Jill Helms

All Publications

• Biomechanical Basis for Bone Healing and Osseointegration of Implants in Sinus Grafts. Clinical implant dentistry and related research Stacchi, C., Coyac, B. R., Helms, J. A. 2024

  Abstract

  A thorough comprehension of the mechanisms controlling new bone formation and implant osseointegration after maxillary sinus floor elevation is crucial for aligning our treatment choices with biological principles and enhancing clinical outcomes. The goal of bone regeneration in sinus lift procedures is to provide a sufficient amount of newly-formed tissue to support implant osseointegration. However, it is still unclear whether there is a minimum quantity of vital bone within the newly-formed tissue required for effective support, though it is generally assumed that vital bone is essential for this process. The source and integration of new bone in maxillary sinus floor elevation procedures remain debated. Most clinical studies suggest a paramount role for sinus floor and bony walls, with a centripetal pattern of new bone formation, while conflicting reports exist regarding the osteogenic role of the Schneiderian membrane. The influence of mechanical input on peri-implant bone formation, mineralization, and maturation is significant, with bone remodeling regulated by mechanical strains generated during loading. Defining optimal loading for implants, particularly in sinus lift procedures, is challenging, as early loading may damage interfacial tissue, interfering with osteogenesis. Differences in osseointegration dynamics between native and augmented bone may arise from biological and mechanical factors, but also from patient-specific factors which should be evaluated in treatment planning. Factors to consider include sinus anatomy, patient and site-specific regenerative potential, and the selection of graft material that matches the osteogenic and mechanical requirements. Tailored approaches integrating patient-specific considerations and refined implant strategies will enhance predictability and longevity of treatment.

  View details for DOI 10.1111/cid.13424

  View details for PubMedID 39637842

• Evaluation of Postoperative Outcomes Following Early and Late Palate Repair: A Preclinical Study. The Journal of craniofacial surgery Aellos, F., Verma, I., Ly, M., Hoy, M., Quach, T., Rosbander, I., Sandoval, A., Wolvis, E., Turkkahraman, H., Helms, J. A. 2024

  Abstract

  To quantitatively assess the impact of early versus late surgical intervention on midfacial growth using a mouse model.A full-thickness mucoperiosteal flap surgery was performed on newborn (P17) mice and on neonatal (P30) mice. High-resolution micro-computed tomographic imaging coupled with histomorphometric analyses was used to assess craniomaxillofacial growth. Histology and immunohistochemical analyses were used to assess cellular and molecular responses postsurgery.Early surgical intervention at P17 resulted in significant midfacial growth arrest, with pronounced maxillary hypoplasia. Histomorphometric analyses revealed significant (P < 0.05) growth disruptions in the mid-palatal suture complex, including premature removal of the cartilaginous growth plate and its replacement by bone. In the suture itself, cell proliferation was significantly reduced (P < 0.05) compared with controls. The same surgical intervention performed in mice at P30 did not lead to significant midfacial growth arrest.Early surgical intervention in a mouse model mirrors the adverse growth outcomes in children undergoing early cleft repair. Molecular and cellular observations accompanying this midfacial growth arrest may inform therapeutic strategies to mitigate midfacial growth disturbances in patients and highlight the need for refined surgical techniques to minimize adverse growth outcomes.

  View details for DOI 10.1097/SCS.0000000000010827

  View details for PubMedID 39499137

• A critical analysis on quality-of-life in women with visible facial disfigurements. Special care in dentistry : official publication of the American Association of Hospital Dentists, the Academy of Dentistry for the Handicapped, and the American Society for Geriatric Dentistry Okine, E., Helms, J. A., Luhrmann, T. 2024

  Abstract

  This study aimed to examine whether surgical treatment for a facial disfigurement influenced an individual's quality of life.One-on-one interviews were conducted with the aim of synthesizing participant's medical experiences into common themes. Additionally, participants completed the World Health Organization's Quality of Life Brief Version (WHOQOL-BREF) questionnaire. The WHOQL-BREF is a standardized testing instrument with four domains of 26 questions, meant to analyze participants' overall quality of health, physical health, psychological status, social relationships, and environmental health. Our study revealed that women with visible facial differences experienced a quality of life below the average of the general population. However, in those who reported above-average quality of life, a key theme emerged: active participation in the choice to undergo surgical treatment. Participants who felt this sense of agency in the decision-making process also reported a more positive healthcare experience. They felt more respected by others, indicating a strong connection between personal agency, surgical choices, and overall well-being.These findings reveal that personal agency plays an important role in the decision-making process for patients undergoing surgical treatment for facial differences, as it improves quality of life and has a positive impact on overall healthcare experience and well-being.

  View details for DOI 10.1111/scd.13017

  View details for PubMedID 38715214

• Dynamic analyses of a soft tissue-implant interface: Biological responses to immediate versus delayed dental implants. Journal of clinical periodontology Aellos, F., Grauer, J. A., Harder, K. G., Dworan, J. S., Fabbri, G., Cuevas, P. L., Yuan, X., Liu, B., Brunski, J. B., Helms, J. A. 2024

  Abstract

  To qualitatively and quantitatively evaluate the formation and maturation of peri-implant soft tissues around 'immediate' and 'delayed' implants.Miniaturized titanium implants were placed in either maxillary first molar (mxM1) fresh extraction sockets or healed mxM1 sites in mice. Peri-implant soft tissues were evaluated at multiple timepoints to assess the molecular mechanisms of attachment and the efficacy of the soft tissue as a barrier. A healthy junctional epithelium (JE) served as positive control.No differences were observed in the rate of soft-tissue integration of immediate versus delayed implants; however, overall, mucosal integration took at least twice as long as osseointegration in this model. Qualitative assessment of Vimentin expression over the time course of soft-tissue integration indicated an initially disorganized peri-implant connective tissue envelope that gradually matured with time. Quantitative analyses showed significantly less total collagen in peri-implant connective tissues compared to connective tissue around teeth around implants. Quantitative analyses also showed a gradual increase in expression of hemidesmosomal attachment proteins in the peri-implant epithelium (PIE), which was accompanied by a significant inflammatory marker reduction.Within the timeframe examined, quantitative analyses showed that connective tissue maturation never reached that observed around teeth. Hemidesmosomal attachment protein expression levels were also significantly reduced compared to those in an intact JE, although quantitative analyses indicated that macrophage density in the peri-implant environment was reduced over time, suggesting an improvement in PIE barrier functions. Perhaps most unexpectedly, maturation of the peri-implant soft tissues was a significantly slower process than osseointegration.

