P., Longaker, M. 2014; 133 (3): 151-?

  View details for DOI 10.1097/01.prs.0000444962.89785.8e

  View details for PubMedID 25942246

• Abstract 161: identification of cell-intrinsic mechanisms and differentially regulated genetic pathways responsible for the age-related functional decline in aged skeletal stem cells. Plastic and reconstructive surgery McArdle, A., Chan, C., Seita, J., Senarath-Yapa, K., Hu, M., Walmsley, G. G., Zielins, E., Atashroo, D., Tevlin, R., Weissman, I., Longaker, M. T. 2014; 133 (3): 178-?

  View details for DOI 10.1097/01.prs.0000444990.75431.f1

  View details for PubMedID 25942271

• Abstract 140: identification, characterization, and prospective isolation of a fibroblast lineage contributing to dermal development, cutaneous scarring, radiation fibrosis, and cancer stroma. Plastic and reconstructive surgery Walmsley, G. G., Rinkevich, Y., Hu, M. S., McArdle, A., Maan, Z. N., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2014; 133 (3): 157-?

  View details for DOI 10.1097/01.prs.0000444968.20280.4d

  View details for PubMedID 25942251

• Abstract 165: Enhanced Adipose-Derived Stromal Cell Osteogenesis through Surface Marker Enrichment and BMP Modulation using Magnet-assisted Transfection. Plastic and reconstructive surgery Chung, M. T., Morrison, S. D., Paik, K. J., McArdle, A., Walmsley, G., Senarath-Yapa, K., Hu, M. S., Tevlin, R., Zielins, E., Atashroo, D., Hong, W. X., Duldulao, C., Wearda, T., Garza, R. M., Momeni, A., Longaker, M. T., Wan, D. C. 2014; 133 (3): 181-182

  View details for DOI 10.1097/01.prs.0000444994.28797.34

  View details for PubMedID 25942275

• Abstract 54: TGF Beta and BMP Signaling Pathways Influence Regenerative Capacity of Calvarial Bones via Cross-Talk and Modulation of Apoptosis: The Potential Therapeutic Role of Small Molecule Inhibitors of TGF Beta Signaling. Plastic and reconstructive surgery Senarath-Yapa, K., Meyer, N., Li, S., Longaker, M. T., Quarto, N. 2014; 133 (3): 65-66

  View details for DOI 10.1097/01.prs.0000445087.68975.92

  View details for PubMedID 25942165

• Abstract 151: short hairpin RNA interference therapy for diabetic murine wound closure and hindlimb ischemia. Plastic and reconstructive surgery Paik, K. J., Rennert, R., Chung, M. T., Sorkin, M., Duscher, D., Atashroo, D., Chen, H., Morrison, S. D., Zimmermann, A., Nauta, A., Ko, S., Tevlin, R., Zielins, E., Hu, M. S., McArdle, A., Walmsley, G., Senarath-Yapa, K., Hong, W. X., Garza, R. M., Duldulao, C., Wearda, T., Momeni, A., Wu, J. C., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2014; 133 (3): 167-168

  View details for DOI 10.1097/01.prs.0000444979.14443.08

  View details for PubMedID 25942261

• Abstract 158: Identification of BMP-Responsive Long Noncoding RNAs in Pluripotent Cells. Plastic and reconstructive surgery Paik, K. J., Qu, K., Hsueh, B., Torre, E. A., Flynn, R. A., Chung, M. T., Spencley, A., Wang, K. C., Wu, J. C., Longaker, M. T., Chang, H. Y., Wan, D. C. 2014; 133 (3): 174-?

  View details for DOI 10.1097/01.prs.0000444987.90678.b2

  View details for PubMedID 25942268

• Induced pluripotent stem cells in regenerative medicine and disease modeling. Current stem cell research & therapy Walmsley, G. G., Hyun, J., McArdle, A., Senarath-Yapa, K., Hu, M. S., Chung, M. T., Wong, V. W., Longaker, M. T., Wan, D. C. 2014; 9 (2): 73-81

  Abstract

  In 2006, Dr. Yamanaka created the induced pluripotent stem cell (iPSC) by reprogramming adult fibroblasts back to an immature, pluripotent state. Effectively bypassing the ethical constraints of human embryonic stem cells, iPSCs have expanded the horizons of regenerative medicine by offering a means to derive autologous patient-matched cells and tissues for clinical transplantation. However, persisting safety concerns must be addressed prior to their widespread clinical application. In this review, we discuss the history of iPSCs, derivation strategies, and current research involving gene therapy and disease modeling. We review the potential of iPSCs for improving a range of cell-based therapies and obstacles to their clinical implementation.

  View details for PubMedID 24359141

• Gene expression in fetal murine keratinocytes and fibroblasts. The Journal of surgical research Hu, M. S., Januszyk, M., Hong, W. X., Walmsley, G. G., Zielins, E. R., Atashroo, D. A., Maan, Z. N., McArdle, A., Takanishi, D. M., Gurtner, G. C., Longaker, M. T., Lorenz, H. P. 2014

  Abstract

  Early fetuses heal wounds without the formation of a scar. Many studies have attempted to explain this remarkable phenomenon. However, the exact mechanism remains unknown. Herein, we examine the predominant cell types of the epidermis and dermis-the keratinocyte and fibroblast-during different stages of fetal development to better understand the changes that lead to scarring wound repair versus regeneration.Keratinocytes and fibroblasts were harvested and cultured from the dorsal skin of time-dated BALB/c fetuses. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was used to select genes with >2-fold expression differences with a false discovery rate <2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways.By comparing the gene expression profile of keratinocytes from E16 versus E18 fetuses, we identified 24 genes that were downregulated at E16. Analysis of E16 and E18 fibroblasts revealed 522 differentially expressed genes. Enrichment analysis showed the top 20 signaling pathways that were downregulated in E16 keratinocytes and upregulated or downregulated in E16 fibroblasts.Our data reveal 546 differentially expressed genes in keratinocytes and fibroblasts between the scarless and scarring transition. In addition, a total of 60 signaling pathways have been identified to be either upregulated or downregulated in these cell types. The genes and pathways recognized by our study may prove to be essential targets that may discriminate between fetal wound regeneration and adult wound repair.

  View details for DOI 10.1016/j.jss.2014.02.030

  View details for PubMedID 24726057

• Paracrine Mechanism of Angiogenesis in Adipose-Derived Stem Cell Transplantation ANNALS OF PLASTIC SURGERY Suga, H., Glotzbach, J. P., Sorkin, M., Longaker, M. T., Gurtner, G. C. 2014; 72 (2): 234-241

  Abstract

  INTRODUCTION: Adipose-derived stem cells (ASCs) have shown potential for cell-based therapy in the field of plastic surgery. However, the fate of ASCs after transplantation and the mechanism(s) of their biologic capabilities remain unclear. METHODS: We isolated and cultured ASCs from transgenic mice that express both luciferase and green fluorescent protein and injected the cells into the inguinal fat pads of wild-type mice. We tested 4 experimental groups, namely, ischemic fat pads with/without ASCs and control fat pads with/without ASCs. RESULTS: Transplanted ASCs were tracked with bioluminescence imaging. The luminescence gradually decreased over 28 days, indicating cell death after transplantation. More ASCs were retained in ischemic fat pads on day 7 compared to control fat pads. On day 14, adipose tissue vascular density was higher in the ASC transplantation groups compared to those without ASCs. On day 28, there was decreased atrophy of adipose tissue in ASC-treated ischemic fat pads. Transplanted ASCs were detected as nonproliferating green fluorescent protein-positive cells, whereas native endothelial cells adjacent to the transplanted ASCs were proliferative. Protein analysis demonstrated higher expression of hepatocyte growth factor and vascular endothelial growth factor in the ASC transplantation groups, suggesting a paracrine mechanism, which was confirmed by in vitro experiments with conditioned media from ASCs. CONCLUSIONS: Transplanted ASCs are preferentially retained in ischemic adipose tissue, although most of the cells eventually undergo cell death. They exert an angiogenic effect on adipose tissue mainly through a paracrine mechanism. Increased understanding of these effects will help develop ASCs as a tool for cell-based therapy.

  View details for DOI 10.1097/SAP.0b013e318264fd6a

  View details for Web of Science ID 000338013700024

• The embrace Device Significantly Decreases Scarring following Scar Revision Surgery in a Randomized Controlled Trial. Plastic and reconstructive surgery Lim, A. F., Weintraub, J., Kaplan, E. N., Januszyk, M., Cowley, C., McLaughlin, P., Beasley, B., Gurtner, G. C., Longaker, M. T. 2014; 133 (2): 398-405

  Abstract

  Mechanically offloading or shielding an incision significantly reduces scarring in both animal and first-in-human studies. Whether or not this strategy would be effective following scar revision surgery was previously unknown. In this article, the authors report that the embrace device, which uses principles of mechanomodulation, significantly improves aesthetic outcomes following scar revision surgery.A prospective, open-label, randomized, single-center study was conducted to evaluate the appearance of scars following revision and embrace treatment. Revision surgery was performed on 12 patients, each acting as his or her own control, and outcomes were assessed at 6 months. A visual analogue scale was used to evaluate each scar, rated by four independent surgeons who were not involved in the study.Evaluation of 6-month scar images by four independent surgeons using the visual analogue scale demonstrated a highly significant improvement in scar appearance following embrace treatment (p < 0.005).The embrace device represents a powerful new technology for significantly improving scar appearance following revision surgery.Therapeutic, II.

  View details for DOI 10.1097/01.prs.0000436526.64046.d0

  View details for PubMedID 24105084

• Concurrent Generation of Functional Smooth Muscle and Endothelial Cells via a Vascular Progenitor. Stem cells translational medicine Marchand, M., Anderson, E. K., Phadnis, S. M., Longaker, M. T., Cooke, J. P., Chen, B., Reijo Pera, R. A. 2014; 3 (1): 91-97

  Abstract

  Smooth muscle cells (SMCs) and endothelial cells (ECs) are typically derived separately, with low efficiencies, from human pluripotent stem cells (hPSCs). The concurrent generation of these cell types might lead to potential applications in regenerative medicine to model, elucidate, and eventually treat vascular diseases. Here we report a robust two-step protocol that can be used to simultaneously generate large numbers of functional SMCs and ECs from a common proliferative vascular progenitor population via a two-dimensional culture system. We show here that coculturing hPSCs with OP9 cells in media supplemented with vascular endothelial growth factor, basic fibroblast growth factor, and bone morphogenetic protein 4 yields a higher percentage of CD31(+)CD34(+) cells on day 8 of differentiation. Upon exposure to endothelial differentiation media and SM differentiation media, these vascular progenitors were able to differentiate and mature into functional endothelial cells and smooth muscle cells, respectively. Furthermore, we were able to expand the intermediate population more than a billionfold to generate sufficient numbers of ECs and SMCs in parallel for potential therapeutic transplantations.

  View details for DOI 10.5966/sctm.2013-0124

  View details for PubMedID 24311701

  View details for PubMedCentralID PMC3902294

• Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance. Nature communications de Almeida, P. E., Meyer, E. H., Kooreman, N. G., Diecke, S., Dey, D., Sanchez-Freire, V., Hu, S., Ebert, A., Odegaard, J., Mordwinkin, N. M., Brouwer, T. P., Lo, D., Montoro, D. T., Longaker, M. T., Negrin, R. S., Wu, J. C. 2014; 5: 3903-?

  Abstract

  The exact nature of the immune response elicited by autologous-induced pluripotent stem cell (iPSC) progeny is still not well understood. Here we show in murine models that autologous iPSC-derived endothelial cells (iECs) elicit an immune response that resembles the one against a comparable somatic cell, the aortic endothelial cell (AEC). These cells exhibit long-term survival in vivo and prompt a tolerogenic immune response characterized by elevated IL-10 expression. In contrast, undifferentiated iPSCs elicit a very different immune response with high lymphocytic infiltration and elevated IFN-γ, granzyme-B and perforin intragraft. Furthermore, the clonal structure of infiltrating T cells from iEC grafts is statistically indistinguishable from that of AECs, but is different from that of undifferentiated iPSC grafts. Taken together, our results indicate that the differentiation of iPSCs results in a loss of immunogenicity and leads to the induction of tolerance, despite expected antigen expression differences between iPSC-derived versus original somatic cells.

  View details for DOI 10.1038/ncomms4903

  View details for PubMedID 24875164

• Wound healing: an update. Regenerative medicine Zielins, E. R., Atashroo, D. A., Maan, Z. N., Duscher, D., Walmsley, G. G., Hu, M., Senarath-Yapa, K., McArdle, A., Tevlin, R., Wearda, T., Paik, K. J., Duldulao, C., Hong, W. X., Gurtner, G. C., Longaker, M. T. 2014; 9 (6): 817-830

  Abstract

  Wounds, both chronic and acute, continue to be a tremendous socioeconomic burden. As such, technologies drawn from many disciplines within science and engineering are constantly being incorporated into innovative wound healing therapies. While many of these therapies are experimental, they have resulted in new insights into the pathophysiology of wound healing, and in turn the development of more specialized treatments for both normal and abnormal wound healing states. Herein, we review some of the emerging technologies that are currently being developed to aid and improve wound healing after cutaneous injury.

  View details for DOI 10.2217/rme.14.54

  View details for PubMedID 25431917

• Skeletal Tissue Engineering PRINCIPLES OF TISSUE ENGINEERING, 4TH EDITION Montoro, D. T., Wan, D. C., Longaker, M. T., Lanza, R., Langer, R., Vacanti, J. 2014: 1289–1302

  View details for DOI 10.1016/B978-0-12-398358-9.00060-4

  View details for Web of Science ID 000427825200065

• Epidermal or dermal specific knockout of PHD-2 enhances wound healing and minimizes ischemic injury. PloS one Zimmermann, A. S., Morrison, S. D., Hu, M. S., Li, S., Nauta, A., Sorkin, M., Meyer, N. P., Walmsley, G. G., Maan, Z. N., Chan, D. A., Gurtner, G. C., Giaccia, A. J., Longaker, M. T. 2014; 9 (4)

  Abstract

  Hypoxia-inducible factor (HIF)-1α, part of the heterodimeric transcription factor that mediates the cellular response to hypoxia, is critical for the expression of multiple angiogenic growth factors, cell motility, and the recruitment of endothelial progenitor cells. Inhibition of the oxygen-dependent negative regulator of HIF-1α, prolyl hydroxylase domain-2 (PHD-2), leads to increased HIF-1α and mimics various cellular and physiological responses to hypoxia. The roles of PHD-2 in the epidermis and dermis have not been clearly defined in wound healing.Epidermal and dermal specific PHD-2 knockout (KO) mice were developed in a C57BL/6J (wild type) background by crossing homozygous floxed PHD-2 mice with heterozygous K14-Cre mice and heterozygous Col1A2-Cre-ER mice to get homozygous floxed PHD-2/heterozygous K14-Cre and homozygous floxed PHD-2/heterozygous floxed Col1A2-Cre-ER mice, respectively. Ten to twelve-week-old PHD-2 KO and wild type (WT) mice were subjected to wounding and ischemic pedicle flap model. The amount of healing was grossly quantified with ImageJ software. Western blot and qRT-PCR was run on protein and RNA from primary cells cultured in vitro.qRT-PCR demonstrated a significant decrease of PHD-2 in keratinocytes and fibroblasts derived from tissue specific KO mice relative to control mice (*p<0.05). Western blot analysis showed a significant increase in HIF-1α and VEGF protein levels in PHD-2 KO mice relative to control mice (*p<0.05). PHD-2 KO mice showed significantly accelerated wound closure relative to WT (*p<0.05). When ischemia was analyzed at day nine post-surgery in a flap model, the PHD-2 tissue specific knockout mice showed significantly more viable flaps than WT (*p<0.05).PHD-2 plays a significant role in the rates of wound healing and response to ischemic insult in mice. Further exploration shows PHD-2 KO increases cellular levels of HIF-1α and this increase leads to the transcription of downstream angiogenic factors such as VEGF.

  View details for DOI 10.1371/journal.pone.0093373

  View details for PubMedID 24695462

  View details for PubMedCentralID PMC3973687

• Osteoclast derivation from mouse bone marrow. Journal of visualized experiments : JoVE Tevlin, R., McArdle, A., Chan, C. K., Pluvinage, J., Walmsley, G. G., Wearda, T., Marecic, O., Hu, M. S., Paik, K. J., Senarath-Yapa, K., Atashroo, D. A., Zielins, E. R., Wan, D. C., Weissman, I. L., Longaker, M. T. 2014

  View details for DOI 10.3791/52056

  View details for PubMedID 25407120

• Osteoclast derivation from mouse bone marrow. Journal of visualized experiments : JoVE Tevlin, R., McArdle, A., Chan, C. K., Pluvinage, J., Walmsley, G. G., Wearda, T., Marecic, O., Hu, M. S., Paik, K. J., Senarath-Yapa, K., Atashroo, D. A., Zielins, E. R., Wan, D. C., Weissman, I. L., Longaker, M. T. 2014

  Abstract

  Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease. As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation. An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

  View details for DOI 10.3791/52056

  View details for PubMedID 25407120

• Wound healing: an update REGENERATIVE MEDICINE Zielins, E. R., Atashroo, D. A., Maan, Z. N., Duscher, D., Walmsley, G. G., Marecic, O., Hu, M., Senarath-Yapa, K., McArdle, A., Tevlin, R., Wearda, T., Paik, K. J., Duldulao, C., Hong, W. X., Gurtner, G. C., Longaker, M. T. 2014; 9 (6): 817-830

  Abstract

  Wounds, both chronic and acute, continue to be a tremendous socioeconomic burden. As such, technologies drawn from many disciplines within science and engineering are constantly being incorporated into innovative wound healing therapies. While many of these therapies are experimental, they have resulted in new insights into the pathophysiology of wound healing, and in turn the development of more specialized treatments for both normal and abnormal wound healing states. Herein, we review some of the emerging technologies that are currently being developed to aid and improve wound healing after cutaneous injury.

  View details for DOI 10.2217/RME.14.54

  View details for Web of Science ID 000345620600012

• Epidermal or Dermal Specific Knockout of PHD-2 Enhances Wound Healing and Minimizes Ischemic Injury. PloS one Zimmermann, A. S., Morrison, S. D., Hu, M. S., Li, S., Nauta, A., Sorkin, M., Meyer, N. P., Walmsley, G. G., Maan, Z. N., Chan, D. A., Gurtner, G. C., Giaccia, A. J., Longaker, M. T. 2014; 9 (4)

  View details for DOI 10.1371/journal.pone.0093373

  View details for PubMedID 24695462

• Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance. Nature communications de Almeida, P. E., Meyer, E. H., Kooreman, N. G., Diecke, S., Dey, D., Sanchez-Freire, V., Hu, S., Ebert, A., Odegaard, J., Mordwinkin, N. M., Brouwer, T. P., Lo, D., Montoro, D. T., Longaker, M. T., Negrin, R. S., Wu, J. C. 2014; 5: 3903-?

  View details for DOI 10.1038/ncomms4903

  View details for PubMedID 24875164

• Biological therapies for the treatment of cutaneous wounds: Phase III and launched therapies EXPERT OPINION ON BIOLOGICAL THERAPY Rennert, R. C., Rodrigues, M., Wong, V. W., Duscher, D., Hu, M., Maan, Z., Sorkin, M., Gurtner, G. C., Longaker, M. T. 2013; 13 (11): 1523-1541

  Abstract

  Normal wound healing mechanisms can be overwhelmed in the setting of complex acute and chronic tissue injury. Biological therapies are designed to augment and/or restore the body's natural wound healing abilities. There are a variety of available and emerging technologies utilizing this approach that have demonstrated the ability to augment wound healing.In this review, the clinical data on launched and emerging biological therapies for wound healing applications are summarized. The methodologies discussed include biological skin equivalents, growth factors/small molecules and stem cell-based therapies.While many products possess convincing clinical data demonstrating their efficacy in comparison to standard treatment options, more robust, controlled studies are needed to determine the relative value among established and emerging biological therapies. Future bioengineering and stem cell-based approaches are of particular interest due to the simultaneous correction of multiple deficiencies present in the nonhealing wound.

  View details for DOI 10.1517/14712598.2013.842972

  View details for Web of Science ID 000325712100007

  View details for PubMedID 24093722

• Biological therapies for the treatment of cutaneous wounds: phase III and launched therapies. Expert opinion on biological therapy Rennert, R. C., Rodrigues, M., Wong, V. W., Duscher, D., Hu, M., Maan, Z., Sorkin, M., Gurtner, G. C., Longaker, M. T. 2013; 13 (11): 1523-1541

  Abstract

  Normal wound healing mechanisms can be overwhelmed in the setting of complex acute and chronic tissue injury. Biological therapies are designed to augment and/or restore the body's natural wound healing abilities. There are a variety of available and emerging technologies utilizing this approach that have demonstrated the ability to augment wound healing.In this review, the clinical data on launched and emerging biological therapies for wound healing applications are summarized. The methodologies discussed include biological skin equivalents, growth factors/small molecules and stem cell-based therapies.While many products possess convincing clinical data demonstrating their efficacy in comparison to standard treatment options, more robust, controlled studies are needed to determine the relative value among established and emerging biological therapies. Future bioengineering and stem cell-based approaches are of particular interest due to the simultaneous correction of multiple deficiencies present in the nonhealing wound.

  View details for DOI 10.1517/14712598.2013.842972

  View details for PubMedID 24093722

• From Germ Theory to Germ Therapy: Skin Microbiota, Chronic Wounds, and Probiotics PLASTIC AND RECONSTRUCTIVE SURGERY Wong, V. W., Martindale, R. G., Longaker, M. T., Gurtner, G. C. 2013; 132 (5): 854E-861E

  Abstract

  Microorganisms living throughout the body comprise the human "microbiota" and play an important role in health and disease. Recent research suggests that alterations in the skin microbiota may underlie chronic wound pathology. Probiotics are bacteria or yeast that confer a health benefit on the host and may have a role in preventing and treating nonhealing wounds by modulating host-microbe interactions.The English literature on skin microbiota, chronic wounds, biofilms, and probiotics is reviewed.Recent evidence indicates that disruption of microbial communities and bacteria-host interactions may contribute to impaired wound healing. Preclinical and human studies highlight the potential of probiotics to prevent or treat various infectious, immune-mediated, and inflammatory diseases.Advances in molecular sequencing and microbiology have shed light on the importance of the human microbiota in development, health, and disease. Probiotics represent a novel approach to altering the microbial environment with beneficial bacteria. Ongoing challenges include the need for better understanding of therapeutic mechanisms, improved regulation of manufacturing practices, and validation in controlled human trials. Current evidence suggests that probiotic-based therapies have considerable potential to exploit host-microbe relationships and improve clinical outcomes.

  View details for DOI 10.1097/PRS.0b013e3182a3c11e

  View details for PubMedID 24165637

• Isolation of human adipose-derived stromal cells using laser-assisted liposuction and their therapeutic potential in regenerative medicine. Stem cells translational medicine Chung, M. T., Zimmermann, A. S., Paik, K. J., Morrison, S. D., Hyun, J. S., Lo, D. D., McArdle, A., Montoro, D. T., Walmsley, G. G., Senarath-Yapa, K., Sorkin, M., Rennert, R., Chen, H., Chung, A. S., Vistnes, D., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2013; 2 (10): 808-817

  Abstract

  Harvesting adipose-derived stromal cells (ASCs) for tissue engineering is frequently done through liposuction. However, several different techniques exist. Although third-generation ultrasound-assisted liposuction has been shown to not have a negative effect on ASCs, the impact of laser-assisted liposuction on the quality and differentiation potential of ASCs has not been studied. Therefore, ASCs were harvested from laser-assisted lipoaspirate and suction-assisted lipoaspirate. Next, in vitro parameters of cell yield, cell viability and proliferation, surface marker phenotype, osteogenic differentiation, and adipogenic differentiation were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic nude mice. Although ASCs isolated from suction-assisted lipoaspirate and laser-assisted lipoaspirate both successfully underwent osteogenic and adipogenic differentiation, the cell yield, viability, proliferation, and frequency of ASCs (CD34(+)CD31(-)CD45(-)) in the stromal vascular fraction were all significantly less with laser-assisted liposuction in vitro (p < .05). In vivo, quantification of osseous healing by micro-computed tomography revealed significantly more healing with ASCs isolated from suction-assisted lipoaspirate relative to laser-assisted lipoaspirate at the 4-, 6-, and 8-week time points (p < .05). Therefore, as laser-assisted liposuction appears to negatively impact the biology of ASCs, cell harvest using suction-assisted liposuction is preferable for tissue-engineering purposes.

  View details for DOI 10.5966/sctm.2012-0183

  View details for PubMedID 24018794

  View details for PubMedCentralID PMC3785265

• Enhancing stem cell survival in vivo for tissue repair BIOTECHNOLOGY ADVANCES Hyun, J. S., Tran, M. C., Wong, V. W., Chung, M. T., Lo, D. D., Montoro, D. T., Wan, D. C., Longaker, M. T. 2013; 31 (5): 736-743

  Abstract

  The ability to use progenitor cells for regenerative medicine remains an evolving but elusive clinical goal. A serious obstacle towards widespread use of stem cells for tissue regeneration is the challenges that face these cells when they are placed in vivo into a wound for therapy. These environments are hypoxic, acidic, and have an upregulation of inflammatory mediators creating a region that is hostile towards cellular survival. Within this environment, the majority of progenitor cells undergo apoptosis prior to participating in lineage differentiation and cellular integration. In order to maximize the clinical utility of stem cells, strategies must be employed to increase the cell's ability to survive in vivo through manipulation of both the stem cell and the surrounding environment. This review focuses on current advances and techniques being used to increase in vivo stem cell survival for the purpose of tissue regeneration.

  View details for DOI 10.1016/j.biotechadv.2012.11.003

  View details for Web of Science ID 000322058900019

  View details for PubMedID 23153460

• Cell surface marker profiling of human adipose derived stem cells using single cell transcriptional analysis identifies heterogeneous subpopulations Sorkin, M., Rennert, R. C., Januszyk, M., Glotzbach, J. P., Chung, M. T., Longaker, M. T., Gurtner, G. C. ELSEVIER SCIENCE INC. 2013: S96–S97

  View details for DOI 10.1016/j.jamcollsurg.2013.07.219

  View details for Web of Science ID 000325577900190

• Molecular analysis and differentiation capacity of adipose-derived stem cells from lymphedema tissue. Plastic and reconstructive surgery Levi, B., Glotzbach, J. P., Sorkin, M., Hyun, J., Januszyk, M., Wan, D. C., Li, S., Nelson, E. R., Longaker, M. T., Gurtner, G. C. 2013; 132 (3): 580-589

  Abstract

  Many breast cancer patients are plagued by the disabling complication of upper limb lymphedema after axillary surgery. Conservative treatments using massage and compression therapy do not offer a lasting relief, as they fail to address the chronic transformation of edema into excess adipose tissue. Liposuction to address the adipose nature of the lymphedema has provided an opportunity for a detailed analysis of the stromal fraction of lymphedema-associated fat to clarify the molecular mechanisms for this adipogenic transformation.Adipose-derived stem cells were harvested from human lipoaspirate of the upper extremity from age-matched patients with lymphedema (n = 3) or subcutaneous adipose tissue from control patients undergoing cosmetic procedures (n = 3). Immediately after harvest, adipose-derived stem cells were analyzed using single-cell transcriptional profiling techniques. Osteogenic, adipogenic, and vasculogenic gene expression and differentiation were assessed by quantitative real-time polymerase chain reaction and standard in vitro differentiation assays.Differential transcriptional clusters of adipose-derived stem cells were found between lymphedema and subcutaneous fat. Interestingly, lymphedema-associated stem cells had a much higher adipogenic gene expression and enhanced ability to undergo adipogenic differentiation. Conversely, they had lower vasculogenic gene expression and diminished capability to form tubules in vitro, whereas the osteogenic differentiation capacity was not significantly altered.Adipose-derived stem cells from extremities affected by lymphedema appear to exhibit transcriptional profiles similar to those of abdominal adipose-derived stem cells; however, their adipogenic differentiation potential is strongly increased and their vasculogenic capacity is compromised. These results suggest that the underlying pathophysiology of lymphedema drives adipose-derived stem cells toward adipogenic differentiation.

  View details for DOI 10.1097/PRS.0b013e31829ace13

  View details for PubMedID 23985633

• Wound healing: a paradigm for regeneration. Mayo Clinic proceedings. Mayo Clinic Wong, V. W., Gurtner, G. C., Longaker, M. T. 2013; 88 (9): 1022-1031

  Abstract

  Human skin is a remarkably plastic organ that sustains insult and injury throughout life. Its ability to expeditiously repair wounds is paramount to survival and is thought to be regulated by wound components such as differentiated cells, stem cells, cytokine networks, extracellular matrix, and mechanical forces. These intrinsic regenerative pathways are integrated across different skin compartments and are being elucidated on the cellular and molecular levels. Recent advances in bioengineering and nanotechnology have allowed researchers to manipulate these microenvironments in increasingly precise spatial and temporal scales, recapitulating key homeostatic cues that may drive regeneration. The ultimate goal is to translate these bench achievements into viable bedside therapies that address the growing global burden of acute and chronic wounds. In this review, we highlight current concepts in cutaneous wound repair and propose that many of these evolving paradigms may underlie regenerative processes across diverse organ systems.

  View details for DOI 10.1016/j.mayocp.2013.04.012

  View details for PubMedID 24001495

• Enhancing in vivo survival of adipose-derived stromal cells through bcl-2 overexpression using a minicircle vector. Stem cells translational medicine Hyun, J., Grova, M., Nejadnik, H., Lo, D., Morrison, S., Montoro, D., Chung, M., Zimmermann, A., Walmsley, G. G., Lee, M., Daldrup-Link, H., Wan, D. C., Longaker, M. T. 2013; 2 (9): 690-702

  Abstract

  Tissue regeneration using progenitor cell-based therapy has the potential to aid in the healing of a diverse range of pathologies, ranging from short-gut syndrome to spinal cord lesions. However, there are numerous hurdles to be overcome prior to the widespread application of these cells in the clinical setting. One of the primary barriers to effective stem cell therapy is the hostile environment that progenitor cells encounter in the clinical injury wound setting. In order to promote cellular survival, stem cell differentiation, and participation in tissue regeneration, relevant cells and delivery scaffolds must be paired with strategies to prevent cell death to ensure that these cells can survive to form de novo tissue. The Bcl-2 protein is a prosurvival member of a family of proteins that regulate the mitochondrial pathway of apoptosis. Using several strategies to overexpress the Bcl-2 protein, we demonstrated a decrease in the mediators of apoptosis in vitro and in vivo. This was shown through the use of two different clinical tissue repair models. Cells overexpressing Bcl-2 not only survived within the wound environment at a statistically significantly higher rate than control cells, but also increased tissue regeneration. Finally, we used a nonintegrating minicircle technology to achieve this in a potentially clinically applicable strategy for stem cell therapy.

  View details for DOI 10.5966/sctm.2013-0035

  View details for PubMedID 23934910

• Molecular analysis and differentiation capacity of adipose-derived stem cells from lymphedema tissue. Plastic and reconstructive surgery Levi, B., Glotzbach, J. P., Sorkin, M., Hyun, J., Januszyk, M., Wan, D. C., Li, S., Nelson, E. R., Longaker, M. T., Gurtner, G. C. 2013; 132 (3): 580-589

  Abstract

  Many breast cancer patients are plagued by the disabling complication of upper limb lymphedema after axillary surgery. Conservative treatments using massage and compression therapy do not offer a lasting relief, as they fail to address the chronic transformation of edema into excess adipose tissue. Liposuction to address the adipose nature of the lymphedema has provided an opportunity for a detailed analysis of the stromal fraction of lymphedema-associated fat to clarify the molecular mechanisms for this adipogenic transformation.Adipose-derived stem cells were harvested from human lipoaspirate of the upper extremity from age-matched patients with lymphedema (n = 3) or subcutaneous adipose tissue from control patients undergoing cosmetic procedures (n = 3). Immediately after harvest, adipose-derived stem cells were analyzed using single-cell transcriptional profiling techniques. Osteogenic, adipogenic, and vasculogenic gene expression and differentiation were assessed by quantitative real-time polymerase chain reaction and standard in vitro differentiation assays.Differential transcriptional clusters of adipose-derived stem cells were found between lymphedema and subcutaneous fat. Interestingly, lymphedema-associated stem cells had a much higher adipogenic gene expression and enhanced ability to undergo adipogenic differentiation. Conversely, they had lower vasculogenic gene expression and diminished capability to form tubules in vitro, whereas the osteogenic differentiation capacity was not significantly altered.Adipose-derived stem cells from extremities affected by lymphedema appear to exhibit transcriptional profiles similar to those of abdominal adipose-derived stem cells; however, their adipogenic differentiation potential is strongly increased and their vasculogenic capacity is compromised. These results suggest that the underlying pathophysiology of lymphedema drives adipose-derived stem cells toward adipogenic differentiation.

  View details for DOI 10.1097/PRS.0b013e31829ace13

  View details for PubMedID 23985633

• Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, C. K., Lindau, P., Jiang, W., Chen, J. Y., Zhang, L. F., Chen, C., Seita, J., Sahoo, D., Kim, J., Lee, A., Park, S., Nag, D., Gong, Y., Kulkarni, S., Luppen, C. A., Theologis, A. A., Wan, D. C., DeBoer, A., Seo, E. Y., Vincent-Tompkins, J. D., Loh, K., Walmsley, G. G., Kraft, D. L., Wu, J. C., Longaker, M. T., Weissman, I. L. 2013; 110 (31): 12643-12648

  Abstract

  Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.

  View details for DOI 10.1073/pnas.1310212110

  View details for PubMedID 23858471

• Wnt3a reestablishes osteogenic capacity to bone grafts from aged animals. journal of bone and joint surgery. American volume Leucht, P., Jiang, J., Cheng, D., Liu, B., Dhamdhere, G., Fang, M. Y., Monica, S. D., Urena, J. J., Cole, W., Smith, L. R., Castillo, A. B., Longaker, M. T., Helms, J. A. 2013; 95 (14): 1278-1288

  Abstract

  Age-related fatty degeneration of the bone marrow contributes to delayed fracture-healing and osteoporosis-related fractures in the elderly. The mechanisms underlying this fatty change are unknown, but they may relate to the level of Wnt signaling within the aged marrow cavity.Transgenic mice were used in conjunction with a syngeneic bone-graft model to follow the fates of cells involved in the engraftment. Immunohistochemistry along with quantitative assays were used to evaluate Wnt signaling and adipogenic and osteogenic gene expression in bone grafts from young and aged mice. Liposomal Wnt3a protein (L-Wnt3a) was tested for its ability to restore osteogenic potential to aged bone grafts in critical-size defect models created in mice and in rabbits. Radiography, microquantitative computed tomography (micro-CT) reconstruction, histology, and histomorphometric measurements were used to quantify bone-healing resulting from L-Wnt3a or a control substance (liposomal phosphate-buffered saline solution [L-PBS]).Expression profiling of cells in a bone graft demonstrated a shift away from an osteogenic gene profile and toward an adipogenic one with age. This age-related adipogenic shift was accompanied by a significant reduction (p < 0.05) in Wnt signaling and a loss in osteogenic potential. In both large and small animal models, osteogenic competence was restored to aged bone grafts by a brief incubation with the stem-cell factor Wnt3a. In addition, liposomal Wnt3a significantly reduced cell death in the bone graft, resulting in significantly more osseous regenerate in comparison with controls.Liposomal Wnt3a enhances cell survival and reestablishes the osteogenic capacity of bone grafts from aged animals.We developed an effective, clinically applicable, regenerative medicine-based strategy for revitalizing bone grafts from aged patients.

  View details for DOI 10.2106/JBJS.L.01502

  View details for PubMedID 23864176

• Wnt3a Reestablishes Osteogenic Capacity to Bone Grafts from Aged Animals JOURNAL OF BONE AND JOINT SURGERY-AMERICAN VOLUME Leucht, P., Jiang, J., Cheng, D., Liu, B., Dhamdhere, G., Fang, M. Y., Monica, S. D., Urena, J. J., Cole, W., Smith, L. R., Castillo, A. B., Longaker, M. T., Helms, J. A. 2013; 95A (14): 1278-1288

  Abstract

  Age-related fatty degeneration of the bone marrow contributes to delayed fracture-healing and osteoporosis-related fractures in the elderly. The mechanisms underlying this fatty change are unknown, but they may relate to the level of Wnt signaling within the aged marrow cavity.Transgenic mice were used in conjunction with a syngeneic bone-graft model to follow the fates of cells involved in the engraftment. Immunohistochemistry along with quantitative assays were used to evaluate Wnt signaling and adipogenic and osteogenic gene expression in bone grafts from young and aged mice. Liposomal Wnt3a protein (L-Wnt3a) was tested for its ability to restore osteogenic potential to aged bone grafts in critical-size defect models created in mice and in rabbits. Radiography, microquantitative computed tomography (micro-CT) reconstruction, histology, and histomorphometric measurements were used to quantify bone-healing resulting from L-Wnt3a or a control substance (liposomal phosphate-buffered saline solution [L-PBS]).Expression profiling of cells in a bone graft demonstrated a shift away from an osteogenic gene profile and toward an adipogenic one with age. This age-related adipogenic shift was accompanied by a significant reduction (p < 0.05) in Wnt signaling and a loss in osteogenic potential. In both large and small animal models, osteogenic competence was restored to aged bone grafts by a brief incubation with the stem-cell factor Wnt3a. In addition, liposomal Wnt3a significantly reduced cell death in the bone graft, resulting in significantly more osseous regenerate in comparison with controls.Liposomal Wnt3a enhances cell survival and reestablishes the osteogenic capacity of bone grafts from aged animals.We developed an effective, clinically applicable, regenerative medicine-based strategy for revitalizing bone grafts from aged patients.

  View details for DOI 10.2106/JBJS.L01502

  View details for Web of Science ID 000321885000005

• A Mechanomodulatory Device to Minimize Incisional Scar Formation. Advances in wound care Wong, V. W., Beasley, B., Zepeda, J., Dauskardt, R. H., Yock, P. G., Longaker, M. T., Gurtner, G. C. 2013; 2 (4): 185-194

  Abstract

  To mechanically control the wound environment and prevent cutaneous scar formation.We subjected various material substrates to biomechanical testing to investigate their ability to modulate skin behavior. Combinations of elastomeric materials, adhesives, and strain applicators were evaluated to develop topical stress-shielding devices. Noninvasive imaging modalities were utilized to characterize anatomic site-specific differences in skin biomechanical properties in humans. The devices were tested in a validated large animal model of hypertrophic scarring. Phase I within-patient controlled clinical trials were conducted to confirm their safety and efficacy in scar reduction in patients undergoing abdominoplasty surgery.Among the tested materials and device applicators, a polymer device was developed that effectively off-loaded high tension wounds and blocked pro-fibrotic pathways and excess scar formation in red Duroc swine. In humans, different anatomic sites exhibit unique biomechanical properties that may correlate with the propensity to form scars. In the clinical trial, utilization of this device significantly reduced incisional scar formation and improved scar appearance for up to 12 months compared with control incisions that underwent routine postoperative care.This is the first device that is able to precisely control the mechanical environment of incisional wounds and has been demonstrated in multiple clinical trials to significantly reduce scar formation after surgery.Mechanomodulatory strategies to control the incisional wound environment can significantly reduce pathologic scarring and fibrosis after surgery.

  View details for PubMedID 24527342

• A Mechanomodulatory Device to Minimize Incisional Scar Formation ADVANCES IN WOUND CARE Wong, V. W., Beasley, B., Zepeda, J., Dauskardt, R. H., Yock, P. G., Longaker, M. T., Gurtner, G. C. 2013; 2 (4): 185–94

  View details for DOI 10.1089/wound.2012.0396

  View details for Web of Science ID 000214927500008

• Paracrine Mechanism of Angiogenesis in Adipose-Derived Stem Cell Transplantation. Annals of plastic surgery Suga, H., Glotzbach, J. P., Sorkin, M., Longaker, M. T., Gurtner, G. C. 2013

  Abstract

  INTRODUCTION: Adipose-derived stem cells (ASCs) have shown potential for cell-based therapy in the field of plastic surgery. However, the fate of ASCs after transplantation and the mechanism(s) of their biologic capabilities remain unclear. METHODS: We isolated and cultured ASCs from transgenic mice that express both luciferase and green fluorescent protein and injected the cells into the inguinal fat pads of wild-type mice. We tested 4 experimental groups, namely, ischemic fat pads with/without ASCs and control fat pads with/without ASCs. RESULTS: Transplanted ASCs were tracked with bioluminescence imaging. The luminescence gradually decreased over 28 days, indicating cell death after transplantation. More ASCs were retained in ischemic fat pads on day 7 compared to control fat pads. On day 14, adipose tissue vascular density was higher in the ASC transplantation groups compared to those without ASCs. On day 28, there was decreased atrophy of adipose tissue in ASC-treated ischemic fat pads. Transplanted ASCs were detected as nonproliferating green fluorescent protein-positive cells, whereas native endothelial cells adjacent to the transplanted ASCs were proliferative. Protein analysis demonstrated higher expression of hepatocyte growth factor and vascular endothelial growth factor in the ASC transplantation groups, suggesting a paracrine mechanism, which was confirmed by in vitro experiments with conditioned media from ASCs. CONCLUSIONS: Transplanted ASCs are preferentially retained in ischemic adipose tissue, although most of the cells eventually undergo cell death. They exert an angiogenic effect on adipose tissue mainly through a paracrine mechanism. Increased understanding of these effects will help develop ASCs as a tool for cell-based therapy.

  View details for DOI 10.1097/SAP.0b013e318264fd6a

  View details for PubMedID 23636112

• Brg1 governs a positive feedback circuit in the hair follicle for tissue regeneration and repair. Developmental cell Xiong, Y., Li, W., Shang, C., Chen, R. M., Han, P., Yang, J., Stankunas, K., Wu, B., Pan, M., Zhou, B., Longaker, M. T., Chang, C. 2013; 25 (2): 169-181

  Abstract

  Hair follicle stem cells (bulge cells) are essential for hair regeneration and early epidermal repair after wounding. Here we show that Brg1, a key enzyme in the chromatin-remodeling machinery, is dynamically expressed in bulge cells to control tissue regeneration and repair. In mice, sonic hedgehog (Shh) signals Gli to activate Brg1 in bulge cells to begin hair regeneration, whereas Brg1 recruits NF-κB to activate Shh in matrix cells to sustain hair growth. Such reciprocal Brg1-Shh interaction is essential for hair regeneration. Moreover, Brg1 is indispensable for maintaining the bulge cell reservoir. Without Brg1, bulge cells are depleted over time, partly through the ectopic expression of the cell-cycle inhibitor p27(Kip1). Also, bulge Brg1 is activated by skin injury to facilitate early epidermal repair. Our studies demonstrate a molecular circuit that integrates chromatin remodeling (Brg1), transcriptional regulation (NF-κB, Gli), and intercellular signaling (Shh) to control bulge stem cells during tissue regeneration.

  View details for DOI 10.1016/j.devcel.2013.03.015

  View details for PubMedID 23602386

• CD90 (Thy-1)-Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A Chung, M. T., Liu, C., Hyun, J. S., Lo, D. D., Montoro, D. T., Hasegawa, M., Li, S., Sorkin, M., Rennert, R., Keeney, M., Yang, F., Quarto, N., Longaker, M. T., Wan, D. C. 2013; 19 (7-8): 989-997

  Abstract

  Stem cell-based bone tissue engineering with adipose-derived stromal cells (ASCs) has shown great promise for revolutionizing treatment of large bone deficits. However, there is still a lack of consensus on cell surface markers identifying osteoprogenitors. Fluorescence-activated cell sorting has identified a subpopulation of CD105(low) cells with enhanced osteogenic differentiation. The purpose of the present study was to compare the ability of CD90 (Thy-1) to identify osteoprogenitors relative to CD(105).Unsorted cells, CD90(+), CD90(-), CD105(high), and CD105(low) cells were treated with an osteogenic differentiation medium. For evaluation of in vitro osteogenesis, alkaline phosphatase (ALP) staining and alizarin red staining were performed at 7 days and 14 days, respectively. RNA was harvested after 7 and 14 days of differentiation, and osteogenic gene expression was examined by quantitative real-time polymerase chain reaction. For evaluation of in vivo osteogenesis, critical-sized (4-mm) calvarial defects in nude mice were treated with the hydroxyapatite-poly(lactic-co-glycolic acid) scaffold seeded with the above-mentioned subpopulations. Healing was followed using micro-CT scans for 8 weeks. Calvaria were harvested at 8 weeks postoperatively, and sections were stained with Movat's Pentachrome.Transcriptional analysis revealed that the CD90(+) subpopulation was enriched for a more osteogenic subtype relative to the CD105(low) subpopulation. Staining at day 7 for ALP was greatest in the CD90(+) cells, followed by the CD105(low) cells. Staining at day 14 for alizarin red demonstrated the greatest amount of mineralized extracellular matrix in the CD90(+) cells, again followed by the CD105(low) cells. Quantification of in vivo healing at 2, 4, 6, and 8weeks postoperatively demonstrated increased bone formation in defects treated with CD90(+) ASCs relative to all other groups. On Movat's Pentachrome-stained sections, defects treated with CD90(+) cells showed the most robust bony regeneration. Defects treated with CD90(-) cells, CD105(high) cells, and CD105(low) cells demonstrated some bone formation, but to a lesser degree when compared with the CD90(+) group.While CD105(low) cells have previously been shown to possess an enhanced osteogenic potential, we found that CD90(+) cells are more capable of forming bone both in vitro and in vivo. These data therefore suggest that CD90 may be a more effective marker than CD105 to isolate a highly osteogenic subpopulation for bone tissue engineering.

  View details for DOI 10.1089/ten.tea.2012.0370

  View details for PubMedID 23216074

• Integration of Multiple Signaling Regulates through Apoptosis the Differential Osteogenic Potential of Neural Crest-Derived and Mesoderm-Derived Osteoblasts PLOS ONE Li, S., Meyer, N. P., Quarto, N., Longaker, M. T. 2013; 8 (3)

  Abstract

  Neural crest-derived (FOb) and mesoderm-derived (POb) calvarial osteoblasts are characterized by distinct differences in their osteogenic potential. We have previously demonstrated that enhanced activation of endogenous FGF and Wnt signaling confers greater osteogenic potential to FOb. Apoptosis, a key player in bone formation, is the main focus of this study. In the current work, we have investigated the apoptotic activity of FOb and POb cells during differentiation. We found that lower apoptosis, as measured by caspase-3 activity is a major feature of neural crest-derived osteoblast which also have higher osteogenic capacity. Further investigation indicated TGF-β signaling as main positive regulator of apoptosis in these two populations of calvarial osteoblasts, while BMP and canonical Wnt signaling negatively regulate the process. By either inducing or inhibiting these signaling pathways we could modulate apoptotic events and improve the osteogenic potential of POb. Taken together, our findings demonstrate that integration of multiple signaling pathways contribute to imparting greater osteogenic potential to FOb by decreasing apoptosis.

  View details for DOI 10.1371/journal.pone.0058610

  View details for Web of Science ID 000317397200008

  View details for PubMedID 23536803

  View details for PubMedCentralID PMC3607600

• Micro-Computed Tomography Evaluation of Human Fat Grafts in Nude Mice TISSUE ENGINEERING PART C-METHODS Chung, M. T., Hyun, J. S., Lo, D. D., Montoro, D. T., Hasegawa, M., Levi, B., Januszyk, M., Longaker, M. T., Wan, D. C. 2013; 19 (3): 227-232

  Abstract

  Although autologous fat grafting has revolutionized the field of soft tissue reconstruction and augmentation, long-term maintenance of fat grafts is unpredictable. Recent studies have reported survival rates of fat grafts to vary anywhere between 10% and 80% over time. The present study evaluated the long-term viability of human fat grafts in a murine model using a novel imaging technique allowing for in vivo volumetric analysis.Human fat grafts were prepared from lipoaspirate samples using the Coleman technique. Fat was injected subcutaneously into the scalp of 10 adult Crl:NU-Foxn1(nu) CD-1 male mice. Micro-computed tomography (CT) was performed immediately following injection and then weekly thereafter. Fat volume was rendered by reconstructing a three-dimensional (3D) surface through cubic-spline interpolation. Specimens were also harvested at various time points and sections were prepared and stained with hematoxylin and eosin (H&E), for macrophages using CD68 and for the cannabinoid receptor 1 (CB1). Finally, samples were explanted at 8- and 12-week time points to validate calculated micro-CT volumes.Weekly CT scanning demonstrated progressive volume loss over the time course. However, volumetric analysis at the 8- and 12-week time points stabilized, showing an average of 62.2% and 60.9% survival, respectively. Gross analysis showed the fat graft to be healthy and vascularized. H&E analysis and staining for CD68 showed minimal inflammatory reaction with viable adipocytes. Immunohistochemical staining with anti-human CB1 antibodies confirmed human origin of the adipocytes.Studies assessing the fate of autologous fat grafts in animals have focused on nonimaging modalities, including histological and biochemical analyses, which require euthanasia of the animals. In this study, we have demonstrated the ability to employ micro-CT for 3D reconstruction and volumetric analysis of human fat grafts in a mouse model. Importantly, this model provides a platform for subsequent study of fat manipulation and soft tissue engineering.

  View details for DOI 10.1089/ten.tec.2012.0371

  View details for Web of Science ID 000314179900006

  View details for PubMedID 22916732

  View details for PubMedCentralID PMC3557441

• Primary cilia act as mechanosensors during bone healing around an implant MEDICAL ENGINEERING & PHYSICS Leucht, P., Monica, S. D., Temiyasathit, S., Lenton, K., Manu, A., Longaker, M. T., Jacobs, C. R., Spilkere, R. L., Guo, H., Brunski, J. B., Helms, J. A. 2013; 35 (3): 392-402

  Abstract

  The primary cilium is an organelle that senses cues in a cell's local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3a(fl/fl) mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective.

  View details for DOI 10.1016/j.medengphy.2012.06.005

  View details for Web of Science ID 000315931400013

  View details for PubMedID 22784673

  View details for PubMedCentralID PMC3517784

• Integration of Multiple Signaling Pathways Determines Differences in the Osteogenic Potential and Tissue Regeneration of Neural Crest-Derived and Mesoderm-Derived Calvarial Bone INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Senarath-Yapa, K., Li, S., Meyer, N. P., Longaker, M. T., Quarto, N. 2013; 14 (3): 5978-5997

  Abstract

  The mammalian skull vault, a product of a unique and tightly regulated evolutionary process, in which components of disparate embryonic origin are integrated, is an elegant model with which to study osteoblast biology. Our laboratory has demonstrated that this distinct embryonic origin of frontal and parietal bones confer differences in embryonic and postnatal osteogenic potential and skeletal regenerative capacity, with frontal neural crest derived osteoblasts benefitting from greater osteogenic potential. We outline how this model has been used to elucidate some of the molecular mechanisms which underlie these differences and place these findings into the context of our current understanding of the key, highly conserved, pathways which govern the osteoblast lineage including FGF, BMP, Wnt and TGFβ signaling. Furthermore, we explore recent studies which have provided a tantalizing insight into way these pathways interact, with evidence accumulating for certain transcription factors, such as Runx2, acting as a nexus for cross-talk.

  View details for DOI 10.3390/ijms14035978

  View details for Web of Science ID 000316609800086

  View details for PubMedID 23502464

  View details for PubMedCentralID PMC3634461

• Commentary on the differential healing capacity of calvarial bone. journal of craniofacial surgery Lo, D. D., McArdle, A., Senarath-Yapa, K., Longaker, M. T. 2013; 24 (2): 344-345

  View details for DOI 10.1097/SCS.0b013e3182802256

  View details for PubMedID 23524689

• Effective Delivery of Stem Cells Using an Extracellular Matrix Patch Results in Increased Cell Survival and Proliferation and Reduced Scarring in Skin Wound Healing TISSUE ENGINEERING PART A Lam, M. T., Nauta, A., Meyer, N. P., Wu, J. C., Longaker, M. T. 2013; 19 (5-6): 738-747

  Abstract

  Wound healing is one of the most complex biological processes and occurs in all tissues and organs of the body. In humans, fibrotic tissue, or scar, hinders function and is aesthetically unappealing. Stem cell therapy offers a promising new technique for aiding in wound healing; however, current findings show that stem cells typically die and/or migrate from the wound site, greatly decreasing efficacy of the treatment. Here, we demonstrate effectiveness of a stem cell therapy for improving wound healing in the skin and reducing scarring by introducing stem cells using a natural patch material. Adipose-derived stromal cells were introduced to excisional wounds created in mice using a nonimmunogenic extracellular matrix (ECM) patch material derived from porcine small-intestine submucosa (SIS). The SIS served as an attractive delivery vehicle because of its natural ECM components, including its collagen fiber network, providing the stem cells with a familiar structure. Experimental groups consisted of wounds with stem cell-seeded patches removed at different time points after wounding to determine an optimal treatment protocol. Stem cells delivered alone to skin wounds did not survive post-transplantation as evidenced by bioluminescence in vivo imaging. In contrast, delivery with the patch enabled a significant increase in stem cell proliferation and survival. Wound healing rates were moderately improved by treatment with stem cells on the patch; however, areas of fibrosis, indicating scarring, were significantly reduced in wounds treated with the stem cells on the patch compared to untreated wounds.

  View details for DOI 10.1089/ten.tea.2012.0480

  View details for Web of Science ID 000314581100015

  View details for PubMedID 23072446

  View details for PubMedCentralID PMC3566655

• Adipose-derived Stromal Cells Overexpressing Vascular Endothelial Growth Factor Accelerate Mouse Excisional Wound Healing MOLECULAR THERAPY Nauta, A., Seidel, C., Deveza, L., Montoro, D., Grova, M., Ko, S. H., Hyun, J., Gurtner, G. C., Longaker, M. T., Yang, F. 2013; 21 (2): 445-455

  Abstract

  Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12-15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10-12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired.

  View details for DOI 10.1038/mt.2012.234

  View details for Web of Science ID 000314434600021

  View details for PubMedID 23164936

  View details for PubMedCentralID PMC3594010

• Discussion: A report of the ASPS Task Force on regenerative medicine: opportunities for plastic surgery. Plastic and reconstructive surgery McArdle, A., Lo, D. D., Hyun, J. S., Senarath-Yapa, K., Chung, M. T., Wan, D. C., Longaker, M. T. 2013; 131 (2): 400-403

  View details for DOI 10.1097/PRS.0b013e318278d88c

  View details for PubMedID 23358002

• The Seed and the Soil Optimizing Stem Cells and Their Environment for Tissue Regeneration ANNALS OF PLASTIC SURGERY Hyun, J. S., Montoro, D. T., Lo, D. D., Flynn, R. A., Wong, V., Chung, M. T., Longaker, M. T., Wan, D. C. 2013; 70 (2): 235-239

  Abstract

  The potential for stem cells to serve as cellular building blocks for reconstruction of complex defects has prompted significant enthusiasm in the field of regenerative medicine. Clinical application, however, is still limited, as implantation of cells into hostile wound environments may greatly hinder their tissue forming capacity. To circumvent this obstacle, novel approaches have been developed to manipulate both the stem cell itself and its surrounding environmental niche. By understanding this paradigm of seed and soil optimization, innovative strategies may thus be developed to harness the true promise of stem cells for tissue regeneration.

  View details for DOI 10.1097/SAP.0b013e31826a18fb

  View details for Web of Science ID 000313964300024

  View details for PubMedID 23295233

• Abnormal Calcium Handling Properties Underlie Familial Hypertrophic Cardiomyopathy Pathology in Patient-Specific Induced Pluripotent Stem Cells CELL STEM CELL Lan, F., Lee, A. S., Liang, P., Sanchez-Freire, V., Nguyen, P. K., Wang, L., Han, L., Yen, M., Wang, Y., Sun, N., Abilez, O. J., Hu, S., Ebert, A. D., Navarrete, E. G., Simmons, C. S., Wheeler, M., Pruitt, B., Lewis, R., Yamaguchi, Y., Ashley, E. A., Bers, D. M., Robbins, R. C., Longaker, M. T., Wu, J. C. 2013; 12 (1): 101-113

  Abstract

  Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca(2+)) imaging indicated dysregulation of Ca(2+) cycling and elevation in intracellular Ca(2+) ([Ca(2+)](i)) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca(2+) homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.

  View details for DOI 10.1016/j.stem.2012.10.010

  View details for Web of Science ID 000313839500014

  View details for PubMedID 23290139

  View details for PubMedCentralID PMC3638033

• Evidence that mast cells are not required for healing of splinted cutaneous excisional wounds in mice. PloS one Nauta, A. C., Grova, M., Montoro, D. T., Zimmermann, A., Tsai, M., Gurtner, G. C., Galli, S. J., Longaker, M. T. 2013; 8 (3)

  Abstract

  Wound healing is a complex biological process involving the interaction of many cell types to replace lost or damaged tissue. Although the biology of wound healing has been extensively investigated, few studies have focused on the role of mast cells. In this study, we investigated the possible role of mast cells in wound healing by analyzing aspects of cutaneous excisional wound healing in three types of genetically mast cell-deficient mice. We found that C57BL/6-Kit(W-sh/W-sh), WBB6F1-Kit(W/W-v), and Cpa3-Cre; Mcl-1(fl/fl) mice re-epithelialized splinted excisional skin wounds at rates very similar to those in the corresponding wild type or control mice. Furthermore, at the time of closure, scars were similar in the genetically mast cell-deficient mice and the corresponding wild type or control mice in both quantity of collagen deposition and maturity of collagen fibers, as evaluated by Masson's Trichrome and Picro-Sirius red staining. These data indicate that mast cells do not play a significant non-redundant role in these features of the healing of splinted full thickness excisional cutaneous wounds in mice.

  View details for DOI 10.1371/journal.pone.0059167

  View details for PubMedID 23544053

  View details for PubMedCentralID PMC3609818

• STEM CELL-BASED BIOENGINEERING OF CRANIOFACIAL BONE STEM CELLS IN CRANIOFACIAL DEVELOPMENT AND REGENERATION Lo, D. D., Montoro, D. T., Grova, M., Hyun, J. S., Chung, M. T., Wan, D. C., Longaker, M. T., Huang, G. T., Thesleff 2013: 379–94

  View details for Web of Science ID 000327996200022

• Murine models of human wound healing. Methods in molecular biology (Clifton, N.J.) Chen, J. S., Longaker, M. T., Gurtner, G. C. 2013; 1037: 265-274

  Abstract

  In vivo wound healing experiments remain the most predictive models for studying human wound healing, allowing an accurate representation of the complete wound healing environment including various cell types, environmental cues, and paracrine interactions. Small animals are economical, easy to maintain, and allow researchers to take advantage of the numerous transgenic strains that have been developed to investigate the specific mechanisms involved in wound healing and regeneration. Here we describe three reproducible murine wound healing models that recapitulate the human wound healing process.

  View details for DOI 10.1007/978-1-62703-505-7_15

  View details for PubMedID 24029941

• Adult stem cells in small animal wound healing models. Methods in molecular biology (Clifton, N.J.) Nauta, A. C., Gurtner, G. C., Longaker, M. T. 2013; 1037: 81-98

  Abstract

  This chapter broadly reviews the use of stem cells as a means to accelerate wound healing, focusing first on the properties of stem cells that make them attractive agents to influence repair, both alone and as vehicles for growth factor delivery. Major stem cell reservoirs are described, including adult, embryonic, and induced pluripotent cell sources, outlining the advantages and limitations of each source as wound healing agents, as well as the possible mechanisms responsible for wound healing acceleration. Finally, the chapter includes a materials and methods section that provides an in-depth description of adult tissue harvest techniques.

  View details for DOI 10.1007/978-1-62703-505-7_5

  View details for PubMedID 24029931

• Absence of endochondral ossification and craniosynostosis in posterior frontal cranial sutures of axin2(-/-) mice. PloS one Behr, B., Longaker, M. T., Quarto, N. 2013; 8 (8)

  Abstract

  During the first month of life, the murine posterior-frontal suture (PF) of the cranial vault closes through endochondral ossification, while other sutures remain patent. These processes are tightly regulated by canonical Wnt signaling. Low levels of active canonical Wnt signaling enable endochondral ossification and therefore PF-suture closure, whereas constitutive activation of canonical Wnt causes PF-suture patency. We therefore sought to test this concept with a knockout mouse model. PF-sutures of Axin2(-/-) mice, which resemble a state of constantly activated canonical Wnt signaling, were investigated during the physiological time course of PF-suture closure and compared in detail with wild type littermates. Histological analysis revealed that the architecture in Axin2(-/-) PF-sutures was significantly altered in comparison to wild type. The distance between the endocranial layers was dramatically increased and suture closure was significantly delayed. Moreover, physiological endochondral ossification did not occur, rather an ectopic cartilage appeared between the endocranial and ectocranial bone layers at P7 which eventually involutes at P13. Quantitative PCR analysis showed the lack of Col10α1 upregulation in Axin2(-/-) PF-suture. Immunohistochemistry and gene expression analysis also revealed high levels of type II collagen as compared to type I collagen and absence of Mmp-9 in the cartilage of Axin2(-/-) PF-suture. Moreover, TUNEL staining showed a high percentage of apoptotic chondrocytes in Axin2(-/-) PF-sutures at P9 and P11 as compared to wild type. These data indicated that Axin2(-/-) PF-sutures lack physiological endochondral ossification, contain ectopic cartilage and display delayed suture closure.

  View details for DOI 10.1371/journal.pone.0070240

  View details for PubMedID 23936395

  View details for PubMedCentralID PMC3731366

• In vivo directed differentiation of pluripotent stem cells for skeletal regeneration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Levi, B., Hyun, J. S., Montoro, D. T., Lo, D. D., Chan, C. K., Hu, S., Sun, N., Lee, M., Grova, M., Connolly, A. J., Wu, J. C., Gurtner, G. C., Weissman, I. L., Wan, D. C., Longaker, M. T. 2012; 109 (50): 20379-20384

  Abstract

  Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.

  View details for DOI 10.1073/pnas.1218052109

  View details for PubMedID 23169671

• Femtosecond plasma mediated laser ablation has advantages over mechanical osteotomy of cranial bone LASERS IN SURGERY AND MEDICINE Lo, D. D., Mackanos, M. A., Chung, M. T., Hyun, J. S., Montoro, D. T., Grova, M., Liu, C., Wang, J., Palanker, D., Connolly, A. J., Longaker, M. T., Contag, C. H., Wan, D. C. 2012; 44 (10): 805-814

  Abstract

  Although mechanical osteotomies are frequently made on the craniofacial skeleton, collateral thermal, and mechanical trauma to adjacent bone tissue causes cell death and may delay healing. The present study evaluated the use of plasma-mediated laser ablation using a femtosecond laser to circumvent thermal damage and improve bone regeneration.Critical-size circular calvarial defects were created with a trephine drill bit or with a Ti:Sapphire femtosecond pulsed laser. Healing was followed using micro-CT scans for 8 weeks. Calvaria were also harvested at various time points for histological analysis. Finally, scanning electron microscopy was used to analyze the microstructure of bone tissue treated with the Ti:Sapphire laser, and compared to that treated with the trephine bur.Laser-created defects healed significantly faster than those created mechanically at 2, 4, and 6 weeks post-surgery. However, at 8 weeks post-surgery, there was no significant difference. In the drill osteotomy treatment group, empty osteocyte lacunae were seen to extend 699 ± 27 µm away from the edge of the defect. In marked contrast, empty osteocyte lacunae were seen to extend only 182 ± 22 µm away from the edge of the laser-created craters. Significantly less ossification and formation of irregular woven bone was noted on histological analysis for drill defects.We demonstrate accelerated bone healing after femtosecond laser ablation in a calvarial defect model compared to traditional mechanical drilling techniques. Improved rates of early regeneration make plasma-mediated ablation of the craniofacial skeleton advantageous for applications to osteotomy.

  View details for DOI 10.1002/lsm.22098

  View details for Web of Science ID 000312941600004

  View details for PubMedID 23184427

• Exogenous Activation of BMP-2 Signaling Overcomes TGF beta-Mediated Inhibition of Osteogenesis in Marfan Embryonic Stem Cells and Marfan Patient-Specific Induced Pluripotent Stem Cells STEM CELLS Quarto, N., Li, S., Renda, A., Longaker, M. T. 2012; 30 (12): 2709-2719

  Abstract

  Marfan syndrome (MFS) is a hereditary disease caused by mutations in the gene encoding Fibrillin-1 (FBN1) and characterized by a number of skeletal abnormalities, aortic root dilatation, and sometimes ectopia lentis. Although the molecular pathogenesis of MFS was attributed initially to a structural weakness of the fibrillin-rich microfibrils within the extracellular matrix, more recent results have documented that many of the pathogenic abnormalities in MFS are the result of alterations in TGFβ signaling. Mutations in FBN1 are therefore associated with increased activity and bioavailability of TGF-β1, which is suspected to be the basis for phenotypical similarities of FBN1 mutations in MFS and mutations in the receptors for TGFβ in Marfan syndrome-related diseases. We have previously demonstrated that unique skeletal phenotypes observed in human embryonic stem cells carrying the monogenic FBN1 mutation (MFS cells) are faithfully phenocopied by cells differentiated from induced pluripotent-stem cells (MFSiPS) derived independently from MFS patient fibroblasts. In this study, we aimed to determine further the biochemical features of transducing signaling(s) in MFS stem cells and MFSiPS cells highlighting a crosstalk between TGFβ and BMP signaling. Our results revealed that enhanced activation of TGFβ signaling observed in MFS cells decreased their endogenous BMP signaling. Moreover, exogenous BMP antagonized the enhanced TGFβ signaling in both MFS stem cells and MFSiPS cells therefore, rescuing their ability to undergo osteogenic differentiation. This study advances our understanding of molecular mechanisms underlying the pathogenesis of bone loss/abnormal skeletogenesis in human diseases caused by mutations in FBN1.

  View details for DOI 10.1002/stem.1250

  View details for PubMedID 23037987

• Introduction: Wound repair SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Longaker, M. T. 2012; 23 (9): 945-945

  View details for DOI 10.1016/j.semcdb.2012.10.002

  View details for Web of Science ID 000311962800001

  View details for PubMedID 23059792

• Soft tissue mechanotransduction in wound healing and fibrosis SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Wong, V. W., Longaker, M. T., Gurtner, G. C. 2012; 23 (9): 981-986

  Abstract

  Recent evidence suggests that mechanical forces can significantly impact the biologic response to injury. Integrated mechanical and chemical signaling networks have been discovered that enable physical cues to regulate disease processes such as pathologic scar formation. Distinct molecular mechanisms control how tensional forces influence wound healing and fibrosis. Conceptual frameworks to understand cutaneous repair have expanded beyond traditional cell-cytokine models to include dynamic interactions driven by mechanical force and the extracellular matrix. Strategies to manipulate these biomechanical signaling networks have tremendous therapeutic potential to reduce scar formation and promote skin regeneration.

  View details for DOI 10.1016/j.semcdb.2012.09.010

  View details for Web of Science ID 000311962800006

  View details for PubMedID 23036529

• Comparison of several attachment methods for human iPS, embryonic and adipose-derived stem cells for tissue engineering JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE Lam, M. T., Longaker, M. T. 2012; 6: s80-s86

  Abstract

  As actual stem cell application quickly approaches tissue engineering and regenerative medicine, aspects such as cell attachment to scaffolds and biomaterials become important and are often overlooked. Here, we compare the effects of several attachment proteins on the adhesion, proliferation and stem cell identity of three promising human stem cell types: human adipose-derived stem cells (hASCs), human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Traditional tissue culture polystyrene plates (TCPS), Matrigel (Mat), laminin (Lam), fibronectin (FN) and poly-L-lysine (PLL) were investigated as attachment protein surfaces. For hASCs typically cultured on TCPS, laminin resulted in the greatest cell attachment and proliferation with largest cell areas, indicating favourability by cell spreading. However, mesenchymal stem cell markers indicative of hASCs were slightly more expressed on surfaces with lowest cell attachment, corresponding to increased cell roundness, a newly observed attribute in hASCs possibly indicating a more stem cell-like character. hESCs preferred Matrigel as a feeder-free culture surface. Interestingly, hiPSCs favoured laminin over Matrigel for colony expansion, shown by larger cell colony area and perimeter lengths, although cell numbers and stem cell marker expression level remained highest on Matrigel. These data provide a practical reference guide for selecting a suitable attachment method for using human induced pluripotent, embryonic or adipose stem cells in tissue engineering and regenerative medicine applications.

  View details for DOI 10.1002/term.1499

  View details for Web of Science ID 000313431100009

  View details for PubMedID 22610948

• Microfluidic Single Cell Analysis Shows Porcine Induced Pluripotent Stem Cell-Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation Gu, M., Nguyen, P., Hu, S., Plews, J. R., Lee, A. S., Huber, B. C., Lee, W., Gong, Y., Almeida, P. E., Connolly, A., Robbins, R. C., Longaker, M. T., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2012

  View details for Web of Science ID 000208885005075

• Models of cranial suture biology. journal of craniofacial surgery Grova, M., Lo, D. D., Montoro, D., Hyun, J. S., Chung, M. T., Wan, D. C., Longaker, M. T. 2012; 23 (7): 1954-1958

  Abstract

  Craniosynostosis is a common congenital defect caused by premature fusion of cranial sutures. The severe morphologic abnormalities and cognitive deficits resulting from craniosynostosis and the potential morbidity of surgical correction espouse the need for a deeper understanding of the complex etiology for this condition. Work in animal models for the past 20 years has been pivotal in advancing our understanding of normal suture biology and elucidating pathologic disease mechanisms. This article provides an overview of milestone studies in suture development, embryonic origins, and signaling mechanisms from an array of animal models including transgenic mice, rats, rabbits, fetal sheep, zebrafish, and frogs. This work contributes to an ongoing effort toward continued development of novel treatment strategies.

  View details for DOI 10.1097/SCS.0b013e318258ba53

  View details for PubMedID 23154351

• Models of Cranial Suture Biology JOURNAL OF CRANIOFACIAL SURGERY Grova, M., Lo, D. D., Montoro, D., Hyun, J. S., Chung, M. T., Wan, D. C., Longaker, M. T. 2012; 23: 1954-1958

  Abstract

  Craniosynostosis is a common congenital defect caused by premature fusion of cranial sutures. The severe morphologic abnormalities and cognitive deficits resulting from craniosynostosis and the potential morbidity of surgical correction espouse the need for a deeper understanding of the complex etiology for this condition. Work in animal models for the past 20 years has been pivotal in advancing our understanding of normal suture biology and elucidating pathologic disease mechanisms. This article provides an overview of milestone studies in suture development, embryonic origins, and signaling mechanisms from an array of animal models including transgenic mice, rats, rabbits, fetal sheep, zebrafish, and frogs. This work contributes to an ongoing effort toward continued development of novel treatment strategies.

  View details for DOI 10.1097/SCS.0b013e318258ba53

  View details for Web of Science ID 000209481500006

• Pierre Robin Sequence and Treacher Collins Hypoplastic Mandible Comparison Using Three-Dimensional Morphometric Analysis JOURNAL OF CRANIOFACIAL SURGERY Chung, M. T., Levi, B., Hyun, J. S., Lo, D. D., Montoro, D. T., Lisiecki, J., Bradley, J. P., Buchman, S. R., Longaker, M. T., Wan, D. C. 2012; 23: 1959-1963

  Abstract

  Pierre Robin sequence and Treacher Collins syndrome are both associated with mandibular hypoplasia. It has been hypothesized, however, that the mandible may be differentially affected. The purpose of this study was to therefore compare mandibular morphology in children with Pierre Robin sequence with children with Treacher Collins syndrome using three-dimensional analysis of computed tomographic scans. A retrospective analysis was performed identifying children with Pierre Robin sequence and Treacher Collins syndrome undergoing computed tomography. Three-dimensional reconstruction was performed, and ramus height, mandibular body length, and gonial angle were measured. These were then compared with those in control children with normal mandibles and with the clinical norms corrected for age and sex based on previously published measurements. Mandibular body length was found to be significantly shorter for children with Pierre Robin sequence, whereas ramus height was significantly shorter for children with Treacher Collins syndrome. This resulted in distinctly different ramus height-mandibular body length ratios. In addition, the gonial angle was more obtuse in both the Pierre Robin sequence and Treacher Collins syndrome groups compared with the controls. Three-dimensional mandibular morphometric analysis in patients with Pierre Robin sequence and Treacher Collins syndrome thus revealed distinctly different patterns of mandibular hypoplasia relative to normal controls. These findings underscore distinct considerations that must be made in surgical planning for reconstruction.

  View details for DOI 10.1097/SCS.0b013e318258bcf1

  View details for Web of Science ID 000209481500007

  View details for PubMedCentralID PMC3544559

• Pierre Robin sequence and Treacher Collins hypoplastic mandible comparison using three-dimensional morphometric analysis. journal of craniofacial surgery Chung, M. T., Levi, B., Hyun, J. S., Lo, D. D., Montoro, D. T., Lisiecki, J., Bradley, J. P., Buchman, S. R., Longaker, M. T., Wan, D. C. 2012; 23 (7): 1959-1963

  Abstract

  Pierre Robin sequence and Treacher Collins syndrome are both associated with mandibular hypoplasia. It has been hypothesized, however, that the mandible may be differentially affected. The purpose of this study was to therefore compare mandibular morphology in children with Pierre Robin sequence with children with Treacher Collins syndrome using three-dimensional analysis of computed tomographic scans. A retrospective analysis was performed identifying children with Pierre Robin sequence and Treacher Collins syndrome undergoing computed tomography. Three-dimensional reconstruction was performed, and ramus height, mandibular body length, and gonial angle were measured. These were then compared with those in control children with normal mandibles and with the clinical norms corrected for age and sex based on previously published measurements. Mandibular body length was found to be significantly shorter for children with Pierre Robin sequence, whereas ramus height was significantly shorter for children with Treacher Collins syndrome. This resulted in distinctly different ramus height-mandibular body length ratios. In addition, the gonial angle was more obtuse in both the Pierre Robin sequence and Treacher Collins syndrome groups compared with the controls. Three-dimensional mandibular morphometric analysis in patients with Pierre Robin sequence and Treacher Collins syndrome thus revealed distinctly different patterns of mandibular hypoplasia relative to normal controls. These findings underscore distinct considerations that must be made in surgical planning for reconstruction.

  View details for DOI 10.1097/SCS.0b013e318258bcf1

  View details for PubMedID 23154353

  View details for PubMedCentralID PMC3544559

• Craniosynostosis: Molecular pathways and future pharmacologic therapy. Organogenesis Senarath-Yapa, K., Chung, M. T., McArdle, A., Wong, V. W., Quarto, N., Longaker, M. T., Wan, D. C. 2012; 8 (4)

  Abstract

  Craniosynostosis describes the premature fusion of one or more cranial sutures and can lead to dramatic manifestations in terms of appearance and functional impairment. Contemporary approaches for this condition are primarily surgical and are associated with considerable morbidity and mortality. The additional post-operative problems of suture refusion and bony relapse may also necessitate repeated surgeries with their own attendant risks. Therefore, a need exists to not only optimize current strategies but also to develop novel biological therapies which could obviate the need for surgery and potentially treat or even prevent premature suture fusion. Clinical studies of patients with syndromic craniosynostosis have provided some useful insights into the important signaling pathways and molecular events guiding suture fate. Furthermore, the highly conserved nature of craniofacial development between humans and other species have permitted more focused and step-wise elucidation of the molecular underpinnings of craniosynostosis. This review will describe the clinical manifestations of craniosynostosis, reflect on our understanding of syndromic and non-syndromic craniosynostoses and outline the different approaches that have been adopted in our laboratory and elsewhere to better understand the pathogenesis of premature suture fusion. Finally, we will assess to what extent our improved understanding of the pathogenesis of craniosynostosis, achieved through laboratory-based and clinical studies, have made the possibility of a non-surgical pharmacological approach both realistic and tangible.

  View details for PubMedID 23249483

• Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Lo, D. D., Hyun, J. S., Chung, M. T., Montoro, D. T., Zimmermann, A., Grova, M. M., Lee, M., Wan, D. C., Longaker, M. T. 2012

  View details for DOI 10.3791/4221

  View details for Web of Science ID 000209225700022

• Craniosynostosis Molecular pathways and future pharmacologic therapy ORGANOGENESIS Senarath-Yapa, K., Chung, M. T., McArdle, A., Wong, V. W., Quarto, N., Longaker, M. T., Wan, D. C. 2012; 8 (4): 103-113

  View details for DOI 10.4161/org.23307

  View details for Web of Science ID 000314500600002

• Regenerative Surgery: Tissue Engineering in General Surgical Practice WORLD JOURNAL OF SURGERY Wong, V. W., Wan, D. C., Gurtner, G. C., Longaker, M. T. 2012; 36 (10): 2288-2299

  Abstract

  Tissue engineering is a broad interdisciplinary field that aims to develop complex tissue and organ constructs through a combination of cell-, biomaterial-, and molecular-based approaches. This approach has the potential to transform the surgical treatment for diseases including trauma, cancer, and congenital malformations. A fundamental knowledge of key concepts in regenerative medicine is imperative for surgeons to maintain a leading role in developing and implementing these technologies. Researchers have started to elucidate the biologic mechanisms that maintain organ homeostasis throughout life, indicating that humans may have the latent capacity to regenerate complex tissues. By exploiting this intrinsic potential of the body, we can move even closer to developing functional, autologous replacement parts for a wide range of surgical diseases.

  View details for DOI 10.1007/s00268-012-1710-1

  View details for PubMedID 22777416

• Microfluidic Single-Cell Analysis Shows That Porcine Induced Pluripotent Stem Cell-Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation CIRCULATION RESEARCH Gu, M., Nguyen, P. K., Lee, A. S., Xu, D., Hu, S., Plews, J. R., Han, L., Huber, B. C., Lee, W. H., Gong, Y., de Almeida, P. E., Lyons, J., Ikeno, F., Pacharinsak, C., Connolly, A. J., Gambhir, S. S., Robbins, R. C., Longaker, M. T., Wu, J. C. 2012; 111 (7): 882-893

  Abstract

  Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models.To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia.Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery.In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.

  View details for DOI 10.1161/CIRCRESAHA.112.269001

  View details for PubMedID 22821929

• Scarless fetal skin wound healing update BIRTH DEFECTS RESEARCH PART C-EMBRYO TODAY-REVIEWS Lo, D. D., Zimmermann, A. S., Nauta, A., Longaker, M. T., Lorenz, H. P. 2012; 96 (3): 237-247

  Abstract

  Scar formation, a physiologic process in adult wound healing, can have devastating effects for patients; a multitude of pathologic outcomes, affecting all organ systems, stems from an amplification of this process. In contrast to adult wound repair, the early-gestation fetal skin wound heals without scar formation, a phenomenon that appears to be intrinsic to fetal skin. An intensive research effort has focused on unraveling the mechanisms that underlie scarless fetal wound healing in an attempt to improve the quality of healing in both children and adults. Unique properties of fetal cells, extracellular matrix, cytokine profile, and gene expression contribute to this scarless repair. Despite the great increase in knowledge gained over the past decades, the precise mechanisms regulating scarless fetal healing remain unknown. Herein, we describe the current proposed mechanisms underlying fetal scarless wound healing in an effort to recapitulate the fetal phenotype in the postnatal environment.

  View details for DOI 10.1002/bdrc.21018

  View details for Web of Science ID 000310475000003

  View details for PubMedID 23109319

• Fetal Mouse Skin Heals Scarlessly in a Chick Chorioallantoic Membrane Model System ANNALS OF PLASTIC SURGERY Carre, A. L., Larson, B. J., Knowles, J. A., Kawai, K., Longaker, M. T., Lorenz, H. P. 2012; 69 (1): 85-90

  Abstract

  In mammals, the early-gestation fetus has the regenerative ability to heal skin wounds without scar formation. This observation was first reported more than 3 decades ago, and has been confirmed in a number of in vivo animal models. Although an intensive research effort has focused on unraveling the mechanisms underlying scarless fetal wound repair, no suitable model of in vitro fetal skin healing has been developed. In this article, we report a novel model for the study of fetal wound healing. Fetal skin from gestational day 16.5 Balb/c mice (total gestation, 20 days) was grafted onto the chorioallantoic membrane of 12-day-old chicken embryos and cultured for up to 7 days. At 48 hours postengraftment, circular wounds (diameter = 1 mm) were made in the fetal skin using a rotating titanium sapphire laser (N = 45). The tissue was examined daily by visual inspection to look for signs of infection and ischemia. The grafts and the surrounding host tissue were examined histologically. In all fetal skin grafts, the wounds completely reepithelialized by postinjury day 7, with regeneration of the dermis. Fetal mouse skin xenografts transplanted onto the chorioallantoic membrane of fertilized chicken eggs provides a useful model for the study of fetal wound healing. This model can be used as an adjunct to traditional in vivo mammalian models of fetal repair.

  View details for DOI 10.1097/SAP.0b013e31822128a9

  View details for Web of Science ID 000305485200020

  View details for PubMedID 21712703

• Large animal induced pluripotent stem cells as pre-clinical models for studying human disease JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Plews, J. R., Gu, M., Longaker, M. T., Wu, J. C. 2012; 16 (6): 1196-1202

  Abstract

  The path to induced pluripotency Discovery of a pan-species pluripotency network Animal iPSCs and disease modelling Issues with large animal iPSCs Conclusions The derivation of human embryonic stem cells and subsequently human induced pluripotent stem cells (iPSCs) has energized regenerative medicine research and enabled seemingly limitless applications. Although small animal models, such as mouse models, have played an important role in the progression of the field, typically, they are poor representations of the human disease phenotype. As an alternative, large animal models should be explored as a potentially better approach for clinical translation of cellular therapies. However, only fragmented information regarding the derivation, characterization and clinical usefulness of pluripotent large animal cells is currently available. Here, we briefly review the latest advances regarding the derivation and use of large animal iPSCs.

  View details for DOI 10.1111/j.1582-4934.2012.01521.x

  View details for Web of Science ID 000304468600005

  View details for PubMedID 22212700

  View details for PubMedCentralID PMC3340484

• Craniofacial Reconstruction With Induced Pluripotent Stem Cells JOURNAL OF CRANIOFACIAL SURGERY Wan, D. C., Wong, V. W., Longaker, M. T. 2012; 23 (3): 623-626

  View details for DOI 10.1097/SCS.0b013e318252f41b

  View details for Web of Science ID 000304479600041

  View details for PubMedID 22627398

  View details for PubMedCentralID PMC3544558

• Rethinking the Blastema PLASTIC AND RECONSTRUCTIVE SURGERY Hyun, J. S., Chung, M. T., Wong, V. W., Montoro, D., Longaker, M. T., Wan, D. C. 2012; 129 (5): 1097-1103

  Abstract

  The phenomenon of tissue regeneration has been well documented across many species. Although some possess the capacity to completely restore an entire amputated limb, others are limited to just the distal digit tip. Initiation of limb regeneration has been described to start with the formation of a blastema, the composition of which has long been thought to consist of undifferentiated pluripotent cells derived through the process of dedifferentiation. Competing theories have been proposed, however, including cellular contributions through transdifferentiation and tissue-specific stem cells. Recent studies have now begun to shed light on this controversy, demonstrating tissue resident stem cells to be an evolutionarily conserved measure for limb regeneration.

  View details for DOI 10.1097/PRS.0b013e31824a2c49

  View details for Web of Science ID 000303497300059

  View details for PubMedID 22544093

• A Comparative Analysis of the Osteogenic Effects of BMP-2, FGF-2, and VEGFA in a Calvarial Defect Model TISSUE ENGINEERING PART A Behr, B., Sorkin, M., Lehnhardt, M., Renda, A., Longaker, M. T., Quarto, N. 2012; 18 (9-10): 1079-1086

  Abstract

  The utilization of growth factors for bone regeneration is a widely studied field. Since the approval of bone morphogenetic protein-2 (BMP-2) for therapeutic use in humans, the concept of utilizing growth factors for bone regeneration in translational medicine has become even more attractive. Despite many studies published on individual growth factors in various bone models, comparative analysis is largely lacking. The aim of our study was to compare three different proosteogenic factors under identical in vivo conditions. Thus, we tested the bone regeneration capacity of the three different growth factors BMP-2, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor A (VEGFA) in a calvarial defect model. We demonstrated that BMP-2 and VEGFA had similar bone healing capacities, resulting in complete calvarial healing as early as week 3. FGF-2 also showed a significantly higher bone regeneration capacity; however, the healing rate was lower than with BMP-2 and VEGFA. Interestingly, these findings were paralleled by an increased angiogenic response upon healing in BMP-2- and VEGFA-treated calvarial defects as compared with FGF-2. Immunohistochemistry for proliferating and osteoprogenitor cells revealed activity at different points after surgery among the groups. In conclusion, we demonstrated an efficient bone regeneration capacity of both BMP-2 and VEGFA, which was superior to FGF-2. Moreover, this study highlights the efficient bone regeneration of VEGFA, which was comparable with BMP-2. These data provide a valuable comparative analysis, which can be used to further optimize growth factor-based strategies in skeletal tissue engineering.

  View details for DOI 10.1089/ten.tea.2011.0537

  View details for Web of Science ID 000303540400019

  View details for PubMedID 22195699

  View details for PubMedCentralID PMC3338108

• Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy SCIENCE TRANSLATIONAL MEDICINE Sun, N., Yazawa, M., Liu, J., Han, L., Sanchez-Freire, V., Abilez, O. J., Navarrete, E. G., Hu, S., Wang, L., Lee, A., Pavlovic, A., Lin, S., Chen, R., Hajjar, R. J., Snyder, M. P., Dolmetsch, R. E., Butte, M. J., Ashley, E. A., Longaker, M. T., Robbins, R. C., Wu, J. C. 2012; 4 (130)

  Abstract

  Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca(2+)), decreased contractility, and abnormal distribution of sarcomeric α-actinin. When stimulated with a β-adrenergic agonist, DCM iPSC-derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric α-actinin distribution. Treatment with β-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.

  View details for DOI 10.1126/scitranslmed.3003552

  View details for Web of Science ID 000303045900004

  View details for PubMedID 22517884

  View details for PubMedCentralID PMC3657516

• Training the Contemporary Surgeon-Scientist PLASTIC AND RECONSTRUCTIVE SURGERY Wan, D. C., Wang, K. C., Longaker, M. T. 2012; 129 (4): 1023-1025

  View details for DOI 10.1097/PRS.0b013e31824421e8

  View details for Web of Science ID 000302227100076

  View details for PubMedID 22456371

• Delivery Strategies for Stem Cell-Based Therapy JOURNAL OF HEALTHCARE ENGINEERING Glotzbach, J. P., Wong, V. W., Levi, B., Longaker, M. T., Gurtner, G. C. 2012; 3 (1): 1-20

  View details for DOI 10.1260/2040-2295.3.1.1

  View details for Web of Science ID 000311674000001

• The angiogenic factor Dell prevents apoptosis of endothelial cells through integrin binding SURGERY Wang, Z., Kundu, R. K., Longaker, M. T., Quertermous, T., Yang, G. P. 2012; 151 (2): 296-305

  Abstract

  Del1 is a secreted protein that is expressed in the endothelium during development and can stimulate angiogenesis through integrin binding and signaling. We were interested in the specific effects of del1 on endothelial cell biology to gain insight into its biologic role during angiogenesis.Primary endothelial cells were treated with a variety of inducers of apoptosis and anoikis followed by assays for numbers of apoptotic cells, and harvest of total protein for immunoblot analysis.Del1 prevented endothelial cell apoptosis in response to TNFα/IFNγ, etoposide, and anoikis, but had no effect on proliferation. The anti-apoptotic effect was mediated specifically through binding of integrin αvβ3 by the RGD motif. FAK/ERK and Akt signaling were both necessary to mediate the anti-apoptotic effect of Del1 with the exception of anoikis, which required only Akt activation.Del1 has been previously shown to promote vascular smooth muscle cell adhesion, migration, and proliferation. We demonstrate here that Del1 prevented apoptosis of endothelial cells in cell culture through integrin binding without any effect on proliferation.

  View details for DOI 10.1016/j.surg.2011.07.013

  View details for Web of Science ID 000299607800019

  View details for PubMedID 21893328

• Skeletogenic phenotype of human Marfan embryonic stem cells faithfully phenocopied by patient-specific induced-pluripotent stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Quarto, N., Leonard, B., Li, S., Marchand, M., Anderson, E., Behr, B., Francke, U., Reijo-Pera, R., Chiao, E., Longaker, M. T. 2012; 109 (1): 215-220

  Abstract

  Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the gene coding for FIBRILLIN-1 (FBN1), an extracellular matrix protein. MFS is inherited as an autosomal dominant trait and displays major manifestations in the ocular, skeletal, and cardiovascular systems. Here we report molecular and phenotypic profiles of skeletogenesis in tissues differentiated from human embryonic stem cells and induced pluripotent stem cells that carry a heritable mutation in FBN1. We demonstrate that, as a biological consequence of the activation of TGF-β signaling, osteogenic differentiation of embryonic stem cells with a FBN1 mutation is inhibited; osteogenesis is rescued by inhibition of TGF-β signaling. In contrast, chondrogenesis is not perturbated and occurs in a TGF-β cell-autonomous fashion. Importantly, skeletal phenotypes observed in human embryonic stem cells carrying the monogenic FBN1 mutation (MFS cells) are faithfully phenocopied by cells differentiated from induced pluripotent-stem cells derived independently from MFS patient fibroblasts. Results indicate a unique phenotype uncovered by examination of mutant pluripotent stem cells and further demonstrate the faithful alignment of phenotypes in differentiated cells obtained from both human embryonic stem cells and induced pluripotent-stem cells, providing complementary and powerful tools to gain further insights into human molecular pathogenesis, especially of MFS.

  View details for DOI 10.1073/pnas.1113442109

  View details for Web of Science ID 000298876500045

  View details for PubMedID 22178754

  View details for PubMedCentralID PMC3252902

• Enhancement of Human Adipose-Derived Stromal Cell Angiogenesis through Knockdown of a BMP-2 Inhibitor PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., Nelson, E. R., Hyun, J. S., Glotzbach, J. P., Li, S., Nauta, A., Montoro, D. T., Lee, M., Commons, G. C., Hu, S., Wu, J. C., Gurtner, G. C., Longaker, M. T. 2012; 129 (1): 53-66

  Abstract

  Previous studies have demonstrated the role of noggin, a bone morphogenetic protein-2 inhibitor, in vascular development and angiogenesis. The authors hypothesized that noggin suppression in human adipose-derived stromal cells would enhance vascular endothelial growth factor secretion and angiogenesis in vitro and in vivo to a greater extent than bone morphogenetic protein-2 alone.Human adipose-derived stromal cells were isolated from human lipoaspirate (n = 6) noggin was knocked down using lentiviral techniques. Knockdown was confirmed and angiogenesis was assessed by tubule formation and quantitative real-time polymerase chain reaction. Cells were seeded onto scaffolds and implanted into a 4-mm critical size calvarial defect. In vivo angiogenic signaling was assessed by immunofluorescence and immunohistochemistry.Human adipose-derived stromal cells with noggin suppression secreted significantly higher amounts of angiogenic proteins, expressed higher levels of angiogenic genes, and formed more tubules in vitro. In vivo, calvarial defects seeded with noggin shRNA human adipose-derived stromal cells exhibited a significantly higher number of vessels in the defect site than controls by immunohistochemistry (p < 0.05). In addition, bone morphogenetic protein-2-releasing scaffolds significantly enhanced vascular signaling in the defect site.Human adipose-derived stromal cells demonstrate significant increases in angiogenesis in vitro and in vivo with both noggin suppression and BMP-2 supplementation. By creating a cell with noggin suppressed and by using a scaffold with increased bone morphogenetic protein-2 signaling, a more angiogenic niche can be created.

  View details for DOI 10.1097/PRS.0b013e3182361ff5

  View details for PubMedID 21915082

• Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling NATURE MEDICINE Wong, V. W., Rustad, K. C., Akaishi, S., Sorkin, M., Glotzbach, J. P., Januszyk, M., Nelson, E. R., Levi, K., Paterno, J., Vial, I. N., Kuang, A. A., Longaker, M. T., Gurtner, G. C. 2012; 18 (1): 148-152

  Abstract

  Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK). Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK-ERK-MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.

  View details for DOI 10.1038/nm.2574

  View details for Web of Science ID 000299018600041

• Stem cell niches for skin regeneration. International journal of biomaterials Wong, V. W., Levi, B., Rajadas, J., Longaker, M. T., Gurtner, G. C. 2012; 2012: 926059-?

  Abstract

  Stem cell-based therapies offer tremendous potential for skin regeneration following injury and disease. Functional stem cell units have been described throughout all layers of human skin and the collective physical and chemical microenvironmental cues that enable this regenerative potential are known as the stem cell niche. Stem cells in the hair follicle bulge, interfollicular epidermis, dermal papillae, and perivascular space have been closely investigated as model systems for niche-driven regeneration. These studies suggest that stem cell strategies for skin engineering must consider the intricate molecular and biologic features of these niches. Innovative biomaterial systems that successfully recapitulate these microenvironments will facilitate progenitor cell-mediated skin repair and regeneration.

  View details for DOI 10.1155/2012/926059

  View details for PubMedID 22701121

• Enhancement of mesenchymal stem cell angiogenic capacity and stemness by a biomimetic hydrogel scaffold BIOMATERIALS Rustad, K. C., Wong, V. W., Sorkin, M., Glotzbach, J. P., Major, M. R., Rajadas, J., Longaker, M. T., Gurtner, G. C. 2012; 33 (1): 80-90

  Abstract

  In this study, we examined the capacity of a biomimetic pullulan-collagen hydrogel to create a functional biomaterial-based stem cell niche for the delivery of mesenchymal stem cells (MSCs) into wounds. Murine bone marrow-derived MSCs were seeded into hydrogels and compared to MSCs grown in standard culture conditions. Hydrogels induced MSC secretion of angiogenic cytokines and expression of transcription factors associated with maintenance of pluripotency and self-renewal (Oct4, Sox2, Klf4) when compared to MSCs grown in standard conditions. An excisonal wound healing model was used to compare the ability of MSC-hydrogel constructs versus MSC injection alone to accelerate wound healing. Injection of MSCs did not significantly improve time to wound closure. In contrast, wounds treated with MSC-seeded hydrogels showed significantly accelerated healing and a return of skin appendages. Bioluminescence imaging and FACS analysis of luciferase+/GFP+ MSCs indicated that stem cells delivered within the hydrogel remained viable longer and demonstrated enhanced engraftment efficiency than those delivered via injection. Engrafted MSCs were found to differentiate into fibroblasts, pericytes and endothelial cells but did not contribute to the epidermis. Wounds treated with MSC-seeded hydrogels demonstrated significantly enhanced angiogenesis, which was associated with increased levels of VEGF and other angiogenic cytokines within the wounds. Our data suggest that biomimetic hydrogels provide a functional niche capable of augmenting MSC regenerative potential and enhancing wound healing.

  View details for DOI 10.1016/j.biomaterials.2011.09.041

  View details for Web of Science ID 000297399700009

  View details for PubMedID 21963148

• Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling. Nature medicine Wong, V. W., Rustad, K. C., Akaishi, S., Sorkin, M., Glotzbach, J. P., Januszyk, M., Nelson, E. R., Levi, K., Paterno, J., Vial, I. N., Kuang, A. A., Longaker, M. T., Gurtner, G. C. 2012; 18 (1): 148-152

  Abstract

  Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK). Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK-ERK-MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.

  View details for DOI 10.1038/nm.2574

  View details for PubMedID 22157678

• Craniofacial Surgery: Innovation, Design, and Strategy JOURNAL OF CRANIOFACIAL SURGERY Warren, S. M., Longaker, M. T. 2012; 23 (1): 7-7

  View details for DOI 10.1097/SCS.0b013e31824209e8

  View details for Web of Science ID 000300234900029

  View details for PubMedID 22337364

• Cranial Suture Biology: From Pathways to Patient Care JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Wan, D. C., Wong, V. W., Nelson, E., Hyun, J., Longaker, M. T. 2012; 23 (1): 13-19

  Abstract

  Craniosynostosis describes the premature pathologic partial or complete fusion of 1 or more of the cranial sutures. Over the past few decades, research on craniosynostosis has progressed from gross description of deformities to an understanding of some of the molecular etiologies behind premature suture fusion. Studies on patients with syndromic craniosynostosis have resulted in the identification of several genes, molecular events, and deformational forces involved in abnormal growth and development of the cranial vault. Conservation of craniofacial development and sequence homology between humans and other species have also led to insightful discoveries in cranial suture development. In this review, we discuss the development of the cranial vault and explain the basic science behind craniosynostosis in humans as well as in animal models and how these studies may lead to future advances in craniosynostosis treatments.

  View details for DOI 10.1097/SCS.0b013e318240c6c0

  View details for Web of Science ID 000300234900033

  View details for PubMedID 22337368

• Repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate. Journal of visualized experiments : JoVE Lo, D. D., Hyun, J. S., Chung, M. T., Montoro, D. T., Zimmermann, A., Grova, M. M., Lee, M., Wan, D. C., Longaker, M. T. 2012

  Abstract

  Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

  View details for DOI 10.3791/4221

  View details for PubMedID 23149856

  View details for PubMedCentralID PMC3499066

• Stem Cells: Update and Impact on Craniofacial Surgery JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Glotzbach, J. P., Wong, V. W., Nelson, E. R., Hyun, J., Wan, D. C., Gurtner, G. C., Longaker, M. T. 2012; 23 (1): 319-322

  View details for DOI 10.1097/SCS.0b013e318241dbaf

  View details for Web of Science ID 000300234900099

  View details for PubMedID 22337434

  View details for PubMedCentralID PMC3282019

• Nonintegrating Knockdown and Customized Scaffold Design Enhances Human Adipose-Derived Stem Cells in Skeletal Repair STEM CELLS Levi, B., Hyun, J. S., Nelson, E. R., Li, S., Montoro, D. T., Wan, D. C., Jia, F. J., Glotzbach, J. C., James, A. W., Lee, M., Huang, M., Quarto, N., Gurtner, G. C., Wu, J. C., Longaker, M. T. 2011; 29 (12): 2018-2029

  Abstract

  An urgent need exists in clinical medicine for suitable alternatives to available techniques for bone tissue repair. Human adipose-derived stem cells (hASCs) represent a readily available, autogenous cell source with well-documented in vivo osteogenic potential. In this article, we manipulated Noggin expression levels in hASCs using lentiviral and nonintegrating minicircle short hairpin ribonucleic acid (shRNA) methodologies in vitro and in vivo to enhance hASC osteogenesis. Human ASCs with Noggin knockdown showed significantly increased bone morphogenetic protein (BMP) signaling and osteogenic differentiation both in vitro and in vivo, and when placed onto a BMP-releasing scaffold embedded with lentiviral Noggin shRNA particles, hASCs more rapidly healed mouse calvarial defects. This study therefore suggests that genetic targeting of hASCs combined with custom scaffold design can optimize hASCs for skeletal regenerative medicine.

  View details for DOI 10.1002/stem.757

  View details for PubMedID 21997852

• Mechanical force prolongs acute inflammation via T-cell-dependent pathways during scar formation FASEB JOURNAL Wong, V. W., Paterno, J., Sorkin, M., Glotzbach, J. P., Levi, K., Januszyk, M., Rustad, K. C., Longaker, M. T., Gurtner, G. C. 2011; 25 (12): 4498-4510

  Abstract

  Mechanical force significantly modulates both inflammation and fibrosis, yet the fundamental mechanisms that regulate these interactions remain poorly understood. Here we performed microarray analysis to compare gene expression in mechanically loaded wounds vs. unloaded control wounds in an established murine hypertrophic scar (HTS) model. We identified 853 mechanically regulated genes (false discovery rate <2) at d 14 postinjury, a subset of which were enriched for T-cell-regulated pathways. To substantiate the role of T cells in scar mechanotransduction, we applied the HTS model to T-cell-deficient mice and wild-type mice. We found that scar formation in T-cell-deficient mice was reduced by almost 9-fold (P < 0.001) with attenuated epidermal (by 2.6-fold, P < 0.01) and dermal (3.9-fold, P < 0.05) proliferation. Mechanical stimulation was highly associated with sustained T-cell-dependent Th2 cytokine (IL-4 and IL-13) and chemokine (MCP-1) signaling. Further, T-cell-deficient mice failed to recruit systemic inflammatory cells such as macrophages or monocytic fibroblast precursors in response to mechanical loading. These findings indicate that T-cell-regulated fibrogenic pathways are highly mechanoresponsive and suggest that mechanical forces induce a chronic-like inflammatory state through immune-dependent activation of both local and systemic cell populations.