  View details for DOI 10.1111/jcpe.13980

  View details for PubMedID 38708491

• The Effect of Osteocyte-Derived RANKL on Bone Graft Remodeling Feher, B., Kampleitner, C., Heimel, P., Tangl, S., Helms, J., Kuchler, U., Gruber, R. OXFORD UNIV PRESS. 2023: 53-54

  View details for Web of Science ID 001266167000166

• The effect of osteocyte-derived RANKL on bone graft remodeling: An in vivo experimental study. Clinical oral implants research Feher, B., Kampleitner, C., Heimel, P., Tangl, S., Helms, J. A., Kuchler, U., Gruber, R. 2023

  Abstract

  Autologous bone is considered the gold standard for grafting, yet it suffers from a tendency to undergo resorption over time. While the exact mechanisms of this resorption remain elusive, osteocytes have been shown to play an important role in stimulating osteoclastic activity through their expression of receptor activator of NF-κB (RANK) ligand (RANKL). The aim of this study was to assess the function of osteocyte-derived RANKL in bone graft remodeling.In Tnfsf11fl/fl ;Dmp1-Cre mice without osteocyte-specific RANKL as well as in Dmp1-Cre control mice, 2.6 mm calvarial bone disks were harvested and transplanted into mice with matching genetic backgrounds either subcutaneously or subperiosteally, creating 4 groups in total. Histology and micro-computed tomography of the grafts and the donor regions were performed 28 days after grafting.Histology revealed marked resorption of subcutaneous control Dmp1-Cre grafts and new bone formation around subperiosteal Dmp1-Cre grafts. In contrast, Tnfsf11fl/fl ;Dmp1-Cre grafts showed effectively neither signs of bone resorption nor formation. Quantitative micro-computed tomography revealed a significant difference in residual graft area between subcutaneous and subperiosteal Dmp1-Cre grafts (p < .01). This difference was not observed between subcutaneous and subperiosteal Tnfsf11fl/fl ;Dmp1-Cre grafts (p = .17). Residual graft volume (p = .08) and thickness (p = .13) did not differ significantly among the groups. Donor area regeneration was comparable between Tnfsf11fl/fl ;Dmp1-Cre and Dmp1-Cre mice and restricted to the defect margins.The results suggest an active function of osteocyte-derived RANKL in bone graft remodeling.

  View details for DOI 10.1111/clr.14187

  View details for PubMedID 37792417

• Linking the Mechanics of Chewing to Biology of the Junctional Epithelium. Journal of dental research Yuan, X., Liu, B., Cuevas, P., Brunski, J., Aellos, F., Petersen, J., Koehne, T., Bröer, S., Grüber, R., LeBlanc, A., Zhang, X., Xu, Q., Helms, J. A. 2023: 220345231185288

  Abstract

  The capacity of a tissue to continuously alter its phenotype lies at the heart of how an animal is able to quickly adapt to changes in environmental stimuli. Within tissues, differentiated cells are rigid and play a limited role in adapting to new environments; however, differentiated cells are replenished by stem cells that are defined by their phenotypic plasticity. Here we demonstrate that a Wnt-responsive stem cell niche in the junctional epithelium is responsible for the capability of this tissue to quickly adapt to changes in the physical consistency of a diet. Mechanical input from chewing is required to both establish and maintain this niche. Since the junctional epithelium directly attaches to the tooth surface via hemidesmosomes, a soft diet requires minimal mastication, and consequently, lower distortional strains are produced in the tissue. This reduced strain state is accompanied by reduced mitotic activity in both stem cells and their progeny, leading to tissue atrophy. The atrophied junctional epithelium exhibits suboptimal barrier functions, allowing the ingression of bacteria into the underlying connective tissues, which in turn trigger inflammation and mild alveolar bone loss. These data link the mechanics of chewing to the biology of tooth-supporting tissues, revealing how a stem cell niche is responsible for the remarkable adaptability of the junctional epithelium to different diets.

  View details for DOI 10.1177/00220345231185288

  View details for PubMedID 37555395

• Mechanical-induced bone remodeling does not depend on Piezo1 in dentoalveolar hard tissue. Scientific reports Nottmeier, C., Lavicky, J., Gonzalez Lopez, M., Knauth, S., Kahl-Nieke, B., Amling, M., Schinke, T., Helms, J., Krivanek, J., Koehne, T., Petersen, J. 2023; 13 (1): 9563

  Abstract

  Mechanosensory ion channels are proteins that are sensitive to mechanical forces. They are found in tissues throughout the body and play an important role in bone remodeling by sensing changes in mechanical stress and transmitting signals to bone-forming cells. Orthodontic tooth movement (OTM) is a prime example of mechanically induced bone remodeling. However, the cell-specific role of the ion channels Piezo1 and Piezo2 in OTM has not been investigated yet. Here we first identify the expression of PIEZO1/2 in the dentoalveolar hard tissues. Results showed that PIEZO1 was expressed in odontoblasts, osteoblasts, and osteocytes, while PIEZO2 was localized in odontoblasts and cementoblasts. We therefore used a Piezo1floxed/floxed mouse model in combination with Dmp1cre to inactivate Piezo1 in mature osteoblasts/cementoblasts, osteocytes/cementocytes, and odontoblasts. Inactivation of Piezo1 in these cells did not affect the overall morphology of the skull but caused significant bone loss in the craniofacial skeleton. Histological analysis revealed a significantly increased number of osteoclasts in Piezo1floxed/floxed;Dmp1cre mice, while osteoblasts were not affected. Despite this increased number of osteoclasts, orthodontic tooth movement was not altered in these mice. Our results suggest that despite Piezo1 being crucial for osteoclast function, it may be dispensable for mechanical sensing of bone remodeling.

  View details for DOI 10.1038/s41598-023-36699-9

  View details for PubMedID 37308580

  View details for PubMedCentralID 3743123

• Wnt/beta-Catenin Signaling in Craniomaxillofacial Osteocytes. Current osteoporosis reports Cuevas, P. L., Aellos, F., Dawid, I. M., Helms, J. A. 2023

  Abstract

  PURPOSE OF REVIEW: There is a growing appreciation within the scientific community that cells exhibit regional variation. Whether the variation is attributable to differences in embryonic origin or anatomical location and mechanical loading has not been elucidated; what is clear, however, is that adult cells carry positional information that ultimately affects their functions. The purpose of this review is to highlight the functions of osteocytes in the craniomaxillofacial (CMF) skeleton as opposed to elsewhere in the body, and in doing so gain mechanistic insights into genetic conditions and chemically-induced diseases that particularly affect this region of our anatomy.RECENT FINDINGS: In the CMF skeleton, elevated Wnt/beta-catenin signaling affects not only bone mass and volume, but also mineralization of the canalicular network and osteocyte lacunae. Aberrant elevation in the Wnt/beta-catenin pathway can also produce micropetrosis and osteonecrosis of CMF bone, presumably due to a disruption in the signaling network that connects osteocytes to one another, and to osteoblasts on the bone surface.