  View details for DOI 10.1096/fj.10-178087

  View details for Web of Science ID 000298138100040

  View details for PubMedID 21911593

• Antimycotic Ciclopirox Olamine in the Diabetic Environment Promotes Angiogenesis and Enhances Wound Healing PLOS ONE Ko, S. H., Nauta, A., Morrison, S. D., Zhou, H., Zimmermann, A., Gurtner, G. C., Ding, S., Longaker, M. T. 2011; 6 (11)

  Abstract

  Diabetic wounds remain a major medical challenge with often disappointing outcomes despite the best available care. An impaired response to tissue hypoxia and insufficient angiogenesis are major factors responsible for poor healing in diabetic wounds. Here we show that the antimycotic drug ciclopirox olamine (CPX) can induce therapeutic angiogenesis in diabetic wounds. Treatment with CPX in vitro led to upregulation of multiple angiogenic genes and increased availability of HIF-1α. Using an excisional wound splinting model in diabetic mice, we showed that serial topical treatment with CPX enhanced wound healing compared to vehicle control treatment, with significantly accelerated wound closure, increased angiogenesis, and increased dermal cellularity. These findings offer a promising new topical pharmacologic therapy for the treatment of diabetic wounds.

  View details for DOI 10.1371/journal.pone.0027844

  View details for Web of Science ID 000297789200029

  View details for PubMedID 22125629

  View details for PubMedCentralID PMC3220686

• CD105 Protein Depletion Enhances Human Adipose-derived Stromal Cell Osteogenesis through Reduction of Transforming Growth Factor beta 1 (TGF-beta 1) Signaling JOURNAL OF BIOLOGICAL CHEMISTRY Levi, B., Wan, D. C., Glotzbach, J. P., Hyun, J., Januszyk, M., Montoro, D., Sorkin, M., James, A. W., Nelson, E. R., Li, S., Quarto, N., Lee, M., Gurtner, G. C., Longaker, M. T. 2011; 286 (45): 39497-39509

  Abstract

  Clinically available sources of bone for repair and reconstruction are limited by the accessibility of autologous grafts, infectious risks of cadaveric materials, and durability of synthetic substitutes. Cell-based approaches for skeletal regeneration can potentially fill this need, and adipose tissue represents a promising source for development of such therapies. Here, we enriched for an osteogenic subpopulation of cells derived from human subcutaneous adipose tissue utilizing microfluidic-based single cell transcriptional analysis and fluorescence-activated cell sorting (FACS). Statistical analysis of single cell transcriptional profiles demonstrated that low expression of endoglin (CD105) correlated with a subgroup of adipose-derived cells with increased osteogenic gene expression. FACS-sorted CD105(low) cells demonstrated significantly enhanced in vitro osteogenic differentiation and in vivo bone regeneration when compared with either CD105(high) or unsorted cells. Evaluation of the endoglin pathway suggested that enhanced osteogenesis among CD105(low) adipose-derived cells is likely due to identification of a subpopulation with lower TGF-β1/Smad2 signaling. These findings thus highlight a potential avenue to promote osteogenesis in adipose-derived mesenchymal cells for skeletal regeneration.

  View details for DOI 10.1074/jbc.M111.256529

  View details for PubMedID 21949130

• Pullulan Hydrogels Improve Mesenchymal Stem Cell Delivery into High-Oxidative-Stress Wounds MACROMOLECULAR BIOSCIENCE Wong, V. W., Rustad, K. C., Glotzbach, J. P., Sorkin, M., Inayathullah, M., Major, M. R., Longaker, M. T., Rajadas, J., Gurtner, G. C. 2011; 11 (11): 1458-1466

  Abstract

  Cell-based therapies for wound repair are limited by inefficient delivery systems that fail to protect cells from the acute inflammatory environment. Here, a biomimetic hydrogel system is described that is based on the polymer pullulan, a carbohydrate glucan known to exhibit potent antioxidant capabilities. It is shown that pullulan hydrogels are an effective cell delivery system and improve mesenchymal stem cell survival and engraftment in high-oxidative-stress environments. The results suggest that glucan hydrogel systems may prove beneficial for progenitor-cell-based approaches to skin regeneration.

  View details for DOI 10.1002/mabi.201100180

  View details for Web of Science ID 000297555500002

  View details for PubMedID 21994074

• Calcium-Based Nanoparticles Accelerate Skin Wound Healing PLOS ONE Kawai, K., Larson, B. J., Ishise, H., Carre, A. L., Nishimoto, S., Longaker, M., Lorenz, H. P. 2011; 6 (11)

  Abstract

  Nanoparticles (NPs) are small entities that consist of a hydroxyapatite core, which can bind ions, proteins, and other organic molecules from the surrounding environment. These small conglomerations can influence environmental calcium levels and have the potential to modulate calcium homeostasis in vivo. Nanoparticles have been associated with various calcium-mediated disease processes, such as atherosclerosis and kidney stone formation. We hypothesized that nanoparticles could have an effect on other calcium-regulated processes, such as wound healing. In the present study, we synthesized pH-sensitive calcium-based nanoparticles and investigated their ability to enhance cutaneous wound repair.Different populations of nanoparticles were synthesized on collagen-coated plates under various growth conditions. Bilateral dorsal cutaneous wounds were made on 8-week-old female Balb/c mice. Nanoparticles were then either administered intravenously or applied topically to the wound bed. The rate of wound closure was quantified. Intravenously injected nanoparticles were tracked using a FLAG detection system. The effect of nanoparticles on fibroblast contraction and proliferation was assessed.A population of pH-sensitive calcium-based nanoparticles was identified. When intravenously administered, these nanoparticles acutely increased the rate of wound healing. Intravenously administered nanoparticles were localized to the wound site, as evidenced by FLAG staining. Nanoparticles increased fibroblast calcium uptake in vitro and caused contracture of a fibroblast populated collagen lattice in a dose-dependent manner. Nanoparticles also increased the rate of fibroblast proliferation.Intravenously administered, calcium-based nanoparticles can acutely decrease open wound size via contracture. We hypothesize that their contraction effect is mediated by the release of ionized calcium into the wound bed, which occurs when the pH-sensitive nanoparticles disintegrate in the acidic wound microenvironment. This is the first study to demonstrate that calcium-based nanoparticles can have a therapeutic benefit, which has important implications for the treatment of wounds.

  View details for DOI 10.1371/journal.pone.0027106

  View details for Web of Science ID 000297154900079

  View details for PubMedID 22073267

  View details for PubMedCentralID PMC3206933

• Pushing Back: Wound Mechanotransduction in Repair and Regeneration JOURNAL OF INVESTIGATIVE DERMATOLOGY Wong, V. W., Akaishi, S., Longaker, M. T., Gurtner, G. C. 2011; 131 (11): 2186-2196

  Abstract

  Human skin is a highly specialized mechanoresponsive interface separating our bodies from the external environment. It must constantly adapt to dynamic physical cues ranging from rapid expansion during embryonic and early postnatal development to ubiquitous external forces throughout life. Despite the suspected role of the physical environment in cutaneous processes, the fundamental molecular mechanisms responsible for how skin responds to force remain unclear. Intracellular pathways convert mechanical cues into biochemical responses (in a process known as mechanotransduction) via complex mechanoresponsive elements that often blur the distinction between physical and chemical signaling. For example, cellular focal adhesion components exhibit dual biochemical and scaffolding functions that are critically modulated by force. Moreover, the extracellular matrix itself is increasingly recognized to mechanically regulate the spatiotemporal distribution of soluble and matrix-bound ligands, underscoring the importance of bidirectional crosstalk between cells and their physical environment. It seems likely that a structural hierarchy exists to maintain both cells and matrix in mechanical homeostasis and that dysregulation of this architectural integrity may underlie or contribute to various skin disorders. An improved understanding of these interactions will facilitate the development of novel biophysical materials and mechanomodulatory approaches to augment wound repair and regeneration.

  View details for DOI 10.1038/jid.2011.212

  View details for Web of Science ID 000296240100012

  View details for PubMedID 21776006

• Role of GSK-3 beta in the Osteogenic Differentiation of Palatal Mesenchyme PLOS ONE Nelson, E. R., Levi, B., Sorkin, M., James, A. W., Liu, K. J., Quarto, N., Longaker, M. T. 2011; 6 (10)

  Abstract

  The function of Glycogen Synthase Kinases 3β (GSK-3β) has previously been shown to be necessary for normal secondary palate development. Using GSK-3ß null mouse embryos, we examine the potential coordinate roles of Wnt and Hedgehog signaling on palatal ossification.Palates were harvested from GSK-3β, embryonic days 15.0-18.5 (e15.0-e18.5), and e15.5 Indian Hedgehog (Ihh) null embryos, and their wild-type littermates. The phenotype of GSK-3β null embryos was analyzed with skeletal whole mount and pentachrome stains. Spatiotemporal regulation of osteogenic gene expression, in addition to Wnt and Hedgehog signaling activity, were examined in vivo on GSK-3β and Ihh +/+ and -/- e15.5 embryos using in situ hybridization and immunohistochemistry. To corroborate these results, expression of the same molecular targets were assessed by qRT-PCR of e15.5 palates, or e13.5 palate cultures treated with both Wnt and Hedgehog agonists and anatagonists.GSK-3β null embryos displayed a 48 percent decrease (*p<0.05) in palatine bone formation compared to wild-type littermates. GSK-3β null embryos also exhibited decreased osteogenic gene expression that was associated with increased Wnt and decreased Hedgehog signaling. e13.5 palate culture studies demonstrated that Wnt signaling negatively regulates both osteogenic gene expression and Hedgehog signaling activity, while inhibition of Wnt signaling augments both osteogenic gene expression and Hedgehog signaling activity. In addition, no differences in Wnt signaling activity were noted in Ihh null embryos, suggesting that canonical Wnt may be upstream of Hedgehog in secondary palate development. Lastly, we found that GSK-3β -/- palate cultures were "rescued" with the Wnt inhibitor, Dkk-1.Here, we identify a critical role for GSK-3β in palatogenesis through its direct regulation of canonical Wnt signaling. These findings shed light on critical developmental pathways involved in palatogenesis and may lead to novel molecular targets to prevent cleft palate formation.

  View details for DOI 10.1371/journal.pone.0025847

  View details for Web of Science ID 000295981600015

  View details for PubMedID 22022457

  View details for PubMedCentralID PMC3194817

• Indian Hedgehog Positively Regulates Calvarial Ossification and Modulates Bone Morphogenetic Protein Signaling GENESIS Lenton, K., James, A. W., Manu, A., Brugmann, S. A., Birker, D., Nelson, E. R., Leucht, P., Helms, J. A., Longaker, M. T. 2011; 49 (10): 784-796

  Abstract

  Much is known regarding the role of Indian hedgehog (Ihh) in endochondral ossification, where Ihh regulates multiple steps of chondrocyte differentiation. The Ihh-/- phenotype is most notable for severely foreshortened limbs and a complete absence of mature osteoblasts. A far less explored phenotype in the Ihh-/- mutant is found in the calvaria, where bones form predominately through intramembranous ossification. We investigated the role of Ihh in calvarial bone ossification, finding that proliferation was largely unaffected. Instead, our results indicate that Ihh is a pro-osteogenic factor that positively regulates intramembranous ossification. We confirmed through histologic and quantitative gene analysis that loss of Ihh results in reduction of cranial bone size and all markers of osteodifferentiation. Moreover, in vitro studies suggest that Ihh loss reduces Bmp expression within the calvaria, an observation that may underlie the Ihh-/- calvarial phenotype. In conjunction with the newly recognized roles of Hedgehog deregulation in craniosynostosis, our study defines Ihh as an important positive regulator of cranial bone ossification.

  View details for DOI 10.1002/dvg.20768

  View details for Web of Science ID 000296420300003

  View details for PubMedID 21557453

• Palatogenesis Engineering, pathways and pathologies ORGANOGENESIS Levi, B., Brugman, S., Wong, V. W., Grova, M., Longaker, M. T., Wan, D. C. 2011; 7 (4): 242-254

  Abstract

  Cleft palate represents the second most common birth defect and carries substantial physiologic and social challenges for affected patients, as they often require multiple surgical interventions during their lifetime. A number of genes have been identified to be associated with the cleft palate phenotype, but etiology in the majority of cases remains elusive. In order to better understand cleft palate and both surgical and potential tissue engineering approaches for repair, we have performed an in-depth literature review into cleft palate development in humans and mice, as well as into molecular pathways underlying these pathologic developments. We summarize the multitude of pathways underlying cleft palate development, with the transforming growth factor beta superfamily being the most commonly studied. Furthermore, while the majority of cleft palate studies are performed using a mouse model, studies focusing on tissue engineering have also focused heavily on mouse models. A paucity of human randomized controlled studies exists for cleft palate repair, and so far, tissue engineering approaches are limited. In this review, we discuss the development of the palate, explain the basic science behind normal and pathologic palate development in humans as well as mouse models and elaborate on how these studies may lead to future advances in palatal tissue engineering and cleft palate treatments.

  View details for DOI 10.4161/org.7.4.17926

  View details for Web of Science ID 000299593000002

  View details for PubMedID 21964245

  View details for PubMedCentralID PMC3265826

• Preclinical Derivation and Imaging of Autologously Transplanted Canine Induced Pluripotent Stem Cells JOURNAL OF BIOLOGICAL CHEMISTRY Lee, A. S., Xu, D., Plews, J. R., Nguyen, P. K., Nag, D., Lyons, J. K., Han, L., Hu, S., Lan, F., Liu, J., Huang, M., Narsinh, K. H., Long, C. T., de Almeida, P. E., Levi, B., Kooreman, N., Bangs, C., Pacharinsak, C., Ikeno, F., Yeung, A. C., Gambhir, S. S., Robbins, R. C., Longaker, M. T., Wu, J. C. 2011; 286 (37): 32697-32704

  Abstract

  Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.

  View details for DOI 10.1074/jbc.M111.235739

  View details for Web of Science ID 000294726800078

  View details for PubMedID 21719696

  View details for PubMedCentralID PMC3173214

• Wound healing and regenerative strategies ORAL DISEASES Nauta, A., Gurtner, G. C., Longaker, M. T. 2011; 17 (6): 541-549

  Abstract

  Wound healing is a complex biological process that affects multiple tissue types. Wounds in the oral cavity are particularly challenging given the variety of tissue types that exist in close proximity to one another. The goal of regenerative medicine is to facilitate the rapid replacement of lost or damaged tissue with tissue that is functional, and physiologically similar to what previously existed. This review provides a general overview of wound healing and regenerative medicine, focusing specifically on how recent advances in the fields of stem cell biology, tissue engineering, and oral disease could translate into improved clinical outcomes.

  View details for DOI 10.1111/j.1601-0825.2011.01787.x

  View details for Web of Science ID 000292985000001

  View details for PubMedID 21332599

• Recommendations on clinical proof of efficacy for potential scar prevention and reduction therapies WOUND REPAIR AND REGENERATION Bush, J. A., McGrouther, D. A., Young, V. L., Herndon, D. N., Longaker, M. T., Mustoe, T. A., Ferguson, M. W. 2011; 19: S32-S37

  Abstract

  Cutaneous scarring is an enormous medical problem with approximately 100 million patients acquiring scars each year. Scar prevention/reduction represents a significant, and largely unmet, clinical need. Research into the prophylactic modulation of scar outcome differs from research into other disease processes as the scar is not present at the start of the study; measurements of changes from baseline are impossible. Final scar morphology is influenced by many variables. A fundamental principle that should be observed in the prospective evaluation of scar prevention/reduction therapies is that, if left untreated, wounds in treatment and control groups should have healed with identical scars. Observation of this principle will allow the detection of true treatment effects. The many variables that influence scar morphology mean that the evaluation of potential pharmaceutical products for this indication favors the use of self-controlled designs in clinical trials. In this article, we review variables that affect scar morphology and recommend the self-controlled design for clinical trials aiming to establish proof of efficacy of scar prevention and reduction pharmaceuticals. With no pharmaceutical products currently licensed for this indication, this represents a new therapeutic area. The principles discussed will also have direct relevance to the wider fields of wound healing and regenerative medicine.

  View details for DOI 10.1111/j.1524-475X.2010.00607.x

  View details for Web of Science ID 000293237400006

  View details for PubMedID 21793964

• Commentary on Role of Apoptosis in Retinoic Acid-Induced Cleft Palate JOURNAL OF CRANIOFACIAL SURGERY Nelson, E. R., Levi, B., Longaker, M. T. 2011; 22 (5): 1572-1573

  View details for DOI 10.1097/SCS.0b013e31822e5ea6

  View details for Web of Science ID 000295398700007

  View details for PubMedID 21959389

• Vascular anastomosis using controlled phase transitions in poloxamer gels NATURE MEDICINE Chang, E. I., Galvez, M. G., Glotzbach, J. P., Hamou, C. D., El-Ftesi, S., Rappleye, C. T., Sommer, K., Rajadas, J., Abilez, O. J., Fuller, G. G., Longaker, M. T., Gurtner, G. C. 2011; 17 (9): 1147-U160

  Abstract

  Vascular anastomosis is the cornerstone of vascular, cardiovascular and transplant surgery. Most anastomoses are performed with sutures, which are technically challenging and can lead to failure from intimal hyperplasia and foreign body reaction. Numerous alternatives to sutures have been proposed, but none has proven superior, particularly in small or atherosclerotic vessels. We have developed a new method of sutureless and atraumatic vascular anastomosis that uses US Food and Drug Administration (FDA)-approved thermoreversible tri-block polymers to temporarily maintain an open lumen for precise approximation with commercially available glues. We performed end-to-end anastomoses five times more rapidly than we performed hand-sewn controls, and vessels that were too small (<1.0 mm) to sew were successfully reconstructed with this sutureless approach. Imaging of reconstructed rat aorta confirmed equivalent patency, flow and burst strength, and histological analysis demonstrated decreased inflammation and fibrosis at up to 2 years after the procedure. This new technology has potential for improving efficiency and outcomes in the surgical treatment of cardiovascular disease.

  View details for DOI 10.1038/nm.2424

  View details for Web of Science ID 000294605100038

  View details for PubMedID 21873986

• Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip NATURE Rinkevich, Y., Lindau, P., Ueno, H., Longaker, M. T., Weissman, I. L. 2011; 476 (7361): 409-U53

  Abstract

  The regrowth of amputated limbs and the distal tips of digits represent models of tissue regeneration in amphibians, fish and mice. For decades it had been assumed that limb regeneration derived from the blastema, an undifferentiated pluripotent cell population thought to be derived from mature cells via dedifferentiation. Here we show that a wide range of tissue stem/progenitor cells contribute towards the restoration of the mouse distal digit. Genetic fate mapping and clonal analysis of individual cells revealed that these stem cells are lineage restricted, mimicking digit growth during development. Transplantation of cyan-fluorescent-protein-expressing haematopoietic stem cells, and parabiosis between genetically marked mice, confirmed that the stem/progenitor cells are tissue resident, including the cells involved in angiogenesis. These results, combined with those from appendage regeneration in other vertebrate subphyla, collectively demonstrate that tissue stem cells rather than pluripotent blastema cells are an evolutionarily conserved cellular mode for limb regeneration after amputation.

  View details for DOI 10.1038/nature10346

  View details for Web of Science ID 000294209400027

  View details for PubMedID 21866153

• Differences in Osteogenic Differentiation of Adipose-Derived Stromal Cells from Murine, Canine, and Human Sources In Vitro and In Vivo PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., Nelson, E. R., Brown, K., James, A. W., Xu, D., Dunlevie, R., Wu, J. C., Lee, M., Wu, B., Commons, G. W., Vistnes, D., Longaker, M. T. 2011; 128 (2): 373-386

  Abstract

  Given the diversity of species from which adipose-derived stromal cells are derived and studied, the authors set out to delineate the differences in the basic cell biology that may exist across species. Briefly, the authors found that significant differences exist with regard to proliferation and osteogenic potentials of adipose-derived stromal cells across species.Adipose-derived stromal cells were derived from human, mouse, and canine sources as previously described. Retinoic acid, insulin-like growth factor-1, and bone morphogenetic protein-2 were added to culture medium; proliferation and osteogenic differentiation were assessed by standardized assays. In vivo methods included seeding 150,000 adipose-derived stromal cells on a biomimetic scaffold and analyzing healing by micro-computed tomography and histology.Adipose-derived stromal cells from all species had the capability to undergo osteogenic differentiation. Canine adipose-derived stromal cells were the most proliferative, whereas human adipose-derived stromal cells were the most osteogenic (p < 0.05). Human cells, however, had the most significant osteogenic response to osteogenic media. Retinoic acid stimulated osteogenesis in mouse and canine cells but not in human adipose-derived stromal cells. Insulin-like growth factor-1 enhanced osteogenesis across all species, most notably in human- and canine-derived cells.Adipose-derived stromal cells derived from human, mouse, and canine all have the capacity to undergo osteogenic differentiation. Canine adipose-derived stromal cells appear to be the most proliferative, whereas human adipose-derived stromal cells appear to be the most osteogenic. Different cytokines and chemicals can be used to modulate this osteogenic response. These results are promising as attempts are made to optimize tissue-engineered bone using adipose-derived stromal cells.

  View details for DOI 10.1097/PRS.0b013e31821e6e49

  View details for PubMedID 21788829

• Improving Cutaneous Scar Formation by Controlling the Mechanical Environment Large Animal and Phase I Studies ANNALS OF SURGERY Gurtner, G. C., Dauskardt, R. H., Wong, V. W., Bhatt, K. A., Wu, K., Vial, I. N., Padois, K., Korman, J. M., Longaker, M. T. 2011; 254 (2): 217-225

  Abstract

  To test the hypothesis that the mechanical environment of cutaneous wounds can control scar formation.Mechanical forces have been recognized to modulate myriad biologic processes, but the role of physical force in scar formation remains unclear. Furthermore, the therapeutic benefits of offloading cutaneous wounds with a device have not been rigorously tested.A mechanomodulating polymer device was utilized to manipulate the mechanical environment of closed cutaneous wounds in red Duroc swine. After 8 weeks, wounds subjected to different mechanical stress states underwent immunohistochemical analysis for fibrotic markers. In a phase I clinical study, 9 human patients undergoing elective abdominal surgery were treated postoperatively with a stress-shielding polymer on one side whereas the other side was treated as standard of care. Professional photographs were taken between 8 and 12 months postsurgery and evaluated using a visual analog scale by lay and professional panels. This study is registered with ClinicalTrials.gov, number NCT00766727.Stress shielding of swine incisions reduced histologic scar area by 6- and 9-fold compared to control and elevated stress states, respectively (P < 0.01 for both) and dramatically decreased the histologic expression of profibrotic markers. Closure of high-tension wounds induced human-like scar formation in the red Duroc, a phenotype effectively mitigated with stress shielding of wounds. In the study on humans, stress shielding of abdominal incisions significantly improved scar appearance (P = 0.004) compared with within-patient controls.These results indicate that mechanical manipulation of the wound environment with a dynamic stress-shielding polymer device can significantly reduce scar formation.

  View details for DOI 10.1097/SLA.0b013e318220b159

  View details for Web of Science ID 000292908700007

  View details for PubMedID 21606834

• Dura Mater Stimulates Human Adipose-Derived Stromal Cells to Undergo Bone Formation in Mouse Calvarial Defects STEM CELLS Levi, B., Nelson, E. R., Li, S., James, A. W., Hyun, J. S., Montoro, D. T., Lee, M., Glotzbach, J. P., Commons, G. W., Longaker, M. T. 2011; 29 (8): 1241-1255

  Abstract

  Human adipose-derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell-cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM-hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC-DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP-2 and anti-BMP-2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM-hASC interaction dramatically reduced calvarial healing with abrogated BMP-2-Smad-1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP-2 pathway, we found that BMP-2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP-2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC-engrafted calvarial defect. Furthermore, DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC mediated repair.

  View details for DOI 10.1002/stem.670

  View details for Web of Science ID 000293133900009

  View details for PubMedID 21656608

  View details for PubMedCentralID PMC4353733

• Fgf-18 Is Required for Osteogenesis But Not Angiogenesis During Long Bone Repair TISSUE ENGINEERING PART A Behr, B., Sorkin, M., Manu, A., Lehnhardt, M., Longaker, M. T., Quarto, N. 2011; 17 (15-16): 2061-2069

  Abstract

  Bone regeneration is a complex event that requires the interaction of numerous growth factors. Fibroblast growth factor (Fgf)-ligands have been previously described for their importance in osteogenesis during development. In the current study, we investigated the role of Fgf-18 during bone regeneration. By utilizing a unicortical tibial defect model, we revealed that mice haploinsufficient for Fgf-18 have a markedly reduced healing capacity as compared with wild-type mice. Reduced levels of Runx2 and Osteocalcin but not Vegfa accompanied the impaired bone regeneration. Interestingly, our data indicated that upon injury angiogenesis was not impaired in Fgf-18(+/-) mice. Moreover, other Fgf-ligands and Bmp-2 could not compensate for the loss of Fgf-18. Finally, application of FGF-18 protein was able to rescue the impaired healing in Fgf-18(+/-) mice. Thus, we identified Fgf-18 as an important mediator of bone regeneration, which is required during later stages of bone regeneration. This study provides hints on how to engineering efficiently programmed bony tissue for long bone repair.

  View details for DOI 10.1089/ten.tea.2010.0719

  View details for Web of Science ID 000293217700015

  View details for PubMedID 21457097

  View details for PubMedCentralID PMC3142654

• An Information Theoretic, Microfluidic-Based Single Cell Analysis Permits Identification of Subpopulations among Putatively Homogeneous Stem Cells PLOS ONE Glotzbach, J. P., Januszyk, M., Vial, I. N., Wong, V. W., Gelbard, A., Kalisky, T., Thangarajah, H., Longaker, M. T., Quake, S. R., Chu, G., Gurtner, G. C. 2011; 6 (6)

  Abstract

  An incomplete understanding of the nature of heterogeneity within stem cell populations remains a major impediment to the development of clinically effective cell-based therapies. Transcriptional events within a single cell are inherently stochastic and can produce tremendous variability, even among genetically identical cells. It remains unclear how mammalian cellular systems overcome this intrinsic noisiness of gene expression to produce consequential variations in function, and what impact this has on the biologic and clinical relevance of highly 'purified' cell subgroups. To address these questions, we have developed a novel method combining microfluidic-based single cell analysis and information theory to characterize and predict transcriptional programs across hundreds of individual cells. Using this technique, we demonstrate that multiple subpopulations exist within a well-studied and putatively homogeneous stem cell population, murine long-term hematopoietic stem cells (LT-HSCs). These subgroups are defined by nonrandom patterns that are distinguishable from noise and are consistent with known functional properties of these cells. We anticipate that this analytic framework can also be applied to other cell types to elucidate the relationship between transcriptional and phenotypic variation.

  View details for DOI 10.1371/journal.pone.0021211

  View details for Web of Science ID 000292033700046

  View details for PubMedID 21731674

  View details for PubMedCentralID PMC3120839

• Unfolded Protein Response Regulation in Keloid Cells JOURNAL OF SURGICAL RESEARCH Butler, P. D., Wang, Z., Ly, D. P., Longaker, M. T., Koong, A. C., Yang, G. P. 2011; 167 (1): 151-157

  Abstract

  Keloids are a common form of pathologic wound healing characterized by excessive production of extracellular matrix. The unfolded protein response (UPR) is a cellular response to hypoxia, a component of the wound microenvironment, capable of protecting cells from the effects of over-accumulation of misfolded proteins. Since keloids have hypersecretion of extracellular matrix, we hypothesized that keloid fibroblasts (KFs) may have enhanced activation of the UPR compared with normal fibroblasts (NFs).KFs and NFs were placed in a hypoxia chamber for 0, 24, and 48h. We also used tunicamycin to specifically up-regulate the UPR. UPR activation was assayed by PCR for xbp-1 splicing and by immunoblotting with specific antibodies for the three UPR transducers. Nuclear localization of XBP-1 protein in KFs was confirmed by immunofluorescence.There is increased activation of XBP-1 protein in KFs compared with NFs following exposure to hypoxia. Pancreatic ER kinase (PERK) and ATF-6, two other pathways activated by the UPR, show comparable activation between KFs and NFs. We confirmed that there is enhanced activation of XBP-1 by demonstrating increased nuclear localization of XBP-1 using immunofluorescence.In contrast to our initial hypothesis that keloids would have broad activation of the UPR, we demonstrate here that there is a specific up-regulation of one facet of the UPR response. This may represent a specific molecular defect in KFs compared with NFs, and also suggests modulation of the UPR can be used in wound healing therapy.

  View details for DOI 10.1016/j.jss.2009.04.036

  View details for Web of Science ID 000288744100029

  View details for PubMedID 19631342

  View details for PubMedCentralID PMC2888625

• Integrating Cultural Competency and Humility Training into Clinical Clerkships: Surgery as a Model JOURNAL OF SURGICAL EDUCATION Butler, P. D., Swift, M., Kothari, S., Nazeeri-Simmons, I., Friel, C. M., Longaker, M. T., Britt, L. D. 2011; 68 (3): 222-230

  Abstract

  Cultural competency is gaining recognition as an essential strategy by which to address health care disparities. A closer examination of medical school curriculums was undertaken to determine how the need for cultural competency and humility (CCH) training in medical education is being addressed.A MEDLINE review of published literature regarding CCH training in medical education was performed. Additionally, key informant interviews with influential faculty members from prominent medical institutions were completed.Many academic medical institutions recognize the need for CCH and have successfully integrated it into the first 2 years of their curriculums. However, there seems to be a uniform deficit in CCH training in the third and fourth years of their education.Recognizing the need for CCH training during the third and fourth years of medical education, we explored the issues inherent to the integration of CCH training in clinical education. Using surgery as a model, we established a set of recommendations to assist clerkship directors and curriculum committees in their efforts to ensure CCH training in the last 2 years of medical education.

  View details for DOI 10.1016/j.jsurg.2011.01.002

  View details for Web of Science ID 000289877700014

  View details for PubMedID 21481809

• Engineered epidermal growth factor mutants with faster binding on-rates correlate with enhanced receptor activation FEBS LETTERS Lahti, J. L., Lui, B. H., Beck, S. E., Lee, S. S., Ly, D. P., Longaker, M. T., Yang, G. P., Cochran, J. R. 2011; 585 (8): 1135-1139

  Abstract

  Receptor tyrosine kinases (RTKs) regulate critical cell signaling pathways, yet the properties of their cognate ligands that influence receptor activation are not fully understood. There is great interest in parsing these complex ligand-receptor relationships using engineered proteins with altered binding properties. Here we focus on the interaction between two engineered epidermal growth factor (EGF) mutants and the EGF receptor (EGFR), a model member of the RTK superfamily. We found that EGF mutants with faster kinetic on-rates stimulate increased EGFR activation compared to wild-type EGF. These findings support previous predictions that faster association rates correlate with enhanced receptor activity.

  View details for DOI 10.1016/j.febslet.2011.03.044

  View details for Web of Science ID 000289505400004

  View details for PubMedID 21439278

  View details for PubMedCentralID PMC3118396

• Concise Review: Adipose-Derived Stromal Cells for Skeletal Regenerative Medicine STEM CELLS Levi, B., Longaker, M. T. 2011; 29 (4): 576-582

  Abstract

  As the average age of the population grows, the incidence of osteoporosis and skeletal diseases continues to rise. Current treatment options for skeletal repair include immobilization, rigid fixation, alloplastic materials, and bone grafts, all which have significant limitations, especially in the elderly. Adipose-derived stromal cells (ASCs) represent a readily available abundant supply of mesenchymal stem cells, which demonstrate the ability to undergo osteogenesis in vitro and in vivo, making ASCs a promising source of skeletal progenitor cells. Current protocols allow for the harvest of over one million cells from only 15 ml of lipoaspirate. Despite the clinical use of ASCs to treat systemic inflammatory diseases, no large human clinical trials exist using ASCs for skeletal tissue engineering. The aim of this review is to define ASCs, to describe the isolation procedure of ASCs, to review the basic biology of their osteogenic differentiation, discuss cell types and scaffolds available for bone tissue engineering, and finally, to explore imaging of ASCs and their potential future role in human skeletal tissue engineering efforts.

  View details for DOI 10.1002/stem.612

  View details for Web of Science ID 000289157100004

  View details for PubMedID 21305671

  View details for PubMedCentralID PMC3323288

• Chemical Control of FGF-2 Release for Promoting Calvarial Healing with Adipose Stem Cells JOURNAL OF BIOLOGICAL CHEMISTRY Kwan, M. D., Sellmyer, M. A., Quarto, N., Ho, A. M., Wandless, T. J., Longaker, M. T. 2011; 286 (13): 11307-11313

  Abstract

  Chemical control of protein secretion using a small molecule approach provides a powerful tool to optimize tissue engineering strategies by regulating the spatial and temporal dimensions that are exposed to a specific protein. We placed fibroblast growth factor 2 (FGF-2) under conditional control of a small molecule and demonstrated greater than 50-fold regulation of FGF-2 release as well as tunability, reversibility, and functionality in vitro. We then applied conditional control of FGF-2 secretion to a cell-based, skeletal tissue engineering construct consisting of adipose stem cells (ASCs) on a biomimetic scaffold to promote bone formation in a murine critical-sized calvarial defect model. ASCs are an easily harvested and abundant source of postnatal multipotent cells and have previously been demonstrated to regenerate bone in critical-sized defects. These results suggest that chemically controlled FGF-2 secretion can significantly increase bone formation by ASCs in vivo. This study represents a novel approach toward refining protein delivery for tissue engineering applications.

  View details for DOI 10.1074/jbc.M110.180042

  View details for Web of Science ID 000288797100043

  View details for PubMedID 21262969

  View details for PubMedCentralID PMC3064187

• Regenerative Medicine CURRENT PROBLEMS IN SURGERY Glotzbach, J. P., Wong, V. W., Gurtner, G. C., Longaker, M. T. 2011; 48 (3): 148-212

  View details for DOI 10.1067/j.cpsurg.2010.11.002

  View details for Web of Science ID 000287543600002

  View details for PubMedID 21295632

• Studies in Adipose-Derived Stromal Cells: Migration and Participation in Repair of Cranial Injury after Systemic Injection PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Nelson, E. R., Hu, S., Sun, N., Peng, M., Wu, J., Longaker, M. T. 2011; 127 (3): 1130-1140

  Abstract

  Adipose-derived stromal cells are a multipotent cell type with the ability to undergo osteogenic differentiation. The authors sought to examine whether systemically administered adipose-derived stromal cells would migrate to and heal surgically created defects of the mouse cranial skeleton.Mouse adipose-derived stromal cells were harvested from luciferase-positive transgenic mice; human adipose-derived stromal cells were harvested from human lipoaspirate and labeled with luciferase and green fluorescent protein. A 4-mm calvarial defect (critical sized) was made in the mouse parietal bone; skin incisions alone were used as a control (n = 5 per group). Adipose-derived stromal cells were injected intravenously (200,000 cells per animal) and compared with saline injection only. Methods of analyses included micro-computed tomographic scanning, in vivo imaging system detection of luciferase activity, and standard histology.Migration of adipose-derived stromal cells to calvarial defect sites was confirmed by accumulation of luciferase activity and green fluorescent protein stain as early as 4 days and persisting up to 4 weeks. Little activity was observed among control groups. Intravenous administration of either mouse or human adipose-derived stromal cells resulted in histologic evidence of bone formation within the defect site, in comparison with an absence of bone among control defects. By micro-computed tomographic analysis, human but not mouse adipose-derived stromal cells stimulated significant osseous healing.Intravenously administered adipose-derived stromal cells migrate to sites of calvarial injury. Thereafter, intravenous human adipose-derived stromal cells contribute to bony calvarial repair. Intravenous administration of adipose-derived stromal cells may be an effective delivery method for future efforts in skeletal regeneration.

  View details for DOI 10.1097/PRS.0b013e3182043712

  View details for Web of Science ID 000287680200013

  View details for PubMedID 21364416

  View details for PubMedCentralID PMC3248272

• Osteogenic Differentiation of Adipose-Derived Stromal Cells in Mouse and Human: In Vitro and In Vivo Methods JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Longaker, M. T. 2011; 22 (2): 388-391

  View details for DOI 10.1097/SCS.0b013e318207b72b

  View details for Web of Science ID 000288535800006

  View details for PubMedID 21415625

• Role of Indian Hedgehog Signaling in Palatal Osteogenesis PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Nelson, E. R., Brugmann, S. A., Sorkin, M., Manu, A., Longaker, M. T. 2011; 127 (3): 1182-1190

  Abstract

  Cleft lip-cleft palate is a common congenital disability and represents a large biomedical burden. Through the use of animal models, the molecular underpinnings of cleft palate are becoming increasingly clear. Indian hedgehog (Ihh) has been shown to be associated with craniofacial development and to be active in the palatine bone. The authors hypothesize that Indian hedgehog activity plays a role in osteogenesis within the secondary palate and that defects in this pathway may inhibit osteogenesis of the secondary palate.Palates were isolated from wild-type mice during the period of palate development (embryonic days 9.5 to 17.5). Quantitative real-time polymerase chain reaction was used for detecting gene expression during osteogenic differentiation and cellular differentiation (Shh, Ihh, Ptc1, Gli1, Gli2, Gli3, Runx2, Alp, and Col1a1). Next, palates were analyzed by hematoxylin and eosin, aniline blue, pentachrome, and in situ hybridization to assess osteogenesis of the palatal shelf and expression of hedgehog pathway genes. Finally, the palates of Indian hedgehog-null mice were analyzed to determine the effect of genetic deficiency on palatal development osteogenesis.Increased Indian hedgehog and osteogenic signaling coincided with ossification and fusion of the palate in wild-type mice. This included a fivefold to 150-fold peak in expression of hedgehog elements, including Ihh, at embryonic day 15.5 as compared with embryonic day 9.5. Contrarily, loss of Indian hedgehog by genetic knockout (Ihh-/-) resulted in decreased secondary palate ossification.The authors' results suggest a role for hedgehog signaling during palatal ossification. The hedgehog pathway is activated during palatal fusion, and deletion of Indian hedgehog leads to diminished ossification of the secondary hard palate.

  View details for DOI 10.1097/PRS.0b013e3182043a07

  View details for Web of Science ID 000287680200019

  View details for PubMedID 21364421

  View details for PubMedCentralID PMC3078688

• Acute Skeletal Injury Is Necessary for Human Adipose-Derived Stromal Cell-Mediated Calvarial Regeneration PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Nelson, E. R., Peng, M., Wan, D. C., Commons, G. W., Lee, M., Wu, B., Longaker, M. T. 2011; 127 (3): 1118-1129

  Abstract

  Studies have demonstrated that human adipose-derived stromal cells (ASCs) are able to repair acute calvarial injuries. The more clinically relevant repair of an established skeletal defect, however, has not been addressed. The authors sought to determine whether human ASCs could heal chronic (established) calvarial defects.Critical-sized (4 mm) mouse parietal defects were created. Human ASCs were engrafted either immediately postoperatively (acute defect) or 8 weeks following defect creation (established defect). Methods of analysis included microcomputer tomography scans, histology, and in situ hybridization. Finally, human ASCs were treated in vitro with platelet-rich plasma to simulate an acute wound environment; proliferation and osteogenic differentiation were assessed (alkaline phosphatase, alizarin red, and quantitative reverse transcriptase polymerase chain reaction).Nearly complete osseous healing was observed when calvarial defects were immediately engrafted with human ASCs. In contrast, when human ASCs were engrafted into established defects, little bone formation occurred. Histological analysis affirmed findings by microcomputer tomography, showing more robust staining for alkaline phosphatase and picrosirius red in an acute than in an established human ASC-engrafted defect. In situ hybridization and quantitative reverse transcriptase polymerase chain reaction showed an increase in bone morphogenetic protein (BMP) expression (BMP-2, BMP-4, and BMP-7) acutely following calvarial defect creation. Finally, in vitro treatment of human ASCs with platelet-rich plasma enhanced osteogenic differentiation and increased BMP-2 expression.Although human ASCs can be utilized to heal an acute mouse calvarial defect, they do not enhance healing of an established (or chronic) defect. Endogenous BMP signaling activated after injury may explain these differences in healing. Platelet-rich plasma enhances osteogenic differentiation of human ASCs in vitro and may prove a promising therapy for future skeletal tissue engineering efforts.

  View details for DOI 10.1097/PRS.0b013e318205f274

  View details for Web of Science ID 000287680200012

  View details for PubMedID 21364415

  View details for PubMedCentralID PMC3073240

• Deleterious Effects of Freezing on Osteogenic Differentiation of Human Adipose-Derived Stromal Cells In Vitro and In Vivo STEM CELLS AND DEVELOPMENT James, A. W., Levi, B., Nelson, E. R., Peng, M., Commons, G. W., Lee, M., Wu, B., Longaker, M. T. 2011; 20 (3): 427-439

  Abstract

  Human adipose-derived stromal cells (hASCs) represent a multipotent stromal cell type with a proven capacity to undergo osteogenic differentiation. Many hurdles exist, however, between current knowledge of hASC osteogenesis and their potential future use in skeletal tissue regeneration. The impact of frozen storage on hASC osteogenic differentiation, for example, has not been studied in detail. To examine the effects of frozen storage, hASCs were harvested from lipoaspirate and either maintained in standard culture conditions or frozen for 2 weeks under standard conditions (90% fetal bovine serum, 10% dimethyl sulfoxide). Next, in vitro parameters of cell morphology (surface electron microscopy [EM]), cell viability and growth (trypan blue; bromodeoxyuridine incorporation), osteogenic differentiation (alkaline phosphatase, alizarin red, and quantitative real-time (RT)-polymerase chain reaction), and adipogenic differentiation (Oil red O staining and quantitative RT-polymerase chain reaction) were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic mice, utilizing a hydroxyapatite (HA)-poly(lactic-co-glycolic acid) scaffold for ASC delivery. Healing was assessed by serial microcomputed tomography scans and histology. Freshly derived ASCs differed significantly from freeze-thaw ASCs in all markers examined. Surface EM showed distinct differences in cellular morphology. Proliferation, and osteogenic and adipogenic differentiation were all significantly hampered by the freeze-thaw process in vitro (*P < 0.01). In vivo, near complete healing was observed among calvarial defects engrafted with fresh hASCs. This was in comparison to groups engrafted with freeze-thaw hASCs that showed little healing (*P < 0.01). Finally, recombinant insulin-like growth factor 1 or recombinant bone morphogenetic protein 4 was observed to increase or rescue in vitro osteogenic differentiation among frozen hASCs (*P < 0.01). The freezing of ASCs for storage significantly impacts their biology, both in vitro and in vivo. The ability of ASCs to successfully undergo osteogenic differentiation after freeze-thaw is substantively muted, both in vitro and in vivo. The use of recombinant proteins, however, may be used to mitigate the deleterious effects of the freeze-thaw process.

  View details for DOI 10.1089/scd.2010.0082

  View details for Web of Science ID 000287798300006

  View details for PubMedID 20536327

  View details for PubMedCentralID PMC3128779

• In brief: Regenerative medicine. Current problems in surgery Glotzbach, J. P., Wong, V. W., Gurtner, G. C., Longaker, M. T. 2011; 48 (3): 142-146

  View details for DOI 10.1067/j.cpsurg.2010.11.001

  View details for PubMedID 21295631

• Engineered Pullulan-Collagen Composite Dermal Hydrogels Improve Early Cutaneous Wound Healing TISSUE ENGINEERING PART A Wong, V. W., Rustad, K. C., Galvez, M. G., Neofyotou, E., Glotzbach, J. P., Januszyk, M., Major, M. R., Sorkin, M., Longaker, M. T., Rajadas, J., Gurtner, G. C. 2011; 17 (5-6): 631-644

  Abstract

  New strategies for skin regeneration are needed to address the significant medical burden caused by cutaneous wounds and disease. In this study, pullulan-collagen composite hydrogel matrices were fabricated using a salt-induced phase inversion technique, resulting in a structured yet soft scaffold for skin engineering. Salt crystallization induced interconnected pore formation, and modification of collagen concentration permitted regulation of scaffold pore size. Hydrogel architecture recapitulated the reticular distribution of human dermal matrix while maintaining flexible properties essential for skin applications. In vitro, collagen hydrogel scaffolds retained their open porous architecture and viably sustained human fibroblasts and murine mesenchymal stem cells and endothelial cells. In vivo, hydrogel-treated murine excisional wounds demonstrated improved wound closure, which was associated with increased recruitment of stromal cells and formation of vascularized granulation tissue. In conclusion, salt-induced phase inversion techniques can be used to create modifiable pullulan-collagen composite dermal scaffolds that augment early wound healing. These novel biomatrices can potentially serve as a structured delivery template for cells and biomolecules in regenerative skin applications.

  View details for DOI 10.1089/ten.tea.2010.0298

  View details for Web of Science ID 000287801600005

  View details for PubMedID 20919949

• Research Training in Plastic Surgery JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Longaker, M. T. 2011; 22 (2): 383-384

  View details for DOI 10.1097/SCS.0b013e318208ba73

  View details for Web of Science ID 000288535800004

  View details for PubMedID 21415623

• Elastic Properties of Induced Pluripotent Stem Cells TISSUE ENGINEERING PART A Hammerick, K. E., Huang, Z., Sun, N., Lam, M. T., Prinz, F. B., Wu, J. C., Commons, G. W., Longaker, M. T. 2011; 17 (3-4): 495-502

  Abstract

  The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications, including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs), a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC), and also the gold standard human embryonic stem cell, we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness, and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly, cells exhibited a noticeable difference in stiffness. From least to most stiff, the order of cell stiffness was as follows: hASC-iPSC, human embryonic stem cell, fibroblast-iPSC, fibroblasts, and, lastly, as the stiffest cell, hASC. In comparing hASC-iPSCs to their origin cell, the hASC, the reprogrammed cell is significantly less stiff, indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence, material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state.

  View details for DOI 10.1089/ten.tea.2010.0211

  View details for Web of Science ID 000286661600023

  View details for PubMedID 20807017

  View details for PubMedCentralID PMC3052278

• Human Adipose-Derived Stromal Cells Stimulate Autogenous Skeletal Repair via Paracrine Hedgehog Signaling with Calvarial Osteoblasts STEM CELLS AND DEVELOPMENT Levi, B., James, A. W., Nelson, E. R., Li, S., Peng, M., Commons, G. W., Lee, M., Wu, B., Longaker, M. T. 2011; 20 (2): 243-257

  Abstract

  Human adipose-derived stromal cells (hASCs) have the proven capacity to ossify skeletal defects. The mechanisms whereby hASCs stimulate bone repair are not fully understood. In this study, we examined the potential for hASCs to stimulate autogenous repair of a mouse calvarial defect. Immunofluoresence, osteogenic stains, and surface electron microscopy were used to demonstrate osteogenic differentiation of hASCs. hASCs were engrafted into 4 mm calvarial defects in athymic mice using an osteoconductive scaffold. Analysis included microcomputed tomography, histology, in situ hybridization, and quantitative real-time-polymerase chain reaction. Next, the in vitro interaction between hASCs and mouse calvarial osteoblasts (mOBs) was assessed by the conditioned medium and coculture assays. The medium was supplemented with Hedgehog signaling modifiers, including recombinant N-terminal Sonic hedgehog, smoothened agonist, and cyclopamine. Finally, cyclopamine was delivered in vivo to hASC-engrafted defects. Significant calvarial healing was observed among hASC-engrafted defects compared with control groups (no treatment or scaffold alone) (*P<0.05). hASCs showed evidence of stimulation of host mouse osteogenesis, including (1) increased expression of bone markers at the defect edge by in situ hybridization, and (2) increased host osteogenic gene expression by species-specific quantitative real-time polymerase chain reaction. Using the conditioned medium or coculture assays, hASCs stimulated mOB osteogenic differentiation, accompanied by Hedgehog signaling activation. N-terminal Sonic hedgehog or smoothened agonist replicated, while cyclopamine reversed, the pro-osteogenic effect of the conditioned medium on mOBs. Finally, cyclopamine injection arrested bone formation in vivo. hASCs heal critical-sized mouse calvarial defects, this is, at least in part, via stimulation of autogenous healing of the host defect. Our studies suggest that hASC-derived Hedgehog signaling may play a paracrine role in skeletal repair.

  View details for DOI 10.1089/scd.2010.0250

  View details for Web of Science ID 000286460000007

  View details for PubMedID 20698749

  View details for PubMedCentralID PMC3128781

• Locally Applied Vascular Endothelial Growth Factor A Increases the Osteogenic Healing Capacity of Human Adipose-Derived Stem Cells by Promoting Osteogenic and Endothelial Differentiation STEM CELLS Behr, B., Tang, C., Germann, G., Longaker, M. T., Quarto, N. 2011; 29 (2): 286-296

  Abstract

  Human adipose-derived stem cells (hASCs) are known for their capability to promote bone healing when applied to bone defects. For bone tissue regeneration, both sufficient angiogenesis and osteogenesis is desirable. Vascular endothelial growth factor A (VEGFA) has the potential to promote differentiation of common progenitor cells to both lineages. To test this hypothesis, the effects of VEGFA on hASCs during osteogenic differentiation were tested in vitro. In addition, hASCs were seeded in murine critical-sized calvarial defects locally treated with VEGFA. Our results suggest that VEGFA improves osteogenic differentiation in vitro as indicated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time polymerase chain reaction analysis. Moreover, local application of VEGFA to hASCs significantly improved healing of critical-sized calvarial defects in vivo. This repair was accompanied by a striking enhancement of angiogenesis. Both paracrine and, to a lesser degree, cell-autonomous effects of VEGFA-treated hASCs were accountable for angiogenesis. These data were confirmed by using CD31(-) /CD45(-) mouse ASCs(GFP+) cells. In summary, we demonstrated that VEGFA increased osteogenic differentiation of hASCS in vitro and in vivo, which was accompanied by an enhancement of angiogenesis. Additionally, we showed that during bone regeneration, the increase in angiogenesis of hASCs on treatment with VEGFA was attributable to both paracrine and cell-autonomous effects. Thus, locally applied VEGFA might prove to be a valuable growth factor that can mediate both osteogenesis and angiogenesis of multipotent hASCs in the context of bone regeneration.

  View details for DOI 10.1002/stem.581

  View details for Web of Science ID 000287698600012

  View details for PubMedID 21732486

  View details for PubMedCentralID PMC3400547

• Differential Expression of Sclerostin in Adult and Juvenile Mouse Calvariae PLASTIC AND RECONSTRUCTIVE SURGERY Kwan, M. D., Quarto, N., Gupta, D. M., Slater, B. J., Wan, D. C., Longaker, M. T. 2011; 127 (2): 595-602

  Abstract

  An understanding of the molecular mechanisms controlling bone formation is central to skeletal tissue engineering efforts. The observation that immature animals are able to heal calvarial defects while adult animals are not has proven to be a useful tool for examining these mechanisms. Thus, the authors compared expression of sclerostin, a bone inhibitor, between the calvariae of juvenile and adult mice.Parietal bone was harvested from juvenile (6-day-old; n = 20) and adult (60-day-old; n = 20) mice. Sclerostin transcript and protein levels were compared between the parietal bone of juvenile and adult mice using polymerase chain reaction, Western blotting, and immunohistochemistry. Finally, osteoblasts from the parietal bone of juvenile and adult mice were harvested and cultured under osteogenic differentiation conditions with and without recombinant sclerostin (200 ng/ml). Terminal osteogenic differentiation was assessed at 21 days with alizarin red staining.Polymerase chain reaction, Western blot analysis, and immunohistochemistry all confirmed greater expression of sclerostin in the parietal bone of adult mice when compared with that of juvenile mice. Osteoblasts, whether from juvenile or adult parietal bones, demonstrated reduced capacity for osteogenic differentiation when exposed to recombinant sclerostin.Given the role of sclerostin in inhibiting bone formation, the authors' findings suggest that differences in expression levels of sclerostin may play a role in the differential regenerative capacity of calvariae from juvenile and adult animals. These findings suggest it as a potential target to abrogate in future tissue engineering studies.

  View details for DOI 10.1097/PRS.0b013e3181fed60d

  View details for Web of Science ID 000286928100014

  View details for PubMedID 21285764

  View details for PubMedCentralID PMC3072034

• Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors NATURE PROTOCOLS Narsinh, K. H., Jia, F., Robbins, R. C., Kay, M. A., Longaker, M. T., Wu, J. C. 2011; 6 (1): 78-88

  Abstract

  Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ∼4 weeks.

  View details for DOI 10.1038/nprot.2010.173

  View details for Web of Science ID 000285965000008

  View details for PubMedID 21212777

  View details for PubMedCentralID PMC3657506

• John Locke's Monetary Argument: An Analysis with Methodological and Historical Implications RHETORIC SOCIETY QUARTERLY Longaker, M. G. 2011; 41 (2): 125-144

  View details for DOI 10.1080/02773945.2010.533148

  View details for Web of Science ID 000289245400002

• Salvage of the Crucified Chin PLASTIC AND RECONSTRUCTIVE SURGERY Wan, D. C., Longaker, M. T., Allam, K. A., Perry, A., Kawamoto, H. K. 2011; 127 (1): 352-354

  View details for DOI 10.1097/PRS.0b013e3181fad3d6

  View details for Web of Science ID 000285992100048

  View details for PubMedID 21200230

• Osteogenic differentiation of human multipotent mesenchymal stromal cells. Methods in molecular biology (Clifton, N.J.) Gupta, D. M., Panetta, N. J., Longaker, M. T. 2011; 698: 201-214

  Abstract

  A comprehensive knowledge of the molecular biology underlying osteogenic differentiation in a controlled, laboratory setting may promise optimization of future cell-based tissue engineering strategies for clinical problems. The scope of this review encompasses a discussion of the methodology utilized to perform such studies. Our laboratory routinely performs both in vitro and in vivo assays underlying osteogenic differentiation, and the widespread use of singular methodology across multiple investigators and institutions promises great advancements for the skeletal tissue engineering community.

  View details for DOI 10.1007/978-1-60761-999-4_16

  View details for PubMedID 21431521

• Craniosynostosis of coronal suture in Twist1(+/-) mice occurs through endochondral ossification recapitulating the physiological closure of posterior frontal suture FRONTIERS IN PHYSIOLOGY Behr, B., Longaker, M. T., Quarto, N. 2011; 2

  Abstract

  Craniosynostosis, the premature closure of cranial suture, is a pathologic condition that affects 1/2000 live births. Saethre-Chotzen syndrome is a genetic condition characterized by craniosynostosis. The Saethre-Chotzen syndrome, which is defined by loss-of-function mutations in the TWIST gene, is the second most prevalent craniosynostosis. Although much of the genetics and phenotypes in craniosynostosis syndromes is understood, less is known about the underlying ossification mechanism during suture closure. We have previously demonstrated that physiological closure of the posterior frontal suture occurs through endochondral ossification. Moreover, we revealed that antagonizing canonical Wnt-signaling in the sagittal suture leads to endochondral ossification of the suture mesenchyme and sagittal synostosis, presumably by inhibiting Twist1. Classic Saethre-Chotzen syndrome is characterized by coronal synostosis, and the haploinsufficient Twist1(+/-) mice represents a suitable model for studying this syndrome. Thus, we seeked to understand the underlying ossification process in coronal craniosynostosis in Twist1(+/-) mice. Our data indicate that coronal suture closure in Twist1(+/-) mice occurs between postnatal day 9 and 13 by endochondral ossification, as shown by histology, gene expression analysis, and immunohistochemistry. In conclusion, this study reveals that coronal craniosynostosis in Twist1(+/-) mice occurs through endochondral ossification. Moreover, it suggests that haploinsufficiency of Twist1 gene, a target of canonical Wnt-signaling, and inhibitor of chondrogenesis, mimics conditions of inactive canonical Wnt-signaling leading to craniosynostosis.

  View details for DOI 10.3389/fphys.2011.00037

  View details for Web of Science ID 000209172600037

  View details for PubMedCentralID PMC3143731

• Craniosynostosis of coronal suture in twist1 mice occurs through endochondral ossification recapitulating the physiological closure of posterior frontal suture. Frontiers in physiology Behr, B., Longaker, M. T., Quarto, N. 2011; 2: 37-?

  Abstract

  Craniosynostosis, the premature closure of cranial suture, is a pathologic condition that affects 1/2000 live births. Saethre-Chotzen syndrome is a genetic condition characterized by craniosynostosis. The Saethre-Chotzen syndrome, which is defined by loss-of-function mutations in the TWIST gene, is the second most prevalent craniosynostosis. Although much of the genetics and phenotypes in craniosynostosis syndromes is understood, less is known about the underlying ossification mechanism during suture closure. We have previously demonstrated that physiological closure of the posterior frontal suture occurs through endochondral ossification. Moreover, we revealed that antagonizing canonical Wnt-signaling in the sagittal suture leads to endochondral ossification of the suture mesenchyme and sagittal synostosis, presumably by inhibiting Twist1. Classic Saethre-Chotzen syndrome is characterized by coronal synostosis, and the haploinsufficient Twist1(+/-) mice represents a suitable model for studying this syndrome. Thus, we seeked to understand the underlying ossification process in coronal craniosynostosis in Twist1(+/-) mice. Our data indicate that coronal suture closure in Twist1(+/-) mice occurs between postnatal day 9 and 13 by endochondral ossification, as shown by histology, gene expression analysis, and immunohistochemistry. In conclusion, this study reveals that coronal craniosynostosis in Twist1(+/-) mice occurs through endochondral ossification. Moreover, it suggests that haploinsufficiency of Twist1 gene, a target of canonical Wnt-signaling, and inhibitor of chondrogenesis, mimics conditions of inactive canonical Wnt-signaling leading to craniosynostosis.

  View details for DOI 10.3389/fphys.2011.00037

  View details for PubMedID 21811467

  View details for PubMedCentralID PMC3143731

• The Role of Stem Cells in Cutaneous Wound Healing: What Do We Really Know? PLASTIC AND RECONSTRUCTIVE SURGERY Ko, S. H., Nauta, A., Wong, V., Glotzbach, J., Gurtner, G. C., Longaker, M. T. 2011; 127 (1): 10S-20S

  Abstract

  Wound repair is a complex process involving the orchestrated interaction of multiple growth factors, cytokines, chemokines, and cell types. Dysregulation of this process leads to problems such as excessive healing in the form of keloids and hypertrophic scars and chronic, nonhealing wounds. These issues have broad global implications. Stem cells offer enormous potential for enhancing tissue repair and regeneration following injury. The rapidly developing fields of stem cell biology and skin tissue engineering create translational opportunities for the development of novel stem cell-based wound-healing therapies.

  View details for DOI 10.1097/PRS.0b013e3181fbe2d8

  View details for Web of Science ID 000286122400003

  View details for PubMedID 21200267

• Surgical Approaches to Create Murine Models of Human Wound Healing JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY Wong, V., Sorkin, M., Glotzbach, J. P., Longaker, M. T., Gurtner, G. C. 2011

  Abstract

  Wound repair is a complex biologic process which becomes abnormal in numerous disease states. Although in vitro models have been important in identifying critical repair pathways in specific cell populations, in vivo models are necessary to obtain a more comprehensive and pertinent understanding of human wound healing. The laboratory mouse has long been the most common animal research tool and numerous transgenic strains and models have been developed to help researchers study the molecular pathways involved in wound repair and regeneration. This paper aims to highlight common surgical mouse models of cutaneous disease and to provide investigators with a better understanding of the benefits and limitations of these models for translational applications.

  View details for DOI 10.1155/2011/969618

  View details for Web of Science ID 000286250000001

  View details for PubMedID 21151647

  View details for PubMedCentralID PMC2995912

• Diathermy or Surgical Scalpel for Abdominal Skin Incisions What Is the Impact On Clinical Practice? ANNALS OF SURGERY Longaker, M. T., Gurtner, G. C. 2011; 253 (1): 14-15

  View details for DOI 10.1097/SLA.0b013e318205718a

  View details for Web of Science ID 000286179100004

  View details for PubMedID 21294284

• Deformational Plagiocephaly: A Look Into the Future JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Wan, D. C., Longaker, M. T., Habal, M. B. 2011; 22 (1): 3-5

  View details for DOI 10.1097/SCS.0b013e3181fb7ee5

  View details for Web of Science ID 000286195600002

  View details for PubMedID 21239916

• Meta Analysis of the Effectiveness of Surgical Scalpel or Diathermy in Making Abdominal Skin Incisions. Annals of surgery Longaker, M. T., Gurtner, G. C. 2010

  View details for DOI 10.1097/SLA.0b013e318205718a

  View details for PubMedID 21135686

• Basic Science Review on Adipose Tissue for Clinicians PLASTIC AND RECONSTRUCTIVE SURGERY Brown, S. A., Levi, B., Lequex, C., Wong, V. W., Mojallal, A., Longaker, M. T. 2010; 126 (6): 1936-1946

  Abstract

  The recognition that fat contains stem cells has driven further examination into the potential uses of fat and adipose-derived stem cells in a wide number of clinical situations. New information about the harvesting, isolation, and subsequent differentiation properties of isolated adipose-derived stem cells has led to new research into novel tissue-engineered constructs and the transformation of adipose-derived stem cells to induced pluripotent stem cells. Clinically, use of fat grafts and adipose-derived stem cells worldwide and in the United States has dramatically increased in parallel to questions concerning the safety and efficacy of adipose-derived stem cell-based treatments. Currently, the U.S. Food and Drug Administration has not approved the use of isolated adipose-derived stem cells for medical indications.

  View details for DOI 10.1097/PRS.0b013e3181f44790

  View details for Web of Science ID 000284832400038

  View details for PubMedID 21124133

• Activation of FGF Signaling Mediates Proliferative and Osteogenic Differences between Neural Crest Derived Frontal and Mesoderm Parietal Derived Bone PLOS ONE Li, S., Quarto, N., Longaker, M. T. 2010; 5 (11)

  Abstract

  As a culmination of efforts over the last years, our knowledge of the embryonic origins of the mammalian frontal and parietal cranial bones is unambiguous. Progenitor cells that subsequently give rise to frontal bone are of neural crest origin, while parietal bone progenitors arise from paraxial mesoderm. Given the unique qualities of neural crest cells and the clear delineation of the embryonic origins of the calvarial bones, we sought to determine whether mouse neural crest derived frontal bone differs in biology from mesoderm derived parietal bone.BrdU incorporation, immunoblotting and osteogenic differentiation assays were performed to investigate the proliferative rate and osteogenic potential of embryonic and postnatal osteoblasts derived from mouse frontal and parietal bones. Co-culture experiments and treatment with conditioned medium harvested from both types of osteoblasts were performed to investigate potential interactions between the two different tissue origin osteoblasts. Immunoblotting techniques were used to investigate the endogenous level of FGF-2 and the activation of three major FGF signaling pathways. Knockdown of FGF Receptor 1 (FgfR1) was employed to inactivate the FGF signaling.Our results demonstrated that striking differences in cell proliferation and osteogenic differentiation between the frontal and parietal bone can be detected already at embryonic stages. The greater proliferation rate, as well as osteogenic capacity of frontal bone derived osteoblasts, were paralleled by an elevated level of FGF-2 protein synthesis. Moreover, an enhanced activation of FGF-signaling pathways was observed in frontal bone derived osteoblasts. Finally, the greater osteogenic potential of frontal derived osteoblasts was dramatically impaired by knocking down FgfR1.Osteoblasts from mouse neural crest derived frontal bone displayed a greater proliferative and osteogenic potential and endogenous enhanced activation of FGF signaling compared to osteoblasts from mesoderm derived parietal bone. FGF signaling plays a key role in determining biological differences between the two types of osteoblasts.

  View details for DOI 10.1371/journal.pone.0014033

  View details for Web of Science ID 000284356900002

  View details for PubMedID 21124973

  View details for PubMedCentralID PMC2987799

• The basic science of vascular biology: implications for the practicing surgeon. Plastic and reconstructive surgery Glotzbach, J. P., Levi, B., Wong, V. W., Longaker, M. T., Gurtner, G. C. 2010; 126 (5): 1528-1538

  Abstract

  A thorough understanding of vascular biology will assist the reconstructive surgeon in both operative planning and development of novel surgical approaches to treat chronic wounds and tissue loss, and to optimize regenerative strategies for tissue reconstruction. In this review, several fundamental concepts of the basic science of vascular biology are discussed, with specific emphasis on the clinical implications most relevant to the reconstructive surgeon. Topics include the vascular physiology of tissue flaps and grafts, the principles of neovascularization including angiogenesis and vasculogenesis, and the basic concepts of bioengineering of vascularized tissue constructs for use in reconstruction. As basic science research increases our collective understanding of vascular physiology--specifically, in the areas of neovascularization and tissue engineering--reconstructive surgeons will be able to improve treatment of the sequelae of ischemic injuries, tissue loss, and chronic wounds.

  View details for DOI 10.1097/PRS.0b013e3181ef8ccf

  View details for PubMedID 21042110

• The Basic Science of Vascular Biology: Implications for the Practicing Surgeon PLASTIC AND RECONSTRUCTIVE SURGERY Glotzbach, J. P., Levi, B., Wong, V. W., Longaker, M. T., Gurtner, G. C. 2010; 126 (5): 1527-1537

  Abstract

  A thorough understanding of vascular biology will assist the reconstructive surgeon in both operative planning and development of novel surgical approaches to treat chronic wounds and tissue loss, and to optimize regenerative strategies for tissue reconstruction. In this review, several fundamental concepts of the basic science of vascular biology are discussed, with specific emphasis on the clinical implications most relevant to the reconstructive surgeon. Topics include the vascular physiology of tissue flaps and grafts, the principles of neovascularization including angiogenesis and vasculogenesis, and the basic concepts of bioengineering of vascularized tissue constructs for use in reconstruction. As basic science research increases our collective understanding of vascular physiology--specifically, in the areas of neovascularization and tissue engineering--reconstructive surgeons will be able to improve treatment of the sequelae of ischemic injuries, tissue loss, and chronic wounds.

  View details for DOI 10.1097/PRS.0b013e3181ef8ccf

  View details for Web of Science ID 000283844700014

• Scarless Fetal Wound Healing: A Basic Science Review PLASTIC AND RECONSTRUCTIVE SURGERY Larson, B. J., Longaker, M. T., Lorenz, H. P. 2010; 126 (4): 1172-1180

  Abstract

  Scar formation is a major medical problem that can have devastating consequences for patients. The adverse physiological and psychological effects of scars are broad, and there are currently no reliable treatments to prevent scarring. In contrast to adult wounds, early gestation fetal skin wounds repair rapidly and in the absence of scar formation. Despite extensive investigation, the exact mechanisms of scarless fetal wound healing remain largely unknown. For some time, it has been known that significant differences exist among the extracellular matrix, inflammatory response, cellular mediators, and gene expression profiles of fetal and postnatal wounds. These differences may have important implications in scarless wound repair.

  View details for DOI 10.1097/PRS.0b013e3181eae781

  View details for Web of Science ID 000282559100004

  View details for PubMedID 20885241

• The Diverse Surgeons Initiative: An Effective Method for Increasing the Number of Under-represented Minorities in Academic Surgery JOURNAL OF THE AMERICAN COLLEGE OF SURGEONS Butler, P. D., Britt, L. D., Green, M. L., Longaker, M. T., Geis, W. P., Franklin, M. E., Ruhalter, A., Fullum, T. M. 2010; 211 (4): 561-566

  View details for DOI 10.1016/j.jamcollsurg.2010.06.019

  View details for Web of Science ID 000282928900019

  View details for PubMedID 20868978

• Opposite Spectrum of Activity of Canonical Wnt Signaling in the Osteogenic Context of Undifferentiated and Differentiated Mesenchymal Cells: Implications for Tissue Engineering TISSUE ENGINEERING PART A Quarto, N., Behr, B., Longaker, M. T. 2010; 16 (10): 3185-3197

  Abstract

  To delineate the competence window in which canonical wingless (Wnt)-signaling can either inhibit or promote osteogenic differentiation, we have analyzed cells with different status, specifically undifferentiated mesenchymal cells, such as adipose-derived stem cells and embryonic calvarial mesenchymal cells, and differentiated mesenchymal cells such as juvenile immature calvarial osteoblasts and adult calvarial osteoblasts. Our analysis indicated that undifferentiated mesenchymal cells and juvenile calvarial osteoblasts are endowed with higher levels of endogenous canonical Wnt signaling compared to fully differentiated adult calvarial osteoblasts, and that different levels of activation inversely correlated with expression levels of several Wnt antagonists. We have observed that activation of canonical Wnt signaling may elicit opposite biological activity in the context of osteogenic differentiation depending on the status of cell, the threshold levels of its activation, and Wnt ligands concentration. The results presented in this study indicate that treatment with Wnt3 and/or expression of constitutively activated β-catenin inhibits osteogenic differentiation of undifferentiated mesenchymal cells, whereas expression of dominant negative transcription factor 4 (Tcf4) and/or secreted frizzled related protein 1 treatment enhances their osteogenic differentiation. Wnt3a treatment also inhibits osteogenesis in juvenile calvarial osteoblasts in a dose-dependent fashion. Conversely, Wnt3a treatment strongly induces osteogenesis in mature calvarial osteoblasts in a dose-dependent manner. Importantly, in vitro data correlated with in vivo results showing that Wnt3a treatment of calvarial defects, created in juvenile mice, promotes calvarial healing and bone regeneration only at low doses, whereas high doses of Wnt3a impairs tissue regeneration. In contrast, high doses of Wnt3a enhance bony tissue regeneration and calvarial healing in adult mice. Therefore, the knowledge of both endogenous activity of canonical Wnt signaling and appropriate concentrations of Wnt3a treatment may lead to significant improvement for bony tissue engineering, as well as for the efficient implement of adipose-derived stem cells in bone regeneration. Indeed, this study has important potential implications for tissue engineering, specifically for repair of juvenile bone defects.

  View details for DOI 10.1089/ten.tea.2010.0133

  View details for Web of Science ID 000282361100017

  View details for PubMedID 20590472

  View details for PubMedCentralID PMC2947420

• Stem Cells PLASTIC AND RECONSTRUCTIVE SURGERY Behr, B., Ko, S. H., Wong, V. W., Gurtner, G. C., Longaker, M. T. 2010; 126 (4): 1163-1171

  Abstract

  Stem cells are self-renewing cells capable of differentiating into multiple cell lines and are classified according to their origin and their ability to differentiate. Enormous potential exists in use of stem cells for regenerative medicine. To produce effective stem cell-based treatments for a range of diseases, an improved understanding of stem cell biology and better control over stem cell fate are necessary. In addition, the barriers to clinical translation, such as potential oncologic properties of stem cells, need to be addressed. With renewed government support and continued refinement of current stem cell methodologies, the future of stem cell research is exciting and promises to provide novel reconstructive options for patients and surgeons limited by traditional paradigms.

  View details for DOI 10.1097/PRS.0b013e3181ea42bb

  View details for Web of Science ID 000282559100003

  View details for PubMedID 20555302

• Depot-Specific Variation in the Osteogenic and Adipogenic Potential of Human Adipose-Derived Stromal Cells PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Glotzbach, J. P., Wan, D. C., Commons, G. W., Longaker, M. T. 2010; 126 (3): 822-834

  Abstract

  Adipose-derived stromal cells hold promise for use in tissue regeneration. However, multiple facets of their biology remain unclear. The authors examined the variations in osteogenesis and adipogenesis in adipose-derived stromal cells between subcutaneous fat depots and potential molecular causes.Adipose-derived stromal cells were isolated from human patients from subcutaneous fat depots, including arm, flank, thigh, and abdomen (n = 5 patients). Osteogenic and adipogenic differentiation was performed (alkaline phosphatase, alizarin red, and oil red O staining, and quantitative real-time polymerase chain reaction). Co-cultures were established to assess the paracrine effect of human adipose-derived stromal cells on mouse osteoblasts. Finally, HOX gene expression was analyzed by quantitative real-time polymerase chain reaction.Subcutaneous fat depots retain markedly different osteogenic and adipogenic potentials. Osteogenesis was most robust in adipose-derived stromal cells from the flank and thigh, as compared with those from the arm and abdomen (p < 0.05 by all markers examined). This was accompanied by elevations of BMP4 and BMPR1B (p < 0.05 by all markers examined). The osteogenic advantage of cells from the flank and thigh was again observed when analyzing the paracrine effects of these cells. Conversely, those cells isolated from the flank had a lesser ability to undergo adipogenic differentiation. Adipose-associated HOX genes were less expressed in flank-derived adipose-derived stromal cells.Variations exist between fat depots in terms of adipose-derived stromal cell osteogenic and adipogenic differentiation. Differences in HOX expression and bone morphogenetic protein signaling may underlie these observations. This study indicates that the choice of fat depot derivation of adipose-derived stromal cells may be an important one for future efforts in tissue engineering.

  View details for DOI 10.1097/PRS.0b013e3181e5f892

  View details for Web of Science ID 000281606700011

  View details for PubMedID 20811215

• Mechanical modulation of inflammation and skin fibrosis through CD4+T cell signaling Wong, V. W., Glotzbach, J. P., Paterno, J., Rustad, K. C., Sorkin, M., Major, M. R., Vial, I. N., Longaker, M. T., Gurtner, G. C. ELSEVIER SCIENCE INC. 2010: S77

  View details for Web of Science ID 000281708600164

• Discussion: Hes1 is required for the development of craniofacial structures derived from ectomesenchymal neural crest cells. journal of craniofacial surgery Levi, B., Brugmann, S., Longaker, M. T. 2010; 21 (5): 1450-1451

  View details for DOI 10.1097/SCS.0b013e3181ecc54b

  View details for PubMedID 20818250

• Tissue Engineering in Plastic Surgery: A Review PLASTIC AND RECONSTRUCTIVE SURGERY Wong, V. W., Rustad, K. C., Longaker, M. T., Gurtner, G. C. 2010; 126 (3): 858-868

  Abstract

  Novel tissue- and organ-engineering strategies are needed to address the growing need for replacement biological parts. Collective progress in stem cell technology, biomaterials, engineering, and molecular medicine has advanced the state of regenerative medicine, yet many hurdles to clinical translation remain. Plastic surgeons are in an ideal position to capitalize on emerging technologies and will be at the forefront of transitioning basic science research into the clinical reconstructive arena. This review highlights fundamental principles of bioengineering, recent progress in tissue-specific engineering, and future directions for this exciting and rapidly evolving area of medicine.

  View details for DOI 10.1097/PRS.0b013e3181e3b3a3

  View details for Web of Science ID 000281606700016

  View details for PubMedID 20811219

• Paracrine Interaction between Adipose-Derived Stromal Cells and Cranial Suture-Derived Mesenchymal Cells PLASTIC AND RECONSTRUCTIVE SURGERY James, A. W., Levi, B., Commons, G. W., Glotzbach, J., Longaker, M. T. 2010; 126 (3): 806-821

  Abstract

  Adipose-derived stromal cells are a potential cell source for the successful healing of skeletal defects. In this study, the authors sought to investigate the potential for cranial suture-derived mesenchymal cells to promote the osteogenic differentiation of adipose-derived stromal cells. Various reports have previously examined the unique in vitro attributes of suture-derived mesenchymal cells; this study sought to extend those findings.Suture-derived mesenchymal cells were isolated from wild-type mice (n = 30) from both fusing posterofrontal and patent sagittal sutures. Cells were placed in Transwell inserts with human adipose-derived stromal cells (n = 5 patients) with osteogenic differentiation medium with or without recombinant Noggin (10 to 400 ng/ml). Specific gene expression of osteogenic markers and Hedgehog pathway were assayed; standard osteogenic assays (alkaline phosphatase and alizarin red staining) were performed. All assays were performed in triplicate.Both posterofrontal and sagittal suture-derived mesenchymal cells induced osteogenic differentiation of adipose-derived stromal cells (p < 0.05). Posterofrontal suture-derived mesenchymal cells induced adipose-derived stromal cell osteogenesis to a greater degree than sagittal suture-derived mesenchymal cells (p < 0.05). This was accompanied by an increase in bone morphogenetic protein expression (p < 0.05). Finally, recombinant Noggin mitigated the pro-osteogenic effects of co-culture accompanied by a reduction in Hedgehog signaling (p < 0.05).Suture-derived mesenchymal cells secrete paracrine factors that induce osteogenic differentiation of multipotent stromal cells (human adipose-derived stromal cells). Cells derived from the fusing posterofrontal suture do this to a significantly greater degree than cells from the patent sagittal suture. Enhanced bone morphogenetic protein and Hedgehog signaling may underlie this paracrine effect.

  View details for DOI 10.1097/PRS.0b013e3181e5f81a

  View details for Web of Science ID 000281606700010

  View details for PubMedID 20811214

  View details for PubMedCentralID PMC2933932

• Hes1 Is Required for the Development of Craniofacial Structures Derived From Ectomesenchymal Neural Crest Cells DISCUSSION JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Brugmann, S., Longaker, M. T. 2010; 21 (5): 1450-1451

  View details for Web of Science ID 000282003300031

• So You Want to Be an Innovator? PLASTIC AND RECONSTRUCTIVE SURGERY Rohrich, R. J., Rosen, J., Longaker, M. T. 2010; 126 (3): 1107-1109

  View details for DOI 10.1097/PRS.0b013e3181e3b854

  View details for Web of Science ID 000281606700045

  View details for PubMedID 20811242

• Differential activation of canonical Wnt signaling determines cranial sutures fate: A novel mechanism for sagittal suture craniosynostosis DEVELOPMENTAL BIOLOGY Behr, B., Longaker, M. T., Quarto, N. 2010; 344 (2): 922-940

  Abstract

  Premature closure of cranial sutures, which serve as growth centers for the skull vault, result in craniosynostosis. In the mouse posterior frontal (PF) suture closes by endochondral ossification, whereas sagittal (SAG) remain patent life time, although both are neural crest tissue derived. We therefore, investigated why cranial sutures of same tissue origin adopt a different fate. We demonstrated that closure of the PF suture is tightly regulated by canonical Wnt signaling, whereas patency of the SAG suture is achieved by constantly activated canonical Wnt signaling. Importantly, the fate of PF and SAG sutures can be reversed by manipulating Wnt signaling. Continuous activation of canonical Wnt signaling in the PF suture inhibits endochondral ossification and therefore, suture closure, In contrast, inhibition of canonical Wnt signaling in the SAG suture, upon treatment with Wnt antagonists results in endochondral ossification and suture closure. Thus, inhibition of canonical Wnt signaling in the SAG suture phenocopies craniosynostosis. Moreover, mice haploinsufficient for Twist1, a target gene of canonical Wnt signaling which inhibits chondrogenesis, have sagittal craniosynostosis. We propose that regulation of canonical Wnt signaling is of crucial importance during the physiological patterning of PF and SAG sutures. Importantly, dysregulation of this pathway may lead to craniosynostosis.

  View details for DOI 10.1016/j.ydbio.2010.06.009

  View details for Web of Science ID 000280787100034

  View details for PubMedID 20547147

• Divergent Modulation of Adipose-Derived Stromal Cell Differentiation by TGF-beta 1 Based on Species of Derivation PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Xu, Y., Commons, G. W., Longaker, M. T. 2010; 126 (2): 412-425

  Abstract

  Adipose-derived stromal cells hold promise for skeletal tissue engineering. However, various studies have observed that adipose-derived stromal cells differ significantly in their biology depending on species of derivation. In the following study, the authors sought to determine the species-specific response of adipose-derived stromal cells to recombinant TGF-beta1 (rTGF-beta1).Adipose-derived stromal cells were derived from mouse and human sources. Recombinant TGF-beta1 was added to culture medium (2.5 to 10 ng/ml); proliferation and osteogenic and adipogenic differentiation were assessed by standardized parameters, including cell counting, alkaline phosphatase, alizarin red, oil red O staining, and quantitative real-time polymerase chain reaction.Recombinant TGF-beta1 was found to significantly repress cellular proliferation in both mouse and human adipose-derived stromal cells (p < 0.01). Recombinant TGF-beta1 was found to significantly repress osteogenic differentiation in mouse adipose-derived stromal cells. In contrast, osteogenic differentiation of human adipose-derived stromal cells proceeded unimpeded in either the presence or the absence of rTGF-beta1. Interestingly, rTGF-beta1 induced expression of a number of osteogenic genes in human adipose-derived stromal cells, including BMP2 and BMP4.The authors' results further detail an important facet in which mouse and human adipose-derived stromal cells differ. Mouse adipose-derived stromal cell osteogenesis is completely inhibited by rTGF-beta1, whereas human adipose-derived stromal cell osteogenesis progresses in the presence of rTGF-beta1. These data highlight the importance of species of derivation in basic adipose-derived stromal cell biology. Future studies will examine in more detail the species-specific differences among adipose-derived stromal cell populations.

  View details for DOI 10.1097/PRS.0b013e3181df64dc

  View details for Web of Science ID 000280143800009

  View details for PubMedID 20679827

• Different endogenous threshold levels of Fibroblast Growth Factor-ligands determine the healing potential of frontal and parietal bones BONE Behr, B., Panetta, N. J., Longaker, M. T., Quarto, N. 2010; 47 (2): 281-294

  Abstract

  In the skull vault, neural crest derived frontal bones have an increased healing capacity and higher expression levels of Fibroblast Growth Factor-ligands as compared to mesoderm-derived parietal bones. Thus, we asked whether Fibroblast Growth Factor-ligands are responsible for the superior healing potential of frontal bones. Parietal defects in juvenile and adult mice treated with Fibroblast Growth Factor-2, -9 and -18 showed increased bone regeneration, comparable to frontal defects. Immunohistochemistry revealed increased recruitment of osteoprogenitors and activation of FGF-signaling pathways in FGF-treated parietal defects. Conversely, calvarial defects in Fgf-9(+/-) and Fgf-18(+/-) mice showed impaired calvarial healing which could be rescued by exogenous Fibroblast Growth Factor-ligands. Moreover, by utilizing Wnt1Cre/R26R mice, the migration and contribution of dura mater and pericranium cells to calvarial healing could be demonstrated. Taken together our results demonstrated that different endogenous threshold levels of Fibroblast Growth Factor-ligands in frontal and parietal bones have a profound impact on calvarial regeneration. The present study thereby opens new avenues for translational medicine.

  View details for DOI 10.1016/j.bone.2010.05.008

  View details for Web of Science ID 000280449300015

  View details for PubMedID 20472108

• Sonic Hedgehog Influences the Balance of Osteogenesis and Adipogenesis in Mouse Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A James, A. W., Leucht, P., Levi, B., Carre, A. L., Xu, Y., Helms, J. A., Longaker, M. T. 2010; 16 (8): 2605-2616

  Abstract

  Adipose-derived stromal cells (ASCs) present a great potential for tissue engineering, as they are capable of differentiating into osteogenic and adipogenic cell types, among others. In this study, we examined the role of Hedgehog signaling in the balance of osteogenic and adipogenic differentiation in mouse ASCs. Results showed that Hedgehog signaling increased during early osteogenic differentiation (Shh, Ptc1, and Gli1), but decreased during adipogenic differentiation. N-terminal Sonic Hedgehog (Shh-N) significantly increased in vitro osteogenic differentiation in mouse ASCs, by all markers examined (*p < 0.01). Concomitantly, Shh-N abrogated adipogenic differentiation, by all markers examined (*p < 0.01). Conversely, blockade of endogenous Hedgehog signaling, with the Hedgehog antagonist cyclopamine, enhanced adipogenesis at the expense of osteogenesis. We next translated these results to a mouse model of appendicular skeletal regeneration. Using quantitative real-time polymerase chain reaction and in situ hybridization, we found that skeletal injury (a monocortical 1 mm defect in the tibia) results in a localized increase in Hedgehog signaling. Moreover, grafting of ASCs treated with Shh-N resulted in significantly increased bone regeneration within the defect site. In conclusion, Hedgehog signaling enhances the osteogenic differentiation of mouse ASCs, at the expense of adipogenesis. These data suggest that Hedgehog signaling directs the lineage differentiation of mesodermal stem cells and represents a promising strategy for skeletal tissue regeneration.

  View details for DOI 10.1089/ten.tea.2010.0048

  View details for Web of Science ID 000280648700018

  View details for PubMedID 20367246

  View details for PubMedCentralID PMC2947454

• Origin Matters: Differences in Embryonic Tissue Origin and Wnt Signaling Determine the Osteogenic Potential and Healing Capacity of Frontal and Parietal Calvarial Bones JOURNAL OF BONE AND MINERAL RESEARCH Quarto, N., Wan, D. C., Kwan, M. D., Panetta, N. J., Li, S., Longaker, M. T. 2010; 25 (7): 1680-1694

  Abstract

  Calvarial bones arise from two embryonic tissues, namely, the neural crest and the mesoderm. In this study we have addressed the important question of whether disparate embryonic tissue origins impart variable osteogenic potential and regenerative capacity to calvarial bones, as well as what the underlying molecular mechanism(s). Thus, by performing in vitro and in vivo studies, we have investigated whether differences exist between neural crest-derived frontal and paraxial mesodermal-derived parietal bone. Of interest, our data indicate that calvarial bone osteoblasts of neural crest origin have superior potential for osteogenic differentiation. Furthermore, neural crest-derived frontal bone displays a superior capacity to undergo osseous healing compared with calvarial bone of paraxial mesoderm origin. Our study identified both in vitro and in vivo enhanced endogenous canonical Wnt signaling in frontal bone compared with parietal bone. In addition, we demonstrate that constitutive activation of canonical Wnt signaling in paraxial mesodermal-derived parietal osteoblasts mimics the osteogenic potential of frontal osteoblasts, whereas knockdown of canonical Wnt signaling dramatically impairs the greater osteogenic potential of neural crest-derived frontal osteoblasts. Moreover, fibroblast growth factor 2 (FGF-2) treatment induces phosphorylation of GSK-3beta and increases the nuclear levels of beta-catenin in osteoblasts, suggesting that enhanced activation of Wnt signaling might be mediated by FGF. Taken together, our data provide compelling evidence that indeed embryonic tissue origin makes a difference and that active canonical Wnt signaling plays a major role in contributing to the superior intrinsic osteogenic potential and tissue regeneration observed in neural crest-derived frontal bone.

  View details for DOI 10.1359/jbmr.091116

  View details for Web of Science ID 000280395900023

  View details for PubMedID 19929441

  View details for PubMedCentralID PMC3154006

• Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271 NATURE Boiko, A. D., Razorenova, O. V., van de Rijn, M., Swetter, S. M., Johnson, D. L., Ly, D. P., Butler, P. D., Yang, G. P., Joshua, B., Kaplan, M. J., Longaker, M. T., Weissman, I. L. 2010; 466 (7302): 133-U155

  Abstract

  The question of whether tumorigenic cancer stem cells exist in human melanomas has arisen in the last few years. Here we show that in melanomas, tumour stem cells (MTSCs, for melanoma tumour stem cells) can be isolated prospectively as a highly enriched CD271(+) MTSC population using a process that maximizes viable cell transplantation. The tumours sampled in this study were taken from a broad spectrum of sites and stages. High-viability cells isolated by fluorescence-activated cell sorting and re-suspended in a matrigel vehicle were implanted into T-, B- and natural-killer-deficient Rag2(-/-)gammac(-/-) mice. The CD271(+) subset of cells was the tumour-initiating population in 90% (nine out of ten) of melanomas tested. Transplantation of isolated CD271(+) melanoma cells into engrafted human skin or bone in Rag2(-/-)gammac(-/-) mice resulted in melanoma; however, melanoma did not develop after transplantation of isolated CD271(-) cells. We also show that in mice, tumours derived from transplanted human CD271(+) melanoma cells were capable of metastatsis in vivo. CD271(+) melanoma cells lacked expression of TYR, MART1 and MAGE in 86%, 69% and 68% of melanoma patients, respectively, which helps to explain why T-cell therapies directed at these antigens usually result in only temporary tumour shrinkage.

  View details for DOI 10.1038/nature09161

  View details for PubMedID 20596026

• Regulation of Human Adipose-Derived Stromal Cell Osteogenic Differentiation by Insulin-Like Growth Factor-1 and Platelet-Derived Growth Factor-alpha PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Wan, D. C., Glotzbach, J. P., Commons, G. W., Longaker, M. T. 2010; 126 (1): 41-52

  Abstract

  Human adipose-derived stromal cells possess a great potential for tissue engineering purposes. The authors' laboratory is interested in harnessing human adipose-derived stromal cells for skeletal tissue regeneration and identifying those factors that enhance human adipose-derived stromal cell osteogenic differentiation. The authors hypothesized that insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) would stimulate human adipose-derived stromal cell osteogenesis and that IGF would stimulate adipogenesis.Adipose-derived stromal cells were harvested from human lipoaspirate. Previously, a microarray analysis examined gene expression throughout osteogenic differentiation. In a candidate fashion, the authors added recombinant IGF-1 and PDGF-alpha individually and in combination. Osteogenesis and adipogenesis were assessed by alkaline phosphatase, alizarin red, and oil red O staining, and quantitative real-time polymerase chain reaction (RUNX2, ALP, OCN, IGF1, PPARG, LPL, AP2, and GCP1). Finally, intersection between IGF and PDGF signaling pathways was evaluated.IGF-1 was observed to increase osteogenic differentiation by all markers (p < 0.01). However, PDGF-alpha when added alone primarily did not affect osteogenic markers. PDGF-alpha positively regulated transcription of IGF1. Addition of PDGF-alpha in combination with or before IGF-1 enhanced osteogenesis more than either alone. IGF-1 increased whereas PDGF-alpha diminished human adipose-derived stromal cell adipogenesis.IGF signaling significantly increased osteogenesis in human adipose-derived stromal cells and may be used for tissue-engineering purposes. The combination of PDGF and IGF may be more beneficial than either alone in driving adipose-derived stromal cell osteogenesis. Future in vivo applications will focus on the combination of adipose-derived stromal cells, biomimetic scaffolds, and recombinant IGF.

  View details for DOI 10.1097/PRS.0b013e3181da8858

  View details for Web of Science ID 000279097500006

  View details for PubMedID 20220555

  View details for PubMedCentralID PMC3016898

• Strategies for organ level tissue engineering ORGANOGENESIS Rustad, K. C., Sorkin, M., Levi, B., Longaker, M. T., Gurtner, G. C. 2010; 6 (3): 151-157

  Abstract

  The field of tissue engineering has made considerable strides since it was first described in the late 1980s. The advent and subsequent boom in stem cell biology, emergence of novel technologies for biomaterial development and further understanding of developmental biology have contributed to this accelerated progress. However, continued efforts to translate tissue-engineering strategies into clinical therapies have been hampered by the problems associated with scaling up laboratory methods to produce large, complex tissues. The significant challenges faced by tissue engineers include the production of an intact vasculature within a tissue-engineered construct and recapitulation of the size and complexity of a whole organ. Here we review the basic components necessary for bioengineering organs-biomaterials, cells and bioactive molecules-and discuss various approaches for augmenting these principles to achieve organ level tissue engineering. Ultimately, the successful translation of tissue-engineered constructs into everyday clinical practice will depend upon the ability of the tissue engineer to "scale up" every aspect of the research and development process.

  View details for DOI 10.4161/org.6.3.12139

  View details for Web of Science ID 000290266200003

  View details for PubMedID 21197216

  View details for PubMedCentralID PMC2946046

• Elucidating Mechanisms of Osteogenesis in Human Adipose-Derived Stromal Cells via Microarray Analysis JOURNAL OF CRANIOFACIAL SURGERY Lee, J., Gupta, D., Panetta, N. J., Levi, B., James, A. W., Wan, D., Commons, G. W., Longaker, M. T. 2010; 21 (4): 1136-1141

  Abstract

  The osteogenic potential of human adipose-derived stromal cells (hASCs), the ease of cell procurement, and the shortcomings of conventional skeletal reconstruction call for further analysis of the molecular mechanisms governing hASC osteogenic differentiation. We have examined the expression profile of the human transcriptome during osteogenic differentiation of ASCs using microarray. Subsequently, we analyzed those genes related to osteogenesis that have not been previously studied about hASCs. We have preliminarily assessed the role of IGFBP3, TGF-B3, TNC, CTGF, DKK-1, and PDGFRB in hASC osteogenic differentiation.We compared the expression profile of undifferentiated hASCs to that of hASCs treated with osteogenic differentiation medium for 1, 3, or 7 days using the Human Exonic Evidence-Based Oligonucleotide chip. Genes significantly overexpress or underexpressed were validated with quantitative reverse transcription-polymerase chain reaction. The osteogenic capability of ASCs was verified by Alizarin Red staining.IGFBP3, TGF-B3, TNC, CTGF, and PDGFRB were all upregulated in early osteogenesis, and TGF-B3, TNC, and PDGFRB were upregulated in late osteogenesis by microarray and quantitative reverse transcription analysis. In contrast, DKK-1 was downregulated in early and late osteogenesis. Alizarin Red staining showed a significant increase in mineralization in hASCs, even after 1 day in osteogenic differentiation medium.Factors that commit hASCs to an osteogenic pathway remain largely unknown. We have described 6 genes that play key roles in hASC osteogenic differentiation. We plan to further exploit these data via in vitro treatment of hASCs with these soluble cytokines and in vivo translation using a nude mouse calvarial defect model.

  View details for DOI 10.1097/SCS.0b013e3181e488d6

  View details for Web of Science ID 000280149100044

  View details for PubMedID 20613589

• Fgf-9 is required for angiogenesis and osteogenesis in long bone repair PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Behr, B., Leucht, P., Longaker, M. T., Quarto, N. 2010; 107 (26): 11853-11858

  Abstract

  Bone healing requires a complex interaction of growth factors that establishes an environment for efficient bone regeneration. Among these, FGFs have been considered important for intrinsic bone-healing capacity. In this study, we analyzed the role of Fgf-9 in long bone repair. One-millimeter unicortical defects were created in tibias of Fgf-9(+/-) and wild-type mice. Histomorphometry revealed that half-dose gene of Fgf-9 markedly reduced bone regeneration as compared with wild-type. Both immunohistochemistry and RT-PCR analysis revealed markedly decreased levels of proliferating cell nuclear antigen (PCNA), Runt-related transcription factor 2 (Runx2), osteocalcin, Vega-a, and platelet endothelial cell adhesion molecule 1 (PECAM-1) in Fgf-9(+/-) defects. muCT angiography indicated dramatic impairment of neovascularization in Fgf-9(+/-) mice as compared with controls. Treatment with FGF-9 protein promoted angiogenesis and successfully rescued the healing capacity of Fgf-9(+/-) mice. Importantly, although other pro-osteogenic factors [Fgf-2, Fgf-18, and bone morphogenic protein 2 (Bmp-2)] still were present in Fgf-9(+/-) mice, they could not compensate for the haploinsufficiency of the Fgf-9 gene. Therefore, endogenous Fgf-9 seems to play an important role in long bone repair. Taken together our data suggest a unique role for Fgf-9 in bone healing, presumably by initiating angiogenesis through Vegf-a. Moreover, this study further supports the embryonic phenotype previously observed in the developing limb, thus promoting the concept that healing processes in adult organisms may recapitulate embryonic skeletal development.

  View details for DOI 10.1073/pnas.1003317107

  View details for Web of Science ID 000279332300037

  View details for PubMedID 20547837

  View details for PubMedCentralID PMC2900703

• Connective Tissue Growth Factor in Regulation of RhoA Mediated Cytoskeletal Tension Associated Osteogenesis of Mouse Adipose-Derived Stromal Cells PLOS ONE Xu, Y., Wagner, D. R., Bekerman, E., Chiou, M., James, A. W., Carter, D., Longaker, M. T. 2010; 5 (6)

  Abstract

  Cytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways.Adipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm(2)), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm(2)) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis.We conclude that CTGF is important in the regulation of cytoskeletal tension mediated ASC osteogenic differentiation.

  View details for DOI 10.1371/journal.pone.0011279

  View details for Web of Science ID 000279135400023

  View details for PubMedID 20585662

  View details for PubMedCentralID PMC2890586

• In vitro effects of direct current electric fields on adipose-derived stromal cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Hammerick, K. E., Longaker, M. T., Prinz, F. B. 2010; 397 (1): 12-17

  Abstract

  Endogenous electric fields play an important role in embryogenesis, regeneration, and wound repair and previous studies have shown that many populations of cells, leukocytes, fibroblasts, epithelial cells, and endothelial cells, exhibit directed migration in response to electric fields. As regenerative therapies continue to explore ways to control mesenchymal progenitor cells to recreate desirable tissues, it is increasingly necessary to characterize the vast nature of biological responses imposed by physical phenomena. Murine adipose-derived stromal cells (mASCs) migrated toward the cathode in direct current (DC) fields of physiologic strength and show a dose dependence of migration rate to stronger fields. Electric fields also caused mASCs to orient perpendicularly to the field vector and elicited a transient increase in cytosolic calcium. Additionally, their galvanotactic response appears to share classic chemotactic signaling pathways that are involved in the migration of other cell types. Galvanotaxis is one predominant result of electric fields on mASCs and it may be exploited to engineer adult stem cell concentrations and locations within implanted grafts or toward sites of wound repair.

  View details for DOI 10.1016/j.bbrc.2010.05.003

  View details for Web of Science ID 000279292800003

  View details for PubMedID 20452327

• Human Adipose Derived Stromal Cells Heal Critical Size Mouse Calvarial Defects PLOS ONE Levi, B., James, A. W., Nelson, E. R., Vistnes, D., Wu, B., Lee, M., Gupta, A., Longaker, M. T. 2010; 5 (6)

  Abstract

  Human adipose-derived stromal cells (hASCs) represent a multipotent cell stromal cell type with proven capacity to differentiate along an osteogenic lineage. This suggests that they may be used to heal defects of the craniofacial or appendicular skeleton. We sought to substantiate the use of undifferentiated hASCs in the regeneration of a non-healing mouse skeletal defect.Human ASCs were harvested from female lipoaspirate. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an apatite coated PLGA scaffold alone, or a scaffold with human ASCs. MicroCT scans were obtained at stratified time points post-injury. Histology, in situ hybridization, and histomorphometry were performed. Near complete healing was observed among hASC engrafted calvarial defects. This was in comparison to control groups that showed little healing (*P<0.01). Human ASCs once engrafted differentiate down an osteogenic lineage, determined by qRT-PCR and histological co-expression assays using GFP labeled cells. ASCs were shown to persist within a defect site for two weeks (shown by sex chromosome analysis and quantified using Luciferase+ ASCs). Finally, rBMP-2 was observed to increase hASC osteogenesis in vitro and osseous healing in vivo.Human ASCs ossify critical sized mouse calvarial defects without the need for pre-differentiation. Recombinant differentiation factors such as BMP-2 may be used to supplement hASC mediated repair. Interestingly, ASC presence gradually dissipates from the calvarial defect site. This study supports the potential translation for ASC use in the treatment of human skeletal defects.

  View details for DOI 10.1371/journal.pone.0011177

  View details for Web of Science ID 000278886300019

  View details for PubMedID 20567510

  View details for PubMedCentralID PMC2887361

• Bone Regeneration and Repair CURRENT STEM CELL RESEARCH & THERAPY Panetta, N. J., Gupta, D. M., Longaker, M. T. 2010; 5 (2): 122-128

  Abstract

  In the face of mounting clinical demand, and armed with reconstructive techniques that are technically challenging and frequently result in suboptimal patient outcomes, increasing focus is being placed on tissue engineering and regenerative medicine as a potential source of novel skeletal reconstructive approaches. Specifically, evidence is accumulating that highlights the promise of osteoprogenitor cell-based reconstructive strategies to meet the needs of an expanding patient population. Historically, the study of cell and molecular biology guiding physiologic and pathologic skeletal development, as well as endogenous bone regeneration following injury, has provided a wealth of information that lends insight toward potential parallel processes that may regulate the osteogenic differentiation of progenitor cells. Multiple progenitor cell populations are now known to possess a capacity to undergo robust osteogenic differentiation in the presence of appropriate environmental cues (hESC, BMSC, ASC, etc.) Recent investigations have put forth multiple advantages of ASC relative to BMSC. Of note, ASC exist in relative abundance, lack the need for in vitro expansion prior to utilization, and can be harvested with relative ease and reduced donor morbidity. Collectively, these factors, paired with promising in vitro and in vivo observations that speak toward the substantial osteogenic potential of ASC, have spurred enthusiasm to pursue the application of ASC in the maturation of skeletal tissue engineering applications. Yet, elucidating what structural and functional properties of scaffolds designed for ASC-mediated skeletal tissue engineering applications (porosity, pore size, composition, mechanical stability, degradation kinetics, etc.), as well as evolving our understanding and capacity to deliver spatiotemporally specific pro-osteogenic targeted molecular manipulation to progenitor cells, remain important hurdles to clear. The scope of this review encompasses the current state of ongoing investigations along these fronts, as well as what future direction will be critical to the transition of cell-based skeletal tissue engineering strategies to the bedside.

  View details for Web of Science ID 000208262700006

  View details for PubMedID 19941457

• Commentary. Aesthetic surgery journal Levi, B., Ko, S. H., Longaker, M. T. 2010; 30 (3): 387-389

  View details for DOI 10.1177/1090820X10374102

  View details for PubMedID 20601561

• Highlights of the proceedings from the 13th International Congress of the International Society of Craniofacial Surgery: ISCFS 2009. journal of craniofacial surgery Bradley, J. P., Warren, S., Longaker, M. T. 2010; 21 (3): 944-946

  View details for DOI 10.1097/SCS.0b013e3181d88125

  View details for PubMedID 20485093

• The SNaP System: Biomechanical and Animal Model Testing of a Novel Ultraportable Negative-Pressure Wound Therapy System PLASTIC AND RECONSTRUCTIVE SURGERY Fong, K. D., Hu, D., Eichstadt, S., Gupta, D. M., Pinto, M., Gurtner, G. C., Longaker, M. T., Lorenz, H. P. 2010; 125 (5): 1362-1371

  Abstract

  Negative-pressure wound therapy is traditionally achieved by attaching an electrically powered pump to a sealed wound bed and applying subatmospheric pressure by means of gauze or foam. The Smart Negative Pressure (SNaP) System (Spiracur, Inc., Sunnyvale, Calif.) is a novel ultraportable negative-pressure wound therapy system that does not require an electrically powered pump.Negative pressure produced by the SNaP System, and a powered pump, the wound vacuum-assisted closure advanced-therapy system (Kinetic Concepts, Inc., San Antonio, Texas), were compared in vitro using bench-top pressure sensor testing and microstrain and stress testing with pressure-sensitive film and micro-computed tomographic scan analysis. In addition, to test in vivo efficacy, 10 rats underwent miniaturized SNaP (mSNaP) device placement on open wounds. Subject rats were randomized to a system activation group (approximately -125 mmHg) or a control group (atmospheric pressure). Wound measurements and histologic data were collected for analysis.Bench measurement revealed nearly identical negative-pressure delivery and mechanical strain deformation patterns between both systems. Wounds treated with the mSNaP System healed faster, with decreased wound size by postoperative day 7 (51 percent versus 12 percent reduction; p < 0.05) and had more rapid complete reepithelialization (21 days versus 32 days; p < 0.05). The mSNaP device also induced robust granulation tissue formation.The SNaP System and an existing electrically powered negative-pressure wound therapy system have similar biomechanical properties and functional wound-healing benefits. The potential clinical efficacy of the SNaP device for the treatment of wounds is supported.

  View details for DOI 10.1097/PRS.0b013e3181d62b25

  View details for Web of Science ID 000276886600008

  View details for PubMedID 20440156

• Retinoic Acid Enhances Osteogenesis in Cranial Suture-Derived Mesenchymal Cells: Potential Mechanisms of Retinoid-Induced Craniosynostosis PLASTIC AND RECONSTRUCTIVE SURGERY James, A. W., Levi, B., Xu, Y., Carre, A. L., Longaker, M. T. 2010; 125 (5): 1352-1361

  Abstract

  In utero retinoid exposure results in numerous craniofacial malformations, including craniosynostosis. Although many malformations associated with retinoic acid syndrome are associated with neural crest defects, the specific mechanisms of retinoid-induced craniosynostosis remain unclear. The authors used the culture of mouse cranial suture-derived mesenchymal cells to probe the potential cellular mechanisms of this teratogen to better elucidate mechanisms of retinoid-induced suture fusion.Genes associated with retinoid signaling were assayed in fusing (posterofrontal) and patent (sagittal, coronal) sutures by quantitative real-time polymerase chain reaction. Cultures of mouse suture-derived mesenchymal cells from the posterofrontal suture were established from 4-day-old mice. Cells were cultured with all-trans retinoic acid (1 and 5 muM). Proliferation, osteogenic differentiation, and specific gene expression were assessed.Mouse sutures were found to express genes necessary for retinoic acid synthesis, binding, and signal transduction, demonstrated by quantitative real-time polymerase chain reaction (Raldh1, Raldh2, Raldh3, and Rbp4). These genes were not found to be differentially expressed in fusing as compared with patent cranial sutures in vivo. Addition of retinoic acid enhanced the osteogenic differentiation of suture-derived mesenchymal cells in vitro, including up-regulation of alkaline phosphatase activity and Runx2 expression. Contemporaneously, cellular proliferation was repressed, as shown by proliferative cell nuclear antigen expression. The pro-osteogenic effect of retinoic acid was accompanied by increased gene expression of several hedgehog and bone morphogenetic protein ligands.Retinoic acid represses proliferation and enhances osteogenic differentiation of suture-derived mesenchymal cells. These in vitro data suggest that retinoid exposure may lead to premature cranial suture fusion by means of enhanced osteogenesis and hedgehog and bone morphogenetic protein signaling.

  View details for DOI 10.1097/PRS.0b013e3181d62980

  View details for Web of Science ID 000276886600007

  View details for PubMedID 20134361

  View details for PubMedCentralID PMC2909493

• Cyclophilin C-Associated Protein/Mac-2 Binding Protein Colocalizes With Calnexin and Regulates the Expression of Tissue Transglutaminase JOURNAL OF CELLULAR PHYSIOLOGY Kong, W., Lin, B. W., Li, S., Longaker, M. T., Lorenz, H. P. 2010; 223 (1): 151-157

  Abstract

  Cyclophilin C-associated protein (CyCAP) or Mac-2 binding protein has been identified as a binding protein for cyclophilin C in mice and for Mac-2 (galectin-3) in human, suggesting its multiple binding activity to proteins. In the present study, using specific anti-rat-CyCAP antibody, we found that CyCAP colocalizes with calnexin at the location near the nuclear envelope, however CyCAP does not have colocalization with calreticulin. In senescent fibroblasts and interferon-gamma (IFNgamma) treated fibroblasts, both calnexin and CyCAP form larger polymers and are released from the endoplasmic reticulum (ER) through the cellular membrane to the extracellular area. Immunoprecipitation studies further confirm that the release of calnexin is through binding to CyCAP. Further, we found that tissue transglutaminase (tTG) protein is decreased, however not at the RNA level, in CyCAP null fibroblasts, which suggests that CyCAP is involved in tTG post-translational modification. Our data give novel evidence that CyCAP regulates the post-translational modification of tTG through its colocalization with calnexin in ER.

  View details for DOI 10.1002/jcp.22020

  View details for Web of Science ID 000275589300017

  View details for PubMedID 20049854

• A nonviral minicircle vector for deriving human iPS cells NATURE METHODS Jia, F., Wilson, K. D., Sun, N., Gupta, D. M., Huang, M., Li, Z., Panetta, N. J., Chen, Z. Y., Robbins, R. C., Kay, M. A., Longaker, M. T., Wu, J. C. 2010; 7 (3): 197-U46

  Abstract

  Owing to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem cells (iPSCs). We report the use of 'minicircle' DNA, a vector type that is free of bacterial DNA and capable of high expression in cells, for this purpose. Here we use a single minicircle vector to generate transgene-free iPSCs from adult human adipose stem cells.

  View details for DOI 10.1038/NMETH.1426

  View details for Web of Science ID 000275058200018

  View details for PubMedID 20139967

  View details for PubMedCentralID PMC2892897

• Pulsed Direct Current Electric Fields Enhance Osteogenesis in Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A Hammerick, K. E., James, A. W., Huang, Z., Prinz, F. B., Longaker, M. T. 2010; 16 (3): 917-931

  Abstract

  Adipose-derived stromal cells (ASCs) constitute a promising source of cells for regenerative medicine applications. Previous studies of osteogenic potential in ASCs have focused on chemicals, growth factors, and mechanical stimuli. Citing the demonstrated role electric fields play in enhancing healing in bone fractures and defects, we investigated the ability of pulsed direct current electric fields to drive osteogenic differentiation in mouse ASCs. Employing 50 Hz direct current electric fields in concert with and without osteogenic factors, we demonstrated increased early osteoblast-specific markers. We were also able to establish that commonly reported artifacts of electric field stimulation are not the primary mediators of the observed effects. The electric fields caused marked changes in the cytoskeleton. We used atomic force microscopy-based force spectroscopy to record an increase in the cytoskeletal tension after treatment with electric fields. We abolished the increased cytoskeletal stresses with the rho-associated protein kinase inhibitor, Y27632, and did not see any decrease in osteogenic gene expression, suggesting that the pro-osteogenic effects of the electric fields are not transduced via cytoskeletal tension. Electric fields may show promise as candidate enhancers of osteogenesis of ASCs and may be incorporated into cell-based strategies for skeletal regeneration.

  View details for DOI 10.1089/ten.tea.2009.0267

  View details for Web of Science ID 000275041500016

  View details for PubMedID 19824802

  View details for PubMedCentralID PMC2862617

• Human iPS cell-based therapy Considerations before clinical applications CELL CYCLE Sun, N., Longaker, M. T., Wu, J. C. 2010; 9 (5): 880-885

  Abstract

  Generation of induced pluripotent stem (iPS) cells has revolutionized the field of regenerative medicine. With the exponential increase in iPS cell research in the past three years, human iPS cells have been derived with different technologies and from various cell types. From a translational perspective, however, a number of issues must be addressed before safe and high quality patient-specific iPS cells can be derived for clinical applications. In addition, iPS cell-based therapies also need to be thoroughly evaluated in pre-clinical animal models before they can be applied to human subjects.

  View details for Web of Science ID 000276307700017

  View details for PubMedID 20160515

• Human Adipose-Derived Stromal Cells Respond to and Elaborate Bone Morphogenetic Protein-2 during In Vitro Osteogenic Differentiation PLASTIC AND RECONSTRUCTIVE SURGERY Panetta, N. J., Gupta, D. M., Lee, J. K., Wan, D. C., Commons, G. W., Longaker, M. T. 2010; 125 (2): 483-493

  Abstract

  Interest in the potential application of adipose-derived stromal cells in cell-mediated tissue engineering of bone and other mesenchymal-derived tissues is growing. This study aimed to investigate the hypothesis that human adipose-derived stromal cells respond to and elaborate bone morphogenetic protein (BMP) 2, which could represent an important target of molecular manipulation to enhance the osteogenic potential of human adipose-derived stromal cells.Human adipose-derived stromal cells were differentiated for 10 days toward the osteogenic lineage in osteogenic differentiation media alone or supplemented with recombinant human BMP2 (rhBMP2). Alizarin red staining was quantified by spectrophotometry. Gene expression analyses were performed using quantitative real-time polymerase chain reaction. BMP2 levels in conditioned media were titered by enzyme-linked immunosorbent assay daily during osteogenic differentiation. Human adipose-derived stromal cells were cultured in complete or partially (50 percent) changed osteogenic differentiation media, or unchanged osteogenic differentiation media, to assay for pro-osteogenic secreted factors. In addition, human adipose-derived stromal cells were cultured in osteogenic differentiation media supplemented with BMP2/BMP4-neutralizing antibody.Exogenous rhBMP2 significantly augmented the in vitro osteogenic potential of human adipose-derived stromal cells in a dose-dependent fashion, and significantly increased transcript levels of RUNX2 and osteocalcin. BMP2, BMP4, BMPR1B, and SMAD1/5 expression was significantly increased during differentiation. Enzyme-linked immunosorbent assay demonstrated significantly increased BMP2 elaboration during differentiation. Culture in conditioned osteogenic differentiation media led to significantly increased matrix mineralization. Mineralization was significantly decreased when osteogenic differentiation media was supplemented with a BMP2/BMP4-neutralizing antibody.These data strongly support that BMP signaling is dynamic and important during normal in vitro osteogenic differentiation of human adipose-derived stromal cells. Thus, BMP2 may be used to enhance the osteogenic differentiation of human adipose-derived stromal cells for bone tissue engineering. Future studies will examine the effect of rhBMP2 on osteogenic differentiation of human adipose-derived stromal cells in vivo.

  View details for DOI 10.1097/PRS.0b013e3181c82d75

  View details for Web of Science ID 000274741700008

  View details for PubMedID 20124834

• Addressing the paucity of underrepresented minorities in academic surgery: can the "Rooney Rule" be applied to academic surgery? AMERICAN JOURNAL OF SURGERY Butler, P. D., Longaker, M. T., Britt, L. D. 2010; 199 (2): 255-262

  View details for DOI 10.1016/j.amjsurg.2009.05.021

  View details for Web of Science ID 000276959700018

  View details for PubMedID 20113702

• Regenerative medicine: a surgeon's perspective 40th Annual Meeting of the American-Pediatric-Surgical-Association Longaker, M. T. W B SAUNDERS CO-ELSEVIER INC. 2010: 11–18

  Abstract

  More than 200 million incisions are made in the world each year on children and adults. They all end up with a scar unless there is an unusual situation where we are operating on an early gestation fetus. The question is, "why do we not regenerate?" and "why do we always heal with either a 'normal amount of scarring' or, approximately 15% of the time, with a pathologic amount of scarring (hypertrophic scar or keloid)?"

  View details for DOI 10.1016/j.jpedsurg.2009.10.004

  View details for Web of Science ID 000274393800002

  View details for PubMedID 20105574

  View details for PubMedCentralID PMC2900786

• From Idea to Bedside: The Process of Surgical Invention and Innovation KEY TOPICS IN SURGICAL RESEARCH AND METHODOLOGY Wall, J., Gurtner, G. C., Longaker, M. T., Athanasiou, T., Debas, H., Darzi, A. 2010: 647–56

  View details for DOI 10.1007/978-3-540-71915-1_52

  View details for Web of Science ID 000274802900052

• A Review of the Therapeutic Potential of Adipose-Derived Stem Cells ADVANCES IN WOUND CARE, VOL 1 Macleod, L. C., James, A. W., Carre, A. L., Longaker, M. T., Lorenz, H. P., Sen, C. K. 2010; 1: 495–501

  View details for DOI 10.1089/awc.2009.0110

  View details for Web of Science ID 000277780600080

• Interaction of Wingless Protein (Wnt), Transforming Growth Factor-beta 1, and Hyaluronan Production in Fetal and Postnatal Fibroblasts PLASTIC AND RECONSTRUCTIVE SURGERY Carre, A. L., James, A. W., Macleod, L., Kong, W., Kawai, K., Longaker, M. T., Lorenz, H. P. 2010; 125 (1): 74-88

  Abstract

  Mammalian fetal skin injury heals scarlessly. The intrinsic differences between embryonic and adult fibroblasts that underlie this observation are poorly understood. Several studies have linked Wnt proteins with skin morphogenesis. The authors' study aimed to establish a correlation between beta-catenin-dependent (canonical) Wnt protein, transforming growth factor (TGF)-beta1, and the expression of hyaluronan synthesis enzymes during scarless versus scarring wound healing.Wnt signaling was quantified after 1.5-mm skin wounds were created in BAT-gal fetal (e16.5) and postnatal (p1) mice. Canonical Wnt signals were localized by X-gal staining and quantified with quantitative real-time polymerase chain reaction. Primary embryonic and postnatal mouse dermal fibroblasts were treated with recombinant Wnt3a or TGF-beta1. Proliferation was assayed by bromodeoxyuridine incorporation. Gene expression of enzymes that regulate hyaluronan production and turnover was examined by quantitative real-time polymerase chain reaction (hyaluronan synthases or HAS1-3, hyaluronadase-2), as well as other target genes for Wnt and TGF-beta (Axin2, TGF-beta1, TGF-beta3, type 1 collagen, proliferating cell nuclear antigen).Canonical Wnt signaling increased following wounding in postnatal, but not fetal, mice. In vitro, rmWnt3a increased postnatal fibroblast proliferation but not in embryonic cells. Both Wnt3a and TGF-beta1 induced HAS2 and HAS3 gene expression in embryonic fibroblasts, while HAS1 and Hyal2 were induced in postnatal fibroblasts. Finally, rmWnt3a significantly increased type I collagen expression, particularly in postnatal fibroblasts, and influenced expression of TGF-beta isoforms.Increased canonical Wnt signaling occurs during postnatal but not fetal cutaneous wound repair. Fetal and postnatal fibroblasts have a disparate response to rmWnt3a in vitro. rmWnt3a affects postnatal fibroblasts in a similar fashion to rhTGF-beta1, a known profibrotic cytokine.

  View details for DOI 10.1097/PRS.0b013e3181c495d1

  View details for Web of Science ID 000273417000011

  View details for PubMedID 20048602

• Unique Modulation of Cadherin Expression Pattern during Posterior Frontal Cranial Suture Development and Closure CELLS TISSUES ORGANS Sahar, D. E., Behr, B., Fong, K. D., Longaker, M. T., Quarto, N. 2010; 191 (5): 401-413

  Abstract

  Cranial suture development involves coordinated expression of multiple genes and tissue contribution from neural crest cells and paraxial mesoderm for timely sutural morphogenesis. Transcription factors, growth factors, and neural crest determinant genes play critical roles in calvarial growth ensuring normal development of the underlying brain. In vitro studies have implicated cell-cell adhesion molecules as a driving force behind suture closure. We performed cDNA microarray to study differential expression of adhesion molecules during the timing of suture closure in a mouse model where only the posterior frontal (PF) suture closes. Our results indicate increased expression of E-cadherin during the period of PF suture closure. Quantitative RT-PCR analysis of E- and N-cadherin in PF closing suture revealed a biphasic expression of N-cadherin, the first phase coinciding with cellular condensation preceding chondrogenesis followed by a second phase coinciding with E-cadherin co-expression and suture closure. Furthermore, expression analysis of the N-cadherin and E-cadherin transcriptional repressors Wnt7a and Snail indicate a specific temporal regulation of these genes, suggesting their potential role as regulators of both E- and N-cadherin during the PF suture development and closure. Finally, given the in vitro evidence of fibroblast growth factor (FGF)-2 as a potential regulator of E- and N-cadherin we investigated the expression of E-cadherin during PF suture closure in Fgf-2 deficient mice. In contrast to in vitrodata previously reported, E-cadherin expression is normal in these animals, and PF suture closure occurs properly, probably due to potential redundancy of FGF ligands ensuring normal temporal expression of E-cadherin and PF suture closure.

  View details for DOI 10.1159/000272318

  View details for Web of Science ID 000276748700007

  View details for PubMedID 20051668

  View details for PubMedCentralID PMC2859230

• Inhibition of Histone Deacetylase Activity in Reduced Oxygen Environment Enhances the Osteogenesis of Mouse Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A Xu, Y., Hammerick, K. E., James, A. W., Carre, A. L., Leucht, P., Giaccia, A. J., Longaker, M. T. 2009; 15 (12): 3697-3707

  Abstract

  Recent studies suggest that oxygen tension has a great impact on the osteogenic differentiation capacity of mesenchymal cells derived from adipose tissue: reduced oxygen impedes osteogenesis. We have found that expansion of mouse adipose-derived stromal cells (mASCs) in reduced oxygen tension (10%) results in increased cell proliferation along with induction of histone deacetylase (HDAC) activity. In this study, we utilized two HDAC inhibitors (HDACi), sodium butyrate (NaB) and valproic acid (VPA), and studied their effects on mASCs expanded in various oxygen tensions (21%, 10%, and 1% O(2)). Significant growth inhibition was observed with NaB or VPA treatment in each oxygen tension. Osteogenesis was enhanced by treatment with NaB or VPA, particularly in reduced oxygen tensions (10% and 1% O(2)). Conversely, adipogenesis was decreased with treatments of NaB or VPA at all oxygen tensions. Finally, NaB- or VPA-treated, reduced oxygen tension-exposed (1% O(2)) ASCs were grafted into surgically created mouse tibial defects and resulted in significantly increased bone regeneration. In conclusion, HDACi significantly promote the osteogenic differentiation of mASCs exposed to reduced oxygen tension; HDACi may hold promise for future clinical applications of ASCs for skeletal regeneration.

  View details for DOI 10.1089/ten.tea.2009.0213

  View details for Web of Science ID 000272528400003

  View details for PubMedID 19505250

  View details for PubMedCentralID PMC2792078

• Craniofacial surgery, from past pioneers to future promise. Journal of maxillofacial and oral surgery Wan, D. C., Kwan, M. D., Kumar, A., Bradley, J. P., Longaker, M. T. 2009; 8 (4): 348-356

  Abstract

  As a surgical subspecialty devoted to restoration of normal facial and calvarial anatomy, craniofacial surgeons must navigate the balance between pathologic states of bone excess and bone deficit. While current techniques employed take root in lessons learned from the success and failure of early pioneers, craniofacial surgery continues to evolve, and novel modalities will undoubtedly arise integrating past and present experiences with future promise to effectively treat craniofacial disorders.This review provides an overview of current approaches in craniofacial surgery for treating states of bone excess and deficit, recent advances in our understanding of the molecular and cellular processes underlying craniosynostosis, a pathological state of bone excess, and current research efforts in cellular-based therapies for bone regeneration.The surgical treatment of bone excess and deficit has evolved to improve both the functional and morphological outcomes of affected patients. Recent progress in elucidating the molecular and cellular mechanisms governing bone formation will be instrumental for developing improved therapies for the treatment of pathological states of bone excess and deficit.While significant advances have been achieved in craniofacial surgery, improved strategies for addressing states of bone excess and bone deficit in the craniofacial region are needed. Investigations on the biomolecular events involved in craniosynostosis and cellular-based bone tissue engineering may soon be added to the armamentarium of surgeons treating craniofacial dysmorphologies.

  View details for DOI 10.1007/s12663-009-0084-x

  View details for PubMedID 23139542

  View details for PubMedCentralID PMC3454104

• TGF-beta 1 RNA Interference in Mouse Primary Dura Cell Culture: Downstream Effects on TGF Receptors, FGF-2, and FGF-R1 mRNA Levels PLASTIC AND RECONSTRUCTIVE SURGERY Ko, S. H., Behr, B., Longaker, M. T. 2009; 124 (5): 1474-1476

  View details for DOI 10.1097/PRS.0b013e3181b989de

  View details for Web of Science ID 000271512700014

• Human skin wounds: A major and snowballing threat to public health and the economy WOUND REPAIR AND REGENERATION Sen, C. K., Gordillo, G. M., Roy, S., Kirsner, R., Lambert, L., Hunt, T. K., Gottrup, F., Gurtner, G. C., Longaker, M. T. 2009; 17 (6): 763-771

  Abstract

  ABSTRACT In the United States, chronic wounds affect 6.5 million patients. An estimated excess of US$25 billion is spent annually on treatment of chronic wounds and the burden is rapidly growing due to increasing health care costs, an aging population and a sharp rise in the incidence of diabetes and obesity worldwide. The annual wound care products market is projected to reach $15.3 billion by 2010. Chronic wounds are rarely seen in individuals who are otherwise healthy. In fact, chronic wound patients frequently suffer from "highly branded" diseases such as diabetes and obesity. This seems to have overshadowed the significance of wounds per se as a major health problem. For example, NIH's Research Portfolio Online Reporting Tool (RePORT; http://report.nih.gov/), directed at providing access to estimates of funding for various disease conditions does list several rare diseases but does not list wounds. Forty million inpatient surgical procedures were performed in the United States in 2000, followed closely by 31.5 million outpatient surgeries. The need for post-surgical wound care is sharply on the rise. Emergency wound care in an acute setting has major significance not only in a war setting but also in homeland preparedness against natural disasters as well as against terrorism attacks. An additional burden of wound healing is the problem of skin scarring, a $12 billion annual market. The immense economic and social impact of wounds in our society calls for allocation of a higher level of attention and resources to understand biological mechanisms underlying cutaneous wound complications.

  View details for DOI 10.1111/j.1524-475X.2009.00543.x

  View details for Web of Science ID 000271314900001

  View details for PubMedID 19903300

  View details for PubMedCentralID PMC2810192

• Discussion. TGF-beta1 RNA interference in mouse primary dura cell culture: downstream effects on TGF receptors, FGF-2, and FGF-R1 mRNA levels. Plastic and reconstructive surgery Ko, S. H., Behr, B., Longaker, M. T. 2009; 124 (5): 1474-1476

  View details for DOI 10.1097/PRS.0b013e3181b989de

  View details for PubMedID 20009833

• From Bedside to Bench and Back Again: Technology Innovation in Plastic Surgery PLASTIC AND RECONSTRUCTIVE SURGERY Gurtner, G. C., Rohrich, R. J., Longaker, M. T. 2009; 124 (4): 1355-1356

  View details for DOI 10.1097/PRS.0b013e3181b8901a

  View details for Web of Science ID 000270332300042

  View details for PubMedID 19935323

• Estrogen/Estrogen Receptor Alpha Signaling in Mouse Posterofrontal Cranial Suture Fusion PLOS ONE James, A. W., Theologis, A. A., Brugmann, S. A., Xu, Y., Carre, A. L., Leucht, P., Hamilton, K., Korach, K. S., Longaker, M. T. 2009; 4 (9)

  Abstract

  While premature suture fusion, or craniosynostosis, is a relatively common condition, the cause is often unknown. Estrogens are associated with growth plate fusion of endochondral bones. In the following study, we explore the previously unknown significance of estrogen/estrogen receptor signaling in cranial suture biology.Firstly, estrogen receptor (ER) expression was examined in physiologically fusing (posterofrontal) and patent (sagittal) mouse cranial sutures by quantitative RT-PCR. Next, the cranial suture phenotype of ER alpha and ER beta knockout (alphaERKO, betaERKO) mice was studied. Subsequently, mouse suture-derived mesenchymal cells (SMCs) were isolated; the effects of 17-beta estradiol or the estrogen antagonist Fulvestrant on gene expression, osteogenic and chondrogenic differentiation were examined in vitro. Finally, in vivo experiments were performed in which Fulvestrant was administered subcutaneously to the mouse calvaria. Results showed that increased ERalpha but not ERbeta transcript abundance temporally coincided with posterofrontal suture fusion. The alphaERKO but not betaERKO mouse exhibited delayed posterofrontal suture fusion. In vitro, addition of 17-beta estradiol enhanced both osteogenic and chondrogenic differentiation in suture-derived mesenchymal cells, effects reversible by Fulvestrant. Finally, in vivo application of Fulvestrant significantly diminished calvarial osteogenesis, inhibiting suture fusion.Estrogen signaling through ERalpha but not ERbeta is associated with and necessary for normal mouse posterofrontal suture fusion. In vitro studies suggest that estrogens may play a role in osteoblast and/or chondrocyte differentiation within the cranial suture complex.

  View details for DOI 10.1371/journal.pone.0007120

  View details for Web of Science ID 000270154400013

  View details for PubMedID 19771170

  View details for PubMedCentralID PMC2743190

• Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sun, N., Panetta, N. J., Gupta, D. M., Wilson, K. D., Lee, A., Jia, F., Hu, S., Cherry, A. M., Robbins, R. C., Longaker, M. T., Wu, J. C. 2009; 106 (37): 15720-15725

  Abstract

  Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.

  View details for DOI 10.1073/pnas.0908450106

  View details for Web of Science ID 000269806600040

  View details for PubMedID 19805220

  View details for PubMedCentralID PMC2739869

• Bone Tissue Engineering Scaffolds of Today and Tomorrow DISCUSSION JOURNAL OF CRANIOFACIAL SURGERY Panetta, N. J., Gupta, D. M., Longaker, M. T. 2009; 20 (5): 1531-1532

  View details for Web of Science ID 000270369000047

  View details for PubMedID 19816291

• Application of FGF-2 mimics bone healing potential of frontal bones in parietal calvarial defects by recruitment of dura mater cells Behr, B., Panetta, N. J., Longaker, M. T., Quarto, N. ELSEVIER SCIENCE INC. 2009: S75

  View details for DOI 10.1016/j.jamcollsurg.2009.06.183

  View details for Web of Science ID 000269755300163

• Tissue Harvest by Means of Suction-Assisted or Third-Generation Ultrasound-Assisted Lipoaspiration Has No Effect on Osteogenic Potential of Human Adipose-Derived Stromal Cells PLASTIC AND RECONSTRUCTIVE SURGERY Panetta, N. J., Gupta, D. M., Kwan, M. D., Wan, D. C., Commons, G. W., Longaker, M. T. 2009; 124 (1): 65-73

  Abstract

  Human adipose-derived stromal cells readily undergo osteogenic differentiation in vitro and in vivo. Thus, interest in their potential role in skeletal tissue engineering continues to escalate. Very little is known regarding the effects that energy delivered by means of third-generation ultrasound-assisted lipoaspiration may have on the osteogenic potential of these cells. The authors investigated whether differences in adipose-derived stromal cell yield, and the in vitro proliferation and osteogenic potential of these cells obtained by suction-assisted lipoaspiration or third-generation ultrasound-assisted lipoaspiration, exist.Adipose-derived stromal cells were harvested from lipoaspiration specimens of patients undergoing elective suction-assisted lipoaspiration and third-generation ultrasound-assisted lipoaspiration. Harvested cells were seeded to evaluate proliferative capacity and in vitro osteogenic potential. Alkaline phosphatase and alizarin red staining were performed to evaluate early and terminal osteogenic differentiation, respectively. Quantitative real-time polymerase chain reaction analysis was used to examine osteogenic gene expression patterns of RUNX2/CFBA1 (early differentiation) and osteocalcin (late differentiation).No significant differences in the proliferative capacity (n = 3), alkaline phosphatase staining (n = 3), or extracellular matrix mineralization (n = 3) of suction-assisted lipoaspiration- or third-generation ultrasound-assisted lipoaspiration-derived cells were appreciated. Transcript levels of markers of early and terminal osteogenic differentiation were not significantly different (n = 3).These findings suggest that exposure of adipose-derived stromal cells to ultrasound energy during tissue harvest by means of third-generation ultrasound-assisted lipoaspiration does not impart a negative consequence toward their proliferative capacity or osteogenic potential. Thus, the cells harvested using third-generation ultrasound-assisted lipoaspiration are comparable to those obtained by means of suction-assisted lipoaspiration for use in the study of osteogenic differentiation and skeletal tissue engineering.

  View details for DOI 10.1097/PRS.0b013e3181ab10cd

  View details for Web of Science ID 000267895000011

  View details for PubMedID 19568046

• The Use of Polymer Scaffolds in Skeletal Tissue Engineering Applications JOURNAL OF CRANIOFACIAL SURGERY Gupta, D. M., Panetta, N. J., Longaker, M. T. 2009; 20 (3): 860-861

  View details for Web of Science ID 000266295400035

  View details for PubMedID 19461326

• Pfeiffer Syndrome Twins: Despite Improved Correction in One Twin, Growth Disturbance Results in Similar Need for Subsequent Monobloc Advancement JOURNAL OF CRANIOFACIAL SURGERY Kohan, E., Longaker, M. T., Nguyen, J., Kawamoto, H., Wexler, A., Cahan, L., Katchikian, H. V., Bradley, J. P. 2009; 20 (3): 811-815

  Abstract

  Twins with Pfeiffer syndrome (or acrocephalosyndactyly) had a similar phenotypic appearance with findings of classic or type 1 Pfeiffer syndrome, including bicoronal and sagittal craniosynostoses, midface hypoplasia, and broad thumbs/toes. We document their treatment with 2 monobloc advancements and discuss growth disturbances in craniofacial dysostosis.At 6 months, both twins underwent release of multisuture (bilateral coronal and sagittal) fusions for improvement of scaphocephalic shape and multisuture release; however, one twin had a more aggressive procedure with advancement of the frontal-orbital region. Despite improved initial correction by one twin, at 5 years of age, both twins presented with midface hypoplasia and exorbitism and underwent a monobloc distraction procedure with similar 20-mm advancements. Comparative analysis by our craniofacial multidisciplinary team included perioperative reports, computed tomographic scans, cephalograms, parent questionnaires, and physician surveys.Both twins had an improved confidence interval scores from 84 to 68 and 82 to 69 postoperatively. In 6-month follow-up, the Whitaker score of the first twin was 2.8, whereas that for the second twin with the frontal-orbital advancement was 1.2. Preschool expressive and receptive tests yielded 97 and 95, and 97 and 98, and developmental testing was similar between the twins. Global evaluations were equivalent to age-matched controls, and memory and attention skills were within normal limits. Parental surveys showed a high level of satisfaction after all procedures in both twins.Our study demonstrates that the phenotypic outcome for both twins remained unchanged when comparing a more aggressive surgery to less aggressive surgery as an infant. The genetic mutation may have overridden the different surgical interventions. Both twins ultimately required 2 subsequent monobloc corrections.

  View details for DOI 10.1097/SCS.0b013e3181a2e545

  View details for Web of Science ID 000266295400023

  View details for PubMedID 19390453

• Ethnic Diversity Remains Scarce in Academic Plastic and Reconstructive Surgery PLASTIC AND RECONSTRUCTIVE SURGERY Butler, P. D., Britt, L. D., Longaker, M. T. 2009; 123 (5): 1618-1627

  Abstract

  Plastic surgery has been dedicated to advancing academic surgery in education, research, innovation, and patient care. Thus, as U.S. health care disparities persist, it would be befitting for plastic surgery to assume the lead in alleviating these disparities. As part of a multifaceted approach to ameliorate health care disparities, increasing diversity in the health care workforce will be imperative. Investigating the demographics of the U.S. plastic surgery residents and faculty can bring attention to a deficit that, if corrected, could benefit the field and improve the entire health care system.Medical students, plastic surgery residents/fellows, and plastic surgery faculty demographic information from 1966 to 2006 was analyzed from the Association of American Medical Colleges' data files.Caucasians encompass 68.7 percent of U.S. plastic surgery residents/fellows, while Asian-, African-, and Latino-Americans encompass 20.9, 3.7, and 6.2 percent, respectively. Caucasians comprise 74.9 percent of academic plastic surgeons, while Asian-, African-, and Latino-Americans comprise 10.9, 1.4, and 3.6 percent, respectively. Caucasians constitute 82.0 percent of tenured full professors, while Asian-, African-, and Latino-Americans constitute 4.9, 1.6, and 4.9 percent, respectively. In 2004, African-Americans and Latino-Americans comprised 3.6 percent and 5.7 percent of all U.S. plastic surgeons, but only 1.5 percent and 4.9 percent of plastic academicians, respectively.Over the last 40 years, plastic surgery has been ineffective in adequately increasing the number of minority residents and faculty. Expanding the number of minority academic plastic surgeons could establish a health care environment more accommodating to minority patients, increase studies highlighting minority health needs, and provide additional role models and mentors.

  View details for DOI 10.1097/PRS.0b013e3181a07610

  View details for Web of Science ID 000266067900029

  View details for PubMedID 19407636

• Untitled AESTHETIC SURGERY JOURNAL Gupta, D. M., Panetta, N. J., Longaker, M. T. 2009; 29 (2): 147-149

  View details for DOI 10.1016/j.asj.2009.01.007

  View details for Web of Science ID 000208138500012

• Craniofacial Autologous Fat Transfer JOURNAL OF CRANIOFACIAL SURGERY Wan, D. C., Lim, A. T., Longaker, M. T. 2009; 20 (2): 273-274

  View details for DOI 10.1097/SCS.0b013e31819921d3

  View details for Web of Science ID 000264570300003

  View details for PubMedID 19305242

• Perspectives in Cell-Based Skeletal Tissue JOURNAL OF CRANIOFACIAL SURGERY Panetta, N. J., Gupta, D. M., Longaker, M. T. 2009; 20 (2): 347-348

  View details for Web of Science ID 000264570300016

  View details for PubMedID 19305243

• Cell Permeant Peptide Analogues of the Small Heat Shock Protein, HSP20, Reduce TGF-beta 1-Induced CTGF Expression in Keloid Fibroblasts JOURNAL OF INVESTIGATIVE DERMATOLOGY Lopes, L. B., Furnish, E. J., Komalavilas, P., Flynn, C. R., Ashby, P., Hansen, A., Ly, D. P., Yang, G. P., Longaker, M. T., Panitch, A., Brophy, C. M. 2009; 129 (3): 590-598

  Abstract

  A growing body of evidence suggests the involvement of connective tissue growth factor (CTGF) in the development and maintenance of fibrosis and excessive scarring. As the expression of this protein requires an intact actin cytoskeleton, disruption of the cytoskeleton represents an attractive strategy to decrease CTGF expression and, consequently, excessive scarring. The small heat-shock-related protein (HSP20), when phosphorylated by cyclic nucleotide signaling cascades, displaces phospho-cofilin from the 14-3-3 scaffolding protein leading to activation of cofilin as an actin-depolymerizing protein. In the present study, we evaluated the effect of AZX100, a phosphopeptide analogue of HSP20, on transforming growth factor-beta-1 (TGF-beta1)-induced CTGF and collagen expression in human keloid fibroblasts. We also examined the effect of AZX100 on scar formation in vivo in dermal wounds in a Siberian hamster model. AZX100 decreased the expression of CTGF and type I collagen induced by TGF-beta1, endothelin, and lysophosphatidic acid. Treatment with AZX100 decreased stress fiber formation and altered the morphology of human dermal keloid fibroblasts. In vivo, AZX100 significantly improved collagen organization in a Siberian hamster scarring model. Taken together, these results suggest the potential use of AZX100 as a strategy to prevent excessive scarring and fibrotic disorders.

  View details for DOI 10.1038/jid.2008.264

  View details for Web of Science ID 000263569500011

  View details for PubMedID 18787533

  View details for PubMedCentralID PMC2740368

• Mesenchymal cells for skeletal tissue engineering PANMINERVA MEDICA Panetta, N. J., Gupta, D. M., Quarto, N., Longaker, M. T. 2009; 51 (1): 25-41

  Abstract

  Today, surgical intervention remains the mainstay of treatment to intervene upon a multitude of skeletal deficits and defects attributable to congenital malformations, oncologic resection, pathologic degenerative bone destruction, and post-traumatic loss. Despite this significant demand, the tools with which surgeons remain equipped are plagued with a surfeit of inadequacies, often resulting in less than ideal patient outcomes. The failings of current techniques largely arise secondary to their inability to produce a regenerate which closely resembles lost tissue. As such, focus has shifted to the potential of mesenchymal stem cell (MSC)-based skeletal tissue engineering. The successful development of such techniques would represent a paradigm shift from current approaches, carrying with it the potential to regenerate tissues which mimic the form and function of endogenous bone. Lessons learned from investigations probing the endogenous regenerative capacity of skeletal tissues have provided direction to early studies investigating the osteogenic potential of MSC. Additionally, increasing attention is being turned to the role of targeted molecular manipulations in augmenting MSC osteogenesis, as well as the development of an ideal scaffold ''vehicle'' with which to deliver progenitor cells. The following discussion presents the authors' current working knowledge regarding these critical aspects of MSC application in cell-based skeletal tissue engineering strategies, as well as provides insight towards what future steps must be taken to make their clinical translation a reality.

  View details for Web of Science ID 000266771000004

  View details for PubMedID 19352307

• Aging and Diabetes Impair the Neovascular Potential of Adipose-Derived Stromal Cells PLASTIC AND RECONSTRUCTIVE SURGERY El-Ftesi, S., Chang, E. I., Longaker, M. T., Gurtner, G. C. 2009; 123 (2): 475-485

  Abstract

  Aging and diabetes are major risk factors for poor wound healing and tissue regeneration that reflect an impaired ability to respond to ischemic insults. The authors explored the intrinsic neovascular potential of adipose-derived stromal cells in the setting of advanced age and in type 1 and type 2 diabetes.Adipose-derived stromal cells isolated from young, aged, streptozotocin-induced, and db/db diabetic mice were exposed to normoxia and hypoxia in vitro. Vascular endothelial growth factor (VEGF) expression, proliferation, and tubulization were measured. Conditioned media harvested from adipose-derived stromal cell cultures were assessed for their ability to stimulate human umbilical vein endothelial cell proliferation (n = 3 and n = 3).Young adipose-derived stromal cells demonstrated significantly higher levels of VEGF production, proliferation, and tubulogenesis than those derived from aged, streptozotocin-induced, and db/db mice in both normoxia and hypoxia. Although aged and diabetic adipose-derived stromal cells retained the ability to up-regulate VEGF secretion, proliferation, and tubulogenesis in response to hypoxia, the response was blunted compared with young controls. Conditioned media derived from these cells cultured in normoxia in vitro also had a significantly greater ability to increase human umbilical vein endothelial cell proliferation compared with media harvested from aged, streptozotocin-induced, and db/db adipose-derived stromal cells. This effect was magnified in conditioned media harvested from hypoxic adipose-derived stromal cell cultures.This study demonstrates that aging and type 1 and type 2 diabetes impair intrinsic adipose-derived stromal cell function; however, these cells may still be a suitable source of angiogenic cells that can potentially improve neovascularization of ischemic tissues.

  View details for DOI 10.1097/PRS.0b013e3181954d08

  View details for Web of Science ID 000265669200006

  View details for PubMedID 19182604

  View details for PubMedCentralID PMC2878769

• The Role of Regional Posterior Frontal Dura Mater in the Overlying Suture Morphology 53rd Annual Meeting of the Plastic-Surgery-Research-Council Slater, B. J., Kwan, M. D., Gupta, D. M., Lee, J. K., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2009: 463–69

  Abstract

  Craniosynostosis, the premature fusion of one or more cranial sutures, is a common developmental disorder resulting in morphologic and functional consequences. The rat model is useful for studying pathologic and normal suture fusion because the posterior frontal suture undergoes fusion but the remaining sutures remain patent. The authors investigated the influence of regional posterior frontal dura mater on the overlying suture morphology and fate.In 8-day-old Sprague-Dawley rats, an 8-mm calvarial disk was excised without disrupting the underlying dura mater (n = 22) and flipped so that the previously ectocranial aspect was adjacent to the dura mater. The animals were humanely killed after 5, 7, 9, 11, and 28 days, and the posterior frontal sutures were analyzed histologically. A comparison was made to control animals in which the disk was excised and then placed back into its anatomical position (n = 5). Immunohistochemistry of the transforming growth factor (TGF)-beta isoforms was performed to investigate their differential, temporal, and spatial expression.Posterior frontal suture fusion occurred on the side adjacent to the dura mater (previously patent ectocranial aspect) in an anterior-to-posterior direction, similar to that in the control group. There was specific expression of the TGF-beta isoforms in the dura mater and suture mesenchyme adjacent to the dura mater.Regional dura mater plays an important role in suture morphology, and the posterior frontal-associated dura mater possesses potent, pro-osteogenic signals that influence the overlying suture fate. The differential expression pattern of TGF-beta signaling from the dura mater further supports the regional paracrine effect of the dura mater.

  View details for DOI 10.1097/PRS.0b013e3181954d21

  View details for Web of Science ID 000265669200004

  View details for PubMedID 19182602

• Cranial Osteogenesis and Suture Morphology in Xenopus laevis: A Unique Model System for Studying Craniofacial Development PLOS ONE Slater, B. J., Liu, K. J., Kwan, M. D., Quarto, N., Longaker, M. T. 2009; 4 (1)

  Abstract

  The tremendous diversity in vertebrate skull formation illustrates the range of forms and functions generated by varying genetic programs. Understanding the molecular basis for this variety may provide us with insights into mechanisms underlying human craniofacial anomalies. In this study, we provide evidence that the anuran Xenopus laevis can be developed as a simplified model system for the study of cranial ossification and suture patterning. The head structures of Xenopus undergo dramatic remodelling during metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. Because of the extended larval period in Xenopus, the molecular basis of these alterations has not been well studied.We examined late larval, metamorphosing, and post-metamorphosis froglet stages in intact and sectioned animals. Using micro-computed tomography (microCT) and tissue staining of the frontoparietal bone and surrounding cartilage, we observed that bone formation initiates from lateral ossification centers, proceeding from posterior-to-anterior. Histological analyses revealed midline abutting and posterior overlapping sutures. To determine the mechanisms underlying the large-scale cranial changes, we examined proliferation, apoptosis, and proteinase activity during remodelling of the skull roof. We found that tissue turnover during metamorphosis could be accounted for by abundant matrix metalloproteinase (MMP) activity, at least in part by MMP-1 and -13.A better understanding of the dramatic transformation from cartilaginous head structures to bony skull during Xenopus metamorphosis may provide insights into tissue remodelling and regeneration in other systems. Our studies provide some new molecular insights into this process.

  View details for DOI 10.1371/journal.pone.0003914

  View details for Web of Science ID 000265481900001

  View details for PubMedID 19156194

  View details for PubMedCentralID PMC2615207

• Ex vivo Model of Cranial Suture Morphogenesis and Fate CELLS TISSUES ORGANS Slater, B. J., Lenton, K. A., James, A., Longaker, M. T. 2009; 190 (6): 336-346

  Abstract

  Craniosynostosis, the premature fusion of cranial sutures, is a common congenital defect. In vivo models for studying cranial suture biology impose inherent restrictions on tissue accessibility and manipulation. The present study was performed to investigate the utility of the renal capsule assay in overcoming these limitations and providing a reproducible model system for studying cranial suture morphogenesis and fate.The posterior frontal suture, which fuses physiologically, and the coronal and sagittal sutures, which remain patent, were dissected from postnatal and embryonic mouse calvaria and placed under the renal capsule of syngeneic recipient mice (n = 72 in total). Sutures were harvested from 1-14 days after transplantation for histological and morphometric analysis. Suture transplants were compared with nonmanipulated sutures at equivalent developmental stages. The derivation of cells associated with the growing transplants was analyzed using beta-actin-GFP (green fluorescent protein) transgenic mice.Sutures transplanted under the renal capsule maintained normal suture morphology and fate with the posterior frontal suture fusing and the coronal and sagittal sutures remaining patent. In posterior frontal suture transplants, the fusion process mimicked in vivo suture fusion with a delay of 1-2 days. In comparison to in vivo suture complexes, transplant thickness and trabeculation were significantly increased. In addition, we found that osteoblasts within the growing transplant were derived from the transplant itself rather than the host.The renal capsule supports the growth of cranial sutures. In this system transplanted sutures recapitulate the anatomical development and fate (fusion or patency) of cranial sutures in vivo. This model system will facilitate controlled ex vivo manipulations of both embryonic and postnatal sutures.

  View details for DOI 10.1159/000228157

  View details for Web of Science ID 000271819700004

  View details for PubMedID 19590164

• Chemical rescue of cleft palate and midline defects in conditional gsk-3beta mice 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Liu, K. J., Arron, J. R., Stankunas, K., Crabtree, G. R., Longaker, M. T. MEDIMOND S R L. 2009: 7–9

  View details for Web of Science ID 000303383400003

• Genomic analysis of osteogenic differentiation of adipose-derived stromal cells 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Lee, J. K., Gupta, D. M., Panetta, N. J., Longaker, M. T. MEDIMOND S R L. 2009: 91–93

  View details for Web of Science ID 000303383400019

• Rapid and efficient feeder-free generation of human adipose stromal cell-derived induced pluripotent stem cells 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Panetta, N. J., Gupta, D. M., Sun, N., Wilson, K. D., Lee, A., Jia, F., Robbins, R. C., Longaker, M. T., Wu, J. C. MEDIMOND S R L. 2009: 159–161

  View details for Web of Science ID 000303383400030

• Human adipose-derived stromal cells elaborate and respond to bone morphogenetic protein-2 (bmp-2) during in vitro osteogenic differentiation 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Gupta, D. M., Panetta, N. J., Wan, D. C., Commons, G. W., Longaker, M. T. MEDIMOND S R L. 2009: 79–81

  View details for Web of Science ID 000303383400016

• Tissue-harvest procedure has no effect on adipose-derived stromal cell-mediated bone tissue engineering in vitro 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Panetta, N. J., Gupta, D. M., Kwan, M. D., Wan, D. C., Commons, G. W., Longaker, M. T. MEDIMOND S R L. 2009: 163–165

  View details for Web of Science ID 000303383400031

• Tissue Turnover in the Xenopus laevis Skull During Metamorphosis 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Slater, B., Liu, K., Kwan, M., Quarto, N., Longaker, M. MEDIMOND S R L. 2009: 5–6

  View details for Web of Science ID 000303383400002

• Taz is a transcriptional modulator of human adipose-derived stromal cell differentiation towards the osteogenic lineage 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Gupta, D. M., Panetta, N. J., Vial, I. N., Lee, J. K., Wan, D. C., Gurtner, G. C., Longaker, M. T. MEDIMOND S R L. 2009: 87–89

  View details for Web of Science ID 000303383400018

• Human adipose-derived stromal cells (hASCs) augment healing of critical-sized calvarial defects 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Panetta, N. J., Gupta, D. M., Lee, J. K., Gupta, A., Levi, B., Commons, G. W., Longaker, M. T. MEDIMOND S R L. 2009: 175–177

  View details for Web of Science ID 000303383400034

• Regional differences in neural crest and paraxial mesoderm-derived in vivo calvarial bone healing exist and persist with age 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Panetta, N. J., Gupta, D. M., Quarto, N., Behr, B., Li, S., Kwan, M. D., Longaker, M. T. MEDIMOND S R L. 2009: 167–169

  View details for Web of Science ID 000303383400032

• Connective tissue growth factor (CTGF/CCN2) and Connexin-43 (Cx43) expression are upregulated in human adipose-derived stromal cells (hASCs) over the course of osteogenic differentiation 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Panetta, N. J., Gupta, D. M., Gupta, A., Lee, J. K., Commons, G. W., Longaker, M. T. MEDIMOND S R L. 2009: 171–173

  View details for Web of Science ID 000303383400033

• Differential FGF Ligands and FGF Receptors Expression Pattern in Frontal and Parietal Calvarial Bones CELLS TISSUES ORGANS Quarto, N., Behr, B., Li, S., Longaker, M. T. 2009; 190 (3): 158-169

  Abstract

  The mammalian skull vault consists mainly of 5 flat bones, the paired frontals and parietals, and the unpaired interparietal. All of these bones are formed by intramembranous ossification within a layer of mesenchyme, the skeletogenic membrane, located between the dermal mesenchyme and the meninges surrounding the brain. While the frontal bones are of neural crest in origin, the parietal bones arise from mesoderm. The present study is a characterization of frontal and parietal bones at their molecular level, aiming to highlight distinct differences between the neural crest-derived frontal and mesodermal-derived parietal bone. We performed a detailed comparative gene expression profile of FGF ligands and their receptors known to play crucial role in skeletogenesis. This analysis revealed that a differential expression pattern of the major FGF osteogenic molecules and their receptors exists between the neural crest-derived frontal bone and the paraxial mesoderm-derived parietal bone. Particularly, the expression of ligands such as Fgf-2, Fgf-9 and Fgf-18 was upregulated in frontal bone on embryonic day 17.5, postnatal day 1 and postnatal day 60 mice. Frontal bone also elaborated higher levels of Fgf receptor 1, 2 and 3 transcripts versus parietal bone. Taken together, these data suggest that the frontal bone is a domain with higher FGF-signaling competence than parietal bone.

  View details for DOI 10.1159/000202789

  View details for Web of Science ID 000268865700004

  View details for PubMedID 19218784

  View details for PubMedCentralID PMC2820336

• Cadherin-11 expression is upregulated in human adipose-derived stromal cells (hasc) over the course of osteogenic differentiation 13th International Congress of the International-Society-of-Craniofacial-Surgery / Paris Distraction Symposium Gupta, D. M., Panetta, N. J., Gupta, A., Lee, J. K., Commons, G. W., Longaker, M. T. MEDIMOND S R L. 2009: 95–97

  View details for Web of Science ID 000303383400020

• Topical Lineage-Negative Progenitor-Cell Therapy for Diabetic Wounds (Invited Discussion) PLASTIC AND RECONSTRUCTIVE SURGERY Gurtner, G. C., Longaker, M. T. 2009; 123 (1): 421-423

  View details for DOI 10.1097/PRS.0b013e318194d2b8

  View details for Web of Science ID 000262317700054

  View details for PubMedID 19116584

• Re: Chen et al. Expression of Inducible Nitric Oxide Synthase and Vascular Endothelial Growth Factor in Ameloblastoma. J Craniofac Surg 2009;20;171-175 DISCUSSION JOURNAL OF CRANIOFACIAL SURGERY Slater, B. J., Kwan, M. D., Longaker, M. T. 2009; 20 (1): 176-177

  View details for Web of Science ID 000262838400046

• FETAL SKIN WOUND HEALING ADVANCES IN CLINICAL CHEMISTRY VOL 48 Buchanan, E. P., Longaker, M. T., Lorenz, H. P. 2009; 48: 137-161

  Abstract

  The developing fetus has the ability to heal wounds by regenerating normal epidermis and dermis with restoration of the extracellular matrix (ECM) architecture, strength, and function. In contrast, adult wounds heal with fibrosis and scar. Scar tissue remains weaker than normal skin with an altered ECM composition. Despite extensive investigation, the mechanism of fetal wound healing remains largely unknown. We do know that early in gestation, fetal skin is developing at a rapid pace and the ECM is a loose network facilitating cellular migration. Wounding in this unique environment triggers a complex cascade of tightly controlled events culminating in a scarless wound phenotype of fine reticular collagen and abundant hyaluronic acid. Comparison between postnatal and fetal wound healing has revealed differences in inflammatory response, cellular mediators, cytokines, growth factors, and ECM modulators. Investigation into cell signaling pathways and transcription factors has demonstrated differences in secondary messenger phosphorylation patterns and homeobox gene expression. Further research may reveal novel genes essential to scarless repair that can be manipulated in the adult wound and thus ameliorate scar.

  View details for DOI 10.1016/S0065-2423(09)48006-5

  View details for Web of Science ID 000270339800006

  View details for PubMedID 19803418

• 5-Fluorouracil Treatment of Problematic Scars Discussion PLASTIC AND RECONSTRUCTIVE SURGERY Yang, G. P., Longaker, M. T. 2009; 123 (1): 149-151

  View details for DOI 10.1097/PRS.0b013e3181904e83

  View details for Web of Science ID 000262317700020

• IFATS Collection: Adipose Stromal Cells Adopt a Proangiogenic Phenotype Under the Influence of Hypoxia STEM CELLS Thangarajah, H., Vial, I. N., Chang, E., El-Ftesi, S., Januszyk, M., Chang, E. I., Paterno, J., Neofytou, E., Longaker, M. T., Gurtner, G. C. 2009; 27 (1): 266-274

  Abstract

  Evolving evidence suggests a possible role for adipose stromal cells (ASCs) in adult neovascularization, although the specific cues that stimulate their angiogenic behavior are poorly understood. We evaluated the effect of hypoxia, a central mediator of new blood vessel development within ischemic tissue, on proneovascular ASC functions. Murine ASCs were exposed to normoxia (21% oxygen) or hypoxia (5%, 1% oxygen) for varying lengths of time. Vascular endothelial growth factor (VEGF) secretion by ASCs increased as an inverse function of oxygen tension, with progressively higher VEGF expression at 21%, 5%, and 1% oxygen, respectively. Greater VEGF levels were also associated with longer periods in culture. ASCs were able to migrate towards stromal cell-derived factor (SDF)-1, a chemokine expressed by ischemic tissue, with hypoxia augmenting ASC expression of the SDF-1 receptor (CXCR4) and potentiating ASC migration. In vivo, ASCs demonstrated the capacity to proliferate in response to a hypoxic insult remote from their resident niche, and this was supported by in vitro studies showing increasing ASC proliferation with greater degrees of hypoxia. Hypoxia did not significantly alter the expression of endothelial surface markers by ASCs. However, these cells did assume an endothelial phenotype as evidenced by their ability to tubularize when seeded with differentiated endothelial cells on Matrigel. Taken together, these data suggest that ASCs upregulate their proneovascular activity in response to hypoxia, and may harbor the capacity to home to ischemic tissue and function cooperatively with existing vasculature to promote angiogenesis.

  View details for DOI 10.1634/stemcells.2008-0276

  View details for Web of Science ID 000263032400030

  View details for PubMedID 18974212

• Differential Effects of TGF-beta 1 and TGF-beta 3 on Chondrogenesis in Posterofrontal Cranial Suture-Derived Mesenchymal Cells In Vitro PLASTIC AND RECONSTRUCTIVE SURGERY James, A. W., Xu, Y., Lee, J. K., Wang, R., Longaker, M. T. 2009; 123 (1): 31-43

  Abstract

  Transforming growth factor (TGF)-beta1 has been associated with cranial suture fusion, whereas TGF-beta3 has been associated with suture patency. The mouse posterofrontal suture, analogous to the human metopic suture, fuses through endochondral ossification.TGF-beta1 and TGF-beta3 expression in the posterofrontal suture was examined by immunohistochemistry. Next, the authors established cultures of suture-derived mesenchymal cells from the posterofrontal suture and examined the cellular responses to TGF-beta1 and TGF-beta3. Proliferation in response to TGF-beta isoforms was examined by bromodeoxyuridine incorporation. High-density micromass culture of posterofrontal mesenchymal cells was used to study the effect of TGF-beta1 and TGF-beta3 on chondrogenic differentiation.TGF-beta1 but not TGF-beta3 protein was highly expressed in chondrocytes within the posterofrontal suture. Significant increases in posterofrontal cell proliferation were observed with TGF-beta3 but not TGF-beta1. TGF-beta1 led to significant increases in chondrogenic-specific gene expression (including Sox9, Col II, Aggrecan, and Col X) as compared with moderate effects of TGF-beta3. TGF-beta1 increased cellular adhesion molecule expression (N-cadherin and fibronectin) and promoted cellular condensation, whereas TGF-beta3 increased cellular proliferation (PCNA expression). Finally, TGF-beta1 and, to a lesser extent, TGF-beta3 induced the expression of fibroblast growth factors (FGF-2 and FGF-18).TGF-beta1 and TGF-beta3 exhibit marked differences in their effects on chondrogenesis in posterfrontal suture-derived mesenchymal cells, influencing different stages of chondrogenic differentiation. TGF-beta3 significantly increased cellular proliferation, whereas TGF-beta1 induced precartilage condensation, promoting chondrocyte differentiation.

  View details for DOI 10.1097/PRS.0b013e3181904c19

  View details for Web of Science ID 000262317700006

  View details for PubMedID 19116522

  View details for PubMedCentralID PMC2748922

• Where Do We Find the Best Evidence? Discussion PLASTIC AND RECONSTRUCTIVE SURGERY Butler, P. D., Longaker, M. T. 2008; 122 (6): 1950-1951

  View details for DOI 10.1097/PRS.0b013e31818d211e

  View details for Web of Science ID 000263721500042

• Gene expression analysis of Dupuytren's disease: the role of TGF-beta2. The Journal of hand surgery, European volume Zhang, A. Y., Fong, K. D., Pham, H., Nacamuli, R. P., Longaker, M. T., Chang, J. 2008; 33 (6): 783-790

  Abstract

  Dupuytren's disease is characterised by nodular fibroblastic proliferation of the palmar fascia leading to contracture of the hand. Transforming growth factor beta (TGF-beta) is thought to play a role in its pathogenesis. We performed a cDNA microarray analysis of Dupuytren's diseased cord tissue with an emphasis on TGF-beta isoforms. Normal-appearing transverse ligament of the palmar fascia from adjacent to the diseased cord and palmar fascia from patients undergoing carpal tunnel release were used as controls. TGF-beta gene expression was confirmed by quantitative real-time polymerase chain reaction. Over 20 unique genes were found to be significantly up-regulated, including several previously reported genes. A dominant increase in TGF-beta2 expression was seen in the cord tissue, whereas TGF-beta1 and TGF-beta3 were found not to be significantly up-regulated. Quantitative real-time polymerase chain reaction confirmed these findings. This gene expression profile allows for further experiments that may eventually lead to gene therapy to block the development and progression of Dupuytren's disease clinically.

  View details for DOI 10.1177/1753193408091352

  View details for PubMedID 18694919

• Transforming Growth Factor-beta 1 Stimulates Chondrogenic Differentiation of Posterofrontal Suture-Derived Mesenchymal Cells In Vitro PLASTIC AND RECONSTRUCTIVE SURGERY Xu, Y., James, A. W., Longaker, M. T. 2008; 122 (6): 1649-1659

  Abstract

  Evidence from animal studies has associated transforming growth factor (TGF)-beta signaling with both normal and premature cranial suture fusion. However, the mechanisms whereby this pleiotropic cytokine mediates suture fusion remain uncertain. The authors established cultures of suture-derived mesenchymal cells from normally fusing (posterofrontal) and patent (sagittal) sutures and examined the in vitro effects of TGF-beta1 on these distinct cell populations.Skulls were harvested from 80 5-day-old mice. Posterofrontal and sagittal sutures were dissected, and cultures of suture-derived mesenchymal cells were established. The mitogenic, osteogenic, and chondrogenic effects of recombinant TGF-beta1 were then assessed on posterofrontal and sagittal suture-derived mesenchymal cells (1 to 10 ng/ml). Quantitative real-time polymerase chain reaction was used to examine the effects of TGF-beta1 on gene expression.TGF-beta1 significantly decreased proliferation of both posterofrontal and sagittal suture-derived mesenchymal cells, by bromodeoxyuridine incorporation assays (n = 6). TGF-beta1 also inhibited osteogenesis in both suture-derived mesenchymal cells determined by alkaline phosphatase activity and mineralization (n = 3 for all assays). During chondrogenic differentiation, TGF-beta1 markedly increased expression of chondrocyte-specific gene markers in posterofrontal suture-derived mesenchymal cells (Sox9, Col II, Aggrecan, and Col X) (p

  View details for DOI 10.1097/PRS.0b013e31818cbf44

  View details for Web of Science ID 000263721500006

  View details for PubMedID 19050517

• GENE EXPRESSION ANALYSIS OF DUPUYTREN'S DISEASE: THE ROLE OF TGF-beta 2 JOURNAL OF HAND SURGERY-EUROPEAN VOLUME Zhang, A. Y., Fong, K. D., Pham, H., Nacamuli, R. P., Longaker, M. T., Chang, J. 2008; 33E (6): 783-790

  View details for DOI 10.1177/1753193408091352

  View details for Web of Science ID 000262331400015

• Differential expression of specific FGF ligands and receptor isoforms during osteogenic differentiation of mouse Adipose-derived Stem Cells (mASCs) recapitulates the in vivo osteogenic pattern GENE Quarto, N., Longaker, M. T. 2008; 424 (1-2): 130-140

  Abstract

  The ability of Adipose-derived Stem Cells (ASCs) to differentiate into various tissues in vitro and in vivo, a function known as "stem cell plasticity", makes them an appealing cell source for tissue engineering. Our laboratory is particularly focused on the potential role of adipose tissue as a readily available postnatal source of osteoprogenitor. Fibroblast growth factors (FGF) and their receptors (FGFR) are important regulators of osteogenesis. The goal of this study was to elucidate how changes in temporal expression patterns of individual components of the fibroblast growth factor (FGF) signaling axis correlate with osteogenic differentiation of mASCs. Our results indicate that FGF ligand genes, such as Fgf-2, -4, -8, and -18, displayed a differential and dynamic profile during mouse ASC (mASC) osteogenesis. Fgf-2 transcript was down-regulated, while Fgf-18 transcript level was strongly up-regulated. Interestingly, a drift in the ratio of different FGF-2 protein forms, with translation favoring the HMWFGF-2 forms, occurred during osteogenic differentiation, whereas, the expression of LMWFGF-2 form was down-regulated. This finding shares similarity with a previous study suggesting that preferential expression of the HMWFGF-2 forms is associated with a more osteogenic differentiated state of calvarial osteoblast. Moreover, a differential expression of Fgf Receptor 1 and 2 resembling that previously found in in vivo osteogenic study was observed. Thus, mASCs undergoing osteogenesis recapitulate the in vivo osteogenic differentiation expression pattern of FGF ligands and receptors of calvarial mesenchymal cells during their own osteogenic differentiation. Indeed, this observation further validates ASCs as a suitable resource for skeletal tissue engineering.

  View details for DOI 10.1016/j.gene.2008.07.029

  View details for Web of Science ID 000260563800018

  View details for PubMedID 18718860

• Plastic Surgery and the Society of University Surgeons: An Expanding Relationship PLASTIC AND RECONSTRUCTIVE SURGERY Longaker, M. T. 2008; 122 (5): 1581-1582

  View details for DOI 10.1097/PRS.0b013e31818824b0

  View details for Web of Science ID 000261138700032

  View details for PubMedID 18971743

• Major Deficit in the Number of Underrepresented Minority Academic Surgeons Persists ANNALS OF SURGERY Butler, P. D., Longaker, M. T., Britt, L. D. 2008; 248 (5): 704-711

  Abstract

  Eliminating health care disparities in the United States will require a multifaceted approach that will include increasing diversity in the health care workforce. Historically, the field of medicine, and particularly surgery, has had an incumbent that grossly misrepresents the patient population. Delineating the exact demographics of the U.S. surgical residents and faculty could provide outstanding information, yielding insight into a possible deficit that, if rectified by the medical education system, could change the face of surgery and the entire health care system.Demographic information regarding medical students, surgical residents, and surgical faculty was retrieved and analyzed from the Association of American Medical Colleges data files dating back to 1966.Whites comprise 64.4% of U.S. surgical residents, whereas Asian Americans, African Americans, and Latino Americans comprise 17.2%, 4.7%, and 5.1%, respectively. Whites comprise 74.1%, of academic surgeons, whereas Asian Americans, African Americans, and Latino Americans comprise 10.8%, 2.9%, and 3.6%, respectively. African Americans and Latino Americans comprise 5.4% and 4.8% of all U.S. surgeons, but only 2.9% and 3.6% of the academic surgeons, respectively. Whites comprise 85.7% of tenured surgical professors, whereas Asian Americans, African Americans, and Latino Americans comprise 4.9%, 1.8%, and 2.7%, respectively.Academic surgery is exceedingly deficient of minority residents, junior faculty, and professors. Correcting this misrepresentation would facilitate establishing a more culturally and ethnically sensitive health care environment for patients who otherwise would not seek care. Additionally, with more minority academic surgeons, there will likely be a commensurate increase in investigative studies highlighting minority specific health care needs and provide additional role models and mentors for future minority surgeons.

  View details for DOI 10.1097/SLA.0b013e31817f2c30

  View details for Web of Science ID 000260726200003

  View details for PubMedID 18948795

• Optimization of flexor tendon tissue engineering with a cyclic strain bioreactor. journal of hand surgery Riboh, J., Chong, A. K., Pham, H., Longaker, M., Jacobs, C., Chang, J. 2008; 33 (8): 1388-1396

  Abstract

  Mechanical manipulation of cultured tendon cells can enhance cell proliferation and matrix production. This study aims to determine the bioreactor strain patterns (amplitude, frequency, and on/off ratio) that favor cellular proliferation, promote collagen production, and maintain morphology in candidate cell lines cultured for flexor tendon tissue engineering, including multipotent stromal cells.We studied epitenon tenocytes (Es), sheath fibroblasts (Ss), bone marrow-derived mesenchymal stem cells (BMSCs), and adipoderived stem cells (ASCs). We examined the effects of 3 patterns of cyclic uniaxial strain on cell proliferation, collagen I production, and cell morphology.Adipoderived stem cells (33% adhesion) and Ss (29%) adhered more strongly to bioreactor membranes than did Es (15%) and BMSCs (7%), p=.04. Continuous cyclic strain (CCS, 8%, 1 Hz) inhibited cell proliferation (p=.01) and increased per-cell collagen production (p=.04) in all cell types. Intermittent cyclic strain (4%, 0.1 Hz, 1 hour on/5 hours off) increased proliferation in ASCs (p=.06) and Ss (p=.04). Intermittent cyclic strain (4%, 0.1 Hz, 1 hour on/2 hours off) increased total collagen production by 25% in ASCs (p=.004) and 20% in Ss (p=.05). Cyclic strain resulted in cell alignment perpendicular to the strain axis, cytoskeletal alignment, and nuclear elongation. These morphological characteristics are similar to those of tenocytes.These results demonstrate that intermittent cyclic strain can increase cell proliferation, promote collagen I production, and maintain tenocyte morphology in vitro. Use of a cell bioreactor might accelerate the in vitro stage of tendon tissue engineering.

  View details for DOI 10.1016/j.jhsa.2008.04.019

  View details for PubMedID 18929207

• Optimization of Flexor Tendon Tissue Engineering With a Cyclic Strain Bioreactor JOURNAL OF HAND SURGERY-AMERICAN VOLUME Riboh, J., Chong, A. K., Pham, H., Longaker, M., Jacobs, C., Chang, J. 2008; 33A (8): 1388-1396

  Abstract

  Mechanical manipulation of cultured tendon cells can enhance cell proliferation and matrix production. This study aims to determine the bioreactor strain patterns (amplitude, frequency, and on/off ratio) that favor cellular proliferation, promote collagen production, and maintain morphology in candidate cell lines cultured for flexor tendon tissue engineering, including multipotent stromal cells.We studied epitenon tenocytes (Es), sheath fibroblasts (Ss), bone marrow-derived mesenchymal stem cells (BMSCs), and adipoderived stem cells (ASCs). We examined the effects of 3 patterns of cyclic uniaxial strain on cell proliferation, collagen I production, and cell morphology.Adipoderived stem cells (33% adhesion) and Ss (29%) adhered more strongly to bioreactor membranes than did Es (15%) and BMSCs (7%), p=.04. Continuous cyclic strain (CCS, 8%, 1 Hz) inhibited cell proliferation (p=.01) and increased per-cell collagen production (p=.04) in all cell types. Intermittent cyclic strain (4%, 0.1 Hz, 1 hour on/5 hours off) increased proliferation in ASCs (p=.06) and Ss (p=.04). Intermittent cyclic strain (4%, 0.1 Hz, 1 hour on/2 hours off) increased total collagen production by 25% in ASCs (p=.004) and 20% in Ss (p=.05). Cyclic strain resulted in cell alignment perpendicular to the strain axis, cytoskeletal alignment, and nuclear elongation. These morphological characteristics are similar to those of tenocytes.These results demonstrate that intermittent cyclic strain can increase cell proliferation, promote collagen I production, and maintain tenocyte morphology in vitro. Use of a cell bioreactor might accelerate the in vitro stage of tendon tissue engineering.

  View details for DOI 10.1016/j.jhsa.2008.04.019

  View details for Web of Science ID 000260049100021

• Global age-dependent differences in gene expression in response to calvarial injuryd JOURNAL OF CRANIOFACIAL SURGERY Wan, D. C., Kwan, M. D., Gupta, D. M., Wang, Z., Slater, B. J., Panetta, N. J., Morrell, N. T., Longaker, M. T. 2008; 19 (5): 1292-1301

  Abstract

  Children less than 2 years of age are capable of healing large calvarial defects, whereas adults have been found to lack this endogenous ability. In this study, we used microarray analysis to compare genomewide expression patterns during active regeneration after injury with calvaria in skeletally immature and mature mice. Parietal bone defects were created in 6-day-old (juvenile) and 60-day-old (adult) mice using a 4-mm trephine bit (n = 20 mice per age group). The calvarial disc was removed, leaving the underlying dura mater intact. Two weeks after injury, the region of regeneration with the underlying dura mater was harvested, and RNA was extracted for microarray analysis. The 25 most differentially upregulated genes in juvenile regenerates compared with adults were listed, as well as selected bone-related genes. In addition, QRT-PCR confirmation of specific genes was performed for validation. Juvenile regenerates expressed significantly greater amounts of BMP-2, -4, -7, as well as FGF-2 and its receptor FGFR-1. Various other growth factors were also noted to be upregulated, including IGF-2 and Ptn. This corresponded with the increased expression of markers for osteogenic differentiation of Sparc and Oc. Markers of osteoclast activity, Acp5, Ctsk, and Mmp2, were noted to be greater in juvenile regenerates compared with adults. The observation of Mmp14 upregulation, however, highlights the importance of balanced osteoclast-mediated bone resorption for ultimate healing. The 2 most differentially regulated genes, transthyretin (Ttr) and prostaglandin D2 synthase (Ptgds), highlight the potential role of retinoic acid signaling and the prostaglandin axis on skeletal regeneration. These findings underscore the multitude of biomolecular mechanisms at play, allowing juvenile calvaria to heal after injury. The identification of various growth factors and cytokines involved also suggests novel therapeutic strategies for tissue-engineering purposes.

  View details for Web of Science ID 000259503400015

  View details for PubMedID 18812854

• Tissue turnover in the Xenopus laevis skull during metamorphosis Slater, B. J., Liu, K. J., Kwan, M. D., Quarto, N., Longaker, M. T. ELSEVIER SCIENCE INC. 2008: S65

  View details for DOI 10.1016/j.jamcollsurg.2008.06.160

  View details for Web of Science ID 000259288500135

• Tunable control of FGF-2 secretion for skeletal tissue engineering Kwan, M. D., Sellmyer, M. A., Quarto, N., Wandless, T. J., Longaker, M. T. ELSEVIER SCIENCE INC. 2008: S63–S64

  View details for DOI 10.1016/j.jamcollsurg.2008.06.157

  View details for Web of Science ID 000259288500132

• Zebrafish maintain regenerative capacity with age 94th Annual Clinical Congress of the American-College-of-Surgeons/63rd Annual Sessions of the Owen H Wangensteen Forum on Fundamental Surgical Problems Panetta, N. J., Ouyang, S., Quarto, N., Chen, J. K., Longaker, M. T. ELSEVIER SCIENCE INC. 2008: S65–S65

  View details for Web of Science ID 000259288500136

• Presidential address: leadership, teamwork, and SUS brand extension SURGERY Longaker, M. T. 2008; 144 (2): 109-118

  View details for DOI 10.1016/j.surg.2008.03.002

  View details for Web of Science ID 000258308400001

  View details for PubMedID 18656615

• Hepatic injury and the kinetics of bone marrow-derived hepatocyte transgene expression JOURNAL OF PEDIATRIC SURGERY Perryman, S. V., Jenkins, D. D., Streetz, K. L., Longaker, M. T., Sylvester, K. G. 2008; 43 (8): 1511-1519

  Abstract

  Numerous congenital and acquired liver diseases could benefit from a successful hepatic cell therapy strategy. Hepatotypic cells derived from bone marrow have been recognized during liver injury, repair, and regeneration. To study this phenomenon, we compared the effect of several modes of experimental hepatic injury on hepatotypic protein expression in a mouse model after bone marrow transplantation.Male mice transgenic for the liver-specific protein human alpha-1 antitrypsin (hAAT) were used as bone marrow donors. Syngeneic wild-type recipient mice were subjected to 1 of 3 hepatic injuries: (1) sublethal irradiation, (2) injection of a hepatotoxic adenoviral construct, and (3) administration of a hepatotoxic diet. Bone marrow-derived hepatotypic (BMdH) transgene expression was determined by serial serum enzyme-linked immunosorbent assay for hAAT.In both acute injury models, hAAT expression was detected as early as 1 week, whereas the control group never elicited hAAT expression. The adenovirus-treated group demonstrated transient hAAT level expression lasting up to 2 weeks postinjury, whereas the irradiated group maintained persistent hAAT expression through 4 months. In the chronic injury (hepatotoxin) model, hAAT expression persisted and was noted to increase over time to 200 to 300 ng/mL.Irradiation favors long-term establishment of BMdH transgene expression, and chronic injury further promotes this phenomenon.

  View details for DOI 10.1016/j.jpedsurg.2007.12.047

  View details for Web of Science ID 000258592500016

  View details for PubMedID 18675644

• Fluid shear stress magnitude, duration, and total applied load regulate gene expression and nitric oxide production in primary calvarial osteoblast cultures PLASTIC AND RECONSTRUCTIVE SURGERY Gonzalez, O., Fong, K. D., Trindade, M. C., Warren, S. M., Longaker, M. T., Smith, R. L. 2008; 122 (2): 419-428

  Abstract

  Successful bone engineering requires an understanding of the effects of mechanical stress on osteoblast differentiation. Therefore, we examined the effects of varying magnitude and duration of fluid shear stress on factors associated with osteoblastic differentiation.Using a cone viscometer, primary neonatal rat calvarial osteoblasts were exposed to continuous fluid shear stress at varying doses: 0.21, 0.43, and 0.85 Pa for varying time periods. Gene expression was analyzed using Northern blots and nitric oxide production was quantified with the colorimetric Griess reaction.Fluid shear stress stimulated comparable transient increases in TGF-beta1 and TGF-beta3 expression by 3 hours. TGF-beta1 expression returned to baseline by 12 hours at all shear doses. In contrast, TGF-beta3 expression decreased by 22 percent and 47 percent at 12 hours in response to 0.43 Pa and 0.85 Pa, respectively. Osteopontin and Msx-2 expression patterns were consistent with a more differentiated phenotype at all shear levels. The maximum level of shear stress increased nitric oxide production 2.5-fold at 12 hours and 6.0-fold at 24 hours.These data demonstrate differential regulation of TGF-beta1 and TGF-beta3 isoforms with fluid shear stress. Furthermore, because osteopontin and Msx-2 changes were consistent with progressive differentiation at all levels of shear stress, dosage appears to be less important than the presence of an effective physical stimulus. Lastly, nitric oxide does not appear to be the primary regulator of early transcriptional changes found in this study.

  View details for DOI 10.1097/PRS.0b013e31817d5ff1

  View details for Web of Science ID 000258136900011

  View details for PubMedID 18626357

• Microarray analysis of the role of regional dura mater in cranial suture fate PLASTIC AND RECONSTRUCTIVE SURGERY Kwan, M. D., Wan, D. C., Wang, Z., Gupta, D. M., Slater, B. J., Longaker, M. T. 2008; 122 (2): 389-399

  Abstract

  Craniosynostosis, the premature fusion of cranial sutures, results in serious neurologic and morphologic abnormalities when left untreated. Surgical excision of the fused sutures and remodeling of the skull remains the standard therapy. Development of novel, minimally invasive therapies for craniosynostosis will undoubtedly be dependent on a more thorough understanding of the molecular mechanisms underlying this abnormality. Significant evidence suggests the influence of regional dura mater on the behavior of the overlying suture complex. The mouse model has been instrumental in investigating this observation because of the natural juxtaposition of the posterior frontal suture, which fuses early in life, with the other cranial sutures, which remain patent.The authors used microarray analysis to compare genomic changes in the dura mater underlying the posterior frontal and sagittal sutures of mice. Suture-associated dura mater was harvested from mice before (postnatal day 5), during (postnatal day 10), and after (postnatal day 20) posterior frontal suture fusion (n = 20 mice for each of the three time points).Microarray results confirmed differential regulation of genes involved in paracrine signaling, extracellular matrix, and bone remodeling between the dura mater underlying the fusing posterior frontal suture and the patent sagittal suture.These data confirm global differences in gene expression between regional dura mater underlying fusing and patent sutures. These results provide further insight into potential molecular mechanisms that may play a role in cranial suture biology.

  View details for DOI 10.1097/PRS.0b013e31817d6244

  View details for Web of Science ID 000258136900008

  View details for PubMedID 18626354

• Regenerative medicine: The next frontier TRANSPLANTATION Kwan, M. D., Longaker, M. T. 2008; 86 (2): 206-207

  View details for DOI 10.1097/TP.0b013e31817f17b2

  View details for Web of Science ID 000257887500006

  View details for PubMedID 18645480

• Proliferation, osteogenic differentiation, and FGF-2 modulation of posterofrontal/sagittal suture-derived mesenchymal cells in vitro PLASTIC AND RECONSTRUCTIVE SURGERY James, A. W., Xu, Y., Wang, R., Longaker, M. T. 2008; 122 (1): 53-63

  Abstract

  Fibroblast growth factor (FGF) signaling is of central importance in premature cranial suture fusion. In the murine skull, the posterofrontal suture normally fuses in early postnatal life, whereas the adjacent sagittal suture remains patent. The authors used a recently developed isolation technique for in vitro culture of suture-derived mesenchymal cells to examine the effects of FGF-2 on proliferation and differentiation of posterofrontal and sagittal suture-derived mesenchymal cells.Skulls were harvested from 40 mice (5-day-old). Posterofrontal and sagittal sutures were dissected, separating sutural mesenchymal tissue from dura mater and pericranium, and cultured. After cell migration from the explant and subculture, differences in proliferation and osteogenic differentiation of these distinct populations were studied. The mitogenic and osteogenic effects of recombinant FGF-2 were then assessed. FGF-2 regulation of gene expression was evaluated.Suture-derived mesenchymal cells isolated from the posterofrontal suture demonstrated significantly higher proliferation rates and a robust mitogenic response to FGF-2 as compared with suture-derived mesenchymal cells isolated from the sagittal suture. Interestingly, posterofrontal suture-derived mesenchymal cells retained a higher in vitro osteogenic potential, as shown by alkaline phosphatase activity and bone nodule formation. FGF-2 significantly diminished osteogenesis in both suture-derived mesenchymal cell populations. Subsequently, Ob-cadherin and Sox9 were found to be differentially expressed in posterofrontal versus sagittal suture-derived mesenchymal cells and dynamically regulated by FGF-2.In vitro osteogenesis of suture-derived mesenchymal cells recapitulates in vivo posterofrontal and sagittal sutural fates. Posterofrontal rather than sagittal suture-derived mesenchymal cells are more responsive to FGF-2 in vitro, in terms of both mitogenesis and osteogenesis.

  View details for DOI 10.1097/PRS.0b013e31817747b5

  View details for Web of Science ID 000257104300006

  View details for PubMedID 18594386

• Mesenchymal cells for skeletal tissue engineering EXPERT OPINION ON BIOLOGICAL THERAPY Slater, B. J., Kwan, M. D., Gupta, D. M., Panetta, N. J., Longaker, M. 2008; 8 (7): 885-893

  Abstract

  Skeletal defects represent a significant socioeconomic burden to the US healthcare system. Current options for reconstructing osseous deficits have shortcomings.To review the use of mesenchymal stem cells for skeletal tissue engineering.We focused on the application of mesenchymal cells in skeletal regeneration, optimization of this technique, tropic effects of multipotent mesenchymal cells, and future directions.A number of cell-based modalities have been investigated. We have been interested in the role of adipose-derived stromal cells in bone regeneration and understanding the mechanisms behind osteogenic differentiation of progenitor cells and acceleration of this process. Future clinical applications of multipotent mesenchymal cells will depend on better understanding of the molecular signaling involved in osteogenic differentiation and maintaining pluripotency.

  View details for DOI 10.1517/14712590802120981

  View details for Web of Science ID 000257408000004

  View details for PubMedID 18549320

• Dissecting the influence of regional dura mater on cranial suture biology PLASTIC AND RECONSTRUCTIVE SURGERY Slater, B. J., Kwan, M. D., Gupta, D. M., Amasha, R. R., Wan, D. C., Longaker, M. T. 2008; 122 (1): 77-84

  Abstract

  Craniosynostosis is a relatively common developmental disorder that leads to a number of serious consequences. Previous studies have shown the influence of dura mater on the overlying cranial suture. This study was conducted to determine the role of regional dura mater versus the intrinsic nature of the suture in directing the overlying suture's fate.The authors examined the effect of regional dura mater on the fate and morphology of the posterofrontal and coronal sutures. In 8-day-old Sprague-Dawley rats, calvarial disks, consisting of the posterofrontal and coronal sutures, were excised and placed in one of three positions: (1) native position (control group), (2) rotated 45 degrees, or (3) rotated 90 degrees (n = 5 animals per group). The animals were euthanized 1 month postoperatively, and the sutures were analyzed histologically.The control group revealed normal suture morphology (n = 5). In the 45-degree rotation group, which placed the posterofrontal and coronal sutures over non-suture-associated dura mater, the posterofrontal sutures fused with thin morphology, and the coronal sutures remained patent (n = 5). In the 90-degree rotation group, the posterofrontal sutures, which were positioned over coronal suture-associated dura mater, were found to be fused with thinner morphology. The coronal sutures of the 90-degree rotation group, which were placed over posterofrontal suture-associated dura mater, remained patent but had acquired a posterofrontal-like morphology (n = 5).This study further elucidates variations in the biology of dura mater, depending on its location. Furthermore, these results illustrate the interplay between regional dura mater and the inherent characteristics of the suture complex in determining suture biology.

  View details for DOI 10.1097/PRS.0b013e318177478c

  View details for Web of Science ID 000257104300008

  View details for PubMedID 18594389

• Advances in science and technology: Impact on craniofacial surgery JOURNAL OF CRANIOFACIAL SURGERY Kwan, M. D., Longaker, M. T. 2008; 19 (4): 1136-1139

  View details for Web of Science ID 000257921300049

  View details for PubMedID 18650748

• Molecular mechanisms of FGF-2 inhibitory activity in the osteogenic context of mouse adipose-derived stem cells (mASCs) BONE Quarto, N., Wan, D. C., Longaker, M. T. 2008; 42 (6): 1040-1052

  Abstract

  Adipose-derived adult stem cells (ASCs), like their bone-marrow derived counterparts, possess the ability to differentiate down osteogenic, chondrogenic, adipogenic, and myogenic pathways. For bone differentiation of mouse ASCs (mASCs), retinoic-acid mediated upregulation of BMPR-IB has been found to be necessary. Interestingly, our previous work has also shown Fibroblast Growth Factor-2 (FGF-2) to strongly inhibit this osteogenic differentiation, even in the presence of retinoic acid. In this report, we investigated the molecular mechanisms underlying FGF-2 mediated osteogenic inhibition, demonstrating that addition of exogenous FGF-2 to mASCs antagonizes upregulation of BMPR-IB gene expression in response to retinoic acid. In addition, constitutive expression of BMPR-IB, but not BMPR-IA or BMPR-II, was found to counteract the inhibitory effects of FGF-2. Finally, p53(-/-) mASCs and human ASCs, both of which express high levels of endogenous BMPR-IB, underwent normal osteogenic differentiation even in the presence of FGF-2. Collectively, our data therefore indicate that FGF-2 antagonizes the response of mASCs to retinoic acid and also suggest that threshold levels of BMPR-IB may play a crucial role both in counteracting the inhibitory role of FGF-2 and in promoting osteogenic differentiation of ASCs in the absence of retinoic acid. Moreover, the present study also indicates that differences exist between mouse and human ASCs in relationship to FGF-2 activity in the osteogenic context.

  View details for DOI 10.1016/j.bone.2008.01.026

  View details for Web of Science ID 000256330000006

  View details for PubMedID 18420480

• Wound repair and regeneration NATURE Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. 2008; 453 (7193): 314-321

  Abstract

  The repair of wounds is one of the most complex biological processes that occur during human life. After an injury, multiple biological pathways immediately become activated and are synchronized to respond. In human adults, the wound repair process commonly leads to a non-functioning mass of fibrotic tissue known as a scar. By contrast, early in gestation, injured fetal tissues can be completely recreated, without fibrosis, in a process resembling regeneration. Some organisms, however, retain the ability to regenerate tissue throughout adult life. Knowledge gained from studying such organisms might help to unlock latent regenerative pathways in humans, which would change medical practice as much as the introduction of antibiotics did in the twentieth century.

  View details for DOI 10.1038/nature07039

  View details for Web of Science ID 000255868400038

  View details for PubMedID 18480812

• Tissue engineering in cleft palate and other congenital malformations PEDIATRIC RESEARCH Panetta, N. J., Gupta, D. M., Slater, B. J., Kwan, M. D., Liu, K. J., Longaker, M. T. 2008; 63 (5): 545-551

  Abstract

  Contributions from multidisciplinary investigations have focused attention on the potential of tissue engineering to yield novel therapeutics. Congenital malformations, including cleft palate, craniosynostosis, and craniofacial skeletal hypoplasias represent excellent targets for the implementation of tissue engineering applications secondary to the technically challenging nature and inherent inadequacies of current reconstructive interventions. Apropos to the search for answers to these clinical conundrums, studies have focused on elucidating the molecular signals driving the biologic activity of the aforementioned maladies. These investigations have highlighted multiple signaling pathways, including Wnt, fibroblast growth factor, transforming growth factor-beta, and bone morphogenetic proteins, that have been found to play critical roles in guided tissue development. Furthermore, a comprehensive knowledge of these pathways will be of utmost importance to the optimization of future cell-based tissue engineering strategies. The scope of this review encompasses a discussion of the molecular biology involved in the development of cleft palate and craniosynostosis. In addition, we include a discussion of craniofacial distraction osteogenesis and how its applied forces influence cell signaling to guide endogenous bone regeneration. Finally, this review discusses the future role of cell-based tissue engineering in the treatment of congenital malformations.

  View details for Web of Science ID 000255311900014

  View details for PubMedID 18427300

• Nitric oxide stimulates proliferation and differentiation of fetal calvarial osteoblasts and dural cells PLASTIC AND RECONSTRUCTIVE SURGERY Gupta, D. M., Li, S., Kwan, M. D., Longaker, M. T. 2008; 121 (5): 1567–69

  View details for DOI 10.1097/PRS.0b013e31816b19d2

  View details for Web of Science ID 000255435200006

• Identification of differentially regulated genes in fetal wounds during regenerative repair WOUND REPAIR AND REGENERATION Colwell, A. S., Longaker, M. T., Lorenz, H. P. 2008; 16 (3): 450-459

  Abstract

  During mammalian skin development, wounds heal with regeneration rather than scar. Genomic microarray analysis of fetal (scarless) and postnatal (scarring) cutaneous wounds was performed to identify genes with differential expression and possible proregenerative function. Differentially expressed genes between the scarless and scarring wound transcriptomes were identified with significance analysis of microarrays. At early time points, the fraction of genes with increased expression was greater in the fetal wounds. Conversely, as time after injury increased, the fraction of genes with increased expression in postnatal wounds increased from 0% at 1 hour to 67% at 24 hours. The fetal 1- and 12-hour wound transcriptomes identified genes important in DNA transcription and repair, cell cycle regulation, protein homeostasis, and intracellular signaling. The predominant expression patterns of these genes from 1 to 24 hours predominantly revealed rapid up-regulation, followed by declining expression at 24 hours. Fewer genes with differential expression between the fetal scarless and postnatal scarring wound transcriptomes were identified at 24 hours, most of which had greater expression in the postnatal wound. Our data suggest that multiple gene products may be necessary for the coordination of skin regeneration during wound repair in the fetus.

  View details for DOI 10.1111/j.1524-475X.2008.00383.x

  View details for Web of Science ID 000255666500016

  View details for PubMedID 18471263

• Increased rate of hair regrowth in mice with constitutive overexpression of Del 1 JOURNAL OF SURGICAL RESEARCH Hsu, G. P., Mathy, J. A., Wang, Z., Xia, W., Sakamoto, G., Kundu, R., Longaker, M. T., Quertermous, T., Yang, G. P. 2008; 146 (1): 73-80

  Abstract

  Developmental endothelial locus (Del)1 is a secreted extracellular matrix-associated protein that stimulates angiogenesis through integrin binding and is implicated in vasculogenesis. We hypothesized that increased expression of an angiogenic factor would lead to enhanced wound healing.Transgenic mice had Del1 cloned behind a keratin 14 promoter (K14-Del1) to drive constitutive expression in basal keratinocytes. Transgenic animals and wild-type litter mates underwent excisional wounding or depilation, and tissues were harvested at various time points. Wound healing and hair regrowth were assessed by photography, histology, and immunohistochemistry. For injection experiments, purified Del1 protein was injected in the flanks of wild-type mice with carrier on the contralateral flank as a control. Del1 expression during hair development was performed using transgenic mice with a LacZ cassette introduced downstream from the native promoter.K14-Del1 animals appeared normal and healed excisional wounds normally but demonstrated an increased rate of hair regrowth after wound healing. Using depilation experiments to specifically address hair follicle growth, we found increased hair regrowth was independent of wounding. This was confirmed by injection of purified Del1 protein. During normal hair anagenesis, Del1 is expressed in the root of the hair follicle.Constitutive expression of Del1 in skin does not affect skin vascularity or improve wound healing. Surprisingly, we found the primary effect of constitutive Del1 expression in the basal keratinocytes was increased hair growth following induction of anagenesis. During normal hair anagenesis, we see expression of Del1 in the root of the hair follicle suggesting it may function there to stimulate hair growth.

  View details for DOI 10.1016/j.jss.2007.02.024

  View details for Web of Science ID 000254798700011

  View details for PubMedID 17764695

• Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium ANNALS OF BIOMEDICAL ENGINEERING Wagner, D. R., Lindsey, D. P., Li, K. W., Tummala, P., Chandran, S. E., Smith, R. L., Longaker, M. T., Carter, D. R., Beaupre, G. S. 2008; 36 (5): 813-820

  Abstract

  This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.

  View details for DOI 10.1007/s10439-008-9448-5

  View details for Web of Science ID 000254755800013

  View details for PubMedID 18266109

• Silva et al: Repair of cranial bone defects with calcium phosphate ceramic implant or autogenous bone graft. journal of craniofacial surgery Amasha, R. R., Kwan, M. D., Longaker, M. T. 2008; 19 (3): 675-677

  View details for DOI 10.1097/SCS.0b013e31815d063b

  View details for PubMedID 18520382

• Blood-derived small Dot cells reduce scar in wound healing EXPERIMENTAL CELL RESEARCH Kong, W., Li, S., Longaker, M. T., Lorenz, H. P. 2008; 314 (7): 1529-1539

  Abstract

  Wounds in fetal skin heal without scar, however the mechanism is unknown. We identified a novel group of E-cadherin positive cells in the blood of fetal and adult mice and named them "Dot cells". The percentage of Dot cells in E16.5 fetal mice blood is more than twenty times higher compared to adult blood. Dot cells also express integrin beta1, CD184, CD34, CD13low and Sca1low, but not CD45, CD44, and CD117. Dot cells have a tiny dot shape between 1 and 7 microm diameters with fast proliferation in vitro. Most of the Dot cells remain positive for E-cadherin and integrin beta1 after one month in culture. Transplantation of Dot cells to adult mice heals skin wounds with less scar due to reduced smooth muscle actin and collagen expression in the repair tissue. Tracking GFP-positive Dot cells demonstrates that Dot cells migrate to wounds and differentiate into dermal cells, which also express strongly to FGF-2, and later lose their GFP expression. Our results indicate that Dot cells are a group of previously unidentified cells that have strong wound healing effect. The mechanism of scarless wound healing in fetal skin is due to the presence of a large number of Dot cells.

  View details for DOI 10.1016/j.yexcr.2008.01.022

  View details for Web of Science ID 000254798600010

  View details for PubMedID 18295204

  View details for PubMedCentralID PMC2692606

• Cell-based therapies for skeletal regenerative medicine HUMAN MOLECULAR GENETICS Kwan, M. D., Slater, B. J., Wan, D. C., Longaker, M. T. 2008; 17: R93-R98

  Abstract

  Skeletal deficits represent a substantial biomedical burden on the US healthcare system. Current strategies for reconstructing bony defects are fraught with inadequacies. Cell-based therapies for skeletal regeneration offer a paradigm shift that may provide alternative solutions. Substantial work has identified a host of cellular sources that possess the potential for osteogenic differentiation. Significant efforts have been devoted toward characterizing the role of postnatal cellular sources that are relatively abundant and easily accessible. Among these, the potential of using adipose-derived stromal cells for skeletal regeneration has garnered much interest. Integral to these efforts directed at characterizing cellular sources are studies that seek to understand the factors that initiate and regulate osteogenic differentiation of progenitor cells. Specifically, focus has been directed on elucidating the role of bone morphogenetic protein and fibroblast growth factor signaling in regulating osteogenic differentiation of osteoprogenitor cells. Concurrent studies in the field of scaffold design have also helped to advance the potential for cell-based therapies.

  View details for DOI 10.1093/hmg/ddn071

  View details for Web of Science ID 000258261600015

  View details for PubMedID 18632703

• Current progress in keloid research and treatment JOURNAL OF THE AMERICAN COLLEGE OF SURGEONS Butler, P. D., Longaker, M. T., Yang, G. P. 2008; 206 (4): 731-741

  View details for DOI 10.1016/j.jamcollsurg.2007.12.001

  View details for Web of Science ID 000254801000017

  View details for PubMedID 18387480

• Cranial Sutures: A Brief Review PLASTIC AND RECONSTRUCTIVE SURGERY Slater, B. J., Lenton, K. A., Kwan, M. D., Gupta, D. M., Wan, D. C., Longaker, M. T. 2008; 121 (4): 170E-178E

  Abstract

  Craniosynostosis, or the premature fusion of one or more cranial sutures, is a relatively common congenital defect that causes a number of morphologic and functional abnormalities. With advances in genetics and molecular biology, research of craniosynostosis has progressed from describing gross abnormalities to understanding the molecular interactions that underlie these cranial deformities. Animal models have been extremely valuable in improving our comprehension of human craniofacial morphogenesis, primarily by human genetic linkage analysis and the development of knock-out animals. This article provides a brief review of perisutural tissue interactions, embryonic origins, signaling molecules and their receptors, and transcription factors in maintaining the delicate balance between proliferation and differentiation of cells within the suture complex that determines suture fate. Finally, this article discusses the potential implications for developing novel therapies for craniosynostosis.

  View details for DOI 10.1097/01.prs.0000304441.99483.97

  View details for Web of Science ID 000207666900003

  View details for PubMedID 18349596

• Effects of mechanical strain on swine wounds - Correlation with human hypertrophic scars Bhatt, K. A., Vial, I. N., Kelantan, M., Wu, K., Park, Y., Paterno, J., Thangarajah, H., Dauskardt, R., Longaker, M. T., Gurtner, G. C. WILEY-BLACKWELL PUBLISHING, INC. 2008: A44–A44

  View details for Web of Science ID 000253761000149

• Replicating ligand domains of fetal matrix to promote tissue regeneration Bhatt, K. A., Rajadas, J., Thangarajah, H., VIAL, N., Chang, E., Kelantan, M., Longaker, M. T., Gurtner, G. C. WILEY-BLACKWELL PUBLISHING, INC. 2008: A38–A38

  View details for Web of Science ID 000253761000128

• Use of organotypic coculture to study keloid biology 17th Annual Meeting of the Society-for-Black-Academic-Surgeons Butler, P. D., Ly, D. P., Longaker, M. T., Yang, G. P. EXCERPTA MEDICA INC-ELSEVIER SCIENCE INC. 2008: 144–48

  Abstract

  Keloids are pathologic scars afflicting a large segment of our population and for which there is no definitive therapy. The lack of an animal model for keloid formation has hampered study. We developed an in vitro organotypic skin model to simulate normal keloid biology, which may allow us to study keloid formation without an animal model.Normal (NFs) and keloid (KFs) human fibroblasts were cultured in a collagen matrix to create a 3-dimensional dermal structure. Normal human keratinocytes (NKs) were cultured as a second layer on top and exposed to an air-fluid interface to allow differentiation into a mature keratinocyte layer. The organotypic skin was maintained for 28 days in Dulbecco's modified eagle medium with 10% fetal calf serum. Samples were collected, processed, sectioned, stained with hematoxylin and eosin, and then measured for qualitative analysis. alpha-smooth-muscle actin was also evaluated by immunoblotting.KF/NK organotypic skin showed increased collagen deposition, based on significantly denser collagen staining, with increased dermal thickness compared with NF/NK organotypic skin. We saw increased contracture in the KF/NK construct, and this correlated with increased organization of alpha-smooth-muscle actin fibers in the dermal layer of KF/NK organotypic skin compared with NF/NK skin.We have shown that coculture of KFs with keloid keratinocytes leads to an increased collagen production and dermal contracture compared with NFs and NKs, consistent with known keloid behavior. Given the lack of an animal model, we believe that organotypic skin culture can serve as a surrogate to study keloid formation.