  View details for DOI 10.1007/s11914-023-00775-w

  View details for PubMedID 36807035

• Corrigendum to "Hormone sensitive lipase ablation promotes bone regeneration" [Biochim. Biophys. Acta Mol. Basis Dis. Volume 1868, Issue 9, 1 September 2022, 166449]. Biochimica et biophysica acta. Molecular basis of disease Shen, W. J., Still, C., Han, L., Yang, P., Chen, J., Wosczyna, M., Rando, T. A., Salmon, B. J., Perez, K. C., Li, J., Cuevas, P. L., Liu, B., Azhar, S., Helms, J., Qi, L. S., Kraemer, F. B. 2022; 1868 (11): 166506

  View details for DOI 10.1016/j.bbadis.2022.166506

  View details for PubMedID 35926435

• A WNT protein therapeutic accelerates consolidation of a bone graft substitute in a pre-clinical sinus augmentation model. Journal of clinical periodontology Coyac, B. R., Wolf, B. J., Bahat, D. J., Arioka, M., Brunski, J. B., Helms, J. A. 2022

  Abstract

  AIM: Autologous bone grafts consolidate faster than bone graft substitutes (BGSs) but resorb over time, which compromises implant support. We hypothesized that differences in consolidation rates affected the mechanical properties of grafts and implant stability, and tested whether a pro-osteogenic protein, liposomal WNT3A (L-WNT3A), could accelerate graft consolidation.MATERIALS AND METHODS: A transgenic mouse model of sinus augmentation with immunohistochemistry, enzymatic assays, and histology were used to quantitatively evaluate the osteogenic properties of autografts and BGSs. Composite and finite element modelling compared changes in the mechanical properties of grafts during healing until consolidation, and secondary implant stability following remodelling activities. BGSs were combined with L-WNT3A and tested for its osteogenic potential.RESULTS: Compared with autografts, BGSs were bioinert and lacked osteoprogenitor cells. While in autografted sinuses, new bone arose evenly from all living autograft particles, new bone around BGSs solely initiated at the sinus floor, from the internal maxillary periosteum. WNT treatment of BGSs resulted in significantly higher expression levels of pro-osteogenic proteins (Osterix, Collagen I, alkaline phosphatase) and lower levels of bone-resorbing activity (tartrate-resistant acid phosphatase activity); together, these features culminated in faster new bone formation, comparable to that of an autograft.CONCLUSIONS: WNT-treated BGSs supported faster consolidation, and because BGSs typically resist resorption, their use may be superior to autografts for sinus augmentation.

  View details for DOI 10.1111/jcpe.13674

  View details for PubMedID 35713219

• Hormone sensitive lipase ablation promotes bone regeneration. Biochimica et biophysica acta. Molecular basis of disease Shen, W. J., Chris Still, I. I., Han, L., Yang, P., Chen, J., Wosczyna, M., Salmon, B. J., Perez, K. C., Li, J., Cuevas, P. L., Liu, B., Azhar, S., Helms, J., Qi, L. S., Kraemer, F. B. 2022: 166449

  Abstract

  There is an inverse relationship between the differentiation of mesenchymal stem cells (MSCs) along either an adipocyte or osteoblast lineage, with lineage differentiation known to be mediated by transcription factors PPARγ and Runx2, respectively. Endogenous ligands for PPARγ are generated during the hydrolysis of triacylglycerols to fatty acids through the actions of lipases such as hormone sensitive lipase (HSL). To examine whether reduced production of endogenous PPARγ ligands would influence bone regeneration, we examined the effects of HSL knockout on fracture repair in mice using a tibial mono-cortical defect as a model. We found an improved rate of fracture repair in HSL-ko mice documented by serial μCT and bone histomorphometry compared to wild-type (WT) mice. Similarly, accelerated rates of bone regeneration were observed with a calvarial model where implantation of bone grafts from HSL-ko mice accelerated bone regeneration at the injury site. Further analysis revealed improved MSC differentiation down osteoblast and chondrocyte lineage with inhibition of HSL. MSC recruitment to the injury site was greater in HSL-ko mice than WT. Finally, we used single cell RNAseq to understand the osteoimmunological differences between WT and HSL-ko mice and found changes in the pre-osteoclast population. Our study shows HSL-ko mice as an interesting model to study improvements to bone injury repair. Furthermore, our study highlights the potential importance of pre-osteoclasts and osteoclasts in bone repair.

  View details for DOI 10.1016/j.bbadis.2022.166449

  View details for PubMedID 35618183

• Experiential factors affecting the empathy of students in their pre-clinical year(s) of 21 universities. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Vigoda, J., Adeniyi, A., Tudor, L., Brassett, C., McWatt, S., Sagoo, M. G., Wingate, R., Chien, C., Traxler, H., Waschke, J., Vielmuth, F., Sigmund, A., Sakurai, T., Yamada, Y., Zeroual, M., Olsen, J., El-Batti, S., Viranta-Kovanen, S., Keay, K., Kitahara, S., Martinez-Abadias, N., Esteban-Torne, M. E., Helms, J., Sforza, C., Gagliano, N., Norris, M., Harmon, D., Yasui, M., Ichiko, M., Lee, S., Reid, S., Lang, A., Kunzel, C., Joseph, M., Buehler, L., Hardy, M., Patel, S., Bernd, P., Kielstein, H., Stewart, W., Kellett, A., Wu, A., Noel, G. 2022; 36 Suppl 1

  Abstract

  Hands-on cadaveric dissection is often considered an important factor in shaping the emotional identity of medical and dental students as healthcare providers. This study explores how demographic and/or experiential factors affect the empathy of students in their pre-clinical year(s) of medical or dental school. In the Summer of 2021 and Fall of 2021, a total of 530 students from 21 universities around the world participating in the International Collaboration and Exchange (ICE) Program, completed a validated questionnaire containing the Santa Clara Brief Compassion (SCBC) Scale and the Toronto Empathy Questionnaire (TEQ). Responses to the SCBC and TEQ were tested for variance and covariance against age group, sex, clinical experience, year of health professional school, format of anatomy education, hours of study on prosections and/or hours of hands-on cadaveric dissection in their respective curricula; and whether their school provides an opportunity for reflection, information about the body donors, a memorial service, and/or addresses empathy in their curricula. Results show that having 40-90 hours of hands-on cadaveric dissection vs 0 hours yielded higher SCBC averages (p = 0.0206) and TEQ scores (p = 0.0031); and having 20-40 hours of hands-on cadaveric dissection vs 0 hours also resulted in higher TEQ scores (p = 0.0105). Comparisons of hours of study on prosections, format of anatomy education, clinical experience, and year of health professional school yielded no significant results in relation to empathy scores. Our study found that across different regions of the world, curricula emphasizing dissection are best at preparing students to become more empathetic healthcare providers. While none of the other curricular factors proved significant, this study confirms the merit of hands-on cadaveric dissection in the emotional development of medical and dental students.

  View details for DOI 10.1096/fasebj.2022.36.S1.R2742

  View details for PubMedID 35556827

• Cultural competency preparedness in medical and health professions students - a collaborative study involving anatomy departments at 20 international universities. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Wu, A., Patel, R., Brassett, C., McWatt, S., Sagoo, M. G., Wingate, R., Chien, C., Traxler, H., Waschke, J., Vielmuth, F., Sigmund, A., Sakurai, T., Yamada, Y., Zeroual, M., Olsen, J., Batti, S. E., Viranta-Kovanen, S., Keay, K., Kitahara, S., Martinez-Abadias, N., Esteban-Torne, M. E., Helms, J., Sforza, C., Gagliano, N., Norris, M., Harmon, D., Yasui, M., Ichiko, M., Lee, S., Reid, S., Lang, A., Kunzel, C., Joseph, M., Buehler, L., Hardy, M., Patel, S., Bernd, P., Kielstein, H., Noel, G., Green, A. 2022; 36 Suppl 1

  Abstract

  INTRODUCTION: Training in cultural competency skills of medical and health professionals has become an important element of school curricula. Evaluation is often performed via self-assessment among student cohorts within one country. Only a few studies utilize any standardized and validated tests. Little is known about global comparisons of baseline levels of cultural competency preparedness among students in various health professions. The aim of the study is to assess the baseline level of cultural competency preparedness in junior medical and health professions students at 20 universities from around the world, utilizing a previously validated and standardized testing tool. Results from this study will aid medical educators in the assessment of the extent of cultural competency required to be included internationally in health education curricula.METHODS: 436 medical and students from various health professions students from 20 universities world-wide participated via an anatomy-based student exchange program (80% preclinical medical students). The students were given a validated questionnaire (1) to assess their preparedness in reference to cultural competency prior to the start of the program. The students were also asked to self-evaluate their cultural competency skills on a 5-point Likert-type scale ("none" to "a lot") encompassing different areas of competency (e.g., knowledge, intrapersonal and interpersonal skills, internal and external outcomes, attitudes). Data were analyzed in Excel for statistical analysis stratified by global region - North America (NA), Europe (EUR), United Kingdom (UK), East Asia (EA), and Australia (AUS) RESULTS: Data are presented as means (M) and their standard deviation. The highest self-assessment mean was for attitudes toward different cultures (4.4 ± 0.7) and lowest for knowledge about other cultures (3.4 ± 0.8). Regarding the question of general preparedness, the average score was 2.93 (± 1.0) in the validated tool (5-point Likert-type scale, "very unprepared" to "well prepared"); 4.6% of students felt "very well prepared", while 24% felt only "well prepared." A comparison by region showed the highest scores were from NA (3.14 ± 1.1), and the lowest scores from the UK (mean 2.74 ± 0.9). Regarding preparedness to evaluate patients from different cultures, 7% of students felt "very well prepared", and 24% felt "well prepared". Comparison between regions showed that the highest scores were found in EUR (3.1± 0.9). Regarding preparedness to treat patients with limited language proficiency, 14% of students felt that they were "very well prepared" (2.6; ± 1.1). A breakdown by regions showed that the highest scores were found in EUR (2.78 ± 1.0). Regarding preparedness to treat patients from ethnic minorities, 17% felt they were "very well prepared", and 31% felt "well prepared" (3.4 ± 1.1), with the UK scoring highest in this category (3.56 ± 1.1).DISCUSSION: Overall, there appears to be a discrepancy among junior students' self-assessments of their cultural competency skills and of their preparedness to treat patients, when compared to standardized test results. Cultural preparedness was similar across the evaluated regions. The data reveal that most regions in the world can benefit from cultural competency training for junior medical and health professions students.REFERENCE: 1. Green AR, Chun MBJ, Cervantes MC, Nudel JD, Duong JV, Krupat E, et al. Measuring Medical Students' Preparedness and Skills to Provide Cross-Cultural Care. Health equity. 2017;1(1):15-22.

  View details for DOI 10.1096/fasebj.2022.36.S1.0R841

  View details for PubMedID 35556020

• An Osteotomy Tool That Preserves Bone Viability: Evaluation in Preclinical and Clinical Settings. Journal of clinical medicine Bahat, O., Yin, X., Holst, S., Zabalegui, I., Berroeta, E., Perez, J., Wohrle, P., Sorgel, N., Brunski, J., Helms, J. A. 2022; 11 (9)

  Abstract

  The main objectives of this work were to assess the efficiency, ease-of-use, and general performance of a novel osseoshaping tool based on first-user clinical experiences and to compare these observations with preclinical data generated in rodents using a miniaturized version of the instrument. All patients selected for the surgery presented challenging clinical conditions in terms of the quality and/or quantity of the available bone. The presented data were collected during the implant placement of 15 implants in 7 patients, and included implant recipient site (bone quality and quantity) and ridge evaluation, intra-operative handling of the novel instrument, and the evaluation of subsequent implant insertion. The instrument was easy to handle and was applied without any complications during the surgical procedure. Its use obviated the need for multiple drills and enabled adequate insertion torque in all cases. This biologically driven innovation in implant site preparation shows improvements in preserving vital anatomical and cellular structures as well as simplifying the surgical protocol with excellent ease-of-use and handling properties.

  View details for DOI 10.3390/jcm11092536

  View details for PubMedID 35566662

• Multiscale analysis of craniomaxillofacial bone repair: A preclinical mini pig study. Journal of periodontology Ticha, P., Pilawski, I., Helms, J. A. 2022

  Abstract

  BACKGROUND: The rate of reparative osteogenesis controls when an implant is sufficiently stable as to allow functional loading. Using a mini pig model, the rate of reparative osteogenesis in two types of implant sites e.g., an osteotomy versus a fresh extraction socket were compared.METHODS: Eight adult mini pigs were used for the study. In Phase I, three premolars were extracted on one side of the oral cavity; 12 weeks later, in Phase II, osteotomies were produced in healed extraction sites, and contralateral premolars were extracted. Animals were sacrificed 1, 5, and 12 weeks after Phase II. Bone repair and remodeling were evaluated using quantitative micro-computed tomographic imaging, histology, and histochemical assays coupled with quantitative dynamic histomorphometry.RESULTS: One week after surgery, extraction sockets and osteotomy sites exhibited similar patterns of new bone deposition. Five weeks after surgery, mineral apposition rates were elevated at the injury sites relative to intact bone. Twelve weeks after surgery, the density of new bone in both injury sites was equivalent to intact bone but quantitative dynamic histomorphometry and cellular activity assays demonstrated bone remodeling was still underway.CONCLUSION(S): The mechanisms and rates of reparative osteogenesis were equivalent between fresh extraction sockets and osteotomies. The volume of new bone required to fill a socket, however, was significantly greater than the volume required to fill an osteotomy. These data provide a framework for estimating the rate of reparative osteogenesis and the time to loading of implants placed in healed sites versus fresh extraction sockets. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/JPER.21-0426