  View details for DOI 10.1016/j.amjsurg.2007.10.003

  View details for Web of Science ID 000252598400002

  View details for PubMedID 18070722

  View details for PubMedCentralID PMC2245861

• Periosteal biaxial residual strains correlate with bone specific growth rates in chick embryos COMPUTER METHODS IN BIOMECHANICS AND BIOMEDICAL ENGINEERING Chen, J. C., Zhao, B., Longaker, M. T., Helms, J. A., Carter, D. R. 2008; 11 (5): 453-461

  Abstract

  It has been proposed that periosteal residual tensile strains influence periosteal bone apposition and endochondral ossification. The role of bone growth rates on the development of residual strains is not well known. This study examined the relationships between specific growth rate and residual strains in chick tibiotarsi. We measured length and circumference during embryonic days 11-20 using microCT. Bones grew faster in length, with longitudinal and circumferential specific growth rates decreasing from 17 to 9% and 14 to 8% per day, respectively. To calculate residual strains, opening dimensions of incisions through the periosteum were analysed using finite element techniques. Results indicate that Poisson's ratio for an isotropic material model is between 0 and 0.04. For the model with Poisson's ratio 0.03, longitudinal and circumferential residual strains decreased from 46.2 to 29.3% and 10.6 to 3.9%, respectively, during embryonic days 14-20. Specific growth rates and residual strains were positively correlated (p<0.05).

  View details for DOI 10.1080/10255840802129817

  View details for Web of Science ID 000260457900004

  View details for PubMedID 18608339

• Formation of in vitro murine cleft palate by abrogation of fibroblast growth factor signaling PLASTIC AND RECONSTRUCTIVE SURGERY Crisera, C., Teng, E., Wasson, K. L., Heller, J., Gabbay, J. S., Sedrak, M. F., Bradley, J. P., Longaker, M. T. 2008; 121 (1): 218-224

  Abstract

  A strong association between fibroblast growth factors (FGFs) and palatal anatomy suggest their role in proper palatal development. The purpose of this study was to establish whether fibroblast growth factor signaling is essential for normal palate development, improve the understanding of the biology of palatal fusion, and create a new in vitro cleft palate model.Palatal pairs excised from embryonic day 13.5 mouse palatal shelves were divided into three equal groups (n = 18 pairs) and cultured with the nasal side down and their medial edge epithelia in close apposition. Controls received vehicle only (n = 6 pairs) or LacZ recombinant virus (n = 6 pairs). The experimental group (n = 6 pairs) received truncated FGF-R1 recombinant virus with hemagglutinin epitope tag (1 x 10(9) plaque-forming units), which abrogated signal transduction by FGF-R1, FGF-R2, and FGF-R3. Tissue sectioning and staining was used to assess palatal continuity at 96 hours and immunohistochemistry was used to localize expression of the truncated receptors.Both groups 1 (control, vehicle only) and 2 (LacZ) showed complete fusion of palatal shelves after 96 hours in five of six specimens and near fusion in the remaining specimen. Beta-galactosidase staining indicated effective delivery of the LacZ virus to targeted epithelial cells. None of the group 3 specimens (FGF-R1) showed histologic resolution of the medial edge epithelia seam. Immunohistochemistry for the hemagglutinin epitope tag indicated infection by the truncated FGF-R1 virus throughout the epithelium and mesenchyme of the epithelium.By abrogating signal transduction by FGF-R1, FGF-R2, and FGF-R3, the authors have demonstrated that such signaling is essential for normal mammalian palate development.

  View details for DOI 10.1097/01.prs.0000293871.78144.28

  View details for Web of Science ID 000252208700027

  View details for PubMedID 18176224

• Confocal laser scanning microscopic analysis of collagen scaffolding patterns in cranial sutures JOURNAL OF CRANIOFACIAL SURGERY Warren, S. M., Walder, B., Dec, W., Longaker, M. T., Ting, K. 2008; 19 (1): 198-203

  Abstract

  Although recent studies indicate that regional dura mater influences the fate of the overlying cranial suture, little is known about the assembly of extracellular matrix (ECM) molecules within the patent and fusing murine cranial suture complexes. Confocal laser scanning microscopy was used to study ECM assembly within patent and fusing cranial suture complexes. Coronal sections (20 microm thick) of patent sagittal (SAG) and fusing posterior frontal (PF) sutures from postnatal 8-, 14-, and 18-day-old Sprague-Dawley rats were scanned in 0.5-microm increments, and images were collected consecutively to create a z-series for three-dimensional reconstruction. Spatial and temporal collagen arrangements were compared between SAG and PF sutures by measuring interfiber distance, fiber thickness, and total collagen surface area at each time point. We demonstrate that on day 8 (before the onset of suture fusion), collagen bundles are randomly arranged in both the SAG and PF sutures. By day 14 (midfusion period), there was a statistically significant reduction in total collagen surface area (80.5% versus 67.4%; P < 0.05) as the collagen bundles were organized into orthogonal lattices along the anterior and endocranial margins of the PF suture. Furthermore, new bone matrix deposition was observed along the edges of these organized collagen bundles. In contrast, collagen within the SAG suture remained randomly arranged and unossified. By day 18 (late fusion period), the PF suture was completely fused except for the posterior-ectocranial portion. This patent section of the PF suture contained a highly organized mineralizing orthogonal collagen lattice. The total collagen surface area in the day-18 PF suture continued to decline compared with the day-8 PF suture (80.5% versus 55.6%; P < 0.05). In the day-18 SAG suture, the collagen bundles remained randomly arranged, and the total surface area did not change. The same analysis was performed in a human pathologic fusing and patent suture. Similar results were observed. The total collagen surface area significantly decreased in the pathologic fusing human suture compared with the patent suture (92.8% versus 60.6%; P < 0.05). Moreover, the pathologically fusing suture contained a highly organized mineralizing orthogonal collagen lattice. This is the first analysis of collagen patterns in patent and fusing cranial sutures.

  View details for Web of Science ID 000252619900033

  View details for PubMedID 18216689

  View details for PubMedCentralID PMC2705761

• Applications of an athymic nude mouse model of nonhealing critical-sized calvarial defects JOURNAL OF CRANIOFACIAL SURGERY Gupta, D. M., Kwan, M. D., Slater, B. J., Wan, D. C., Longaker, M. T. 2008; 19 (1): 192-197

  Abstract

  Calvarial bone defects are a common clinical scenario in craniofacial surgery. Numerous approaches are used to reconstruct skull defects, and each possesses its own inherent disadvantages. This fact underscores the opportunity to develop a novel method to repair osseous defects in craniofacial surgery. Recent literature strongly suggests that cell-based therapies in the form of regenerative medicine may be a developing paradigm in reconstructive surgery. Although numerous studies have probed osteoprogenitor cells from mice, few have explored the biology of human cells in the setting of osteogenesis in an equally rigorous manner. This study proposes a nude mouse model of critical-sized calvarial defects to study the in vivo biology of human osteoprogenitor cells. Critical-sized 4.0-mm calvarial defects were created in nude mice (n = 15) with a custom trephine drill bit outfitted to a dental drill handpiece. During the craniotomy, the dura mater was spared from injury. Gross inspection, routine histology, and micro-computed tomographic scanning were performed at 2, 4, 8, and 16 weeks postoperatively. There was no calvarial healing in any of the animals by 16 weeks. The dura mater remained intact in all subjects. Gross, histologic, and radiographic assays confirmed these findings. Although several studies have implanted human osteoprogenitor cells in vivo in various animal models, few have documented the appropriate controls or conditions necessary to support the potential to translate benchtop findings into clinical applications. We propose in this study that the nude mouse critical-sized calvarial defect model will be valuable with increasing investigations with human osteoprogenitor cells.

  View details for Web of Science ID 000252619900032

  View details for PubMedID 18216688

• Current treatment of craniosynostosis and future therapeutic directions. Frontiers of oral biology Wan, D. C., Kwan, M. D., Lorenz, H. P., Longaker, M. T. 2008; 12: 209-230

  Abstract

  Normal craniofacial development is contingent upon coordinated growth between the brain and overlying calvaria. Craniosynostosis, the premature fusion of one or more cranial sutures, perturbs this natural framework, resulting in dramatic dysmorphology of the skull and face along with a multitude of associated functional abnormalities. Traditional approaches to the treatment of craniosynostosis have employed complex surgical remodeling of the skull vault and facial deformities all aimed at increasing the amount of intracranial volume and restoring a more normal craniofacial appearance. Significant morbidity and mortality, however, have plagued these procedures, driving dramatic evolution in our approach towards the treatment of pathologically fused sutures. Recent clinical and genetic studies have identified multiple forms of human craniosynostosis, each associated with mutations within various cytokine signaling pathways. Knowledge garnered from these investigations bear promise for the future development of alternative strategies to enhance or perhaps even replace contemporary approaches for the treatment of craniosynostosis.

  View details for DOI 10.1159/0000115043

  View details for PubMedID 18391503

• Dura mater-derived FGF-2 mediates mitogenic signaling in calvarial osteoblasts AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Li, S., Quarto, N., Longaker, M. T. 2007; 293 (6): C1834-C1842

  Abstract

  Although dura mater tissue is believed to have an important role in calvarial reossification in many in vivo studies, few studies have shown the direct effect of dura mater cells on osteoblasts. In addition, no reports have yet identified the potential factor(s) responsible for various biological activities exerted by dura mater on calvarial reossification (e.g., cell proliferation). In this study, we tested the effect of dura mater on calvarial-derived osteoblasts by performing both heterotypic coculture and by culturing osteoblast cells with conditioned media harvested from dura mater cells of juvenile (3-day-old) and adult (30-day-old) mice. The results presented here demonstrate that cellular proliferation of juvenile osteoblast cells was significantly increased by juvenile dura mater either in the coculture system or when dura mater cell-conditioned medium was applied to the osteoblast cells. Moreover, high levels of FGF-2 protein were detected in juvenile dura mater cells and their conditioned medium. In contrast, low levels of FGF-2 protein were detected in adult dura mater cells, whereas FGF-2 protein was not detectable in their conditioned medium. Abrogation of the mitogenic effect induced by juvenile dura mater cell-conditioned medium was achieved by introducing a neutralizing anti-FGF-2 antibody, thus indicating that FGF-2 may be responsible for the mitogenic effect of the juvenile dura mater. Moreover, data obtained by exploring the three major FGF-2 signaling pathways further reinforced the idea that FGF-2 might be an important paracrine signaling factor in vivo supplied by the underlying dura mater to the overlying calvarial osteoblasts.

  View details for DOI 10.1152/ajpcell.00135.2007

  View details for Web of Science ID 000251562200013

  View details for PubMedID 17913846

• Revitalizing the training of clinical scientists in surgery PLASTIC AND RECONSTRUCTIVE SURGERY Wan, D. C., Longaker, M. T. 2007; 120 (7): 2073–75

  View details for DOI 10.1097/01.prs.0000287387.63772.5e

  View details for Web of Science ID 000251668400043

• In vitro expansion of adipose-derived adult stromal cells in hypoxia enhances early chondrogenesis TISSUE ENGINEERING Xu, Y., Malladi, P., Chiou, M., Bekerman, E., Giaccia, A. J., Longaker, M. T. 2007; 13 (12): 2981-2993

  Abstract

  Cartilage is an avascular tissue, and chondrocytes in vivo experience a severely hypoxic environment. Using a defined in vitro model of early chondrogenesis, we attempted to enrich for cells with an enhanced ability for chondrogenic differentiation by pre-exposure of mouse adipose-derived adult stromal cells (ADASs) to a hypoxic (2% oxygen) environment. ADASs were subsequently expanded in 2% or 21% oxygen environments, resulting in 2 groups of cells, and then early chondrogenic differentiation was induced at 21% oxygen tension using a 3-dimensional micromass culture system. ADAS chondrogenesis was assessed using Alcian Blue staining for proteoglycans and quantification of sulfated glycosaminoglycans. Osteogenesis of the 2 cell groups was also studied. Two percent oxygen tension profoundly increased the proliferation of ADASs. ADASs expanded in 2% oxygen tension exhibited enhanced early chondrogenic differentiation and diminished osteogenesis, suggesting that the reduced oxygen environment may favor chondroprogenitors. Gene expression analysis suggested that matrix metalloproteinase synthesis was inhibited in cells expanded in 2% oxygen. Furthermore, re-oxygenation of the 2% oxygen-expanded ADASs before differentiation did not significantly affect early chondrogenesis. Thus, priming ADASs with 2% oxygen may have selected for chondrogenic progenitors with an enhanced ability to survive and differentiate. This study is relevant for the future application of cell-based therapies involving cartilage tissue regeneration.

  View details for DOI 10.1089/ten.2007.0050

  View details for Web of Science ID 000251788400018

  View details for PubMedID 17916040

• Molecular and cellular characterization of mouse calvarial osteoblasts derived from neural crest and paraxial mesoderm PLASTIC AND RECONSTRUCTIVE SURGERY Xu, Y., Malladi, P., Zhou, D., Longaker, M. T. 2007; 120 (7): 1783-1795

  Abstract

  Cranial skeletogenic mesenchyme is derived from two distinct embryonic sources: mesoderm and cranial neural crest. Previous studies have focused on molecular and cellular differences of juvenile and adult osteoblasts.To further understand the features of mouse-derived juvenile osteoblasts, the authors separated calvarial osteoblasts by their developmental origins: frontal bone-derived osteoblasts from cranial neural crest, and parietal bone-derived osteoblasts from paraxial mesoderm. Cells were harvested from a total of 120 mice.Interestingly, the authors observed distinct morphologies and proliferation potential of the two populations of osteoblasts. Osteogenic genes such as alkaline phosphatase, osteopontin, collagen I, and Wnt5a, which was recently identified as playing a role in skeletogenesis, were abundantly expressed in parietal bone-derived osteoblasts versus frontal bone-derived osteoblasts. In addition, fibroblast growth factor (FGF) receptor 2, and FGF-18 were more highly expressed in the parietal bone-derived osteoblasts, suggesting a more differentiated phenotype. In contrast, FGF-2, and adhesion molecules osteoblast cadherins and bone morphogenetic protein receptor IB, the bone tissue-specific type receptor were overexpressed in frontal bone-derived osteoblasts compared with parietal bone-derived osteoblasts.The authors observed that although neural crest-derived osteoblasts represented a population of less differentiated, faster growing cells, they formed bone nodules more rapidly than parietal bone-derived osteoblasts. This in vitro study suggests that embryonic tissue derivations influence postnatal in vitro calvarial osteoblast cell biology.

  View details for DOI 10.1097/01.prs.0000279491.48283.51

  View details for Web of Science ID 000251668400005

  View details for PubMedID 18090740

• Increased CCN2 transcription in keloid fibroblasts requires cooperativity between AP-1 and SMAD binding sites ANNALS OF SURGERY Xia, W., Kong, W., Wang, Z., Phan, T., Lim, I. J., Longaker, M. T., Yang, G. P. 2007; 246 (5): 886-895

  Abstract

  We examined the transcriptional response to serum stimulation as an in vitro model of wound healing in keloid fibroblasts to identify molecular mechanisms leading to their aberrant growth.Keloids are proliferative dermal growths representing a pathologic wound healing response. Although several groups have shown increased expression of profibrotic factors in keloids, there is little known about why they are expressed at higher levels than normal.Fibroblasts derived from keloids and normal scar were subjected to serum stimulation as an in vitro model to mimic a component of the wound microenvironment to examine differential gene expression in keloid derived fibroblasts versus normal human fibroblasts. A promoter analysis was performed to identify specific enhancers involved in mediating the differential response of connective tissue growth factor (CTGF, CCN2). Point mutations in the enhancers were performed to confirm their role. Finally, we examined activation of transcription factors known to bind the targeted enhancers.Transcription of CCN2 after serum stimulation was significantly higher in keloid versus normal fibroblasts. Promoter analysis demonstrates the fragment from -625/-140 conferred increased serum responsiveness. Mutational analysis showed an AP-1 and SMAD binding site were both necessary for serum responsiveness. Preventing activation of either transcriptional complex will block CCN2 transcription. Additional experiments suggest that a single complex that includes components of the AP-1 and SMAD binding complexes is responsible for transactivation in response to serum. The key difference between keloid and normal fibroblasts appears to be the degree of activation of c-Jun.We suggest that altered responsiveness to cellular stress, based upon current data using serum stimulation and past data on response to mechanical strain, is a key defect leading to keloid formation.

  View details for DOI 10.1097/SLA.0b013e318070d54f

  View details for Web of Science ID 000250773400028

  View details for PubMedID 17968183

• Gupta et al: TGF Alpha has a low protein expression in nonsyndromic clefts - Discussion JOURNAL OF CRANIOFACIAL SURGERY Longaker, M. T., Gupta, D. M., Liu, K. J. 2007; 18 (6): 1281–83

  View details for Web of Science ID 000251517500006

• Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis FASEB JOURNAL Aarabi, S., Bhatt, K. A., Shi, Y., Paterno, J., Chang, E. I., Loh, S. A., Holmes, J. W., Longaker, M. T., Yee, H., Gurtner, G. C. 2007; 21 (12): 3250-3261

  Abstract

  Hypertrophic scars occur following cutaneous wounding and result in severe functional and esthetic defects. The pathophysiology of this process remains unknown. Here, we demonstrate for the first time that mechanical stress applied to a healing wound is sufficient to produce hypertrophic scars in mice. The resulting scars are histopathologically identical to human hypertrophic scars and persist for more than six months following a brief (one-week) period of augmented mechanical stress during the proliferative phase of wound healing. Resulting scars are structurally identical to human hypertrophic scars and showed dramatic increases in volume (20-fold) and cellular density (20-fold). The increased cellularity is accompanied by a four-fold decrease in cellular apoptosis and increased activation of the prosurvival marker Akt. To clarify the importance of apoptosis in hypertrophic scar formation, we examine the effects of mechanical loading on cutaneous wounds of animals with altered pathways of cellular apoptosis. In p53-null mice, with down-regulated cellular apoptosis, we observe significantly greater scar hypertrophy and cellular density. Conversely, scar hypertrophy and cellular density are significantly reduced in proapoptotic BclII-null mice. We conclude that mechanical loading early in the proliferative phase of wound healing produces hypertrophic scars by inhibiting cellular apoptosis through an Akt-dependent mechanism.

  View details for DOI 10.1096/fj.07-8218com

  View details for Web of Science ID 000249781600025

  View details for PubMedID 17504973

• Noggin suppression enhances in vitro osteogenesis and accelerates in vivo bone formation JOURNAL OF BIOLOGICAL CHEMISTRY Wan, D. C., Pomerantz, J. H., Brunet, L. J., Kim, J., Chou, Y., Wu, B. M., Harland, R., Blau, H. M., Longaker, M. T. 2007; 282 (36): 26450-26459

  Abstract

  Several investigations have demonstrated a precise balance to exist between bone morphogenetic protein (BMP) agonists and antagonists, dictating BMP signaling and osteogenesis. We report a novel approach to manipulate BMP activity through a down-regulation of the potent BMP antagonist Noggin, and examined the effects on the bone forming capacity of osteoblasts. Reduction of noggin enhanced BMP signaling and in vitro osteoblast bone formation, as demonstrated by both gene expression profiles and histological staining. The effects of noggin suppression on in vivo bone formation were also investigated using critical-sized calvarial defects in mice repaired with noggin-suppressed osteoblasts. Radiographic and histological analyses revealed significantly more bone regeneration at 2 and 4 weeks post-injury. These findings strongly support the concept of enhanced osteogenesis through a down-regulation in Noggin and suggest a novel approach to clinically accelerate bone formation, potentially allowing for earlier mobilization of patients following skeletal injury or surgical resection.

  View details for DOI 10.1074/jbc.M703282200

  View details for Web of Science ID 000249239600050

  View details for PubMedID 17609215

• Hypertrophic scar formation following burns and trauma: New approaches to treatment PLOS MEDICINE Aarabi, S., Longaker, M. T., Gurtner, G. C. 2007; 4 (9): 1464-1470

  View details for DOI 10.1371/journal.pmed.0040234

  View details for Web of Science ID 000249768100011

  View details for PubMedID 17803351

  View details for PubMedCentralID PMC1961631

• Regulation of the unfolded protein response in keloid fibroblasts Butler, P. D., Wang, Z., Koong, A., Longaker, M. T., Yang, G. P. ELSEVIER SCIENCE INC. 2007: S56–S57

  View details for DOI 10.1016/j.jamcollsurg.2007.06.136

  View details for Web of Science ID 000249397300115

• Ex vivo model of cranial suture fate Slater, B. J., Lenton, K., Longaker, M. T. ELSEVIER SCIENCE INC. 2007: S59–S60

  View details for DOI 10.1016/j.jamcollsurg.2007.06.145

  View details for Web of Science ID 000249397300123

• Inhibition of an extracellular matrix protein increases survival in orthotopic nude mouse models Ham, C. M., Erler, J. T., Giaccia, A. J., Longaker, M. T., Yang, G. P. ELSEVIER SCIENCE INC. 2007: S95

  View details for DOI 10.1016/j.jamcollsurg.2007.06.237

  View details for Web of Science ID 000249397300206

• Differential expression of sclerostin in the calvaria of young and adult mice 93rd Annual Clinical Congress of the American-College-of-Surgeons Kwan, M. D., Slater, B., Deepak, G., Wan, D., Longaker, M. T. ELSEVIER SCIENCE INC. 2007: S59–S59

  View details for Web of Science ID 000249397300121

• Geometric morphometric analysis of craniofacial deformity in the noggin mutant 93rd Annual Clinical Congress of the American-College-of-Surgeons Gupta, D. M., Young, N. M., Wan, D. C., Brunet, L. J., Harland, R. M., Helms, J. A., Longaker, M. T. ELSEVIER SCIENCE INC. 2007: S60–S60

  View details for Web of Science ID 000249397300124

• Use of organotypic skin culture to study keloid biology 93rd Annual Clinical Congress of the American-College-of-Surgeons Butler, P. D., Ly, D., Wang, Z., Longaker, M. T., Yang, G. P. ELSEVIER SCIENCE INC. 2007: S57–S57

  View details for Web of Science ID 000249397300117

• Del1 protects chondrocytes from apoptosis 93rd Annual Clinical Congress of the American-College-of-Surgeons Wang, Z., Nie, X., Longaker, M. T., Yang, G. P. ELSEVIER SCIENCE INC. 2007: S45–S45

  View details for Web of Science ID 000249397300086

• Thermoreversible poloxamers and applications for vascular biology 93rd Annual Clinical Congress of the American-College-of-Surgeons Chang, E. I., Hamou, C. D., Galvez, M. G., Longaker, M. T., Gurtner, G. C. ELSEVIER SCIENCE INC. 2007: S108–S108

  View details for Web of Science ID 000249397300235

• Live imaging of Smad2/3 signaling in mouse skin wound healing WOUND REPAIR AND REGENERATION Chong, A. K., Satterwhite, T., Pham, H. M., Costa, M. A., Luo, J., Longaker, M. T., Wyss-Coray, T., Chang, J. 2007; 15 (5): 762-766

  Abstract

  Biophotonics and real-time imaging are novel technologies that can greatly enhance the study of complex biological processes. We applied this technology in a transgenic mouse with a luciferase reporter gene fused to a transforming growth factor-beta (TGF-beta) responsive Smad2/3-binding element to study bioluminescence after skin wounding. Two dorsal midline excisional skin wounds were made using a biopsy punch. One wound was randomized to suture closure and the other allowed to heal by secondary intention (n=8 each wound). Bioluminescence was measured at fixed time points following surgery. Phospho-Smad2/3 immunohistochemistry was performed to localize expression in skin wound samples. In vivo bioluminescence increased following skin wounding. Peak activity occurred on day 17 and was fourfold that of baseline (p<0.05). Subgroup analysis of primary and secondary healing showed that primarily sutured wounds had peak activities earlier than those with secondary healing, although this did not reach statistical significance. Intense phospho-Smad2/3 staining was found in the hair follicles. In vivo bioluminescence tracks Smad2/3-dependent TGF-beta signaling in the in vivo wound healing process. Our findings suggest that signaling increases after wound healing, which contrasts with other studies that show raised TGF-beta signaling in the initial days following wounding.

  View details for DOI 10.1111/j.1524-475X.2007.00299.x

  View details for Web of Science ID 000249846800019

  View details for PubMedID 17971023

• In vitro analysis of transforming growth factor-beta 1 inhibition in novel Transgenic SBE-luciferase mice ANNALS OF PLASTIC SURGERY Satterwhite, T. S., Chong, A. K., Luo, J., Pham, H., Costa, M., Longaker, M. T., Wyss-Coray, T., Chang, J. 2007; 59 (2): 207-213

  Abstract

  Transforming growth factor beta1 (TGF-beta1) expression correlates with scarring. A novel transgenic mouse model with a Smad2/3-responsive luciferase reporter construct (SBE-luc) has been developed. We hypothesized that bioluminescence in SBE-luc dermal fibroblasts could be measured to assess TGF-beta1 inhibition.Cultured dermal fibroblasts from SBE-luc mice were treated simultaneously with TGF-beta1 and increasing doses of either neutralizing antibody to TGF-beta (NA-TGFbeta) or SB-431542, a novel TGF-beta receptor kinase inhibitor. Fibroblasts were measured for luciferase activity. SBE-luc fibroblasts underwent Western blot analysis for collagen type I production.TGF-beta1 produced maximal luciferase activity in SBE-luc fibroblasts at 0.1 ng/mL (P < 0.05). NA-TGFbeta and SB-431542 inhibited luciferase activity in a dose-dependent fashion, with complete inhibition achieved by 0.1 microg/mL and 1 microM, respectively (P < 0.05). NA-TGFbeta and SB-431542 inhibited collagen type I production.Our in vitro results provide validation for further in vivo real-time imaging studies using the SBE-luc mouse as a novel wound-healing model.

  View details for DOI 10.1097/01.sap.0000252732.25168.34

  View details for Web of Science ID 000248363400017

  View details for PubMedID 17667417

• Early fetal healing as a model for adult organ regeneration TISSUE ENGINEERING Yannas, I. V., Kwan, M. D., Longaker, M. T. 2007; 13 (8): 1789-1798

  Abstract

  Evidence is provided pointing out certain basic similarities, though not an identity, between the mechanisms of early fetal regeneration and induced organ regeneration in adults. These similarities favor a model of induced organ regeneration in which biologically active scaffolds block wound contraction and scar formation.

  View details for DOI 10.1089/ten.2006.0054

  View details for Web of Science ID 000248742200001

  View details for PubMedID 17518739

• Analysis of the material properties of early chondrogenic differentiated adipose-derived stromal cells (ASC) using an in vitro three-dimensional micromass culture system BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Xu, Y., Balooch, G., Chiou, M., Bekerman, E., Ritchie, R. O., Longaker, M. T. 2007; 359 (2): 311-316

  Abstract

  Cartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro have poor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a great potential for application to tissue engineering due to their ability to differentiate into cartilage, bone, and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model system of early chondrogenesis with ASC. The material properties of these micromasses showed a significant increase in dynamic and static elastic modulus during the early chondrogenic differentiation process. These data suggest that the 3D micromass culture system represents an in vitro model of early chondrogenesis with dynamic cell signaling interactions associated with the mechanical properties of chondrocyte differentiation.

  View details for DOI 10.1016/j.bbrc.2007.05.098

  View details for Web of Science ID 000247494400020

  View details for PubMedID 17543281

  View details for PubMedCentralID PMC2677915

• Refining retinoic acid stimulation for osteogenic differentiation of murine adipose-derived adult stromal cells TISSUE ENGINEERING Wan, D. C., Siedhoff, M. T., Kwan, M. D., Nacamuli, R. P., Wu, B. M., Longaker, M. T. 2007; 13 (7): 1623-1631

  Abstract

  Murine adipose-derived adult stromal cells (ADAS) seeded onto appropriate scaffolds and pre-incubated with retinoic acid have been shown to generate in vivo bone rapidly. Prompt resorption ensues, however, as a result of osteoclastogenesis, likely secondary to retinoic acid carryover. In this study, we determined the effects of abbreviated retinoic acid exposure on ADAS osteogenic differentiation. Histological staining and gene expression analysis revealed that longer retinoic acid exposure resulted in better in vitro bone differentiation. However, significant osteogenesis was observed in ADAS after just 15 days of retinoic acid supplementation, suggesting that continual culture with retinoic acid is unnecessary for initiation of the osteogenic program. This was confirmed using ADAS pre-incubated in monolayer with an abbreviated 15 days of retinoic acid exposure before implantation into critical-sized calvarial defects. Similar rates of regeneration were observed between ADAS exposed to for 15 days or for a full 25-day course of retinoic acid before defect repair. Furthermore, by limiting retinoic acid exposure to ADAS in monolayer without scaffold, accelerated bone formation was observed without concomitant osteoclastic resorption. These data suggest that skeletal regeneration may be improved by modulating retinoic acid exposure before implantation, markedly accelerating the repair of bone defects using ADAS.

  View details for DOI 10.1089/ten.2006.0283

  View details for Web of Science ID 000248035500022

  View details for PubMedID 17518707

• Accelerated bone repair after plasma laser corticotomies ANNALS OF SURGERY Leucht, P., Lam, K., Kim, J., Mackanos, M. A., Simanovskii, D. M., Longaker, M. T., Contag, C. H., Schwettman, H. A., Helms, J. A. 2007; 246 (1): 140-150

  Abstract

  To reveal, on a cellular and molecular level, how skeletal regeneration of a corticotomy is enhanced when using laser-plasma mediated ablation compared with conventional mechanical tissue removal.Osteotomies are well-known for their most detrimental side effect: thermal damage. This thermal and mechanical trauma to adjacent bone tissue can result in the untoward consequences of cell death and eventually in a delay in healing.Murine tibial corticotomies were performed using a conventional saw and a Ti:Sapphire plasma-generated laser that removes tissue with minimal thermal damage. Our analyses began 24 hours after injury and proceeded to postsurgical day 6. We investigated aspects of wound repair ranging from vascularization, inflammation, cell proliferation, differentiation, and bone remodeling.Histology of mouse corticotomy sites uncovered a significant difference in the onset of bone healing; whereas laser corticotomies showed abundant bone matrix deposition at postsurgical day 6, saw corticotomies only exhibited undifferentiated tissue. Our analyses uncovered that cutting bone with a saw caused denaturation of the collagen matrix due to thermal effects. This denatured collagen represented an unfavorable scaffold for subsequent osteoblast attachment, which in turn impeded deposition of a new bony matrix. The matrix degradation induced a prolonged inflammatory reaction at the cut edge to create a surface favorable for osteochondroprogenitor cell attachment. Laser corticotomies were absent of collagen denaturation, therefore osteochondroprogenitor cell attachment was enabled shortly after surgery.In summary, these data demonstrate that corticotomies performed with Ti:Sapphire lasers are associated with a reduced initial inflammatory response at the injury site leading to accelerated osteochondroprogenitor cell migration, attachment, differentiation, and eventually matrix deposition.

  View details for DOI 10.1097/01.sla.0000258559.07435.b3

  View details for Web of Science ID 000247672300022

  View details for PubMedID 17592303

  View details for PubMedCentralID PMC1899222

• Hypoxia inducible factor-1 alpha deficiency affects chondrogenesis of adipose-derived adult stromal cells TISSUE ENGINEERING Malladi, P., Xu, Y., Chiou, M., Giaccia, A. J., Longaker, M. T. 2007; 13 (6): 1159-1171

  Abstract

  Increased cartilage-related disease, poor regeneration of cartilage tissue, and limited treatment options have led to intense research in tissue engineering of cartilage. Adipose-derived adult stromal cells (ADAS) are a promising cell source for skeletal tissue engineering; understanding ADAS cellular signaling and chondrogenesis will advance cell-based therapies in cartilage repair. Chondrocytes are unique-they are continuously challenged by a hypoxic microenvironment. Hypoxia inducible factor-1-alpha (HIF-1alpha), a critical mediator of a cell's response to hypoxia, plays a significant role in chondrocyte survival, growth arrest, and differentiation. By using an established in vitro 3-dimensional micromass system, we investigated the role of HIF-1alpha in chondrogenesis. Targeted deletion of HIF-1alpha in ADAS substantially inhibited the chondrogenic pathway specifically. In marked contrast, deletion of HIF-1alpha did not affect osteogenic differentiation but enhanced adipogenic differentiation. This study demonstrates the critical and specific interplay between HIF-1alpha and chondrogenesis in vitro.

  View details for DOI 10.1089/ten.2006.0265

  View details for Web of Science ID 000247237600003

  View details for PubMedID 17518738

• Hif-1 alpha regulates differentiation of limb bud mesenchyme and joint development JOURNAL OF CELL BIOLOGY Provot, S., Zinyk, D., Gunes, Y., Kathri, R., Le, Q., Kronenberg, H. M., Johnson, R. S., Longaker, M. T., Giaccia, A. J., Schipani, E. 2007; 177 (3): 451-464

  Abstract

  Recent evidence suggests that low oxygen tension (hypoxia) may control fetal development and differentiation. A crucial mediator of the adaptive response of cells to hypoxia is the transcription factor Hif-1alpha. In this study, we provide evidence that mesenchymal condensations that give origin to endochondral bones are hypoxic during fetal development, and we demonstrate that Hif-1alpha is expressed and transcriptionally active in limb bud mesenchyme and in mesenchymal condensations. To investigate the role of Hif-1alpha in mesenchymal condensations and in early chondrogenesis, we conditionally inactivated Hif-1alpha in limb bud mesenchyme using a Prx1 promoter-driven Cre transgenic mouse. Conditional knockout of Hif-1alpha in limb bud mesenchyme does not impair mesenchyme condensation, but alters the formation of the cartilaginous primordia. Late hypertrophic differentiation is also affected as a result of the delay in early chondrogenesis. In addition, mutant mice show a striking impairment of joint development. Our study demonstrates a crucial, and previously unrecognized, role of Hif-1alpha in early chondrogenesis and joint formation.

  View details for DOI 10.1083/jcb.200612023

  View details for Web of Science ID 000246580000009

  View details for PubMedID 17470636

  View details for PubMedCentralID PMC2064828

• Proposition 71 and CIRM - assessing the return on investment NATURE BIOTECHNOLOGY Longaker, M. T., Baker, L. C., Greely, H. T. 2007; 25 (5): 513-521

  Abstract

  Given that Californian voters authorized state coffers to sell $3 billion in bonds to fund the California Institute for Regenerative Medicine (CIRM) with the expectation of health and financial benefits, what benchmarks should be used to measure the initiative's success?

  View details for Web of Science ID 000246369400014

  View details for PubMedID 17483831

• Keratinocytes modulate fetal and postnatal fibroblast transforming growth factor-beta and Smad expression in co-culture PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Yun, R., Krummel, T. M., Longaker, M. T., Lorenz, H. P. 2007; 119 (5): 1440-1445

  Abstract

  The mechanism of fetal scarless wound repair is poorly understood but is thought to involve unique characteristics and behavior patterns of the fetal dermal fibroblast. The authors hypothesized that keratinocytes may differentially modulate expression of key growth factors expressed during wound healing in fetal and postnatal fibroblasts.Murine E17 fetal (n = 12 animals) and newborn (n = 8 animals) fibroblasts were grown in isolation and co-culture with newborn keratinocytes (n = 12 animals). Quantitative real-time polymerase chain reaction was performed for transforming growth factor (TGF)-beta isoform, receptor, and signaling molecule (Smad) gene expression in each group under both conditions.At baseline, fetal fibroblasts have 1.8-fold greater TGF-beta3 expression than postnatal fibroblasts (p < 0.01). Keratinocytes induce a further increase of TGF-beta3 expression (p < 0.01) but decreased TGF-beta1, TGF-beta2, TGF-beta receptor (R)-I, and TGF-betaR-II expression in fetal fibroblasts. Keratinocytes also induce an increase in TGF-beta3 (p < 0.01) and a decrease TGF-beta2, TGF-betaR-I, and TGF-betaR-II expression in postnatal fibroblasts; however, TGF-beta1 expression is unchanged. Fetal fibroblasts have lower baseline expression of Smad3 and Smad4 than postnatal fibroblasts (p < 0.05). Keratinocytes decrease Smad3 and increase Smad7 expression in both fetal and postnatal fibroblasts (p < 0.01). In contrast, keratinocytes decrease Smad2 only in fetal fibroblasts (p < 0.05).Keratinocytes have an overall antifibrotic influence on both fetal and postnatal fibroblasts in co-culture conditions. These data further characterize intrinsic differences between fetal and postnatal fibroblasts.

  View details for DOI 10.1097/01.prs.0000256049.53562.39

  View details for Web of Science ID 000245711700007

  View details for PubMedID 17415238

• Engineering epidermal growth factor mutant proteins for wound healing Experimental Biology 2007 Annual Meeting Beck, S. E., Ly, D., Lee, S., Longaker, M., Yang, G., Cochran, J. FEDERATION AMER SOC EXP BIOL. 2007: A251–A251

  View details for Web of Science ID 000245708502050

• Chemical rescue of cleft palate and midline defects in conditional GSK-3 beta mice NATURE Liu, K. J., Arron, J. R., Stankunas, K., Crabtree, G. R., Longaker, M. T. 2007; 446 (7131): 79-82

  Abstract

  Glycogen synthase kinase-3beta (GSK-3beta) has integral roles in a variety of biological processes, including development, diabetes, and the progression of Alzheimer's disease. As such, a thorough understanding of GSK-3beta function will have a broad impact on human biology and therapeutics. Because GSK-3beta interacts with many different pathways, its specific developmental roles remain unclear. We have discovered a genetic requirement for GSK-3beta in midline development. Homozygous null mice display cleft palate, incomplete fusion of the ribs at the midline and bifid sternum as well as delayed sternal ossification. Using a chemically regulated allele of GSK-3beta (ref. 6), we have defined requirements for GSK-3beta activity during discrete temporal windows in palatogenesis and skeletogenesis. The rapamycin-dependent allele of GSK-3beta produces GSK-3beta fused to a tag, FRB* (FKBP/rapamycin binding), resulting in a rapidly destabilized chimaeric protein. In the absence of drug, GSK-3beta(FRB)*(/FRB)* mutants appear phenotypically identical to GSK-3beta-/- mutants. In the presence of drug, GSK-3betaFRB* is rapidly stabilized, restoring protein levels and activity. Using this system, mutant phenotypes were rescued by restoring endogenous GSK-3beta activity during two distinct periods in gestation. This technology provides a powerful tool for defining windows of protein function during development.

  View details for DOI 10.1038/nature05557

  View details for Web of Science ID 000244525600041

  View details for PubMedID 17293880

• Cyclic mechanical strain increases production of regulators of bone healing in cultured murine osteoblasts JOURNAL OF THE AMERICAN COLLEGE OF SURGEONS Singh, S. P., Chang, E. I., Gossain, A. K., Mehara, B. J., Galiano, R. D., Jensen, J., Longaker, M. T., Gurtner, G. C., Saadeh, P. B. 2007; 204 (3): 426-434

  Abstract

  The adaptive response of bone to mechanical strain, for which angiogenesis is required, is underscored during fracture healing. Vascular endothelial growth factor (VEGF) and transforming growth factor beta-1 (TGF-beta1) are critical regulators of angiogenesis. The purpose of this study was to examine the effect of strain on the production of VEGF and TGF-beta1.MC3T3-E1 mouse osteoblasts underwent cyclic strain (low, 0.1 Hz, or high, 0.2 Hz) for 24 or 48 hours. VEGF and TGF-beta1 protein levels were determined by ELISA, and Northern blot analysis was performed for VEGF mRNA. Alkaline phosphatase (an osteoblast differentiation marker) activity was determined by functional enzymatic assay. All measurements were standardized for cell number by crystal violet colorimetric assay. Statistical significance was determined by t-test, ANOVA, and the Tukey-Kramer test.Protein production of VEGF and TGF-beta1 was dose-dependently elevated by strain (p < 0.05); alkaline phosphatase did not rise significantly. Northern blot analysis of strained osteoblast cells demonstrated increased VEGF mRNA. Cyclic strain was found to be progressively destructive in a dose-dependent manner, causing 51% and 70% decreases in cell number under low and high strain, respectively (p < 0.01).We demonstrated simultaneous, dose-dependent increases in VEGF and TGF-beta1 protein production by osteoblastic cells in response to increasing strain. VEGF mRNA also increased in response to strain. This strain-induced increase in angiogenic cytokines suggests a potential mechanism by which injured bone may recruit a new blood supply. But we also found increasing strain to increase cellular toxicity, suggesting that cyclic mechanical strain may select for a subpopulation of osteoblasts.

  View details for DOI 10.1016/j.jamcollsurg.2006.11.019

  View details for Web of Science ID 000244825200012

  View details for PubMedID 17324777

• Transforming growth factor-beta, smad, and collagen expression patterns in fetal and adult keratinocytes PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Faudoa, R., Krummel, T. M., Longaker, M. T., Lorenz, H. P. 2007; 119 (3): 852-857

  Abstract

  The transforming growth factor (TGF)-beta family regulates cellular proliferation, differentiation, and migration. To better define the influence of keratinocyte-derived TGF-beta during development and repair, the authors examined the TGF-beta isoform, receptor, signal messenger Smad, and collagen type I expression in fetal and postnatal keratinocytes.Sprague-Dawley rat keratinocytes were isolated in primary culture from fetal E17 (n = 6), newborn (n = 4), and 6-week-old adults (n = 4). Under serum-free conditions, quantitative polymerase chain reaction was performed for TGF-beta1, TGF-beta2, and TGF-beta3 ligands; TGF-beta receptor 1 (RI) and TGF-beta receptor 2 (RII); Smad4 and Smad7; and collagen type I expression.Total TGF-beta isoform expression increased 1.7-fold from E17 to newborn (p < 0.05) and adult (p < 0.01) ages. TGF-beta1 expression was 25-fold greater than TGF-beta2 and 10-fold greater than TGF-beta3 in fetal keratinocytes (p < 0.01 for each). The expression of TGF-beta1 was fivefold greater compared with TGF-beta2 and TGF-beta3 in newborn and adult keratinocytes (p < 0.01). TGF-beta-RI expression increased more than twofold (p < 0.01), whereas TGF-beta-RII expression increased by 25 percent (p < 0.01) from E17 to adult age. Smad4 increased more than twofold (p < 0.01), whereas Smad7 did not change appreciably. Collagen type I expression increased over 100-fold from E17 to adult (p < 0.005).The TGF-beta system and collagen type I have increased expression with increasing gestational age in keratinocytes. This suggests an increased profibrotic TGF-beta response and collagen type I production in keratinocytes during skin differentiation at ages associated with scarring.

  View details for DOI 10.1097/01.prs.0000255541.39993.66

  View details for Web of Science ID 000244438700011

  View details for PubMedID 17312487

• Re: Differential effects of FGFR2 mutation in ophthalniologic findings in Apert syndrome. Discussion JOURNAL OF CRANIOFACIAL SURGERY Kwan, M. D., Wan, D. C., Lorenz, H. P., Longaker, M. T. 2007; 18 (2): 459-460

  View details for Web of Science ID 000245424400039

  View details for PubMedID 17414305

• Isolation and characterization of posterofrontal/sagittal suture mesenchymal cells in vitro PLASTIC AND RECONSTRUCTIVE SURGERY Xu, Y., Malladi, P., Chiou, M., Longaker, M. T. 2007; 119 (3): 819-829

  Abstract

  Craniosynostosis, the premature fusion of cranial sutures, affects one in 2500 children. In the mouse, the posterofrontal suture is programed to fuse postnatally, but the adjacent sagittal suture remains patent throughout life. To study the cellular process of suture fusion, the authors isolated and studied suture-derived mesenchymal cells.Skulls were harvested from 80 mice (2 to 5 days old), and posterofrontal and sagittal sutures were dissected meticulously. Suture mesenchymal tissue was separated from the underlying dura mater and overlying pericranium and cultured in growth media. After the cells migrated from the explant tissues, the morphologies of the two cell populations were studied carefully, and quantitative real-time polymerase chain reaction was performed to evaluate gene expression.Both posterofrontal and sagittal cells exhibited highly heterogeneous morphologies, and the posterofrontal cells migrated faster than the sagittal cells. Accordingly, growth factors such as transforming growth factor-beta1 and fibroblast growth factor (FGF)-2 were expressed significantly more highly in posterofrontal compared with sagittal suture mesenchymal cells. In contrast, FGF receptor 2 and FGF-18 were expressed significantly more in sagittal than in posterofrontal suture cells. Importantly, bone morphogenic protein-3, the only osteogenic inhibitor in the bone morphogenic protein family, and noggin, a bone morphogenic protein antagonist, were expressed significantly more in sagittal than in posterofrontal suture cells, suggesting a possible mechanism of suture patency.To the authors' knowledge, this is the first analysis of mouse suture-derived mesenchymal cells. The authors conclude that isolation of suture-derived mesenchymal cells will provide a useful in vitro system with which to study the mechanisms underlying suture biology.

  View details for DOI 10.1097/01.prs.0000255540.91987.a0

  View details for Web of Science ID 000244438700007

  View details for PubMedID 17312483

• Microvascular reconstruction of the pediatric mandible PLASTIC AND RECONSTRUCTIVE SURGERY Warren, S. M., Borud, L. J., Brecht, L. E., Longaker, M. T., Siebert, J. W. 2007; 119 (2): 649-661

  Abstract

  Free tissue transfer for adult mandibular reconstruction is a well-established technique; however, there are few reports of pediatric microvascular lower jaw reconstruction.This retrospective study was undertaken to review the range of indications, choices, safety, and efficacy of pediatric free tissue transfer to the lower jaw. All patients underwent a parascapular, scapular, or fibula free tissue transfer. Flap choice was based on preoperative clinical examination, radiographic findings, need for linear or multiplanar mandibular reconstruction, need for dental restoration, severity of soft-tissue deficit, and peroneal artery anatomy.Over a 10-year period (1989 to 1999), we performed eight free tissue transfers to reconstruct the mandibles of seven children, aged 6 to 17 years. Indications included radiation-induced hypoplasia (n = 1), postsurgical resection of fibrous dysplasia (n = 1), hemifacial microsomia (n = 3), Robin sequence with severe micrognathia (n = 1), and osteomyelitis (n = 1). The authors transferred four parascapular osseocutaneous, two scapular osseocutaneous, one fibular osseocutaneous, and one fibular osseous flap to reconstruct five ramus, four condyle, and two subtotal mandibular defects. All bony defects were successfully bridged and all osseous flaps successfully integrated. Postoperatively, mandibular symmetry and Angle class I occlusion were restored in all patients throughout the 10.5-year follow-up period (range, 9 to 14 years). Two patients received osseointegrated dental implants. Our only complication was the partial loss of a skin paddle.Microvascular reconstruction of the pediatric mandible, in selected patients, is a safe, reliable procedure that provides the bone stock and soft tissue necessary to restore normal maxillomandibular growth and dental rehabilitation.

  View details for DOI 10.1097/01.prs.0000246482.36624.bd

  View details for Web of Science ID 000243584300028

  View details for PubMedID 17230103

• Progress and potential for regenerative medicine ANNUAL REVIEW OF MEDICINE Gurtner, G. C., Callaghan, M. J., Longaker, M. T. 2007; 58: 299-312

  Abstract

  Regenerative medicine focuses on new therapies to replace or restore lost, damaged, or aging cells in the human body to restore function. This goal is being realized by collaborative efforts in nonmammalian and human development, stem cell biology, genetics, materials science, bioengineering, and tissue engineering. At present, understanding existing reparative processes in humans and exploring the latent ability to regenerate tissue remains the focus in this field. This review covers recent work in limb regeneration, fetal wound healing, stem cell biology, somatic nuclear transfer, and tissue engineering as a foundation for developing new clinical therapies to augment and stimulate human regeneration.

  View details for DOI 10.1146/annurev.med.58.085405.095329

  View details for Web of Science ID 000244461500020

  View details for PubMedID 17076602

• Adipose tissue engineering from human adult stem cells: Clinical implications in plastic and reconstructive surgery PLASTIC AND RECONSTRUCTIVE SURGERY Wan, D. C., Longaker, M. T. 2007; 119 (1): 84–85

  View details for DOI 10.1097/01.prs.0000246803.35586.5d

  View details for Web of Science ID 000243094700012

• Upregulation of neurodevelopmental genes during scarless healing 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Antony, A. K., Kong, W. Y., Longaker, M. T., Lorenz, H. P. MEDIMOND S R L. 2007: 25–27

  View details for Web of Science ID 000257312900007

• Influence of the fetal keratinocyte in scarless repair 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Antony, A. K., Kong, W., Longaker, M. T., Lorenz, H. P. MEDIMOND S R L. 2007: 33–36

  View details for Web of Science ID 000257312900009

• Retinoic acid induces differential expression of MMP-1 and 9 in fetal and adult fibroblasts 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Antony, A. K., Bari, S., Kong, W., Longaker, M. T., Lorenz, H. P. MEDIMOND S R L. 2007: 29–31

  View details for Web of Science ID 000257312900008

• Wnt signaling pathway critical for fetal wound healing 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Knowles, J. A., Hantash, B. M., Longaker, M. T., Lorenz, H. P. MEDIMOND S R L. 2007: 69–72

  View details for Web of Science ID 000257312900018

• Gene expression differences between the dura mater of fusing and patent sutures in the mouse model 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Kwan, M. D., Wan, D. C., Wang, Z., Gupta, D. M., Slater, B. J., Beck, D., Longaker, M. T. MEDIMOND S R L. 2007: 21–24

  View details for Web of Science ID 000257312900006

• Geometric morphometric analysis identifies craniofacial deformity in the noggin mutant 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Gupta, D. M., Young, N. M., Wan, D. C., Kwan, M. D., Slater, B. J., Brunet, L. J., Harland, R. M., Helms, J. A., Longaker, M. T. MEDIMOND S R L. 2007: 13–15

  View details for Web of Science ID 000257312900004

• Osteogenic differentiation of mouse adipose-derived stromal cells requires retinoic acid and bone morphogenetic protein type IB signaling 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Wan, D. C., Shi, Y. Y., Nacamull, R. P., Quarto, N., Kwan, M. D., Gupta, D. M., Slater, B. J., LYONS, K. M., Longaker, M. T. MEDIMOND S R L. 2007: 45–47

  View details for Web of Science ID 000257312900012

• Applications of an Athymic nude mouse model of nonhealing critical-sized calvarial defects 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Gupta, D. M., Kwan, M. D., Slater, B. J., Wan, D. C., Longaker, M. T. MEDIMOND S R L. 2007: 53–55

  View details for Web of Science ID 000257312900014

• Noggin suppression enhances in vivo bone formation 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Wan, D. C., Pomerantz, J. H., Brunet, L. J., Kim, J., Chou, Y., Kwan, M. D., Slater, B. J., Gupta, D. M., Wu, B. M., Harland, R. M., Blau, H. M., Longaker, M. T. MEDIMOND S R L. 2007: 49–52

  View details for Web of Science ID 000257312900013

• Microarray analysis of differential gene expression between juvenile and adult calvarial defects 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Wan, D. C., Kwan, M. D., Wang, Z., Slater, B. J., Gupta, D. M., Longaker, M. T. MEDIMOND S R L. 2007: 61–63

  View details for Web of Science ID 000257312900016

• Refining retinoic acid stimulation of murine adipose-derived stromal cells accelerates in vivo bone formation 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Wan, D. C., Siedhoff, M. T., Kwan, M. D., Nacamuli, R. P., Slater, B. J., Gupta, D. M., Wu, B. M., Longaker, M. T. MEDIMOND S R L. 2007: 57–59

  View details for Web of Science ID 000257312900015

• Differential expression of sclerostin between juvenile and adult mice 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Kwan, M. D., Gupta, D. M., Slater, B. J., Wan, D. C., Quarto, N., Longaker, M. T. MEDIMOND S R L. 2007: 17–19

  View details for Web of Science ID 000257312900005

• Rotation of cranial sutures to determine the role of regional dura mater on cranial suture fate 12th Biennial Meeting of the International-Society-Craniofacial-Surgery Slater, B. J., Kwan, M. D., Gupta, D. M., Amasha, R. R., Wan, D. C., Longaker, M. T. MEDIMOND S R L. 2007: 65–67

  View details for Web of Science ID 000257312900017

• Cyclophilin C-associated protein is up-regulated during wound healing JOURNAL OF CELLULAR PHYSIOLOGY Kong, W., Li, S., Longaker, M. T., Lorenz, H. P. 2007; 210 (1): 153-160

  Abstract

  Cyclophilin C-associated protein (CyCAP) is identified from macrophages. It locates in intracellular, membrane bound and extracellular, suggesting it has an important role, however both of its regulation and function have not been elucidated. The expression of CyCAP in skin and during wound healing is also unknown. We demonstrate that CyCAP is expressed in both dermal fibroblasts and keratinocytes. In the dermis, the majority of CyCAP protein is located intracellular in a filamentous protein form while a lesser amount is in the extracellular matrix (ECM). CyCAP gene and protein expression is increased 1 day after skin wound healing in both fetal and adult rats and remains elevated level up to 1 week in adult rats. Immunohistochemistry studies demonstrate that the increased CyCAP expression locates mainly to inflammatory cells, including macrophages, monocytes and lymphocytes during wound healing. Interferon-gamma increases CyCAP gene and protein expression in cultured rat fibroblasts. We also found that wound healing is slower and less collagen is expressed in skin of CyCAP null mice. These data are the first observations of CyCAP expression in skin and during wound repair. Our data indicates that CyCAP is regulated by IFNgamma and may function on immune defense in macrophages, lymphocytes, dermal fibroblasts and keratinocytes during wound healing.

  View details for DOI 10.1002/jcp.20830

  View details for Web of Science ID 000242568200016

  View details for PubMedID 16998803

• Epithelial-mesenchymal transition occurs after epidermal development in mouse skin EXPERIMENTAL CELL RESEARCH Kong, W., Li, S., Liu, C., Bari, A. S., Longaker, M. T., Lorenz, H. P. 2006; 312 (19): 3959-3968

  Abstract

  In the present study, we studied epithelial-mesenchymal transition (EMT) with fetal and postnatal serial skin sections. E-cadherin, occludin and zonula occludens 1 (ZO-1)-expressing cells appear in the dermal area from E18.5 to postnatal day 9 (P9), with highest expression from P2 to P5. The co-expression of mesenchymal marker alpha-smooth muscle (alpha-SMA), fibronectin and vimentin with E-cadherin in these dermal cells was further examined. Almost no dermal cells express alpha-SMA before P0. From P2 to P6, cells expressing both E-cadherin and alpha-SMA appear in the dermis. In contrast, fibronectin-releasing cells were detected in the dermis as early as on E15.5, although on P5, some dermal cells was found weakly expressing both fibronectin and E-cadherin, most cells strongly expressing fibronectin did not express E-cadherin. Vimentin was mainly expressed in both endothelial and blood-derived cells and did not show co-expression with E-cadherin. Confocal microscopy studies further found that during EMT, E-cadherin appears intracellularly, while the expression of alpha-SMA starts from the membrane area and moves to the cytosol of the cells. Our data are the first in vivo evidence that EMT occurs during mouse skin development. Dermal cells are derived from EMT and other origins, including blood, during skin development.

  View details for DOI 10.1016/j.yexcr.2006.08.029

  View details for Web of Science ID 000241790000023

  View details for PubMedID 17027753

• Mannose-6-phosphate, an inhibitor of transforming growth factor-beta, improves range of motion after flexor tendon repair. journal of bone and joint surgery. American volume Bates, S. J., Morrow, E., Zhang, A. Y., Pham, H., Longaker, M. T., Chang, J. 2006; 88 (11): 2465-2472

  Abstract

  Adhesion formation between the flexor tendon and its surrounding fibro-osseous sheath results in a decreased postoperative range of motion in the hand. Transforming growth factor-beta (TGF-beta) is a key cytokine in the pathogenesis of tissue fibrosis. In this study, the effects of two natural inhibitors of TGF-beta, decorin and mannose-6-phosphate, were investigated in vitro and in vivo.In the in vitro investigation, primary cell cultures from rabbit flexor tendon sheath, epitenon, and endotenon were established and each was supplemented with TGF-beta along with increasing doses of decorin or mannose-6-phosphate. Collagen-I production was measured with enzyme-linked immunosorbent assay (ELISA). For the in vivo study, rabbit zone-II flexor tendons were transected and then immediately repaired. Single intraoperative graded doses of decorin, mannose-6-phosphate, or phosphate-buffered saline solution (control) were added to the repair sites, and the forepaws were tested for the range of motion and repair strength at eight weeks postoperatively.Decorin and mannose-6-phosphate both reduced TGF-beta upregulated collagen production. Intraoperative application of low-dose mannose-6-phosphate significantly improved the range of motion of the operatively treated digits. The effect on breaking strength of the tendon repair was inconclusive.Mannose-6-phosphate is effective in reducing TGF-beta upregulated collagen production in an in vitro model. This finding correlated with our in vivo finding that a single intraoperative dose of mannose-6-phosphate improved the postoperative range of motion.

  View details for PubMedID 17079405

• Mannose-6-phosphate, an inhibitor of transforming growth factor-beta, improves range of motion after flexor tendon repair JOURNAL OF BONE AND JOINT SURGERY-AMERICAN VOLUME Bates, S. J., Morrow, E., Zhang, A. Y., Pham, H., Longaker, M. T., Chang, J. 2006; 88A (11): 2465-2472

  Abstract

  Adhesion formation between the flexor tendon and its surrounding fibro-osseous sheath results in a decreased postoperative range of motion in the hand. Transforming growth factor-beta (TGF-beta) is a key cytokine in the pathogenesis of tissue fibrosis. In this study, the effects of two natural inhibitors of TGF-beta, decorin and mannose-6-phosphate, were investigated in vitro and in vivo.In the in vitro investigation, primary cell cultures from rabbit flexor tendon sheath, epitenon, and endotenon were established and each was supplemented with TGF-beta along with increasing doses of decorin or mannose-6-phosphate. Collagen-I production was measured with enzyme-linked immunosorbent assay (ELISA). For the in vivo study, rabbit zone-II flexor tendons were transected and then immediately repaired. Single intraoperative graded doses of decorin, mannose-6-phosphate, or phosphate-buffered saline solution (control) were added to the repair sites, and the forepaws were tested for the range of motion and repair strength at eight weeks postoperatively.Decorin and mannose-6-phosphate both reduced TGF-beta upregulated collagen production. Intraoperative application of low-dose mannose-6-phosphate significantly improved the range of motion of the operatively treated digits. The effect on breaking strength of the tendon repair was inconclusive.Mannose-6-phosphate is effective in reducing TGF-beta upregulated collagen production in an in vitro model. This finding correlated with our in vivo finding that a single intraoperative dose of mannose-6-phosphate improved the postoperative range of motion.

  View details for DOI 10.2106/JBJS.E.00143

  View details for Web of Science ID 000241769800020

• Craniofacial tissue engineering by stem cells JOURNAL OF DENTAL RESEARCH Mao, J. J., Giannobile, W. V., Helms, J. A., Hollister, S. J., Krebsbach, P. H., Longaker, M. T., Shi, S. 2006; 85 (11): 966-979

  Abstract

  Craniofacial tissue engineering promises the regeneration or de novo formation of dental, oral, and craniofacial structures lost to congenital anomalies, trauma, and diseases. Virtually all craniofacial structures are derivatives of mesenchymal cells. Mesenchymal stem cells are the offspring of mesenchymal cells following asymmetrical division, and reside in various craniofacial structures in the adult. Cells with characteristics of adult stem cells have been isolated from the dental pulp, the deciduous tooth, and the periodontium. Several craniofacial structures--such as the mandibular condyle, calvarial bone, cranial suture, and subcutaneous adipose tissue--have been engineered from mesenchymal stem cells, growth factor, and/or gene therapy approaches. As a departure from the reliance of current clinical practice on durable materials such as amalgam, composites, and metallic alloys, biological therapies utilize mesenchymal stem cells, delivered or internally recruited, to generate craniofacial structures in temporary scaffolding biomaterials. Craniofacial tissue engineering is likely to be realized in the foreseeable future, and represents an opportunity that dentistry cannot afford to miss.

  View details for Web of Science ID 000241520600001

  View details for PubMedID 17062735

• Innovation in surgery - A historical perspective ANNALS OF SURGERY Riskin, D. J., T Longaker, M., Gertner, M., Krummel, T. M. 2006; 244 (5): 686-693

  Abstract

  To describe the field of surgical innovation from a historical perspective, applying new findings from research in technology innovation.While surgical innovation has a rich tradition, as a field of study it is embryonic. Only a handful of academic centers of surgical innovation exist, all of which have arisen within the last 5 years. To this point, the field has not been well defined, nor have future options to promote surgical innovation been thoroughly explored. It is clear that surgical innovation is fundamental to surgical progress and has significant health policy implications. A process of systematically evaluating and promoting innovation in surgery may be critical in the evolving practice of medicine.A review of the academic literature in technology innovation was undertaken. Articles and books were identified through technical, medical, and business sources. Luminaries in surgical innovation were interviewed to develop further relevance to surgical history. The concepts in technology innovation were then applied to innovation in surgery, using the historical example of surgical endoscopy as a representative area, which encompasses millennia of learning and spans multiple specialties of care.The history of surgery is comprised largely of individual, widely respected surgeon innovators. While respecting individual accomplishments, surgeons as a group have at times hindered critical innovation to the detriment of our profession and patients. As a clinical discipline, surgery relies on a tradition of research and attracting the brightest young minds. Innovation in surgery to date has been impressive, but inconsistently supported.A body of knowledge on technology innovation has been developed over the last decade but has largely not been applied to surgery. New surgical innovation centers are working to define the field and identify critical aspects of surgical innovation promotion. It is our responsibility as a profession to work to understand innovation in surgery, discover, translate, and commercialize advances to address major clinical problems, and to support the future of our profession consistently and rationally.

  View details for DOI 10.1097/01.sla.0000242706.91771.ce

  View details for Web of Science ID 000242278300012

  View details for PubMedID 17060760

  View details for PubMedCentralID PMC1856601

• The ethics of innovation in pediatric surgery. Seminars in pediatric surgery Riskin, D. J., Longaker, M. T., Krummel, T. M. 2006; 15 (4): 319-323

  Abstract

  Ethical issues in pediatric research have long been debated, and experimentation in pediatric surgery is under intense scrutiny. Extensive legislation and institutional systems that attempt to protect children while supporting necessary research are at times ineffective. Pediatric surgery has less funding and resources for innovation than fields with higher clinical volume. Not unlike pediatrics in general, innovation in pediatric surgery must be beyond criticism. And yet, for the sake of patients, innovation should not only be maintained, but must be encouraged.

  View details for PubMedID 17055963

• Early-gestation fetal scarless wounds have less lysyl oxidase expression PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Krummel, T. M., Longaker, M. T., Lorenz, H. P. 2006; 118 (5): 1125-1129

  Abstract

  Lysyl oxidase cross-links collagen and elastin. Because cross-linking likely influences collagen architecture, the authors compared lysyl oxidase expression during scarless and scarring fetal dermal wound repair.Excisional dermal wounds were made on E17 (gestational day 16.5) and E19 (gestational day 18.5) mouse fetuses. Skin and wound RNA was collected at 8, 12, and 24 hours. Quantitative real-time polymerase chain reaction was performed for lysyl oxidase. The effect of transforming growth factor (TGF)-beta1 on lysyl oxidase expression in fetal fibroblasts was tested. Confluent primary fetal and postnatal fibroblast cultures were stimulated with TGF-beta1 for 24 hours, and lysyl oxidase expression was quantitated by performing real-time polymerase chain reaction. Lysyl oxidase expression was also quantitated in unwounded fetal skin to determine its expression profile during development.E17 and E19 fetal skin had approximately 2-fold greater lysyl oxidase expression than postnatal skin (p < 0.01), and fetal fibroblasts had greater baseline lysyl oxidase expression than postnatal fibroblasts. After TGF-beta1 stimulation, fetal and postnatal fibroblasts responded with increases in lysyl oxidase expression. In E17 early-gestation scarless fetal wounds, lysyl oxidase had small increases (<1.5-fold) in expression from 1 to 12 hours. In late-gestation E19 scarring fetal wounds, lysyl oxidase increased 1.8-fold at 8 hours and 2-fold at 12 hours, which was significantly greater than the changes observed in E17 scarless wounds (p < 0.01 for each).Lysyl oxidase has greater expression in E19 late-gestation wounds that heal with scar compared with E17 early-gestation scarless wounds. This suggests a role for lysyl oxidase in scar formation.

  View details for DOI 10.1097/01.prs.0000221056.27536.db

  View details for Web of Science ID 000241327600008

  View details for PubMedID 17016177

• Differential gene expression between juvenile and adult dura mater: A window into what genes play a role in the regeneration of membranous bone PLASTIC AND RECONSTRUCTIVE SURGERY Wan, D. C., Aalami, O. O., Wang, Z., Nacamuli, R. P., Lorget, F., Derynck, R., Longaker, M. T. 2006; 118 (4): 851-861

  Abstract

  Although reossification of large calvarial defects is possible in children, adults lack this tissue engineering capacity. In this study, the authors compared the differences in gene expression between juvenile and adult dura mater using a mouse cDNA microarray with 42,000 unique elements.Non-suture-associated parietal bone was harvested from 6-day-old and 60-day-old mice. The dura mater was carefully dissected from the calvarial disk and snap-frozen. RNA was extracted from pooled dura mater for microarray analysis. The 25 most differentially expressed genes were listed, as were selected bone-related genes. In addition, quantitative real-time reverse-transcriptase polymerase chain reaction confirmation of selected genes-BMP-2, BMP-4, and BMP-7; and osteopontin (OP), osteocalcin (OC), and FGFR-1-was performed.Juvenile dura mater expressed significantly greater amounts of BMP-2 and OP. Minimal difference in OC expression was observed between juvenile and adult dura mater. Extracellular matrix proteins (Col3a1, 5a1, 6a1, and fibronectin 1), osteoblast differentiation markers (Runx2/Cbfa1, Itm2a, and FGFR-1), and the growth factor Ptn were among other genes with greater expression in juvenile dura mater. Markers of osteoclasts (Acp5, MMP9, Ctsk) and the multiple candidate gene Ntrk2 were also expressed at higher levels in the juvenile dura mater.These findings suggest a more differentiated osteoprogenitor population to exist along with a greater presence of osteoclasts in the juvenile dura mater relative to adults. In addition to establishing a baseline difference in gene expression between juvenile and adult dura mater, new genes potentially critical to the regenerative potential of juvenile calvaria were identified.

  View details for DOI 10.1097/01.prs.0000232366.23897.2b

  View details for Web of Science ID 000240700100004

  View details for PubMedID 16980845

• Novel techniques in hernia repair. Current surgery Antony, A. K., Herrera, F. A., Easter, D. W., Longaker, M. T., Lorenz, H. P. 2006; 63 (5): 306-309

  View details for PubMedID 16971199

• Hypoxia induced pre-reperfusion response is critical for ischernic acute tubular necrosis Nie, X., Wang, Z., Rodriguez, E., Chi, A., Longaker, M., Yang, G. ELSEVIER SCIENCE INC. 2006: S25

  View details for DOI 10.1016/j.jamcollsurg.2006.05.062

  View details for Web of Science ID 000240406800039

• Retinoic acid induces differential expression of MMP-1&-9 in fetal and adult fibroblasts Antony, A. K., Bari, S., Longaker, M., Lorenz, H. ELSEVIER SCIENCE INC. 2006: S37

  View details for DOI 10.1016/j.jamcollsurg.2006.05.092

  View details for Web of Science ID 000240406800064

• Upregulation of WNT-4 expression in keloid fibroblasts after TGF-B1&-B3 stimulation Antony, A. K., Colwell, A., Longaker, M. T., Lorenz, H. ELSEVIER SCIENCE INC. 2006: S57

  View details for DOI 10.1016/j.jamcollsurg.2006.05.149

  View details for Web of Science ID 000240406800115

• BMP antagonism via noggin in adult dura mater 61st Annual Session of the Surgical Forum 2006 Clinical Congress Kwan, M. D., Wan, D., Longaker, M. ELSEVIER SCIENCE INC. 2006: S55–S55

  View details for Web of Science ID 000240406800108

• Cellular response of keloid-derived keratinocytes to physical stress 61st Annual Session of the Surgical Forum 2006 Clinical Congress Wang, Z., Phan, T., Lim, I., Longaker, M., Yang, G. ELSEVIER SCIENCE INC. 2006: S54–S55

  View details for Web of Science ID 000240406800107

• Fluorescence-activated cell sorting with CD105 identifies osteoprogenitors within mouse and human-derived AMCs 61st Annual Session of the Surgical Forum 2006 Clinical Congress Wan, D. C., Kwan, M., Nacamuli, R., Tataria, M., Longaker, M. ELSEVIER SCIENCE INC. 2006: S42–S42

  View details for Web of Science ID 000240406800076

• Differential transcriptional responses of keloid and normal keratinocytes to serum stimulation JOURNAL OF SURGICAL RESEARCH Xia, W., Phan, T., Lim, I. J., Longaker, M. T., Yang, G. P. 2006; 135 (1): 156-163

  Abstract

  Keloids are benign tumors that occur only in response to injury, for which there is no effective treatment. We demonstrated previously that keloid keratinocytes (KKs) promote fibroblast proliferation more than normal keratinocytes (NKs) and that transforming growth factor (TGF)-beta is a component of that signal. We used the transcriptional response to serum stimulation to examine how TGF-beta expression is stimulated in KKs.Quiescent KKs and NKs were stimulated using serum; harvested using RNA at 0, 1, 6, 12, and 24 h; and analyzed using quantitative real-time polymerase chain reaction. TGF-beta activity in the conditioned medium was measured with an MLEC/PAI-luciferase assay. Inhibition of ERK1/2, p38 kinase, and JNK pathways was performed with PD98059, SB203580, and SP600125, respectively.Increased transcription of TGF-beta2 occurs within 1 h of serum stimulation in KKs but not in NKs. In contrast, TGF-beta3 transcription was suppressed in KKs compared with NKs. No significant differences were observed in the transcriptional response of TGF-beta1. Increased TGF-beta2 mRNA correlated with increased TGF-beta biological activity in the conditioned medium. Inhibition of the ERK, p38 kinase or JNK signal transduction pathways blocked the transcriptional up-regulation of TGF-beta2, TbetaR1, and TbetaR2 in KKs.KKs produce more TGF-beta2 mRNA than NKs in response to serum stimulation, resulting in increased TGF-beta activity in conditioned medium. Combining these results with our previous data lead us to propose a model of keloid formation characterized by an exaggerated response to cellular stress and abnormal epithelial-mesenchymal signaling promoting keloid formation.

  View details for DOI 10.1016/j.jss.2006.01.031

  View details for Web of Science ID 000240203500023

  View details for PubMedID 16564547

• Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wan, D. C., Shi, Y., Nacamuli, R. P., Quarto, N., Lyons, K. M., Longaker, M. T. 2006; 103 (33): 12335-12340

  Abstract

  Although the multilineage potential of human adipose-derived adult stromal cells (ADAS) has been well described, few published studies have investigated the biological and molecular mechanisms underlying osteogenic differentiation of mouse ADAS. We report here that significant osteogenesis, as determined by gene expression and histological analysis, is induced only when mouse ADAS are cultured in the presence of retinoic acid with or without recombinant human bone morphogenetic protein (BMP)-2 supplementation. Furthermore, a dynamic expression profile for the BMP receptor (BMPR) isoform IB was observed, with dramatic up-regulation during osteogenesis. Western blot analysis revealed that retinoic acid enhanced levels of BMPR-IB protein during the first 7 days of osteogenic differentiation and that RNAi-mediated suppression of BMPR-IB dramatically impaired the ability of ADAS to form bone in vitro. In contrast, absence of BMPR-IA did not significantly diminish ADAS osteogenesis. Our data therefore demonstrate that the osteogenic commitment of multipotent mouse ADAS requires retinoic acid, which enhances expression of the critical BMPR-IB isoform.

  View details for DOI 10.1073/pnas.0604849103

  View details for Web of Science ID 000239867500026

  View details for PubMedID 16894153

  View details for PubMedCentralID PMC1567881

• Functions of vitamin D, retinoic acid, and dexamethasone in mouse adipose-derived mesenchymal cells TISSUE ENGINEERING Malladi, P., Xu, Y., Yang, G. P., Longaker, M. T. 2006; 12 (7): 2031-2040

  Abstract

  Adipose-derived mesenchymal cells (AMCs) offer great promise for tissue engineering of bone. Previously, 1,25-dihydroxyvitamin D3, retinoic acid (RA), and dexamethasone had been shown to promote osteogenesis in bone marrow-derived mesenchymal cells (BMSCs). To study the osteogenic characteristics of mouse AMCs, we applied these 3 hormones alone and in combination to the AMCs and examined markers of osteogenic differentiation. Interestingly, vitamin D and RA demonstrated a consistent, dose-dependent enhancement of osteogenesis and upregulated osteoblast specific markers including osteopontin and osteocalcin. However, in AMCs, dexamethasone clearly inhibited osteogenic differentiation in a dose dependent fashion and greatly increased the adipogenic marker peroxisome proliferator activated receptor gamma (PPAgamma). In summary, we show in vitro that vitamin D and RA are potential candidates to serve as enhancers of osteogenesis of AMCs and may be incorporated into future cell-based strategies for bone tissue engineering.

  View details for Web of Science ID 000239571800029

  View details for PubMedID 16889531

• Tissue engineering cartilage with aged articular chondrocytes in vivo PLASTIC AND RECONSTRUCTIVE SURGERY Malladi, P., Xu, Y., Longaker, M. T. 2006; 118 (1): 50-53

  View details for DOI 10.1097/01.prs.0000231940.88501.83

  View details for Web of Science ID 000239048700008

• Connective tissue growth factor-specific monoclonal antibody therapy inhibits pancreatic tumor growth and metastasis CANCER RESEARCH Dornhoefer, N., Spong, S., Bennewith, K., Salim, A., Klaus, S., Kambham, N., Wong, C., Kaper, F., Sutphin, P., Nacalumi, R., Hoeckel, M., Le, Q., Longaker, M., Yang, G., Koong, A., Giaccia, A. 2006; 66 (11): 5816-5827

  Abstract

  Pancreatic cancer is highly aggressive and refractory to most existing therapies. Past studies have shown that connective tissue growth factor (CTGF) expression is elevated in human pancreatic adenocarcinomas and some pancreatic cancer cell lines. To address whether and how CTGF influences tumor growth, we generated pancreatic tumor cell lines that overexpress different levels of human CTGF. The effect of CTGF overexpression on cell proliferation was measured in vitro in monolayer culture, suspension culture, or soft agar, and in vivo in tumor xenografts. Although there was no effect of CTGF expression on proliferation in two-dimensional cultures, anchorage-independent growth (AIG) was enhanced. The capacity of CTGF to enhance AIG in vitro was linked to enhanced pancreatic tumor growth in vivo when these cells were implanted s.c. in nude mice. Administration of a neutralizing CTGF-specific monoclonal antibody, FG-3019, had no effect on monolayer cell proliferation, but blocked AIG in soft agar. Consistent with this observation, anti-CTGF treatment of mice bearing established CTGF-expressing tumors abrogated CTGF-dependent tumor growth and inhibited lymph node metastases without any toxicity observed in normal tissue. Together, these studies implicate CTGF as a new target in pancreatic cancer and suggest that inhibition of CTGF with a human monoclonal antibody may control primary and metastatic tumor growth.

  View details for DOI 10.1158/0008-5472.CAN-06-0081

  View details for Web of Science ID 000238003100038

  View details for PubMedID 16740721

• Wnt-4 expression is increased in fibroblasts after TGF-beta 1 stimulation and during fetal and postnatal wound repair PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Krummel, T. M., Longaker, M. T., Lorenz, H. P. 2006; 117 (7): 2297-2301

  Abstract

  Wnt-4 is a mitogen expressed during postnatal repair and scar formation; however, its expression profile during scarless repair is unknown. Transforming growth factor (TGF)-beta1 has high expression during healing with scar formation. Whether TGF-beta1 directly influences Wnt-4 expression in fetal or postnatal fibroblasts has not been examined.Primary fetal and postnatal mouse fibroblasts were stimulated with TGF-beta1 and Wnt-4 expression quantitated by real-time polymerase chain reaction. Fetal E17 and postnatal mouse excisional wounds were also analyzed for Wnt-4 expression by real-time polymerase chain reaction.In E17 fibroblasts after TGF-beta1 stimulation, Wnt-4 expression increased 4-fold at 1 hour (p < 0.05) and peaked with an 11-fold increase at 2 hours (p < 0.05). By 24 hours, expression decreased to 2-fold baseline levels (p < 0.05). In postnatal fibroblasts, Wnt-4 expression also increased after TGF-beta stimulation, but peak expression was larger and relatively delayed, with a 17-fold increase at 12 hours (p < 0.005). Expression levels at 24 hours were still 4-fold greater than baseline (p < 0.05). In E17 fetal skin, Wnt-4 expression was 3.5-fold greater compared with 3-week-old mice (p < 0.005). Small increases in Wnt-4 expression (less than 2-fold) occurred during both fetal scarless and postnatal scarring mouse wound repair.The authors' data suggest that TGF-beta directly increases Wnt-4 expression in fetal and postnatal fibroblasts and that Wnt-4 is increased in both fetal and postnatal repair.

  View details for DOI 10.1097/01.prs.0000218708.16909.31

  View details for Web of Science ID 000238431500029

  View details for PubMedID 16772932

• FGF-2 inhibits osteogenesis in mouse adipose tissue-derived stromal cells and sustains their proliferative and osteogenic potential state TISSUE ENGINEERING Quarto, N., Longaker, M. T. 2006; 12 (6): 1405-1418

  Abstract

  In addition to adult bone marrow-derived mesenchymal stem cells, more recently, a second large stromal compartment found in adipose tissue has received attention and is believed to contain multipotent cells. In vitro, adipose tissue-derived stromal cells (ADS) can differentiate down osteogenic, chondrogenic, myogenic, adipose, and even neuronal pathways. In this article, we explore the effect of fibroblast growth factors 2 (FGF-2) on ADS cells. Our results demonstrated that FGF-2 inhibits osteogenesis in ADS cells. The osteogenic inhibitory effects is dose-dependent and reversible, thus suggesting that the lack of osteogenesis observed in ADS cells exposed to FGF-2 is not due to a negative selection triggered by this factor on a subpopulation of osteoblast progenitors. Furthermore, either overexpression of FGF-2, or continuous FGF-2 treatment sustain the proliferative and osteogenic potential state of ADS cells. Therefore, FGF-2 appears to be a positive regulator of osteoprogenitor cells and a negative regulator of osteoblast differentiation in ADS cells. These FGF-2 functional characteristics may assist with cell selection and enrichment for the purpose of bone tissue engineering.

  View details for Web of Science ID 000239570400003

  View details for PubMedID 16846339

• Fetal and adult fibroblasts have similar TGF-beta-mediated, Smad-dependent signaling pathways 90th Annual Clinical Congress of the American-College-of-Surgeons Colwell, A. S., Krummel, T. M., Longaker, M. T., Lorenz, H. P. LIPPINCOTT WILLIAMS & WILKINS. 2006: 2277–83

  Abstract

  The scarless fetal skin-healing mechanism is mediated in part by the fibroblast and involves differential expression of transforming growth factor (TGF)-beta isoforms 1 and 3. The authors hypothesized that fetal and adult fibroblasts respond differently to TGF-beta isoform-specific stimulation, which may influence whether wounds scar. Connective tissue growth factor (CTGF), Smad3, and Smad7 are TGF-beta target genes. Expression of these targets was quantitated after TGF-beta1 and -beta3 stimulation of fetal and adult fibroblasts.Primary mouse fibroblast cultures at gestational day 16.5 (E17), 18.5 (E19), and 6 weeks (adult) were stimulated with TGF-beta1 or TGF-beta3. Quantitative polymerase chain reaction was performed for CTGF, Smad3, and Smad7 expression.CTGF was reduced four-fold in E17 and E19 compared with adult fibroblasts (p < 0.005). After TGF-beta1 stimulation, CTGF expression increased more than 60-fold in both E17 and E19 (p < 0.01), which was three-fold greater than that in adult fibroblasts (p < 0.005). TGF-beta3 induced more than 70-fold, 50-fold, and 20-fold increases in CTGF expression in E17, E19, and adult fibroblasts, respectively (p < 0.01 for each). Both TGF-beta1 and -beta3 decreased Smad3 expression and increased Smad7 expression in each fibroblast type, suggesting that intact TGF-beta-mediated signaling pathways were present.Fetal (E17 and E19) fibroblasts have lower CTGF expression compared with adult fibroblasts. However, fetal fibroblasts have larger increases in CTGF expression after TGF-beta1 or -beta3 stimulation. Fetal and adult mouse fibroblasts have similar TGF-beta1 and TGF-beta3 transcriptional regulation of Smad3 and Smad7. This suggests that scarless healing is likely not mediated by different Smad-dependent transcriptional responses to TGF-beta isoforms in the fetal E17 fibroblast.

  View details for DOI 10.1097/01.prs.0000224299.16523.76

  View details for Web of Science ID 000238431500026

  View details for PubMedID 16772929

• An in vivo mouse excisional wound model of scarless healing PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Krummel, T. M., Longaker, M. T., Lorenz, H. P. 2006; 117 (7): 2292-2296

  Abstract

  The purpose of this study was to develop a reproducible murine model of fetal scarless wound healing.One-millimeter excisional wounds were made in fetal skin at gestational days 16.5 (E17) and 18.5 (E19) (term = day 21.5, or E22) and marked with India ink. Fetal mortality was less than 30 percent in E17 fetuses and 0 percent in E19 fetuses. Control postnatal 2-mm open wounds were made in 3-week-old mice.At 48 hours, E17 skin wounds had healed completely. E19 skin wounds also healed but were marked by skin irregularity at the wound site. Histologically, E17 wounds had fine reticular collagen architecture by trichrome staining and hair follicle regeneration. In contrast, E19 wounds healed with collagen deposition and scar formation and no hair follicle regeneration.The authors have developed a reliable mouse model of fetal scarless repair to help elucidate the mechanism of scarless wound healing to take advantage of genetically modified animals. The knowledge gained may be used to manipulate scarring in the adult to produce a more fetal-like wound.

  View details for DOI 10.1097/01.prs.0000219340.47232.eb

  View details for Web of Science ID 000238431500028

  View details for PubMedID 16772931

• Mitogenic and chondrogenic effects of fibroblast growth factor-2 in adipose-derived mesenchymal cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Chiou, M., Xu, Y., Longaker, M. T. 2006; 343 (2): 644-652

  Abstract

  Adipose-derived mesenchymal cells (AMCs) have demonstrated a great capacity for differentiating into bone, cartilage, and fat. Studies using bone marrow-derived mesenchymal cells (BMSCs) have shown that fibroblast growth factor (FGF)-2, a potent mitogenic factor, plays an important role in tissue engineering due to its effects in proliferation and differentiation for mesenchymal cells. The aim of this study was to investigate the function of FGF-2 in AMC chondrogenic differentiation and its possible contributions to cell-based therapeutics in skeletal tissue regeneration. Data demonstrated that FGF-2 significantly promoted the proliferation of AMCs and enhanced chondrogenesis in three-dimensional micromass culture. Moreover, priming AMCs with treatment of FGF-2 at 10 ng/ml demonstrated that cells underwent chondrogenic phenotypic differentiation, possibly by inducing N-Cadherin, FGF-receptor 2, and transcription factor Sox9. Our results indicated that FGF-2 potentiates chondrogenesis in AMCs, similar to its functions in BMSCs, suggesting the versatile potential applications of FGF-2 in skeletal regeneration and cartilage repair.

  View details for DOI 10.1016/j.bbrc.2006.02.171

  View details for PubMedID 16554022

• Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Malladi, P., Xu, Y., Chiou, M., Giaccia, A. J., Longaker, M. T. 2006; 290 (4): C1139-C1145

  Abstract

  Recent studies have demonstrated that adipose-derived mesenchymal cells (AMCs) offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage, and fat. Given that cartilage is an avascular tissue and that mesenchymal cells experience hypoxia during prechondrogenic condensation in endochondral ossification, the goal of this study was to understand the influence of oxygen tension on AMC differentiation into bone and cartilage. In vitro chondrogenesis was induced using a three-dimensional micromass culture model supplemented with TGF-beta1. Collagen II production and extracellular matrix proteoglycans were assessed with immunohistochemistry and Alcian blue staining, respectively. Strikingly, micromasses differentiated in reduced oxygen tension (2% O(2)) showed markedly decreased chondrogenesis. Osteogenesis was induced using osteogenic medium supplemented with retinoic acid or vitamin D and was assessed with alkaline phosphatase activity and mineralization. AMCs differentiated in both 21 and 2% O(2) environments. However, osteogenesis was severely diminished in a low-oxygen environment. These data demonstrated that hypoxia strongly inhibits in vitro chondrogenesis and osteogenesis in AMCs.

  View details for DOI 10.1152/ajpcell.00415.2005

  View details for PubMedID 16291817

• Craniofacial bone tissue engineering. Dental clinics of North America Wan, D. C., Nacamuli, R. P., Longaker, M. T. 2006; 50 (2): 175-?

  Abstract

  Repair and reconstruction of the craniofacial skeleton represents a significant biomedical burden, with thousands of procedures per-formed annually secondary to injuries and congenital malformations. Given the multitude of current approaches, the need for more effective strategies to repair these bone deficits is apparent. This article explores two major modalities for craniofacial bone tissue engineering: distraction osteogenesis and cellular based therapies. Current understanding of the guiding principles for each of these modalities is elaborated on along with the knowledge gained from clinical and investigative studies. By laying this foundation, future directions for craniofacial distraction and cell-based bone engineering have emerged with great promise for the advancement of clinical practice.

  View details for PubMedID 16530056

• Absence of the p53 tumor suppressor gene promotes osteogenesis in mesenchymal stem cells 57th Annual Meeting of the American-Academy-of-Pediatrics Tataria, M., Quarto, N., Longaker, M. T., Sylvester, K. G. W B SAUNDERS CO-ELSEVIER INC. 2006: 624–32

  Abstract

  Osteosarcoma arises predominantly in the metaphyseal growth plate of children during the growth spurt years. These tumors develop during physiological growth from an expanding cell population, suggesting that the transformed cell is a bone-forming progenitor. An absence of the p53 oncogene has been implicated in the origin and progression of osteosarcoma, and because mesenchymal stem cells (MSCs) are the physiological osteogenic progenitor cell population, we hypothesized that a p53-/- mutation would enhance bone differentiation of MSC in a mouse model of in vitro osteogenesis.Clonal MSC populations were derived from p53-/- mice. P53-/- and wild-type cells were placed in osteogenic culture and assessed via Alizarin Red quantification and alkaline phosphatase staining. The osteogenic marker genes Cbfa1, osteopontin, and osteocalcin were assessed by quantitative real time polymerase chain reaction during differentiation.Bone nodule formation and alkaline phosphatase staining was accelerated and enhanced in the p53-/- cells. The early and intermediate osteogenic markers, Cbfa1 and osteopontin, were upregulated in p53-/- MSCs compared with wild-type cells during osteogenesis. The terminal osteogenic marker gene osteocalcin was paradoxically lower in p53-/- MSCs indicating impaired terminal differentiation.The p53-/- mutation enhances and accelerates early osteogenesis in MSCs, but prevents terminal differentiation toward a mature osteocyte phenotype. These findings may have important implications for the regulation of the MSC compartment during the derivation of osteosarcoma in children.

  View details for DOI 10.1016/j.jpedsurg.2005.12.001

  View details for Web of Science ID 000237015800002

  View details for PubMedID 16567167

• Guided tissue regeneration enhances bone formation in a rat model of failed osteogenesis PLASTIC AND RECONSTRUCTIVE SURGERY Fang, T. D., Nacamuli, R. P., Song, H. J., Fong, K. D., Shi, Y. Y., Longaker, M. T. 2006; 117 (4): 1177-1185

  Abstract

  Guided tissue regeneration is a technique that uses barrier materials to enhance tissue regeneration. Although previously demonstrated to be an effective way of enhancing craniofacial osteogenesis in several animal models, the ability of guided tissue regeneration to augment bone formation in the context of distraction osteogenesis is unknown. In the current study, the authors applied the principle of guided tissue regeneration to their rat mandibular distraction osteogenesis model in an attempt to enhance bone regeneration.Twelve (n = 6 per group) adult Sprague-Dawley rats underwent routine gradual distraction (5 days' latency, 4-mm distraction over 8 days, 4 to 6 weeks of consolidation) and acute distraction (immediate lengthening to 4 mm, 6 to 8 weeks of consolidation). An additional 10 animals underwent acute distraction followed by application of bioabsorbable Gore Resolut XT membranes (acute distraction plus guided tissue regeneration). Membranes were completely wrapped around the distraction gap. Animals were killed 6 and 8 weeks postoperatively and mandibles analyzed radiographically and histologically.Quantitative histomorphometric analyses were performed to compare relative bone formation between all three groups. Gradual distraction mandibles achieved bony union by 6 weeks with 86 percent bone formation, which increased to 98 percent by 8 weeks. Acute distraction mandibles healed with a fibrous nonunion and only 37 percent bone formation by 8 weeks. In contrast, acute distraction plus guided tissue regeneration-treated mandibles formed significantly more bone than acute distraction mandibles by 6 weeks (57 percent) and achieved bony bridging by 8 weeks, with 88 percent new bone formation.The authors' data demonstrate that guided tissue regeneration can significantly enhance bone formation in a fibrous nonunion model of mandibular distraction osteogenesis.

  View details for DOI 10.1097/01.prs.0000204581.59190.53

  View details for Web of Science ID 000236697800016

  View details for PubMedID 16582784

• Gene expression programs in response to hypoxia: Cell type specificity and prognostic significance in human cancers PLOS MEDICINE Chi, J. T., Wang, Z., Nuyten, D. S., Rodriguez, E. H., Schaner, M. E., Salim, A., Wang, Y., Kristensen, G. B., Helland, A., Borresen-Dale, A. L., Giaccia, A., Longaker, M. T., Hastie, T., Yang, G. P., Van de Vijver, M. J., Brown, P. O. 2006; 3 (3): 395-409

  Abstract

  Inadequate oxygen (hypoxia) triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1alpha protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases.We analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1alpha RNA in renal cells, and it could be diminished by reducing HIF-1alpha expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use.The transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis in breast and ovarian cancer.

  View details for DOI 10.1371/journal.pmed.0030047

  View details for Web of Science ID 000236897500020

  View details for PubMedID 16417408

  View details for PubMedCentralID PMC1334226

• The ISCFS: A body for clinical, educational, and research innovation JOURNAL OF CRANIOFACIAL SURGERY Longaker, M. T., Wan, D. C. 2006; 17 (2): 215-216

  View details for Web of Science ID 000236747300002

  View details for PubMedID 16633164

• Relationships between tissue dilatation and differentiation in distraction osteogenesis MATRIX BIOLOGY Morgan, E. F., Longaker, M. T., Carter, D. R. 2006; 25 (2): 94-103

  Abstract

  Mechanical factors modulate the morphogenesis and regeneration of mesenchymally derived tissues via processes mediated by the extracellular matrix (ECM). In distraction osteogenesis, large volumes of new bone are created through discrete applications of tensile displacement across an osteotomy gap. Although many studies have characterized the matrix, cellular and molecular biology of distraction osteogenesis, little is known about relationships between these biological phenomena and the local physical cues generated by distraction. Accordingly, the goal of this study was to characterize the local physical environment created within the osteotomy gap during long bone distraction osteogenesis. Using a computational approach, we quantified spatial and temporal profiles of three previously identified mechanical stimuli for tissue differentiation-pressure, tensile strain and fluid flow-as well as another candidate stimulus-tissue dilatation (volumetric strain). Whereas pressure and fluid velocity throughout the regenerate decayed to less than 31% of initial values within 20 min following distraction, tissue dilatation increased with time, reaching steady state values as high as 43% strain. This dilatation created large reductions and large gradients in cell and ECM densities. When combined with previous findings regarding the effects of strain and of cell and ECM densities on cell migration, proliferation and differentiation, these results indicate two mechanisms by which tissue dilatation may be a key stimulus for bone regeneration: (1) stretching of cells and (2) altering cell and ECM densities. These results are used to suggest experiments that can provide a more mechanistic understanding of the role of tissue dilatation in bone regeneration.

  View details for DOI 10.1016/j.matbio.2005.10.006

  View details for Web of Science ID 000236135400005

  View details for PubMedID 16330195

  View details for PubMedCentralID PMC2040040

• P38 MAP kinase mediates transforming growth factor-beta 2 transcription in human keloid fibroblasts AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY Xia, W., Longaker, M. T., Yang, G. P. 2006; 290 (3): R501-R508

  Abstract

  Keloids are abnormal fibrous growths of the dermis that develop only in response to wounding and represent a form of benign skin tumor. Previous studies have shown increased protein levels of TGF-beta in keloid tissue, suggesting a strong association with keloid formation leading us to examine mechanisms for why it is more highly expressed in keloids. Here, we use serum stimulation as an in vitro model to mimic a component of the wound microenvironment and examine differential gene expression in keloid human fibroblasts (KFs) vs. normal human fibroblasts (NFs). Transcription of TGF-beta2 was rapid and peaked between 1 and 6 h after serum stimulation in KFs vs. NFs. We confirmed increased TGF-beta activity in the conditioned medium from KFs, but not NFs. Inhibition of second messenger signaling pathways demonstrated that only the p38 MAPK inhibitor SB-203580 could block upregulation of TGF-beta2 following serum stimulation in KFs. Immunoblotting demonstrated that p38 MAPK was phosphorylated within 15 min and was maintained at a high level in KFs but not in NFs. The transcription factors activating transcription factor-2 and Elk-1 are activated by p38 MAPK, and also showed rapid and prolonged phosphorylation kinetics in KFs but not in NFs. In conclusion, increased TGF-beta2 transcription in response to serum stimulation in KFs appears to be mediated by the p38 MAPK pathway. This suggests the mechanism of keloid pathogenesis may be due in part to an inherent difference in how the fibroblasts respond to wounding.

  View details for DOI 10.1152/ajpregu.00472.2005

  View details for Web of Science ID 000235210300003

  View details for PubMedID 16467496

• Increased transcriptional response to mechanical strain in keloid fibroblasts due to increased focal adhesion complex formation JOURNAL OF CELLULAR PHYSIOLOGY Wang, Z., Fong, K. D., Phan, T. T., Lim, I. J., Longaker, M. T., Yang, G. P. 2006; 206 (2): 510-517

  Abstract

  Clinicians have observed that keloids preferentially form in body areas subject to increased skin tension. We hypothesized a difference exists in the transcriptional response of keloid fibroblasts to mechanical strain compared with normal fibroblasts. Normal and keloid fibroblasts were seeded in a device calibrated to deliver a known level of equibiaxial strain. We examined the transcriptional response of TGF-beta isoforms and collagen Ialpha, genes differentially expressed in keloids. Keloid fibroblasts produced more mRNA for TGF-beta1, TGF-beta2, and collagen Ialpha after mechanical strain compared to normals, and this was correlated with protein production. Inhibiting the major mechanical signal transduction pathway with the ERK inhibitor, U0126, blocked upregulation of gene expression. In addition, keloid fibroblasts formed more focal adhesion complexes as measured by immunofluorescence for focal adhesion kinase, integrin beta1, and vinculin. Finally, there is increased activation of focal adhesion kinase when we detected the phosphorylated form of focal adhesion kinase with immunofluorescence and immunoblotting. In summary, keloid fibroblasts have an exaggerated response to mechanical strain compared to normal fibroblasts leading to increased production of pro-fibrotic growth factors. This may be one molecular mechanism for the development of keloids.

  View details for DOI 10.1002/jcp.20486

  View details for Web of Science ID 000234458300028

  View details for PubMedID 16155910

• Expression of a novel gene, MafB, in Dupuytren's disease JOURNAL OF HAND SURGERY-AMERICAN VOLUME Lee, L. C., Zhang, A. Y., Chong, A. K., Pham, H., Longaker, M. T., Chang, J. 2006; 31A (2): 211-218

  Abstract

  Dupuytren's disease (DD) is characterized by fibroblastic proliferation of the palmar fascia, often leading to flexion contracture in the hand. Although there is a strong genetic component the genome-wide expression of novel genes is not known. The purpose of this study was to use DNA microarray technology to identify upregulated genes in DD.Human tissue samples were harvested from 3 patient sources: DD cord tissue (n = 20), normal-appearing adjacent control fascia (n = 15), and palmar fascia from patients having carpal tunnel release (n = 15). DNA microarray analysis was performed on amplified sample RNA. Novel genes were compared with known gene functions. A candidate gene of interest was studied further by using immunohistochemistry on DD tissue samples and controls.Several novel genes not described previously in the study of DD were upregulated significantly, including MafB, collagen type V, alpha-2 (COL5A2), collagen type VIII, alpha-1 (COL8A1), contactin I (CNTN1), and leucine-rich repeat containing 17 (LRRC17). These upregulated genes were compared with their known gene-expression profiles in other tissues and their purported functions. MafB was found to be of particular interest because of its prominent role in tissue development and cellular differentiation. MafB immunohistochemistry showed positive staining in 50% of the DD specimens but complete absence of MafB in all control tissues (adjacent control fascia, carpal tunnel fascia). Co-localization experiments with MafB and alpha-smooth muscle actin showed staining properties in similar regions but these 2 proteins were not confined solely to the same cells.Microarray analysis of DD tissue has identified significant upregulated gene expression of MafB. MafB protein also is found in Dupuytren's cords but not in control fascia. Co-localization data suggest that the association of MafB with DD is not related exclusively to myofibroblast proliferation. Because of its role in fibroblastic transformation in other models MafB and its relationship to the pathogenesis of DD deserves further study.

  View details for DOI 10.1016/j.jhsa.2005.09.007

  View details for Web of Science ID 000235558900007

• Expression of a novel gene, MafB, in Dupuytren's disease. journal of hand surgery Lee, L. C., Zhang, A. Y., Chong, A. K., Pham, H., Longaker, M. T., Chang, J. 2006; 31 (2): 211-218

  Abstract

  Dupuytren's disease (DD) is characterized by fibroblastic proliferation of the palmar fascia, often leading to flexion contracture in the hand. Although there is a strong genetic component the genome-wide expression of novel genes is not known. The purpose of this study was to use DNA microarray technology to identify upregulated genes in DD.Human tissue samples were harvested from 3 patient sources: DD cord tissue (n = 20), normal-appearing adjacent control fascia (n = 15), and palmar fascia from patients having carpal tunnel release (n = 15). DNA microarray analysis was performed on amplified sample RNA. Novel genes were compared with known gene functions. A candidate gene of interest was studied further by using immunohistochemistry on DD tissue samples and controls.Several novel genes not described previously in the study of DD were upregulated significantly, including MafB, collagen type V, alpha-2 (COL5A2), collagen type VIII, alpha-1 (COL8A1), contactin I (CNTN1), and leucine-rich repeat containing 17 (LRRC17). These upregulated genes were compared with their known gene-expression profiles in other tissues and their purported functions. MafB was found to be of particular interest because of its prominent role in tissue development and cellular differentiation. MafB immunohistochemistry showed positive staining in 50% of the DD specimens but complete absence of MafB in all control tissues (adjacent control fascia, carpal tunnel fascia). Co-localization experiments with MafB and alpha-smooth muscle actin showed staining properties in similar regions but these 2 proteins were not confined solely to the same cells.Microarray analysis of DD tissue has identified significant upregulated gene expression of MafB. MafB protein also is found in Dupuytren's cords but not in control fascia. Co-localization data suggest that the association of MafB with DD is not related exclusively to myofibroblast proliferation. Because of its role in fibroblastic transformation in other models MafB and its relationship to the pathogenesis of DD deserves further study.