  View details for PubMedID 35194780

• Wnt/beta-catenin Signaling Controls Maxillofacial Hyperostosis. Journal of dental research Chen, J., Cuevas, P. L., Dworan, J. S., Dawid, I., Turkkahraman, H., Tran, K., Delgado-Calle, J., Bellido, T., Gorski, J. P., Liu, B., Brunski, J. B., Helms, J. A. 1800: 220345211067705

  Abstract

  The roles of Wnt/beta-catenin signaling in regulating the morphology and microstructure of craniomaxillofacial (CMF) bones was explored using mice carrying a constitutively active form of beta-catenin in activating Dmp1-expressing cells (e.g., dabetacatOt mice). By postnatal day 24, dabetacatOt mice exhibited midfacial truncations coupled with maxillary and mandibular hyperostosis that progressively worsened with age. Mechanistic insights into the basis for the hyperostotic facial phenotype were gained through molecular and cellular analyses, which revealed that constitutively activated beta-catenin in Dmp1-expressing cells resulted in an increase in osteoblast number and an increased rate of mineral apposition. An increase in osteoblasts was accompanied by an increase in osteocytes, but they failed to mature. The resulting CMF bone matrix also had an abundance of osteoid, and in locations where compact lamellar bone typically forms, it was replaced by porous, woven bone. The hyperostotic facial phenotype was progressive. These findings identify for the first time a ligand-independent positive feedback loop whereby unrestrained Wnt/beta-catenin signaling results in a CMF phenotype of progressive hyperostosis combined with architecturally abnormal, poorly mineralized matrix that is reminiscent of craniotubular disorders in humans.

  View details for DOI 10.1177/00220345211067705

  View details for PubMedID 35114849

• Clinically relevant preclinical animal models for testing novel cranio-maxillofacial bone 3D-printed biomaterials. Clinical and translational medicine Hatt, L. P., Thompson, K., Helms, J. A., Stoddart, M. J., Armiento, A. R. 2022; 12 (2): e690

  Abstract

  Bone tissue engineering is a rapidly developing field with potential for the regeneration of craniomaxillofacial (CMF) bones, with 3D printing being a suitable fabrication tool for patient-specific implants. The CMF region includes a variety of different bones with distinct functions. The clinical implementation of tissue engineering concepts is currently poor, likely due to multiple reasons including the complexity of the CMF anatomy and biology, and the limited relevance of the currently used preclinical models. The 'recapitulation of a human disease' is a core requisite of preclinical animal models, but this aspect is often neglected, with a vast majority of studies failing to identify the specific clinical indication they are targeting and/or the rationale for choosing one animal model over another. Currently, there are no suitable guidelines that propose the most appropriate animal model to address a specific CMF pathology and no standards are established to test the efficacy of biomaterials or tissue engineered constructs in the CMF field. This review reports the current clinical scenario of CMF reconstruction, then discusses the numerous limitations of currently used preclinical animal models employed for validating 3D-printed tissue engineered constructs and the need to reduce animal work that does not address a specific clinical question. We will highlight critical research aspects to consider, to pave a clinically driven path for the development of new tissue engineered materials for CMF reconstruction.

  View details for DOI 10.1002/ctm2.690

  View details for PubMedID 35170248

• Effects of masticatory loading on bone remodeling around teeth vs. implants: insights from a preclinical model. Clinical oral implants research Tian, Y., Sadowsky, S. J., Brunski, J. B., Yuan, X., Helms, J. A. 2022

  Abstract

  Teeth connect to bone via a periodontal ligament whereas implants connect to bone directly. Consequently, masticatory loads are distributed differently to periodontal versus peri-implant bone. Our objective was to determine how masticatory loading of an implant versus a tooth affected peri-implant versus periodontal bone remodeling. Our hypothesis was that strains produced by functional loading of an implant would be elevated compared to the strains around teeth, and that this would stimulate a greater degree of bone turnover around implants versus in periodontal bone.Sixty skeletally mature mice were divided into two groups. In the Implant group, maxillary first molars (mxM1) were extracted, and after socket healing, titanium alloy implants were positioned sub-occlusally. After osseointegration, implants were exposed, resin crowns were placed, and masticatory loading was initiated. In a Control group the dentition was left intact. Responses of peri-implant and periodontal bone were measured using micro-CT, histology, bone remodeling assays, and quantitative histomorphometry while bone strains were estimated using finite element (FE) analyses.When a submerged osseointegrated implant is exposed to masticatory forces peri-implant strains are elevated, and peri-implant bone undergoes significant remodeling that culminates in new bone accrual. The accumulation of new bone functions to reduce both peri-implant strains and bone remodeling activities, equivalent to those observed around the intact dentition.

  View details for DOI 10.1111/clr.13894

  View details for PubMedID 35051302

• Targeting Notch inhibitors to the myeloma bone marrow niche decreases tumor growth and bone destruction without gut toxicity. Cancer research Sabol, H. M., Ferrari, A. J., Adhikari, M., Amorim, T., McAndrews, K., Anderson, J., Vigolo, M., Lehal, R., Cregor, M., Khan, S., Cuevas, P. L., Helms, J. A., Kurihara, N., Srinivasan, V., Ebetino, F. H., Boeckman, R. K., Roodman, G. D., Bellido, T., Delgado-Calle, J. 2021

  Abstract

  Systemic inhibition of Notch with gamma-secretase inhibitors (GSI) decreases multiple myeloma (MM) tumor growth, but the clinical use of GSI is limited due to its severe gastrointestinal toxicity. In this study, we generated a GSI Notch inhibitor specifically directed to the bone (BT-GSI). BT-GSI administration decreased Notch target gene expression in the bone marrow, but it did not alter Notch signaling in intestinal tissue or induce gut toxicity. In mice with established human or murine MM, treatment with BT-GSI decreased tumor burden and prevented the progression of MM-induced osteolytic disease by inhibiting bone resorption more effectively than unconjugated GSI at equimolar doses. These findings show that BT-GSI has dual anti-myeloma and anti-restorative properties, supporting the therapeutic approach of bone-targeted Notch inhibition for the treatment of MM and associated bone disease.

  View details for DOI 10.1158/0008-5472.CAN-21-0524

  View details for PubMedID 34348968

• Accelerating Socket Repair via WNT3A Curtails Alveolar Ridge Resorption. Journal of dental research Arioka, M., Dawid, I. M., Cuevas, P. L., Coyac, B. R., Leahy, B., Wang, L., Yuan, X., Li, Z., Zhang, X., Liu, B., Helms, J. A. 2021: 220345211019922

  Abstract

  Tooth extraction triggers alveolar ridge resorption, and when this resorption is extensive, it can complicate subsequent reconstructive procedures that use dental implants. Clinical data demonstrate that the most significant dimensional changes in the ridge occur soon after tooth extraction. Here, we sought to understand whether a correlation existed between the rate at which an extraction socket heals and the extent of alveolar ridge resorption. Maxillary molars were extracted from young and osteoporotic rodents, and quantitative micro-computed tomographic imaging, histology, and immunohistochemistry were used to simultaneously follow socket repair and alveolar ridge resorption. Extraction sockets rapidly filled with new bone via the proliferation and differentiation of Wnt-responsive osteoprogenitor cells and their progeny. At the same time that new bone was being deposited in the socket, tartrate-resistant acid phosphatase-expressing osteoclasts were resorbing the ridge. Significantly faster socket repair in young animals was associated with significantly more Wnt-responsive osteoprogenitor cells and their progeny as compared with osteoporotic animals. Delivery of WNT3A to the extraction sockets of osteoporotic animals restored the number of Wnt-responsive cells and their progeny back to levels seen in young healthy animals and accelerated socket repair in osteoporotic animals back to rates seen in the young. In cases where the extraction socket was treated with WNT3A, alveolar ridge resorption was significantly reduced. These data demonstrate a causal link between enhancing socket repair via WNT3A and preserving alveolar ridge dimensions following tooth extraction.

  View details for DOI 10.1177/00220345211019922

  View details for PubMedID 34157887

• Biology of sinus floor augmentation with an autograft vs. a bone graft substitute in a preclinical in vivo experimental model. Clinical oral implants research Coyac, B. R., Wu, M., Bahat, D. J., Wolf, B. J., Helms, J. A. 2021

  Abstract

  OBJECTIVES: Compared to autografts, bone graft substitutes are slower to consolidate. If we understood why, this might open strategies to accelerate new bone formation and thus shorten the time to implant placement. In this study, we aimed at comparing autologous bone graft with a bovine bone graft substitute in a preclinical sinus lift model.MATERIALS AND METHODS: The murine posterior paranasal sinus served as a recipient site for grafting. Autograft from the oral cavity was compared against bone graft substitute using molecular, cellular, and histological analyses conducted on post-grafting days (PSD) 0, 9, 18, and 120.RESULTS: Either autografts or bone graft substitutes were positioned on the sinus floor and remained in situ throughout the study. At the time of grafting and until day 9, bone graft substitutes were devoid of cells and alkaline phosphatase (ALP) activity while autografts were comprised of viable cells and showed strong ALP (mineralization) activity. Consequently, new bone formed faster in autografts compared to bone graft substitutes (140.21±41.21m vs. 41.70±10.09m, respectively, PSD9, p=0.0143). By PSD18, osteogenesis was evident in autografted and xenografted sites. Osteoclasts identified by tartrate resistant acid phosphatase attached to, but did not resorb the bone graft substitute matrix. Autograft matrix, however, underwent extensive resorption. Transgenic mice revealed that Wnt-responsive osteoprogenitor cells originated primarily from the maxillary bone internal periosteum and not from the Schneiderian membrane.CONCLUSION: Autografts produce new bone sooner, but bovine bone graft substitutes eventually consolidate and then resist resorption. Enhancing osteoprogenitor cell recruitment to a bone graft substitute constitutes a viable strategy for accelerating bone formation in a sinus lift procedure.

  View details for DOI 10.1111/clr.13781

  View details for PubMedID 34031931

• Comparative analyses of the soft tissue interfaces around teeth and implants: Insights from a pre-clinical implant model. Journal of clinical periodontology Yuan, X., Pei, X., Chen, J., Zhao, Y., Brunski, J. B., Helms, J. A. 2021

  Abstract

  AIM: To evaluate the similarities and differences in barrier function of a peri-implant epithelium (PIE) versus a native junctional epithelium (JE).MATERIALS AND METHODS: A mouse model was used wherein titanium implants were placed sub-occlusally in healed extraction sites. The PIE was examined at multiple timepoints after implant placement, to capture and understand the temporal nature of its assembly and homeostatic status. Mitotic activity, hemidesmosomal attachment apparatus, and inflammatory responses in the PIE were compared against a JE. Additionally, we evaluated whether the PIE developed a Wnt-responsive stem cell niche like a JE.RESULTS: The PIE developed from oral epithelium (OE) that had, by the time of implant placement, lost all characteristics of a JE. Compared with a JE, an established PIE had more proliferating cells, exhibited lower expression of attachment proteins, and had significantly more inflammatory cells in the underlying connective tissue. Wnt-responsive cells in the OE contributed to an initial PIE, but Wnt-responsive cells and their descendants were lost as the PIE matured.CONCLUSIONS: Although histologically similar, the PIE lacked a Wnt-responsive stem cell niche and exhibited characteristics of a chronically inflamed tissue. Both features contributed to suboptimal barrier functions of the PIE compared with a native JE.

  View details for DOI 10.1111/jcpe.13446

  View details for PubMedID 33713489

• Drill Hole Models to Investigate Bone Repair. Methods in molecular biology (Clifton, N.J.) Li, Z., Helms, J. A. 2021; 2221: 193–204

  Abstract

  Our understanding of the mechanisms underlying fracture healing is rapidly developing and is contributing to new therapeutic strategies to enhance repair. To gain new insights, animal models must also evolve. From initially imprecise, uncontrolled bone defects we now have precise injury models that still capture all of the stages and phases of bone repair yet do so in a highly reproducible manner. The simple mono-cortical defect model allows assessment of bone repair through a cartilage intermediate, e.g., endochondral ossification, as well as direct bone repair, e.g., intramembranous healing. Cellular contributions of the periosteum can be distinguished from contributions originating in the bone marrow. In this chapter, we focus on the advantages of this bone repair model, as well as its limitations.

  View details for DOI 10.1007/978-1-0716-0989-7_12

  View details for PubMedID 32979205

• A novel cryo-embedding method for in-depth analysis of craniofacial mini pig bone specimens. Scientific reports Ticha, P., Pilawski, I., Yuan, X., Pan, J., Tulu, U. S., Coyac, B. R., Hoffmann, W., Helms, J. A. 2020; 10 (1): 19510

  Abstract

  The disconnect between preclinical and clinical results underscores the imperative for establishing good animal models, then gleaning all available data on efficacy, safety, and potential toxicities associated with a device or drug. Mini pigs are a commonly used animal model for testing orthopedic and dental devices because their skeletons are large enough to accommodate human-sized implants. The challenge comes with the analyses of their hard tissues: current methods are time-consuming, destructive, and largely limited to histological observations made from the analysis of very few tissue sections. We developed and employed cryo-based methods that preserved the microarchitecture and the cellular/molecular integrity of mini pig hard tissues, then demonstrated that the results of these histological, histochemical, immunohistochemical, and dynamic histomorphometric analyses e.g., mineral apposition rates were comparable with similar data from preclinical rodent models. Thus, the ability to assess static and dynamic bone states increases the translational value of mini pig and other large animal model studies. In sum, this method represents logical means to minimize the number of animals in a study while simultaneously maximizing the amount of information collected from each specimen.