  View details for PubMedID 16473681

• Skin wounds in the MRL/MPJ mouse heal with scar WOUND REPAIR AND REGENERATION Colwell, A. S., Krummel, T. M., Kong, W., Longaker, M. T., Lorenz, H. P. 2006; 14 (1): 81-90

  Abstract

  Adult MRL/MpJ mice regenerate cartilage during repair of through-and-through ear punch wounds. However, the ability of this mouse strain to heal isolated cutaneous wounds by regeneration or with scar is unknown. The purpose of this study was to characterize the rate of reepithelialization and collagen architecture in dermal wounds from MRL/MpJ mice compared with C57bl/6 and Balb/c strains. Full-thickness incisional (5 mm) and excisional (2 mm diameter) skin wounds were made on the dorsum of 7-week-old MRL/MpJ, C57bl/6, and Balb/c mice. Ear punch wounds were made simultaneously on each animal. Reepithelialization was complete by 48 hours for incisional skin wounds in each strain. All excisional wounds showed incomplete reepithelialization at 24, 48, and 72 hours. At 14 days, all skin wounds had grossly healed. In contrast to the ear wounds made in C57bl/6 and Balb/c mice, MRL/MpJ ear wounds were completely healed by day 28. Dorsal skin wound sections at 14 and 28 days revealed dense collagen deposition and similar degrees of fibrosis between the three strains of mice. In conclusion, in contrast to wound healing in the ear, MRL/MpJ mouse dorsal cutaneous wounds heal similarly to C57bl/6 and Balb/c mice with dermal collagen deposition and scar formation.

  View details for DOI 10.1111/j.1743-6109.2005.00092.x

  View details for Web of Science ID 000235220000012

  View details for PubMedID 16476076

• Murine models of mandibular distraction osteogenesis: Towards defining the mechanical environment and the role of angiogenesis 5th International Congress of the Maxillofacial and Craniofacial Distraction Kwan, M. D., Wan, D. C., Loboa, E. G., Fang, T. D., Longaker, M. T. MEDIMOND S R L. 2006: 1–4

  View details for Web of Science ID 000243535300001

• Adipose-derived mesenchymal cells (AMCs): a promising future for skeletal tissue engineering. Biotechnology & genetic engineering reviews Xu, Y., Malladi, P., Wagner, D. R., Tataria, M., Chiou, M., Sylvester, K. G., Longaker, M. T. 2006; 23: 291-308

  View details for PubMedID 22530513

• The osteogenic potential of adipose-derived mesenchymal cells is maintained with aging PLASTIC AND RECONSTRUCTIVE SURGERY Shi, Y. Y., Nacamuli, R. P., Salim, A., Longaker, M. T. 2005; 116 (6): 1686-1696

  Abstract

  Adipose-derived mesenchymal cells are multipotent progenitor cells derived from the vascular-stromal compartment of adipose tissue. Although we have recently shown that these cells, from both juvenile and adult animals, are capable of forming bone in vivo, a detailed examination of the differences in the biology of these two populations (and in particular their ability to form bone) has not been performed.Adipose-derived mesenchymal cells were harvested from juvenile (6-day-old) and adult (60-day-old) mice. Differences in cellular attachment, proliferation, and proliferating cell nuclear antigen production were assessed. The ability of cells to undergo adipogenic differentiation was determined by Oil Red O staining. Early osteogenic differentiation was determined with alkaline phosphatase staining, and terminal differentiation with von Kossa staining as well as determination of extracellular matrix calcium content. All experiments were performed in triplicate.Greater attachment, proliferation, and proliferating cell nuclear antigen production were seen in juvenile as compared with adult adipose-derived mesenchymal cells. The juvenile cells underwent significantly greater adipogenic differentiation than did adult cells (p < 0.001). Interestingly, the adult cells were capable of robust early and terminal osteogenic differentiation, with levels of all three osteo-genic assays being similar to those seen in juvenile cells. Differences were not statistically significant.Although biologic differences exist between adipose-derived mesenchymal cells from juveniles and adults, the osteogenic capacity of these cells appears to be minimally affected by donor age. This suggests that these cells may be a particularly useful cellular resource in the design of cell-based therapies for skeletal regeneration in an aging population.

  View details for DOI 10.1097/01.prs.0000185606.03222.a9

  View details for Web of Science ID 000233119900017

  View details for PubMedID 16267433

• Age-dependent properties and quasi-static strain in the rat sagittal suture JOURNAL OF BIOMECHANICS Henderson, J. H., Chang, L. Y., Song, H. M., Longaker, M. T., Carter, D. R. 2005; 38 (11): 2294-2301

  Abstract

  We measured the morphology of and performed tensile tests on sagittal sutures from rats of postnatal age 2 to 60 days. Using the properties measured ex vivo and a pressure vessel-based analysis, we estimated the quasi-static strain that had existed in the suture in vivo from 2 to 60 days. Sutural thickness, width, and stiffness per length were notable properties found to be age dependent. Sutural thickness increased 4.5-fold (0.11-0.50mm) between 2 and 60 days. Sutural width increased transiently between 2 and 20 days, peaking around 8 days; at 8 days, mean sutural width was 75% larger than mean sutural width at two days (0.35+/-0.07 (SD) vs. 0.20+/-0.06 mm). Sutural stiffness per length increased 4.4-fold (8.77-38.3N/mm/mm) between 2 and 60 days. The quasi-static sutural strain estimated to exist in vivo averaged 270+/-190 muepsilon between 2 and 60 days and was not age dependent. These findings provide data on the age-dependent sutural properties of infant to mature rats and provide the first estimate of quasi-static sutural strain in vivo in the rat. The findings show that during development the rat sagittal suture, as a structure, changes significantly and is exposed to quasi-static tensile strain in vivo due to intracranial pressure.

  View details for DOI 10.1016/j.jbiomech.2004.07.037

  View details for PubMedID 16154417

• Bone induction in craniofacial defects. Orthodontics & craniofacial research Nacamuli, R. P., Longaker, M. T. 2005; 8 (4): 259-266

  Abstract

  Reconstruction of craniofacial bony deficiencies, whether acquired through trauma or as a result of treatment for disease, is a chronic problem. Although numerous approaches utilizing a wide array of materials ranging from alloplastic materials to autogenous bone grafts have been employed to achieve bony replacement, no ideal clinical approach exists. In this brief review, we will provide an overview of current approaches to treating craniofacial bony defects. We will then discuss advances being made in the design of scaffolding materials and potential candidate cell types with which to design tissue-engineered constructs for craniofacial skeletal repair.

  View details for PubMedID 16238606

• Expression and possible mechanisms of regulation of BMP3 in rat cranial sutures PLASTIC AND RECONSTRUCTIVE SURGERY Nacamuli, R. P., Fong, K. D., Lenton, K. A., Song, H. M., Fang, T. D., Salim, A., Longaker, M. T. 2005; 116 (5): 1353-1362

  Abstract

  Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial sutures and the factors regulating its expression in vitro.Microarray analysis was performed on rat posterior frontal and sagittal cranial sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor beta1, or cultured with primary rat nonsuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction.BMP3 expression in the posterior frontal suture decreased over the time course analyzed, whereas it increased in the sagittal suture. Notably, BMP3 expression was higher in the patent sagittal suture during the window of posterior frontal suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01).BMP3 is expressed in rat cranial sutures in a temporal pattern suggesting involvement in cranial suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial suture biology.

  View details for DOI 10.1097/01.prs.0000182223.85978.34

  View details for Web of Science ID 000232421100023

  View details for PubMedID 16217479

• Smad3 signalling plays an important role in keloid pathogenesis via epithelial-mesenchymal interactions JOURNAL OF PATHOLOGY Phan, T. T., Lim, I. J., Aalami, O., Lorget, F., Khoo, A., Tan, E. K., Mukhopadhyay, A., Longaker, M. T. 2005; 207 (2): 232-242

  Abstract

  Smad signalling plays important roles in developmental and cancer biology as well as in fibropathogenesis. Its role in keloid biology is not known. Epithelial-mesenchymal interactions, originally described in normal skin, have recently been established to play a significant role in keloid pathogenesis, and demonstrate the important influence of keratinocyte paracrine factor signalling on fibroblast behaviour. The present study investigated the role of downstream Smad cascade induction in this interaction. Normal fibroblasts (NF) and keloid fibroblasts (KF) were co-cultured in serum-free medium with normal keratinocytes (NK) or keloid keratinocytes (KK) for 5 days, after which fibroblast cell lysates were subjected to western blot and immunoprecipitation analysis to quantify the levels of Smad and Smad2/3/4 binding complex. In another set of experiments, wild-type (wt), Smad2-null (Smad2-/-) and Smad3-null (Smad3-/-) mouse embryonic fibroblasts (MEF) were assayed for cell proliferation and collagen production after serum-free co-culture with KK or exposure to conditioned media collected from serum-free KK/KF co-culture. Compared to normal skin, keloids expressed high basal levels of TGFbetaR1 and TGFbetaR2, Smad2, 3 and 4 and phospho-Smad2. Upregulation of TGFbetaR1 and TGFbetaR2, Smad3 and p-Smad2 was observed in KF co-cultured with KK, together with enhanced Smad3 phosphorylation and Smad2/3/4 binding complex production. When MEF-wt, MEF-Smad2-/- or MEF-Smad3-/- were co-cultured with KK or exposed to KK/KF co-culture conditioned media, enhanced proliferation and collagen production were seen in MEF-wt and MEF-Smad2-/- but not in MEF-Smad3-/- cells. The activation of Smad signalling, importantly that of Smad3, appears to be one facet of the complex epithelial-mesenchymal interactions in keloid pathogenesis, resulting in active KF proliferation and collagen-ECM production in co-culture with KK. This finding suggests the suppression of Smad signalling as a novel approach in keloid therapy.

  View details for DOI 10.1002/path.1826

  View details for Web of Science ID 000232213500012

  View details for PubMedID 16052471

• Hypertrophic scar fibroblasts have increased connective tissue growth factor expression after transforming growth factor-beta stimulation 49th Annual Meeting of the Plastic-Surgery-Research-Council Colwell, A. S., Phan, T. T., Kong, W., Longaker, M. T., Lorenz, P. H. LIPPINCOTT WILLIAMS & WILKINS. 2005: 1387–90

  Abstract

  Hypertrophic scars and keloids respond to dermal disruption with excessive collagen deposition and increased transforming growth factor (TFG)-beta expression. Connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta activity that is associated with scar and fibrosis. The authors hypothesize that there is increased expression of CTGF by hypertrophic scar and keloid fibroblasts in response to TGF-beta stimulation.Primary fibroblasts were isolated in culture from human hypertrophic scar (n = 2), keloid (n = 2), and normal skin (n = 2). After 18 hours of serum starvation, the cells were stimulated with 10 ng/ml of TGF-beta1, TGF-beta2, and TGF-beta3 for 24 hours. Quantitative real-time polymerase chain reaction was performed on extracted RNA samples to assay for CTGF mRNA expression.Baseline CTGF expression was increased 20-fold in unstimulated hypertrophic scar fibroblasts and 15-fold in keloid fibroblasts compared with normal fibroblasts. CTGF expression increased greater than 150-fold when stimulated with TGF-beta1 (p < 0.002) and greater than 100-fold when stimulated by TGF-beta2 or TGF-beta3 compared with normal fibroblasts (p < 0.02 and p < 0.002, respectively). CTGF expression was greatest after TGF-beta1 stimulation in hypertrophic scar fibroblasts compared with TGF-beta2 (p < 0.04) and TGF-beta3 (p < 0.02). Keloid fibroblast CTGF expression also increased greater than 100-fold after stimulation with TGF-beta1 (p = 0.16) and greater than 75-fold after addition of TGF-beta2 and TGF-beta3 (p = 0.06 and p = 0.22, respectively).Hypertrophic scar fibroblasts have both intrinsic up-regulation of CTGF transcription and an exaggerated capacity for CTGF transcription in response to TGF-beta stimulation. These data suggest that blockage of CTGF activity may reduce pathologic scar formation.

  View details for DOI 10.1097/01.prs.0000182343.99694.28

  View details for Web of Science ID 000232421100027

  View details for PubMedID 16217483

• Microarray analysis of mechanical shear effects on flexor tendon cells PLASTIC AND RECONSTRUCTIVE SURGERY Fong, K. D., Trindade, M. C., Wang, Z., Nacamuli, R. P., Pham, H., Fang, T. D., Song, H. J., Smith, L., Longaker, M. T., Chang, J. 2005; 116 (5): 1393-1404

  Abstract

  Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. The authors hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing.To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis.Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, the authors also found up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with the authors' microarray data.The authors demonstrate an overall antifibrotic expression pattern in response to shear stress in tendon cells that may provide insight into the mechanisms by which early mobilization decreases adhesion formation without impaired tendon healing.

  View details for DOI 10.1097/01.prs.0000182345.86453.4f

  View details for Web of Science ID 000232421100029

  View details for PubMedID 16217485

• Noggin suppression enhances osteogenesis of murine osteoblasts 91st Annual Clinical Congress of the American-College-of-Surgeons Wan, D. C., Pomerantz, J., Nacamuli, R., Siedhoff, M., Blau, H., Longaker, M. T. ELSEVIER SCIENCE INC. 2005: S62–S62

  View details for Web of Science ID 000231745800129

• Isolation and characterization of posterior-frontal/sagittal suture mesenchymal cells 91st Annual Clinical Congress of the American-College-of-Surgeons Xu, Y., Malladi, P., Longaker, M. T. ELSEVIER SCIENCE INC. 2005: S61–S61

  View details for Web of Science ID 000231745800126

• Effect of the angiogenic factor Del-1 on endothelial cell apoptosis 91st Annual Clinical Congress of the American-College-of-Surgeons Wang, Z., Longaker, M. T., Yang, G. ELSEVIER SCIENCE INC. 2005: S104–S104

  View details for Web of Science ID 000231745800234

• Gene profiling of cells expressing different FGF-2 forms GENE Quarto, N., Fong, K. D., Longaker, M. T. 2005; 356: 49-68

  Abstract

  Fibroblast Growth Factor-2 (FGF-2) induces cell proliferation, cell migration, embryonic development, cell differentiation, angiogenesis and malignant transformation. The four forms of FGF-2 (Low Molecular Weight) and (High Molecular Weights) are alternative translation products, and have a different subcellular localization: the high molecular weight (HMWFGF-2) forms are nuclear while the low molecular weight form, (LMWFGF-2) is mainly cytoplasmic. Our previous work demonstrated NIH 3T3 cells expressing different FGF-2 forms, displayed a different phenotype, suggesting that nuclear and cytoplasmic forms of FGF-2 may have different functions. Here we report a cDNA microarray-based study in NIH 3T3 fibroblasts expressing different FGF-2 forms. Several candidate genes that affect cell-cycle, tumor suppression, adhesion and transcription were identified as possible mediators of the HMWFGF-2 phenotype and signaling pattern. These results demonstrated that HMWFGF-2 and LMWFGF-2 target the expression of different genes. Particularly, our data suggest that HMWFGF-2 forms may function as inducers of growth inhibition and tumor suppression activities.

  View details for DOI 10.1016/j.gene.2005.05.014

  View details for Web of Science ID 000231750700006

  View details for PubMedID 16023796

• Adipose-derived mesenchymal cells as a potential cell source for skeletal regeneration CURRENT OPINION IN MOLECULAR THERAPEUTICS Xu, Y., Malladi, P., Wagner, D. R., Longaker, M. T. 2005; 7 (4): 300-305

  Abstract

  Recent studies suggest that adipose tissue contains pluripotent cells that are similar to those derived from other tissues, such as bone marrow. Mesenchymal cells isolated from adipose tissue are capable of differentiating along osteogenic, chondrogenic, myogenic, adipogenic and possibly neuronal lineages. Current knowledge of adipose-derived mesenchymal cells is reviewed, with a particular focus on efforts to direct these cells towards bone formation. Cell-based therapies using adipose tissue are anticipated to be of great clinical interest for skeletal tissue repair and regeneration.

  View details for PubMedID 16121695

• Angiogenesis is required for successful bone induction during distraction osteogenesis JOURNAL OF BONE AND MINERAL RESEARCH Fang, T. D., Salim, A., Xia, W., Nacamuli, R. P., Guccione, S., Song, H. M., Carano, R. A., Filvaroff, E. H., Bednarski, M. D., Giaccia, A. J., Longaker, M. T. 2005; 20 (7): 1114-1124

  Abstract

  The role of angiogenesis during mechanically induced bone formation is incompletely understood. The relationship between the mechanical environment, angiogenesis, and bone formation was determined in a rat distraction osteogenesis model. Disruption of either the mechanical environment or endothelial cell proliferation blocked angiogenesis and bone formation. This study further defines the role of the mechanical environment and angiogenesis during distraction osteogenesis.Whereas successful fracture repair requires a coordinated and complex transcriptional program that integrates mechanotransductive signaling, angiogenesis, and osteogenesis, the interdependence of these processes is not fully understood. In this study, we use a system of bony regeneration known as mandibular distraction osteogenesis (DO) in which a controlled mechanical stimulus promotes bone induction after an osteotomy and gradual separation of the osteotomy edges to examine the relationship between the mechanical environment, angiogenesis, and osteogenesis.Adult Sprague-Dawley rats were treated with gradual distraction, gradual distraction plus the angiogenic inhibitor TNP-470, or acute distraction (a model of failed bony regeneration). Animals were killed at the end of distraction (day 13) or at the end of consolidation (day 41) and examined with muCT, histology, and immunohistochemistry for angiogenesis and bone formation (n = 4 per time-point per group). An additional group of animals (n = 6 per time-point per group) was processed for microarray analysis at days 5, 9, 13, 21, and 41.Either TNP-470 administration or disruption of the mechanical environment prevented normal osteogenesis and resulted in a fibrous nonunion. Subsequent analysis of the regenerate showed an absence of angiogenesis by gross histology and immunohistochemical localization of platelet endothelial cell adhesion molecule in the groups that failed to heal. Microarray analysis revealed distinct patterns of expression of genes associated with osteogenesis, angiogenesis, and hypoxia in each of the three groups. Our findings confirm the interdependence of the mechanical environment, angiogenesis, and osteogenesis during DO, and suggest that induction of proangiogenic genes and the proper mechanical environment are both necessary to support new vasculature for bone induction in DO.

  View details for DOI 10.1359/JBMR.050301

  View details for Web of Science ID 000230134100005

  View details for PubMedID 15940364

• Age-dependent residual tensile strains are present in the dura mater of rats JOURNAL OF THE ROYAL SOCIETY INTERFACE Henderson, J. H., Nacamuli, R. P., Zhao, B., Longaker, M. T., Carter, D. R. 2005; 2 (3): 159-167

  Abstract

  The objectives of this study were to determine whether residual tensile strains exist in the dura mater of mammals in vivo, and whether the strains are age-dependent. We made incisions in the parietal dura mater of immature and mature rats, and measured the retraction of the dura mater from each incision. We then used a finite-element model to calculate the strain present in the parietal dura mater of each rat. We found that age-dependent residual tensile strains are present in the dura mater of rats. The mean average residual strain of the immature rats was significantly larger than that of the mature rats (4.96+/-1.54% (s.d.) versus 0.39+/-0.13%, p<0.0001), with the mean strain calculated in the mature rats of the order of the minimum measurement that could be made using our experimental approach. In addition, in the immature rats mean residual strain in the longitudinal direction was significantly larger than mean residual strain in the transverse direction (6.11+/-3.62% versus 3.82+/-2.64%, p=0.0218). Our findings show that age-dependent residual tensile strains exist in the dura mater of rats. We speculate that these strains may reflect the rate and direction of cranial growth and may also influence cranial healing.

  View details for DOI 10.1098/rsif.2005.0035

  View details for PubMedID 16849176

• Differential transcriptional expression profiles of juvenile and adult calvarial bone PLASTIC AND RECONSTRUCTIVE SURGERY Aalami, O. O., Nacamuli, R. P., Salim, A., Fong, K. D., Lenton, K. A., Song, H. M., Fang, T. D., Longaker, M. T. 2005; 115 (7): 1986-1994

  Abstract

  It has widely been observed that young children are capable of reossifying large calvarial defects, while adults lack this endogenous tissue-engineering capacity. The ability of juvenile animals to regenerate calvarial defects has been investigated in multiple animal models, including mice. In this study, the authors used cDNA microarrays to investigate the expression of osteogenesis-associated genes upstream and downstream of Runx2 in juvenile and adult mouse calvaria.Nonsuture-associated parietal bone discs were harvested from 6-day-old (n = 50) and 60-day-old (n = 35) male CD-1 mice. After separation of the underlying dura mater and overlying pericranium, the calvarial discs were snap-frozen and RNA was extracted from pooled samples of calvaria for microarray analysis. Genes analyzed included cytokines, receptors, and cell-surface and matrix proteins both upstream and downstream of Runx2.Genes associated with the Runx2 pathway had notably higher levels in the juvenile versus adult calvaria. All genes except for osteocalcin were expressed at least twofold higher in the juvenile calvaria. This pattern was validated with quantitative real-time polymerase chain reaction. In addition, mRNA for potent osteoinductive growth factors was present at higher levels in the juvenile compared with the adult calvaria.These findings reflect a genomic environment of active osteoblast differentiation and ossification in the juvenile calvaria compared with the adult "quiescent" calvarial tissue. These data suggest that a decreased osteogenic potential of adult calvarial osteoblasts may, in part, explain the inability of adult animals to heal calvarial defects.

  View details for DOI 10.1097/01.PRS.0000163323.66318.73

  View details for Web of Science ID 000229497700025

  View details for PubMedID 15923847

• Innovation: A sustainable competitive advantage for plastic and reconstructive surgery PLASTIC AND RECONSTRUCTIVE SURGERY Longaker, M. T., Rohrich, R. J. 2005; 115 (7): 2135–36

  View details for DOI 10.1097/01.PRS.0000168495.95560.EB

  View details for Web of Science ID 000229497700050

  View details for PubMedID 15923869

• Mechanobiology of mandibular distraction osteogenesis: finite element analyses with a rat model JOURNAL OF ORTHOPAEDIC RESEARCH Loboa, E. G., Fang, T. D., Parker, D. W., Warren, S. M., Fong, K. D., Longaker, M. T., Carter, D. R. 2005; 23 (3): 663-670

  Abstract

  Three-dimensional finite element (FE) analyses were performed to characterize the local mechanical environment created within the tissue regenerate during mandibular distraction osteogenesis (DO) in a rat model. Finite element models were created from three-dimensional computed tomography image data of rat hemi-mandibles at four different time points during an optimal distraction osteogenesis protocol (i.e., most successful protocol for bone formation): end latency (post-operative day (POD) 5), distraction day 2 (POD 7), distraction day 5 (POD 10), and distraction day 8 (POD 13). A 0.25 mm distraction was simulated and the resulting hydrostatic stresses and maximum principal tensile strains were determined within the tissue regenerate. When compared to previous histological findings, finite element analyses showed that tensile strains up to 13% corresponded to regions of new bone formation and regions of periosteal hydrostatic pressure with magnitudes less than 17 kPa corresponded to locations of cartilage formation. Tensile strains within the center of the gap were much higher, leading us to conclude that tissue damage would occur there if the tissue was not compliant enough to withstand such high strains, and that this damage would trigger formation of new mesenchymal tissue. These data were consistent with histological evidence showing mesenchymal tissue present in the center of the gap throughout distraction. Finite element analyses performed at different time points during distraction were instrumental in determining the changes in hydrostatic stress and tensile strain fields throughout distraction, providing a mechanical environment rationale for the different levels of bone formation in end latency, and distraction day 2, 5, and 8 specimens.

  View details for DOI 10.1016/j.orthres.2004.09.010

  View details for Web of Science ID 000229375000022

  View details for PubMedID 15885489

• Sox9 neural crest determinant gene controls patterning and closure of the posterior frontal cranial suture DEVELOPMENTAL BIOLOGY Sahar, D. E., Longaker, M. T., Quarto, N. 2005; 280 (2): 344-361

  Abstract

  Cranial suture development involves a complex interaction of genes and tissues derived from neural crest cells (NCC) and paraxial mesoderm. In mice, the posterior frontal (PF) suture closes during the first month of life while other sutures remain patent throughout the life of the animal. Given the unique NCC origin of PF suture complex (analogous to metopic suture in humans), we performed quantitative real-time PCR and immunohistochemistry to study the expression pattern of the NCC determinant gene Sox9 and select markers of extracellular matrix. Our results indicated a unique up-regulated expression of Sox9, a regulator of chondrogenesis, during initiation of PF suture closure, along with the expression of specific cartilage markers (Type II Collagen and Type X Collagen), as well as cartilage tissue formation in the PF suture. This process was followed by expression of bone markers (Type I Collagen and Osteocalcin), suggesting endochondral ossification. Moreover, we studied the effect of haploinsufficiency of the NCC determinant gene Sox9 in the NCC derived PF suture complex. A decrease in dosage of Sox9 by haploinsufficiency in NCC-derived tissues resulted in delayed PF suture closure. These results demonstrate a unique development of the PF suture complex and the role of Sox9 as an important contributor to timely and proper closure of the PF suture through endochondral ossification.

  View details for DOI 10.1016/j.ydbio.2005.01.022

  View details for Web of Science ID 000228377600007

  View details for PubMedID 15882577

• FGF-2 acts through an ERK1/2 intracellular pathway to affect osteoblast differentiation PLASTIC AND RECONSTRUCTIVE SURGERY Spector, J. A., Mathy, J. A., Warren, S. M., Nacamuli, R. P., Song, H. M., Lenton, K., Fong, K. D., Fang, D. T., Longaker, M. T. 2005; 115 (3): 838-852

  Abstract

  An abundance of genetic and experimental data have suggested that fibroblast growth factor (FGF) signaling plays a central role in physiological and pathological cranial suture fusion. Although alterations in the differentiation and proliferation of sutural osteoblasts may be a key mediator of this process, the mechanisms by which FGF signaling regulates osteoblast differentiation remain incompletely understood. In the current study, the authors show that recombinant human FGF-2 alters osteoblastic expression of bone morphogenetic protein-2 and Msx-2 in vitro to favor cellular differentiation and osteoinduction. The ERK1/2 intracellular signaling cascade was shown to be necessary for recombinant human FGF-2-mediated bone morphogenetic protein-2 transcriptional changes. Furthermore, the cellular production of an intermediate transcriptional modifier was found to be necessary for the recombinant human FGF-2-mediated gene expression changes in bone morphogenetic protein-2 and Msx-2. Together, these findings offer new insight into the mechanisms by which FGF-2 modulates osteoblast biology.

  View details for DOI 10.1097/01.PRS.0000153035.73507.7B

  View details for Web of Science ID 000227432100023

  View details for PubMedID 15731686

• Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover TISSUE ENGINEERING Cowan, C. M., Aalami, O. O., Shi, Y. Y., Chou, Y. F., Mari, C., Thomas, R., Quarto, N., Nacamuli, R. P., Contag, C. H., Wu, B., Longaker, M. T. 2005; 11 (3-4): 645-658

  Abstract

  Reconstruction of craniofacial defects presents a substantial biomedical burden, and requires complex surgery. Interestingly, children after age 2 years and adults are unable to heal large skull defects. This nonhealing paradigm provides an excellent model system for craniofacial skeletal tissueengineering strategies. Previous studies have documented the in vivo osteogenic potential of adipose-derived stromal (ADS) cells and bone marrow-derived stromal (BMS) cells. This study investigates the ability to accelerate in vivo osteogenesis on ex vivo recombinant human bone morphogenetic protein 2 (BMP-2) and retinoic acid stimulation. Mouse osteoblasts, ADS cells, and BMS cells were seeded onto apatite-coated PLGA scaffolds, stimulated with rhBMP-2 and retinoic acid ex vivo for 4 weeks, and subsequently implanted into critically sized (4 mm) calvarial defects. Samples were harvested after 2, 4, 8, and 12 weeks. Areas of complete bony bridging were noted as early as 2 weeks in vivo; however, osteoclasts were attracted to the scaffold as identified by calcitonin receptor staining and tartrate-resistant acid phosphatase activity staining. Although the optimal method of in vitro osteogenic priming for mesenchymal cells remains unknown, these results provide evidence that BMP-2 and retinoic acid stimulation of multipotent cells ex vivo can subsequently induce significant quantities of bone formation within a short time period in vivo.

  View details for PubMedID 15869441

• New developments in pediatric plastic surgery research CLINICS IN PLASTIC SURGERY Nacamuli, R. P., Wan, D. C., Lenton, K. A., Longaker, M. T. 2005; 32 (1): 123-?

  Abstract

  Pediatric plastic surgery research is a rapidly expanding field. Unique in many ways, researchers in this field stand at the union of multiple scientific specialties, including biomedical engineering, tissue engineering, polymer science, molecular biology, developmental biology, and genetics. The goal of this scientific effort is to translate research advances into improved treatments for children with congenital and acquired defects. Although the last decade has seen a dramatic acceleration in research related to pediatric plastic surgery, the next 10 years will no doubt lead to novel treatment strategies with improved clinical outcomes.

  View details for DOI 10.1016/j.cps.2004.10.003

  View details for Web of Science ID 000226935600013

  View details for PubMedID 15636770

• Noggin suppression enhances osteogenesis of murine osteoblasts 11th International Congress of the International-Society-of-Craniofacial-Surgery Wan, D. C., Pomerants, J. H., Nacamuli, R. P., Kim, J. B., Blau, H. M., Longaker, M. T. MEDIMOND S R L. 2005: 3–5

  View details for Web of Science ID 000243381600001

• Gene expression profile of the WNT family in cranial suture development 11th International Congress of the International-Society-of-Craniofacial-Surgery Malladi, P., Xu, Y., Nusse, R., Longaker, M. MEDIMOND S R L. 2005: 263–263

  View details for Web of Science ID 000243381600065

• Cranial suture biology CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY, VOL 66 Lenton, K. A., Nacamuli, R. P., Wan, D. C., Helms, J. A., Longaker, M. T. 2005; 66: 287-?

  View details for Web of Science ID 000228212000009

  View details for PubMedID 15797457

• Increased angiogenesis and expression of vascular endothelial growth factor during scarless repair PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Beanes, S. R., Soo, C., Dang, C., Ting, K., Longaker, M. T., Atkinson, J. B., Lorenz, H. P. 2005; 115 (1): 204-212

  Abstract

  Vascular endothelial growth factor (VEGF) is a dimeric heparin-binding glycoprotein that is a potent endothelial cell-specific mitogen with increased expression during adult cutaneous wound healing. VEGF activity is mediated by two receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), which are expressed primarily in vascular endothelial cells. Initiation of profibrotic cytokine expression likely coordinates the transition from scarless healing to scar formation in fetal wounds. Angiogenesis is an important component of the scarring repair process, but the function of VEGF and degree of angiogenesis during scarless repair has not been investigated. We hypothesize that VEGF and its receptors are differentially expressed in scarless compared with scarring fetal wounds because VEGF is implicated in angiogenesis during skin development and adult wound healing. Excisional wounds were created on fetal rats at gestational ages 16.5 days (E16) and 18.5 days (E18) (term = 21.5 days). Wounds were harvested at 24 and 72 hours (n = 12 wounds per time point). Nonwounded fetal skin (E17, E19, and E21) was used as control. Reduced-cycle, specific-primer, reverse-transcriptase polymerase chain reaction was performed to determine the expression of VEGF and its receptors, VEGFR-1 and VEGFR-2. Wounds at 72 hours and fetal skin controls were examined under high-power microscopy for blood vessel counts. Unpaired two-tailed t test was used (p < 0.05 was considered significant). VEGF expression increased 2.4-fold (p < 0.001) during normal skin development from E17 to E19. In scarless wounds (E16), VEGF expression increased 2.8-fold (p < 0.02) at 72 hours. No increased expression occurred in the scarring wounds (E18). VEGFR-1 and VEGFR-2 expression increased over 2-fold during normal skin development from E17 to E21. However, each was down-regulated 30 to 50 percent in scarless (E16) and scarring (E18) wounds. There is a 2-fold increase in mean vessel counts per high-power field in scarless (E16) wounds at 72 hours compared with age-matched control skin (p < 0.02) and a 1.7-fold increase in mean vessel count in scarring fetal wounds (E18) compared with age-matched control skin (p < 0.05). There is no difference in the total number of vessels found in scarless versus scarring wounds or between 19.5-day versus 21.5-day fetal skin. VEGF and its receptors, VEGFR-1 and VEGFR-2, increase expression during skin development and dermal differentiation. VEGF expression quickly elevates during scarless compared with scarring repair, which likely contributes to the more rapid scarless fetal repair rate. Similar numbers of new ves-sels are formed during scarless and scarring fetal repair.

  View details for DOI 10.1097/01.PRS.0000138252.51581.22

  View details for Web of Science ID 000226058400028

  View details for PubMedID 15622252

• The zebrafish (Danio rerio): a model system for cranial suture Patterning CELLS TISSUES ORGANS Quarto, N., Longaker, M. T. 2005; 181 (2): 109-118

  Abstract

  The zebrafish (Danio rerio) is an alluring model system currently used to study early embryonic development, organogenesis and gene functional analysis. However, few studies have been devoted to post-embryonic development. We have explored the possibility of using this organism to analyze how cranial suture patterning occurs. This study reports on the establishment of the zebrafish skull vault anatomy, calvarial osteogenesis, and cranial suture morphology. Our results demonstrate that the anatomy of the zebrafish cranial vault and cranial sutures is very similar to that of mammalian organisms. Indeed, the zebrafish represents a versatile and valuable model system for the study of the biogenesis of cranial sutures.

  View details for DOI 10.1159/000091100

  View details for Web of Science ID 000235916100005

  View details for PubMedID 16534205

• Mammalian fetal organ regeneration REGENERATIVE MEDICINE I: THEORIES, MODELS AND METHODS Colwell, A. S., Longaker, M. T., Lorenz, H. P. 2005; 93: 83-100

  Abstract

  The developing fetus has the remarkable ability to heal dermal skin wounds by regenerating normal epidermis and dermis with restoration of the extracellular matrix architecture, strength, and function. The biology responsible for scarless wound healing in skin is a paradigm for ideal tissue repair. This regenerative capacity is lost in late gestation when fetal wounds heal with fibrosis and scar. Early in gestation, fetal skin is developing at a rapid pace in a unique environment. Investigation of normal skin embryogenesis and comparison between early scarless and late scarring fetal wounds has revealed distinct differences in inflammatory response, cellular mediators, wound contraction, cytokines, growth factors, and extracellular matrix modulators. The knowledge gained from comparative observational studies has served as a base for experimental interventions in animal models to induce or ameliorate scar. Although much progress has been made over the past decade, the mechanism of fetal wound healing remains largely unknown and attempts to mimic the scarless wound phenotype have not been completely successful. Identification of more key genes involved in skin regeneration may have implications in adult skin wounds and repair in other organ systems.

  View details for DOI 10.1007/b99972

  View details for Web of Science ID 000228468300004

  View details for PubMedID 15791945

• High-dose retinoic acid modulates rat calvarial osteoblast biology JOURNAL OF CELLULAR PHYSIOLOGY Song, H. M., Nacamuli, R. P., Xia, W., Bari, A. S., Shi, Y. Y., Fang, T. D., Longaker, M. T. 2005; 202 (1): 255-262

  Abstract

  Retinoic acid has been shown to adversely affect craniofacial development. Cleft palate and craniosynostosis are two examples of craniofacial defects associated with prenatal exposure to this agent. Although the effects of retinoic acid on cephalic neural crest-derived tissues have previously been studied, the specific effects of retinoic acid on the cellular biology of osteoblasts remain unclear. The purpose of this study was to analyze in detail the effects of pharmacologic doses of retinoic acid on the differentiation and proliferation of osteoblasts derived from an intramembranous source. Primary rat calvarial osteoblasts were established in culture and treated with 1 or 10 microM all-trans-retinoic acid. Retinoic acid treatment markedly increased expression of osteopontin up to 48 h after stimulation. Consistent with this early stage of differentiation, both mRNA and protein analysis of FGF receptor isoforms demonstrated a switch in predominance from fibroblast growth factor receptor 2 (fgfr2) to fgfr1. Analysis of PCNA protein confirmed inhibition of proliferation by retinoic acid. To determine whether these alterations in osteoblast biology would lead to increased differentiation, we examined short term [alkaline phosphatase (AP) activity] and long term (von Kossa staining) surrogates of bone formation in vitro. These assays confirmed that retinoic acid increased osteogenesis, with a 4-fold increase in bone nodule formation in cells treated with 10 microM retinoic acid after 28 days. Overall, our results demonstrated that pharmacologic doses of all-trans-retinoic acid decreased osteoblast proliferation and increased differentiation, suggesting that retinoic acid may effect craniofacial development by pathologically enhancing osteogenesis.

  View details for DOI 10.1002/jcp.20115

  View details for Web of Science ID 000225514300027

  View details for PubMedID 15389522

• Cyclophilin C-associated protein is a mediator for fibronectin fragment-induced matrix metalloproteinase-13 expression JOURNAL OF BIOLOGICAL CHEMISTRY Kong, W. Y., Longaker, M. T., Lorenz, H. P. 2004; 279 (53): 55334-55340

  Abstract

  The function of cyclophilin C-associated protein (CyC-AP) on expression of extracellular matrix and matrix metalloproteinases (MMPs) was studied in CyC-AP-null mice. Fibronectin showed increased expression of the 53- and 29-kDa fragments in skin and wounds from CyC-AP-null mice. Type I collagen had an initial degraded pattern in the skin of CyC-AP-null mice, which did not occur in wild-type mice. MMP-3, MMP-13, MMP-14, and tumor necrosis factor-alpha (TNFalpha) had a higher expression in CyC-AP-null skin. During wound healing, MMP-13 and TNFalpha were stimulated to an even higher level, suggesting they are regulated by multiple factors. To understand the regulatory mechanisms of the up-regulated MMPs, the direct effects of TNFalpha, IL-1beta, 45-kDa fibronectin fragment (FN-45), and the 70-kDa fibronectin fragments (FN-70) on the expression of MMPs were studied. MMP-13 expression increased significantly in both CyC-AP-null and wild-type dermal fibroblasts after treatment with IL-1beta or with TNFalpha. However, MMP-13 expression did not increase in CyC-AP-null fibroblasts but did increase only in wild-type fibroblasts after FN-45 and FN-70 treatment. MMP-3 activation was induced by FN-45 and did not show a difference between CyC-AP-null and wild-type fibroblasts, suggesting different regulatory pathways for FN-45 on MMP-13 and MMP-3 expression. Our data are the first to demonstrate that deletion of CyC-AP can abolish fibronectin fragment-induced MMP-13 expression through an unknown mechanism. CyC-AP is an important factor for the regulation of MMP-13 expression.

  View details for DOI 10.1074/jbc.M410804200

  View details for Web of Science ID 000225960800045

  View details for PubMedID 15507443

• Quantitative transcriptional analysis of fusing and nonfusing cranial suture complexes in mice PLASTIC AND RECONSTRUCTIVE SURGERY Nacamuli, R. P., Song, H. J., Fang, T. D., Fong, K. D., Mathy, J. A., Shi, Y. Y., Salim, A., Longaker, M. T. 2004; 114 (7): 1818-1825

  Abstract

  Previous studies have documented the differences in expression of various genes associated with the process of osteogenesis in fusing and nonfusing cranial sutures, including growth factors, growth factor receptors, and extracellular matrix molecules. Most of these studies were performed in rats, and although the biology regulating cranial suture fusion in mice and rats is presumed to be similar, studies are needed to verify these expression patterns as mice become increasingly utilized for scientific inquiry into the molecular biology of suture fusion and patency. The purpose of this study was to determine the differences in expression of several genes known to be critical to osteoblast biology. Posterior frontal and sagittal suture complexes (including the associated dura mater, suture mesenchyme, and osteogenic fronts) were isolated from 5-, 15-, 25-, 35-, and 45-day-old male CD-1 mice (n = 8 per age; n = 40 total). Total cellular RNA was extracted and converted to cDNA. Quantitative real-time reverse transcriptase polymerase chain reaction was then performed for the following genes: transforming growth factor beta1 and beta3, fibroblast growth factor receptor 1, Runx2,Osteopontin, and Osteocalcin. Expression of all genes examined was increased significantly in the posterior frontal suture as compared with the sagittal suture. Peak expression for all genes was observed on day 25. These data demonstrate that the expression of osteogenic growth factors, growth factor receptors, transcription factors, and extracellular matrix molecules is increased in the fusing posterior frontal suture in mice.

  View details for DOI 10.1097/01.PRS.0000143578.41666.2C

  View details for Web of Science ID 000225578900018

  View details for PubMedID 15577353

• Molecular and cellular characterization of mouse calvarial osteoblasts derived from neural crest and paraxial-mesoderm 44th Annual Meeting of the American-Society-for-Cell-Biology Xu, Y., Malladi, P., Zhou, D., Helms, J. A., Longaker, M. T. AMER SOC CELL BIOLOGY. 2004: 351A–351A

  View details for Web of Science ID 000224648803015

• Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts JOURNAL OF BIOLOGICAL CHEMISTRY Salim, A., Nacamuli, R. P., Morgan, E. F., Giaccia, A. J., Longaker, M. T. 2004; 279 (38): 40007-40016

  Abstract

  Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O(2) (ambient oxygen), 2% O(2) (hypoxia), and <0.02% O(2) (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts.

  View details for DOI 10.1074/jbc.M403715200

  View details for Web of Science ID 000223791500098

  View details for PubMedID 15263007

• Applications of a mouse model of calvarial healing: Differences in regenerative abilities of juveniles and adults PLASTIC AND RECONSTRUCTIVE SURGERY Aalami, O. O., Nacamuli, R. P., Lenton, K. A., Cowan, C. M., Fang, T. D., Fong, K. D., Shi, Y. Y., Song, H. M., Sahar, D. E., Longaker, M. T. 2004; 114 (3): 713-720

  Abstract

  Young children are capable of healing large calvarial defects, whereas adults lack this endogenous osseous tissue-engineering capacity. Despite the important clinical implications, little is known about the molecular and cell biology underlying this differential ability. Traditionally, guinea pig, rabbit, and rat models have been used to study the orchestration of calvarial healing. To harness the research potential of knockout and transgenic mice, the authors developed a mouse model for calvarial healing. Nonsuture-associated parietal defects 3, 4, and 5 mm in diameter were made in both juvenile (6-day-old, n = 15) and adult (60-day-old, n = 15) mice. Calvariae were harvested after 8 weeks and analyzed radiographically and histologically. Percentage of healing was quantified using Scion Image software analysis of calvarial radiographs. A significant difference in the ability to heal calvarial defects was seen between 6-day-old and 60-day-old mice when 3-, 4-, or 5-mm defects were created. The authors' analysis revealed that juvenile mice healed a significantly greater percentage of their calvarial defects than adult mice (juvenile mean percentage of healing: 3-mm defects, 59 percent; 4-mm defects, 65 percent; 5-mm defects, 44 percent; adult mean percentage of healing: <5 percent in all groups; p < 0.05). All three defect sizes were found to be critical in the adult, whereas significant healing was seen regardless of the size of the defect in juvenile mice. The establishment of this model will facilitate further, detailed evaluation of the molecular biology underlying the different regenerative abilities of juvenile versus adult mice and enhance research into membranous bone induction by making available powerful tools such as knockout and transgenic animals.

  View details for DOI 10.1097/01.PRS.0000131016.12754.30

  View details for Web of Science ID 000223436200015

  View details for PubMedID 15318051

• Cranial neural crest-derived cells participate in craniofacial skeletal repair 90th Annual Clinical Congress of the American-College-of-Surgeons Nacamuli, R. P., Fang, T. D., Salim, A., Song, H., Shi, Y. Y., Hu, D., Miclau, T., Helms, J., Longaker, M. T. ELSEVIER SCIENCE INC. 2004: S48–S49

  View details for Web of Science ID 000223760800097

• Increased CTGF transcription in keloid fibroblasts requires cooperativity between AP-1 and SMAD binding sites 90th Annual Clinical Congress of the American-College-of-Surgeons Xia, W., Kong, W. Y., Phan, T. T., Lim, I., Longaker, M. T., Yang, G. ELSEVIER SCIENCE INC. 2004: S57–S57

  View details for Web of Science ID 000223760800118

• Complex epithelial-mesenchymal interactions modulate transforming growth factor-beta expression in keloid-derived cells WOUND REPAIR AND REGENERATION Xia, W., Phan, T. T., Lim, I. J., Longaker, M. T., Yang, G. P. 2004; 12 (5): 546-556

  Abstract

  Keloids are proliferative dermal growths representing a pathologic wound healing response. We have previously demonstrated that coculture of fibroblasts derived from either keloid or normal skin have an elevated proliferation rate when cocultured with keloid-derived keratinocytes vs. normal keratinocytes. In these studies, we examined the contribution of transforming growth factor-beta (TGF-beta) to this phenomenon using a two-chamber coculture system. Fibroblast proliferation in coculture was slower with the addition of a pan-TGF-beta neutralizing antibody. Keloid keratinocytes in coculture expressed more TGF-beta1, -beta3, and TGF-beta receptor 1 than normal keratinocytes. Keloid fibroblasts cocultured with keloid keratinocytes expressed more mRNA for TGF-beta1, -beta2, TGF-beta receptor 1, and Smad2. Keloid fibroblasts also produced more type I collagen, connective tissue growth factor, and insulin-like growth factor-II/mannose-6-phosphate receptor when cocultured with keloid keratinocytes vs. normal keratinocytes. Levels of total and activated TGF-beta activity increased when fibroblasts were cocultured with keratinocytes, correlating with the changes in transcriptional activity of TGF-beta. In conclusion, we find a complex paracrine interaction regulates TGF-beta mRNA expression and activation between keratinocytes and fibroblasts. These data suggest that keloid pathogenesis may result from both an increased TGF-beta production and activation by the keloid keratinocyte, and elevated TGF-beta expression, utilization, and signaling in keloid fibroblasts.

  View details for Web of Science ID 000224025400007

  View details for PubMedID 15453837

• Donor-derived, liver-specific protein expression after bone marrow transplantation TRANSPLANTATION Jenkins, D. D., Streetz, K., Tataria, M., Sahar, D., Kurobe, M., Longaker, M. T., Kay, M. A., Sylvester, K. G. 2004; 78 (4): 530-536

  Abstract

  Bone marrow transplantation (BMT) may represent a novel mechanism to deliver a functional gene to a deficient liver. Bone marrow-derived hepatocytes are rare and without a defined contribution to liver function. Consequently, the clinical significance of BMT to treat liver disease is unclear. We sought to quantify bone marrow-derived hepatocyte protein expression after BMT and determine whether the process is inducible with liver injury.Mice transgenic for human alpha-1 antitrypsin (hAAT) under a hepatocyte-specific promoter were used as bone marrow donors. Adenoviral transduction of modified urokinase plasminogen activator (Ad-muPA) was used to induce liver injury. Eight weeks after lethal irradiation and BMT, recipients were stratified into two groups: BMT alone (n = 5) and BMT + Ad-muPA (n= 10). Both groups of animals were bled before (t = 0) and at 2, 4, 8, and 16 weeks after Ad-muPA administration, and the serum samples were assessed for hAAT by enzyme-linked immunosorbent assay.Transgenic donor mice expressed 5 to 10 mg/mL of hAAT. Recipients of BMT alone expressed less than 80 ng/mL of hAAT over all time periods. Animals receiving BMT + Ad-muPA showed sustained and stable hAAT expression of approximately 200 ng/mL. Differences were statistically significant at each time point.Serum protein levels from liver-specific transgene expression are detectable and persist after BMT. Expression is low, but inducible with liver injury. We are currently developing strategies to augment donor-derived, liver-specific protein expression after BMT.

  View details for DOI 10.1097/01.TP.0000130180.42573.BI

  View details for Web of Science ID 000223486500008

  View details for PubMedID 15446311

• Modulation of FAK, Akt, and p53 by stress release of the fibroblast-populated collagen matrix (Retracted Article. See vol 121, pg 151, 2004) Carlson, M. A., Longaker, M. T., Thompson, J. S. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2004: 171–77

  Abstract

  Fibroblast survival in a three-dimensional collagen matrix is dependent in part upon the rigid anchorage of the matrix to tissue culture plastic. We hypothesized that focal adhesion kinase (FAK) and protein kinase B (Akt) would be activated and that the p53 level would be low in the rigidly anchored (attached) collagen matrix; loss of anchorage (detachment) was hypothesized to have the opposite effects.Human foreskin fibroblasts were cultured in attached bovine collagen matrices for 48 h before detachment as free-floating matrices. At various time points postrelease, matrix lysates were blotted for the proteins of interest, and the terminal deoxynucleotidyltransferase-mediated dUTP nick-end label assay was performed on both whole matrices and cytospin preparations. Irradiated monolayer fibroblasts were used as positive controls for the amount of p53 protein.Terminal deoxynucleotidyltransferase-mediated dUTP nick-end label positivity in attached versus detached matrices (at 24 h post detachment) was 0.7 +/- 03 versus 5.3 +/- 1.7% (P < 0.05, unpaired t test). FAK and Akt were phosphorylated (activated) in the attached matrix; there was a near complete of loss of both activated forms within 4 h of matrix detachment. Irradiated monolayer fibroblasts had increased levels of p53, mdm2, and p21. In contrast, the p53, mdm2, and p21 levels were just at the level of detection in the attached matrix, but were induced 5- to 10-fold within 2-4 h after matrix detachment.FAK and Akt are activated in the attached fibroblast-populated collagen matrix whereas the p53 level is relatively low; matrix detachment downregulates FAK and Akt activity and induces p53. The state of mechanical anchorage of the collagen matrix regulates the survival of embedded fibroblasts through a mechanism which may involve FAK.

  View details for DOI 10.1016/j.jss.2003.12.002

  View details for Web of Science ID 000222504300002

  View details for PubMedID 15234210

• Stem cell differentiation NATURE BIOTECHNOLOGY Salim, A., Giaccia, A. J., Longaker, M. T. 2004; 22 (7): 804-805

  View details for DOI 10.1038/nbt0704-803d

  View details for Web of Science ID 000222533000013

  View details for PubMedID 15229534

• Creation and characterization of a mouse model of mandibular distraction osteogenesis BONE Fang, T. D., Nacamuli, R. P., Song, H. M., Fong, K. D., Warren, S. M., Salim, A., Carano, R. A., Filvaroff, E. H., Longaker, M. T. 2004; 34 (6): 1004-1012

  Abstract

  While the histological and ultrastructural changes associated with distraction osteogenesis have been extensively characterized using various animal models, the molecular mechanisms governing this technique remain poorly understood. In the current study, for the first time, we describe a mouse mandibular distraction osteogenesis model. Development of this model will allow assessment of factors involved in normal vs. abnormal healing (especially in non-unions) of craniofacial skeletal elements. Complete osteotomies were created on the right hemimandibles of 51 adult male CD-1 mice and customized distraction devices attached. Thirty-three animals underwent gradual distraction (5 days latency, distraction at 0.2 mm BID x 8 days, 28 days consolidation), while the remaining 18 mice underwent acute lengthening (immediate distraction to 3.2 mm) at the time of surgery. Mandibles were harvested at time points corresponding to the latent (POD 5), distraction (POD 9, 13), and consolidation (POD 28, 41) periods and processed for histological or quantitative real-time RT-PCR analysis. Specimens from each group were processed for microCT analysis. Histological and radiological data demonstrated that all mandibles undergoing gradual distraction achieved complete bony union by the end of consolidation, while those undergoing acute lengthening formed a fibrous non-union. Quantitative real-time RT-PCR demonstrated upregulation of mRNA for VEGF, FGF-2, collagen I, and osteopontin during gradual distraction but not during acute lengthening. These data validate our novel mouse mandibular distraction model and demonstrate its utility in elucidating the molecular mechanisms regulating bone formation during distraction osteogenesis as compared to those that are expressed during the formation of fibrous non-unions.

  View details for DOI 10.1016/j.bone.2004.02.011

  View details for Web of Science ID 000222219600009

  View details for PubMedID 15193546

• Apoptosis in a rodent model of cranial suture fusion: In situ imaging and gene expression analysis PLASTIC AND RECONSTRUCTIVE SURGERY Fong, K. D., Song, H. M., Nacamuli, R. P., Franc, B. L., Mari, C., Fang, T. D., Warren, S. M., Contag, C. H., Blankenberg, F. G., Longaker, M. T. 2004; 113 (7): 2037-2047

  Abstract

  Craniosynostosis, the premature fusion of cranial sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-frontal suture fusion and sagittal suture patency to globally examine apoptosis in cranial sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m-labeled annexin V at time points before, during, and after the period of predicted posterior-frontal suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-frontal and sagittal suture complexes during the period of predicted posterior-frontal suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-frontal and sagittal sutures during the period of predicted posterior-frontal suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-frontal suture during fusion, activation of genes associated with the death receptor-mediated apoptotic pathway predominated in the patent sagittal suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing sutures is similar, the pathways mediating apoptosis within each suture are distinct.

  View details for DOI 10.1097/01.prs.000012118201199.c1

  View details for PubMedID 15253194

• Adipose-derived adult stromal cells heal critical-size mouse calvarial defects NATURE BIOTECHNOLOGY Cowan, C. M., Shi, Y. Y., Aalami, O. O., Chou, Y. F., Mari, C., Thomas, R., Quarto, N., Contag, C. H., Wu, B., Longaker, M. T. 2004; 22 (5): 560-567

  Abstract

  In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84-99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.

  View details for DOI 10.1038/nbt958

  View details for PubMedID 15077117

• Highlights of the proceedings from the 10th International Congress of the International Society of Craniofacial Surgery. journal of craniofacial surgery Lorenz, H. P., Longaker, M. T. 2004; 15 (3): 533-537

  View details for PubMedID 15111825

• Mechanisms of murine cranial suture patency mediated by a dominant negative transforming growth factor-beta receptor adenovirus PLASTIC AND RECONSTRUCTIVE SURGERY Song, H. J., Fong, D., Nacamuli, R. P., Warren, S. M., Fang, T. D., Mathy, J. A., Cowan, C. M., Aalami, O. O., Longaker, M. T. 2004; 113 (6): 1685-1697

  Abstract

  Using a physiologic model of mouse cranial suture fusion, the authors' laboratory has previously demonstrated that transforming growth factor (TGF)-betas appear to be more abundantly expressed in the suture complex of the fusing posterior frontal compared with the patent sagittal suture. Furthermore, the authors have shown that by blocking TGF-beta signaling with a replication-deficient adenovirus encoding a defective, dominant negative type II TGF-beta receptor (AdDN-TbetaRII), posterior frontal suture fusion was inhibited. In this study, the authors attempt to further elucidate the role of TGF-beta in cranial suture fusion by investigating possible mechanisms of AdDN-TbetaRII-mediated cranial suture patency using both an established organ culture model and a novel in vitro co-culture system that recapitulates the in vivo anatomic dura mater/cranial suture relationship. In this article, the authors demonstrate that blocking TGF-beta signaling with the AdDN-TbetaRII construct led to inhibition of cellular proliferation in the suture mesenchyme and subjacent dura mater during the early period of predicted posterior frontal suture fusion. Interestingly, co-culture experiments revealed that transfecting osteoblasts with AdDN-TbetaRII led to alterations in the gene expression levels of two important bone-related molecules (Msx2 and osteopontin). Inhibiting TGF-beta signaling prevented time-dependent suppression of Msx2 and prevented induction of osteopontin, thereby retarding osteoblast differentiation. Furthermore, the authors demonstrated that the AdDN-TbetaRII construct was capable of blocking TGF-beta -mediated up-regulation of collagen IalphaI, an extracellular matrix molecule important for bone formation. Collectively, these data strongly suggest that AdDN-TbetaRII maintains posterior frontal patency, in part by altering early events in de novo bone formation, including cellular proliferation and early extracellular matrix production.

  View details for DOI 10.1097/001.PRS.000117363.43699.5B

  View details for Web of Science ID 000221141000019

  View details for PubMedID 15114130

• In vitro murine posterior frontal suture fate is age-dependent: Implications for cranial suture biology PLASTIC AND RECONSTRUCTIVE SURGERY Song, H. J., Sahar, D. E., Fong, K. D., Nacamuli, R. P., Fang, T. D., Mathy, J. A., Aalami, O. O., Warren, S. M., Longaker, M. T. 2004; 113 (4): 1192-1204

  Abstract

  In CD-1 mice, the posterior frontal suture (analogous to the human metopic suture) fuses while all other cranial sutures remain patent. In an in vitro organ culture model, the authors previously demonstrated that posterior frontal sutures explanted immediately before the onset of suture fusion (at 25 days old) mimic in vivo physiologic fusion. In the first portion of this study, the authors defined how early in development the posterior frontal suture fuses in their tension-free, serum-free organ culture system by serially analyzing posterior frontal suture fusion from calvariae explanted at different stages of postnatal development. Their results revealed a divergence of suture fate leading to abnormal patency or physiologic fusion between the first and second weeks of life, respectively, despite viability and continued growth of the calvarial explants in vitro. From these data, the authors postulated that the gene expression patterns present in the suture complex at the time of explant may determine whether the posterior frontal suture fuses or remains patent in organ culture. Therefore, to elucidate potentially important differences in gene expression within this "window of opportunity," they performed a cDNA microarray analysis on 5-day-old and 15-day-old posterior frontal and sagittal whole suture complexes corresponding to the age ranges for unsuccessful (1 to 7 days old) and successful (14 to 21 days old) in vitro posterior frontal suture fusion. Overall, their microarray results reveal interesting differential expression patterns of candidate genes in different categories, including angiogenic cytokines and mechanosensitive genes potentially important in cranial suture biology.

  View details for DOI 10.1097/01.PRS.0000110203.90911.63

  View details for Web of Science ID 000220618600011

  View details for PubMedID 15083020

• Inhibition of TGF-beta-induced collagen production in rabbit flexor tendons. journal of hand surgery Zhang, A. Y., Pham, H., Ho, F., Teng, K., Longaker, M. T., Chang, J. 2004; 29 (2): 230-235

  Abstract

  Postoperative adhesions frequently compromise the success of flexor tendon repair. Manipulation of growth factors responsible for scar formation may be a method of decreasing adhesion formation. Transforming growth factor beta (TGF-beta) is a key cytokine in the pathogenesis of tissue fibrosis. The purpose of this study was to examine the effectiveness of TGF-beta neutralizing antibody in blocking TGF-beta-induced collagen I production in rabbit flexor tendons in vitro.Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF-beta along with increasing doses of TGF-beta neutralizing antibody (0.1-2.0 microg/mL). Collagen I production was measured by enzyme-linked immunoabsorbent assay and TGF-beta bioactivity was measured by the luciferase assay. Results were compared with TGF-beta alone and unsupplemented controls.The addition of neutralizing antibody significantly reduced TGF-beta-induced collagen I production in a dose-dependent manner in all 3 cell cultures. TGF-beta bioactivity was also reduced by its neutralizing antibody.This study shows that TGF-beta inhibition through its neutralizing antibody was effective in cultured flexor tendon cells. The results encourage further experiments that use such agents to modulate flexor tendon wound healing in in vivo models in the hope of eventually blocking the effect of TGF-beta on flexor tendons clinically.

  View details for PubMedID 15043894

• The fibroblast-populated collagen matrix as a model of wound healing: a review of the evidence WOUND REPAIR AND REGENERATION Carlson, M. A., Longaker, M. T. 2004; 12 (2): 134–47

  Abstract

  The fibroblast-populated collagen matrix (FPCM) has been utilized as an in vitro model of wound healing for more than 2 decades. It offers a reasonable approximation of the healing wound during the phases of established granulation tissue and early scar. The gross and microscopic morphology of the FPCM and the healing wound are similar at analogous phases. The processes of proliferation, survival/apoptosis, protein synthesis, and contraction act in similar directions in these two models, and the response to exogenous agents also is consistent between them. If its limitations are respected, then the FPCM can be used as a model of the healing wound.

  View details for DOI 10.1111/j.1067-1927.2004.012208.x

  View details for Web of Science ID 000220852800003

  View details for PubMedID 15086764

• Inhibition of TGF-beta-induced collagen production in rabbit flexor tendons JOURNAL OF HAND SURGERY-AMERICAN VOLUME Zhang, A. Y., Pham, H., Ho, F., Teng, K., Longaker, M. T., Chang, J. 2004; 29A (2): 230-235

  Abstract

  Postoperative adhesions frequently compromise the success of flexor tendon repair. Manipulation of growth factors responsible for scar formation may be a method of decreasing adhesion formation. Transforming growth factor beta (TGF-beta) is a key cytokine in the pathogenesis of tissue fibrosis. The purpose of this study was to examine the effectiveness of TGF-beta neutralizing antibody in blocking TGF-beta-induced collagen I production in rabbit flexor tendons in vitro.Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF-beta along with increasing doses of TGF-beta neutralizing antibody (0.1-2.0 microg/mL). Collagen I production was measured by enzyme-linked immunoabsorbent assay and TGF-beta bioactivity was measured by the luciferase assay. Results were compared with TGF-beta alone and unsupplemented controls.The addition of neutralizing antibody significantly reduced TGF-beta-induced collagen I production in a dose-dependent manner in all 3 cell cultures. TGF-beta bioactivity was also reduced by its neutralizing antibody.This study shows that TGF-beta inhibition through its neutralizing antibody was effective in cultured flexor tendon cells. The results encourage further experiments that use such agents to modulate flexor tendon wound healing in in vivo models in the hope of eventually blocking the effect of TGF-beta on flexor tendons clinically.

  View details for DOI 10.1016/j.jhsa.2003.11.005

  View details for Web of Science ID 000220362100009

• Sutural bone deposition rate and strain magnitude during cranial development BONE Henderson, J. H., Longaker, M. T., Carter, D. R. 2004; 34 (2): 271-280

  Abstract

  It is widely believed that rapid growth of the human brain generates tensile strain in cranial sutures, and that this strain influences the rate of bone deposition at the sutural margins during development. We developed general theoretical techniques for estimating sutural bone deposition rate and strain magnitude during mammalian cranial development. A geometry-based analysis was developed to estimate sutural bone deposition rate. A quasi-static stress analysis was developed to estimate sutural strain magnitude. We applied these techniques to the special case of normal cranial development in humans. The results of the bone deposition rate analysis indicate that average human sutural bone deposition rate is on the order of 100 microm/day at 1 month of age and decreases in an approximately exponential fashion during the first 4 years of life. The results of the strain analysis indicate that sutural strain magnitude is highly dependent on the assumed stiffness of the sutures, with estimated strain at 1 month of age ranging from approximately 20 to 400 microstrain. Regardless of the assumed stiffness of the sutures, the results indicate that sutural strain magnitude is small and decreases in an approximately exponential fashion during the first 4 years of life. The finding that both sutural bone deposition rate and strain magnitude decrease with increasing age is consistent with quasi-static tensile strain in sutures influencing sutural osteoblast activity in a dose-dependent manner. However, the small magnitude of the predicted strains suggests that tissue level strains in sutures may be too small to directly influence osteoblast biology. In light of these results, we suggest other biomechanical mechanisms, such as a tension-induced angiogenic environment in the sutures or mechanotransduction in the underlying dura mater, through which tension across sutures may regulate the rate of bone deposition in sutures.

  View details for DOI 10.1016/j.bone.2003.10.007

  View details for PubMedID 14962805

• [The molecular biology of distraction osteogenesis]. Revue de stomatologie et de chirurgie maxillo-faciale Boulétreau, P., Longaker, M. T. 2004; 105 (1): 23-25

  Abstract

  Distraction osteogenesis has become a mainstay in bone engineering and the recent application of this technique to the membranous craniofacial skeleton has significantly improved our armamentarium for reconstructive craniomaxillofacial procedures. However, if the biomechanical, histological and ultrastructural changes associated with distraction osteogenesis have been widely described, the molecular mechanisms governing the formation of new bone in the interfragmental gap of gradually distracted bone segments remain largely unclear. Recently, our laboratory has described a rat mandibular distraction model that provides an excellent environment for deciphering the molecular mechanisms that mediate distraction osteogenesis. In this Article, we present the hypotheses and current research that have furthered our knowledge of the molecular mechanisms that govern distraction osteogenesis. Recent studies have implicated a growing number of cytokines that are intimately involved in the regulation of bone synthesis and turnover. The gene regulation of numerous cytokines (Transforming Growth Factor-B, Bone Morphogenetic Proteins, Insulin-like Growth Factor-1, Fibroblast Growth Factor-2) during distraction osteogenesis have been best characterized and will be discussed in this text. We believe that novel systems like the rat model will facilitate our understanding of the biomolecular mechanisms that mediate membranous distraction osteogenesis and will ultimately guide the development of targeted-strategies designed to accelerate bone healing.

  View details for PubMedID 15041867

• Mechanobiology of mandibular distraction osteogenesis: experimental analyses with a rat model BONE Loboa, E. G., Fang, T. D., Warren, S. M., Lindsey, D. P., Fong, K. D., Longaker, M. T., Carter, D. R. 2004; 34 (2): 336-343

  Abstract

  We analyzed mechanobiological influences on successful distraction osteogenesis (DO). Mandibular distraction surgeries were performed on 15 adult male Sprague-Dawley rats. Animals underwent gradual distraction (GD), progressive lengthening by small increments (5-day latency followed by 0.25 mm distractions twice daily for 8 days followed by 28-day maturation period). Distracted hemimandibles were harvested on postoperative days (POD) 5, 7, 10, 13, and 41. Load-displacement curves were then recorded for ex vivo distractions of 0.25 mm and stresses determined. Histologically, new bone formation appeared in GD specimens on distraction day 2 (POD 7), filling 50-60% of the gap by distraction day 8 (POD 13), with nearly complete bony bridging at end maturation (POD 41). Average tensile strains imposed by each incremental distraction ranged from approximately 10% to 12.5% during distraction days 2-8 and were associated with bone apposition rates of about 260 microm/day. Because this GD protocol was previously determined to be optimal for DO, we conclude that strains within this range provide an excellent environment for de novo bone apposition. Distraction caused tissue damage in distraction day 2, 5, and 8 specimens as evidenced by distinct drops in the load/displacement curves. Taken together, our interpretation of these data is that daily distractions cause daily tissue damage which triggers new mesenchymal tissue formation.

  View details for DOI 10.1016/j.bone.2003.10.012

  View details for Web of Science ID 000220014000011

  View details for PubMedID 14962812

• Flexor tendon wound healing in vitro: Lactate up-regulation of TGF-beta expression and functional activity 47th Annual Meeting of the Plastic-Surgery-Research-Council Yalamanchi, N., Klein, M. B., Pham, H. M., Longaker, M. T., Chang, J. LIPPINCOTT WILLIAMS & WILKINS. 2004: 625–32

  Abstract

  Flexor tendon wound healing in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. These adhesions can significantly alter tendon gliding and ultimately hand function. Lactate and transforming growth factor-beta (TGF-beta) are two important mediators of wound healing that have been demonstrated to independently increase collagen production by cells of the tendon sheath, epitenon, and endotenon. This study examined the effects of lactate on TGF-beta peptide and receptor production by flexor tendon cells. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. Cell cultures were supplemented with 50 mM lactate, and the expression of three TGF-beta peptide isoforms (beta1, beta2, and beta3) and three receptor isoforms (R1, R2, and R3) was quantified with enzyme-linked immunosorbent assays. TGF-beta functional activity was also assessed with the addition of tendon cell conditioned media to mink lung epithelial cells transfected with a luciferase reporter gene expression construct responsive to TGF-beta. Supplementation of the cell culture medium with lactate significantly (p < 0.05) increased the expression of all TGF-beta peptide and receptor isoforms in all three cell lines. Tendon sheath fibroblasts exhibited the greatest increases in beta1 and beta2 peptide isoform expression (30 and 23 percent, respectively), whereas endotenon tenocytes demonstrated the greatest increase in beta3 peptide expression (32 percent). Epitenon tenocytes exhibited the greatest increases in receptor isoform R1 and R2 expression (17 and 19 percent, respectively). All three tendon cell types demonstrated significant (p < 0.05) increases in TGF-beta functional activity when exposed to lactate. Epitenon tenocytes demonstrated the greatest increase in activity (>4 times control values), whereas tendon sheath fibroblasts demonstrated the highest overall levels of total TGF-beta functional activity. Lactate significantly increased TGF-beta peptide (beta1, beta2, and beta3) expression, receptor (R1, R2, and R3) expression, and functional activity, suggesting a common pathway regulating tendon cell collagen production. Modulation of lactate and TGF-beta levels may provide a means of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.

  View details for DOI 10.1097/01.PRS.0000101529.47062.34

  View details for Web of Science ID 000220063300023

  View details for PubMedID 14758225

• Tissue-engineered bone using mesenchymal stem cells and a biodegradable scaffold JOURNAL OF CRANIOFACIAL SURGERY Warren, S. M., Nacamuli, R. K., Song, H. J., Longaker, M. T. 2004; 15 (1): 34-37

  View details for Web of Science ID 000225848500011

  View details for PubMedID 14704559

• Stem cells - Review and update ARCHIVES OF SURGERY Sylvester, K. G., Longaker, M. T. 2004; 139 (1): 93-99

  Abstract

  Regenerative medicine and emerging biotechnologies stand to revolutionize the practice of medicine. Advancements in stem cell biology, including embryonic and postnatal somatic stem cells, have made the prospect of tissue regeneration a potential clinical reality. Short of reproductive cloning, these same technologies, properly used, could allow for the creation of replacement tissue for the deficient host. To provide a concise review for surgeons on the current science and biology of stem cells, we surveyed the scientific literature, MEDLINE, and relevant political headlines that illuminate the stem cell discussion; the issues are summarized in this review. Building on this conceptual framework, the related issues of clinical promise and the political debate enveloping this emerging technology are examined. A basic understanding of stem cell biology is paramount to stay informed of this emerging technology and the national debate.

  View details for Web of Science ID 000187998500022

  View details for PubMedID 14718284

• From scarless fetal wounds to keloids: Molecular studies in wound healing WOUND REPAIR AND REGENERATION Yang, G. P., Lim, I. J., Phan, T. T., Lorenz, H. P., Longaker, M. T. 2003; 11 (6): 411-418

  Abstract

  Surgical researchers were among the first to describe the different phases of wound healing and the events in tissue repair and regeneration that were taking place during each phase. The understanding of these events has been significantly enhanced in recent years by modern techniques in molecular and cellular biology. In this article, we discuss new findings in scarless fetal repair, angiogenesis in wound healing, and keloid pathogenesis. This serves to highlight the advances that have been made and also how much remains to be understood.

  View details for Web of Science ID 000186779200006

  View details for PubMedID 14617279

• Mechanical strain affects dura mater biological processes: Implications for immature calvarial healing PLASTIC AND RECONSTRUCTIVE SURGERY Fong, K. D., Warren, S. M., Loboa, E. G., Henderson, J. H., Fang, T. D., Cowan, C. M., Carter, D. R., Longaker, M. T. 2003; 112 (5): 1312-1327

  Abstract

  The human brain grows rapidly during the first 2 years of life. This growth generates tensile strain in the overlying dura mater and neurocranium. Interestingly, it is largely during this 2-year growth period that infants are able to reossify calvarial defects. This clinical observation is important because it suggests that calvarial healing is most robust during the period of active intracranial volume expansion. With a rat model, it was previously demonstrated that immature dura mater proliferates more rapidly and produces more osteogenic cytokines and markers of osteoblast differentiation than does mature dura mater. It was therefore hypothesized that mechanical strain generated by the growing brain induces immature dura mater proliferation and increases osteogenic cytokine expression necessary for growth and healing of the overlying calvaria. Human and rat (n = 40) intracranial volume expansion was calculated as a function of age. These calculations demonstrated that 83 percent of human intracranial volume expansion is complete by 2 years of age and 90 percent of Sprague-Dawley rat intracranial volume expansion is achieved by 2 months of age. Next, the maximal daily circumferential tensile strains that could be generated in immature rat dura mater were calculated, and the corresponding daily biaxial tensile strains in the dura mater during this 2-month period were determined. With the use of a three-parameter monomolecular growth curve, it was calculated that rat dura mater experiences daily equibiaxial strains of at most 9.7 percent and 0.1 percent at birth (day 0) and 60 days of age, respectively. Because it was noted that immature dural cells may experience tensile strains as high as approximately 10 percent, neonatal rat dural cells were subjected to 10 percent equibiaxial strain in vitro, and dural cell proliferation and gene expression profiles were analyzed. When exposed to mechanical strain, immature dural cells rapidly proliferated (5.8-fold increase in proliferating cell nuclear antigen expression at 24 hours). Moreover, mechanical strain induced marked up-regulation of dural cell osteogenic cytokine production; transforming growth factor-beta1 messenger RNA levels increased 3.4-fold at 3 hours and fibroblast growth factor-2 protein levels increased 4.5-fold at 24 hours and 5.6-fold at 48 hours. Finally, mechanical strain increased dural cell expression of markers of osteoblast differentiation (2.8-fold increase in osteopontin levels at 3 hours). These findings suggest that mechanical strain can induce changes in dura mater biological processes and gene expression that may play important roles in coordinating the growth and healing of the neonatal calvaria.

  View details for DOI 10.1097/01.PRS.0000079860.14734.D6

  View details for PubMedID 14504515

• Markers of osteoblast differentiation in fusing and nonfusing cranial sutures PLASTIC AND RECONSTRUCTIVE SURGERY Nacamuli, R. P., Fong, K. D., Warren, S. M., Fang, T. D., Song, H. M., Helms, J. A., Longaker, M. T. 2003; 112 (5): 1328-1335

  Abstract

  Accumulating clinical genetic data support the hypothesis that alterations in osteoblast differentiation are closely associated with craniosynostoses. Gain-of-function mutations in FGFR1, FGFR2, FGFR3, and Msx2 and loss-of-function mutations in Twist are examples of such alterations. Several studies have examined how these mutations alter the expression patterns for transcription factors such as Runx2 and noncollagenous extracellular matrix molecules such as osteopontin and osteocalcin. One limitation of such studies is that they examine samples derived from craniosynostotic patients with sutures that have already fused, thus missing the dynamic osteogenic process of suture fusion. In this study, in situ hybridization was used to localize Runx2, osteopontin, and osteocalcin expression in the sagittal and posterior frontal sutures in mice (n = 20), before (day 13), during (days 23, 33, and 43), and after (day 53) the period of physiological posterior frontal suture fusion. The data demonstrated similar patterns of expression in fusing (posterior frontal) and nonfusing (sagittal) sutures. The expression of all three genes was primarily concentrated in the osteogenic fronts of both sutures and decreased with time. Notably, none of the three genes was expressed in the mesenchyme of either fusing or nonfusing sutures. The data suggest that the molecular signals leading to bone formation along the osteogenic fronts in fusing and nonfusing sutures are similar, raising the possibility that other factors, such as antagonists of osteogenesis, might have a role in maintaining suture patency.

  View details for DOI 10.1097/01.PRS.0000079826.24086.CD

  View details for Web of Science ID 000220062700015

  View details for PubMedID 14504516

• Tissue engineering and regenerative medicine CLINICS IN PLASTIC SURGERY Jenkins, D. D., Yang, G. P., Lorenz, H. P., Longaker, M. T., Sylvester, K. G. 2003; 30 (4): 581-?

  Abstract

  Regenerative medicine is evolving toward a powerful new paradigm of functional restoration. With the ethical use of gene therapy or through the manipulation of autologous tissues, improved tissue replacements may soon be available. The promise of engineered whole organs, although fraught with technical hurdles, remains on the horizon. As these advances occur, physicians and surgeons of the twenty-first century will possess ever more powerful tools to restore form and function.

  View details for DOI 10.1016/S0094-1298(03)00076-2

  View details for Web of Science ID 000186313400011

  View details for PubMedID 14621306

• Mechanical signal transduction in normal versus keloid fibroblasts 89th Annual Clinical Congress of the American-College-of-Surgeons Wang, Z., Phan, T. T., Lim, I., Yang, G., Longaker, M. ELSEVIER SCIENCE INC. 2003: S52–S52

  View details for Web of Science ID 000185248100127

• Age-associated differences in adipose-derived mesenchymal stromal cells 89th Annual Clinical Congress of the American-College-of-Surgeons Shi, Y. Y., Nacamuli, R., Fong, K., Salim, A., Fang, T., Song, M., Cowan, K., Longaker, M. ELSEVIER SCIENCE INC. 2003: S46–S46

  View details for Web of Science ID 000185248100109

• High dose retinoic acid modulates rat calvarial osteoblast biology 89th Annual Clinical Congress of the American-College-of-Surgeons Song, M. H., Nacamuli, R., Bari, S., Fong, K., Fang, T., Shi, Y. Y., Warren, S., Longaker, M. ELSEVIER SCIENCE INC. 2003: S47–S47

  View details for Web of Science ID 000185248100112

• Signal transduction in keloid versus normal human fibroblasts following serum stimulation 89th Annual Clinical Congress of the American-College-of-Surgeons Xia, W., Phan, T. T., Lim, I., Yang, G., Longaker, M. ELSEVIER SCIENCE INC. 2003: S53–S53

  View details for Web of Science ID 000185248100128

• Age-related changes in the biomolecular mechanisms of clvarial osteoblast biology affect fibroblast growth factor-2 signaling and osteogenesis JOURNAL OF BIOLOGICAL CHEMISTRY Cowan, C. M., Quarto, N., Warren, S. M., Salim, A., Longaker, M. T. 2003; 278 (34): 32005-32013

  Abstract

  The ability of immature animals to orchestrate successful calvarial ossification has been well described. This capacity is markedly attenuated in mature animals and humans greater than 2 years of age. Few studies have investigated biological differences between juvenile and adult osteoblasts that mediate successful osteogenesis. To identify possible mechanisms for this clinical observation, we investigated cellular and molecular differences between primary osteoblasts derived from juvenile (2-day-old) and adult (60-day-old) rat calvaria. Data demonstrated that juvenile osteoblasts contain a subpopulation of less differentiated cells as observed by spindle-like morphology and decreased osteocalcin production. Juvenile, compared with adult, osteoblasts showed increased proliferation and adhesion. Furthermore, following rhFGF-2 stimulation juvenile osteoblasts increased expression of collagen I alpha 1 (5-fold), osteopontin (13-fold), and osteocalcin (16-fold), compared with relatively unchanged adult osteoblasts. Additionally, juvenile osteoblasts organized and produced more matrix proteins and formed 41-fold more bone nodules. Alternatively, adult osteoblasts produced more FGF-2 and preferentially translated the high molecular weight (22 kDa) form. Although adult osteoblasts transcribed more FGF-R1 and juvenile osteoblasts transcribed more FGF-R2 at baseline levels, juvenile osteoblasts translated more FGF-R1 and -R2 and showed increased phosphorylation. Collectively, these findings begin to explain why juvenile, but not adult, osteoblasts successfully heal calvarial defects.

  View details for DOI 10.1074/jbc.M304698200

  View details for Web of Science ID 000184782100070

  View details for PubMedID 12788918

• FGF-2 stimulation affects calvarial osteoblast biology: Quantitative analysis of nine genes important for cranial suture biology by real-time reverse transcription polymerase chain reaction PLASTIC AND RECONSTRUCTIVE SURGERY Mathy, J. A., Lenton, K., Nacamuli, R. P., Fong, K. D., Song, H. M., Fang, T. D., Yang, G. P., Longaker, M. T. 2003; 112 (2): 528-539

  Abstract

  Appropriately timed closure of the cranial sutures is a critical factor in normal postnatal morphogenesis of the cranial vault. Suture patency is necessary to permit rapid neonatal expansion of the cerebral hemispheres, and later ossification is important for bony protection of the cerebrum. Premature suture ossification (craniosynostosis) leads to myriad adverse functional and developmental consequences. Several murine studies have implicated dura-derived fibroblast growth factor-2 (FGF-2) paracrine signaling as a critical factor promoting physiologic posterior frontal suture fusion. In this study, the authors used real-time reverse transcription polymerase chain reaction (RT-PCR) to study an in vitro system that models the in vivo stimulation of suture calvarial osteoblasts by dura-derived FGF-2. The authors advocate real-time RT-PCR as a powerful and rapid technique that offers advantages in the highly sensitive, specific, and reproducible analyses of nine genes known to be important in cranial suture biology. The genes studied were growth factors [FGF-2, transforming growth factor (TGF)-beta 1, TGF-beta 2, and TGF-beta 3], growth factor receptors (FGF-R1, FGF-R2, TGF-beta RI, and TGF-beta RII), and a marker of osteoblast differentiation (Co1-I alpha I). These analyses provide a "snapshot" of several important genes involved in suture fusion that is more inclusive and quantitative than that which has been previously reported.

  View details for DOI 10.1097/01.PRS.0000070729.05978.BB

  View details for Web of Science ID 000184532700020

  View details for PubMedID 12900611

• The influence of temperature on the degradation rate of LactoSorb Copolymer. Pietrzak WS, Kumar M, Eppley BL. J Craniofac Surg 2003; 14:176-183. journal of craniofacial surgery Fong, K. D., Nacamuli, R. P., Longaker, M. T. 2003; 14 (4): 594-595

  View details for PubMedID 12947922

• Abstinence from smoking reduces incisional wound infection: A randomized, controlled triol ANNALS OF SURGERY Yang, G. P., Longaker, M. T. 2003; 238 (1): 6–8

  View details for DOI 10.1097/01.SLA.0000074966.51219.eb

  View details for Web of Science ID 000185834900002

  View details for PubMedID 12832960

  View details for PubMedCentralID PMC1422653

• Porous polymethylmethacrylate as bone substitute in the craniofacial area. Bruens ML, Pieterman H, de Wijn JR, et al. J Craniofac Surg 2003; 14:63-68. journal of craniofacial surgery Lenton, K. A., Nacamuli, R. P., Longaker, M. T. 2003; 14 (4): 596-598

  View details for PubMedID 12947924

• In utero surgery for cleft lip/palate: Minimizing the "ripple effect" of scarring JOURNAL OF CRANIOFACIAL SURGERY Lorenz, H. P., Longaker, M. T. 2003; 14 (4): 504-511

  Abstract

  Surgical intervention is currently performed on highly selected fetuses with anatomical deformities that have a high mortality or severe morbidity when treated postnatally. In the future, in utero surgical intervention for non-life-threatening disease may become possible as fetal surgery becomes safer for the mother and fetus. Fetal cleft repair is an attractive intervention for plastic surgeons because it affords the potential to provide a scarless repair and correct the primary deformity. Furthermore, scarless fetal lip and palate repairs may prevent the ripple effect of postnatal scarring with its resultant secondary dentoalveolar and midface growth deformities. These potential benefits can dramatically reduce the number of postnatal reconstructive procedures in children with facial clefts. The rationale for a prenatal treatment approach to the patient with cleft lip/palate and the experimental evidence to support in utero intervention are discussed in this article.

  View details for Web of Science ID 000184409100019

  View details for PubMedID 12867864

• Molecular cloning and expression of keratinocyte proline-rich protein, a novel squamous epithelial marker isolated during skin development JOURNAL OF BIOLOGICAL CHEMISTRY Kong, W. Y., Longaker, M. T., Lorenz, H. P. 2003; 278 (25): 22781-22786

  Abstract

  We describe a novel rat cDNA named keratinocyte proline-rich protein (KPRP) isolated by RNA differential display during skin development. We determine that KPRP is expressed in stratified squamous epithelium, and its approximately 2.8-kb cDNA encodes a 699-amino acid protein with high proline content (19%). KPRP is an insoluble protein, similar to most epidermal terminal differentiation-associated proteins. Immunoblot of the protein lysate from keratinocytes, using strong reducing conditions, demonstrates two KPRP bands of approximately 76 and 55 kDa size. KPRP is expressed in stratified squamous epithelia of skin, tongue, and esophagus. The initiation of KPRP expression in fetal rat skin at E17, E18, E19, E20, and E21 was analyzed by reverse transcription-PCR. Fetal skin at E19 and later expresses KPRP. In situ hybridization of skin from E18, E19, and 4-day-old neonatal rats demonstrates that interfollicular and follicular keratinocytes express KPRP. Anti-KPRP antibody demonstrates KPRP protein localizes to all layers of stratified epithelia in skin, tongue, and esophagus. In cultured dermal keratinocytes, KPRP is diffusely distributed throughout the cytoplasm with denser staining adjacent to the nuclear and plasma membranes. Additionally, immunoreactive intracellular granules are observed during keratinocyte detachment from their plastic substrate. Rat KPRP has 89% homology to a mouse genomic DNA sequence and 56% homology to a human hypothetical protein. We conclude that KPRP may be a new epidermal terminal differentiation-related protein expressed in stratified squamous epithelia. KPRP is expressed by fetal dermal keratinocytes during late gestation and is a new marker of maturing epidermis during fetal skin development.

  View details for DOI 10.1074/jbc.M210488200

  View details for Web of Science ID 000183503900081

  View details for PubMedID 12668678

• New strategies for craniofacial repair and replacement: A brief review JOURNAL OF CRANIOFACIAL SURGERY Fong, K. D., Nacamuli, R. P., Song, H. J., Warren, S. M., Lorenz, H. P., Longaker, M. T. 2003; 14 (3): 333-339

  Abstract

  Craniofacial anomalies can severely affect the appearance, function, and psychosocial well being of patients; thus, tissue engineers are developing new techniques to functionally and aesthetically rebuild craniofacial structures. In the past decade, there have been tremendous advances in the field of tissue engineering that will substantially alter how surgeons approach craniofacial reconstruction. In this brief review, we highlight some of the preclinical recombinant protein, gene transfer, and cell-based strategies currently being developed to augment endogenous tissue repair or create structures for replacement. In addition, we discuss the importance of studying endogenous models of tissue induction and present some of the current in vitro and in vivo approaches to growing complex tissues/organs for craniofacial reconstruction.

  View details for Web of Science ID 000183221300011

  View details for PubMedID 12826804

• Scientific foundations - Equibiaxial tensile strain affects calvarial osteoblast biology JOURNAL OF CRANIOFACIAL SURGERY Fong, K. D., Nacamuli, R. P., Loboa, E. G., Henderson, J. H., Fang, T. D., Song, H. M., Cowan, C. M., Warren, S. M., Carter, D. R., Longaker, M. T. 2003; 14 (3): 348-355

  Abstract

  Mechanical tensile strain is believed to play an important role in regulating calvarial morphogenesis. To better understand the effects of mechanical strain on pathologic calvarial growth, we applied 10% constant equibiaxial tensile strain to neonatal rat calvarial osteoblast cultures and examined cellular proliferation, cytokine production, and extracellular matrix molecule expression. Mechanical strain markedly increased osteoblast proliferation as demonstrated by increased proliferating cell nuclear antigen (PCNA) protein. In addition, both transforming growth factor-beta1 (TGF-beta1) mRNA expression and fibroblast growth factor-2 (FGF-2) protein production were increased with exposure to strain. Moreover, mechanical strain induced expression of the extracellular matrix molecule collagen IalphaI. To further explore the relationship between mechanotransduction, osteogenesis, and angiogenesis, we examined the effect of mechanical strain on calvarial osteoblast expression of vascular endothelial growth factor (VEGF). Interestingly, we found that mechanical strain induced a rapid (within 3 hrs) increase in osteoblast VEGF expression. These data suggest that constant equibiaxial tensile strain-induced mechanotransduction can influence osteoblasts to assume an "osteogenic" and "angiogenic" phenotype, and these findings may have important implications for understanding the mechanisms of pathologic strain-induced calvarial growth.

  View details for Web of Science ID 000183221300013

• Regional dura mater differentially regulates osteoblast gene expression JOURNAL OF CRANIOFACIAL SURGERY Warren, S. M., Greenwald, J. A., Nacamuli, R. P., Fong, K. D., Song, H. J., Fang, T. D., Mathy, J. A., Longaker, M. T. 2003; 14 (3): 363-370

  Abstract

  Recent studies have suggested that regionally differentiated dura mater regulates murine cranial suture fate by providing growth factors to the osteoblasts in the overlying suture complex. To determine if regionally differentiated dura mater is capable of effecting changes in osteoblast gene expression, an in vitro coculture system was established in which osteoblast-enriched cell cultures derived from neonatal rat calvaria were grown in serum-free media in the presence of dural cells derived from posterior frontal (PF) or sagittal (SAG) dural tissues, recapitulating the in situ relation between the underlying dura mater and the osteoblasts in the overlying cranial suture. In this study, the changes in osteoblast gene expression induced by signaling from regional dura mater were examined by analyzing total cellular RNA isolated from osteoblasts cocultured with PF or SAG dural cells. The expression of extracellular matrix molecules (alkaline phosphatase, bone sialoprotein, osteopontin, and osteocalcin) and the transcription factor Msx2 was assessed. Consistent with previous data, the findings demonstrate that osteoblasts cocultured with dural cells undergo changes in gene expression indicative of a more differentiated osteoblast. Additionally, the data suggest that regionally differentiated dura mater isolated from the PF suture enhances the expression of osteogenic genes to a greater extent than SAG suture-derived dural cells. These data support an osteoinductive role for suture-derived dural cells in vitro that may have implications for suture biology in vivo.

  View details for Web of Science ID 000183221300015

  View details for PubMedID 12826808

• Equibiaxial tensile strain affects calvarial osteoblast biology. journal of craniofacial surgery Fong, K. D., Nacamuli, R. P., Loboa, E. G., Henderson, J. H., Fang, T. D., Song, H. M., Cowan, C. M., Warren, S. M., Carter, D. R., Longaker, M. T. 2003; 14 (3): 348-355

  Abstract

  Mechanical tensile strain is believed to play an important role in regulating calvarial morphogenesis. To better understand the effects of mechanical strain on pathologic calvarial growth, we applied 10% constant equibiaxial tensile strain to neonatal rat calvarial osteoblast cultures and examined cellular proliferation, cytokine production, and extracellular matrix molecule expression. Mechanical strain markedly increased osteoblast proliferation as demonstrated by increased proliferating cell nuclear antigen (PCNA) protein. In addition, both transforming growth factor-beta1 (TGF-beta1) mRNA expression and fibroblast growth factor-2 (FGF-2) protein production were increased with exposure to strain. Moreover, mechanical strain induced expression of the extracellular matrix molecule collagen IalphaI. To further explore the relationship between mechanotransduction, osteogenesis, and angiogenesis, we examined the effect of mechanical strain on calvarial osteoblast expression of vascular endothelial growth factor (VEGF). Interestingly, we found that mechanical strain induced a rapid (within 3 hrs) increase in osteoblast VEGF expression. These data suggest that constant equibiaxial tensile strain-induced mechanotransduction can influence osteoblasts to assume an "osteogenic" and "angiogenic" phenotype, and these findings may have important implications for understanding the mechanisms of pathologic strain-induced calvarial growth.

  View details for PubMedID 12826806

• The BMP antagonist noggin regulates cranial suture fusion NATURE Warren, S. M., Brunet, L. J., Harland, R. M., Economides, A. N., Longaker, M. T. 2003; 422 (6932): 625-629

  Abstract

  During skull development, the cranial connective tissue framework undergoes intramembranous ossification to form skull bones (calvaria). As the calvarial bones advance to envelop the brain, fibrous sutures form between the calvarial plates. Expansion of the brain is coupled with calvarial growth through a series of tissue interactions within the cranial suture complex. Craniosynostosis, or premature cranial suture fusion, results in an abnormal skull shape, blindness and mental retardation. Recent studies have demonstrated that gain-of-function mutations in fibroblast growth factor receptors (fgfr) are associated with syndromic forms of craniosynostosis. Noggin, an antagonist of bone morphogenetic proteins (BMPs), is required for embryonic neural tube, somites and skeleton patterning. Here we show that noggin is expressed postnatally in the suture mesenchyme of patent, but not fusing, cranial sutures, and that noggin expression is suppressed by FGF2 and syndromic fgfr signalling. Since noggin misexpression prevents cranial suture fusion in vitro and in vivo, we suggest that syndromic fgfr-mediated craniosynostoses may be the result of inappropriate downregulation of noggin expression.

  View details for DOI 10.1038/nature01545

  View details for Web of Science ID 000182111400045

  View details for PubMedID 12687003

• Tools and techniques for craniofacial tissue engineering TISSUE ENGINEERING Warren, S. M., Fong, K. D., Chen, C. M., Loboa, E. G., Cowan, C. M., Lorenz, H. P., Longaker, M. T. 2003; 9 (2): 187-200

  Abstract

  Craniofacial surgery is an important conduit for tissue-engineering applications. As interdisciplinary collaborations improve, we can expect to see remarkable progress in de novo tissue synthesis, replacement, and repair. Ultimately, we may one day find that gene-modified cell-based tissue-engineering strategies will succeed today's reconstructive strategies. In this review, we highlight the major gene- and cell-based preclinical tools and techniques that are currently being developed to solve common craniofacial problems.

  View details for Web of Science ID 000182336200001

  View details for PubMedID 12740082

• Reversed latissimus dorsi muscle flap for repair of recurrent congenital diaphragmatic hernia 33rd Annual Meeting of the American-Pediatric-Surgical-Association Sydorak, R. M., Hoffman, W., LEE, H., Yingling, C. D., Longaker, M., Chang, J., Smith, B., Harrison, M. R., Albanese, C. T. W B SAUNDERS CO-ELSEVIER INC. 2003: 296–300

  Abstract

  Neonates with large congenital diaphragmatic hernias (CDH) require prosthetic patch closure of the defect because of the paucity of native diaphragmatic tissue. As the child grows, patch separation can occur necessitating reoperation. Use of vascularized autologous tissue may decrease the incidence of reherniation as tissue incorporation and growth may be improved. The authors report our early experience using a local muscle advancement flap with microneural anastomosis for those children in whom reherniation develops after prosthetic patch placement.Seven patients with CDH (6 left and 1 right) whose synthetic diaphragmatic patch separated from the chest wall resulting in a clinically significant recurrent hernia were followed up with prospectively. After dissecting the ipsilateral latissimus dorsi off the chest wall and dividing the thoracodorsal neurovascular bundle (based on its lumbar blood supply), the synthetic patch was removed via an eighth intercostal incision. The muscle flap was placed into the hemithorax through the bed of the tenth rib and sutured in place over a Vicryl mesh scaffold. The thoracodorsal nerve was anastomosed to the phrenic nerve. Functional analysis of the flap was performed in 4 patients.Age at placement of the muscle graft ranged from 2 months to 48 months (median, 24 months). There has been no evidence of reherniation after placement of the muscle graft. Long-term outcome and functional analysis of the flap was available in 4 patients (mean, 19 months). Two infants had fluoroscopic and sonographic evidence of nonparadoxical neodiaphragmatic motion. In one of these, electromyographic evidence of function was documented with a phrenic nerve conduction velocity of 22 meters per second. The third infant showed no evidence of neodiaphragmatic motion, and the fourth infant had paradoxical motion.This is the first direct documentation of phrenic nerve function in an infant with CDH. An innervated reversed latissimus dorsi (RLD) flap reconstruction for recurrent CDH provides an alternative to prosthetic patch repair. This technique offers the advantages of autologous vascularized tissue with potential phrenic nerve innervation and physiologic neodiaphragmatic motion.

  View details for DOI 10.1053/jpsu.2003.50097

  View details for Web of Science ID 000181293000007

  View details for PubMedID 12632338

• Wound splinting regulates granulation tissue survival Carlson, M. A., Longaker, M. T., Thompson, J. S. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2003: 304–9

  Abstract

  Fibroblast survival within an in vitro collagen matrix is dependent on matrix anchorage to a rigid substratum. The purpose of this study was to determine whether granulation tissue survival in vivo also is dependent on matrix anchorage. We hypothesized that splinting an excisional wound (i.e., anchoring the wound edges) would promote granulation tissue survival and that desplinting a splinted wound would produce granulation tissue apoptosis.Eighteen Wistar rats (3 months, 350 g) underwent excisional wounding (2 x 2 cm, dorsal skin) with immediate wound splinting (a metal template affixed with sutures) on day 0. On day 6, rats (n = 6 per group) underwent splint removal (desplinted), splint removal with circumferential incision of the wound edge (desplint/release), or no intervention (splinted); sacrifice of all animals was on day 7. Frozen sections of granulation tissue were stained with TUNEL or H and E; data were analyzed with ANOVA and the unpaired t test.The cross-sectional and surface area of the desplinted and desplint/release granulation tissue both decreased compared to the splinted granulation tissue (*P < 0.05). The nuclear density of the desplint/release granulation tissue was 25% less compared to the splinted granulation tissue (*P < 0.05). The desplinted and desplint/release apoptotic rates were twice and >10x greater than the splinted apoptotic rate, respectively (*P < 0.05).The rate of cell death in a splinted wound (an in vivo equivalent of an anchored FPCM) is minimal to nil, which is consistent with our hypothesis. Desplinting and releasing the wound edge of a previously splinted wound (the in vivo equivalent of a detached FPCM) results in granulation tissue regression and a large increase in apoptosis. Desplinting a wound alone results in changes somewhat intermediate to the splinted and desplint/release conditions. Loss of wound anchorage acutely promotes granulation tissue apoptosis.

  View details for DOI 10.1006/jsre.2002.6602

  View details for Web of Science ID 000182342400018

  View details for PubMedID 12697415

• [Inhibiting scar formation in rat cutaneous wounds by blocking TGF-beta signaling]. Zhonghua yi xue za zhi Liu, W., Chua, C. H., Wu, X., Wang, D., Yin, D., Cui, L., Cao, Y., Longaker, M. T. 2003; 83 (1): 31–36

  Abstract

  OBJECTIVE: TGF-beta plays a key role in wound scarring. This study explored the possibility of using gene therapy to inhibit wound scarring by blocking TGF-beta signaling.METHODS: In vitro, human normal dermal fibroblasts were infected with recombinant adenoviruses of truncated TGF-beta receptor II (tTGF-betaRII, 100 pfu/cell) and beta-galactosidase (beta-gal, 100 pfu/cell), and their effects on regulating TGF-beta1 gene expression were analyzed by Northern blot. For gene therapy, beta-gal and tTGF-betaRII viruses (1 x 10(9) pfu)were injected intradermally at left and right side of dorsal skin of newborn Sprague-Dawley rats (n = 15) respectively. A full-thickness incisional wound (0.5 cm long) was created at the injection sites of each rat 2 days post-injection. An incisional wound was similarly created in the middle part of the dorsal skin of tTGF-betaRII transgenic mice (n = 5) and control mice (n = 5). Wound tissues of rats and mice were harvested at various time points post-wounding for histological and immunohistochemical analysis. Scar area in tissue section was measured by Image-Pro Plus software.RESULTS: Over-expression of tTGF-betaRII markedly reduced TGF-beta1 gene expression in dermal fibroblasts. Adenovirus mediated gene expression in skin reached a peak level 2 - 3 days post-injection, and decreased gradually at 5 - 7 days. Two weeks post-wounding, histology and quantitative analysis demonstrated that relative scar area in the wounds of transgenic mice and control mice were 136,969.8 +/- 66,339 and 474,641.6 +/- 227,396 respectively, the scar area of transgenic wounds was 29 percent of control area (P < 0.05). In all rats, wounds transfected with tTGF-betaRII gene healed with much less scarring (relative scar area 128,311.2 +/- 36,764.6) than control wounds (251,189.1 +/- 62,544.7) of the same rat, with a 45% reduction of scar area in average (P < 0.001). In addition, the tTGF-betaRII expression also decreased inflammation and TGF-beta1 production in treated wounds, and promoted the repair of panniculus muscle in treated wounds.CONCLUSIONS: Adenovirus mediated over-expression of tTGF-betaRII can block TGF-beta signaling and inhibit wound scarring, and thus can serve as a gene therapy strategy to control wound scarring.

  View details for PubMedID 12757642

• Methods for investigating fetal tissue repair. Methods in molecular medicine Peled, Z. M., Warren, S. M., Bouletreau, P. J., Longaker, M. T. 2003; 78: 149-159

  View details for PubMedID 12825269

• The molecular and biological mechanisms of bone distraction 4th International Congress of Maxillofacial and Craniofacial Distraction Bouletreau, P., Longaker, M. T. MEDIMOND S R L. 2003: 9–14

  View details for Web of Science ID 000188281300002

• Retinoic acid-mediated effects on calvarial osteoblast differentiation and proliferation: Implications for retinoic acid-induced craniofacial abnormalities 10th International Congress of the International-Society-of-Craniofacial-Surgery Song, H. M., Nacamuli, R. P., Xia, W., Bari, S., Fong, K. D., Fang, T. D., Shi, Y. Y., Warren, S. M., Longaker, M. T. MEDIMOND PUBLISHING CO. 2003: 493–495

  View details for Web of Science ID 000227470500103

• In utero models of craniofacial surgery WORLD JOURNAL OF SURGERY Harling, T. R., Stelnicki, E. J., Hedrick, M. H., Longaker, M. T. 2003; 27 (1): 108-116

  Abstract

  Fetal surgery is finding ever-increasing applications. At present, fetal surgical techniques are used only to treat diseases associated with high mortality risk or severe morbidity if left untreated prior to birth. Yet as a discipline, in utero therapy is attractive to plastic surgeons because it affords the potential to (1) provide a scarless repair, (2) correct the primary deformity, (3) prevent secondary deformities, and (4) give the parents a "normal"-appearing child at birth. Each of these potential benefits is particularly important in patients with craniofacial anomalies. This article will both review the animal models that have been used to study the potential for intrauterine plastic surgery and provide a synopsis of the benefits that fetal therapy may provide in the treatment of selected craniofacial anomalies.

  View details for DOI 10.1007/s00268-002-6745-2

  View details for Web of Science ID 000180270800017

  View details for PubMedID 12557046

• Medial canthal reconstruction using a medially based upper eyelid myocutaneous flap PLASTIC AND RECONSTRUCTIVE SURGERY Jelks, G. W., Glat, P. M., Jelks, E. B., Longaker, M. T. 2002; 110 (7): 1636-1643

  Abstract

  Periorbital reconstruction following skin cancer ablation represents a challenging problem. A thorough understanding of the complex periorbital anatomy is necessary to preserve lid function and protect the ocular surface. The medial canthal region represents the most difficult periorbital zone to reconstruct. This area has a complex anatomy involving both the medial canthus itself and the lacrimal apparatus. The authors present their experience with a versatile technique for reconstruction of the medial canthal periorbital region, namely, a medially based upper eyelid myocutaneous flap. In the 10 patients in whom this procedure was used, there was one partial and no complete flap losses. The authors believe that the medially based upper lid myocutaneous flap offers an excellent solution to the difficult problem of medial canthal periorbital reconstruction.

  View details for DOI 10.1097/01.PRS.0000033020.40691.B2

  View details for Web of Science ID 000179622400002

  View details for PubMedID 12447042

• Matrix metalloproteinases and the ontogeny of scarless repair: The other side of the wound healing balance PLASTIC AND RECONSTRUCTIVE SURGERY Peled, Z. M., Phelps, E. D., Updike, D. L., Chang, J., Krummel, T. M., Howard, E. W., Longaker, M. T. 2002; 110 (3): 801-811

  Abstract

  Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process. In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3. Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.

  View details for DOI 10.1097/01.PRS.0000019915.20203.EC

  View details for Web of Science ID 000177332700013

  View details for PubMedID 12172142

• Adenovirus-mediated transmission of a dominant negative transforming growth factor-beta receptor inhibits in vitro mouse cranial suture fusion PLASTIC AND RECONSTRUCTIVE SURGERY Mehrara, B. J., Spector, J. A., Greenwald, J. A., Ueno, H., Longaker, M. T. 2002; 110 (2): 506-514

  Abstract

  Recent studies have implicated the transforming growth factor (TGF)-beta family in the regulation of pathological sporadic cranial suture fusion. In addition, these studies have shown that TGF-beta is highly expressed by the dura mater underlying fusing murine cranial sutures. The purpose of the present experiments was to analyze the effects of disrupting TGF-beta signaling during programmed mouse cranial suture fusion. Using recombinant DNA technology, a replication-deficient adenovirus encoding a defective TGF-beta receptor (Ad.DN-TbetaRII) capable of blocking TGF-beta biological activity was constructed. Mouse posterior frontal sutures were harvested before the initiation of suture fusion (postnatal day 25), and the dura mater underlying the suture was infected with vehicle, Ad.DN-TbetaRII, or control virus (Ad.LacZ; n = 10 each). Sutures were cultured for 14 or 30 days in an organ culture system and analyzed macroscopically and histologically.X-gal staining of Ad.LacZ-infected sutures 14 days after culture revealed strong staining of cells localized to the dura mater. Macroscopic analysis revealed complete sutural fusion in vehicle and Ad.LacZ-infected sutures. In contrast, Ad.DN-TBRII-infected sutures demonstrated nearly complete patency. Histological analysis confirmed our macroscopic observations with sutural fusion in 81.3 +/- 10 percent and 74.5 +/- 9 percent of vehicle and Ad.LacZ-infected sutures, respectively, versus 38.1 +/- 12 percent (p < 0.001) in Ad.DN-TbetaRII-infected sutures. In addition, transfection with the Ad.DN-TbetaRII virus resulted in a significant attenuation of anterior-to-posterior suture fusion, with the majority of fused sections localized to anterior sections. These data strongly implicate TGF-beta biological activity in the dura mater underlying the posterior frontal suture in the regulation of programmed sutural fusion. In addition, this study demonstrates the utility of adenovirus-mediated gene transfer in preventing programmed sutural fusion.

  View details for Web of Science ID 000177043900022

  View details for PubMedID 12142669

• Flexor tendon healing in vitro: effects of TGF-beta on tendon cell collagen production. journal of hand surgery Klein, M. B., Yalamanchi, N., Pham, H., Longaker, M. T., Chang, J. 2002; 27 (4): 615-620

  Abstract

  Flexor tendon healing is complicated by adhesions to the surrounding sheath. Transforming growth factor beta (TGF-beta) is a cytokine with numerous activities related to wound healing. We examined the effects of TGF-beta-1, -2 and -3 on tendon cell proliferation and collagen production. Three separate cell lines--sheath fibroblasts, epitenon and endotenon tenocytes--were isolated from rabbit flexor tendons and cultured separately. Cell culture media was supplemented with 1 or 5 ng/mL of TGF-beta-1, -2, or -3. Cell number and collagen I and III production were measured and compared with unsupplemented control cultures. The addition of TGF-beta to cell culture media resulted in a decrease in cell number in all 3 lines that did not reach statistical significance. There was a significant increase (p <.05) in collagen I and III production with the addition of all 3 TGF-beta isoforms. Modulation of TGF-beta production may provide a mechanism to modulate adhesion formation clinically.

  View details for PubMedID 12132085

• Flexor tendon healing in vitro: Effects of TGF-beta on tendon cell collaizen production JOURNAL OF HAND SURGERY-AMERICAN VOLUME Klein, M. B., Yalamanchi, N., Pham, H., Longaker, M. T., Chang, J. 2002; 27A (4): 615-620

  Abstract

  Flexor tendon healing is complicated by adhesions to the surrounding sheath. Transforming growth factor beta (TGF-beta) is a cytokine with numerous activities related to wound healing. We examined the effects of TGF-beta-1, -2 and -3 on tendon cell proliferation and collagen production. Three separate cell lines--sheath fibroblasts, epitenon and endotenon tenocytes--were isolated from rabbit flexor tendons and cultured separately. Cell culture media was supplemented with 1 or 5 ng/mL of TGF-beta-1, -2, or -3. Cell number and collagen I and III production were measured and compared with unsupplemented control cultures. The addition of TGF-beta to cell culture media resulted in a decrease in cell number in all 3 lines that did not reach statistical significance. There was a significant increase (p <.05) in collagen I and III production with the addition of all 3 TGF-beta isoforms. Modulation of TGF-beta production may provide a mechanism to modulate adhesion formation clinically.

  View details for DOI 10.1053/jhsu.2002.34004

  View details for Web of Science ID 000176906500008

• Fibroblasts cocultured with keloid keratinocytes: normal fibroblasts secrete collagen in a keloidlike manner 87th Annual Clinical Congress of the American-College-of-Surgeons Lim, I. J., Phan, T. T., Bay, B. H., Qi, R., Huynh, H., Tan, W. T., LEE, S. T., Longaker, M. T. AMER PHYSIOLOGICAL SOC. 2002: C212–C222

  Abstract

  Keloid scars represent a pathological response to cutaneous injury, reflecting a new set point between synthesis and degradation biased toward extracellular matrix (ECM) collagen accumulation. Using a serum-free two-chamber coculture model, we recently demonstrated a significant increase in normal fibroblast proliferation when cocultured with keloid-derived keratinocytes. We hypothesized that similar keratinocyte-fibroblast interactions might influence fibroblast collagen production and examined conditioned media and cell lysate from coculture for collagen I and III production by Western blot, allied with Northern analysis for procollagen I and III mRNA. Normal fibroblasts cocultured with keloid keratinocytes produced increased soluble collagen I and III with a corresponding increase in procollagen I and III mRNA transcript levels. This was associated with decreased insoluble collagen from cell lysate. When keloid fibroblasts were cocultured with keloid keratinocytes, both soluble and insoluble collagen were increased with associated procollagen III mRNA upregulation. Transmission electron microscopy of normal fibroblasts cocultured with keloid keratinocytes showed an ECM appearance similar to in vivo keloid tissue, an appearance not seen when normal fibroblasts were cocultured with normal keratinocytes.

  View details for DOI 10.1152/ajpcell.00555.2001

  View details for Web of Science ID 000176070800023

  View details for PubMedID 12055090

• Factors in the fracture microenvironment induce primary osteoblast angiogenic cytokine production PLASTIC AND RECONSTRUCTIVE SURGERY Bouletreau, P. J., Warren, S. M., Spector, J. A., Steinbrech, D. S., Mehrara, B. J., Longaker, M. T. 2002; 110 (1): 139-148

  Abstract

  Neoangiogenesis is essential for successful wound repair. Platelets are among the earliest cells recruited to a site of skeletal injury and are thought to provide numerous factors critical to successful repair. The release of platelet-derived growth factor (PDGF) after skeletal injury increases osteoblast proliferation, chemotaxis, and collagen synthesis; however, its angiogenic effect on osteoblast biology remains unknown. The purpose of this study was to investigate the effect of recombinant human (rh)PDGF-BB on the synthesis of vascular endothelial growth factor (VEGF) by primary neonatal rat calvarial osteoblasts. Furthermore, the authors investigated whether PDGF works in concert with hypoxia, another component of the fracture microenvironment, to additively or synergistically induce VEGF production. Osteoblast cultures were stimulated with varying concentrations of rhPDGF-BB (1, 10, 50, and 100 ng/ml) in normoxic and hypoxic (<1% oxygen) conditions for 0, 3, 6, 12, and 24 hours, and VEGF gene expression was analyzed by Northern blot analysis. To determine whether rhPDGF-BB-induced VEGF messenger RNA (mRNA) expression was transcriptionally mediated or required de novo protein synthesis, transcription, and translation, studies were performed using actinomycin D and cycloheximide, respectively. Treatment with 50 ng/ml rhPDGF-BB resulted in a 2.4-fold increase in VEGF mRNA expression after 3 hours. Interestingly, rhPDGF-BB and hypoxia seemed to have an additive effect, resulting in a 3.7-fold increase in VEGF mRNA expression after 6 hours in primary neonatal rat calvarial osteoblasts. Furthermore, by using actinomycin D and cycloheximide, the authors demonstrated that the rhPDGF-BB-induced VEGF mRNA expression was transcriptionally mediate and not dependent on de novo protein synthesis. These data demonstrate that rhPDGF-BB transcriptionally increases osteoblasts VEGF mRNA expression in vitro. Furthermore, the semiquantitative results suggest that rhPDGF-BB and hypoxia act additively to increase VEGF mRNA expression. It is postulated that similar mechanisms may occur in vivo, at a site of skeletal injury, to induce neoangiogenesis and promote fracture repair.

  View details for Web of Science ID 000176399000025

  View details for PubMedID 12087245

• Re: Sequence analysis of fibroblast growth factor receptor 2 (FGFR2) in Japanese patients with craniosynostosis. Sakai et al. J Craniofac. Surg. 2001, 12: 580-585. journal of craniofacial surgery Warren, S. M., Longaker, M. T. 2002; 13 (4): 597-599

  View details for PubMedID 12140430

• Hypoxia and VEGF up-regulate BMP-2 mRNA and protein expression in microvascular endothelial cells: Implications for fracture healing PLASTIC AND RECONSTRUCTIVE SURGERY Bouletreau, P. J., Warren, S. M., Spector, J. A., Peled, Z. M., Gerrets, R. P., Greenwald, J. A., Longaker, M. T. 2002; 109 (7): 2384-2397

  Abstract

  The endothelium is a metabolically active secretory tissue, capable of responding to a wide array of environmental stimuli. Hypoxia and vascular endothelial growth factor (VEGF) are two components of the putative fracture microenvironment. This study investigated the role of hypoxia and VEGF on endothelial cell activation as it relates to the bone repair process. It was hypothesized that endothelial cells may have an important osteogenic role in fracture healing through the production of bone morphogenetic protein-2 (BMP-2), an osteogenic cytokine at the fracture site. Therefore, BMP-2 mRNA and protein expression in endothelial cells under hypoxia and/or VEGF treatment was studied. The authors observed a 2-fold to 3-fold up-regulation of BMP-2 mRNA expression in bovine capillary endothelial cells and human microvascular endothelial cells stimulated with hypoxia or rhVEGF. Furthermore, the combined effects of hypoxia and rhVEGF appeared to be additive on BMP-2 mRNA expression in bovine capillary endothelial cells. Actinomycin D and cycloheximide studies suggested that the increased mRNA expression was transcriptionally regulated. BMP-2 protein expression was up-regulated after 24 and 48 hours of treatment with either hypoxia or rhVEGF in bovine capillary endothelial cells. Surprisingly, the data suggest that endothelial cells may play not only an angiogenic role but also an osteogenic role by a direct stimulation of the osteoblasts, through the enhanced expression of a potent osteogenic factor, BMP-2, at the fracture site.

  View details for Web of Science ID 000175761500033

  View details for PubMedID 12045566

• Transport distraction osteogenesis: A new method to heal adult calvarial defects PLASTIC AND RECONSTRUCTIVE SURGERY Bouletreau, P. J., Warren, S. M., Paccione, M. F., Spector, J. A., McCarthy, J. G., Longaker, M. T. 2002; 109 (3): 1074-1084

  Abstract

  Popularized by Gavril Ilizarov in the 1960s, monofocal distraction osteogenesis has become a well-established method of endogenous bone engineering. This revolutionary surgical technique has significantly augmented the available reconstructive orthopedic and craniomaxillofacial procedures. Bifocal distraction osteogenesis, or bone transportation, is a modification of monofocal distraction that involves moving a free segment of living bone to fill an intercalary bone defect. Bifocal distraction has been applied successfully to reconstruct complex mandibular and long bone defects. Because traumatic or postsurgical calvarial defects do not spontaneously heal in humans older than 18 to 24 months of age, we hypothesized that bifocal distraction osteogenesis could be applied to the skull to close critical size calvarial defects. Critical size (15 x 15 mm) calvarial defects were created in eight New Zealand White rabbits. Next, a 15-mm x 10-mm calvarial box osteotomy was created just anterior to the skull defect. This osteotomy created a free bone segment that could be transported. A custom-made transport distraction device was fixed into place and the skin incision was closed. After a 4-day latency period, the distraction device was activated (0.5 mm once daily for 30 days) in seven animals; the distraction device in one animal was not activated and served as a control. All animals underwent 30 days of consolidation and were then killed. Radiographs and computed tomographic scans were performed at the following time points: end of latency period (postoperative day 4), mid-distraction (postoperative day 19), and end of consolidation period (postoperative day 64). Gross and histologic analysis was performed to evaluate the quality of the bony regenerate. The control animal healed with a fibrous union. Complete closure of the skull defects was observed in five of seven rabbits at the end of the consolidation period. One animal was removed from the study because of an early loosening of the distraction device, and one was removed because of device failure. Of the remaining five animals that completed the distraction protocol, radiographs and computerized tomographic scans showed successful ossification in all five rabbits at the end of the consolidation period. This study suggests that transport distraction osteogenesis is a promising technique that may be applied to a variety of commonly encountered craniofacial problems such as nonhealing calvarial defects.

  View details for Web of Science ID 000174211900046

  View details for PubMedID 11884839

• Granulation tissue regression induced by musculocutaneous advancement flap coverage Carlson, M. A., Longaker, M. T., Thompson, J. S. MOSBY, INC. 2002: 332–37

  Abstract

  Clinical experience suggests that granulation tissue may be inhibited by coverage with a musculocutaneous flap. We hypothesized that coverage of an open wound with a musculocutaneous flap would result in regression and apoptosis of the wound's granulation tissue.In the first experiment, 32 rats underwent excisional wounding; 16 underwent musculocutaneous flap coverage of their granulation tissue on postwounding day 8, and then 16 rats (8 controls + 8 flaps) were killed on both postwounding days 10 and 12 (2 and 4 days after the flap procedure, respectively). In the second experiment, 18 rats were wounded, and on postwounding day 5 the rats underwent flap coverage (n = 6), wound edge release/mobilization (the first step of the flap procedure) without flap coverage (n = 6), or dressing change only (n = 6); all rats were killed on postwounding day 6 (24 hours after the secondary intervention). Apoptosis was quantified with the terminal deoxynucleotidyl transferase-mediated nick-end labeling assay.Placement of a musculocutaneous flap over an 8-day-old excisional wound in the first experiment increased the apoptotic rate in the granulation tissue from 0% to 1% (controls) to 5% to 10% at both 2 and 4 days after flap coverage (P <.05). Cell population density decreased 50% in the flap-covered granulation tissue compared with the controls (P <.05). In the second experiment, circumferential release of the granulation tissue resulted in an equivalent increase in granulation tissue apoptosis over controls compared to that induced by the full flap procedure.Coverage of established granulation tissue with a musculocutaneous flap resulted in histologic regression of the wound's granulation tissue after 2 to 4 days of flap coverage and induced at least a 5-fold increase in the apoptotic rate of the granulation tissue. Releasing the wound edge increased granulation tissue apoptosis to a level equivalent to that produced by the musculocutaneous flap procedure, suggesting that alteration of the wound's mechanical environment is responsible for the acute induction of apoptosis in this model.

  View details for DOI 10.1067/msy.2002.120673

  View details for Web of Science ID 000174528400014

  View details for PubMedID 11894039

• Co-culture of osteoblasts, with immature dural cells causes an increased rate and degree of osteoblast differentiation PLASTIC AND RECONSTRUCTIVE SURGERY Spector, J. A., Greenwald, J. A., Warren, S. M., Bouletreau, P. J., Crisera, F. E., Mehrara, B. J., Longaker, M. T. 2002; 109 (2): 631-642

  Abstract

  For decades surgeons have exploited the ability of infants to reossify large calvarial defects. To demonstrate the role of dura mater-osteoblast communication during the process of calvarial reossification, the authors used a novel in vitro system that recapitulates the in vivo anatomic relationship of these cell populations. Primary cultures of osteoblast cells from 2-day-old Sprague-Dawley rat pups were grown on six-well plates, and cultures of immature, non-suture-associated dura mater cells from 6-day-old Sprague-Dawley rat pups were grown on Transwell inserts. When the osteoblast and dura mater cell cultures reached confluence, they were combined. This Transwell co-culture system permitted the two cell populations to grow together in the same well, but it prevented direct cell-to-cell contact. Therefore, the authors were able to determine, for the first time, whether paracrine signaling from immature, non-suture-associated dura mater could influence the biologic activity of osteoblasts. Osteoblasts co-cultured with dural cells proliferated significantly faster after 2 days (2.1 x 10(5) +/- 2.4 x 10(4) versus 1.4 x 10(5) +/- 2.2 x 10(4), p < or = 0.05) and 4 days (3.1 x 10(5) +/- 5 x 10(4) versus 2.2 x 10(5) +/- 4.0 x 10(4), p < or = 0.01) than did osteoblasts cultured alone. After 20 days, co-cultured osteoblasts expressed greater amounts of mRNA for several markers of osteoblast differentiation, including collagen I alpha I (4-fold), alkaline phosphatase (2.5-fold), osteopontin (3-fold), and osteocalcin (4-fold), than did osteoblasts cultured alone. After 30 days, co-cultured osteoblasts produced bone nodules that were significantly greater both in number (324 +/- 29 nodules versus 252 +/- 29 nodules per well, p , < or = 0.04) and total area of nodules (65 +/- 11 mm(2) versus 24 +/- 1.6 mm(2), p < or = 0.003) than osteoblasts cultured alone. To begin to understand how dural cells effect changes in osteoblast gene expression, the authors compared the expression of candidate genes, transforming growth factor beta 1 and fibroblast growth factor 2, in dural cells and osteoblasts before and after 5 days of culture. Interestingly, the dura mater produced marked amounts of these osteogenic cytokines compared with osteoblasts.The described co-culture system demonstrated that co-cultured osteoblasts proliferated more rapidly and experienced an increased rate and degree of cellular maturation than did osteoblasts cultured alone. The authors hypothesize that this effect was due to paracrine signaling (e.g., transforming growth factor beta 1 and fibroblast growth factor 2) from the dura mater, and they are investigating those mechanisms in ongoing experiments. Collectively these data verify that immature, non-suture-associated dura mater can influence the biologic activity of osteoblasts. Moreover, the production of cytokines derived from the dura mater (e.g., transforming growth factor beta 1 and fibroblast growth factor 2), and they may begin to explain why immature animals and infants with intact dura mater can reossify large calvarial defects.

  View details for Web of Science ID 000173678000033

  View details for PubMedID 11818846

• Dura mater biology: Autocrine and paracrine effects of fibroblast growth factor 2 PLASTIC AND RECONSTRUCTIVE SURGERY Spector, J. A., Greenwald, J. A., Warren, S. M., Bouletreau, P. J., Detch, R. C., Fagenholz, P. J., Crisera, F. E., Longaker, M. T. 2002; 109 (2): 645-654

  Abstract

  The dura mater, the outermost layer of the meninges, is thought to be essential for calvarial morphogenesis, postnatal suture fusion, and osseous repair of calvarial defects. Despite numerous studies illustrating the fundamental role of the dura mater, there is little information about the autocrine and paracrine mechanisms regulating dural cell biology during calvarial ossification. Previous work conducted in the authors' laboratory demonstrated that non-suture-associated dural cells from 6-day-old rat pups expressed high levels of fibroblast growth factor 2 (FGF-2), whereas dural cells from 60-day-old adult rats expressed very little FGF-2. Because young mammals can successfully heal large calvarial defects, the authors sought to investigate the autocrine and/or paracrine effects of FGF-2 on the proliferation, gene expression, and alkaline phosphatase production of dural cells. Cultures of non-suture-associated dural cells were established from 6-day-old Sprague-Dawley rat pups and then stimulated with recombinant human FGF-2 (rhFGF-2; 10 ng/ml). Dural cells stimulated with rhFGF-2 proliferated significantly faster than untreated dural cells at 24 hours (2.1 x 10(5) +/- 3.2 x 10(4) versus 1.1 x 10(5) +/- 1.8 x 10(4), p < or = 0.001) and 48 hours (2.3 x 10(5) +/- 4.2 x 10(4) versus 1.2 x 10(5) +/- 1.3 x 10(4), p < or = 0.001). Moreover, dural cells stimulated with rhFGF-2 expressed 7-fold more proliferating cell nuclear antigen than did control cultures. Treatment with rhFGF-2 increased dural cell expression of genes important for skeletal repair: FGF-2 (7-fold), transforming growth factor beta 1 (3-fold), transforming growth factor beta 3 (4-fold), and type I collagen (4-fold). Furthermore, rhFGF-2 increased dural cell expression of osteopontin (2-fold), a "late" marker of osteoblastic differentiation. Interestingly, dural cell alkaline phosphatase activity, an "earlier" marker of osteoblast differentiation, was significantly decreased by treatment with rhFGF-2 compared with control cultures at 24 hours (0.005 +/- 0.001 versus 0.01 +/- 0.003, p < or = 0.01) and 48 hours (0.004 +/- 0.0009 versus 0.01 +/- 0.0009). Together these data provide insight into the autocrine and paracrine effects of FGF-2 on the biology of the dura mater.

  View details for Web of Science ID 000173678000035

  View details for PubMedID 11818848

• The molecular biology of distraction osteogenesis JOURNAL OF CRANIO-MAXILLOFACIAL SURGERY Bouletreau, P. J., Warren, S. M., Longaker, M. T. 2002; 30 (1): 1-11

  Abstract

  Distraction osteogenesis has become a mainstay in bone tissue engineering and has significantly improved our armamentarium for reconstructive craniomaxillofacial procedures. However, although the biomechanical, histological, and ultrastructural changes associated with distraction osteogenesis have been widely described, the molecular mechanisms governing the formation of new bone in the interfragmental gap of gradually distracted bone segments remain largely unclear. Recently, a rat model of mandibular distraction was described that provides an excellent environment for deciphering the molecular mechanisms that mediate distraction osteogenesis. This article presents the hypotheses and current research that have furthered knowledge of the molecular mechanisms that govern distraction osteogenesis. Recent studies have implicated a growing number of cytokines that are intimately involved in the regulation of bone synthesis and turnover. The gene regulation of numerous cytokines (transforming growth factor-beta1, -beta2, -beta3, bone morphogenetic proteins, insulin-like growth factor-1, fibroblast growth factor-2) and extracellular matrix proteins (osteonectin, osteopontin) during distraction osteogenesis have been best characterized and are discussed in this article. It is believed that understanding the biomolecular mechanisms that mediate membranous distraction osteogenesis may guide the development of targeted strategies designed to improve distraction osteogenesis and accelerate bone healing.

  View details for DOI 10.1054/jems.2001.0263

  View details for Web of Science ID 000177084200001

  View details for PubMedID 12064876

• Overhealing, underhealing, and skin regeneration: a new perspective on wound healing. Asian journal of surgery Peled, Z. M., Galiano, R., Chin, G. S., Chang, J., Krummel, T. M., Longaker, M. T. 2002; 25 (1): 102-110

  View details for PubMedID 17585454

• The pathogenesis of craniosynostosis in the fetus YONSEI MEDICAL JOURNAL Warren, S. M., Longaker, M. T. 2001; 42 (6): 646-659

  Abstract

  Craniosynostosis occurs in approximately 1:2000 live births. It may affect the coronal, sagittal, metopic and lambdoid sutures in isolation or in combination. Although non-syndromic synostoses are more common, over 150 genetic syndromes have been identified. Recent advances in genetic mapping have linked chromosomal mutations with craniosynostotic syndromes. Despite the identification of these genetic mutations, the fundamental biomolecular mechanisms mediating cranial suture biology remain unknown. Today, many laboratories are investigating murine cranial suture biology as a model for human cranial suture development and fusion. Normal murine cranial suture biology is very complex, but evidence suggests that the dura mater provides the biomolecular blueprints (e.g. the soluble growth factors), which guide the fate of the pleuripotent osteogenic fronts. While our knowledge of these dura-derived signals has increased dramatically in the last decade, we have barely begun to understand the fundamental mechanisms that mediate cranial suture fusion or patency. Interestingly, recent advances in both premature human and programmed murine suture fusion have revealed unexpected results, and have generated more questions than answers.

  View details for Web of Science ID 000173016100009

  View details for PubMedID 11754148

• Gene expression of transforming growth factor beta isoforms in interposition nerve grafting. journal of hand surgery Karanas, Y. L., Bogdan, M. A., Lineaweaver, W. C., Hentz, V. R., Longaker, M. T., Chang, J. 2001; 26 (6): 1082-1087

  Abstract

  Scar production and neuroma formation at nerve graft coaptation sites may limit axonal regeneration and impair functional outcome. Transforming growth factor beta (TGF-beta) is a family of growth factors that is involved in scar formation, wound healing, and nerve regeneration. Fifteen adult Sprague-Dawley rats underwent autogenous nerve grafting. The nerve grafts were analyzed by in situ hybridization to determine the temporal and spatial expression of TGF-beta1 and TGF-beta3 messenger RNA (mRNA). The grafted nerves showed increased expression of TGF-beta1 and TGF-beta3 mRNA in the nerve and the surrounding connective tissue during the first postoperative week. These data suggest that modulation of TGF-beta levels in the first postoperative week may be effective in helping to control scar formation and improve nerve regeneration.

  View details for PubMedID 11721255

• Gene expression of transforming growth factor beta isoforms in interposition nerve grafting JOURNAL OF HAND SURGERY-AMERICAN VOLUME Karanas, Y. L., Bogdan, M. A., Lineaweaver, W. C., Hentz, V. R., Longaker, M. T., Chang, J. 2001; 26A (6): 1082-1087

  Abstract

  Scar production and neuroma formation at nerve graft coaptation sites may limit axonal regeneration and impair functional outcome. Transforming growth factor beta (TGF-beta) is a family of growth factors that is involved in scar formation, wound healing, and nerve regeneration. Fifteen adult Sprague-Dawley rats underwent autogenous nerve grafting. The nerve grafts were analyzed by in situ hybridization to determine the temporal and spatial expression of TGF-beta1 and TGF-beta3 messenger RNA (mRNA). The grafted nerves showed increased expression of TGF-beta1 and TGF-beta3 mRNA in the nerve and the surrounding connective tissue during the first postoperative week. These data suggest that modulation of TGF-beta levels in the first postoperative week may be effective in helping to control scar formation and improve nerve regeneration.

  View details for Web of Science ID 000172412500014

• Fetal wound healing: Progress report and future directions SURGERY Longaker, N. T., Peled, Z. M., Chang, J., Krummel, T. M. 2001; 130 (5): 785-787

  View details for Web of Science ID 000172026000002

  View details for PubMedID 11685186

• The ontogeny of scarless healing II: EGF and PDGF-B gene expression in fetal rat skin and fibroblasts as a function of gestational age ANNALS OF PLASTIC SURGERY Peled, Z. M., Rhee, S. J., HSU, M., Chang, J., Krummel, T. M., Longaker, M. T. 2001; 47 (4): 417-424

  Abstract

  Twenty years ago, surgeons noted the ability of early-gestation fetal skin to heal in a scarless manner. Since that time, numerous investigators have attempted to elucidate the mechanisms behind this phenomenon. As a result of this effort, it is now well established that many animals undergo a transition late in development from scarless cutaneous healing to a scar-forming, adultlike phenotype. The authors have been interested in the role played by cytokines known to be involved in the adult wound-healing process and how they relate to scarless repair. They therefore asked the following question: Are genes for epidermal growth factor (EGF) and platelet-derived growth factor-B (PDGF-B) expressed differentially as a function of gestational age in fetal rat skin and dermal fibroblasts? To answer this question, skin from fetal Sprague-Dawley rats (N = 56) at time points that represented both the scarless and scar-forming periods of rat gestation was harvested. In addition, fibroblasts derived from fetal rat skin were cultured in vitro at similar times. These cells were expanded in culture and, when confluent, total ribonucleic acid from both fibroblasts and whole skin was extracted and subjected to Northern blot analysis with probes for EGF and PDGF-B. Results demonstrated that neither EGF nor PDGF-B gene expression changed markedly as a function of gestational age in fetal fibroblasts alone. In whole skin, however, both EGF and PDGF-B demonstrated a marked decrease in gene expression with increasing gestational age. Furthermore, the most striking decrease in gene expression for both cytokines came between 16 and 18 days of gestation-the transition point between scarless and scar-forming repair in the fetal rat. These data suggest that EGF and PDGF may play a role in the mechanism of scarless cutaneous repair. Moreover, it appears that fetal fibroblasts are not the cell type responsible for this differential gene expression. These results raise questions about the unique cytokine milieu likely to be present during the time of scarless healing and the cells that ultimately guide the mechanisms leading to skin regeneration.

  View details for Web of Science ID 000171407400010

  View details for PubMedID 11601578

• New directions in plastic surgery research CLINICS IN PLASTIC SURGERY Warren, S. M., Longaker, M. T. 2001; 28 (4): 719-?

  Abstract

  Plastic surgery research affords tremendous opportunities in a variety of affluent mode systems. Only recently have researchers applied molecular biologic techniques to common plastic surgery problems. For example, investigating the fundamental biomolecular mechanisms of normal palate and cranial suture morphogenesis will improve the understanding of the etiopathogenesis of CLP and craniosynostosis and facilitate the development of biologically-based interventions. Furthermore, as interdisciplinary collaborations improve, surgeons can expect to see remarkable progress in de novo tissue synthesis, replacement, and repair. Ultimately, they may one day find that gene-modified endogenous tissue engineering will succeed today's biocompatible scaffolds and allogeneic or zenogeneic replacement strategies. In general, plastic surgeons can look forward to the development of highly effective biomolecular treatments for clinical problems such as complex wound repair, prolific scarring, bone deficits (or surpluses), and organ system replacement or repair. Researchers believe that biologically-based strategies like these will be combined with technical advances that harness minimally invasive approaches. Together, clinicians expect these new tactics will reduce morbidity and improve the results of clinical problems treated by plastic surgeons.

  View details for Web of Science ID 000171901500008

  View details for PubMedID 11727856

• Differential expression of transforming growth factor-beta receptors in a rabbit zone II flexor tendon wound healing model 69th Annual Meeting of the American-Society-of-Plastic-and-Reconstructive-Surgeons Ngo, M., Pham, H., Longaker, M. T., Chang, J. LIPPINCOTT WILLIAMS & WILKINS. 2001: 1260–67

  Abstract

  Flexor tendon repair in zone II is complicated by adhesions that impair normal postoperative gliding. Transforming growth factor-beta (TGF-beta) is a family of growth factors that has been implicated in scar formation. The TGF-beta family of proteins binds to three distinct classes of membrane receptors, termed RI, RII, and RIII. In this study, we analyzed the temporal and spatial distribution of TGF-beta receptor isoforms (RI, RII, and RIII) in a rabbit zone II flexor tendon wound healing model.Twenty-eight adult New Zealand White rabbit forepaws underwent isolation of the middle digit flexor digitorum profundus tendon in zone II. The tendons underwent transection in zone II and immediate repair. The tendons were harvested at increasing time points: 1, 3, 7, 14, 28, and 56 days postoperatively (n = 4 at each time point). The control flexor tendons were harvested without transection and repair (n = 4). Immunohistochemical analysis was used to detect the expression patterns for TGF-beta receptors RI, RII, and RIII. Immunohistochemical staining of the transected and repaired tendons demonstrated up-regulation of TGF-beta RI, RII, and RIII protein levels. TGF-beta receptor production in the experimental group (transection and repair) was concentrated in the epitenon and along the repair site. Furthermore, the TGF-beta receptor expression levels peaked at day 14 and decreased by day 56 postoperatively. In contrast, minimal receptor expression was observed in the untransected and unrepaired control tendons. These data provide evidence that (1) TGF-beta receptors are up-regulated after injury and repair; (2) peak levels of TGF-beta receptor expression occurred at day 14 and decreased by day 56 after wounding and repair; and (3) both the tendon sheath and epitenon have the highest receptor expression, and both may play critical roles in flexor tendon wound healing. Understanding the up-regulation of TGF-beta isoforms and the up-regulation of their corresponding receptors during flexor tendon wound healing provides new targets for biomolecular modulation of postoperative scar formation.

  View details for Web of Science ID 000171428900025

  View details for PubMedID 11604629

• A mouse model of mandibular osteotomy healing JOURNAL OF CRANIOFACIAL SURGERY Paccione, M. F., Warren, S. M., Spector, J. A., Greenwald, J. A., Bouletreau, P. J., Longaker, M. T. 2001; 12 (5): 444-450

  Abstract

  The purpose of this study was to establish a novel mouse model of membranous osteotomy healing. By applying this model to transgenic mice or using in situ hybridization techniques, we can subsequently investigate candidate genes that are believed to be important in membranous osteotomy healing. In the current study, 20 adult male CD-1 mice underwent a full-thickness osteotomy between the second and third molars of the right hemimandible using a 3-mm diamond disc and copious irrigation. Compo-Post pins were secured into the mandible, 2 mm anterior and posterior to the osteotomy. After the soft tissues were reapproximated and the skin was closed, an acrylic external fixator was attached to the exposed posts for stabilization. The animals were killed on postoperative day number 7, 10, 14, and 28 (n=5 animals per time point). The right hemimandibles were decalcified and embedded in paraffin for histologic evaluation or immunohistochemistry localizing osteocalcin. At 7 days after the osteotomy, early intramembranous bone formation could be seen extending from either edge of the osteotomized bone. By 10 days, an increasing number of small blood vessels could be seen within and around the osteotomy. At 14 days, the bone edges were in close approximation, and by 28 days the callus had been replaced by actively remodeling woven bone in all specimens examined. Immunohistochemistry demonstrated that osteocalcin expression correlated temporally with the transition from a soft to a hard callus. Furthermore, osteocalcin was spatially confined to osteoblasts actively laying down new osteoid or remodeling bone. This study describes a novel mouse model of membranous osteotomy healing that can be used as a paradigm for future osteotomy healing studies investigating candidate genes critical for osteogenesis and successful bone repair.

  View details for Web of Science ID 000170944000008

  View details for PubMedID 11572249

• Differential expression of transforming growth factor-beta receptors I and II and activation of Smad 3 in keloid fibroblasts 85th Annual Meeting of the American-College-of-Surgeons Surgical Forum Chin, G. S., Liu, W., Peled, Z., Lee, T. Y., Steinbrech, D. S., HSU, M., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2001: 423–29

  Abstract

  Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially collagen. However, the molecular mechanisms regulating this pathologic collagen deposition still remain to be elucidated. A previous study by this group demonstrated that transforming growth factor (TGF)-beta1 and -beta2 ligands were expressed at greater levels in keloid fibroblasts when compared with normal human dermal fibroblasts (NHDFs), suggesting that TGF-beta may play a fibrosis-promoting role in keloid pathogenesis.To explore the biomolecular mechanisms of TGF-beta in keloid formation, the authors first compared the expression levels of the type I and type II TGF-beta receptors in keloid fibroblasts and NHDFs. Next, they investigated the phosphorylation of Smad 3, an intracellular TGF-beta signaling molecule, in keloid fibroblasts and NHDFs. Finally, they examined the regulation of TGF-beta receptor II by TGF-beta1, TGF-beta2, and TGF-beta3 ligands. Our findings demonstrated an increased expression of TGF-beta receptors (types I and II) and increased phosphorylation of Smad 3 in keloid fibroblasts relative to NHDFs. These data support a possible role of TGF-beta and its receptors as fibrosis-inducing growth factors in keloids. In addition, all three isoforms of recombinant human TGF-beta proteins could further stimulate the expression of TGF-beta receptor II in both keloids and NHDFs. Taken together, these results substantiate the hypothesis that the elevated levels of TGF-beta ligands and receptors present in keloids may support increased signaling and a potential role for TGF-beta in keloid pathogenesis.

  View details for Web of Science ID 000170096900022

  View details for PubMedID 11496185

• Importance of the nasal-to-cervical relationship to the profile in rhinoplasty surgery PLASTIC AND RECONSTRUCTIVE SURGERY Greer, S. E., Matarasso, A., Wallach, S. G., Simon, G., Longaker, M. T. 2001; 108 (2): 522-531

  Abstract

  There is general agreement that when discussing surgery with the prospective rhinoplasty patient, one may also include discussion of the chin because of the important interrelationship between these two regions. It is apparent that on the profile-lateral view, the four prominences-the forehead, nose, chin, and neck-balance and complement one another. The cervical region, the fourth dimension, was examined to estimate the aesthetic significance of the nasal-to-cervical relationship and to determine the implications to rhinoplasty surgery. Part I of the study was a survey to test the hypothesis that the cervical region affects the perceived impression of the nose. Four standard facial-profile black-and-white photographs were chosen to represent varying degrees of nasal dorsum hump and cervical ptosis problems. Using computer-altering software, only the cervical regions were altered to create a pair of photographs for each of the four profiles: one with a youthful-looking non-ptotic cervical region, the other with a ptotic aged-looking cervical region. Raters were asked to give their first-impression opinions of which nose subjectively appeared "better." Raters consistently (84 percent of the time) chose the nose on the faces with the less ptotic neck as being better. Therefore, a less ptotic neck improved the perceived appearance of the nose. Part II was a retrospective chart review of the rhinoplasty patients of a single surgeon by independent raters. To estimate the aesthetic significance of the nasal-to-cervical relationship, the proportion of patients undergoing rhinoplasty surgery who could have potentially benefited from a youth-restoring neck procedure was determined. Criteria originally described by Ellenbogen and Karlin for judging the results of youth-restoring neck procedures were used as relative indications for neck surgery. An average of 27.2 percent of the patients did not have visible criteria and therefore by definition had relative indications for neck-rejuvenating procedures when undergoing rhinoplasty surgery. As demonstrated in part I of the study, improving the neck could improve the perceived results of the rhinoplasty. Part III of the study validated the Ellenbogen and Karlin criteria. The present authors found that the original criteria were probably based on female patients, that male and older patients had more indications for surgery, and that there was significant interrater agreement with the youthful criteria. In summary, the authors established that a strong nasal-to-cervical relationship exists whereby the perceived appearance of the nose is affected by the neck. The significance of this relationship to rhinoplasty surgery was determined, and it was found that more than 27 percent of rhinoplasty patients could obtain better perceived nasal results with a concomitant neck-rejuvenating procedure. Consequently, discussing neck-rejuvenating procedures with the rhinoplasty patient is valuable.

  View details for Web of Science ID 000170096900037

  View details for PubMedID 11496199

• Retinoid signaling directs secondary lineage selection in pancreatic organogenesis JOURNAL OF PEDIATRIC SURGERY Kadison, A., Kim, J., Maldonado, T., Crisera, C., Prasadan, K., Manna, P., Preuett, B., Hembree, M., Longaker, M., Gittes, G. 2001; 36 (8): 1150-1156

  Abstract

  Retinoid signaling plays an important role in many differentiation pathways. Retinoid signaling has been implicated in the induction of differentiation by pancreatic ductal cancer cell lines and in patients with pancreatic cancer. The authors wished to better understand the role of retinoid signaling in pancreatic development.Embryonic pancreas was harvested from mice at serial gestational ages and immunohistochemical analysis was performed for retinoic acid receptors (RAR-alpha, RAR-beta, RAR-gamma), and retinoid X receptors (RXR-alpha, RXR-beta, and RXR-gamma). Also, early embryonic pancreases were cultured for 7 days with exogenous 9-cis retinoic acid (9cRA) or all-trans retinoic acid (atRA) and analyzed histologically and immunohistochemically.Retinoid receptors were expressed in a lineage-specific distribution, with stronger expression for many in the exocrine compartment. The receptors were not often expressed until late gestation. Exogenous 9cRA induced predominantly ducts instead of acini, plus more mature endocrine (islet) architecture. Exogenous atRA induced predominantly acini instead of ducts, with no apparent endocrine effect.Retinoids may have an important role in pancreatic differentiation, with a particular effect on secondary lineage selection between ductal and acinar phenotype. Because the control of ductal versus acinar differentiation has been implicated strongly in the pathogenesis of pancreatic ductal carcinoma, these results may lay the groundwork for studies in the mechanism of induced differentiation of pancreatic ductal cancer by retinoids.

  View details for Web of Science ID 000170120300009

  View details for PubMedID 11479845

• Role of TGF-beta signaling in the regulation of programmed cranial suture fusion JOURNAL OF CRANIOFACIAL SURGERY Longaker, M. T. 2001; 12 (4): 389-390

  View details for Web of Science ID 000169712600016

  View details for PubMedID 11482626

• Quantitative assessment of cranial defect healing and correlation with the expression of TGF-beta JOURNAL OF CRANIOFACIAL SURGERY Gosain, A. K., Song, L. S., Yu, P. R., Mehrara, B. J., Maeda, C. Y., Gold, L. I., Longaker, M. T. 2001; 12 (4): 401-404

  Abstract

  Circular parietal defects from 3 to 12 mm in diameter were made in 45 6-month old skeletally mature guinea pigs, and animals were sacrificed after survival periods of 3 days to 12 weeks. The original defect was harvested in continuity with a rim of surrounding bone and the adjacent dura and pericranium. After 12 weeks, all 3 and 5 mm defects were completely covered by a bridge of bone, while residual defects were noted within the 8 and 12 mm wounds. Percentage of new bone formation was significantly higher within 3 mm defects, than in all larger defects at each time interval from 1 week on (P < .05), reaching a mean of 93% in 3 mm defects and remaining below a mean of 31% in the remaining defect sizes. Immunolocalization demonstrated an osteogenic front in which the osteoblasts stained strongly for all isoforms of TGF-beta, with the intensity decreasing after the majority of the defects had reossified; this front was located at the advancing bone edge of the defect as well as the endocranial side adjacent to the dura. In conclusion, isoforms of TGF-beta are upregulated during a limited "window" of time corresponding to the period of calvarial reossification, and are localized to osteoblasts within an osteogenic front at the periphery and dural surfaces of the defects.

  View details for Web of Science ID 000169712600019

  View details for PubMedID 11482629

• Hypoxia regulates osteoblast gene expression JOURNAL OF SURGICAL RESEARCH Warren, S. M., Steinbrech, D. S., Mehrara, B. J., Saadeh, P. B., Greenwald, J. A., Spector, J. A., Bouletreau, P. J., Longaker, M. T. 2001; 99 (1): 147-155

  Abstract

  Vascular disruption secondary to fracture creates a hypoxic gradient of injury wherein the oxygen tension at the center of the wound is very low. In vivo this hypoxic microenvironment stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. In order to begin to dissect this complex system, we have examined the effects of hypoxia on isolated osteoblast gene expression in vitro. Understanding gene expression in this system may facilitate the development of targeted therapeutic modalities designed to accelerate fracture repair and reduce complications. Using an established model of in vitro hypoxia, we have analyzed the expression of genes involved in bone matrix production and turnover. Subconfluent neonatal rat calvarial osteoblasts were exposed to hypoxia (pO(2) = 35-40 mm Hg) and total cellular RNA was collected at 0, 3, 6, 24, and 48 h. Northern analysis was used to analyze the expression patterns of (1) transforming growth factors (TGFs)-beta1, -beta2, and -beta3 and their type I receptor; (2) collagens I and III; and (3) tissue inhibitor of metalloproteinase-1. We have demonstrated a marked elevation of TGF-beta1 gene expression within 3 h of hypoxia. Although neither TGF-beta2 nor TGF-beta3 expression was affected by hypoxia, the TGF-beta type I receptor was substantially upregulated within 6 h. In addition, extracellular matrix scaffolding molecules (collagens I and III) were markedly, but differentially, upregulated. Finally, we have demonstrated that the expression of an inhibitor of extracellular matrix turnover, the tissue inhibitor of metalloproteinase-1, was strikingly decreased in response to hypoxia. These results imply that hypoxia can affect osseous healing by altering the expression of cytokines, bone-specific extracellular matrix molecules, and their regulators.

  View details for DOI 10.1006/jsre.2001.6128

  View details for Web of Science ID 000169838100021

  View details for PubMedID 11421617

• Ontogeny of expression of transforming growth factor-beta 1 (TGF-beta 1), TGF-beta 3, and TGF-beta receptors I and II in fetal rat fibroblasts and skin PLASTIC AND RECONSTRUCTIVE SURGERY HSU, M., Peled, Z. M., Chin, G. S., Liu, W., Longaker, M. T. 2001; 107 (7): 1787-1794

  Abstract

  Fetal cutaneous wounds that occur in early gestation heal without scar formation. Although much work has been done to characterize the role of transforming growth factor-beta (TGF-beta) isoforms in the adult wound repair process, their function in fetal scarless wound repair is not well understood. The authors hypothesized that the pattern of expression for TGF-beta isoforms and their receptors may influence the phenotypic transition from scarless to scar-forming repair observed during fetal gestation. Using time-dated fetal Sprague-Dawley rat fibroblasts and unwounded skin at gestational ages 14, 16, 18, and 21 days postcoitum of the scarless (< or =16 days) and scar-forming (>16 days) periods of gestation (term = 21.5 days), the authors analyzed the endogenous messenger RNA (mRNA) levels of TGF-beta 1 and TGF-beta 3 and their signaling receptors TGF-beta-RI and TGF-beta-RII. Northern blot analyses in both fibroblasts and unwounded skin revealed that levels of TGF-beta 1 were not differentially expressed, whereas more TGF-beta 3 mRNA transcript was found in early than in late gestation. Fibroblast expression of TGF-beta-RI showed no substantial differences, whereas expression of TGF-beta-RII increased during gestation. In contrast, expression of both TGF-beta-RI and TGF-beta-RII in unwounded skin showed decreasing levels as a function of gestational age. The differential levels of TGF-beta 1 and TGF-beta 3 suggest that the ratio of these cytokines may provide a predominantly antiscarring or profibrotic signal upon wounding during the scar-free or scar-forming periods of gestation, respectively. Furthermore, lower amounts of the ligand-binding TGF-beta-RII seen in early gestation fibroblasts suggest a decreased ability to perceive ligand during the period of scarless repair.

  View details for Web of Science ID 000169013300023

  View details for PubMedID 11391201

• Distraction osteogenesis of the craniofacial skeleton PLASTIC AND RECONSTRUCTIVE SURGERY McCarthy, J. G., Stelnicki, E. J., Mehrara, B. J., Longaker, M. T. 2001; 107 (7): 1812-1827

  Abstract

  Distraction osteogenesis is becoming the treatment of choice for the surgical correction of hypoplasias of the craniofacial skeleton. Its principle is based on the studies of Ilizarov, who showed that osteogenesis can be induced if bone is expanded (distracted) along its long axis at the rate of 1 mm per day. This process induces new bone formation along the vector of pull without requiring the use of a bone graft. The technique also provides the added benefit of expanding the overlying soft tissues, which are frequently deficient in these patients. This article reviews the authors' 11-year clinical and research experience with mandibular distraction osteogenesis. It highlights the indications and contraindications of the technique and emphasizes the critical role that basic science research has played in its evolution.

  View details for Web of Science ID 000169013300029

  View details for PubMedID 11391207

• Auricular reconstruction: Indications for autogenous and prosthetic techniques PLASTIC AND RECONSTRUCTIVE SURGERY Thorne, C. H., Brecht, L. E., Bradley, J. P., Levine, J. P., Hammerschlag, P., Longaker, M. T. 2001; 107 (5): 1241-1251

  Abstract

  After studying this article, the participant should be able to: 1. Describe the alternatives for auricular reconstruction. 2. Discuss the pros and cons of autogenous reconstruction of total or subtotal auricular defects. 3. Enumerate the indications for prosthetic reconstruction of total or subtotal auricular defects. 4. Understand the complexity of and the expertise required for prosthetic reconstruction of auricular defects. The indications for autogenous auricular reconstruction versus prosthetic reconstruction with osseointegrated implant-retained prostheses were outlined in Plastic and Reconstructive Surgery in 1994 by Wilkes et al. of Canada, but because of the relatively recent Food and Drug Administration approval (1995) of extraoral osseointegrated implants, these indications had not been examined by a surgical unit in the United States. The purpose of this article is to present an evolving algorithm based on an experience with 98 patients who underwent auricular reconstruction over a 10-year period. From this experience, the authors conclude that autogenous reconstruction is the procedure of choice in the majority of pediatric patients with microtia. Prosthetic reconstruction of the auricle is considered in such pediatric patients with congenital deformities for the following three relative indications: (1) failed autogenous reconstruction, (2) severe soft-tissue/skeletal hypoplasia, and/or (3) a low or unfavorable hairline. A fourth, and in our opinion the ideal, indication for prosthetic ear reconstruction is the acquired total or subtotal auricular defect, most often traumatic or ablative in origin, which is usually encountered in adults. Although prosthetic reconstruction requires surgical techniques that are less demanding than autogenous reconstruction, construction of the prosthesis is a time-consuming task requiring experience and expertise. Although autogenous reconstruction presents a technical challenge to the surgeon, it is the prosthetic reconstruction that requires lifelong attention and may be associated with late complications. This article reports the first American series of auricular reconstruction containing both autogenous and prosthetic methods by a single surgical team.

  View details for Web of Science ID 000167844800024

  View details for PubMedID 11373570

• Matrix metalloproteinase involvement in fetal wound healing Phelps, E. D., Peled, Z., Longaker, M. T., Howard, E. W. FEDERATION AMER SOC EXP BIOL. 2001: A26

  View details for Web of Science ID 000167438100143

• Discoidin domain receptors and their ligand, collagen, are temporally regulated in fetal rat fibroblasts in vitro PLASTIC AND RECONSTRUCTIVE SURGERY Chin, G. S., Lee, S., HSU, M., Liu, W., Kim, W. J., Levinson, H., Longaker, M. T. 2001; 107 (3): 769-776

  Abstract

  The biochemical regulation of collagen deposition during adult cutaneous wound repair is poorly understood. Likewise, how collagen is perceived and modulated in fetal scarless healing remains unknown. Recently, discoidin domain receptors-1 and 2 (DDR1 and DDR2) with tyrosine kinase activity have been identified as novel receptors for collagen. In light of these findings, it was speculated that the production of collagen receptors DDR1 and DDR2 by fetal fibroblasts may be temporally regulated to correlate with the ontogeny of embryonic scar formation. More specifically, because DDRs directly bind collagen and transmit the signals intracellularly, it was hypothesized that they may play an important role in fetal scarless healing by ultimately regulating and modulating collagen production and organization. As part of a fundamental assessment to elucidate the role of DDRs in scarless fetal wound repair, the endogenous expression of DDR1, DDR2, collagen I, and total collagen, as a function of fetal Sprague-Dawley rat skin fibroblasts of different gestational ages, representing scar-free (E16.5) periods was determined. Using explanted dermal fibroblasts of gestational days E13.5, E16.5, E18.5, and E21.5 (term gestation = 21.5 days) fetuses (n = 92), [3H]proline incorporation assay and Northern and Western blotting analysis were performed to compare the expressions of these molecules with scar-free and scar-forming stages of embryonic development. These results revealed a pattern of increasing collagen production with increasing gestational ages, whereas DDR1 expression decreased with increasing gestational age. This observation suggests that elevated levels of DDR1 may play an important role in scarless tissue regeneration by early gestation fetal fibroblasts. In contrast, DDR2 was expressed by fetal rat fibroblasts at a similar level throughout gestation. These data demonstrate for the first time the temporal expression of collagen and DDR tyrosine kinases in fetal rat fibroblasts as a function of gestational ages. Overall, these data suggest that differential temporal expression of the above-mentioned molecules during fetal skin development may play an important role in the ontogeny of scar formation. Future studies will involve the characterization of the biomolecular functions of these receptor kinases during fetal wound repair.

  View details for PubMedID 11304604

• Subatmospheric pressure dressing for saphenous vein donor-site complications ANNALS OF THORACIC SURGERY Greer, S. E., Grossi, E. A., Chin, D., Longaker, M. T. 2001; 71 (3): 1038-1040

  Abstract

  Newer endoscopic techniques have been successful at reducing saphenous vein donor-site wound complications, but not entirely eliminating them. Tissue necrosis with superimposed infection is typically treated with antibiotics and surgical debridement. Typically, primary reclosure is not possible and the open leg wound is allowed to slowly granulate with dressing changes until a skin graft can be performed. This report describes an alternative treatment using subatmospheric pressure dressing to promote granulation tissue and wound closure in saphenous vein donor-site wounds.

  View details for Web of Science ID 000167439200077

  View details for PubMedID 11269429

• Rat mandibular distraction osteogenesis: Latency, rate, and rhythm determine the adaptive response JOURNAL OF CRANIOFACIAL SURGERY Paccione, M. F., Mehrara, B. J., Warren, S. M., Greenwald, J. A., Spector, J. A., Luchs, J. S., Longaker, M. T. 2001; 12 (2): 175-182

  Abstract

  Distraction osteogenesis is a well-established technique of endogenous tissue engineering. The biomechanical factors thought to affect the quality of the distraction regenerate include the latency, rate, rhythm, and consolidation period. In an effort to understand the impact of these parameters on regenerate bone formation, this study was designed to decipher the most adaptive response in a rat model of mandibular distraction osteogenesis. Ninety-six adult Sprague-Dawley rats were divided into 16 subgroups (n = 6 per subgroup) based on variations in the distraction parameters (i.e., latency, rate, and rhythm). After a 28-day consolidation period, the mandibles were harvested, decalcified, and sectioned. A standardized histologic ranking system was used to evaluate the effect of each protocol on the adaptive response of the regenerate bone. In this study, we have demonstrated that the latency period dramatically affects the success of distraction osteogenesis. Furthermore, distraction rates up to 0.50 mm per day stimulated excellent regenerate bone formation, whereas greater distraction rates produced a fibrous union. Finally, higher frequency distraction (i.e., increased rhythm) appeared to accelerate regenerate bone formation. We believe that defining the critical parameters of this model will improve future analysis of gene expression during rat mandibular distraction osteogenesis and may facilitate the development of biologically based strategies designed to enhance regenerate bone formation.

  View details for Web of Science ID 000170020400015

  View details for PubMedID 11314629

• Osteoblast gene expression is differentially regulated by TGF-beta isoforms JOURNAL OF CRANIOFACIAL SURGERY Fagenholz, P. J., Warren, S. M., Greenwald, J. A., Bouletreau, P. J., Spector, J. A., Crisera, F. E., Longaker, M. T. 2001; 12 (2): 183-190

  Abstract

  The transforming growth factor beta (TGF-beta) superfamily encompasses a number of important growth factors including several TGF-beta isoforms, the bone morphogenetic proteins, activins, inhibins, and growth and differentiation factors. TGF-beta 1, -beta 2, and -beta 3 are three closely related isoforms that are widely expressed during skeletal morphogenesis and bone repair. Numerous studies suggest that each isoform has unique in vivo functions; however, the effects of these TGF-beta isoforms on osteoblast gene expression and maturation have never been directly compared. In the current study, we treated undifferentiated neonatal rat calvaria osteoblast-enriched cell cultures with 2.5 ng/ml of each TGF-beta isoform and analyzed gene expression at 0, 3, 6, and 24 hours. We demonstrated unique isoform-specific regulation of endogenous TGF-beta 1 and type I collagen mRNA transcription. To assess the effects of extended TGF-beta treatment on osteoblast maturation, we differentiated osteoblast cultures in the presence of 2.5 ng/ml of each TGF-beta isoform. Analysis of collagen I, alkaline phosphatase, and osteocalcin demonstrated that each TGF-beta isoform uniquely suppressed the transcription of these osteoblast differentiation markers. Interestingly, TGF-beta isoform treatment increased osteopontin expression in primary osteoblasts after 4 and 10 days of differentiation. To our knowledge, these data provide the first direct comparison of the effects of the TGF-beta isoforms on osteoblast gene expression in vitro. Furthermore, these data suggest that TGF-beta isoforms may exert their unique in vivo effects by differentially regulating osteoblast cytokine secretion, extracellular matrix production, and the rate of cellular maturation.

  View details for Web of Science ID 000170020400016

  View details for PubMedID 11314630

• New developments in cranial suture research PLASTIC AND RECONSTRUCTIVE SURGERY Warren, S. M., Greenwald, J. A., Spector, J. A., Bouletreau, P., Mehrara, B. J., Longaker, M. T. 2001; 107 (2): 523-540

  View details for Web of Science ID 000166825100034

  View details for PubMedID 11214072

• Rat mandibular distraction osteogenesis: Part III. Gradual distraction versus acute lengthening PLASTIC AND RECONSTRUCTIVE SURGERY Warren, S. M., Mehrara, B. J., Steinbrech, D. S., Paccione, M. F., Greenwald, J. A., Spector, J. A., Longaker, M. T. 2001; 107 (2): 441-453

  Abstract

  Distraction osteogenesis is a well-established method of endogenous tissue engineering. This technique has significantly augmented our armamentarium of reconstructive craniofacial procedures. Although the histologic and ultrastructural changes associated with distraction osteogenesis have been extensively described, the molecular mechanisms governing successful membranous distraction remain unknown. Using an established rat model, the molecular differences between successful (i.e., osseous union with gradual distraction) and ineffective (i.e., fibrous union with acute lengthening) membranous bone lengthening was analyzed. Herein, the first insight into the molecular mechanisms of successful membranous bone distraction is provided. In addition, these data provide the foundation for future targeted therapeutic manipulations designed to improve osseous regeneration. Vertical mandibular osteotomies were created in 52 adult male Sprague-Dawley rats, and the animals were fitted with customized distraction devices. Twenty-six animals underwent immediate acute lengthening (3 mm; a length previously shown to result in fibrous union) and 26 animals were gradually distracted (after a 3-day latency period, animals were distracted 0.25 mm twice daily for 6 days; total = 3 mm). Four mandibular regenerates were harvested from each group for RNA analysis on 5, 7, 9, 23, and 37 days postoperatively (n = 40). Two mandibular regenerates were also harvested from each group and prepared for immunohistochemistry on postoperative days 5, 7, and 37 (n = 12). In addition to the 52 experimental animals, 4 control rats underwent sham operations (skin incision only) and mandibular RNA was immediately collected. Control and experimental specimens were analyzed for collagen I, osteocalcin, tissue inhibitor of metalloproteinase-1, and vascular endothelial growth factor mRNA and protein expression. In this study, marked elevation of critical extracellular matrix molecules (osteocalcin and collagen I) during the consolidation phase of gradual distraction compared with acute lengthening is demonstrated. In addition, the expression of an inhibitor of extracellular matrix turnover, tissue inhibitor of metalloproteinase-1, remained strikingly elevated in gradually distracted animals. Finally, this study demonstrated that neither gradual distraction nor acute lengthening appreciably alters vascular endothelial growth factor expression. These results suggest that gradual distraction osteogenesis promotes successful osseous bone repair by regulating the expression of bone-specific extracellular matrix molecules. In contrast, decreased production or increased turnover of bone scaffolding proteins (i.e., collagen) or regulators of mineralization (i.e., osteocalcin) may lead to fibrous union during acute lengthening.

  View details for Web of Science ID 000166825100021

  View details for PubMedID 11214060

• In vivo modulation of FGF biological activity alters cranial suture fate AMERICAN JOURNAL OF PATHOLOGY Greenwald, J. A., Mehrara, B. J., Spector, J. A., Warren, S. M., Fagenholz, P. J., Smith, L. P., Bouletreau, P. J., Crisera, F. E., Ueno, H., Longaker, M. T. 2001; 158 (2): 441-?

  Abstract

  Gain-of-function mutations in fibroblast growth factor receptors have been identified in numerous syndromes associated with premature cranial suture fusion. Murine models in which the posterior frontal suture undergoes programmed fusion after birth while all other sutures remain patent provide an ideal model to study the biomolecular mechanisms that govern cranial suture fusion. Using adenoviral vectors and targeted in utero injections in rats, we demonstrate that physiological posterior frontal suture fusion is inhibited using a dominant-negative fibroblast growth factor receptor-1 construct, whereas the normally patent coronal suture fuses when infected with a construct that increases basic fibroblast growth factor biological activity. Our data may facilitate the development of novel, less invasive treatment options for children with craniosynostosis.

  View details for Web of Science ID 000166925900014

  View details for PubMedID 11159182

  View details for PubMedCentralID PMC1850306

• Expression of bone morphogenetic proteins during membranous bone healing PLASTIC AND RECONSTRUCTIVE SURGERY Spector, J. A., Luchs, J. S., Mehrara, B. J., Greenwald, J. A., Smith, L. P., Longaker, M. T. 2001; 107 (1): 124-134

  Abstract

  For the reconstructive plastic surgeon, knowledge of the molecular biology underlying membranous fracture healing is becoming increasingly vital. Understanding the complex patterns of gene expression manifested during the course of membranous fracture repair will be crucial to designing therapies that augment poor fracture healing or that expedite normal osseous repair by strategic manipulation of the normal course of gene expression. In the current study, we present a rat model of membranous bone repair. This model has great utility because of its technical simplicity, reproducibility, and relatively low cost. Furthermore, it is a powerful tool for analysis of the molecular regulation of membranous bone repair by immunolocalization and/or in situ hybridization techniques. In this study, an osteotomy was made within the caudal half of the hemimandible, thus producing a stable bone defect without the need for external or internal fixation. The healing process was then catalogued histologically in 28 Sprague-Dawley rats that were serially killed at 1, 2, 3, 4, 5, 6, and 8 weeks after operation. Furthermore, using this novel model, we analyzed, within the context of membranous bone healing, the temporal and spatial expression patterns of several members of the bone morphogenetic protein (BMP) family, known to be critical regulators of cells of osteoblast lineage. Our data suggest that BMP-2/-4 and BMP-7, also known as osteogenic protein-1 (OP-1), are expressed by osteoblasts, osteoclasts, and other more primitive mesenchymal cells within the fracture callus during the early stages of membranous fracture healing. These proteins continue to be expressed during the process of bone remodeling, albeit less prominently. The return of BMP-2/-4 and OP-1 immunostaining to baseline intensity coincides with the histological appearance of mature lamellar bone. Taken together, these data underscore the potentially important regulatory role played by the bone morphogenetic proteins in the process of membranous bone repair.

  View details for Web of Science ID 000166100000018

  View details for PubMedID 11176610

• Development of a device for the delivery of agents to bone during distraction osteogenesis JOURNAL OF CRANIOFACIAL SURGERY Grayson, B. H., Rowe, N. M., Hollier, L. H., Williams, J. K., McCormick, S., Longaker, M. T., McCarthy, J. G. 2001; 12 (1): 19-25

  Abstract

  Various agents have been theoretically and experimentally implicated as mediators of distraction osteogenesis (DO). The purpose of this study was to develop a vehicle for the potential delivery of these factors to the region of the distraction site in an attempt to manipulate this biologic process. Three adult mongrel dogs (12 months old) had oblique osteotomies performed bilaterally through the gonial regions. In group I, the external distracter was affixed to the right hemimandible of two dogs (n = 2 hemimandibles) with cannulated pins (external diameter = 1.5 mm; lumen diameter = 1.0 mm; length = 60 mm), whereas the distracter on the left was affixed with standard, noncannulated pins of the same dimensions. In group II, cannulated pins were used to affix the external distracter to both hemimandibles (n = 2 hemimandibles) of a dog. The devices were activated after a 5-day latency period and were lengthened at a rate of 1 mm/day for 20 days. During the distraction period, 0.1 ml/d of sterile india ink was injected into the cannulated pins, after which the sterile stylet was replaced. The activation protocol was followed by 28 days of fixation (consolidation period). The hemimandibles from group I underwent removal of soft tissues, acetone fixation, and gross examination/photography, whereas the hemimandibles from group II were prepared for histologic evaluation (whole mount, hematoxylin and eosin staining). All dogs survived to the end of the study and demonstrated successful DO without evidence of complications. Hemimandibles in group I displayed evidence of india ink on both the lingual and buccal cortex around the cannulated pin site, in the regenerate and on the neocortices of the distracted segment. Hemimandibles of group II showed histologic evidence of the india ink being deposited densely around the cannulated pin site and extending in a radial fashion around the pin site into the regenerate. This study demonstrates for the first time a vehicle device for the delivery of an inert dye to the regenerate site during distraction osteogenesis. This vehicle offers the potential of delivery of various factors implicated in distraction osteogenesis (i.e., mitogens) in an attempt to alter this process and also substances (i.e., chemotherapy, antibiotics, etc.) for use in the treatment of various osteopathies.

  View details for Web of Science ID 000167445100004

  View details for PubMedID 11314182

• Endogenous cytokine levels as a function of gestational age in fetal rat fibroblasts: Implications for scarless cleft lip/palate repair 9th International Congress of the International-Society-of-Craniofacial-Surgery Peled, Z., Chin, G., HSU, M., Liu, W., Levinson, H., Lee, S., Longaker, M. MEDIMOND S R L. 2001: 105–107

  View details for Web of Science ID 000174770400029

• In vitro "Cleft palate" model with mesenchymal rescue 9th International Congress of the International-Society-of-Craniofacial-Surgery Erfani, S., Crisera, C., Maldonado, T., Warren, S., Bouletreau, P., Peled, Z., Longaker, M. MEDIMOND S R L. 2001: 77–79

  View details for Web of Science ID 000174770400022

• Nasal tip hemangiomas: A new subunit approach 9th International Congress of the International-Society-of-Craniofacial-Surgery Zide, B., Warren, S., Longaker, M. MEDIMOND S R L. 2001: 277–279

  View details for Web of Science ID 000174770400075

• Osteoblast expression of vascular endothelial growth factor is modulated by the extracellular microenvironment Annual Meeting of the American-Association-of-Academic-Surgeons Spector, J. A., Mehrara, B. J., Greenwald, J. A., Saadeh, P. B., Steinbrech, D. S., Bouletreau, P. J., Smith, L. P., Longaker, M. T. AMER PHYSIOLOGICAL SOC. 2001: C72–C80

  Abstract

  Angiogenesis, the formation of new blood vessels, is crucial to the process of fracture healing. Vascular disruption after osseous injury results in an acidic, hypoxic wound environment. We have previously shown that osteoblasts can produce vascular endothelial growth factor (VEGF) in response to a variety of stimuli. In this study we examined pH and lactate concentration, two components of the putative fracture extracellular microenvironment, and determined their relative contribution to regulation of rat calvarial osteoblast VEGF production under both normoxic and hypoxic conditions. Our results demonstrate that pH and lactate concentration do independently affect osteoblast VEGF mRNA and protein production. Acidic pH (7.0) significantly decreased VEGF production, under normoxic and hypoxic conditions (P < 0.05), compared with neutral pH (7.4). This decrease was primarily transcriptionally regulated, because the rate of VEGF mRNA degradation was unchanged at pH 7.0 vs. 7.4. Similarly, an elevated lactate concentration (22 mM) also depressed osteoblast elaboration of VEGF at both neutral and acidic pH (P < 0.001). Furthermore, the effects of increasing acidity and elevated lactate appeared to be additive.

  View details for Web of Science ID 000165930100008

  View details for PubMedID 11121378

• A novel model of mouse mandibular osteotomy healing 9th International Congress of the International-Society-of-Craniofacial-Surgery Saadeh, P., Paccione, M., Warren, S., Bouletreau, P., Longaker, M. MEDIMOND S R L. 2001: 113–115

  View details for Web of Science ID 000174770400031

• In vivo modification of FGF-biologic activity alters cranial suture fate 9th International Congress of the International-Society-of-Craniofacial-Surgery Longaker, M., Greenwald, J., Mehrara, B., Spector, J., Warren, S., Fagenholz, P., Smith, L., Bouletreau, P., Crisera, F., Ueno, H. MEDIMOND S R L. 2001: 93–96

  View details for Web of Science ID 000174770400026

• Understanding the molecular mechanisms governing distraction osteogenesis 9th International Congress of the International-Society-of-Craniofacial-Surgery Mehrara, B., Warren, S., Bouletreau, P., Paccione, M., Longaker, M. MEDIMOND S R L. 2001: 97–100

  View details for Web of Science ID 000174770400027

• Immavure dura mater paracrine signaling increases osteoblast proliferation and differentiation 9th International Congress of the International-Society-of-Craniofacial-Surgery Fagenholz, P., Spector, J., Greenwald, J., Warren, S., Detch, R., Mehrara, B., Longaker, M. MEDIMOND S R L. 2001: 81–84

  View details for Web of Science ID 000174770400023

• The autocrine and paracrine effects of FGF-2 on dura mater: Implications for calvarial growth and re-ossification 9th International Congress of the International-Society-of-Craniofacial-Surgery Smith, L., Spector, J., Greenwald, J., Warren, S., Bouletreau, P., Detch, R., Fagenholz, P., Crisera, F., Longaker, M. MEDIMOND S R L. 2001: 121–124

  View details for Web of Science ID 000174770400033

• Regional dura mater determines fate of overlying cranial suture 9th International Congress of the International-Society-of-Craniofacial-Surgery Greenwald, J., Mehrara, B., Spector, J., Warren, S., Crisera, F., Fagenholz, P., Bouletreau, P., Longaker, M. MEDIMOND S R L. 2001: 89–92

  View details for Web of Science ID 000174770400025

• Latency, rate, and rhythm determine the adaptive response in a rat model of mandibular distraction osteogenesis 9th International Congress of the International-Society-of-Craniofacial-Surgery Paccione, M., Warren, S., Mehrara, B., Bouletreau, P., Longaker, M. MEDIMOND S R L. 2001: 173–175

  View details for Web of Science ID 000174770400046

• Regional differentiation of cranial suture-associated dura mater in vivo and in vitro: Implications for suture fusion and patency JOURNAL OF BONE AND MINERAL RESEARCH Greenwald, J. A., Mehrara, B. J., Spector, J. A., Warren, S. M., Crisera, F. E., Fagenholz, P. J., Bouletreau, P. J., Longaker, M. T. 2000; 15 (12): 2413-2430

  Abstract

  Despite its prevalence, the etiopathogenesis of craniosynostosis is poorly understood. To better understand the biomolecular events that occur when normal craniofacial growth development goes awry, we must first investigate the mechanisms of normal suture fusion. Murine models in which the posterior frontal (PF) suture undergoes programmed sutural fusion shortly after birth provide an ideal model to study these mechanisms. In previous studies, our group and others have shown that sutural fate (i.e., fusion vs. patency) is regulated by the dura mater (DM) directly underlying a cranial suture. These studies have led to the hypothesis that calvarial DM is regionally differentiated and that this differentiation guides the development of the overlying suture. To test this hypothesis, we evaluated the messenger RNA (mRNA) expression of osteogenic cytokines (transforming growth factor beta1 [TGF-beta1] and TGF-beta3) and bone-associated extracellular matrix (ECM) molecules (collagen I, collagen III, osteocalcin, and alkaline phosphatase) in freshly isolated, rat dural tissues associated with the PF (programmed to fuse) or sagittal (SAG; remains patent) sutures before histological evidence of sutural fusion (postnatal day 6 [N6]). In addition, osteocalcin protein expression and cellular proliferation were localized using immunohistochemical staining and 5-bromo-2'deoxyuridine (BrdU) incorporation, respectively. We showed that the expression of osteogenic cytokines and bone-associated ECM molecules is potently up-regulated in the DM associated with the PF suture. In addition, we showed that cellular proliferation in the DM associated with the fusing PF suture is significantly less than that found in the patent SAG suture just before the initiation of sutural fusion N6. Interestingly, no differences in cellular proliferation rates were noted in younger animals (embryonic day 18 [E18] and N2). To further analyze regional differentiation of cranial suture-associated dural cells, we established dural cell cultures from fusing and patent rat cranial sutures in N6 rats and evaluated the expression of osteogenic cytokines (TGF-beta1 and fibroblast growth factor 2 [FGF-2]) and collagen I. In addition, we analyzed cellular production of proliferating cell nuclear antigen (PCNA). These studies confirmed our in vivo findings and showed that dural cell cultures derived from the fusing PF suture expressed significantly greater amounts of TGF-beta1, FGF-2, and collagen I. In addition, similar to our in vivo findings, we showed that PF suture-derived dural cells produced significantly less PCNA than SAG suture-derived dural cells. Finally, coculture of dural cells with fetal rat calvarial osteoblastic cells (FRCs) revealed a statistically significant increase in proliferation (*p < 0.001) in FRCs cocultured with SAG suture-derived dural cells as compared with FRCs cocultured alone or with PF suture-derived dural cells. Taken together, these data strongly support the hypothesis that the calvarial DM is regionally differentiated resulting in the up-regulation of osteogenic cytokines and bone ECM molecules in the dural tissues underlying fusing but not patent cranial sutures. Alterations in cytokine expression may govern osteoblastic differentiation and ECM molecule deposition, thus regulating sutural fate. Elucidation of the biomolecular events that occur before normal cranial suture fusion in the rat may increase our understanding of the events that lead to premature cranial suture fusion.

  View details for Web of Science ID 000165463300014

  View details for PubMedID 11127206

• Gene expression of transforming growth factor-beta 3 and tissue inhibitor of metalloproteinase type 1 during membranous bone healing in rats JOURNAL OF CRANIOFACIAL SURGERY Bouletreau, P. J., Steinbrech, D., Spector, J. A., Warren, S. M., Greenwald, J. A., Mehrara, B. J., Detch, R. C., Longaker, M. T. 2000; 11 (6): 521-526

  Abstract

  A number of growth factors have been implicated in fracture repair. Transforming growth factor-beta 3 (TGF-beta 3) is believed to be involved in osteoblast proliferation, chemotaxis, and collagen synthesis. The collagens act as the scaffolding for new bone matrix formation, whereas tissue inhibitors of metalloproteinases (TIMPs) may help regulate matrix remodeling in bone repair. Despite their hypothesized integral role in fracture repair, the temporal expression of these molecules in membranous bone fracture healing remains unknown. The objective of this study was to assess the temporal pattern of TGF-beta 3 and TIMP type 1 (TIMP-1) expression in rat mandibular fracture healing. Twenty-eight adult male Sprague-Dawley rats underwent a mandibular osteotomy, and the healing regenerate was harvested on postoperative days 3, 5, 7, 9, 23, and 37. Total cellular ribonucleic acid was isolated, and Northern analysis was performed. TGF-beta 3 expression was downregulated dramatically 3 days after the osteotomy and remained less than 20% of control levels throughout repair. In marked contrast, TIMP-1 gene expression, low during early repair, increased more than twofold over control at later time points. Understanding the temporal pattern of gene expression during membranous fracture healing has important clinical implications because elucidating these mechanisms may lead to appropriate biomolecular approaches to augment membranous bone fracture healing.

  View details for Web of Science ID 000167444900003

  View details for PubMedID 11314491

• The effects of ionizing radiation on osteoblast-like cells in vitro PLASTIC AND RECONSTRUCTIVE SURGERY Dudziak, M. E., Saadeh, P. B., Mehrara, B. J., Steinbrech, D. S., Greenwald, J. A., Gittes, G. K., Longaker, M. T. 2000; 106 (5): 1049-1061

  Abstract

  The well-described detrimental effects of ionizing radiation on the regeneration of bone within a fracture site include decreased osteocyte number, suppressed osteoblast activity, and diminished vascularity. However, the biologic mechanisms underlying osteoradionecrosis and the impaired fracture healing of irradiated bone remain undefined. Ionizing radiation may decrease successful osseous repair by altering cytokine expression profiles resulting from or leading to a change in the osteoblastic differentiation state. These changes may, in turn, cause alterations in osteoblast proliferation and extracellular matrix formation. The purpose of this study was to investigate the effects of ionizing radiation on the proliferation, maturation, and cytokine production of MC3T3-E1 osteoblast-like cells in vitro. Specifically, the authors examined the effects of varying doses of ionizing radiation (0, 40, 400, and 800 cGy) on the expression of transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and alkaline phosphatase. In addition, the authors studied the effects of ionizing radiation on MC3T3-E1 cellular proliferation and the ability of conditioned media obtained from control and irradiated cells to regulate the proliferation of bovine aortic endothelial cells. Finally, the authors evaluated the effects of adenovirus-mediated TGF-beta1 gene therapy in an effort to "rescue" irradiated osteoblasts. The exposure of osteoblast-like cells to ionizing radiation resulted in dose-dependent decreases in cellular proliferation and promoted cellular differentiation (i.e., increased alkaline phosphatase production). Additionally, ionizing radiation caused dose-dependent decreases in total TGF-beta1 and VEGF protein production. Decreases in total TGF-beta1 production were due to a decrease in TGF-beta1 production per cell. In contrast, decreased total VEGF production was secondary to decreases in cellular proliferation, because the cellular production of VEGF by irradiated osteoblasts was moderately increased when VEGF production was corrected for cell number. Additionally, in contrast to control cells (i.e., nonirradiated), conditioned media obtained from irradiated osteoblasts failed to stimulate the proliferation of bovine aortic endothelial cells. Finally, transfection of control and irradiated cells with a replication-deficient TGF-beta1 adenovirus before irradiation resulted in an increase in cellular production of TGF-beta1 protein and VEGF. Interestingly, this intervention did not alter the effects of irradiation on cellular proliferation, which implies that alterations in TGF-beta1 expression do not underlie the deficiencies noted in cellular proliferation. The authors hypothesize that ionizing radiation-induced alterations in the cytokine profiles and differentiation states of osteoblasts may provide insights into the cellular mechanisms underlying osteoradionecrosis and impaired fracture healing.

  View details for Web of Science ID 000089824900015

  View details for PubMedID 11039376

• "Ring" lipoma causing extensor tenosynovitis PLASTIC AND RECONSTRUCTIVE SURGERY Sharma, S., Tiwari, P., Longaker, M. T. 2000; 106 (5): 1072-1074

  View details for Web of Science ID 000089824900018

  View details for PubMedID 11039379

• A molecular analysis of the isolated rat posterior frontal and sagittal sutures: Differences in gene expression PLASTIC AND RECONSTRUCTIVE SURGERY Spector, J. A., Mehrara, B. J., Greenwald, J. A., Saadeh, P. B., Steinbrech, D. S., Smith, L. P., Longaker, M. T. 2000; 106 (4): 852-861

  Abstract

  Although it is one of the most commonly occurring craniofacial congenital disabilities, craniosynostosis (the premature fusion of cranial sutures) is nearly impossible to prevent because the molecular mechanisms that regulate the process of cranial suture fusion remain largely unknown. Recent studies have implicated the dura mater in determining the fate of the overlying cranial suture; however, the molecular biology within the suture itself has not been sufficiently investigated. In the murine model of cranial suture fusion, the posterior frontal suture is programmed to begin fusing by postnatal day 12 in rats (day 25 in mice), reliably completing bony union by postnatal day 22 (day 45 in mice). In contrast, the sagittal suture remains patent throughout the life of the animal. Using this model, this study sought to examine for the first time what differences in gene expression--if any--exist between the two sutures with opposite fates. For each series of experiments, 35 to 40 posterior frontal and sagittal suture complexes were isolated from 6-day-old Sprague-Dawley rat pups. Suture-derived cell cultures were established, and ribonuicleic acid was derived from snap-frozen, isolated suture tissue. Results demonstrated that molecular differences between the posterior frontal and sagittal suture complexes were readily identified in vivo, although these distinctions were lost once the cells comprising the suture complex were cultured in vitro. Hypothetically, this change in gene expression resulted from the loss of the influence of the underlying dura mater. Significant differences in the expression of genes encoding extracellular matrix proteins existed in vivo between the posterior frontal and sagittal sutures. However, the production of the critical, regulatory cytokine transforming growth factor beta-1 was equal between the two suture complexes, lending further support to the hypothesis that dura mater regulates the fate of the overlying cranial suture.

  View details for Web of Science ID 000089084200017

  View details for PubMedID 11007400

• Immature versus mature dura mater: II. Differential expression of genes important to calvarial reossification PLASTIC AND RECONSTRUCTIVE SURGERY Greenwald, J. A., Mehrara, B. J., Spector, J. A., Fagenholz, P. J., Saadeh, P. B., Steinbrech, D. S., Gittes, G. K., Longaker, M. T. 2000; 106 (3): 630-638

  Abstract

  The ability of immature animals and newborns to orchestrate successful calvarial reossification is well described. This capacity is markedly attenuated in mature animals and in humans greater than 2 years of age. Previous studies have implicated the dura mater as critical to successful calvarial reossification. The authors have previously reported that immature, but not mature, dural tissues are capable of elaborating a high expression of osteogenic growth factors and extracellular matrix molecules. These findings led to the hypothesis that a differential expression of osteogenic growth factors and extracellular matrix molecules by immature and mature dural tissues may be responsible for the clinically observed phenotypes (i.e., immature animals reossify calvarial defects; mature animals do not). This study continues to explore the hypothesis through an analysis of transforming growth factor (TGF)-beta3, collagen type III, and alkaline phosphatase mRNA expression. Northern blot analysis of total RNA isolated from freshly harvested immature (n = 60) and mature (n = 10) dural tissues demonstrated a greater than three-fold, 18-fold, and nine-fold increase in TGF-beta3, collagen type III, and alkaline phosphatase mRNA expression, respectively, in immature dural tissues as compared with mature dural tissues. Additionally, dural cell cultures derived from immature (n = 60) and mature dura mater (n = 10) were stained for alkaline phosphatase activity to identify the presence of osteoblast-like cells. Alkaline phosphatase staining of immature dural cells revealed a significant increase in the number of alkaline phosphatase-positive cells as compared with mature dural tissues (p < 0.001). In addition to providing osteogenic humoral factors (i.e., growth factors and extracellular matrix molecules), this finding suggests that immature, but not mature, dura mater may provide cellular elements (i.e., osteoblasts) that augment successful calvarial reossification. These studies support the hypothesis that elaboration of osteogenic growth factors (i.e., TGF-beta33) and extracellular matrix molecules (i.e., collagen type III and alkaline phosphatase) by immature, but not mature, dural tissues may be critical for successful calvarial reossification. In addition, these studies suggest for the first time that immature dural tissues may provide cellular elements (i.e., osteoblasts) to augment this process.

  View details for Web of Science ID 000088925700016

  View details for PubMedID 10987470

• Differential expression of fibromodulin, a transforming growth factor-beta modulator, in fetal skin development and scarless repair 83rd Annual Clinical Congress of the American-College-of-Surgeons Soo, C., Hu, F. Y., Zhang, X. L., Wang, Y., Beanes, S. R., Lorenz, H. P., Hedrick, M. H., Mackool, R. J., Plaas, A., Kim, S. J., Longaker, M. T., Freymiller, E., Ting, K. ELSEVIER SCIENCE INC. 2000: 423–33

  Abstract

  Transforming growth factor-beta (TGF-beta1, -beta2, and -beta3) has been implicated in the ontogenetic transition from scarless fetal repair to adult repair with scar. Generally, TGF-beta exerts its effects through type I and II receptors; however, TGF-beta modulators such as latent TGF-beta binding protein-1 (LTBP-1), decorin, biglycan, and fibromodulin can bind and potentially inhibit TGF-beta activity. To more fully explore the role of TGF-beta ligands, receptors, and potential modulators during skin development and wound healing, we have used a rat model that transitions from scarless fetal-type repair to adult-type repair with scar between days 16 and 18 of gestation. We showed that TGF-beta ligand and receptor mRNA levels did not increase during the transition to adult-type repair in fetal skin, whereas LTBP-1 and fibromodulin expression decreased. In addition, TGF-beta1 and -beta3; type I, II, and III receptors; as well as LTBP-1, decorin, and biglycan were up-regulated during adult wound healing. In marked contrast, fibromodulin expression was initially down-regulated in adult repair. Immunostaining demonstrated significant fibromodulin induction 36 hours after injury in gestation day 16, but not day 19, fetal wounds. This inverse relationship between fibromodulin expression and scarring in both fetal and adult rat wound repair suggests that fibromodulin may be a biologically relevant modulator of TGF-beta activity during scar formation.

  View details for Web of Science ID 000088625100011

  View details for PubMedID 10934147

• The true hanging columella: Simplified diagnosis and treatment using a modified direct approach PLASTIC AND RECONSTRUCTIVE SURGERY Matarasso, A., Greer, S. E., Longaker, M. T. 2000; 106 (2): 469-474

  Abstract

  An imbalance between the alar rim and the columella border can be a disturbing aesthetic deformity. If the cause is a pseudohanging columella, the therapy should be directed to the alar rims. When the deformity is a true hanging columella with unusually wide medial crural cartilages, balance can be restored by excising a C-shaped crescent of cartilage from the cranial border of the medial crura of the alar cartilages in a direct approach. This condition was present in approximately 15 percent of the patients reviewed. The treatment of a true hanging columella adds a subtle beneficial enhancement to the results of a rhinoplasty. The authors describe a simplified diagnostic method and present their experience treating the true hanging columella using a modified "direct approach" through a closed endonasal rhinoplasty.

  View details for Web of Science ID 000088631000037

  View details for PubMedID 10946947

• Reconstruction of a tibial defect with microvascular transfer of a previously fractured fibula ANNALS OF PLASTIC SURGERY Sharma, S., Tiwari, P., Kasabian, A. K., Longaker, M. T. 2000; 45 (2): 202-206

  Abstract

  A 43-year-old man sustained severe injuries to his lower limbs with extensive soft-tissue damage and bilateral tibial-fibular fractures. Acutely, the patient underwent external fixation and a free latissimus dorsi flap for soft-tissue coverage of the left leg. However, the tibia had a nonviable butterfly fragment that left a 7-cm defect after debridement. Subsequently, the contralateral fractured fibula was used as a bridging vascularized graft for this tibial defect. The transfer of a fibula containing the zone of injury from a previous high-energy fracture has not been reported. This case demonstrates the successful microvascular transfer of a previously fractured fibula for the repair of a contralateral tibial bony defect.

  View details for Web of Science ID 000088658800020

  View details for PubMedID 10949352

• Mechanisms of fibroblast growth factor-2 modulation of vascular endothelial growth factor expression by osteoblastic cells ENDOCRINOLOGY Saadeh, P. B., Mehrara, B. J., Steinbrech, D. S., Spector, J. A., Greenwald, J. A., Chim, G. S., Ueno, H., Gittes, G. K., Longaker, M. T. 2000; 141 (6): 2075-2083

  Abstract

  Normal bone growth and repair is dependent on angiogenesis. Fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGFbeta) have all been implicated in the related processes of angiogenesis, growth, development, and repair. The purpose of this study was to investigate the relationships between FGF-2 and both VEGF and TGFbeta in nonimmortalized and clonal osteoblastic cells. Northern blot analysis revealed 6-fold peak increases in VEGF mRNA at 6 h in fetal rat calvarial cells and MC3T3-E1 osteoblastic cells after stimulation with FGF-2. Actinomycin D inhibited these increases in VEGF mRNA, whereas cycloheximide did not. The stability ofVEGF mRNA was not increased after FGF-2 treatment. Furthermore, FGF-2 induced dose-dependent increases in VEGF protein levels (P < 0.01). Although in MC3T3-E1 cells, TGFbeta1 stimulates a 6-fold peak increase in VEGF mRNA after 3 h of stimulation, we found that both TGFbeta2 and TGFbeta3 yielded 2- to 3-fold peak increases in VEGF mRNA levels noted after 6 h of stimulation. Similarly, both TGFbeta2 and TGFbeta3 dose dependently increased VEGF protein production. To determine whether FGF-2-induced increases in VEGF mRNA may have occurred independently of TGFbeta, we disrupted TGFbeta signal transduction (using adenovirus encoding a truncated form of TGFbeta receptor II), which attenuated TGFbeta1 induction of VEGF mRNA, but did not impede FGF-2 induction ofVEGF mRNA. In summary, FGF-2-induced VEGF expression by osteoblastic cells is a dose-dependent event that may be independent of concomitant FGF-2-induced modulation of TGFbeta activity.

  View details for Web of Science ID 000088386100025

  View details for PubMedID 10830293

• Traumatic abdominal wall pseudolipoma following suction-assisted lipectomy PLASTIC AND RECONSTRUCTIVE SURGERY Sharma, S., Perrotti, J. A., Longaker, M. T. 2000; 105 (7): 2589-2592

  View details for Web of Science ID 000087277300048

  View details for PubMedID 10845316

• The impact of biomolecular medicine and tissue engineering on plastic surgery in the 21st century PLASTIC AND RECONSTRUCTIVE SURGERY Lorenz, H. P., Hedrick, M. H., Chang, J., Mehrara, B. J., Longaker, M. T. 2000; 105 (7): 2467-2481

  View details for Web of Science ID 000087277300027

  View details for PubMedID 10845305

• In vitro validation of duct differentiation in developing embryonic mouse pancreas JOURNAL OF SURGICAL RESEARCH Kadison, A. S., Maldonado, T. S., Crisera, C. A., Longaker, M. T., Gittes, G. K. 2000; 90 (2): 126-130

  Abstract

  Early embryonic pancreatic epithelia have the capacity for either endocrine or exocrine lineage commitment. Recent studies demonstrated the pluripotential nature of these undifferentiated cells. Isolated pancreatic epithelia grown under the renal capsule formed primarily islets. However, when these same epithelia were grown in a basement-membrane-rich gel (Matrigel) they formed mostly ducts. Currently, there is no model for in vitro pancreatic duct formation and therefore, the mechanism of duct morphogenesis has never been described. The purpose of this study was to provide such a model by characterizing the expression of two duct markers, carbonic anhydrase II (CAII) and the cystic fibrosis transmembrane conductance regulator (CFTR), in isolated undifferentiated pancreatic epithelia grown in vitro.We microdissected embryonic pancreases at Embryonic Days (E)9.5-11.5 and performed RT-PCR for CAII and CFTR on E9.5 whole pancreases, E10. 5 and E11.5 epithelia, as well as E11.5 epithelia grown for 7 days in Matrigel. Next we performed in situ hybridization for CAII and CFTR and immunohistochemistry for CAII on E11.5 epithelia grown for 7 days in Matrigel.Early, undifferentiated embryonic pancreatic epithelium does not express CAII and CFTR by RT-PCR. When E11.5 epithelia were grown for 7 days in Matrigel, however, gene expression for both markers is upregulated as ducts form. Furthermore, CAII was seen by IHC and both CAII and CFTR were seen by in situ hybridization in the ducts after 7 days in Matrigel.These data validate our in vitro system as a model for studying the mechanism of normal pancreatic duct differentiation and may potentially help us to understand the faulty mechanism involved in pancreatic ductal carcinogenesis.

  View details for Web of Science ID 000087240000006

  View details for PubMedID 10792952

• Gene expression of TGF-beta, TGF-beta receptor, and extracellular matrix proteins during membranous bone healing in rats PLASTIC AND RECONSTRUCTIVE SURGERY Steinbrech, D. S., Mehrara, B. J., Rowe, N. M., Dudziak, M. E., Luchs, J. S., Saadeh, P. B., Gittes, G. K., Longaker, M. T. 2000; 105 (6): 2028-2038

  Abstract

  Poorly healing mandibular fractures and osteotomies can be troublesome complications of craniomaxillofacial trauma and reconstructive surgery. Gene therapy may offer ways of enhancing bone formation by altering the expression of desired growth factors and extracellular matrix molecules. The elucidation of suitable candidate genes for therapeutic intervention necessitates investigation of the endogenously expressed patterns of growth factors during normal (i.e., successful) fracture repair. Transforming growth factor beta1 (TGF-beta1), its receptor (Tbeta-RII), and the extracellular matrix proteins osteocalcin and type I collagen are thought to be important in long-bone (endochondral) formation, fracture healing, and osteoblast proliferation. However, the spatial and temporal expression patterns of these molecules during membranous bone repair remain unknown. In this study, 24 adult rats underwent mandibular osteotomy with rigid external fixation. In addition, four identically treated rats that underwent sham operation (i.e., no osteotomy) were used as controls. Four experimental animals were then killed at each time point (3, 5, 7, 9, 23, and 37 days after the procedure) to examine gene expression of TGF-beta1 and Tbeta-RII, osteocalcin, and type I collagen. Northern blot analysis was used to compare gene expression of these molecules in experimental animals with that in control animals (i.e., nonosteotomized; n = 4). In addition, TGF-beta1 and T-RII proteins were immunolocalized in an additional group of nine animals killed on postoperative days 3, 7, and 37. The results of Northern blot analysis demonstrated a moderate increase (1.7 times) in TGF-beta1 expression 7 days postoperatively; TGF-beta1 expression returned thereafter to near baseline levels. Tbeta-RII mRNA expression was downregulated shortly after osteotomy but then increased, reaching a peak of 1.8 times the baseline level on postoperative day 9. Osteocalcin mRNA expression was dramatically downregulated shortly after osteotomy and remained low during the early phases of fracture repair. Osteocalcin expression trended slowly upward as healing continued, reaching peak expression by day 37 (1.7 times the control level). In contrast, collagen type IalphaI mRNA expression was acutely downregulated shortly after osteotomy, peaked on postoperative days 5, and then decreased at later time points. Histologic samples from animals killed 3 days after osteotomy demonstrated TGF-beta1 protein localized to inflammatory cells and extracellular matrix within the fracture gap, periosteum, and peripheral soft tissues. On postoperative day 7, TGF-beta1 staining was predominantly localized to the osteotomized bone edges, periosteum, surrounding soft tissues, and residual inflammatory cells. By postoperative day 37, complete bony healing was observed, and TGF-beta1 staining was localized to the newly formed bone matrix and areas of remodeling. On postoperative day 3, Tbeta-RII immunostaining localized to inflammatory cells within the fracture gap, periosteal cells, and surrounding soft tissues. By day 7, Tbeta-RII staining localized to osteoblasts of the fracture gap but was most intense within osteoblasts and mesenchymal cells of the osteotomized bone edges. On postoperative day 37, Tbeta-RII protein was seen in osteocytes, osteoblasts, and the newly formed periosteum in the remodeling bone. These observations agree with those of previous in vivo studies of endochondral bone formation, growth, and healing. In addition, these results implicate TGF-beta1 biological activity in the regulation of osteoblast migration, differentiation, and proliferation during mandibular fracture repair. Furthermore, comparison of these data with gene expression during mandibular distraction osteogenesis may provide useful insights into the treatment of poorly healing fractures because distraction osteogenesis has been shown to be effective in the management of these difficult clinical cases.

  View details for Web of Science ID 000086725000018

  View details for PubMedID 10839400

• The use of newborn rats and an adenoviral gene delivery vector as a model system for wound-healing research ANNALS OF PLASTIC SURGERY Liu, W., Mehrara, B. J., Chin, G. S., HSU, M., Peled, Z., Longaker, M. T. 2000; 44 (5): 543-551

  Abstract

  An attractive experimental method to elucidate the role of growth factors and cytokines in cutaneous wound healing would be to overexpress or "knock out" a molecule using a gene delivery vector and observe the impact on the wound repair process. As a first step toward developing an adenoviral gene delivery procedure to study wound repair, the authors injected beta-galactosidase (beta-gal) adenoviruses either subcutaneously or intradermally into the dorsal skin of 10-day-old postnatal Sprague-Dawley rats. Histological analysis and beta-gal staining were used to determine the expression and localization of the transferred gene. Beta-gal expression was observed as early as day 1 and up to day 7 postintradermal injection and day 9 postsubcutaneous injection, with no obvious inflammatory reaction detected at the injection sites. Furthermore, as expected, greater beta-gal expression was observed in the dermis of intradermally injected rats compared with the dermis of subcutaneously injected rats. Next, the authors sought to determine whether cutaneous wounds would heal before dissipation of the transferred gene. They created incisional and excisional wounds on the backs of similar-age rats. They found that incisional wounds closed by day 5 postwounding, whereas excisional wounds closed by day 14 postwounding. Their study demonstrated that an adenoviral vector delivered a gene efficiently into newborn rat skin and maintained the gene expression for at least as long as it would take for an incisional wound to heal. The combined use of newborn rat wound models and an adenoviral vector may provide a useful in vivo system to define the biological roles of growth factors and cytokines involved in the wound repair process. These discoveries may lead to the development of gene therapy approaches for abnormal wound healing.

  View details for Web of Science ID 000087001700025

  View details for PubMedID 10805307

• Ontogeny of activin B and follistatin in developing embryonic mouse pancreas: Implications for lineage selection JOURNAL OF GASTROINTESTINAL SURGERY Maldonado, T. S., Kadison, A. S., Crisera, C. A., Grau, J. B., Alkasab, S. L., Longaker, M. T., Gittes, G. K. 2000; 4 (3): 269-275

  Abstract

  Activin, a member of the transforming growth factor-beta superfamily, has been shown to be a critical regulator in exocrine and endocrine pancreas formation. The purpose of our study was to describe the ontogeny of activin B and its inhibitor, follistatin, in developing pancreas and to elucidate potential mechanisms for exocrine and endocrine lineage selection. Mouse embryonic pancreata were dissected at various ages (day 10 [E10.5] to birth [E18.5]), sectioned, and immunostained for activin B (one of two existing isomers, A and B), follistatin, insulin, and glucagon. In addition, reverse transcriptase-polymerase chain reaction was employed to determine the messenger RNA expression of follistatin in isolated pancreatic epithelia and mesenchyme of various ages. Activin B was first detected at E12.5 in epithelial cells coexpressing glucagon. At E16.5 these coexpressors appeared as clusters in close proximity to early ducts. By E18.5 activin B was localized to forming islets where cells coexpressed glucagon and were arranged in the mantle formation characteristic of mature alpha cells. Follistatin was found to be ubiquitous in pancreatic mesenchyme at early ages by immunohistochemical analysis, disappearing sometime after E12.5. Follistatin reappeared in E18.5 islets and remains expressed in adult islets. Follistatin messenger RNA was first detected in epithelium at E11.5, preceding its protein expression in islets later in gestation. We propose that mesenchyme-derived follistatin inhibits epithelium-derived activin at early embryonic ages allowing for unopposed exocrine differentiation and relative suppression of endocrine differentiation. At later ages the decrease in the amount of mesenchyme relative to epithelium and the subsequent drop in follistatin levels liberates epithelial activin to allow differentiation of endocrine cells to form mature islets by the time of birth.