  View details for DOI 10.1038/s41598-020-76336-3

  View details for PubMedID 33177543

• Molecular Basis for Craniofacial Phenotypes Caused by Sclerostin Deletion. Journal of dental research Chen, J., Yuan, X., Pilawski, I., Liu, X., Delgado-Calle, J., Bellido, T., Turkkahraman, H., Helms, J. A. 2020: 22034520963584

  Abstract

  Some genetic disorders are associated with distinctive facial features, which can aid in diagnosis. While considerable advances have been made in identifying causal genes, relatively little progress has been made toward understanding how a particular genotype results in a characteristic craniofacial phenotype. An example is sclerosteosis/van Buchem disease, which is caused by mutations in the Wnt inhibitor sclerostin (SOST). Affected patients have a high bone mass coupled with a distinctive appearance where the mandible is enlarged and the maxilla is foreshortened. Here, mice carrying a null mutation in Sost were analyzed using quantitative micro-computed tomographic (CT) imaging and histomorphometric analyses to determine the extent to which the size and shape of craniofacial skeleton were altered. Sost-/- mice exhibited a significant increase in appositional bone growth, which increased the height and width of the mandible and reduced the diameters of foramina. In vivo fluorochrome labeling, histology, and immunohistochemical analyses indicated that excessive bone deposition in the premaxillary suture mesenchyme curtailed overall growth, leading to midfacial hypoplasia. The amount of bone extracellular matrix produced by Sost-/- cells was significantly increased; as a consequence, osteoid seams were evident throughout the facial skeleton. Collectively, these analyses revealed a remarkable fidelity between human characteristics of sclerosteosis/van Buchem disease and the Sost-/- phenotype and provide clues into the conserved role for sclerostin signaling in modulating craniofacial morphology.

  View details for DOI 10.1177/0022034520963584

  View details for PubMedID 33078679

• Pro-osteogenic Effects of WNT in a Mouse Model of Bone Formation Around Femoral Implants. Calcified tissue international Li, Z., Yuan, X., Arioka, M., Bahat, D., Sun, Q., Chen, J., Helms, J. A. 2020

  Abstract

  Wnt signaling maintains homeostasis in the bone marrow cavity: if Wnt signaling is inhibited then bone volume and density would decline. In this study, we identified a population of Wnt-responsive cells as osteoprogenitor in the intact trabecular bone region, which were responsible for bone development and turnover. If an implant was placed into the long bone, this Wnt-responsive population and their progeny contributed to osseointegration. We employed Axin2CreCreERT2/+;R26mTmG/+ transgenic mouse strain in which Axin2-positive, Wnt-responsive cells, and their progeny are permanently labeled by GFP upon exposure to tamoxifen. Each mouse received femoral implants placed into a site prepared solely by drilling, and a single-dose liposomal WNT3A protein was used in the treatment group. A lineage tracing strategy design allowed us to identify cells actively expressing Axin2 in response to Wnt signaling pathway. These tools demonstrated that Wnt-responsive cells and their progeny comprise a quiescent population residing in the trabecular region. In response to an implant placed, this population becomes mitotically active: cells migrated into the peri-implant region, up-regulated the expression of osteogenic proteins. Ultimately, those cells gave rise to osteoblasts that produced significantly more new bone in the peri-implant region. Wnt-responsive cells directly contributed to implant osseointegration. Using a liposomal WNT3A protein therapeutic, we showed that a single application at the time of implant placed was sufficient to accelerate osseointegration. The Wnt-responsive cell population in trabecular bone, activated by injury, ultimately contributes to implant osseointegration. Liposomal WNT3A protein therapeutic accelerates implant osseointegration in the long bone.

  View details for DOI 10.1007/s00223-020-00757-5

  View details for PubMedID 32990765

• Formation and regeneration of a Wnt-responsive junctional epithelium. Journal of clinical periodontology Yuan, X., Chen, J., Van Brunt, L. A., Grauer, J., Xu, Q., Pei, X., Wang, L., Zhao, Y., Helms, J. A. 2020

  Abstract

  AIM: To identify the molecular mechanisms mediating the persistent defensive functions of the self-renewing junctional epithelium (JE).MATERIALS AND METHODS: Two strains of Wnt reporter mice, Axin2CreErt2/+ ;R26RmTmG/+ and Axin2LacZ/+ , were employed, along with three clinically relevant, experimental scenarios where the function of the JE is disrupted: after tooth extraction, after a partial gingivectomy and after a complete circumferential gingivectomy.RESULTS: Using transgenic Wnt reporter strains of mice, we established the JE is a Wnt-responsive epithelium beginning at the time of its formation, and that it maintains this status into adulthood. After tooth extraction, progeny of the initial Wnt-responsive JE population directly contributed to healing, and ultimately adopted an oral epithelium (OE) phenotype. In the traditional partial gingivectomy model, the JE completely regenerated and did so via progeny of the original Wnt-responsive population. However, following circumferential gingivectomy, the OE was incapable of reestablishing a functional JE.CONCLUSIONS: A Wnt-responsive niche at the interface between tooth and oral epithelia is required for a functional JE.

  View details for DOI 10.1111/jcpe.13371

  View details for PubMedID 32991010

• The Junctional Epithelium Is Maintained by a Stem Cell Population. Journal of dental research Yuan, X., Chen, J., Gauer, J., Xu, Q., Van Brunt, L. A., Helms, J. A. 2020: 22034520960125

  Abstract

  The most fundamental function of an epithelial tissue is to act as a barrier, regulating interactions between the external environment and the body. This barrier function typically requires a contiguous cell layer but since teeth penetrate the oral epithelium, a modified barrier has evolved, called the junctional epithelium (JE). In health, the JE attaches to the tooth, sealing the inside of the body against oral micro-organisms. Breakdown of the JE barrier results in periodontal ligament (PDL) disintegration, alveolar bone resorption, and ultimately tooth loss. Using lineage tracing and DNA pulse-chase analyses, we identified an anatomical location in the JE that supported both fast- and slow-cycling Wnt-responsive stem cells that contributed to self-renewal of the tissue. Stem cells produced daughter cells with an extraordinarily high rate of turnover that maintained JE integrity for 1.4 y in mice. Blocking cell proliferation via a chemotherapeutic agent 5-fluorouracil (5-Fu) eliminated fast-cycling stem cells, which caused JE degeneration, PDL destruction, and bone resorption. Upon removal of 5-Fu, slow-cycling stem cells regenerated both the structure and barrier function of the JE. Taken together, our studies identified a stem cell population in the JE and have potential clinical implications for prevention and treatment of periodontitis.