  View details for Web of Science ID 000087138800012

  View details for PubMedID 10769089

• Defective epithelial-mesenchymal interactions dictate the organogenesis of tracheoesophageal fistula PEDIATRIC SURGERY INTERNATIONAL Crisera, C. A., Grau, J. B., Maldonado, T. S., Kadison, A. S., Longaker, M. T., Gittes, G. K. 2000; 16 (4): 256-261

  Abstract

  We have previously suggested that the fistula tract in esophageal atresia with tracheoesophageal fistula (EA/TEF) arises from a trifurcation of the embryonic lung bud. Thus, it appears to be a respiratory-derived structure, and expresses the lung-specific transcription factor TTF-1 in its epithelium. The fistula tract does not give rise to lungs like the other branches from the bud. It grows caudally until it fistulizes with the stomach. We hypothesized that epithelial-mesenchymal interactions (EMI) dictate the differential pattern of growth of the respiratory-derived fistula tract in EA/TEF. EA/TEF was induced in rat embryos via prenatal exposure to adriamycin. Microdissection was performed on E13.5 embryos to isolate developing lung bud, fistula tract, or esophagus from adriamycin-treated or control animals, respectively. The mesenchyme and epithelium from each of these foregut structures were separated. The individual epithelia were recombined with each of the various mesenchymes and grown in culture. They were assayed for relative degrees of branching. Isolated lung-bud epithelia (LBE) or fistula epithelium were also cultured in Matrigel with exogenous fibroblast growth factors (FGF) and subsequently assayed for branching. The fistula-tract mesenchyme relatively inhibited branching of lung epithelium. The epithelium of the fistula tract could be induced to branch by non-fistula (lung or esophageal) mesenchyme. The fistula-tract and adriamycin-treated LBE both branched in response to FGF1. In contrast, neither responded to FGF7 or FGF10. EMI are defective in the developing EA/TEF. The inability to respond to FGF7 and FGF10 suggests an epithelial defect involving the receptor FGF2R-IIIb, to which these mesenchymal factors obligately bind. Thus, the mesenchyme around the developing fistula tract may lack an FGF branching morphogen(s), such as FGF1. Hence, this mesenchyme is unable to induce branching of respiratory epithelia and allows the middle branch of the embryonic tracheal trifurcation to grow caudally as an unbranched tube until it fistulizes into the stomach.

  View details for Web of Science ID 000087910600007

  View details for PubMedID 10898225

• "Pumping the regenerate": An evaluation of oscillating distraction osteogenesis in the rodent mandible 16th Annual Meeting of the Northeastern-Society-of-Plastic-Surgeons Greenwald, J. A., Luchs, J. S., Mehrara, B. J., Spector, J. A., Mackool, R. J., McCarthy, J. G., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2000: 516–21

  Abstract

  Mandibular distraction osteogenesis (DO) has become an important technique to lengthen the hypoplastic mandible and to reconstruct osseous defects after ablative surgery. The hallmark of successful DO is the creation of new bone within the distraction gap. Several anecdotal reports have described alternating compressing and lengthening protocols (i.e., "pumping the regenerate") to augment regenerate bone formation. The purpose of this experiment was to analyze formally the effects of an alternating compression/distraction protocol with a traditional distraction protocol. Ten adult male rats underwent unilateral mandibular osteotomy with placement of a custom distractor. After a latency period of 5 days, distraction was initiated at a rate of 0.25 mm twice daily. Animals in the control group (N = 5) were distracted to a length of 5.0 mm for 10 days at a rate of 0.25 mm twice daily. In contrast, animals in the experimental group (N = 5) were distracted to a length of 2.5 mm (at a rate of 0.25 mm twice daily) for 5 days, then compressed 1.0 mm for a 2-day period, and redistracted to a length of 5.0 mm. Regenerate cross-sectional area was evaluated by computed tomography performed after 5 weeks of consolidation. Gross examination and histological analysis were performed by a panel of experienced reviewers. Radiological as well as histological analysis of regenerate cross-sectional area demonstrated no significant differences between experimental (i.e., "pumped") and control groups. Both groups demonstrated excellent regenerate bone formation with no evidence of fibrous union. This study represents the first attempt to investigate the anecdotal technique of pumping the mandibular regenerate. The authors have demonstrated that pumping the regenerate leads to no substantial differences in radiological or histological appearance of regenerate bone formation.

  View details for Web of Science ID 000087001700018

  View details for PubMedID 10805303

• Expression of adenovirally delivered gene products in healing osseous tissues 16th Annual Meeting of the Northeastern-Society-of-Plastic-Surgeons Spector, J. A., Mehrara, B. J., Luchs, J. S., Greenwald, J. A., Fagenholz, P. J., Saadeh, P. B., Steinbrech, D. S., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2000: 522–28

  Abstract

  Gene therapy has moved from the promise of laboratory investigation to the reality of clinical practice in just the last decade. Various methods for delivery of genes to host cells have been developed and utilized both in vitro and in vivo. From the perspective of the plastic surgeon, gene therapy holds the promise to augment healing in clinical situations that remain difficult to treat, such as chronic wounds, osteoradionecrosis, or possibly to expedite current clinical practices, such as distraction osteogenesis. The authors chose to investigate the potential for gene therapy in osseous tissues using a replication-deficient adenovirus vector to deliver the marker transgene beta-galactosidase. An adenovirus vector is ideal for use in situations in which transgene expression is desired for only a relatively short period of time, such as wound and fracture healing. Utilizing a rat mandibular osteotomy model, they demonstrated that, using an adenoviral vector, foreign genes can be delivered in a simple fashion and can be expressed in a reliable manner within and around the osteotomy site for at least 10 days. Furthermore, there was no evidence of transfection of distant tissues associated with local application of the adenovirus vector. With this information, clinicians may now attempt to deliver osteogenic and angiogenic genes in a site-specific fashion to improve and expedite osseous healing.

  View details for Web of Science ID 000087001700019

  View details for PubMedID 10805304

• Hypoxia increases insulinlike growth factor gene expression in rat osteoblasts 16th Annual Meeting of the Northeastern-Society-of-Plastic-Surgeons Steinbrech, D. S., Mehrara, B. J., Saadeh, P. B., Greenwald, J. A., Spector, J. A., Gittes, G. K., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2000: 529–34

  Abstract

  Vascular disruption secondary to fracture leads to a hypoxic zone of injury where the oxygen tension at the center of the wound is quite low. In this dynamic microenvironment, a number of growth factors are elaborated to stimulate the synthetic processes of fracture repair. Previously the authors have shown the hypoxia-induced increase of vascular endothelial growth factor expression in osteoblasts. The purpose of these experiments was to examine osteoblast expression of insulinlike growth factors (IGF) I and II--cytokines believed to play a role in increased collagen synthesis, chemotaxis, and proliferation of osteoblasts in response to hypoxia. Primary cell cultures of osteoblasts isolated from neonatal rat calvaria were subjected to hypoxia (PO2 = 35 mmHg) for 0, 3, 6, 24, and 48 hours. Northern blot analysis of ribonucleic acid (RNA) from resulting cultures demonstrated a more than 60% increase in IGF-II messenger RNA (mRNA) expression after 3 hours of hypoxia. IGF-II mRNA expression continued to increase through later time points to 200% and 260% of baseline at 24 and 48 hours respectively. In contrast, IGF-I demonstrated no significant change in mRNA expression compared with baseline control (normoxia) cultures. In these experiments the authors have demonstrated a hypoxia-induced increase in IGF-II but not IGF-I in primary osteoblasts. The differential expression of these two growth factors may underscore important differences in the behavior of osteoblasts in the hypoxic fracture microenvironment. Taken together, these data add additional support to the theory that hypoxia induces gene-specific changes in expression of molecules important to extracellular matrix formation for successful bone healing.

  View details for Web of Science ID 000087001700021

  View details for PubMedID 10805305

• VEGF expression in an osteoblast-like cell line is regulated by a hypoxia response mechanism AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Steinbrech, D. S., Mehrara, B. J., Saadeh, P. B., Greenwald, J. A., Spector, J. A., Gittes, G. K., Longaker, M. T. 2000; 278 (4): C853-C860

  Abstract

  Angiogenesis is essential for the increased delivery of oxygen and nutrients required for the reparative processes of bone healing. Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, has been implicated in this process. We have previously shown that hypoxia specifically and potently regulates the expression of VEGF by osteoblasts. However, the molecular mechanisms governing this interaction remain unknown. In this study, we hypothesized that the hypoxic regulation of VEGF expression by osteoblasts occurs via an oxygen-sensing mechanism similar to the regulation of the erythropoietin gene (EPO). To test this hypothesis, we examined the kinetics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt on the expression of VEGF in osteoblastic cells because these metallic ions mimic hypoxia by binding to the heme portion of oxygen-sensing molecules. Our results indicated that hypoxia potently stimulates VEGF mRNA expression. In addition, we found that nickel and cobalt both stimulate VEGF gene expression in a similar time- and dose-dependent manner, suggesting the presence of a hemelike oxygen-sensing mechanism similar to that of the EPO gene. Moreover, actinomycin D, cycloheximide, dexamethasone, and mRNA stabilization studies collectively established that this regulation is predominantly transcriptional, does not require de novo protein synthesis, and is not likely mediated by the transcriptional activator AP-1. These studies demonstrate that hypoxia, nickel, and cobalt regulate VEGF expression in osteoblasts via a similar mechanism, implicating the involvement of a heme-containing oxygen-sensing molecule. This may represent an important mechanism of VEGF regulation leading to increased angiogenesis in the hypoxic microenvironment of healing bone.

  View details for Web of Science ID 000086325900025

  View details for PubMedID 10751333

• Biomolecular mechanisms of calvarial bone induction: Immature versus mature dura mater PLASTIC AND RECONSTRUCTIVE SURGERY Greenwald, J. A., Mehrara, B. J., Spector, J. A., Chin, G. S., Steinbrech, D. S., Saadeh, P. B., Luchs, J. S., Paccione, M. F., Gittes, G. K., Longaker, M. T. 2000; 105 (4): 1382-1392

  Abstract

  The ability of newborns and immature animals to reossify calvarial defects has been well described. This capacity is generally lost in children greater than 2 years of age and in mature animals. The dura mater has been implicated as a regulator of calvarial reossification. To date, however, few studies have attempted to identify biomolecular differences in the dura mater that enable immature, but not mature, dura to induce osteogenesis. The purpose of these studies was to analyze metabolic characteristics, protein/gene expression, and capacity to form mineralized bone nodules of cells derived from immature and mature dura mater. Transforming growth factor beta-1, basic fibroblast growth factor, collagen type IalphaI, osteocalcin, and alkaline phosphatase are critical growth factors and extracellular matrix proteins essential for successful osteogenesis. In this study, we have characterized the proliferation rates of immature (6-day-old rats, n = 40) and mature (adult rats, n = 10) dura cell cultures. In addition, we analyzed the expression of transforming growth factor beta-1, basic fibroblast growth factor-2, proliferating cell nuclear antigen, and alkaline phosphatase. Our in vitro findings were corroborated with Northern blot analysis of mRNA expression in total cellular RNA isolated from snap-frozen age-matched dural tissues (6-day-old rats, n = 60; adult rats, n = 10). Finally, the capacity of cultured dural cells to form mineralized bone nodules was assessed. We demonstrated that immature dural cells proliferate significantly faster and produce significantly more proliferating cell nuclear antigen than mature dural cells (p < 0.01). Additionally, immature dural cells produce significantly greater amounts of transforming growth factor beta-1, basic fibroblast growth factor-2, and alkaline phosphatase (p < 0.01). Furthermore, Northern blot analysis of RNA isolated from immature and mature dural tissues demonstrated a greater than 9-fold, 8-fold, and 21-fold increase in transforming growth factor beta-1, osteocalcin, and collagen IalphaI gene expression, respectively, in immature as compared with mature dura mater. Finally, in keeping with their in vivo phenotype, immature dural cells formed large calcified bone nodules in vitro, whereas mature dural cells failed to form bone nodules even with extended culture. These studies suggest that differential expression of growth factors and extracellular matrix molecules may be a critical difference between the osteoinductive capacity of immature and mature dura mater. Finally, we believe that the biomolecular bone- and matrix-inducing phenotype of immature dura mater regulates the ability of young children and immature animals to heal calvarial defects.

  View details for Web of Science ID 000085995500018

  View details for PubMedID 10744229

• Lip service for the stiff upper lip PLASTIC AND RECONSTRUCTIVE SURGERY Zide, B. M., Bradley, J. P., Longaker, M. T. 2000; 105 (3): 1154-1158

  Abstract

  Lip augmentation procedures can restore volume and shape to the aging, thin upper lip, but some patients may develop problematic lip tightness. This stiff upper lip is manifested by a restricted smile and an adynamic central upper lip. We have had success in treating postreconstruction and postaugmentation stiff upper lip with a therapeutic device and treatment regimen. This therapy alleviated tightness and inability to smile. Also, the change in lip commissure-to-commissure distance in repose and when smiling improved after treatment.

  View details for Web of Science ID 000085787600050

  View details for PubMedID 10724277

• Controlled multiplanar distraction of the mandible part III: Laboratory studies of sagittal (anteroposterior) and horizontal (mediolateral) movements JOURNAL OF CRANIOFACIAL SURGERY Hollier, L. H., Rowe, N. M., Mackool, R. J., Williams, J. K., Kim, J. H., Longaker, M. T., Grayson, B. H., McCarthy, J. G. 2000; 11 (2): 83-95

  Abstract

  Distraction osteogenesis has proven to be an effective technique for the correction of mandibular deficiencies. However, problems have been encountered in achieving a final, idealized form of the mandible when using distraction devices capable of moving the bone segments in only one dimension (uniplanar). Specifically, occlusal irregularities and deficiencies in lower facial contour have been seen following uniplanar distraction. To address these problems, a distraction device capable of independent movements in three planes (multiplanar) was developed. Previously reported studies in a canine model have demonstrated that this device can successfully distract the mandible along both the sagittal axis (anteroposterior or z-axis) and the vertical axis (superoinferior or y-axis). This study examines the ability of the multiplanar device to distract along the sagittal and horizontal axes (mediolateral or x-axis). A total of 12 dogs were included in the study. All animals underwent unilateral or bilateral mandibular distraction using an external multiplanar device. After a latency period of 5 days, primary distraction along the anteroposterior axis at a rate of 1 mm/day for 10 days (10 mm total) was performed. During the following 10 days, along with an additional 11 mm to 20 mm of anteroposterior axis distraction, concomitant secondary distraction was performed along the horizontal (mediolateral) axis at a rate of 5 degrees/day (50 degrees total). Cephalometric radiographs were obtained preoperatively and at the conclusion of both anteroposterior and combined anteroposterior-mediolateral distraction. Computed tomography (CT) scans were obtained preoperatively and at the end of consolidation (28 days), after which all animals were sacrificed and the dry skulls examined. In all animals, distraction along the mediolateral or x-axis was found to change the anteroposterior projection of the mandible. Varus angulation of the device with respect to the midline of the mandible caused compression of the distracted segments and reduced the anteroposterior thrust of the mandible. In contrast, valgus positioning of the device, with respect to the midline of the mandible, created the opposite effect, increasing the distracted length in the anteroposterior direction. The bone (mandibular) segments being distracted assumed the orientation of the device only for valgus positioning of the device (producing a decrease in the bigonial distance). Conversely, there was no effect from the mediolateral angulation on the distracted segments during varus positioning of the device. A possible explanation for this finding may be a greater resistance to an increase in the bigonial distance (varus positioning of the device) posed by obstruction of lateral movement of the condyle. This stands in contrast to a decrease in the bigonial distance observed following valgus positioning of the device. These findings confirm the clinical impression that distraction along the anteroposterior or sagittal axis remains the critical or keystone therapeutic maneuver in distraction of the mandible. Mediolateral or horizontal axis distraction is best used only as a supplementary movement; in essence, it only affects the anteroposterior dimension with little impact on clinically relevant changes to the bigonial distance.

  View details for Web of Science ID 000085997900003

  View details for PubMedID 11314135

• Differential expression of matrix metalloproteinases and their tissue-derived inhibitors in cutaneous wound repair PLASTIC AND RECONSTRUCTIVE SURGERY Soo, C., Shaw, W. W., Zhang, X. L., Longaker, M. T., Howard, E. W., Ting, K. 2000; 105 (2): 638-647

  Abstract

  Wound extracellular matrix is a key regulator of cell adhesion, migration, proliferation, and differentiation during cutaneous repair. The amount and organization of normal wound extracellular matrix are determined by a dynamic balance among overall matrix synthesis, deposition, and degradation. Matrix metalloproteinases (MMPs) are one family of structurally related enzymes that have the collective ability to degrade nearly all extracellular matrix components. The MMPs are broadly categorized into collagenases, gelatinases, stromelysins, and membrane-type MMPs by their substrate specificity. The aim of this study was to characterize the temporal changes in mRNA profiles for rat collagenase [matrix metalloproteinase-1 (MMP-1)], gelatinase A (MMP-2), matrilysin (MMP-7), gelatinase B (MMP-9), and membrane type 1-MMP (MT1-MMP), as well as tissue inhibitor of metalloproteinases-1 (TIMP-1), TIMP-2, and TIMP-3 during the inflammatory, granulation, and early remodeling phases of excisional skin repair. Eight full-thickness skin wounds were made on the backs of each rat (7-mm2 wounds; 16 rats; n = 128 wounds). Two animals at a time were reanesthetized, and all eight wounds on each animal were excised at 12 and 24 hours and at 2, 3, 5, 7, 10, and 14 days after injury. Six wounds from each animal were excised for RNA isolation, whereas two wounds were excised for histology. Controls consisted of nonwounded skin from identical locations in four animals. Total RNA from each time point was isolated and relative mRNA quantitation performed by using reduced-cycle reverse transcription-polymerase chain reaction. Correct polymerase chain reaction product amplification was confirmed by probing the blotted polymerase chain reaction product with a 32P-labeled oligonucleotide specific for a given MMP or TIMP. We demonstrated that the majority of MMP and TIMP mRNA induction and peak expression coincided temporally with the well-characterized inflammatory and granulation stages of repair. In conclusion, there is a distinct pattern of MMP and TIMP expression during normal excisional wound repair.

  View details for Web of Science ID 000085332700024

  View details for PubMedID 10697171

• Studies in flexor tendon wound healing: Neutralizing antibody to TGF-beta 1 increases postoperative range of motion Annual Scientific Meeting of the American-College-of-Surgeons Chang, J., Thunder, R., Most, D., Longaker, M. T., Lineaweaver, W. C. LIPPINCOTT WILLIAMS & WILKINS. 2000: 148–55

  Abstract

  The postoperative outcome of hand flexor tendon repair remains limited by tendon adhesions that prevent normal range of motion. Recent studies using in situ hybridization techniques have implicated transforming growth factor beta-1 (TGF-beta1) in both intrinsic and extrinsic mechanisms of repair. TGF-beta1 is a growth factor that plays multiple roles in wound healing and has also been implicated in the pathogenesis of excessive scar formation. The purpose of this study was to examine the effect of neutralizing antibody to TGF-beta1 in a rabbit zone II flexor tendon wound-healing model. Twenty-two adult New Zealand White rabbits underwent complete transection of the middle digit flexor digitorum profundus tendon in zone II. The tendons were immediately repaired and received intraoperative infiltration of one of the following substances: (1) control phosphate-buffered saline; (2) 50 microg neutralizing antibody to TGF-beta1; (3) 50 microg each of neutralizing antibody to TGF-beta1 and to TGF-beta2. Eight rabbits that had not been operated on underwent analysis for determination of normal flexion range of motion at their proximal and distal interphalangeal joints, using a 1.2-N axial load applied to the flexor digitorum profundus tendon. All rabbits that had been operated on were placed in casts for 8 weeks to allow maximal tendon adhesion and were then killed to determine their flexion range of motion. Statistical analysis was performed using the Student's unpaired t test. When a 1.2-N load was used on rabbit forepaws that had not been operated on, normal combined flexion range of motion at the proximal and distal interphalangeal joints was 93+/-6 degrees. Previous immobilization in casts did not reduce the range of motion in these forepaws (93+/-4 degrees). In the experimental groups, complete transection and repair of the flexor digitorum profundus tendon with infiltration of control phosphate-buffered saline solution resulted in significantly decreased range of motion between the proximal and distal phalanges [15+/-6 degrees (n = 8)]. However, in the tendon repairs infiltrated with neutralizing antibody to TGF-beta1, flexion range of motion increased to 32+/-9 degrees (n = 7; p = 0.002). Interestingly, a combination of neutralizing antibody to TGF-beta1 and that to TGF-beta2 did not improve postoperative range of motion [18+/-4 degrees (n = 7; p = 0.234)]. These data demonstrate that (1) the rabbit flexor tendon repair model is useful for quantifying tendon scar formation on the basis of degrees of flexion between proximal and distal phalanges; (2) intraoperative infiltration of neutralizing antibody to TGF-beta1 improves flexor tendon excursion; and (3) simultaneous infiltration of neutralizing antibody to TGF-beta2 nullifies this effect. Because TGF-beta1 is thought to contribute to the pathogenesis of excessive scar formation, the findings presented here suggest that intraoperative biochemical modulation of TGF-beta1 levels limits flexor tendon adhesion formation.

  View details for Web of Science ID 000084513700025

  View details for PubMedID 10626983

• A rat model of gingivoperiosteoplasty JOURNAL OF CRANIOFACIAL SURGERY Mehrara, B. J., Saadeh, P. B., Steinbrech, D. S., Dudziak, M., Grayson, B. H., Cutting, C. B., McCarthy, J. G., Gittes, G. K., Longaker, M. T. 2000; 11 (1): 54-58

  Abstract

  The ability to avoid a subsequent bone graft makes the use of gingivoperiosteoplasty (GPP) at the time of cleft lip repair an attractive technique. The use of GPP, in combination with presurgical orthodontics, has been shown to result in successful bony union in the majority of patients. However, secondary bone grafting is still necessary in 30% to 40% of patients due to persistent alveolar bony defects. The elucidation of methods to improve the success rates of these procedures has been hampered by the lack of reproducible animal models. The purpose of this study was, therefore, to develop a rodent model of GPP that would facilitate the investigation of methods to improve osteogenesis in alveolar defects. We report a surgically produced rat model (9 x 5 x 3-mm alveolar defect) that is reproducible, inexpensive (relative to large-animal models), and simple technically. In addition, healing in this model occurs in a predictable manner during a 12-week period, thus enabling analysis of methods designed to accelerate or facilitate osseous regeneration.

  View details for Web of Science ID 000084778600010

  View details for PubMedID 11314101

• Chin surgery: II. Submental ostectomy and soft-tissue excision PLASTIC AND RECONSTRUCTIVE SURGERY Zide, B. M., Longaker, M. T. 1999; 104 (6): 1854-1860

  Abstract

  At the present time, surgical reduction of an isolated large chin is not a simple procedure. Essentially, two surgical procedures exist for chin reduction: osteotomy with setback or prominence reduction by burring. Both of these procedures have potential negative aesthetic sequelae, including mental nerve injuries, bony contour irregularities, increasing submental soft-tissue fullness, and chin pad ptosis. In this report, the authors present a new approach to chin reduction: submental ostectomy with soft-tissue excision. This technique reduces the prominent chin and avoids ptosis by soft-tissue adjustment.

  View details for Web of Science ID 000083340000039

  View details for PubMedID 10541192

• The use of subatmospheric pressure dressing for the coverage of radial forearm free flap donor-site exposed tendon complications ANNALS OF PLASTIC SURGERY Greer, S. E., Longaker, M. T., Margiotta, M., Mathews, A. J., Kasabian, A. 1999; 43 (5): 551-554

  Abstract

  Since its description in China in 1978, the radial forearm free flap has become a workhorse for the reconstructive surgeon. However, the flap has known disadvantages in complications of the wrist donor site. Skin graft breakdown with exposure of the flexor tendons of the wrist is the most common. The authors describe in a patient series a new treatment for this complication. They used subatmospheric pressure dressing to stimulate granulation tissue coverage of the tendon and to facilitate epithelialization. As many as one third of all patients undergoing radial forearm free flaps develop exposed tendon complications and may benefit from Vacuum Assisted Closure (VAC) therapy.

  View details for Web of Science ID 000083557300018

  View details for PubMedID 10560875

• Chin surgery: I. Augmentation - The allures and the alerts PLASTIC AND RECONSTRUCTIVE SURGERY Zide, B. M., Pfeifer, T. M., Longaker, M. T. 1999; 104 (6): 1843-1853

  Abstract

  The correction of sagittal deformities of the chin presents a seemingly simple surgical challenge. However, several authors have reported negative sequelae from such chin surgery, During the past 11 years, the senior author (B.M.Z.) has evaluated more than 100 such cases of adverse results after chin augmentation. Many surgeons, it seems, use chin implants unnecessarily and, thus, get into trouble. Because alloplastic chin augmentation is deceptively easy, it tends to be overused in certain situations. Either the surgeon's evaluation is too narrowly focused or his/her abilities to perform other types of surgery (e.g., osseous genioplasty) are limited. Herein, the authors present a diagnostic evaluation protocol, QUAC (Quick Analysis of the Chin), to assist in avoiding simple mistakes in alloplastic chin augmentation. This protocol will alert the surgeon to situations that, if unrecognized, will cause problems and create an unhappy patient. This article will specifically focus on (1) lower lip analysis; (2) the effect of the labiomental fold; (3) chin pad evaluation, both static and dynamic; (4) the anatomy of the cleft chin; (5) special situations; and (6) how to troubleshoot three common problems. The accompanying article, Chin Surgery II, will present a new operation that treats a chin problem that was previously difficult to correct.

  View details for Web of Science ID 000083340000038

  View details for PubMedID 10541191

• Transforming growth factor-beta 1 modulates the expression of vascular endothelial growth factor by osteoblasts AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Saadeh, P. B., Mehrara, B. J., Steinbrech, D. S., Dudziak, M. E., Greenwald, J. A., Luchs, J. S., Spector, J. A., Ueno, H., Gittes, G. K., Longaker, M. T. 1999; 277 (4): C628-C637

  Abstract

  Angiogenesis is essential to both normal and pathological bone physiology. Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis, whereas transforming growth factor-beta1 (TGF-beta1) modulates bone differentiation, matrix formation, and cytokine expression. The purpose of this study was to investigate the relationship between TGF-beta1 and VEGF expression in osteoblasts and osteoblast-like cells. Northern blot analysis revealed an early peak of VEGF mRNA (6-fold at 3 h) in fetal rat calvarial cells and MC3T3-E1 osteoblast-like cells after stimulation with TGF-beta1 (2.5 ng/ml). The stability of VEGF mRNA in MC3T3-E1 cells was not increased after TGF-beta1 treatment. Actinomycin D inhibited the TGF-beta1-induced peak in VEGF mRNA, whereas cycloheximide did not. Blockade of TGF-beta1 signal transduction via a dominant-negative receptor II adenovirus significantly decreased TGF-beta1 induction of VEGF mRNA. Additionally, TGF-beta1 induced a dose-dependent increase in VEGF protein expression by MC3T3-E1 cells (P < 0.01). Dexamethasone similarly inhibited VEGF protein expression. Both TGF-beta1 mRNA and VEGF mRNA were concurrently present in rat membranous bone, and both followed similar patterns of expression during rat mandibular fracture healing (mRNA and protein). In summary, TGF-beta1-induced VEGF expression by osteoblasts and osteoblast-like cells is a dose-dependent event that may be intimately related to bone development and fracture healing.

  View details for Web of Science ID 000083919100005

  View details for PubMedID 10516092

• Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro PLASTIC AND RECONSTRUCTIVE SURGERY Steinbrech, D. S., Mehrara, B. J., Saadeh, P. B., Chin, G., Dudziak, M. E., Gerrets, R. P., Gittes, G. K., Longaker, M. T. 1999; 104 (3): 738-747

  Abstract

  Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.

  View details for Web of Science ID 000082004500019

  View details for PubMedID 10456527

• New developments in craniofacial surgery research CLEFT PALATE-CRANIOFACIAL JOURNAL Mehrara, B. J., Longaker, M. T. 1999; 36 (5): 377-387

  Abstract

  The recent explosion in our understanding of developmental biology and genetics has enhanced our understanding of craniofacial biology. While it is not possible to summarize all new developments in craniofacial research, this article will review three areas: fetal models and surgery for craniofacial disorders, the biology of distraction osteogenesis, and the molecular mechanisms of cranial suture fusion. Numerous models of craniofacial disorders have been described, including small, short gestation and large, long gestation. The benefits and shortcomings of each are discussed. In addition, we discuss recent studies investigating the molecular mechanisms of mandibular distraction osteogenesis. Finally, we present a review of recent advances in the understanding of mechanisms of craniosynostosis, with particular emphasis on the biology of programmed cranial suture fusion in rodents.

  View details for Web of Science ID 000082621300001

  View details for PubMedID 10499398

• Regional differentiation of rat cranial suture-derived dural cells is dependent on association with fusing and patent cranial sutures PLASTIC AND RECONSTRUCTIVE SURGERY Mehrara, B. J., Greenwald, J., Chin, G. S., Dudziak, M., Sagrioglu, J., Steinbrech, D. S., Saadeh, P. B., Gittes, G. K., Longaker, M. T. 1999; 104 (4): 1003-1013

  Abstract

  A significant body of literature supports a role for the dura mater underlying cranial sutures in the regulation of sutural fate. These studies have implicated regional differentiation of the dura mater based on association with fusing and patent rat cranial sutures. The purpose of these experiments was to isolate and characterize dural cells associated with fusing (posterior frontal) and patent (sagittal) rat cranial sutures. Six-day-old rats were killed, and the dura mater underlying the posterior frontal and sagittal sutures was harvested. Dural cells were briefly trypsinized and allowed to reach confluence. Two litters (10 animals per litter) were used for each set of experiments. Cells were harvested after the first and fifth passages for analysis of vimentin and desmoplakin expression (characteristic of human meningeal cells), cellular proliferation, density at confluence (a measure of cellular contact inhibition), and alkaline phosphatase production. In addition, bone nodule formation and collagen I production were analyzed in first passage cells. The results indicate that suture-derived dural cells can be established and that these cells coexpress vimentin and desmoplakin. In addition, it is demonstrated that first-passage sagittal suture-derived dural cells proliferate significantly faster and have decreased cellular contact inhibition than posterior frontal suture-derived cells (p < 0.01). Finally, it is shown that suture-derived dural cells have osteoblast-like properties, including alkaline phosphatase production, collagen I expression, and bone nodule formation in vitro. The possible mechanisms by which regional differentiation of suture-derived dural cells occur are discussed.

  View details for Web of Science ID 000082302500016

  View details for PubMedID 10654740

• Basic fibroblast growth factor and transforming growth factor beta-1 expression in the developing dura mater correlates with calvarial bone formation PLASTIC AND RECONSTRUCTIVE SURGERY Mehrara, B. J., Most, D., Chang, J., Bresnick, S., Turk, A., Schendel, S. A., Gittes, G. K., Longaker, M. T. 1999; 104 (2): 435-444

  Abstract

  Numerous studies have found dura mater-calvarial mesenchyme interactions during calvarial bone induction; however, the exact molecular mechanisms governing these inductive events remain unknown. Recent studies have implicated basic fibroblast growth factor (FGF-2) and transforming growth factor-beta1 (TGF-beta1) in regulating bone formation. The purpose of this study was, therefore, to investigate the expression of FGF-2 and TGF-beta1 during calvarial bone formation in rats. Eight rats were killed on embryonic days 14, 18, and 20 and neonatal day 1 (n = 32). Four animals at each time point were analyzed by in situ hybridization, and the remainder were analyzed by immunohistochemistry. The results indicated that the dura mater underlying the developing calvarial bone strongly expressed FGF-2 and TGF-beta1 mRNA at all time points examined. In contrast, minimal growth factor expression was noted in the overlying calvarial mesenchyme until embryonic day 18, but it increased significantly with increasing age. Importantly, FGF-2 and TGF-beta1 mRNA expression in the dura mater underlying the developing calvarium preceded and was significantly greater than expression in the calvarium mesenchyme (p < 0.05). Interestingly, minimal expression of FGF-2 and TGF-beta1 mRNA was noted for all time points in the dura mater underlying the posterior frontal suture and within the posterior frontal suture connective tissue (p < 0.01 when compared with the dura mater underlying the developing calvarium). Immunohistochemical findings closely paralleled mRNA expression, with intense staining for FGF-2 and TGF-beta1 in the dura mater underlying the developing calvarial mesenchyme. Increasing FGF-2 and TGF-beta1 staining was noted within calvarial osteoblasts with increasing age, particularly in cells located near the endocranial surface (i.e., in contact with the developing dura mater). These findings, together with the known biologic functions of FGF-2 and TGF-beta1, implicate these growth factors in the regulation of calvarial bone growth by the developing dura mater. The possible mechanisms of this interaction are discussed.

  View details for Web of Science ID 000081608500017

  View details for PubMedID 10654687

• Molecular approaches to understanding organogenesis. Seminars in pediatric surgery Crisera, C. A., Longaker, M. T., Gittes, G. K. 1999; 8 (3): 109-118

  Abstract

  The elucidation of the molecular mechanisms of mammalian organogenesis is the foundation on which we can build an improved understanding of organ pathology and pathophysiology. This paper uses the lung and the pancreas as paradigms to demonstrate how advances in basic molecular developmental biology research has translated into new appreciation of, and even novel potential treatment strategies for, congenital anomalies and mature diseases of these organs.

  View details for PubMedID 10461323

• Fetal wound repair: where do we go from here? Seminars in pediatric surgery Stelnicki, E. J., Chin, G. S., Gittes, G. K., Longaker, M. T. 1999; 8 (3): 124-130

  Abstract

  In contrast to adult wound healing, early-gestation fetal skin wound healing occurs rapidly, in a regenerative fashion, and without scar formation. The accelerated rate of healing, relative lack of an acute inflammatory response, and an absence of neovascularization distinguishes fetal from adult wound healing. However, this remarkable ability of the fetus to heal without scarring still remains poorly understood. The uncertainties include the role of cytokines, extracellular matrix components, homeobox genes, and certain cell types in the scarless wound repair process. Nevertheless, some strides have been made within the last two decades. This report, discusses the current knowledge of the mechanisms and characteristics of scarless fetal wound healing. Furthermore, to shy away from being just another all inclusive review, the authors point out deficiencies in the knowledge base on this important topic. Last, the future direction of research is discussed that may elucidate the mechanisms regulating the scarless repair phenomena.

  View details for PubMedID 10461325

• Adenovirus-mediated gene therapy of osteoblasts in vitro and in vivo JOURNAL OF BONE AND MINERAL RESEARCH Mehrara, B. J., Saadeh, P. B., Steinbrech, D. S., Dudziak, M., Spector, J. A., Greenwald, J. A., Gittes, G. K., Longaker, M. T. 1999; 14 (8): 1290-1301

  Abstract

  Modulation of biological pathways governing osteogenesis may accelerate osseous regeneration and reduce the incidence of complications associated with fracture healing. Transforming growth factor beta1 (TGF-beta1) is a potent growth factor implicated in the regulation of osteogenesis and fracture repair. The use of recombinant proteins, however, has significant disadvantages and has limited the clinical utility of these molecules. Targeted gene therapy using adenovirus vectors is a technique that may circumvent difficulties associated with growth factor delivery. In this study, we investigate the efficacy of replication-deficient adenoviruses containing the human TGF-beta1 and the bacterial lacZ genes in transfecting osteoblasts in vitro and osseous tissues in vivo. We demonstrate that adenovirus-mediated gene therapy efficiently transfects osteoblasts in vitro with the TGF-beta1 virus causing a marked up-regulation in TGF-beta1 mRNA expression even 7 days after transfection. Increased TGF-beta1 mRNA expression was efficiently translated into protein production and resulted in approximately a 46-fold increase in TGF-beta1 synthesis as compared with control cells (vehicle- or B-galactosidase-transfected). Moreover, virally produced TGF-beta1 was functionally active and regulated the expression of collagen IalphaI (5-fold increase) and the vascular endothelial growth factor (2.5-fold increase). Using an adenovirus vector encoding the Escherichia coli LacZ gene, we demonstrated that adenovirus-mediated gene transfer efficiently transfects osteoblasts and osteocytes in vivo and that transfection can be performed by a simple percutaneous injection. Finally, we show that delivery of the hTGF-beta1 gene to osseous tissues in vivo results in significant changes in the epiphyseal plate primarily as a result of increased thickness of the provisional calcification zone.

  View details for Web of Science ID 000081682000005

  View details for PubMedID 10457261

• Fibroblast response to hypoxia: The relationship between angiogenesis and matrix regulation Annual Meeting of the Association-for-Academic-Surgery Steinbrech, D. S., Longaker, M. T., Mehrara, B. J., Saadeh, P. B., Chin, G. S., Gerrets, R. P., Chau, D. C., Rowe, N. M., Gittes, G. K. ACADEMIC PRESS INC ELSEVIER SCIENCE. 1999: 127–33

  Abstract

  A number of studies have demonstrated the critical role of angiogenesis for successful wound repair in the surgical patient. Vascular disruption from tissue injury due to trauma or surgery leads to a hypoxic zone in the healing wound. In this dynamic process, angiogenesis is vital for the delivery of oxygen, nutrients, and growth factors necessary to initiate the synthetic processes of wound healing. Fibroblasts, invading the wound early in the healing process, are involved in extracellular matrix (ECM) deposition as well as wound contraction. However, the exact mechanisms by which important genes are regulated remain unknown. In order to examine these processes, we studied the effects of hypoxia on fibroblasts for the expression of VEGF, type IalphaI collagen, and matrix-metalloproteinase-3, three genes essential for the regulation of angiogenesis, ECM deposition, and ECM degradation in wound healing. Primary cell cultures of normal human dermal fibroblasts (NHDFs) were placed in hypoxia for varying periods of time. Northern blot hybridization was performed with [alpha32P]dCTP-labeled cDNA probes for VEGF, type IalphaI collagen, and MMP-3. The results demonstrated a time-dependent VEGF mRNA upregulation (470% of baseline) under hypoxia. Type IalphaI collagen increased (170% of baseline) at 24 h, but was then abruptly downregulated to 3.8% of baseline at 48 h. MMP-3 was incrementally downregulated to 2.2% of baseline at 48 h. These experiments focused on the effect of hypoxia on genes thought to play a role in wound repair. VEGF upregulation in the hypoxic microenvironment of the early wound may serve to stimulate angiogenesis. Type IalphaI collagen, though upregulated early on, was abruptly downregulated at 48 h. This downregulation may reflect the in vivo requirement for angiogenesis to deliver oxygen for successful hydroxylation and collagen synthesis in the wound. MMP-3, also downregulated at 48 h, may also implicate the need for angiogenesis. These data support the theory that hypoxia-driven angiogenesis is critical for ECM formation and remodeling in successful soft tissue repair. Furthermore, they may represent the role of hypoxia as an important regulator to efficiently balance these complex processes in the healing wound.

  View details for Web of Science ID 000081004400002

  View details for PubMedID 10357908

• Angiogenesis during mandibular distraction osteogenesis Meeting of the Northeastern-Society-of-Plastic-Surgeons Rowe, N. M., Mehrara, B. J., Luchs, J. S., Dudziak, M. E., Steinbrech, D. S., Illei, P. B., Fernandez, G. J., Gittes, G. K., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 1999: 470–75

  Abstract

  Recruitment of a blood supply is critical for successful bone induction and fracture healing. Despite the clinical success of distraction osteogenesis (DO), an analysis of angiogenesis during membranous bone DO has not been performed. The purpose of this study was to evaluate the temporal and spatial pattern of angiogenesis during mandibular DO. The right hemimandible of adult male rats was osteotomized, and a customized distraction device was applied. Following a 3-day latency period, distraction was begun at a rate of 0.25 mm twice daily for 6 days (3.0 mm total; 12% increase in mandibular length). Three animals each were sacrificed on days 2, 4, and 6 of distraction (D1, D2, and D3 respectively), or after 1, 2, or 4 weeks of consolidation (C1, C2, and C3 respectively). Two experienced pathologists reviewed the regenerate histology, and angiogenesis was assessed by counting the number of blood vessels per intermediate-power field (IPF). Statistical analysis was performed using analysis of variance, with p < or = 0.05 considered significant. Results demonstrate that mandibular DO was associated with an intense vascular response during the early stages of distraction (D1). On average, 31.5+/-7.9 vessels were noted in each IPF examined during this time point. The number of blood vessels in the distraction regenerate decreased significantly during the later distraction time points, with approximately 14.0+/-2.0 and 14.7+/-3.5 blood vessels per IPF in sections obtained after days 4 and 6 of distraction (D2, D3) respectively. However, blood vessels at these time points took on a more mature histological pattern. During the consolidation period, the number of blood vessels noted in the regenerate decreased with 8.0+/-2.6, 9.3+/-2.1, and 4.0+/-2.0 vessels per IPF in sections obtained after 1, 2, or 4 weeks of consolidation (C1, C2, C3) respectively (p < 0.05 compared with vessel counts during the earliest distraction time point). This study demonstrates for the first time that an intense vascular response associated with mandibular DO occurs primarily during the early stages of distraction. The authors hypothesize that as distraction continues, newly formed vessels likely undergo consolidation, thus forming more mature vessels capable of withstanding distraction forces. Future studies will assess the effects of therapeutic interventions designed to increase angiogenesis during DO on bony regenerate formation.

  View details for Web of Science ID 000080223600002

  View details for PubMedID 10340853

• Hypoxia upregulates VEGF production in keloid fibroblasts ANNALS OF PLASTIC SURGERY Steinbrech, D. S., Mehrara, B. J., Chau, D., Rowe, N. M., Chin, G., Lee, T., Saadeh, P. B., Gittes, G. K., Longaker, M. T. 1999; 42 (5): 514-519

  Abstract

  The etiology of keloid formation is diverse. They are characterized grossly as thick scar tissue that extends beyond the boundaries of the original wound. Histologically, keloids are composed of excessive collagen with an abnormally large number of partially or totally occluded microvessels. This occlusion of keloid microvessels has been hypothesized to contribute to a hypoxic microenvironment within these pathological scars. Vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen, and proangiogenic cytokine have been implicated in normal and pathological wound healing. The purpose of this study was to evaluate the amount of VEGF protein production by fibroblast cell lines derived from keloids and normal human dermal skin in hypoxic compared with normoxic culture conditions. By enzyme-linked immunosorbent protein assay, VEGF was increased in both keloid and normal human dermal fibroblasts in hypoxia over normoxic controls. There was not, however, a significant difference between upregulation of VEGF protein when comparing the keloid and normal fibroblast groups. As the result of the data, alternative hypotheses for hypoxia-induced keloid formation were explored: (1) downstream modulation or signal transduction of VEGF, (2) VEGF production from cells other than fibroblasts, (3) the importance of matrix accumulation stimulated by hypoxia, or (4) increased migration of cells (other than fibroblasts) specific to keloid biology. These hypotheses may help explain the possible role of hypoxia in the pathogenesis of keloid formation. Future studies involving in situ hybridization or immunohistochemical analysis may offer greater insight into the mechanisms underlying keloid formation. Ultimately, our therapeutic goal is the utilization of biomolecular approaches for the suppression of keloid formation.

  View details for Web of Science ID 000080223600015

  View details for PubMedID 10340860

• Thrombospondin 1 and its specific cysteine-serine-valine-threonine-cysteine-glycine receptor in fetal wounds ANNALS OF PLASTIC SURGERY Roth, J. J., Sung, J. J., Granick, M. S., Solomon, M. P., Longaker, M. T., Rothman, V. L., Nicosia, R. F., Tuszynski, G. P. 1999; 42 (5): 553-563

  Abstract

  Thrombospondin 1 (TSP-1), an adhesive glycoprotein, plays an important role in platelet adhesion, inflammation, cell-cell interaction, and angiogenesis. TSP-1 is expressed by endothelial cells, fibroblasts, and macrophages. The unique cysteine-serinevaline-threonine-cysteine-glycine (CSVTCG) binding domain of TSP-1 also plays an important role in cell binding and modulation of cellular processes. The purpose of this study was to evaluate histologically and quantitatively TSP-1 and its CSVTCG receptor in fetal skin wounds over time. Pregnant ewes underwent laparotomy and hysterotomy. At 65 days gestation (term, 145 days), incisional and excisional wounds were created on the fetal back in a similar position on each animal. The uterus and laparotomy were closed. The wounds were harvested on days 1, 3, 7, 21, and 28. Expression of TSP-1 and its CSVTCG receptor was evaluated immunohistochemically and quantitated by computer image analysis in units of absorbance. Immunoglobulin G (negative) controls were performed and subtracted from the TSP-1 sample to eliminate background absorbance readings. Serum (negative) control was used for the CSVTCG receptor. Platelet concentrates were used as the positive control: TSP-1, 63.43; CSVTCG, 58.72. Results are expressed as absorbance+/-SEM. Results of TSP-1 are as follows: day 1, 33.02+/-0.26; day 3, 22.21+/-0.14; day 7, 20.56+/-1.07; day 21, 7.76+/-0.40; and day 28, 5.99+/-0.03. TSP-1 displays an early peak during fetal skin repair, followed by a steep decrease over the viewed time period. Results of CSVTCG receptor are as follows: day 1, 26.19+/-2.43; day 3, 30.20+/-0.64; day 7, 24.56+/-0.80; day 21, 24.70+/-0.40; and day 28, 21.65+/-1.39. Thus, CSVTCG receptor displays a slowed decrease in expression over time during fetal repair. No significant differences were noted between incisional and excisional samples. Temporal and histological differences exist in the localization and expression of TSP-1 and its CSVTCG receptor during fetal wound repair. TSP-1 is upregulated in tissues early. This corresponds with the known role of TSP-1 in cell-cell interaction, including potentiation of growth factor activity. TSP-1 also modulates matrix, allowing scar-free provisional matrix in the earlier stages of repair deposited by platelets. The potentiation of cell-associated protease activity by TSP-1 can support tissue and matrix turnover. This activity of TSP-1 may contribute to the formation of a scarless wound. TSP-1 destabilizes extracellular matrix contacts, and facilitates mitosis and migration. The action of TSP-1 as an adhesive protein allows numerous different cells to adhere to the extracellular membrane. CSVTCG receptor expression decreases during fetal repair as the cells migrate to the epithelial surface, suggesting a significant role of the CSVTCG receptor in keratinocytic maturation, differentiation, and epithelization.

  View details for Web of Science ID 000080223600025

  View details for PubMedID 10340867

• Analysis of TGF-beta production by fusing and nonfusing mouse cranial sutures in vitro ANNALS OF PLASTIC SURGERY Sagiroglu, J. S., Mehrara, B. J., Chau, D., Saadeh, P. B., Gittes, G. K., Longaker, M. T. 1999; 42 (5): 496-501

  Abstract

  The role of transforming growth factor beta (TGF-beta) in the regulation of cranial suture fusion has been studied by various qualitative techniques such as in situ hybridization and immunohistochemistry. Although the relative expression of TGF-beta isoforms has been assessed in these studies, increased expression of TGF-beta has not been demonstrated in a quantitative fashion. Therefore, the purpose of this study was to quantify TGF-beta production by fusing (posterofrontal [PF]) and nonfusing (sagittal) mouse sutures using two different quantitative TGF-beta assays. The PF and sagittal sutures of 25-day-old mice were harvested and cultured separately in vitro. Culture media conditioned for 48 hours were collected after 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days of culture, and total TGF-beta production was assessed using a TGF-beta bioassay. For a quantitative TGF-beta1 immunoassay, media conditioned for 48 hours were collected after 3, 5, 7, 9, 14, 22, and 28 days of culture. The TGF-beta bioassay revealed large amounts of total TGF-beta activity in both PF and sagittal sutures during the first week of culture, with decreasing amounts thereafter. Absolute TGF-beta activity in conditioned media collected from PF sutures at several early time points was higher than those obtained from sagittal sutures; however, these differences were not statistically significant. The results of the TGF-beta1 immunoassay (enzyme-linked immunosorbent assay) were similar to the bioassay in that the highest TGF-beta1 levels were noted during the first week of culture period and decreased thereafter. Analysis of variance of these samples, however, revealed significantly more TGF-beta1 protein in samples collected from the PF suture compared with the sagittal suture on days 3 and 5 of culture (p < 0.05). TGF-beta1 levels in the conditioned media obtained from PF sutures remained elevated compared with the sagittal suture on days 7 and 9; however, these differences were not statistically significant. Increased production of TGF-beta in the conditioned media of fusing PF sutures is the first such quantitative demonstration of growth factor upregulation during suture fusion and supports the hypothesis that TGF-beta expression may be important in cranial suture fusion.

  View details for Web of Science ID 000080223600010

  View details for PubMedID 10340857

• Epithelio-mesenchymal interactions in the developing mouse pancreas: Morphogenesis of the adult architecture JOURNAL OF PEDIATRIC SURGERY Rose, M. I., Crisera, C. A., Colen, K. L., Connelly, P. R., Longaker, M. T., Gittes, G. K. 1999; 34 (5): 774-779

  Abstract

  The mammalian pancreas is thought to develop through a complex interaction between the budding epithelium and the surrounding mesenchyme. The exact nature of this interaction is unclear. Most of what is known to date of these interactions comes from a series of organ culture experiments done in the late 1960s. Nevertheless, these important experiments may have been confounded by less-defined culture media and organ dissection techniques, because the results are not reproducible in our hands. The authors undertook a study to reexplore these basic epithelio-mesenchymal interactions.Using previously described organ dissection and culture techniques the authors examined the basic interactions between the embryonic pancreatic epithelium and its mesenchyme with histological and immunohistological techniques.The authors found that, contrary to previous reports, the earliest pancreatic anlage did not possess the intrinsic signaling necessary to support normal growth and differentiation in vitro. Intimate contact between the epithelium and the mesenchyme may be necessary until E11.5 for normal growth and differentiation. The age of the mesenchyme seemed to correlate with the degree of acinar differentiation, and proximity of mesenchyme was important for acinar differentiation.Previous investigations into the basic epithelio-mesenchymal interactions in the developing mammalian pancreas may have had confounding factors. Extrinsic signals seem necessary for complete pancreatic differentiation, and mesenchymal factors appear important for acinar differentiation.

  View details for Web of Science ID 000080447500045

  View details for PubMedID 10359180

• Vascular development in the mouse embryonic pancreas and lung JOURNAL OF PEDIATRIC SURGERY Colen, K. L., Crisera, C. A., Rose, M. I., Connelly, P. R., Longaker, M. T., Gittes, G. K. 1999; 34 (5): 781-785

  Abstract

  The purpose of this study was to analyze the formation of blood vessels in the developing mouse pancreas and lung by studying two ligands, angiopoietin-1 (ang1) and angiopoietin-2 (ang2), which are thought to play a role as angiogenesis-activating factors in development. Understanding the role of vasculogenic peptides in normal embryonic development also may have important implications for common clinical problems regarding neonatal pulmonary vasculature.Reverse transcriptase-polymerase chain reaction (RT-PCR) as well as Southern blotting was used to determine the ontogeny of angiopoietin-1 and angiopoietin-2 gene expression in the embryonic mouse pancreas and lung. Immunohistochemistry was performed for von Willebrand factor, a known marker of endothelial cells, to chronicle the development of the vasculature in these organs.The authors determined the temporal expression of angiopoietin-1 and angiopoietin-2 as a function of gestational age. RT-PCR data demonstrated expression of ang1 and ang2 in the developing mouse lung between gestational day E9.5 and postnatal day 1, and in the developing pancreas between gestational days E12.5 and E18.5. Southern blot analysis confirmed PCR data for ang2 expression in both the lung and pancreas. The authors also traced the spatial development of the vascular system by von Willebrand factor staining. For both lung and pancreas specimens, no blood vessels were identifiable by immunohistochemistry until embryonic day 12.5. With increased gestational age, the blood vessel networks grew larger.The authors have demonstrated that ang1 and ang2 may be involved in the mechanisms of vascular development in the embryonic mouse lung and pancreas.

  View details for Web of Science ID 000080447500047

  View details for PubMedID 10359181

• Craniofacial distraction osteogenesis JOURNAL OF CRANIOFACIAL SURGERY Hollier, L. H., Gosain, A., Stelnecki, E., Longaker, M., McCarthy, J. G. 1999; 10 (3): 268-282

  View details for Web of Science ID 000080170800016

• The ontogeny of TGF-beta 1, -beta 2, -beta 3, and TGF-beta receptor-II expression in the pancreas: Implications for regulation of growth and differentiation JOURNAL OF PEDIATRIC SURGERY Crisera, C. A., Rose, M. I., Connelly, P. R., Li, M., Colen, K. L., Longaker, M. T., Gittes, G. K. 1999; 34 (5): 689-693

  Abstract

  The transforming growth factor-beta (TGF-beta) cytokines are important regulators of growth and differentiation in multiple mammalian organ systems. Recent studies suggest that they may play a significant role in the regulation of pancreatic organogenesis. The authors proposed to examine the ontogeny of expression of the TGF-beta cytokine isoforms (TGF-beta1, beta2, and beta3), as well as that of the type II TGF-beta receptor (TbetaRII), in the pancreas. We hypothesized that their patterns of expression might help to clarify the manner in which they influence the development of this organ.Embryos from pregnant CD-1 mice were harvested on gestational days 12.5, 15.5, and 18.5. Microdissection was performed on the embryos to isolate their pancreases. The pancreases were fixed, frozen embedded, and sectioned with a cryostat. Immunohistochemistrywas performed using polyclonal antibodies to TGF-beta1, beta2, and beta3, and TbetaRII.The patterns of expression of TGF-beta1, beta2, and beta3 were similar throughout gestation. They were all present, though weakly, early in the development of the pancreas, in the E12.5 epithelial cells. Their expression persisted and became localized to the acinar cells later in gestation. TbetaRII staining was present in both the E12.5 epithelial cells and the surrounding mesenchyme. As the pancreas developed, TbetaRII became strongly expressed in the ductal epithelial cells with only minimal staining in the acinar and endocrine cells.TGF-betas may play a role in regulating pancreatic organogenesis. Our data suggest that they may be required for the normal development of acini. As in other cell systems, TGF-beta1 may act as a suppressor of pancreatic cellular growth and differentiation. The localization of TbetaRII to the mature ductal epithelium may indicate a need for ongoing regulation of growth and differentiation in the pancreatic ducts beyond the fetal period.

  View details for Web of Science ID 000080447500015

  View details for PubMedID 10359165

• Chondrocyte extraction, proliferation, and characterization for construct development Meeting of the Northeastern-Society-of-Plastic-Surgeons Saadeh, P. B., Brent, B., Mehrara, B. J., Steinbrech, D. S., Ting, V., Gittes, G. K., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 1999: 509–13

  View details for Web of Science ID 000080223600013

• Gene expression of insulin-like growth factors I and II in rat membranous osteotomy healing Meeting of the Northeastern-Society-of-Plastic-Surgeons Steinbrech, D. S., Mehrara, B. J., Rowe, N. M., Dudziak, M. E., Saadeh, P. B., Gittes, G. K., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 1999: 481–87

  Abstract

  Poorly healing mandibular osteotomies can be a difficult problem in reconstructive surgery. Many therapies have been attempted to augment the healing of mandibular fractures, defects, or osteotomies, but these methods have substantial drawbacks or have been ineffective. The difficulty in treating poorly healing bony defects has led to the exploration of gene therapy as a possible approach to supplement or accelerate mandibular fracture healing. To understand at what point the introduction of a suitable gene candidate might be of benefit in mandibular healing, it is imperative to examine the temporal expression of bone growth factors in a model of membranous bone healing. Insulinlike growth factors (IGFs) I and II are two such bone growth factor candidates because of their known potent in vitro as well as in vivo effects on bone formation. In this study the authors demonstrate the temporal pattern of IGF I and IGF II gene expression during mandibular osteotomy healing using a rat model. Their data reveal that IGF I and IGF II were elevated 7 days after a mandibular osteotomy that was held in external fixation. The upregulation of IGF I and IGF II during mandibular bone healing underscores the importance of these growth factors in bone repair. Gene therapy utilizing recombinant viral constructs containing IGFs I and II may be of benefit during mandibular bone healing in an effort to augment clinical scenarios of poor or retarded bony repair.

  View details for Web of Science ID 000080223600006

  View details for PubMedID 10340855

• Human cartilage engineering: chondrocyte extraction, proliferation, and characterization for construct development. Annals of plastic surgery Saadeh, P. B., Brent, B., Mehrara, B. J., Steinbrech, D. S., Ting, V., Gittes, G. K., Longaker, M. T. 1999; 42 (5): 509-513

  Abstract

  To date, many efforts to engineer cartilage have focused on matrix construction with the goal of producing a durable construct as cartilage replaces the resorbing matrix. However, the importance of matrix construction is at least matched by the challenge of efficient chondrocyte extraction, culture expansion, and prevention of dedifferentiation. This challenge is underscored by the large number of chondrocytes needed for a clinically significant construct such as an ear. Because human rib provides a large, readily available source of hyaline cartilage, the authors evaluated human rib chondrocyte extraction and found that maximum viable cell yield occurred after a 6-hour digestion. They also evaluated human microtic auricular remnant chondrocyte extraction and identified fibroblast contamination as a shortcoming of this potential source of chondrocytes. Initially, rib chondrocytes proliferated in vitro with a doubling time of approximately 1 week. As the cells were passaged, proliferation decreased such that the cells stopped proliferating and adopted a large, spindle-shaped morphology by passage 6. Interestingly, no increase in proliferation was noted when rib chondrocytes were stimulated with transforming growth factor beta 1, bone morphogenetic protein 2, and basic fibroblast growth factor. The major obstacles to the use of autologous rib chondrocytes in matrix construction are the low cell yield from a small piece of rib and the limited proliferation that these cells will undergo in vitro. Further investigation of culture systems and mitogenic cytokines may help resolve these limitations.

  View details for PubMedID 10340859

• Expression of high-affinity receptors for TGF-beta during rat cranial suture fusion Meeting of the Northeastern-Society-of-Plastic-Surgeons Mehrara, B. J., Steinbrech, D. S., Saadeh, P. B., Gittes, G. K., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 1999: 502–8

  Abstract

  The etiology of craniosynostosis is unknown. The elucidation of the biological pathways responsible for this disorder has been hampered by an inability to evaluate cranial sutures before, during, and after cranial suture fusion. The programmed fusion of the rat posterofrontal (PF) suture postnatally provides an excellent model to study the molecular events that occur during cranial suture fusion. Previous experiments have implicated transforming growth factor beta (TGF-beta) growth factors in the regulation of PF suture fusion. The purpose of these experiments was to localize the expression of high-affinity receptors for these growth factors during cranial suture fusion. Four rats were sacrificed on postnatal days 8, 12, 17, and 40 (N = 16). The PF and sagittal sutures were harvested and prepared for immunohistochemical localization of TGF-beta receptor 1 and receptor 2 (Tbeta-RI, Tbeta-RII) protein. Results indicate that immunostaining for Tbeta-RI and Tbeta-RII is markedly increased in the dura mater and osteoblasts of the sutural margin of the PF suture during active suture fusion (on postnatal days 12, 17, and 40) compared with the osteoblasts and dura mater underlying the patent sagittal suture. These results, in combination with the authors' previous findings as well as studies supporting a role for TGF-beta molecules in the regulation of osteogenesis, implicate TGF-beta signaling in the regulation of suture fusion. The possible mechanisms of ligand-receptor interaction are discussed.

  View details for Web of Science ID 000080223600011

  View details for PubMedID 10340858

• Primary and secondary orbit surgery: The transconjunctival approach 7th International Congress of the International-Society-for-Craniofacial-Surgery Lorenz, H. P., Longaker, M. T., Kawamoto, H. K. LIPPINCOTT WILLIAMS & WILKINS. 1999: 1124–28

  Abstract

  The transconjunctival approach to the orbit is underutilized because of concern regarding inadequate exposure and higher postoperative rates of lower eyelid shortening and ectropion. All patients who had a transconjunctival incision performed for orbital surgery over the last 6 years (1990 to 1996) were studied. Patients who had a transconjunctival blepharoplasty were excluded. A total of 35 patients, average age 32 years, had 45 transconjunctival incisions performed. Lateral canthotomy or cantholysis was not done. Operations fell into three categories: fracture plating alone, 10 (22 percent); split-calvarial bone graft placement with or without plating, 26 (58 percent); and orbital decompression, 9 (20 percent). The overall incidence of ectropion was 6.7 percent (3 of 45). One patient (2 percent) had transient ectropion, and two patients (4 percent) had persistent ectropion, which required surgical correction. Ectropion occurred only in those lower eyelids that had a previous transcutaneous incision (3 of 18 = 17 percent). None occurred in those eyelids that had no prior incision or only a previous transconjunctival incision. The transconjunctival approach without a lateral canthotomy provides safe access to the orbit in eyelids that have not had a previous transconjunctival incision.

  View details for Web of Science ID 000079107900003

  View details for PubMedID 10088496

• Rat mandibular distraction osteogenesis: II. Molecular analysis of transforming growth factor beta-1 and osteocalcin gene expression PLASTIC AND RECONSTRUCTIVE SURGERY Mehrara, B. J., Rowe, N. M., Steinbrech, D. S., Dudziak, M. E., Saadeh, P. B., McCarthy, J. G., Gittes, G. K., Longaker, M. T. 1999; 103 (2): 536-547

  Abstract

  Distraction osteogenesis is a powerful technique capable of generating viable osseous tissue by the gradual separation of osteotomized bone edges. Although the histologic and ultrastructural changes associated with this process have been extensively delineated, the molecular events governing these changes remain essentially unknown. We have devised a rat model of mandibular distraction osteogenesis that facilitates molecular analysis of this process. Such information has significant clinical implications because it may enable targeted therapeutic manipulations designed to accelerate osseous regeneration. In this study, we have evaluated the expression of transforming growth factor beta-1, a major regulator of osteogenesis during endochondral bone formation and development, and osteocalcin, an abundant noncollagenous extracellular matrix protein implicated in the regulation of mineralization and bone turnover. The right hemimandible of 36 adult male rats was osteotomized, and a customized distraction device was applied. Animals were allowed to recover and, after a 3-day latency period, were distracted at a rate of 0.25 mm twice daily for 6 days followed by a 2- or 4-week consolidation period. Distraction regenerate was harvested after the latency period, days 2, 4, or 6 of distraction, and after 2 or 4 weeks of consolidation and processed for Northern analysis (n = 4 at each time point) and immunohistochemical localization of TGF-beta1 (n = 2 at each time point). Six sham-operated animals (i.e., skin incision without osteotomy) were also killed (immediately postoperatively), and the mandibles were harvested and prepared in a similar fashion. Equal loading and transfer of RNA for Northern analysis was ensured by stripping and probing membranes with a probe against GAPDH (a housekeeping gene). Our results demonstrate that the spatial and temporal patterns of TGF-beta1 mRNA expression and protein production coincide with osteoblast migration, differentiation, and extracellular matrix synthesis. In addition, we demonstrate that TGF-beta1 production may be an important regulator of vasculogenesis during mandibular distraction osteogenesis. Finally, we have shown that osteocalcin gene expression coincides temporally with mineralization during rat mandibular distraction osteogenesis.

  View details for Web of Science ID 000078331800026

  View details for PubMedID 9950542

• Large arteriovenous malformations of the face: Aesthetic results with recurrence control PLASTIC AND RECONSTRUCTIVE SURGERY Bradley, J. P., Zide, B. M., Berenstein, A., Longaker, M. T. 1999; 103 (2): 351-361

  Abstract

  Large facial arteriovenous malformations are problematic for patients because of grotesque disfigurement, risk of rapid enlargement, and life-threatening rupture. Successful treatment of these relentless complex lesions is one of the most difficult challenges facing plastic surgeons. From a series of 300 large facial arteriovenous malformations, 85 patients were treated with embolization and excision; six of these cases (representing six separate anatomic regions: labial, auricular, eyelid, cheek, chin, and occipitoparietal) were selected for review. The purpose of this article was to look critically at the management of these six facial arteriovenous malformations, including patient presentation, angiographic procedures, surgical planning and technique, and postoperative long-term follow-up care. Lessons learned from the six representative cases provide clues for the management of large facial arteriovenous malformations and demonstrate the possibilities of recurrence and their occasionally relentless behavior. The cases show that long-term control of these lesions with acceptable aesthetic results can be achieved. The mainstay of treatment includes the following: (1) selective intra-arterial embolization with fine catheters and direct lesional embolization; (2) judicious resection and reconstruction with local or expanded tissue flaps; and (3) careful follow-up with serial examinations, duplex, and arteriography.

  View details for Web of Science ID 000078331800001

  View details for PubMedID 9950519

• Proliferative hemangiomas: Analysis of cytokine gene expression and angiogenesis 66th Annual Meeting of the American-Society-of-Plastic-and-Reconstructive-Surgeons Chang, J., Most, D., Bresnick, S., Mehrara, B., Steinbrech, D. S., Reinisch, J., Longaker, M. T., Turk, A. E. LIPPINCOTT WILLIAMS & WILKINS. 1999: 1–9

  Abstract

  Hemangiomas are benign vascular tumors of childhood that can lead to disfigurement and/or life-threatening consequences. The pathogenesis of hemangioma formation is likely to involve increased angiogenesis. Basic fibroblast growth factor and vascular endothelial growth factor are cytokines that stimulate angiogenesis in multiple in vivo and in vitro models. Proliferative hemangiomas have been found to have elevated levels of basic fibroblast growth factor and vascular endothelial growth factor protein, but the gene expression of these cytokines in human specimens has not been previously studied. We examined the gene expression and spatial distribution of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA in proliferative versus involuted human hemangioma specimens using nonisotopic in situ hybridization techniques. Thirteen hemangioma specimens were harvested during initial surgical excision. In situ hybridization was performed on frozen sections of both proliferative and involuted hemangioma specimens using genetically engineered antisense probes specific for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA. Controls were an interleukin-6 sense sequence and a transforming growth factor-beta 1 antisense sequence. A large number of cells within the specimens of proliferative hemangiomas revealed localized gene expression of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (626 +/- 129 and 1660 +/- 371 cells/mm2, respectively). The majority of the cells were endothelial in origin. In contrast, involuted hemangioma specimens revealed significantly lower numbers of cells staining positive for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (44 +/- 11 and 431 +/- 76 cells/mm2, respectively; p < 0.05). Transforming growth factor-beta 1 messenger RNA was slightly more expressed by involuted hemangiomas (117 +/- 30 cells/mm2). There were very low levels of transforming growth factor-beta 1 gene expression from proliferative hemangiomas (37 +/- 24 cells/mm2; p < 0.02). These data demonstrate that (1) in situ hybridization allows identification and relative quantitation of cells expressing messenger RNA for specific growth factors in human hemangioma specimens; (2) basic fibroblast growth factor and vascular endothelial growth factor messenger RNA are up-regulated in proliferative hemangiomas; and (3) transforming growth factor-beta 1 messenger RNA remains low in both proliferative and involuted hemangiomas. Because basic fibroblast growth factor and vascular endothelial growth factor messenger RNA have been implicated in the pathobiology of human hemangioma formation, biochemical modulation of these angiogenic cytokines may eventually help inhibit proliferation and promote regression of hemangiomas.

  View details for Web of Science ID 000077707200001

  View details for PubMedID 9915157

• Esophageal atresia with tracheoesophageal fistula: Suggested mechanism in faulty organogenesis JOURNAL OF PEDIATRIC SURGERY Crisera, C. A., Connelly, P. R., Marmureanu, A. R., Colen, K. L., Rose, M. I., Li, M., Longaker, M. T., Gittes, G. K. 1999; 34 (1): 204-208

  Abstract

  The organogenesis of esophageal atresia with tracheoesophageal fistula (EA-TEF) is unknown. Using an established model for EA-TEF in rats, the authors proposed to study this aberrancy of development in the hope of gaining insight into its mechanism of formation.Pregnant Sprague-Dawley rats were injected with 2.2 mg/kg of Adriamycin intraperitoneally on days 6 through 9 of gestation. Using microdissection, the trachea, blind-ending esophagus, TEF, and stomach were isolated from embryos of various gestional ages. The specimens were analyzed histologically with routine H&E staining.The classic EA-TEF developed in the embryos, with proximal EA and distal TEF. As expected, the atresia formed as a blind-ending pouch, but the distal fistula began as an apparent equal trifurcation of the tracheal anlage into two mainstem bronchi and the fistula tract leading to the stomach. Histological analysis of the fistula tract showed respiratorylike pseudostratified columnar epithelium.TEF develops as the middle branch of a tracheal trifurcation. EA-TEF occurs by a primary atresia of the esophagus. As a secondary phenomenon, the distal foregut anlage is switched toward the pulmonary phenotype. It trifurcates, and its middle branch grows caudally to fistulize into the stomach.

  View details for Web of Science ID 000078137400074

  View details for PubMedID 10022173

• Natural history of fetuses with cleft lip PLASTIC AND RECONSTRUCTIVE SURGERY Lopoo, J. B., Hedrick, M. H., Chasen, S., Montgomery, L., Chervenak, F. A., Goldstein, R., Hoffman, W. Y., Harrison, M. R., Longaker, M. T. 1999; 103 (1): 34-38

  Abstract

  The prospect of fetal surgery for cleft lip is predicated on our ability to accurately identify fetuses with clefts and exclude those that have associated anomalies. Prenatal ultrasound is currently the most appropriate means with which to do this. We reviewed the ultrasonographic data from two large perinatal referral institutions to determine the natural history of fetuses with cleft lip who may be candidates for fetal surgery. Forty fetuses had a cleft lip diagnosed prenatally by ultrasound. In this group, severe associated anomalies were common (30 of the 40) and multiple (23 of the 40) in a majority of fetuses. Life-threatening anomalies, such as central nervous system and cardiac anomalies, were the most common defects. As a result, many fetuses were aborted therapeutically or died in the perinatal period. Out of 12 surviving fetuses, only six had isolated clefts, and two surviving fetuses, diagnosed with isolated cleft lip, had no defect identified postnatally. This information has important implications for the perinatal management of fetuses with cleft lip and the potential role of fetal intervention.

  View details for Web of Science ID 000077707200007

  View details for PubMedID 9915161

• Molecular studies in flexor tendon wound healing: the role of basic fibroblast growth factor gene expression. journal of hand surgery Chang, J., Most, D., Thunder, R., Mehrara, B., Longaker, M. T., Lineaweaver, W. C. 1998; 23 (6): 1052-1058

  Abstract

  Basic fibroblast growth factor (bFGF) is a cytokine that plays a fundamental role in angiogenesis. This study examines bFGF messenger RNA (mRNA) expression in a rabbit flexor tendon wound healing model. Thirty-four New Zealand white rabbit forepaws underwent transection and repair of the middle digit flexor digitorum profundus tendon in zone II. Tendons were harvested at increasing time intervals and analyzed by in situ hybridization and immunohistochemistry. Few tenocytes and tendon sheath cells expressed bFGF mRNA in unwounded tendons. In contrast, tendons subjected to transection and repair exhibited an increased signal for bFGF mRNA in both resident tenocytes concentrated along the epitenon and infiltrating fibroblasts and inflammatory cells from the tendon sheath. These data demonstrate that (1) normal tenocytes and tendon sheath cells are capable of bFGF production, (2) bFGF mRNA is upregulated in the tendon wound environment, and (3) the upregulation of this angiogenic cytokine occurs in tenocytes as well as in tendon sheath fibroblasts and inflammatory cells.

  View details for PubMedID 9848558

• Molecular studies in flexor tendon wound healing: The role of basic fibroblast growth factor gene expression JOURNAL OF HAND SURGERY-AMERICAN VOLUME Chang, J., Most, D., Thunder, R., Mehrara, B., Longaker, M. T., Lineaweaver, W. C. 1998; 23A (6): 1052-1058

  View details for Web of Science ID 000077120700015

• Rat mandibular distraction osteogenesis: Part I. Histologic and radiographic analysis PLASTIC AND RECONSTRUCTIVE SURGERY Rowe, N. M., Mehrara, B. J., Dudziak, M. E., Steinbreck, D. S., Mackool, R. J., Gittes, G. K., McCarthy, J. G., Longaker, M. T. 1998; 102 (6): 2022-2032

  Abstract

  The application of distraction osteogenesis to craniofacial surgery has altered the approach and treatment of congenital and acquired craniofacial defects. Although the histologic and ultrastructural changes associated with distraction osteogenesis have been described extensively, relatively little is known about the molecular regulation of this process. The elucidation of the molecular mechanisms of distraction osteogenesis has important clinical implications because it may facilitate the use of recombinant proteins or gene therapy to accelerate bone regeneration. Molecular analysis of distraction osteogenesis has been hindered by the use of large animal models in which only limited genetic information is available. In this study, a rat model of mandibular distraction osteogenesis is described. This report includes a pilot study (n = 50) to develop an appropriate distraction device and to determine the optimal placement of the osteotomy. The study subsequently included 80 animals, 35 of which were distracted at a rate of 0.25 mm per day for 6 days (1.5 mm total) and 35 that were distracted at a rate of 0.25 mm twice per day (3.0 mm total). These animals were killed at various time points (after latency and during the distraction and consolidation periods) and displayed histologic and radiographic findings of membranous bone distraction osteogenesis that were consistent with those in large ,animal and clinical models. In addition, five animals each were acutely lengthened 1.5 mm and 3.0 mm and demonstrated a fibrous nonunion. Furthermore, the utility of this model is demonstrated in the analysis of the molecular mechanisms of distraction osteogenesis by applying the polymerase chain reaction to total cellular RNA isolated from normal and distracted rat mandibles. In conclusion, it is believed that the rat model of distraction osteogenesis has significant advantages over traditional models, including decreased costs and facilitation of molecular analysis.

  View details for Web of Science ID 000076924100033

  View details for PubMedID 9811000

• The combination of endoscopy and distraction osteogenesis in the development of a canine midface advancement model JOURNAL OF CRANIOFACIAL SURGERY Levine, J. P., Rowe, N. M., Bradley, J. P., Williams, J. K., Mackool, R. J., Longaker, M. T., McCarthy, J. G. 1998; 9 (5): 423-432

  Abstract

  The requirements for reconstruction in patients with midface hypoplasia can be formidable: a bicoronal scalp incision, Le Fort III or monobloc skeletal advancement, harvesting and insertion of bone grafts, application of rigid (and occasionally intermaxillary) fixation, blood transfusions, and prolonged operative time and hospitalization. The introduction of the endoscope offers the possibility of minimally invasive surgery with improved visualization of the osteotomy sites. The development of distraction osteogenesis as a surgical technique allows controlled and gradual advancement of the osteotomized skeletal segment and associated soft tissue. The purpose of this study was to develop a canine model of an endoscopically assisted Le Fort III osteotomy with attendant midface distraction. Four mongrels (20 kg in weight) were study subjects. Three 2-cm skin incisions were made (two perpendicular to the zygomaticomaxillary suture and one perpendicular to the nasofrontal suture). The soft tissue and periosteum were evaluated bluntly. Retractors specially designed for the project created a space for endoscopic visualization. Bilateral zygomatic, nasofrontal, and medial orbital wall osteotomies, corticotomies, or both were performed under endoscopic visualization using a reciprocating saw; the medial orbital wall sectioning was specifically not completed (i.e., corticotomy) to avoid laceration of the mucosa and attendant bleeding. The pterygomaxillary osteotomy was completed with an osteotome and mallet. Finally, the nasal septum was only partially divided with an osteotome to avoid excessive blood loss. Four distraction devices were placed across the above-noted osteotomies (two across the nasofrontal osteotomy and one across each lateral osteotomy). The animals were distracted 1 mm per day for 16 to 40 days after surgery (16-40 mm of linear distraction). Cephalograms and computed tomography scans were obtained before and after distraction. The animals were killed after remaining in fixation for 4 to 6 weeks after distraction. All soft tissue was removed and the skull was examined. Photos were obtained throughout the experiment for documentation. The study demonstrated that Le Fort III osteotomies can be performed successfully via small incisions with endoscopic assistance in canine subjects with excellent visualization and minimal bleeding. The advancement of the midface segment can be achieved by activation of an external distraction device.

  View details for Web of Science ID 000075751600004

  View details for PubMedID 9780909

• Scarless healing - The fetal wound CLINICS IN PLASTIC SURGERY Mackool, R. J., Gittes, G. K., Longaker, M. T. 1998; 25 (3): 357-?

  Abstract

  Fetal wounds heal without a scar early in gestation, and may hold the key to scarless repair. Several important concepts central to the fetal wound-healing response have been determined. The fetal fibroblast modulates the wound-healing response through collagen deposition, extracellular matrix deposition, and growth factor secretion. Fetal repair is both gestational-age and wound-size dependent, with a transition from scarless to scarring repair occurring during fetal life. Fetal fibroblasts manifest a decreased ability to induce dermal appendage formation from fetal epithelium at the same time that scarring in the fetus begins, suggesting that epithelial-mesenchymal interactions play an important role in scarless fetal repair. The fetal immune response during wound healing differs from the adult response, with a primarily mononuclear cell infiltrate and decreased activity and presence of polymorphonuclear leukocytes, whereas the cytokine profile of the fetal wound differs markedly from that of the adult wound. Patterning genes (homeobox genes) involved in organogenesis may prove integral to fetal healing, and are emerging as an active area of research. Once the biology of fetal wound healing is fully determined, attempts to manipulate the adult wound undoubtedly will progress rapidly, and scarless repair may become a clinical reality in children and adults.

  View details for Web of Science ID 000075153600004

  View details for PubMedID 9696898

• Periorbital melanocytic lesions: Excision and reconstruction in 40 patients PLASTIC AND RECONSTRUCTIVE SURGERY Glat, P. M., Longaker, M. T., Jelks, E. B., Spector, J. A., Roses, D. F., Shapiro, R. A., Zide, P. M., Jelks, G. W. 1998; 102 (1): 19-27

  Abstract

  The treatment of melanoma arising in the periorbital region is a difficult reconstructive problem. The abundance of vital structures in close proximity to one another makes the resection and subsequent reconstructive procedures extremely challenging. Reported here is experience with periorbital melanocytic lesions in 40 patients with the emphasis on the types of reconstruction performed. Forty patients with periorbital melanocytic lesions were treated between 1984 and 1995. The periorbital region was subdivided into five zones. These zones are the following: zone I, upper eyelid; zone II, lower eyelid; zone III, medial canthus; zone IV, lateral canthus; and zone V, contiguous structures. Ocular melanomas were not included in this study. The distribution of the lesions in our 40 patients was zone I (n = 1), zone II (n = 14), zone III (n = 1), zone IV (n = 9), and zone V (n = 31). The ages of the patients ranged from 3 to 84 years at the time of reconstruction, with an average age of 57 years. Resection and reconstruction were performed simultaneously in all patients. Thirty-six of the patients were reconstructed with one procedure, three patients required two procedures, and one patient required five procedures. The tumor type was superficial spreading melanoma in 15 patients, melanoma in situ in 17 patients, malignant spindle cell neoplasm in 2 patients, desmoplastic melanoma in 2 patients, amelanocytic melanoma in 1 patient, epithelioid melanoma in 1 patient, and atypical melanocytic nevus in 2 patients in which an early, evolving melanoma could not be excluded. Elective lymph node dissection was performed in four patients for intermediate thickness lesions (1.5 to 4.0 mm). The types of reconstructions performed included full-thickness skin grafts, upper lid myocutaneous flaps, cheek advancement flaps, cervicofacial flaps, inferiorly based nasolabial flaps, tarsoconjunctival flaps, frontalis muscle flaps, medial transposition Z-plasty, and primary closure. The resection of periorbital melanomas can be difficult because of the number of important anatomic structures in the region. The challenge to the surgeon in handling head and neck melanomas in general lies in the need to provide the best functional and aesthetic result while still resecting the primary lesion with the intent of effecting a cure. We present our series to demonstrate that the adequacy of margins of resection need not be compromised to facilitate reconstruction and that excellent results are obtainable with reconstructive procedures performed after adequate resections. Several different types of flaps and grafts can be used, with the indications varying depending on the location of the lesion and the extent of resection. The major reconstructive options will be reviewed in detail.

  View details for Web of Science ID 000074472100003

  View details for PubMedID 9655402

• Studies in cranial suture biology: Regional dura mater determines overlying suture biology PLASTIC AND RECONSTRUCTIVE SURGERY Levine, J. P., Bradley, J. P., Roth, D. A., McCarthy, J. G., Longaker, M. T. 1998; 101 (6): 1441-1447

  Abstract

  The influence of dura mater on adjacent cranial sutures is significant. By better understanding the mechanisms of normal suture fusion and the role of the dura mater, it may be possible to delineate the events responsible for the premature suture fusion seen in craniosynostosis. In the Sprague-Dawley rat, the posterior frontal suture normally fuses between 12 and 20 days of postnatal life and has proved to be an excellent model to describe normal suture fusion. The purpose of this study was to document the critical role that the dura mater-suture complex may play on cranial suture biology. Forty Sprague-Dawley rats at 8 days of age were divided into two groups of 20 animals each. The control group (group A) had surgical disruption of the dura mater-calvarial interface. This was accomplished by elevating a strip of cranium inclusive of the posterior frontal and sagittal sutures and replacement of the cranial strip back to its anatomic position, all with the dura mater left intact. The experimental group (group B) had the same calvarial elevation (strip craniectomy), but the sutural anatomy/alignment was rotated 180 degrees. This rotation placed the posterior frontal suture into the sagittal suture's anatomic position and the sagittal suture into the posterior frontal suture's anatomic position. All of these procedures were accomplished by leaving the underlying dura mater intact. Animals were killed at 20, 30, 40, and 50 days (12, 22, 32, and 42 days postoperatively), and tissue sections were examined with hematoxylin and eosin staining. Group A (control) showed normal but delayed suture activity. The posterior frontal suture fused, and the sagittal suture remained patent. Fusion was delayed, not beginning before 20 days (12 days postoperative) and showing complete fusion between 30 and 40 days. Group B (180-degree calvarial rotation) demonstrated that the suture in the posterior frontal anatomic position (actual sagittal suture) fused between 20 and 40 days, whereas the suture in the sagittal anatomic position (actual posterior-frontal suture) remained patent throughout the study. This study demonstrates that the location of the dura mater-suture complex is important in determining either suture patency or closure in this model. Normal closure of the suture overlying the posterior frontal dura mater demonstrates that the dura mater itself, or forces derived in specific cranial locations, determines the overlying suture biology.

  View details for Web of Science ID 000073423800002

  View details for PubMedID 9583471

• In vitro prefabrication of human cartilage shapes using fibrin glue and human chondrocytes ANNALS OF PLASTIC SURGERY Ting, V., Sims, C. D., Brecht, L. E., McCarthy, J. G., Kasabian, A. K., Connelly, P. R., Elisseeff, J., Gittes, G. K., Longaker, M. T. 1998; 40 (4): 413-420

  Abstract

  We report the first generation of human cartilage from fibrin glue using a technique of molding chondrocytes in fibrin glue developed in our laboratory. Human costal chondrocytes were suspended in cryoprecipitate and polymerized into a human nasal shape with bovine thrombin. After culture in vitro for 4 weeks, this construct was implanted subcutaneously into a nude mouse. The final construct harvested after 4 weeks in vivo demonstrated some preservation of its original features. Histological analysis showed features of native cartilage, including matrix synthesis and viable chondrocytes by nuclear staining. Biochemical analysis demonstrated active matrix production. Biomechanical testing was performed. To our knowledge this is the first reported creation of human cartilage from fibrin glue, and the first creation of human cartilage in vitro. This technique may become a promising means of engineering precisely designed autogenous cartilage for human reconstruction.

  View details for Web of Science ID 000072782100027

  View details for PubMedID 9555998

• Vascular lip enlargement .2. Port-wine macrocheilia - Tenets of therapy based on normative values PLASTIC AND RECONSTRUCTIVE SURGERY Zide, B. M., Glat, P. M., Stile, F. L., Longaker, M. T. 1997; 100 (7): 1674-1681

  Abstract

  Port-wine (capillary) vascular malformations that enlarge the lips (port-wine macrocheilia) are challenging reconstructive problems which, as a result, often go untreated. The surgical management of these lesions is not straightforward. Scarification by laser to diminish the discoloration has been performed with good results in some cases. However, laser treatment does little to correct three-dimensional tissue deformities such as macrocheilia, which must be addressed surgically. We present our experience with the treatment of port-wine macrocheilia in 11 patients over the 10-year period between 1983 and 1994. Basic principles for surgical and nonsurgical treatment of these patients are also discussed. Normative data about lip dimensions are important to surgical planning. We used 40 male and female volunteers, all less than 30 years of age, as a source for measuring normal lip dimensions, thereby creating a normative database. Preestablished points in the labial and perioral region were marked. Measurements were taken and then averaged. This information was used as a guide for surgical excision of large defects in some patients. In addition, in both the lower and the upper lip, if the opposite side is uninvolved, this database could serve as a template for reconstruction of the affected side. Between 1983 and 1994, 11 patients underwent surgery for port-wine macrocheilia. Of the 11 patients, 1 had previous treatment consisting of laser scarification. Four patients had port-wine vascular malformations involving the upper lip alone, four involved the lower lip alone, and three involved both lips. In six patients, other areas of the face and body were also involved. Our experience has led us to perform earlier surgical intervention than has previously been described for these patients. Basic reconstructive surgical principles and planning based on normative data and templates can lead to excellent results.

  View details for Web of Science ID A1997YK10700005

  View details for PubMedID 9393463

• Vascular lip enlargement .1. Hemangiomas - Tenets of therapy PLASTIC AND RECONSTRUCTIVE SURGERY Zide, B. M., Glat, P. H., Stile, F. L., Longaker, M. T. 1997; 100 (7): 1664-1673

  Abstract

  Vascular lesions involving the lips pose a difficult problem for both the surgeon and patient. Their removal by surgery may result in greater disfigurement and impairment than the original lesion. When nonsurgical modalities fail, using a well-planned strategy of sequential procedures can provide excellent results. Many hemangioma patients require judicious serial debulking of excess tissue mass, whereas enlargement from port-wine lesions may require direct aggressive surgery. Over a 10-year period, 38 patients underwent surgery for treatment of vascular lip enlargement. In 27 patients, the lip deformities were caused by hemangiomas. The remaining 11 patients had macrocheilia associated with port-wine vascular malformations. This paper specifically addresses hemangiomas of the lips, tenets for their removal, and reduction strategies. Of the 27 patients with hemangiomas involving the lips, 12 had had some form of previous treatment including corticosteroids (4 patients), embolization (3 patients), laser (3 patients), and interferon (2 patients). All 12 of these patients had unsatisfactory results. Specific tenets for the surgical management of these patients are presented. The distribution of the facial hemangiomas was as follows: 15 patients had isolated involvement of the upper lip, 7 lesions involved the lower lip alone and 5 involved both upper and lower lips. Additionally, 10 of these lesions involved the cheek(s), nose, or chin to some degree. Six patients experienced some form of functional impairment before our evaluation including difficulty with eating or drinking, visual obstruction, and psychosocial problems. All operations were performed following several principles established by the senior surgeon (B.M.Z.). By following the tenets presented in this report, he has achieved near-normal lip form, giving the patient marked improvement in appearance and function.

  View details for Web of Science ID A1997YK10700004

  View details for PubMedID 9393462

• Wound healing is accelerated by agonists of adenosine A(2) (G(alpha s)-linked) receptors JOURNAL OF EXPERIMENTAL MEDICINE Montesinos, M. C., Gadangi, P., Longaker, M., Sung, J., Levine, J., Nilsen, D., Reibman, J., Li, M., Jiang, C. K., Hirschhorn, R., Recht, P. A., Ostad, E., Levin, R. I., Cronstein, B. N. 1997; 186 (9): 1615-1620

  Abstract

  The complete healing of wounds is the final step in a highly regulated response to injury. Although many of the molecular mediators and cellular events of healing are known, their manipulation for the enhancement and acceleration of wound closure has not proven practical as yet. We and others have established that adenosine is a potent regulator of the inflammatory response, which is a component of wound healing. We now report that ligation of the G alpha s-linked adenosine receptors on the cells of an artificial wound dramatically alters the kinetics of wound closure. Excisional wound closure in normal, healthy mice was significantly accelerated by topical application of the specific A2A receptor agonist CGS-21680 (50% closure by day 2 in A2 receptor antagonists. In rats rendered diabetic (streptozotocin-induced diabetes mellitus) wound healing was impaired as compared to nondiabetic rats; CGS-21680 significantly increased the rate of wound healing in both nondiabetic and diabetic rats. Indeed, the rate of wound healing in the CGS-21680-treated diabetic rats was greater than or equal to that observed in untreated normal rats. These results appear to constitute the first evidence that a small molecule, such as an adenosine receptor agonist, accelerates wound healing in both normal animals and in animals with impaired wound healing.

  View details for Web of Science ID A1997YE94200024

  View details for PubMedID 9348321

• Evolution of the lateral canthoplasty: Techniques and indications PLASTIC AND RECONSTRUCTIVE SURGERY Glat, P. M., Jelks, G. W., Jelks, E. B., Wood, M., Gadangi, P., Longaker, M. T. 1997; 100 (6): 1396-1405

  Abstract

  Lateral canthoplasty is a useful method to restore eyelid function and to protect the ocular surfaces. The success of the procedure depends on the proper analysis of periorbital anatomy as it relates to the specific indication for lateral canthoplasty. We report the experience with 1565 lateral canthoplasties with emphasis on the evaluation of newer techniques that better address anatomic and functional requirements. Between 1981 and 1994, 1565 lateral canthoplasties were performed in 684 patients. Of these, 1369 "reconstructive" lateral canthoplasties were performed in 586 patients and 196 "cosmetic" lateral canthoplasties were performed in 98 patients. All operations were performed by a single surgeon (Jelks), and follow-up ranged from 1 to 14 years. The evolution of the operative technique for lateral canthoplasty has been toward an operation that corresponds with the anatomy of the individual. Indications for the procedure include lateral canthal dystopia, horizontal lid laxity, ectropion, entropion, lid margin eversion, lid retraction with or without soft-tissue deficiency, and aesthetic improvement. The types of procedures performed will be reviewed in detail. The evaluation of the newer forms of lateral canthoplasty as unique reconstructive tools and as adjuncts to aesthetic surgery will be discussed.

  View details for Web of Science ID A1997YH20400003

  View details for PubMedID 9385950

• The inferior retinacular lateral canthoplasty: A new technique PLASTIC AND RECONSTRUCTIVE SURGERY Jelks, G. W., Glat, P. M., Jelks, E. B., Longaker, M. T. 1997; 100 (5): 1262-1270

  Abstract

  Lateral canthoplasty is a useful method of restoring lower eyelid position and thereby protecting the ocular surfaces. The success of the lateral canthoplasty procedure depends on the proper analysis of periorbital anatomy. Newer lateral canthoplasty techniques have become progressively refined in an attempt to avoid the drawbacks and pitfalls of older procedures. We present the inferior retinacular lateral canthoplasty, developed to effectively address the problems associated with lower lid laxity and/or malposition. The inferior retinacular lateral canthoplasty is a versatile reconstructive procedure that also can be used as an adjunct to aesthetic surgery. The evolution of the inferior retinacular lateral canthoplasty from over 15 years of clinical experience is discussed.

  View details for Web of Science ID A1997XY99600030

  View details for PubMedID 9326791

• Gene expression of transforming growth factor beta-1 in rabbit zone II flexor tendon wound healing: Evidence for dual mechanisms of repair Annual Meeting of the American-Society-of-Plastic-and-Reconstructive-Surgeons Chang, J., Most, D., Stelnicki, E., Siebert, J. W., Longaker, M. T., Hui, K., Lineaweaver, W. C. LIPPINCOTT WILLIAMS & WILKINS. 1997: 937–44

  Abstract

  The postoperative outcome of hand flexor tendon repair can be complicated by adhesions between the repair site and surrounding tissue. To date, the biology of hand flexor tendon wound healing remains controversial--both intrinsic (resident tenocyte) and extrinsic (tendon sheath fibroblast and inflammatory cell) processes may contribute to repair. Transforming growth factor beta-1 is a cytokine that plays multiple roles in wound healing but is also implicated in the pathogenesis of excessive scar formation. This study examines the activation of transforming growth factor beta-1 mRNA in a rabbit zone II flexor tendon wound-healing model. Forty New Zealand White rabbit forepaws underwent complete transection and repair of the middle digit flexor digitorum profundus tendon in zone II. Tendons were harvested at increasing time intervals (1, 3, 7, 14, 28, and 56 days) and analyzed by in situ hybridization and immunohistochemistry to determine the expression patterns of transforming growth factor beta-1. A small number of tenocytes exhibited expression of transforming growth factor beta-1 mRNA at baseline in nonwounded control tendon specimens. The surrounding tendon sheath in these control specimens also revealed low numbers of fibroblasts and inflammatory cells expressing transforming growth factor beta-1 mRNA. In contrast, flexor tendons subjected to transection and repair exhibited increased signal for transforming growth factor beta-1 mRNA in both resident tenocytes and infiltrating fibroblasts and inflammatory cells from the tendon sheath. These data demonstrate that (1) normal unwounded tenocytes and tendon sheath cells are capable of transforming growth factor beta-1 production, (2) this cytokine is activated in the tendon wound environment, as evidenced by mRNA upregulation, and (3) the upregulation of this cytokine in both "intrinsic" tenocytes and "extrinsic" tendon sheath fibroblasts and inflammatory cells supports dual mechanisms for tendon repair. Because transforming growth factor beta-1 is thought to contribute to the pathogenesis of excessive scar formation, the findings presented here suggest that perioperative biochemical modulation of transforming growth factor beta-1 levels may help limit flexor tendon adhesion formation.

  View details for Web of Science ID A1997XW23900016

  View details for PubMedID 9290662

• Intracranial hypertension in a patient with craniofacial synostosis and patent sutures JOURNAL OF CRANIOFACIAL SURGERY Williams, J. K., Longaker, M. T., Wisoff, J., McCarthy, J. G. 1997; 8 (5): 373-378

  Abstract

  Craniosynostosis is often associated with restrictive cranial vault deformities, diminished intracranial volume, and intracranial hypertension. Advances in imaging techniques have provided a method of intracranial volume analysis that has demonstrated a more complicated relationship between craniofacial abnormalities and elevated intracranial pressures. Studies have confirmed a decrease in intracranial volume in the presence of craniosynostosis, but this association is not found in every patient. We report an unusual case of elevated intracranial pressure in a patient with the clinical stigmata of Crouzon's syndrome but with patent cranial vault sutures.

  View details for Web of Science ID A1997XW32200008

  View details for PubMedID 9482078

• Reduced expression of PDGF and PDGF receptors during impaired wound healing JOURNAL OF INVESTIGATIVE DERMATOLOGY Beer, H. D., Longaker, M. T., Werner, S. 1997; 109 (2): 132-138

  Abstract

  A series of studies has shown that application of platelet-derived growth factor (PDGF) to a wound enhances the process of wound repair, especially in animals with wound-healing defects. In the current study, we investigated the regulation of PDGF A and PDGF B and their receptors during wound repair in mice. Both ligands and both types of receptor were expressed in normal and wounded skin, whereby PDGF A and PDGF B proteins were found at different sites in the healing wound. Surprisingly, no significant induction of these genes was observed after skin injury in normal mice, and expression levels were similar at all stages of the repair process. To determine a possible role of endogenous PDGF in normal wound healing, we subsequently analyzed the regulation of PDGF and PDGF receptors during wound healing in healing-impaired animals. Genetically diabetic db/db mice showed a significant reduction in PDGF A and A-type receptor expression in nonwounded and wounded back skin. Furthermore, expression of the B-type receptor was also reduced during the repair process. Systemic glucocorticoid treatment caused a severe defect in wound repair that was accompanied by reduced expression of PDGF A and B and of the B-type receptor in the early phase of wound healing. These results provide an explanation for the beneficial effect of exogenous PDGF in the treatment of wound-healing disorders. Furthermore, our data suggest that a certain expression level of PDGF and its receptors is essential for normal repair.

  View details for Web of Science ID A1997XM19500002

  View details for PubMedID 9242497

• Wound size and gestational age modulate scar formation in fetal wound repair 43rd Annual International Congress of the British-Association-of-Paediatric-Surgeons Cass, D. L., Bullard, K. M., Sylvester, K. G., Yang, E. Y., Longaker, M. T., Adzick, N. S. W B SAUNDERS CO-ELSEVIER INC. 1997: 411–15

  Abstract

  The early-gestation fetus heals incisional skin wounds rapidly and scarlessly. The morphology with which the fetus heals excisional skin wounds remains unclear. To characterize excisional fetal wound repair, and to determine whether there is a developmentally regulated wound-size threshold beyond which fetal skin heals with scar, the authors created excisional wounds in fetal lambs of varying gestational age. Time-mated pregnant ewes carrying 22 fetuses at 60 to 90 days' gestation (term, 145 days) underwent laparotomy and hysterotomy. An incisional wound and four circular, punch biopsy wounds of 2, 4, 6, and 10 mm in diameter were placed on the back of each fetal lamb and marked with India ink. The wounds were harvested at 14 days' postwounding and examined grossly and microscopically after serial sectioning and histological staining. Morphological features of all wounds were graded. By 14 days' postwounding all fetal wounds had healed completely. for lambs at each gestational age, increasing wound size was strongly associated with an increase in the frequency of scar. Also, as gestational age increased from 60 to 90 days' gestation the frequency of scarless repair decreased. By understanding the cellular and molecular processes that mediate scar formation with increasing wound size and advancing gestational age, the authors hope to gain further insight into the mechanisms of scarless fetal wound repair.

  View details for Web of Science ID A1997WP14100008

  View details for PubMedID 9094005

• Studies in cranial suture biology .1. Increased immunoreactivity for TGF-beta isoforms (beta 1, beta 2, and beta 3) during rat cranial suture fusion JOURNAL OF BONE AND MINERAL RESEARCH Roth, D. A., Longaker, M. T., McCarthy, J. G., Rosen, D. M., McMullen, H. F., Levine, J. P., Sung, J., Gold, L. I. 1997; 12 (3): 311-321

  Abstract

  The mechanisms involved in normal cranial suture development and fusion as well as the pathophysiology of craniosynostosis, a premature fusion of the cranial sutures, are not well understood. Transforming growth factor-beta isoforms (TGF-beta 1, beta 2, and beta 3) are abundant in bone and stimulate calvarial bone formation when injected locally in vivo. To gain insight into the role of these factors in normal growth and development of cranial sutures and the possible etiology of premature cranial suture fusion, we examined the temporal and spatial expression of TGF-beta isoforms during normal cranial suture development in the rat. In the Sprague-Dawley rat, only the posterior frontal cranial suture undergoes fusion between 12 and 22 days of age, while all other cranial sutures remain patent. Therefore, immunohistochemical analysis of the fusing posterior frontal suture was compared with the patent sagittal suture at multiple time points from the fetus through adult. Whereas the intensity of immunostaining was the same in the posterior frontal and sagittal sutures in the fetal rat, there was increased immunoreactivity for TGF-beta isoforms in the actively fusing posterior frontal suture compared with the patent sagittal suture starting 2 days after birth and continuing until approximately 20 days. There were intensely immunoreactive osteoblasts present during fusion of the posterior frontal suture. In contrast, the patent sagittal suture was only slightly immunoreactive. A differential immunostaining pattern was observed among the TGF-beta isoforms; TGF-beta 2 was the most immunoreactive isoform and was also most strongly associated with osteoblasts adjacent to the dura and the margin of the fusing suture. Since the increased expression of TGF-beta 2 during suture fusion suggested a possible regulatory role, recombinant TGF-beta 2 was added directly to the posterior frontal and sagittal sutures in vivo to determine if suture fusion could be initiated. Exogenously added TGF-beta 2 stimulated fusion of the ectocranial surface of the posterior frontal suture. These data provide evidence for a regulatory role for these growth factors in cranial suture development and fusion. Additionally, the intense immunostaining for TGF-beta 2 in the dura mater underlying the fusing suture supports a role for the dura mater in suture fusion. It is possible that premature or excessive expression of these factors may be involved in the etiopathogenesis of craniosynostosis and that modulation of the growth factor profile at the suture site may have potential therapeutic value.

  View details for Web of Science ID A1997WN14800002

  View details for PubMedID 9076573

• Reconstruction of breast asymmetry in Poland's chest-wall deformity using microvascular free flaps PLASTIC AND RECONSTRUCTIVE SURGERY Longaker, M. T., Glat, P. M., Colen, L. B., Siebert, J. W. 1997; 99 (2): 429-436

  Abstract

  Poland's syndrome comprises a spectrum of chest-wall deformities affecting, to various degrees, the rib cage, the chest-wall muscles, the neurovascular structures of the ipsilateral arm, and the overlying breast. This study details our experience with nine female Poland's syndrome patients who had chest-wall and breast asymmetry corrected by microvascular free-tissue transfer. Nine female patients with Poland's chest deformity underwent 12 microvascular free flaps between 1989 and 1994. Donor sites for free-tissue transfer included eight transverse rectus abdominis flaps, two superior gluteal flaps, one inferior gluteal flap, and one contralateral latissimus dorsi flap. Recipient vessels were branches of the subscapular vascular axis in all patients. Patients' ages ranged from 18 to 47 years at the time of reconstruction. Chest-wall and breast asymmetry varied from accompanying severe pectus and rib cage deformities to isolated breast involvement. Complications were limited to a superior gluteal flap loss due to anomalous subscapular venous drainage. This patient underwent a successful second superior gluteal flap reconstruction utilizing the cephalic venous outflow system. Chest-wall and breast symmetry was restored in all patients. This study demonstrates that microsurgical reconstruction of chest-wall and breast asymmetry in Poland's syndrome yields excellent results with a high degree of patient satisfaction. Careful intraoperative assessment of the recipient vessels prior to flap transfer is mandatory. Because Poland's chest-wall deformity may include anomalies of the vascular system, preoperative vascular assessment with duplex ultrasonography should be considered in all patients, and use of preoperative angiography or venography in selected patients also appears justified.

  View details for Web of Science ID A1997WG44100017

  View details for PubMedID 9030150

• Immunolocalization of transforming growth factor beta 1, beta 2, and beta 3 and insulin-like growth factor I in premature cranial suture fusion PLASTIC AND RECONSTRUCTIVE SURGERY Roth, D. A., Gold, L. I., Han, V. K., McCarthy, J. G., Sung, J. J., Wisoff, J. H., Longaker, M. T. 1997; 99 (2): 300-309

  Abstract

  The etiology of craniosynostosis remains unknown. The beta group of transforming growth factors (TGF-beta) and insulin-like growth factors (IGF-I and IGF-II) are known to induce new bone formation and, when added exogenously, cause accelerated closure of calvarial defects. The possible roles of these bone growth factors in premature cranial suture fusion in humans have not been explored. We analyzed a total of 20 cranial suture biopsy samples (10 synostotic and 10 normal) from 10 infants with single-suture craniosynostosis undergoing cranial vault remodeling. Using isoform-specific antibodies for TGF-beta 1, -beta 2, and -beta 3 and IGF-I, we demonstrated immunoreactivity of these growth factors were present in human cranial sutures; the TGF-beta 2 isoform was the most intensely immunoreactive. Most importantly, the TGF-beta isoforms and IGF-I showed more intense immunoreactivity in the actively fusing craniosynostotic sutures compared with the control patent sutures. Specifically, the TGF-beta isoforms and IGF-I were intensely localized in the osteoblasts synthesizing new bone at the suture margin. It is noteworthy that although the patent sutures were less immunoreactive for TGF-beta isoforms than fused sutures, there was a distinct pattern of the TGF-beta 3 isoform that was immunolocalized to the margin of the normal patent sutures. This suggests a possible role for TGF-beta 3 in maintaining cranial suture patency. The increased immunoreactivity of both TGF-beta 2 and IGF-I in the actively fusing sutures compared with the patent control sutures indicates that these growth factors may play a role in the biology underlying premature suture closure. To our knowledge, this is the first study showing the presence of TGF-beta 1, -beta 2, and -beta 3 and IGF-I in prematurely fusing human cranial sutures. In the future, manipulating the local expression of these growth factors at the suture site may enable plastic surgeons to modulate premature suture fusion.

  View details for Web of Science ID A1997WG44100002

  View details for PubMedID 9030135

• A new rat model for the study of mandibular distraction osteogenesis 7th International Congress of the International-Society-for-Craniofacial-Surgery Rowe, N. M., Mehrara, B. J., Mackool, R. J., Gittes, G. K., McCarthy, J. G., Longaker, M. T. MONDUZZI EDITORE. 1997: 337–339

  View details for Web of Science ID 000076426900095

• Enophthalmos revisited: Zygoma mobilization and repositioning through minimal exposure 7th International Congress of the International-Society-for-Craniofacial-Surgery Longaker, M. T., Kawamoto, H. K. MONDUZZI EDITORE. 1997: 215–216

  View details for Web of Science ID 000076426900061

• Transcranial Doppler analysis of intra-cranial pressure in craniosynostosis 7th International Congress of the International-Society-for-Craniofacial-Surgery Stotland, M. A., Westra, S. J., Kawamoto, H. K., Longaker, M. T., Lazareff, J. MONDUZZI EDITORE. 1997: 97–99

  View details for Web of Science ID 000076426900030

• A new in utero model for lateral-oblique facial clefts 7th International Congress of the International-Society-for-Craniofacial-Surgery Stelnicki, E. J., Hoffman, W. Y., Vanderwall, K., Harrison, M. R., Foster, R., Longaker, M. T. MONDUZZI EDITORE. 1997: 353–354

  View details for Web of Science ID 000076426900100

• The effect of bilateral distraction osteogenesis utilizing a multiplanar device on the temporomandibular joint (TMJ): A canine study 7th International Congress of the International-Society-for-Craniofacial-Surgery Stucki-McCormick, S. U., Grayson, B. H., Longaker, M. T., Williams, J. K., Mackool, R. J., Rowe, N., McCarthy, J. G. MONDUZZI EDITORE. 1997: 35–37

  View details for Web of Science ID 000076426900012

• A new in utero sheep model for unilateral coronal craniosynostosis that demonstrates the importance of suture fusion in the development of the associated craniofacial abnormalities 7th International Congress of the International-Society-for-Craniofacial-Surgery Stelnicki, E. J., Vanderwall, K., Hoffman, W. Y., Harrison, M. R., Glowacki, J., Longaker, M. T. MONDUZZI EDITORE. 1997: 351–352

  View details for Web of Science ID 000076426900099

• Engineering of cartilaginous human shapes in vitro using plasma derived polymer substances and chondrocytes 7th International Congress of the International-Society-for-Craniofacial-Surgery Ting, V., Sims, C. D., Brecht, L. E., McCarthy, J. G., Kasabian, A. K., Connelly, P. R., Gittes, G. K., Longaker, M. T. MONDUZZI EDITORE. 1997: 39–40

  View details for Web of Science ID 000076426900013

• Reconstruction of severe periorbital deformities using microsurgical free tissue transfers 7th International Congress of the International-Society-for-Craniofacial-Surgery Siebert, J. W., Jelks, G. W., Brecht, L. E., Longaker, M. T. MONDUZZI EDITORE. 1997: 259–261

  View details for Web of Science ID 000076426900073

• Reconstruction of Tessier No. 4 clefts revisited 7th International Congress of the International-Society-for-Craniofacial-Surgery Longaker, M. T., Kawamoto, H. K. MONDUZZI EDITORE. 1997: 177–179

  View details for Web of Science ID 000076426900052

• Volumetric quantification of the changes in the human mandible after distraction: A new analytic technique 7th International Congress of the International-Society-for-Craniofacial-Surgery Mackool, R. J., Mehrara, B. J., Grayson, B. H., Longaker, M. T., McCarthy, J. G. MONDUZZI EDITORE. 1997: 79–81

  View details for Web of Science ID 000076426900026

• The in utero correction of unilateral coronal craniosynostosis 7th International Congress of the International-Society-for-Craniofacial-Surgery Stelnicki, E. J., Vanderwall, K., Harrison, M. R., Longaker, M. T., Kaban, L. B., Hoffman, W. Y. MONDUZZI EDITORE. 1997: 41–42

  View details for Web of Science ID 000076426900014

• Monobloc fronto-facial advancement: The UCLA experience 1988-1996 7th International Congress of the International-Society-for-Craniofacial-Surgery Benhaim, P., Longaker, M. T., Kawamoto, H. K. MONDUZZI EDITORE. 1997: 119–120

  View details for Web of Science ID 000076426900037

• Transconjunctival approach to orbital surgery: Ectropion incidence increases in lids with a previous subciliary incision 7th International Congress of the International-Society-for-Craniofacial-Surgery Lorenz, H. P., Longaker, M. T., Kawamoto, H. K. MONDUZZI EDITORE. 1997: 243–245

  View details for Web of Science ID 000076426900069

• The inframammary extended circumflex scapular flap: An aesthetic improvement of the parascapular flap PLASTIC AND RECONSTRUCTIVE SURGERY Siebert, J. W., Longaker, M. T., Angrigiani, C. 1997; 99 (1): 70-77

  Abstract

  Parascapular free flaps traditionally have been designed obliquely across the back, corresponding to the descending branch of the circumflex scapular artery. The donor site of this workhorse flap has the drawback of a widened and frequently hypertrophic scar. In searching for aesthetic improvements in the donor site, we have progressively rotated the axis of this flap in an anterior direction. The end result of this modification is the flap we report here: the inframammary extended circumflex scapular flap. This flap has a longitudinal axis of rotation lying curvilinearly from the inframammary fold to the circumflex scapular artery within the triangular anatomic space. We have used this flap in 20 patients over the past 2 years. The vast majority of these cases were deepithelialized flaps with customized extensions of dorsal thoracic fascia to correct facial asymmetry. We feel that the inframammary extended circumflex scapular artery flap donor-site scar is well hidden within the inframammary fold, and that the unavoidable widening and hypertrophy of parascapular and scapular flap donor-site scars were minimized compared with traditional flap designs.

  View details for Web of Science ID A1997WA11700010

  View details for PubMedID 8982188

• Deep-plane cervicofacial ''hike'': Anatomic basis with dog-ear blepharoplasty PLASTIC AND RECONSTRUCTIVE SURGERY Longaker, M. T., Glat, P. M., Zide, B. M. 1997; 99 (1): 16-21

  Abstract

  The temporal and cheek areas are particularly vulnerable to sun damage and therefore to skin cancers. Rotation-advancement flaps are used commonly in the reconstruction of these regions following resection of skin cancers. Such flaps usually are modifications of the Mustardé, cervicofacial, and Juri flaps. The drawbacks to these flaps relate to a random, unpredictable perfusion with skin loss at the distal flap tip, a vertically oriented dog-ear that predictably is located on the cheek, and the risk of gravitational and cicatricial forces acting on the lower eyelid causing ectropion. The deep-plane technique for raising cheek flaps, as has been described recently for use in rhytidectomy, allows the plastic surgeon to address both drawbacks of the standard cervicofacial flaps. The vertical "hike" deep-plane approach addresses both drawbacks of the rotation-advancement flaps by including better perfusion and superior mobility after release of restraining ligaments. We describe the anatomic rationale for the deep-plane dissection of the cheek in cadaver studies and present our clinical technique with a vertical "hike" cheek advancement with removal of the horizontally oriented dog-ear as in a blepharoplasty. This single-stage technique will be called the deep-plane "hike" flap. The vertically advanced flap must be slightly overcorrected by anchoring the flap to the periosteum just above the recipient defect. This deep fixation removes all tension from the skin and prevents ectropion. This single-stage reconstruction affords excellent cosmetic results without compromising any future reconstructive efforts.

  View details for Web of Science ID A1997WA11700003

  View details for PubMedID 8982182

• Acute biceps compartment syndrome associated with the use of a noninvasive blood pressure monitor ANESTHESIA AND ANALGESIA Sutin, K. M., Longaker, M. T., Wahlander, S., Kasabian, A. K., Capan, L. M. 1996; 83 (6): 1345-1346

  View details for Web of Science ID A1996VV54300040

  View details for PubMedID 8942614

• The anti-inflammatory mechanism of sulfasalazine is related to adenosine release at inflamed sites JOURNAL OF IMMUNOLOGY Gadangi, P., Longaker, M., NAIME, D., Levin, R. I., Recht, P. A., Montesinos, M. C., Buckley, M. T., Carlin, G., Cronstein, B. N. 1996; 156 (5): 1937-1941

  Abstract

  The anti-inflammatory mechanism of sulfasalazine is not well understood. It has recently been shown that sulfasalazine inhibits 5-aminoimidazole-4-carboxamidoribonucleotide (AICAR) transformylase, an enzyme involved in de novo purine biosynthesis. We recently demonstrated that methotrexate promotes intracellular AICAR accumulation, thereby increasing adenosine release and diminishing inflammation, so we tested the hypothesis that sulfasalazine similarly promotes intracellular AICAR accumulation. We studied adenosine release and the state of inflammation in in vitro and in vivo models of the inflammatory process. The adhesion of stimulated neutrophils (FMLP) to endothelial cells preincubated with sulfasalazine was inhibited in a dose-dependent manner. Elimination of extracellular adenosine by addition of adenosine deaminase or inhibition of adenosine by the adenosine A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) completely reversed the anti-inflammatory effect of sulfasalazine (at concentrations <1 microM in this in vitro model. To determine whether this phenomenon was relevant to inhibition of inflammation in vivo, we studied the effect of sulfasalazine (100 mg/kg/day by gastric gavage for 3 days) on leukocyte accumulation in the murine air pouch model of inflammation. Treatment with sulfasalazine markedly decreased the number of leukocytes that accumulated in the inflamed (carrageenan, 2 mg/ml) air pouch. Injection of either adenosine deaminase or DMPX, but not the A1 receptor antagonist 8-cyclopentyl-dipropylxanthine, significantly reversed the anti-inflammatory effects of sulfasalazine treatment. Sulfasalazine increased the exudate adenosine concentration from 127 +/- 64 nM to 869 +/- 47 nM. Moreover, sulfasalazine treatment promoted a marked increase in splenocyte AICAR concentration from 35 +/- 6 to 96 +/- 3 pmols/10(6) splenocytes, which is consistent with the in vitro observation that sulfasalazine inhibits AICAR transformylase. These results indicate that sulfasalazine, like methotrexate, enhances adenosine release at an inflamed site and that adenosine diminishes inflammation via occupancy of A2 receptors on inflammatory cells. Our studies provide evidence that sulfasalazine and methotrexate may be described as a newly recognized family of anti-inflammatory agents that share the property of using adenosine as an antagonist of inflammation.

  View details for Web of Science ID A1996TW69900031

  View details for PubMedID 8596047

• Microsurgical reconstruction of the lower extremity using the 3M microvascular coupling device in venous anastomoses ANNALS OF PLASTIC SURGERY Denk, M. J., Longaker, M. T., BASNER, A. L., Glat, P. M., Karp, N. S., Kasabian, A. K. 1995; 35 (6): 601-606

  Abstract

  Microsurgical reconstruction of the lower extremity presents a difficult problem to plastic surgeons; the rate of failure is higher than any other anatomical site. We reviewed our recent experience with lower extremity microsurgical reconstruction using the 3M vascular coupling device. We believe the excellent patency rate of the coupler may minimize the well-described problem of venous thrombosis in this challenging group of patients. This study involves a consecutive series of 11 patients who presented for reconstructive microsurgery of the lower extremity at NYU Medical Center hospitals between June 1 and September 1, 1994. Ten of 11 patients had free flap transfer to traumatic lower extremity injuries, whereas the remaining reconstruction was in a diabetic individual with a chronic wound. Fifteen microvascular venous anastomoses were performed; all but 1 was performed using the 3M coupler. Our experience with 11 patients, involving 14 mechanically coupled venous anastomoses, demonstrated successful use of the coupler. No intraoperative or postoperative vascular complications occurred. The overall success rate of the 3M coupler for venous anastomoses was 100%, and all microvascular free flaps were successful. We recommend using the 3M coupling device for venous anastomoses during reconstructive microsurgery of the lower extremity. Our series demonstrates the safety and effectiveness of the 3M coupler in this challenging group of patients. In addition, a secondary benefit of the 3M coupler is a significant reduction in operative time.

  View details for Web of Science ID A1995TK91000008

  View details for PubMedID 8748342

• SALVAGE OF TRAUMATIC BELOW-KNEE AMPUTATION STUMPS UTILIZING THE FILET OF FOOT FREE-FLAP - CRITICAL-EVALUATION OF 6 CASES PLASTIC AND RECONSTRUCTIVE SURGERY Kasabian, A. K., Glat, P. M., Eidelman, Y., COLEN, S., Longaker, M. T., Attinger, C., Shaw, W. 1995; 96 (5): 1145-1153

  Abstract

  Over a 12-year period between 1979 and 1991, 27 patients were operated on at the New York University Medical Center for salvage of below-knee amputation stumps utilizing free flaps. Six different donor sites were used. In 6 patients, the amputated foot was the donor site for a free flap to cover the tibial stump. There were 3 males and 3 females in this group. Five of the patients underwent immediate filet of foot reconstructions, while 1 patient had a reconstruction performed 69 days after injury, electively, when it was determined that below-knee amputation was the best option. All foot flaps survived and ultimately provided the major soft-tissue coverage for the below-knee amputation stump. The length of hospitalization ranged from 24 to 118 days. The time required from foot filet procedure to ambulation was 2, 4, 6, 7, 9, and 12 months in the 6 patients. Five of the 6 patients have resumed work or school after their injury. Foot flaps were based on the posterior tibial artery, anterior tibial artery, or both vessels. Nerve anastomosis of the posterior tibial nerve was performed in 5 patients. In 1 patient it was possible to maintain the continuity of the posterior tibial nerve. Five of the 6 patients were tested over a year after the flap, and all have good cold, pressure, and vibration sensation. Two of the 5 patients have heat sensation, and all 5 patients have at least protective pressure sensation. All the patients ambulate well with a below-knee prosthesis.

  View details for Web of Science ID A1995RX59300023

  View details for PubMedID 7568492

• HEMODYNAMIC-STUDY OF DIFFERENT ANGLED END-TO-SIDE ANASTOMOSES MICROSURGERY Zhang, L., Moskovitz, M., PISCATELLI, S., Longaker, M. T., Siebert, J. W. 1995; 16 (2): 114-117

  Abstract

  This study assessed the postoperative microvascular hemodynamics in end-to-side arterial anastomoses of varied angles in order to determine whether the angle of the anastomosis affects arterial flow. Thirty male rats were divided into three groups of ten. Carotid to carotid end-to-side anastomoses were performed with anastomotic angles of 45, 90, and 135 degrees. Postoperative flow was assessed using high frequency pulsed Doppler ultrasound (HFPDU) at 30 and 120 min postoperatively. Measurements at 30 min demonstrated significant differences in flow, with the 45 degrees grafted vessels utilizing 56.5% of total carotid flow, and the 90 degrees and 135 degrees anastomoses appropriating 46.5% and 43.2% of flow, respectively (comparing 45 degrees to both 90 degrees, P < 0.05, and 135 degrees, P < 0.002). The change from baseline in 45 degrees and 90 degrees groups dissipated over a 2 hr postoperative period, but flow in the grafted vessels in the 135 degrees group continued well below 50% at 39.9% (comparing to both 45 degrees and 90 degrees, P < 0.001), thereby displaying a significant difference in the postoperative arterial flow of varied angled microanastomoses.

  View details for Web of Science ID A1995QQ67700012

  View details for PubMedID 7783602

• Regional dura mater determines cranial suture fusion 6th International Congress of the International-Society-of-Cranio-Facial-Surgery Bradley, J. P., Levine, J. P., Roth, D. A., McCarthy, J. G., Longaker, M. T. MONDUZZI EDITORE. 1995: 87–89

  View details for Web of Science ID A1995BH77C00024

• Microsurgical correction of facial contour in congenital craniofacial malformations: The marriage of hard and soft tissue 6th International Congress of the International-Society-of-Cranio-Facial-Surgery Longaker, M. T., Siebert, J. W. MONDUZZI EDITORE. 1995: 337–339

  View details for Web of Science ID A1995BH77C00095

• Cleft lip and palate repair in fetal lambs 6th International Congress of the International-Society-of-Cranio-Facial-Surgery Hedrick, M. H., Hoffman, W. Y., Longaker, M. T. MONDUZZI EDITORE. 1995: 13–15

  View details for Web of Science ID A1995BH77C00004

• Wound healing in the fetus. Possible role for inflammatory macrophages and transforming growth factor-beta isoforms. Wound repair and regeneration Longaker, M. T., Bouhana, K. S., Harrison, M. R., DANIELPOUR, D., ROBERTS, A. B., Banda, M. J. 1994; 2 (2): 104-112

  Abstract

  Macrophages are believed to play a crucial role in wound healing by synthesizing and secreting numerous cytokines. Some of these cytokines, such as transforming growth factor-beta and tumor necrosis factor-alpha, promote fibrosis and repair. We have shown that macrophages are recruited to sterile fetal wounds and have the potential to regulate repair by synthesizing transforming growth factor-beta(1), transforming growth factor-beta(2), and tumor necrosis factor-alpha. Transforming growth factor-beta was present in fetal lamb wounds in higher amounts than in adult sheep wounds. Furthermore, the concentrations and ratios of the transforming growth factor-beta isoforms in wounds that healed without scarring were different from those in wounds that scarred; transforming growth factor-beta(2) was highest in fetal wounds that did not scar and lowest in adult wounds. These data suggest that concentrations of transforming growth factor-beta isoforms rather than total transforming growth factor-beta concentration may be important in the regulation of fibrosis in prenatal and postnatal wound healing.

  View details for PubMedID 17134379

• HYALURONATE METABOLISM UNDERGOES AN ONTOGENIC TRANSITION DURING FETAL DEVELOPMENT - IMPLICATIONS FOR SCAR-FREE WOUND-HEALING JOURNAL OF PEDIATRIC SURGERY Estes, J. M., Adzick, N. S., Harrison, M. R., Longaker, M. T., Stern, R. 1993; 28 (10): 1227-1231

  Abstract

  Wound healing in the fetus occurs by a different process from that in the adult. Instead of healing with scar formation, fetal cutaneous wounds heal by regeneration that results in complete restoration of normal skin architecture. The mechanisms responsible for this remarkable phenomenon involve factors in the fetal environment and properties intrinsic to fetal cells. Hyaluronic acid (HA) is a major component of the fetal extracellular matrix (ECM) and is believed to play an important role in this process. In this study, HA and HA-stimulating activity (HASA) in fetal and adult wound fluid were examined using sensitive, newly developed assays. In an ovine model, higher levels of HA and HASA were observed in fetal as compared with adult wound fluid. This difference was most prominent in wound fluid from fetal lambs at 75 and 100 days gestation (term = 145 days); these samples contained persistently elevated HA and HASA levels for up to 2 weeks after wounding (HA peak levels 145 micrograms/mL and 110 micrograms/mL, respectively). In contrast, wound fluid from 120-day-gestation fetuses had significantly lower levels (P < .001) that were transient and similar to that in the adult (HA peak levels 70 micrograms/mL and 10 micrograms/mL, respectively). These observations confirm an ontogenic transition in wound HA metabolism from a fetal to an adult-like phenotype. Levels of HASA as a function of time after wounding correlated with levels of HA, suggesting a role for HASA in controlling HA deposition during tissue repair. Two patterns of HASA and HA synthesis were noted.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1993MD71600002

  View details for PubMedID 8263679

• ARE PATIENTS WITH NEOPLASIA AT AN INCREASED RISK FOR CARDIAC MYXOMAS HUMAN PATHOLOGY Suman, V. J., Tazelaar, H. D., Bailey, K., Melton, J., Longaker, M. T., Stern, R., Finkbeiner, W. E. 1993; 24 (9): 1008-1011

  Abstract

  A case-control study was undertaken to assess whether a significant association exists between the presence of a neoplasm or malignancy and the presence of a cardiac myxoma. We identified 13 patients seen at our institution between 1935 and 1990 whose autopsies revealed a cardiac myxoma that was undetected during the patient's lifetime. For each patient four control subjects were selected among the autopsied patients who had never had a cardiac myxoma; these were matched for age at death, gender, and year of autopsy. The institutional medical records and autopsy reports revealed that 46% of the patients and 65% of their controls had a neoplasm diagnosed prior to or at autopsy. The estimated odds ratio was 0.34 (95% confidence interval, 0.05 to 1.84). There was insufficient evidence to conclude that an association exists between the presence of a neoplasm and the presence of a cardiac myxoma (P = .2722).

  View details for Web of Science ID A1993LX83000012

  View details for PubMedID 8253455

• FETAL CLEFT-LIP REPAIR IN RABBITS - LONG-TERM CLINICAL AND CEPHALOMETRIC RESULTS CLEFT PALATE-CRANIOFACIAL JOURNAL Kaban, L. B., Dodson, T. B., Longaker, M. T., Stern, M., Umeda, H., Adzick, S. 1993; 30 (1): 13-21

  Abstract

  We have developed a model for fetal cleft lip (CL) repair in rabbits. To date, the in utero CL procedure has been performed on 174 fetuses in 98 pregnant does. Details of the model, wound healing characteristics, and early growth results have been published previously. In this study, we report long-term clinical and cephalometric findings in 23 fetuses who underwent the fetal CL procedure, were born alive, and survived until completion of growth. The surgically created and repaired CL in fetal rabbits described here resulted in healing without scar formation. The deformity varies from an incomplete to a severe complete cleft, resembling the clinical spectrum of spontaneous clefts in humans. Cephalometric studies indicate that there were no statistically significant differences in premaxillary width, anterior maxillary length, or anterior and posterior maxillary width among control, unrepaired, and repaired animals. Documentation of this phenomenon in higher animals is necessary before the technique can be applied to humans with cleft lip.

  View details for Web of Science ID A1993KH78000002

  View details for PubMedID 8418867

• NEW TECHNIQUES IN FETAL SURGERY 23RD ANNUAL MEETING OF THE CANADIAN ASSOC OF PAEDIATRIC SURGEONS Jennings, R. W., Adzick, N. S., Longaker, M. T., Lorenz, H. P., Estes, J. M., Harrison, M. R. W B SAUNDERS CO-ELSEVIER INC. 1992: 1329–33

  Abstract

  Optimal fetal management during and after fetal surgery has been limited by an inability to reliably monitor the fetal heart rate and temperature, and by a lack of access to the fetal circulation. In order to solve these problems, we used early third trimester fetal sheep to develop: (1) an implantable radiotelemetry device that transmits the fetal electrocardiogram and temperature, and (2) an intraosseous access technique. A miniaturized radiotelemeter was implanted subcutaneously in the axilla of four fetal sheep. Safe implantation of the radiotelemeter was technically feasible and the device reliably recorded the fetal electrocardiogram and temperature both intraoperatively and postoperatively. Although many possible routes for access to the fetal circulation have been tried experimentally and clinically for both resuscitation and blood sampling, none have proven satisfactory. We assessed the use of intraosseous access in fetal sheep (n = 6) for both infusion and blood sampling. Access with an intraosseous needle was obtained in both sheep fetuses and human fetal cadavers. Blood gas values (pH, PCO2, and PO2) obtained from the medullary cavity of fetal sheep accurately reflected peripheral venous values. Resuscitation drugs reached the fetal circulation via the intraosseous route: sodium bicarbonate elevated venous bicarbonate levels from 28.4 +/- 1.7 to 31.8 +/- 2.1 mEq/dL (P < .05); injected glucose increased venous glucose levels by 520 +/- 108 mg/dL (P < .01); and injection of calcium chloride elevated venous calcium levels from 11.8 +/- 0.7 to 15.2 +/- 2.2 mEq/dL (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1992JR78000021

  View details for PubMedID 1403515

• An in utero model of craniosynostosis. journal of craniofacial surgery Duncan, B. W., Adzick, N. S., Moelleken, B. R., Chua, J., Bradley, S. M., Longaker, M. T., LEVINSOHN, D. G., Harrison, M. R., Kaban, L. B. 1992; 3 (2): 70-78

  Abstract

  To define better the pathophysiology of craniosynostosis, we developed an in utero model in rabbits. Premature fusion of the sutures was achieved by osteoinduction using demineralized bone matrix (DBM). Thirty-six fetuses from 18 time-dated pregnant rabbits underwent coronal strip craniectomies and implantation of DBM at 25 days of gestation (term = 31 days). Seventeen (47%) survived vaginal delivery and 10 (28%) survived for long-term follow-up. Surviving, operated animals demonstrated bony fusion across the coronal sutures presumably due to osteoinduction by the DBM. Coronal computed tomographic scans with sagittal reconstructions revealed that experimental animals had taller and wider skulls than nonoperated control animals. We conclude that employing DBM to create craniosynostosis in the fetal rabbit is technically feasible. Such a model may be used to more clearly define the pathophysiology of craniosynostosis and to evaluate methods of treatment.

  View details for PubMedID 1290786

• FETAL FRACTURE-HEALING IN A LAMB MODEL PLASTIC AND RECONSTRUCTIVE SURGERY Longaker, M. T., MOELLEKEN, B. R., Cheng, J. C., Jennings, R. W., Adzick, N. S., MINTOROVICH, J., LEVINSOHN, D. G., Gordon, L., Harrison, M. R., Simmons, D. J. 1992; 90 (2): 161-171

  Abstract

  A large animal model to assess fetal fracture repair and the ability to close excisional bony defects is presented. Incisional and excisional ulnar fractures were made in 14 midgestation fetal lambs, harvested at serial time points, and subjected to high-resolution low-kilovolt magnification radiographs, magnetic resonance imaging scans, and histologic analysis. Fetal fracture healing was characterized by early closure of excisional defects and rapid fracture healing with minimal or no soft-tissue inflammation or callus formation. Magnetic resonance imaging scans of the fractures revealed a characteristic pattern compatible with the histologic findings, namely, minimal inflammation in soft tissue adjacent to the fracture site. Histologic and magnification radiographic findings indicated that complete bony repair occurred within 21 days in incisional defects and within 40 days in excisional defects. In both cases, healed fetal bone resembled normal bone matrix. Excisional defects, including periosteum, of greater than three times the width of the bony cortex closed rapidly with virtually normal-appearing bony matrix and with minimal or no callus formation.

  View details for Web of Science ID A1992JG69400001

  View details for PubMedID 1631210

• NECROTIZING FASCIITIS IN 2 CHILDREN WITH ACUTE LYMPHOBLASTIC-LEUKEMIA JOURNAL OF PEDIATRIC SURGERY Duncan, B. W., Adzick, N. S., DeLorimier, A. A., Longaker, M. T., Ferrell, L. D., Zoger, S., Harrison, M. R. 1992; 27 (5): 668-671

  Abstract

  Necrotizing fasciitis is a severe, soft tissue infection, and is an unusual condition in children. The cornerstone of therapy is prompt, aggressive surgical treatment. Despite vigorous treatment, mortality rates are high. We report the occurrence of necrotizing fasciitis in two children during the granulocytopenic phase of induction chemotherapy for acute lymphoblastic leukemia. The diagnosis and treatment of necrotizing fasciitis in these two children was made more difficult by their underlying disease and its chemotherapy. The successful treatment of their infections relied on a multimodality approach. Aggressive surgical debridement was the mainstay of therapy. Adjuvant therapy was vital to the successful outcomes and included meticulous wound care, intravenous hyperalimentation, appropriate antibiotics, and granulocyte transfusions.

  View details for Web of Science ID A1992HT46000036

  View details for PubMedID 1625147

• THE EFFECT OF TISSUE EXPANSION ON DERMAL FIBROBLAST CONTRACTION ANNALS OF PLASTIC SURGERY Chang, B., TUCHLER, R. E., Siebert, J. W., Longaker, M. T., Burd, D. A. 1992; 28 (4): 315-319

  Abstract

  Tissue expansion alters the function of skin cells. We studied the effects of expansion on the contractile function of dermal fibroblasts using an in vitro model, the fibroblast-populated collagen lattice (FPCL). Spherical expanders were placed dorsally in 30 Sprague-Dawley rats; one-half were serially inflated. One, 2, and 4 weeks later, 5 rats from each group were killed. Fibroblasts were cultured from dermis overlying the expanders and mixed with collagen, medium, and serum in petri dishes to form FPCL. Fibroblasts from 5 rats that had not undergone surgery were cultured to make control FPCL. Contraction was assessed by measuring the areas of the FPCL. At 48 hours, FPCL containing expanded fibroblasts had contracted significantly less than those containing unexpanded or control fibroblasts. Four weeks of expansion resulted in less contraction than 1 or 2 weeks. Tissue expansion inhibits the in vitro contractile function of dermal fibroblasts in the rat in a time-related fashion.

  View details for Web of Science ID A1992HN46700003

  View details for PubMedID 1596063

• SCARLESS FETAL HEALING - THERAPEUTIC IMPLICATIONS ANNALS OF SURGERY Adzick, N. S., Longaker, M. T. 1992; 215 (1): 3-7

  Abstract

  The purpose of this report is to call attention to the fetal wound healing process as a blueprint for ideal tissue repair. Wound healing in the fetus is fundamentally different from healing in the adult. Fetal tissue repair occurs rapidly and in the absence of scar formation. Because scarring and fibrosis dominate some diseases in every area of medicine, an understanding of fetal wound healing should help develop therapeutic strategies to avert the devastating consequences of excessive scar formation.

  View details for Web of Science ID A1992GY75700002

  View details for PubMedID 1731647

• FETAL SURGERY FOR CLEFT-LIP - A PLEA FOR CAUTION PLASTIC AND RECONSTRUCTIVE SURGERY Longaker, M. T., Whitby, D. J., Adzick, N. S., Kaban, L. B., Harrison, M. R. 1991; 88 (6): 1087-1092

  View details for Web of Science ID A1991GR65500025

  View details for PubMedID 1946761

• SHEEP AMNIOTIC-FLUID HAS A PROTEIN FACTOR WHICH STIMULATES HUMAN FIBROBLAST POPULATED COLLAGEN LATTICE CONTRACTION JOURNAL OF CELLULAR PHYSIOLOGY Rittenberg, T., Longaker, M. T., Adzick, N. S., Ehrlich, H. P. 1991; 149 (3): 444-450

  Abstract

  Sutured incisional wounds made in fetal sheep and rabbits heal without scarring. Fetal sheep excisional wounds can close by contraction, but those in fetal rabbits do not. In vivo and in vitro evidence suggests that rabbit amniotic fluid inhibits wound contraction. The question arises: does sheep amniotic fluid promote wound contraction because their fetal wounds close by contraction? Sheep amniotic fluid (SAF) from 100 and 125 days gestation was tested in fibroblast populated collagen lattice (FPCL) system, an in vitro model of wound contraction. SAF stimulated FPCL contraction in a dose responsive manner. SAF from a 100 day fetus was more stimulating than a 125 day SAF. SAF enhanced FPCL contraction in the presence or absence of serum. SAF was fractionated by size, using column chromatography. It yielded a fraction with an estimated molecular weigh near 40,000 daltons, which stimulated FPCL contraction. The factor was inactivated by proteolytic digestion and heat denaturation. This protein fraction which stimulates FPCL contraction is not related to 1) actin-myosin filaments enhanced contraction by ATP-induced cell contraction, 2) promotion of fibroblast elongation on glass surface or in collagen, or 3) increased cell number by enhanced fibroblast duplication in a collagen matrix. A mechanism for SAF promotion of FPCL contraction was investigated but not identified.

  View details for Web of Science ID A1991GU23700012

  View details for PubMedID 1744173

• HYALURONIDASE LEVELS IN URINE FROM WILMS-TUMOR PATIENTS JOURNAL OF THE NATIONAL CANCER INSTITUTE Stern, M., Longaker, M. T., Adzick, N. S., Harrison, M. R., Stern, R. 1991; 83 (21): 1569-1574

  Abstract

  The pathophysiology of Wilms' tumor is associated with major alterations in hyaluronic acid metabolism. Elevated levels of both hyaluronic acid and a hyaluronic acid-stimulating activity occur in the urine and serum of patients with this tumor. In the current study, we describe elevated levels of urinary hyaluronidase in five patients with Wilms' tumor. Following surgical removal of the tumor, enzyme levels decreased toward normal. Characterization of enzyme activity indicates that hyaluronidase may be produced by the tumor itself. Alternatively, normal renal tissue may also be producing enzyme in a compensatory response to the elevated hyaluronic acid levels in these patients. We suggest that urinary hyaluronidase can be used as an additional marker for Wilms' tumor.

  View details for Web of Science ID A1991GM85900017

  View details for PubMedID 1660075

• ANIMAL-MODELS FOR THE STUDY OF FETAL TISSUE-REPAIR JOURNAL OF SURGICAL RESEARCH Adzick, N. S., Longaker, M. T. 1991; 51 (3): 216-222

  Abstract

  Recent experimental and clinical evidence suggests that the fetus responds to injury in a fashion fundamentally different from that of the adult. Acute inflammation is almost always absent, hyaluronic acid is a prominent component of the wound matrix, and collagen is deposited in a scarless manner. Using a variety of animal models and techniques, numerous investigators have begun to analyze the constituents of the fetal wound healing process in an attempt to understand the control mechanisms that endow the fetus with unique healing abilities. Since scarring and fibrosis dominate some diseases in almost every medical specialty, the ultimate clinical aim is to delineate the biological principles of fetal wound healing and then apply them to modulate adult wound healing problems.

  View details for Web of Science ID A1991GD58000007

  View details for PubMedID 1908924

• MIDGESTATIONAL EXCISIONAL FETAL LAMB WOUNDS CONTRACT INUTERO JOURNAL OF PEDIATRIC SURGERY Longaker, M. T., Burd, D. A., GOWN, A. M., YEN, T. S., Jennings, R. W., Duncan, B. W., Harrison, M. R., Adzick, N. S. 1991; 26 (8): 942-948

  Abstract

  Clinical observations and experimental data suggest that fetal wound healing is very different from adult wound healing. An understanding of the biology of scarless fetal wound healing has tremendous clinical potential for modulating postnatal wound problems. In this study, the fetal lamb model was used to assess excisional fetal skin wound contraction in utero. Full-thickness 9-mm punch biopsy wounds were created on fetal lambs at 100 days' gestation (term, 145 days). Half of the wounds remained exposed to amniotic fluid, whereas the other half were covered by a silastic patch to exclude amniotic fluid. Wounds were harvested 3, 7, or 14 days later and wound areas were calculated. Exposure to amniotic fluid retarded wound contraction significantly at 3 days, but by 14 days all wounds had completely contracted and reepithelialized. Myofibroblasts are an important cellular element of wound contraction. The presence of wound myofibroblasts was documented by both transmission electronmicroscopy and immunocytochemistry with antimuscle actin antibody. It is concluded that fetal lamb wounds contract in utero and exposure to amniotic fluid appears to retard fetal skin wound contraction only during the early healing process.

  View details for Web of Science ID A1991GA10200012

  View details for PubMedID 1919988

• ONTOGENY OF FETAL SHEEP POLYMORPHONUCLEAR LEUKOCYTE PHAGOCYTOSIS JOURNAL OF PEDIATRIC SURGERY Jennings, R. W., Adzick, N. S., Longaker, M. T., Duncan, B. W., Scheuenstuhl, H., Hunt, T. K. 1991; 26 (7): 853-855

  Abstract

  Premature infants and neonates are vulnerable to bacterial sepsis. This susceptibility may be due to the relative immaturity of their immune systems. To determine if neonates and, in particular, premature infants have decreased polymorphonuclear leukocyte (PMN) phagocytosis, we tested PMN phagocytosis of Staphylococcus aureus as a function of gestational age in the fetal lamb model. Because phagocytosis is made more efficient by the presence of opsonins in plasma, fetal and postnatal PMN phagocytosis were also measured after exposure to fetal and adult plasma. PMNs were isolated from fetal lambs at 104, 114, 124, and 141 days' gestation (term gestation for the fetal lamb is 145 days), as well as from 10-day-old neonatal sheep and adult sheep. Labeled S aureus were opsonized by incubation in either fetal or adult plasma, or left unopsonized for baseline values. Phagocytosis was measured as a percent of adult PMN phagocytosis after adult plasma opsonization. It was found that fetal PMN function is limited by two factors during the early third trimester: a primary defect in the ability of the PMN to phagocytose S aureus despite adequate opsonization, and the diminished ability of autologous fetal plasma to opsonize bacteria. The defect in PMN phagocytosis disappears late in the third trimester, but the inability of the fetal plasma to opsonize effectively continues until after birth.

  View details for Web of Science ID A1991FU99300025

  View details for PubMedID 1895198

• INUTERO ARTERIAL EMBOLISM FROM RENAL-VEIN THROMBOSIS WITH SUCCESSFUL POSTNATAL THROMBOLYTIC THERAPY JOURNAL OF PEDIATRIC SURGERY Duncan, B. W., Adzick, N. S., Longaker, M. T., Edwards, J. R., Nelson, R. M., Koerper, M. A. 1991; 26 (6): 741-743

  Abstract

  Thromboembolic events in the pediatric age group occur most commonly in neonates, and newborns of diabetic mothers are particularly at risk. We report a newborn with right renal vein and inferior vena cava thrombosis who apparently embolized across the foramen ovale antenatally with resultant right brachial artery occlusion. The baby was delivered by cesarean section from an insulin-dependent diabetic mother. At the time of birth, there was severe right arm ischemia with absent brachial and radial pulses. There was clinical evidence of distal embolization with a "trash" lesion of the distal right middle finger as well as a midforearm area of full-thickness skin loss. Ultrasound demonstrated a right renal vein thrombosis and a 95% occlusion of the inferior vena cava. Regional urokinase therapy was instituted through a lower extremity vein with a 5,000 U/kg bolus and then 5,000 U/kg/h continuous infusion. Twelve hours of infusion of urokinase led to clinical resolution of the right arm ischemia, with return of pulses. Follow-up ultrasound showed the right renal vein thrombosis and inferior vena cava clot to be completely resolved. The right middle finger and forearm lesions subsequently have healed primarily. We report this as a case of in utero arterial embolization with successful postnatal therapy using regional urokinase infusion.

  View details for Web of Science ID A1991FP68800024

  View details for PubMedID 1941470

• INTRAOPERATIVE RADIATION-THERAPY FOR WILMS-TUMOR INSITU OR EXVIVO CANCER Halberg, F. E., Harrison, M. R., Salvatierra, O., Longaker, M. T., Wara, W. M., Phillips, T. L. 1991; 67 (11): 2839-2843

  Abstract

  Patients with bilateral Wilms' tumor who have local recurrence after undergoing maximum-dose multitechnique therapy are problematic. The role of surgery in the management of these patients is changing from ablation to preservation of renal tissue. Bilateral nephrectomy is reserved as a last resort. Intraoperative radiation therapy (IORT) is advantageous to conservative management, as illustrated by the current cases of two children with recurrent Wilms' tumors in their remaining kidneys. Both children underwent limited surgery and precisely directed IORT. In one patient the nephron-sparing surgery and IORT were performed in situ. In the other the kidney was removed, treated with ex vivo bench surgery and radiation surgery, and then reimplanted. The adjuvant use of IORT, either in situ or ex vivo, in nephron-sparing surgery permits complete obliteration of gross and microscopic disease while maximizing residual renal function.

  View details for Web of Science ID A1991FM14000021

  View details for PubMedID 1851049

• FETAL CLEFT-LIP REPAIR IN RABBITS - POSTNATAL FACIAL GROWTH AFTER REPAIR JOURNAL OF ORAL AND MAXILLOFACIAL SURGERY Dodson, T. B., Schmidt, B., Longaker, M. T., Kaban, L. B. 1991; 49 (6): 603-611

  Abstract

  We have previously described a model for in utero cleft lip repair in rabbits. Cleft lip and alveolus (CL) were created in fetal rabbits at 24 days gestation (term, 31 days). In this study, postnatal maxillary growth was evaluated in three groups of animals: 1) unoperated controls, 2) unrepaired CL, and 2) repaired CL. The animals were killed at 4, 12, and 26 weeks after birth. Direct cephalometry was performed on dry skulls to evaluate premaxillary width, anterior maxillary length and width, and posterior maxillary width. The results of this study indicate that rabbits that undergo an in utero CL procedure, with or without repair, exhibit no significant decrease in maxillary length and width when compared with controls.

  View details for Web of Science ID A1991FN44400009

  View details for PubMedID 2037916

• INVITRO FETAL WOUND CONTRACTION - THE EFFECT OF AMNIOTIC-FLUID BRITISH JOURNAL OF PLASTIC SURGERY Burd, D. A., Longaker, M. T., Rittenberg, T., Adzick, N. S., Harrison, M. R., Ehrlich, H. P. 1991; 44 (4): 302-305

  Abstract

  This study looks at the remodelling of an in vitro system of foetal wound healing. Contraction is confirmed and the stimulatory role of amniotic fluid demonstrated.

  View details for Web of Science ID A1991FN31500014

  View details for PubMedID 2059789

• STUDIES IN FETAL WOUND-HEALING .5. A PROLONGED PRESENCE OF HYALURONIC-ACID CHARACTERIZES FETAL WOUND FLUID ANNALS OF SURGERY Longaker, M. T., Chiu, E. S., Adzick, N. S., Stern, M., Harrison, M. R., Stern, R. 1991; 213 (4): 292-296

  Abstract

  Midgestation fetal wound healing is characterized by healing without fibrosis or scar formation. The mechanisms that underlie this remarkable process are mediated in part through a fetal wound extracellular matrix rich in hyaluronic acid. In this study a newly developed assay was used to determine the hyaluronic acid levels in fetal and adult wound fluid. Adult wound fluid had a rapid increase in hyaluronic acid, which peaked at 3 days and decreased to 0 by 7 days. In contrast levels of hyaluronic acid in fetal wound fluid increased rapidly and remained significantly elevated for 3 weeks. This prolonged presence of hyaluronic acid in the matrix of fetal wounds creates a 'permissive' wound environment that promotes fetal fibroblast movement and proliferation and inhibits cytodifferentiation. Such a matrix environment promotes healing by regeneration rather than by scarring. This observation has therapeutic implications. The prolonged application of hyaluronic acid or hyaluronate protein complexes to wounds in children or adults may modulate healing in a manner that makes the wounds more fetal-like.

  View details for Web of Science ID A1991FG13900003

  View details for PubMedID 2009010

• FETAL DIAPHRAGMATIC WOUNDS HEAL WITH SCAR FORMATION JOURNAL OF SURGICAL RESEARCH Longaker, M. T., Whitby, D. J., Jennings, R. W., Duncan, B. W., Ferguson, M. W., Harrison, M. R., Adzick, N. S. 1991; 50 (4): 375-385

  Abstract

  Fetal wound healing is fundamentally different from wound healing in the adult. Although experimental work in mice, rats, rabbits, monkeys, and sheep has demonstrated that fetal healing occurs without inflammation and scarring, all of these studies have been limited to fetal skin wounds. Whether all fetal tissues heal in a regenerative-like fashion is unknown. Amniotic fluid exposure may play an important role in scarless fetal skin wound healing, but the effect of amniotic fluid on fetal mesothelial wound healing has not been characterized. To investigate these questions we created bilateral linear diaphragmatic wounds in 100-day gestation fetal lambs (term = 145 days). The right thoracotomy was closed to exclude amniotic fluid. In contrast, the left thoracotomy was fashioned into an Eloesser flap which permitted the left diaphragmatic wound to be continually bathed in amniotic fluid. Wounds were harvested after 1, 2, 7, or 14 days and analyzed by light microscopy and immunohistochemistry with antibodies to collagen types I, III, IV, and VI. Whether bathed in or excluded from amniotic fluid, the mesothelial-lined diaphragm healed with scar formation and without evidence of muscle regeneration. Interestingly, diaphragmatic wounds exposed to amniotic fluid were covered by a thick fibrous collagen peel similar to that seen in gastroschisis bowel. These findings indicate that not all fetal tissues share the unique scarless healing properties of fetal skin.

  View details for Web of Science ID A1991FK30700013

  View details for PubMedID 2020189

• THE BIOLOGY OF FETAL WOUND-HEALING - A REVIEW PLASTIC AND RECONSTRUCTIVE SURGERY Longaker, M. T., Adzick, N. S. 1991; 87 (4): 788-798

  View details for Web of Science ID A1991FE05900032

  View details for PubMedID 2008482

• MATERNAL OUTCOME AFTER OPEN FETAL SURGERY - A REVIEW OF THE 1ST 17 HUMAN CASES JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION Longaker, M. T., Golbus, M. S., Filly, R. A., Rosen, M. A., Chang, S. W., Harrison, M. R. 1991; 265 (6): 737-741

  Abstract

  A few fetal diseases may benefit from surgical treatment before birth, but hysterotomy and subsequent delivery by cesarean section pose a risk to the otherwise unaffected mother. To assess maternal risk of mortality, morbidity, and reproductive potential after fetal surgery, we reviewed our experience with 17 highly selected women who underwent fetal surgery. Fifteen of these procedures were performed for one of two congenital anomalies: severe bilateral hydronephrosis and congenital diaphragmatic hernia. There were no deaths or serious maternal injuries. In the 14 women who continued pregnancy after hysterotomy, uterine irritability and preterm labor were frequent complications, requiring early confinement in most cases. There has been no detectable effect on future fertility, as indicated by eight subsequent normal pregnancies. We conclude that hysterotomy for fetal surgery can be accomplished without unduly endangering the mother's life or her future reproductive potential. However, morbidity related to premature labor remains a serious problem, and our ability to control uterine contractions after hysterotomy remains the limiting factor in human fetal surgery.

  View details for Web of Science ID A1991EW47900025

  View details for PubMedID 1990189

• IMMUNOLOGICAL EVALUATION OF HEMATOPOIETIC CHIMERIC RHESUS-MONKEYS 13TH INTERNATIONAL CONGRESS OF THE TRANSPLANTATION SOC Duncan, B. W., Harrison, M. R., Zanjani, E. D., TARANTAL, A. F., Adzick, N. S., Bradley, S. M., Longaker, M. T., Jennings, R. W., Roberts, L. J., Bigler, M. E., Roncarolo, M. G. ELSEVIER SCIENCE INC. 1991: 841–43

  View details for Web of Science ID A1991EV39000338

  View details for PubMedID 1671308

• SCARLESS WOUND-HEALING IN THE FETUS - THE ROLE OF THE EXTRACELLULAR-MATRIX 3RD INTERNATIONAL CONF ON TISSUE REPAIR Adzick, N. S., Longaker, M. T. WILEY-LISS, INC. 1991: 177–192

  View details for Web of Science ID A1991BT24A00015

  View details for PubMedID 1862130

• Update on the status of fetal surgery. Surgery annual Longaker, M. T., Adzick, N. S., Harrison, M. R. 1991; 23: 53-68

  View details for PubMedID 1858008

• HYALURONIC-ACID IN A CARDIAC MYXOMA - A BIOCHEMICAL AND HISTOLOGICAL ANALYSIS VIRCHOWS ARCHIV A-PATHOLOGICAL ANATOMY AND HISTOPATHOLOGY Longaker, M. T., Chiu, E. S., Hendin, B., Finkbeiner, W. E., Stern, R. 1991; 418 (5): 435-437

  Abstract

  Cardiac myxoma is the most common primary tumor of the heart. This tumor has a gelatinous stroma that is thought to be composed of glycosaminoglycans, the classical acid mucopolysaccharide ground substance. We examined both biochemically and histologically the hyaluronic acid in a case of cardiac myxoma using a newly developed hyaluronic acid-binding protein probe. We observed that hyaluronic acid was localized in the amorphous stroma and occurred at levels 30 times that found in normal atrial septum.

  View details for Web of Science ID A1991FN37900009

  View details for PubMedID 2035257

• ETIOLOGY OF INTESTINAL DAMAGE IN GASTROSCHISIS .2. TIMING AND REVERSIBILITY OF HISTOLOGICAL-CHANGES, MUCOSAL FUNCTION, AND CONTRACTILITY 21ST ANNUAL MEETING OF THE CANADIAN ASSOC OF PAEDIATRIC SURGEONS Langer, J. C., Bell, J. G., Castillo, R. O., Crombleholme, T. M., Longaker, M. T., Duncan, B. W., Bradley, S. M., Finkbeiner, W. E., Verrier, E. D., Harrison, M. R. W B SAUNDERS CO. 1990: 1122–26

  Abstract

  Previous work in the fetal lamb examined the relative effects of amniotic fluid and bowel constriction in the etiology of bowel damage in gastroschisis. The present study used the same model to assess the timing and reversibility of these changes during gestation. Gastroschisis was created at 80 days' gestation, and a tape was placed around the bowel to cause gradual constriction with growth. Lambs were killed at 100 days, 120 days, and term. Bowel damage was assessed using histology, mucosal enzyme activity, and in vitro motility. In an additional "repaired" group, the constrictor was removed at 120 days, a silastic pouch placed over the bowel, and bowel damage assessed at term. Normal fetuses at each gestational age were used as controls. A fibrous peel was observed at all gestational ages. Mucosal villous atrophy and mesenteric venous and lymphatic dilation were mild at 100 and 120 days, but severe at term. These changes were present but mild in repaired animals at term. Mucosal enzyme activity decreased gradually with gestational age; inhibition of maltase activity was maximal at term, and was significantly reversed by repair, whereas inhibition of aminooligopeptidase activity was maximal at 120 days, and was not affected by repair. Protein/DNA, DNA/weight, and protein/weight ratios showed that repaired mucosal cells were significantly more proliferative, smaller, and less mature than control or gastroschisis cells. In vitro motility studies demonstrated a mild decrease in contractility at 100 and 120 days, and a large decrease at term. This deleterious effect at the end of gestation was only partially reversed by repair in utero.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1990EH58400005

  View details for PubMedID 2148773

• INFECTIVE ENDOCARDITIS AND AN EMBOLOMYCOTIC ANEURYSM IN A 25-MONTH-OLD CHILD JOURNAL OF CARDIOVASCULAR SURGERY Cave, C., Longaker, M. T., Merrick, S., Teitel, D. F., Goldstone, J., Verrier, E. D. 1990; 31 (6): 805-808

  Abstract

  Infective endocarditis in young children is uncommon, especially where there is no underlying structural heart disease. While septic embolization in adults occurs in up to 43% of the cases of endocarditis, there is little data on systemic embolization in cases of children. We present an unusual case of a 25-month-old child with infective endocarditis and an embolomycotic aneurysm treated by mitral valve replacement and aortoiliac reconstruction.

  View details for Web of Science ID A1990EP22500025

  View details for PubMedID 2262512

• TAKAYASUS-ARTERITIS (TYPE-III) IN AN 8-YEAR-OLD GIRL - A MULTIMODALITY APPROACH TO STAGED REVASCULARIZATION OF THE HEART, HEAD, AND ABDOMINAL VISCERA - A CASE-REPORT VASCULAR SURGERY Longaker, M. T., Hanley, F. L., Goldstone, J., Teitel, D. F., Finkbeiner, W. E., Verrier, E. D. 1990; 24 (8): 616-622

  View details for Web of Science ID A1990EF32000014

• FETAL WOUND-HEALING IN A LARGE ANIMAL-MODEL - THE DEPOSITION OF COLLAGEN IS CONFIRMED BRITISH JOURNAL OF PLASTIC SURGERY Burd, D. A., Longaker, M. T., Adzick, N. S., Harrison, M. R., Ehrlich, H. P. 1990; 43 (5): 571-577

  Abstract

  Foetal wound healing occurs without scarring. A scar is a collagen-rich repair tissue, and the absence of scarring in the foetus has raised questions concerning the presence and nature of collagen deposition in foetal wounds. Studies of collagen deposition in foetal wounds in small animals, performed late in gestation, have been equivocal. In this study, using a large animal with a long gestational period, the sheep, the deposition of collagen is confirmed.

  View details for Web of Science ID A1990DZ64400010

  View details for PubMedID 2224353

• CAROTID-ARTERY RECONSTRUCTION FOLLOWING EXTRACORPOREAL MEMBRANE-OXYGENATION AMERICAN JOURNAL OF DISEASES OF CHILDREN Crombleholme, T. M., Adzick, N. S., DeLorimier, A. A., Longaker, M. T., Harrison, M. R., CHARLTON, V. E. 1990; 144 (8): 872-874

  Abstract

  Right hemispheric brain injury has been noted in surviving infants treated with venoarterial extracorporeal membrane oxygenation (ECMO). This phenomenon may be secondary to permanent ligation of the right carotid artery. At our institution, conventional ventilatory therapy failed in five neonates with respiratory insufficiency, and they were treated successfully with ECMO. In four of the five neonates, the right carotid artery was reconstructed at the time of decannulation. At discharge, all newborns with carotid artery repair showed no signs of unilateral brain injury and had excellent antegrade flow in the right carotid artery as assessed by both duplex and transcranial Doppler ultrasound scanning. Carotid artery reconstruction after ECMO is a technically simple procedure that may reduce the incidence of right hemispheric brain injury and long-term consequences of marginal cerebral perfusion.

  View details for Web of Science ID A1990DT47400021

  View details for PubMedID 2198805

• FETAL WOUND-HEALING - AN INVITRO EXPLANT MODEL JOURNAL OF PEDIATRIC SURGERY Burd, D. A., Longaker, M. T., Adzick, N. S., COMPTON, C. C., Harrison, M. R., Siebert, J. W., Ehrlich, H. P. 1990; 25 (8): 898-901

  Abstract

  The ability of fetal skin wounds to heal without scar formation is remarkable. The mechanisms that endow the fetus with this unique healing ability remain unknown. We have developed an in vitro explant model using fetal sheep skin to investigate fetal wound healing. This model eliminates the complex systemic mechanisms that modulate in vivo wound healing. We demonstrated that using an enriched medium, midgestation fetal sheep skin explants following wounding reepithelialized within 4 days. By 7 days after wounding the confluent epidermis was thicker, but the dermal wound remained open. This model demonstrates that it is possible to achieve conditions in culture that maintain tissue viability and support reepithelialization. This model may allow us to resolve some of the individual components that participate in the process of scarless fetal skin healing.

  View details for Web of Science ID A1990DT71100014

  View details for PubMedID 2401945

• MEDIASTINAL PANCREATIC PSEUDOCYSTS IN CHILDREN JOURNAL OF PEDIATRIC SURGERY Crombleholme, T. M., DeLorimier, A. A., Adzick, N. S., Longaker, M. T., Harrison, M. R., Cox, K. L., Heyman, M. B. 1990; 25 (8): 843-845

  Abstract

  Mediastinal pseudocyst is an unusual complication of pancreatitis, with only four cases previously reported in children. The extent of the pseudocyst can be defined by computed tomography or magnetic resonance imaging scan and preoperative aspiration of cyst fluid for amylase level can establish the diagnosis. Endoscopic retrograde cholangiopancreatography to define ductal anatomy can help plan the appropriate drainage procedure. Although exceedingly rare, the diagnosis of pseudocyst should be considered for any cystic mass in the abdomen or thorax, even in the absence of elevated amylase or history suggesting pancreatitis.

  View details for Web of Science ID A1990DT71100003

  View details for PubMedID 1698218

• PULMONARY VASCULAR-RESISTANCE IN NEONATAL SWINE - RESPONSE TO RIGHT PULMONARY-ARTERY OCCLUSION, ISOPROTERENOL, AND PROSTAGLANDIN-E1 JOURNAL OF PEDIATRIC SURGERY Crombleholme, T. M., Adzick, N. S., Longaker, M. T., Bradley, S. M., Duncan, B. W., Jennings, R., Verrier, E. D., Harrison, M. R. 1990; 25 (8): 861-866

  Abstract

  The pulmonary physiological response of adults to unilateral pulmonary artery (PA) occlusion has been well-characterized as resulting in a decrease in the pulmonary vascular resistance (PVR), in order to maintain the same PA pressure and accommodate the entire cardiac output (CO). We evaluated the response of the neonate to unilateral PA occlusion and how this response is altered by infusions of Isoproterenol (Isuprel) and prostaglandin E1 (PGE1) in the neonatal swine model. Twenty farm piglets (five at 1 day, three at 5 days, seven at 14 days, and five at 60 days as controls) underwent left lateral thoracotomy and measurement of PA and left atrial (LA) pressures, CO, and PVR with the right PA open and occluded. To determine if neonatal PVR could be influenced by a vasodilator (indicating the vascular capacity is not fixed) or by an inotrope (indicating the lung is not maximally recruited) this experiment was then repeated with infusions of PGE1 (a vasodilator) at doses of 0.1, 0.5, and 1.0 micrograms/kg/min and subsequently with Isuprel (an inotrope and vasodilator) at doses of 0.1, 0.5, 1.0 micrograms/kg/min. Control measurements taken without unilateral PA occlusion showed that PVR is high at 1 day of age but progressively decreases to a level 89% lower by 60 days of age. The vascular capacity of the neonatal lung is fixed and responds to unilateral PA occlusion with a dramatic increase in PVR. This response cannot be altered by either a vasodilator (PGE1) or an inotrope (Isuprel) thereby limiting the utility of these drugs in treating neonatal pulmonary hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1990DT71100007

  View details for PubMedID 2401941

• A RABBIT MODEL FOR FETAL CLEFT-LIP REPAIR JOURNAL OF ORAL AND MAXILLOFACIAL SURGERY Longaker, M. T., Dodson, T. B., Kaban, L. B. 1990; 48 (7): 714-719

  Abstract

  Recent clinical and experimental data indicate that fetal wound healing occurs without the accompanying inflammation and scar formation typical of postnatal wounds. The lack of scar tissue may have significant implications for craniomaxillofacial surgery. This report documents the development of a model for fetal cleft lip repair in rabbits. The survival rate for the first 47 fetuses was 76.6%.

  View details for Web of Science ID A1990DL73600010

  View details for PubMedID 2358948

• FETAL TRAUMA - RELATION TO MATERNAL INJURY JOURNAL OF PEDIATRIC SURGERY Farmer, D. L., Adzick, N. S., Crombleholme, W. R., Crombleholme, T. M., Longaker, M. T., Harrison, M. R. 1990; 25 (7): 711-714

  Abstract

  Conventional surgical wisdom is that fetal death is a predictable consequence of severe maternal injury. In order to define the natural history of maternal-fetal trauma and better formulate management strategies, we reviewed our recent experience with 32 cases of maternal trauma at a major trauma center. There were three cases of fetal death; two were associated with severe maternal injury, but one had no significant injury to the mother. All cases had placental injury. Conversely, there were five cases of severe maternal trauma but only two unsuccessful pregnancy outcomes. We conclude that the extent of maternal injury does not necessarily correlate with the degree of fetal injury. Lethal placental or direct fetal injury can occur even in the absence of significant maternal injury. In selected cases, fetal salvage after maternal-fetal trauma may be achieved by early delivery and prompt pediatric surgical intervention.

  View details for Web of Science ID A1990DN58300002

  View details for PubMedID 2380886

• TESTING THE LIMITS OF NEONATAL TRACHEAL RESECTION JOURNAL OF PEDIATRIC SURGERY Longaker, M. T., Harrison, M. R., Adzick, N. S. 1990; 25 (7): 790-792

  Abstract

  Obstructive lesions of the trachea in infants can be life-threatening because secretions or mucosal inflammation can easily obstruct the tiny compromised airway, and distal obstruction cannot be relieved by an endotracheal or tracheostomy tube. The principles of tracheal resection in adults are well established. The limits of tracheal resection in neonates are unknown. We present a case in which at least two-thirds of the trachea was resected successfully in a neonate.

  View details for Web of Science ID A1990DN58300021

  View details for PubMedID 2380898

• THE MODIFIED PUESTOW PROCEDURE FOR CHRONIC RELAPSING PANCREATITIS IN CHILDREN 22ND ANNUAL MEETING OF THE PACIFIC ASSOC OF PEDIATRIC SURGEONS Crombleholme, T. M., DeLorimier, A. A., Way, L. W., Adzick, N. S., Longaker, M. T., Harrison, M. R. W B SAUNDERS CO. 1990: 749–54

  Abstract

  Chronic relapsing pancreatitis in children is an unusual condition that often goes undiagnosed and untreated for years. In light of recent reports in adults that endocrine and exocrine function may be preserved by early pancreaticojejunostomy, we reviewed our experience with this procedure (one Duval, 10 Puestows) in 10 children between 1969 and 1989. The underlying etiology was familial pancreatitis in four patients, one case of unknown etiology, congenital ductal anomalies in four (one pancreas divisum, one annular pancreas, one choledochal cyst, and one ductal stenosis), and posttraumatic in one. All 10 had intractable recurrent abdominal pain. Preoperatively, only three patients evidenced exocrine insufficiency and none had endocrine insufficiency. There was complete resolution of pain in eight patients and improvement in two during a mean observation period of 4 years (range, 7 months to 19.75 years). Exocrine insufficiency resolved in two patients but has persisted in the third patient now on Viokase. Endocrine insufficiency has developed during follow-up in one patient. Pancreaticojejunostomy provides excellent relief of recurrent pain in chronic relapsing pancreatitis in children. Endoscopic retrograde cholangiopancreatography (ERCP) is indicated when the diagnosis of chronic relapsing pancreatitis is suspected to define the ductal anatomy. Pancreaticojejunostomy may prevent the progression of exocrine and endocrine insufficiency if performed early in the course of the disease.

  View details for Web of Science ID A1990DN58300011

  View details for PubMedID 2380891

• COMPLETE HEART-BLOCK IN FETAL LAMBS .1. TECHNIQUE AND ACUTE PHYSIOLOGICAL-RESPONSE JOURNAL OF PEDIATRIC SURGERY Crombleholme, T. M., Harrison, M. R., Longaker, M. T., Langer, J. C., Adzick, N. S., Bradley, S., Duncan, B., Verrier, E. D. 1990; 25 (6): 587-593

  Abstract

  Prenatally diagnosed complete heart block (CHB), without associated cardiac anomalies, is often refractory to attempts to increase heart rate and cardiac output by medical therapy, and results in fetal death. We developed a model of CHB in fetal lambs in order to evaluate the acute physiological consequences of CHB and to assess the feasibility of fetal cardiac ventricular (V) and atrioventricular (AV) pacing. After hysterotomy and fetal sternotomy AV-sequential pacing leads were placed on the right atrium and ventricle (RV). Electromagnetic flow transducers were placed around the pulmonary artery and the ascending aorta to measure combined ventricular output (CVO) in normal sinus rhythm (NSR), CHB, AV, and V pacing. CHB block was successfully induced in 19 of 21 (90%) fetal lambs at 100 to 110 days' gestation by injection of formalin into the interventricular septum. Two fetuses were refractory to CHB despite multiple injections. The mean ventricular escape rate was 52 +/- 3 (+/- SEM) beats/min (range, 45 to 60 beats/min). CHB lasted for the period of study (1 to 8 hours), and spontaneously reverted to NSR in one fetus after 3 hours. Combined ventricular output in NSR was 470 +/- 31 mL/min (+/- SEM), which fell to 213 +/- 13 mL/min in CHB, 45% of normal CVO (P less than .0001). CVO during AV-sequential and V pacing was 77% (P = .02) and 62% (P less than .001), respectively, of combined ventricular output in NSR. Both RV (27%) and left ventricles (LV) (37%) showed compensatory increase in stroke volume during CHB.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1990DH10500001

  View details for PubMedID 2358988

• FETAL INTERVENTION IN OBSTRUCTIVE UROPATHY - PROGNOSTIC INDICATORS AND EFFICACY OF INTERVENTION AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY Crombleholme, T. M., Harrison, M. R., Golbus, M. S., Longaker, M. T., Langer, J. C., Callen, P. W., Anderson, R. L., Goldstein, R. B., Filly, R. A. 1990; 162 (5): 1239-1244

  Abstract

  Management of the fetus with bilateral hydronephrosis is controversial; ability to predict outcome and efficacy of prenatal intervention are unknown. We studied 40 fetuses referred for ultrasonography, examination of fetal urine, and possible therapy. We retrospectively assigned fetuses to a good prognosis group if fetal urine was hypotonic (sodium less than 100 mEq/L, chloride less than 90 mEq/L, osmolarity less than 210 mOsm/L) and there was no ultrasonographic evidence of dysplasia; we assigned fetuses to a poor prognosis group if even one criterion was abnormal. Survival was greater in the good prognosis group than in the poor prognosis group (81% vs 12.5%; 87% vs 30%, excluding abortions) (p less than 0.005). We then attempted to assess the efficacy of prenatal urinary decompression by comparing outcome within the good and poor prognosis groups. Survival with intervention was greater in both the good prognosis group and the poor prognosis group (89% vs 70% and 30% vs 0%). In 6 of the 8 survivors in the good prognosis group, severe oligohydramnios was reversed by decompression. We conclude the fetal urine electrolyte levels and ultrasonographic appear helpful in predicting residual fetal renal function and neonatal outcome and that prenatal decompression may prevent the development of fatal pulmonary hypoplasia.

  View details for Web of Science ID A1990DD89300026

  View details for PubMedID 2187354

• STUDIES IN FETAL WOUND-HEALING, .7. FETAL WOUND-HEALING MAY BE MODULATED BY HYALURONIC-ACID STIMULATING ACTIVITY IN AMNIOTIC-FLUID JOURNAL OF PEDIATRIC SURGERY Longaker, M. T., Adzick, N. S., Hall, J. L., STAIR, S. E., Crombleholme, T. M., Duncan, B. W., Bradley, S. M., Harrison, M. R., Stern, R. 1990; 25 (4): 430-433

  Abstract

  Fetal wound healing occurs rapidly and without inflammation, fibrosis, or scar formation. It is a process fundamentally different from adult wound healing. The mechanisms that underlie such unique healing properties are unknown. However, hyaluronic acid, a glycosaminoglycan component of the extracellular matrix, is prominent throughout the course of fetal wound healing, and is thought to play a major role in the healing process. Amniotic fluid contains high levels of hyaluronic acid. Amniotic fluid also contains a number of potent growth factors that are critical for fetal development. In this report, a new factor in amniotic fluid that stimulates deposition of hyaluronic acid is described. This activity is measured in an in vitro assay system in which cultured fibrosarcoma cells are used as indicator cells. Amniotic fluid thus provides two separate mechanisms for the deposition of hyaluronic acid. One is by exogenous application directly onto fetal skin wounds; the second is by providing a factor to increase the production of hyaluronic acid endogenously, by stimulating cells around the wound site. The resulting hyaluronic acid-rich area may support the ability of the fetal wound to heal with its unique properties.

  View details for Web of Science ID A1990CX79100014

  View details for PubMedID 2329459

• NEPHRON-SPARING APPROACH TO BILATERAL WILMS-TUMOR - INSITU OR EXVIVO SURGERY AND RADIATION-THERAPY JOURNAL OF PEDIATRIC SURGERY Longaker, M. T., Harrison, M. R., Adzick, N. S., Crombleholme, T. M., Langer, J. C., Duncan, B. W., Halberg, F. E., Salvatierra, O. 1990; 25 (4): 411-414

  Abstract

  Bilateral Wilms' tumor patients, who experience local recurrence after maximal multimodality therapy, present a difficult surgical problem. The role of surgery in the management of these patients has changed from ablation to preservation of renal tissue, with bilateral nephrectomy as a last resort. Two children who had recurrent tumors in the remaining kidney underwent nephron-sparing surgery and focal intraoperative radiation therapy. In one case, this nephron-sparing surgery and intraoperative radiation therapy were performed in situ, in the other kidney was removed and reimplanted (ex vivo bench surgery and irradiation). These techniques may allow complete obliteration of gross and microscopic disease, while maximizing residual renal function.

  View details for Web of Science ID A1990CX79100009

  View details for PubMedID 2158540

• HYALURONIC ACID-STIMULATING ACTIVITY IN THE PATHOPHYSIOLOGY OF WILMS-TUMORS JOURNAL OF THE NATIONAL CANCER INSTITUTE Longaker, M. T., Adzick, N. S., SADIGH, D., Hendin, B., STAIR, S. E., Duncan, B. W., Harrison, M. R., SPENDLOVE, R., Stern, R. 1990; 82 (2): 135-139

  Abstract

  Markedly elevated levels of hyaluronic acid occur in the serum and urine of some patients with Wilms' tumor. We have recently described a glycoprotein factor in fetal serum that stimulates deposition of hyaluronic acid. In a survey of bovine fetal tissue extracts, we have identified the fetal kidney as the source of this circulating activity. Wilms' tumors arise from transformed "rests" of fetal kidney. We demonstrate here that such tumors continue to produce this fetal factor and that the hyaluronic acid-stimulating activity is found in the urine of children with Wilms' tumors. In the three patients with Wilms' tumor who were followed, elevated levels of hyaluronic acid-stimulating activity were found in their urine before treatment. By 2 months after surgical removal of their tumors, these levels had returned to baseline. We propose that hyaluronic acid-stimulating activity is the mechanism for the elevated levels of hyaluronic acid in the sera and urine of these patients. The activity is an oncofetal protein and the first for which a function has been identified. It also is a marker for this common childhood solid tumor and has the potential for identifying children at increased risk.

  View details for Web of Science ID A1990CH50000010

  View details for PubMedID 2152946

• REDUCED-SIZE LUNG TRANSPLANTATION IN NEONATAL SWINE - TECHNIQUE AND SHORT-TERM PHYSIOLOGICAL-RESPONSE 25TH ANNIVERSARY MEETING OF THE SOC OF THORACIC SURGEONS Crombleholme, T. M., Adzick, N. S., Longaker, M. T., Bradley, S., Duncan, B. W., Verrier, E. D., Harrison, M. R. ELSEVIER SCIENCE INC. 1990: 55–60

  Abstract

  Lung transplantation is now a clinical reality in adults but is limited by the scarcity of appropriate donors. The donor shortage is even more acute for neonatal and pediatric patients. Reduced-size lung grafts would expand the pool of appropriate cadaveric donors and allow HLA-matched living related lobar or segmental lung transplants. To evaluate this experimentally, we developed a model of pulmonary lobar transplantation in neonatal pigs and studied the acute hemodynamic response after transplanting the left lower lobe from a more mature donor to a neonatal recipient. Technical considerations included using the recipient atrial appendage for the pulmonary venous anastomosis. Nine pairs of pigs underwent left lateral thoracotomy. The recipient left atrial and pulmonary arterial pressures, cardiac output, and pulmonary vascular resistance were measured before pneumonectomy and after left lower lobe transplantation. Although the left atrial and pulmonary arterial pressures remained unchanged after transplantation, there was a 15% increase in pulmonary vascular resistance and a 23% reduction in cardiac output. Neither change was statistically significant. The distribution of blood flow through the left and right pulmonary arteries was unchanged after transplantation. We conclude that lobar transplantation is technically feasible in immature animals and that the pulmonary venous anastomosis to the left atrial appendage facilitates the procedure. This model may prove useful in studying lung transplantation in immature recipients and expedite implementation of reduced-size lung transplantation in neonatal and pediatric patients.

  View details for Web of Science ID A1990CJ81000010

  View details for PubMedID 2297276

• PULMONARY LOBAR TRANSPLANTATION IN NEONATAL SWINE - A MODEL FOR TREATMENT OF CONGENITAL DIAPHRAGMATIC-HERNIA JOURNAL OF PEDIATRIC SURGERY Crombleholme, T. M., Adzick, N. S., Hardy, K., Longaker, M. T., Bradley, S. M., Duncan, B. W., Verrier, E. D., Harrison, M. R. 1990; 25 (1): 11-18

  Abstract

  Congenital diaphragmatic hernia (CDH) babies born with severe pulmonary hypoplasia are unsalvageable despite maximal therapy including extracorporeal membrane oxygenation (ECMO). Lung transplantation is a potential treatment for these otherwise doomed infants using ECMO as a bridge to transplantation. Cadaveric, or living related donation of a more mature reduced size lung (pulmonary lobe or segment) may help solve the critical donor shortage problem. We evaluated the physiological response of mature left lower lobe (LLL) transplants in neonatal swine with the hemodynamic conditions of CDH simulated by occlusion of the right pulmonary artery (PA), and also studied the pulmonary function of the mature lobar graft compared with the neonatal lung. LLL transplantation was well tolerated and resulted in minimal alteration in hemodynamic parameters. The response to right PA occlusion was similar pre- and posttransplantation with a fall in cardiac output and a significant rise in pulmonary vascular resistance. Compared with the contralateral native lung, the lobar graft was preferentially ventilated with resultant higher pH (7.65 +/- 0.17 v 7.41 +/- 0.08, P less than .01) and lower pCO2 (17 +/- 6 v 36 +/- 5, P less than .001). The more mature lobar graft was preferentially ventilated due to the increased compliance compared with the neonatal right lung (8.16 +/- 1.28 v 5.48 +/- 0.82 mL/cm, P less than .0001). Reduced size lung transplantation is technically feasible and may help solve the donor problem for severe CDH neonates for whom no effective therapy is currently available.

  View details for Web of Science ID A1990CH03800003

  View details for PubMedID 2299534

• STUDIES IN FETAL WOUND-HEALING, .6. 2ND AND EARLY 3RD TRIMESTER FETAL WOUNDS DEMONSTRATE RAPID COLLAGEN DEPOSITION WITHOUT SCAR FORMATION JOURNAL OF PEDIATRIC SURGERY Longaker, M. T., Whitby, D. J., Adzick, N. S., Crombleholme, T. M., Langer, J. C., Duncan, B. W., Bradley, S. M., Stern, R., Ferguson, M. W., Harrison, M. R. 1990; 25 (1): 63-69

  Abstract

  The mechanisms that underlie the lack of scarring in fetal wounds are unknown, but probably relate to the control of collagen fibrillogenesis. The role of collagen in the fetal wound matrix is controversial, and several wound implant models have been used to evaluate collagen deposition in fetal wounds. Unfortunately, these models create an artificial wound environment and may thereby affect the results. In order to study fetal wound collagen deposition in linear wounds without artificially altering the wound environment, we applied a highly sensitive immunohistochemical technique that uses antibodies to collagen types I, III, IV, and VI. We found that collagen was deposited in fetal wounds much more rapidly than in adult wounds. Wound collagen deposition occurred in a normal dermal and mesenchymal pattern in second and early third trimester fetal lambs. These findings are consistent with the observation that the fetus heals rapidly and without scar formation. In contrast, wounds in late gestation fetal lambs showed some evidence of scar formation. Further studies may suggest ways to alter the adult wound so that it heals in a fetal manner.

  View details for Web of Science ID A1990CH03800012

  View details for PubMedID 2299547

• Hyaluronic acid deposition in cardiac myxomas: localization using a hyaluronate-specific binding protein. The American journal of cardiovascular pathology Hendin, B. N., Longaker, M. T., Finkbeiner, W. E., Roberts, L. J., Stern, R. 1990; 3 (3): 209-215

  Abstract

  Myxomas are the most common primary tumors of the heart. These lesions contain a gelatinous, ground-substancelike material which has been described as glycosaminoglycan in nature. Using a newly developed, cartilage-derived hyaluronic acid-binding protein and a modification of the avidin-biotin immunostaining procedure, we demonstrate that hyaluronic acid is contained in the jellylike material of cardiac myxomas.

  View details for PubMedID 1710472

• STUDIES IN FETAL WOUND-HEALING .4. HYALURONIC ACID-STIMULATING ACTIVITY DISTINGUISHES FETAL WOUND FLUID FROM ADULT WOUND FLUID ANNALS OF SURGERY Longaker, M. T., Chiu, E. S., Harrison, M. R., Crombleholme, T. M., Langer, J. C., Duncan, B. W., Adzick, N. S., Verrier, E. D., Stern, R. 1989; 210 (5): 667-672

  Abstract

  Recent clinical and experimental evidence suggests that the fetus responds to injury in a fashion fundamentally different from that of the adult. Our initial experience with human open fetal surgery reinforces experimental observations that the fetal wounds heal without the scarring, inflammation, and contraction that often accompany adult wounds. In this study we examine fetal wound fluid in an attempt to elucidate the control mechanisms that endow the fetus with unique healing properties. The extracellular matrix of fetal wounds is rich in hyaluronic acid, a glycosaminoglycan found in high concentrations whenever there is tissue proliferation, regeneration, and repair. We establish that wound fluid from the fetus contains high levels of hyaluronic acid-stimulating activity that may underlie the elevated deposition of hyaluronic acid in the fetal wound matrix. In contrast there was no hyaluronic acid-stimulating activity present in adult wound fluid. Hyaluronic acid, in turn, fosters an extracellular environment permissive for cell motility and proliferation that may account for the unique properties observed in fetal wound healing.

  View details for Web of Science ID A1989AX42200016

  View details for PubMedID 2818035

• ETIOLOGY OF INTESTINAL DAMAGE IN GASTROSCHISIS .1. EFFECTS OF AMNIOTIC-FLUID EXPOSURE AND BOWEL CONSTRICTION IN A FETAL LAMB MODEL JOURNAL OF PEDIATRIC SURGERY Langer, J. C., Longaker, M. T., Crombleholme, T. M., Bond, S. J., Finkbeiner, W. E., Rudolph, C. A., Verrier, E. D., Harrison, M. R. 1989; 24 (10): 992-997

  Abstract

  Gastroschisis is often complicated by damage to the herniated small bowel, resulting in motility and absorption disturbances and occasional intestinal necrosis and atresia. To study the pathophysiology of this process, a model of gastroschisis was developed in fetal lambs. At 80 days' gestation, the anterior abdominal wall was partially excised to create a small peritoneal cavity, and the small bowel was extruded through a Silastic ring to create a defect of uniform size. In one experimental group, a tie was placed around the herniated bowel at the level of the abdominal wall to provide gradual constriction as the fetus grew. In a second group, no tie was placed. Control animals had a simple laparotomy and no abdominal wall defect; some also had a constrictor placed around the base of the bowel. The animals were delivered near term, and the bowel was evaluated histologically and by an in-vitro bowel motility assay. Histologic examination showed normal ganglion cells in all groups and no evidence of ischemic injury. A fibrous peel was seen only in bowel exposed to amniotic fluid, with or without a constrictor. Lymphatic and venous dilation, smooth-muscle thickening, and focal mucosal blunting were seen in bowel subjected to chronic obstruction by a constrictor, regardless of whether it was exposed to amniotic fluid. Both constriction of the bowel and amniotic fluid exposure were associated with a decrease in motility; these two effects were independent and additive.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1989AV52600011

  View details for PubMedID 2530329

• STUDIES IN FETAL WOUND-HEALING .1. A FACTOR IN FETAL SERUM THAT STIMULATES DEPOSITION OF HYALURONIC-ACID JOURNAL OF PEDIATRIC SURGERY Longaker, M. T., Harrison, M. R., Crombleholme, T. M., Langer, J. C., Decker, M., Verrier, E. D., SPENDLOVE, R., Stern, R. 1989; 24 (8): 789-792

  Abstract

  Fetal wound healing without scar formations, fibrosis, or contracture might be accompanied by major differences in the wound extracellular matrix. The matrix of fetal wounds is rich in hyaluronic acid, a glycosaminoglycan found in high concentrations whenever there is tissue proliferation, regeneration, and repair. Although hyaluronic acid is a critical molecule for both embryonic development and wound healing, no factor has yet been identified that modulates hyaluronic acid in a consistent manner. We describe here a substance present in fetal sheep serum that stimulates hyaluronic acid synthesis by cultured fibroblasts. This glycoprotein factor appears to be ubiquitous, present in fetal sheep and bovine serum, reaching a peak in each at 40% of the way through gestation. This factor is also present in amniotic fluid. It might control hyaluronic acid deposition. In turn, hyaluronic acid, by creating an extracellular environment permissive for cell motility and proliferation, might be critical for fetal development. We suggest that the same sequence of events underlie the unique properties observed in fetal wound healing.

  View details for Web of Science ID A1989AK08500014

  View details for PubMedID 2769547

• STUDIES IN FETAL WOUND-HEALING .2. A FETAL ENVIRONMENT ACCELERATES FIBROBLAST MIGRATION INVITRO JOURNAL OF PEDIATRIC SURGERY Longaker, M. T., Harrison, M. R., Langer, J. C., Crombleholme, T. M., Verrier, E. D., SPENDLOVE, R., Stern, R. 1989; 24 (8): 793-798

  Abstract

  We have used an in vitro model of wound healing using scratches made in a confluent monolayer of fibroblasts. The effects of fetal calf and postnatal calf serum on the migration of fibroblasts were compared. Differences between fetal and calf serum-incubated fibroblasts grown on coverslips were observed within 15 minutes of exposure. Cells in fetal serum began to change both shape and orientation and to move into the trough created by the scratch. The fibroblasts incubated in fetal calf serum completely filled in the trough within 16 hours while those incubated in calf serum did not do so even after 24 hours. We estimate that, at any point, there was a 50% lag time in the migration of the fibroblasts in the presence of postnatal calf serum. This difference in migration and filling was not a function of mitogenesis; the mitogenicity of the two sera were comparable. The results suggest that fibroblast migration in vitro is accelerated by the fetal serum. A similar mechanism may occur in vivo and may underlie the ability of the fetal wound to heal more rapidly.

  View details for Web of Science ID A1989AK08500015

  View details for PubMedID 2769548

• APPENDICOVESICOSTOMY - A NEW TECHNIQUE FOR BLADDER DIVERSION DURING RECONSTRUCTION OF CLOACAL EXSTROPHY JOURNAL OF PEDIATRIC SURGERY Longaker, M. T., Harrison, M. R., Langer, J. C., Crombleholme, T. M. 1989; 24 (7): 639-641

  Abstract

  Urinary tract diversion in the reconstruction of cloacal exstrophy presents a difficult problem. Many methods have been used, but none has been found to be ideal. We describe a simple technique that incorporates the ileocecal plate in the bladder augmentation, and uses the appendix as a conduit for urinary diversion. This technique offers the advantages of primary closure, effective urinary drainage without tubes, and simplicity of bowel reconstruction with preservation of maximal absorptive surface.

  View details for Web of Science ID A1989AE75300005

  View details for PubMedID 2754579

• NEONATAL MODEL OF HETEROTOPIC HEART-TRANSPLANTATION IN PIGS JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Verrier, E. D., Crombleholme, T. M., SAUER, L., Longaker, M., Langer, J. C., Flake, A. W., Dae, M., Brem, H., Harrison, M. R., Starnes, V. 1989; 98 (1): 127-136

  Abstract

  To investigate the long-term success of heart transplantation in newborn infants who have complex congenital heart disease, we have developed a model of heterotopic heart transplantation in immature pigs. We chose the heterotopic technique because it is simple, does not require cardiopulmonary bypass or heparin, allows for significant size disparity between the recipient and donor hearts, and allows for experimental comparisons between the two hearts. Small newborn piglet hearts are harvested, prepared, and then transplanted into the left chest of larger weanling pigs to augment or substitute for the native left ventricle. Preliminary data from transplants into 49 pigs suggest that the technique is technically possible, the pigs can be immunosuppressed over the long term, and the donor heart can contribute hemodynamically. Experimentally, the model is well designed for the investigation of issues critical for the long-term success of heart transplantation in infants and children, including growth and development, optimal long-term immunosuppression, differences in immunotolerance, and the study of coronary obliterative disease. Clinically, the model has potential applicability in congenital heart anomalies if one native functioning atrium and ventricle are present.

  View details for Web of Science ID A1989AE79300016

  View details for PubMedID 2661923

• BILIARY APPENDICO-DUODENOSTOMY - A NONREFLUXING CONDUIT FOR BILIARY RECONSTRUCTION JOURNAL OF PEDIATRIC SURGERY Crombleholme, T. M., Harrison, M. R., Langer, J. C., Longaker, M. T. 1989; 24 (7): 665-667

  Abstract

  Biliary conduits constructed during operations for choledochal cysts or biliary atresia are frequently complicated by reflux of gastrointestinal contents, stasis, and obstruction with resulting cholangitis. We have used the appendix as a biliary conduit for cases of biliary atresia and choledochal cyst, adapting the urologic technique of a tunneled, nonrefluxing anastomosis for reconstruction of the biliary tree--biliary appendico-duodenostomy (BAD). From our preliminary experience with this technique, it appears promising.

  View details for Web of Science ID A1989AE75300011

  View details for PubMedID 2754583

• HYALURONIC ACID-STIMULATING ACTIVITY IN SERA FROM THE BOVINE FETUS AND FROM BREAST-CANCER PATIENTS CANCER RESEARCH Decker, M., Chiu, E. S., Dollbaum, C., Moiin, A., Hall, J., SPENDLOVE, R., Longaker, M. T., Stern, R. 1989; 49 (13): 3499-3505

  Abstract

  The sine qua non of malignancy is the ability of tumor cells to migrate and invade surrounding tissue. There are many substances that have been described that enhance cell motility and hyaluronic acid is prominent among these. Hyaluronic acid is a high molecular weight alternating disaccharide polymer found in abundance in extracellular matrices whenever rapid cell proliferation or tissue regeneration and repair occur. It creates a permissive environment for cell motility during embryogenesis, and high levels of hyaluronic acid also correlate with increased tumor cell invasion and aggressiveness. Little is known about the regulation of hyaluronic acid production, either in normal tissue or in malignancy. In this study, we characterize a hyaluronic acid-stimulating activity in fetal calf serum and describe a similar activity in the sera of breast cancer patients. The stimulating activity was measured by placing aliquots of test substance on fibrosarcoma cells. These indicator cells, which synthesize copious quantities of hyaluronic acid, respond to stimulation in a time- and dose-dependent fashion. The fetal calf serum hyaluronic acid-stimulating activity is maximum early in gestation and then falls rapidly to essentially no activity at term. This activity was partially purified from 120-day fetal calf serum by concanavalin A-Sepharose affinity and ion exchange chromatography and is accounted for by a glycoprotein with a molecular weight of 150,000 on gel filtration under native conditions. The sera of breast cancer patients with measurable burden of disease also contained hyaluronic acid-stimulating activity, which was not present in normal serum donors or in breast cancer patients without evidence of disease. The production of this stimulating activity may contribute to the development of the malignant phenotype by inducing hyaluronic acid-rich microenvironments that are permissive to tumor cell invasion and metastases.

  View details for Web of Science ID A1989AB72700010

  View details for PubMedID 2731171

• PRIMARY FETAL HYDROTHORAX - NATURAL-HISTORY AND MANAGEMENT JOURNAL OF PEDIATRIC SURGERY Longaker, M. T., LaBerge, J. M., Dansereau, J., Langer, J. C., Crombleholme, T. M., Callen, P. W., Golbus, M. S., Harrison, M. R. 1989; 24 (6): 573-576

  Abstract

  Primary fetal hydrothorax presents a wide spectrum of severity ranging from small, harmless effusions, to life-threatening thoracic compression. To define natural history and management, we reviewed 32 cases seen at two large perinatal centers from 1980 to 1987. Spontaneous resolution of the effusions was seen in three fetuses, all of whom survived. Three fetuses were electively terminated. The overall mortality was 53%. In the 24 untreated fetuses, sex and the presence of polyhydramnios did not influence mortality, but hydrops, gestational age less than 35 weeks at delivery, and bilateral effusions were associated with a poor prognosis. Five fetuses underwent in utero decompression. In four, thoracentesis was performed, with rapid reaccumulation of the effusion. All four died from pulmonary insufficiency. In the fifth fetus, a thoracoamniotic shunt permanently decompressed the effusion, with resolution of the hydrops, and delivery of a normal viable infant. We conclude that (1) primary fetal hydrothorax may resolve or progress to hydrops, necessitating close follow-up with ultrasound; (2) pulmonary hypoplasia as a result of undrained large pleural effusions may result in neonatal mortality; (3) the gestational age at both diagnosis and delivery, the development of hydrops, and bilaterality of effusions are important prognostic predictors; and (4) the fetus with large effusions and hydrops has a poor prognosis, and thoracic decompression with a thoracoamniotic shunt may prove life saving.

  View details for Web of Science ID A1989U944100015

  View details for PubMedID 2661800

• FETAL HYDROPS AND DEATH FROM SACROCOCCYGEAL TERATOMA - RATIONALE FOR FETAL SURGERY AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY Langer, J. C., Harrison, M. R., Schmidt, K. G., Silverman, N. H., Anderson, R. L., Goldberg, J. D., Filly, R. A., Crombleholme, T. M., Longaker, M. T., Golbus, M. S. 1989; 160 (5): 1145-1150

  Abstract

  Most sacrococcygeal teratomas diagnosed before birth can be managed by planned delivery and postnatal surgery. However, large tumors early in gestation may result in placentomegaly, hydrops, and fetal death and a preeclampsia-like syndrome in the mother. This chain of events may result from high output cardiac failure in the fetus caused by arteriovenous shunting through the tumor. We recently encountered this situation in a fetus at 21 weeks' gestation and performed fetal surgery in an attempt to reverse the process. Excision of the teratoma resulted in reversal of hydrops, diminution of descending aortic flow on Doppler echocardiography, and decrease in placental thickness. Despite these changes, uterine irritability after hysterotomy resulted in labor and delivery of a nonviable premature infant. This case demonstrates that when fetal sacrococcygeal teratoma becomes very large early in gestation, high output cardiac failure can endanger both fetus and mother. In the future, use of Doppler echocardiography may allow appropriate selection of high-risk fetuses. Intervention to prevent arteriovenous shunting through the tumor may offer these fetuses an improved chance for survival.

  View details for Web of Science ID A1989U693200026

  View details for PubMedID 2658603

• SYSTEMIC-TO-PULMONARY ARTERY SHUNT USING THE INTERNAL MAMMARY ARTERY ANNALS OF THORACIC SURGERY Longaker, M. T., Merrick, S., Crombleholme, T. M., Langer, J. C., Verrier, E. D., Turley, K. 1989; 47 (3): 464-465

  Abstract

  Systemic-to-pulmonary artery shunts may be useful for palliation of cyanotic congenital heart disease. We report the case of a 5-year-old boy in whom the internal mammary artery was used to create a systemic-to-pulmonary artery shunt after failure of a previous Blalock-Taussig shunt. This technique may have distinct advantages in selected cases and should be considered as an alternative during investigation of the older child who requires a systemic-to-pulmonary artery shunt.

  View details for Web of Science ID A1989U019800027

  View details for PubMedID 2467633

• BILATERAL RENAL AUTO-TRANSPLANTATION WITH PYELOVESICOSTOMY - A SURGICAL-TREATMENT OF REFRACTORY ENTERIC HYPEROXALURIA SURGERY Salvatierra, O., Longaker, M., Crombleholme, T. 1989; 105 (3): 430-435

  Abstract

  A 38-year-old man, with only 3 feet of small bowel remaining after multiple resections because of chronic inflammatory bowel disease, had severe symptomatic calcium oxalate nephroureterolithiasis. Because of the refractory symptoms, he was successfully treated with bilateral autotransplantation of the kidneys, totally bypassing the ureters. Anatomically effective urinary tract continuity was reestablished by means of bilateral pyelovesicostomies with concomitant rectus muscle vesicofixation to create direct stone-dumping channels into the urinary bladder. The patient is now completely without symptoms 18 months after surgery. A description and rationale for this surgical treatment is provided. Bilateral autotransplantation of the kidneys with direct drainage into the urinary bladder may be an attractive and viable therapeutic option in complicated patients with short-gut syndrome and severe refractory calcium oxalate nephroureterolithiasis.

  View details for Web of Science ID A1989T496600014

  View details for PubMedID 2522248

• EARLY EXPERIENCE WITH OPEN FETAL SURGERY FOR CONGENITAL HYDRONEPHROSIS JOURNAL OF PEDIATRIC SURGERY Crombleholme, T. M., Harrison, M. R., Langer, J. C., Longaker, M. T., Anderson, R. L., SLOTNICK, N. S., Filly, R. A., Callen, P. W., Goldstein, R. B., Golbus, M. S. 1988; 23 (12): 1114-1121

  Abstract

  The fetus with severe bilateral hydronephrosis and associated oligohydramnios in the second trimester is doomed at birth by ongoing pulmonary and renal damage. Since decompression with percutaneously placed catheters anesthetic, surgical, and tocolytic techniques for open fetal anesthetic, surgical, and tocolytic techniques for open fetal urinary tract decompression in animals, and have now applied those techniques to a small group of five patients. One had bilateral ureterostomies and the subsequent four had marsupialization of the bladder. All pregnancies proceeded to cesarean delivery at 32 to 35 weeks' gestation. There was no long-term maternal morbidity, and two mothers have since experienced normal pregnancies. Three fetuses had return of normal amniotic fluid dynamics, and all three had adequate pulmonary function at birth, suggesting that fatal pulmonary hypoplasia associated with early severe oligohydramnios had been reversed. Two neonates died at birth with pulmonary hypoplasia. One had no amniotic fluid even after decompression, and the other had some amniotic fluid after decompression but a tiny chest cavity due to the long period of severe oligohydramnios before decompression. Of the three surviving infants, one had normal renal function when she died of unrelated causes at 9 months of age. One has normal renal function at 23 months and the third had failing renal function at 2 1/2 years and has grown and developed normally, but will require renal transplantation. We have now developed selection criteria that would exclude from treatment the two fetuses who died of pulmonary hypoplasia and the one who developed renal failure.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1988R253700005

  View details for PubMedID 3236176

• PRENATAL-DIAGNOSIS AND MANAGEMENT OF BILATERAL HYDRONEPHROSIS PEDIATRIC NEPHROLOGY Crombleholme, T. M., Harrison, M. R., Longaker, M. T., Langer, J. C. 1988; 2 (3): 334-342

  Abstract

  This report reviews the management of the fetus with congenital hydronephrosis (CH), a challenging diagnostic and therapeutic problem. Experimental models of obstructive uropathy have produced histologic changes similar to those seen in kidneys of human neonates with congenital hydronephrosis. Relief of obstruction in utero in these models has been shown to prevent some of the dysplastic changes caused by obstruction. These studies have formed the theoretical basis for in utero decompression to restore amniotic fluid dynamics to prevent death from pulmonary hypoplasia, and reverse or arrest dysplastic morphogenesis. The development of prognostic criteria has greatly aided in selection of appropriate fetuses for intervention. These criteria include: (1) Na less than 100 mEq/l; (2) Cl less than 90 mEq/l; (3) osmolarity less than 210 mosmol; (4) sonographic appearance of the fetal kidneys; (5) amniotic fluid status; (6) urine output at fetal bladder catheterization. All fetuses should have ultrasonography to exclude other anomalies, and karyotype analysis to exclude chromosomal abnormality. If amniotic fluid volume is normal, the pregnancy is followed with serial ultrasound examinations. If oligohydramnios develops, a prognostic evaluation is performed, including fetal bladder catheterization. If the fetus has poor residual renal function, on the basis of prognostic criteria, appropriate counseling may be given. If the fetus has good residual renal function, depending on lung maturity, it can be delivered early for corrective surgery. If diagnosed prior to lung maturity in utero, decompression by either vesicoamniotic shunting or open fetal surgery may be attempted in the highly selected case.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1988P159100014

  View details for PubMedID 3153038

• Perinatal management of the fetus with an abdominal wall defect. Fetal therapy Langer, J. C., Harrison, M. R., Adzick, N. S., Longaker, M. T., Crombleholme, T. M., Golbus, M. S., Anderson, R. L., Callen, P. W., Filly, R. A. 1987; 2 (4): 216-221

  Abstract

  The antenatal diagnosis of abdominal wall defects has allowed improved perinatal management. For fetuses with associated anomalies, the options of elective termination or minimal intervention can be offered. Our ability to predict the extent of bowel damage in gastroschisis based on the ultrasound findings enables us to offer early delivery to those fetuses who are at high risk. The data are not clear at the present time whether cesarean section offers any advantage. These fetuses should, however, be delivered at a center which is capable of providing high level medical and surgical care to these potentially ill infants. Initial resuscitation of these neonates requires early insertion of an intravenous line and a nasogastric tube, the administration of antibiotics, sterile coverage of the eviscerated bowel, and careful attention to temperature instability. Neonates with gastroschisis should be operated on as soon as they are stable, whereas infants with omphalocele can be investigated for associated anomalies prior to surgery. Primary fascial closure is performed whenever possible. Where this is not possible, a staged repair using a silastic chimney achieves closure within 3-6 days. Skin coverage alone or nonoperative management is reserved for the few cases with giant omphalocele, associated anomalies, or poor operative risk. Decisions about primary versus delayed closure, while usually dictated by clinical judgement, can be aided by indirect measurement of intraabdominal pressure. Postoperative ventilation, and consideration of long-term nutritional needs, are also important parts of the perioperative management.

  View details for PubMedID 2978282