  View details for DOI 10.1177/0022034520960125

  View details for PubMedID 32985318

• Mechano-adaptive Responses of Alveolar Bone to Implant Hyper-loading in a pre-clinical in vivo model. Clinical oral implants research Tian, Y., Li, Z., Chen, J., Yuan, X., Sadowsky, S. J., Coyac, B. R., Brunski, J. B., Helms, J. A. 2020

  Abstract

  OBJECTIVES: Oral implants transmit biting forces to peri-implant bone. In turn, those forces subject peri-implant bone to mechanical stresses and strains. Here, our objective was to understand how peri-implant bone responded to conditions of normal vs. hyper-loading in a mouse model.MATERIAL AND METHODS: Sixty-six mice were randomly assigned to 2 groups; both groups underwent bilateral maxillary first molar extraction followed by complete healing. Titanium alloy implants were placed in healed sites and positioned below the occlusal plane. After osseointegration, a composite crown was affixed to the implant so masticatory loading would ensue. In controls, the remaining dentition was left intact but in the hyper-loaded (test) group, the remaining molars were extracted. 3D finite element analyses (FEA) calculated peri-implant strains resulting from normal and hyper-loading. Peri-implant tissues were analyzed at multiple time points using micro-computed tomography (CT) imaging, histology, enzymatic assays of bone remodeling, and vital dye labeling to evaluate bone accrual.RESULTS: Compared to controls, hyper-loaded implants experienced a 3.6-fold increase in occlusal force, producing higher peri-implant strains. Bone formation and resorption were both significantly elevated around hyper-loaded implants, eventually culminating in a significant increase in peri-implant bone volume/total volume (BV/TV). In our mouse model, masticatory hyper-loading of an osseointegrated implant was associated with increased peri-implant strain, increased peri-implant bone remodeling, and a net gain in bone deposition.CONCLUSION: Hyper-loading results in bone strain with catabolic and anabolic bone responses, leading to a net gain in bone deposition.

  View details for DOI 10.1111/clr.13662

  View details for PubMedID 32882082

• Bone formation around unstable implants is enhanced by a WNT protein therapeutic in a preclinical in vivo model. Clinical oral implants research Coyac, B. R., Leahy, B., Li, Z., Salvi, G., Yin, X., Brunski, J. B., Helms, J. A. 2020

  Abstract

  OBJECTIVES: Our objective was to test the hypothesis that local delivery of a WNT protein therapeutic would support osseointegration of an unstable implant placed into an oversized osteotomy and subjected to functional loading.MATERIALS AND METHODS: Using a split-mouth design in an ovariectomized (OVX) rat model, 50 titanium implants were placed in oversized osteotomies. Implants were subjected to functional loading. One-half of the implants were treated with a liposomal formulation of WNT3A protein (L-WNT3A); the other half received an identical liposomal formulation containing phosphate buffered saline (PBS). Finite element modeling estimated peri-implant strains caused by functional loading. Histological, molecular, cellular, and quantitative micro-computed tomographic (CT) imaging analyses were performed on samples from post-implant days (PID) 3, 7, and 14. Lateral implant stability was quantified at PID7 and 14.RESULTS: Finite element analyses predicted levels of peri-implant strains incompatible with new bone formation. Micro-CT imaging, histological, and quantitative immunohistochemical (IHC) analyses confirmed that PBS-treated implants underwent fibrous encapsulation. In those cases where the peri-implant environment was treated with L-WNT3A, CT imaging, histological, and quantitative IHC analyses demonstrated a significant increase in expression of proliferative (PCNA) and osteogenic (Runx2, Osterix) markers. One week after L-WNT3A treatment, new bone formation was evident and two weeks later, L-WNT3A treated gaps had a stiffer interface compared to PBS-treated gaps.CONCLUSION: In a rat model, unstable implants undergo fibrous encapsulation. If the same unstable implants are treated with L-WNT3A at the time of placement, then it results in significantly more peri-implant bone and greater interfacial stiffness.

  View details for DOI 10.1111/clr.13659

  View details for PubMedID 32881143

• Interspecies Comparison of Alveolar Bone Biology, Part I: Morphology and Physiology of Pristine Bone. JDR clinical and translational research Pilawski, I., Tulu, U. S., Ticha, P., Schupbach, P., Traxler, H., Xu, Q., Pan, J., Coyac, B. R., Yuan, X., Tian, Y., Liu, Y., Chen, J., Erdogan, Y., Arioka, M., Armaro, M., Wu, M., Brunski, J. B., Helms, J. A. 2020: 2380084420936979

  Abstract

  INTRODUCTION: Few interspecies comparisons of alveolar bone have been documented, and this knowledge gap raises questions about which animal models most accurately represent human dental conditions or responses to surgical interventions.OBJECTIVES: The objective of this study was to employ state-of-the-art quantitative metrics to directly assess and compare the structural and functional characteristics of alveolar bone among humans, mini pigs, rats, and mice.METHODS: The same anatomic location (i.e., the posterior maxillae) was analyzed in all species via micro-computed tomographic imaging, followed by quantitative analyses, coupled with histology and immunohistochemistry. Bone remodeling was evaluated with alkaline phosphatase activity and tartrate-resistant acid phosphatase staining to identify osteoblast and osteoclast activities. In vivo fluorochrome labeling was used as a means to assess mineral apposition rates.RESULTS: Collectively, these analyses demonstrated that bone volume differed among the species, while bone mineral density was equal. All species showed a similar density of alveolar osteocytes, with a highly conserved pattern of collagen organization. Collagen maturation was equal among mouse, rat, and mini pig. Bone remodeling was a shared feature among the species, with morphologically indistinguishable hemiosteonal appearances, osteocytic perilacunar remodeling, and similar mineral apposition rates in alveolar bone.CONCLUSIONS: Our analyses demonstrated equivalencies among the 4 species in a plurality of the biological features of alveolar bone. Despite contradictory results from older studies, we found no evidence for the superiority of pig models over rodent models in representing human bone biology.Knowledge Transfer Statement: Animal models are extensively used to evaluate bone tissue engineering strategies, yet there are few state-of-the-art studies that rigorously compare and quantify the factors influencing selection of a given animal model. Consequently, there is an urgent need to assess preclinical animal models for their predictive value to dental research. Our article addresses this knowledge gap and, in doing so, provides a foundation for more effective standardization among animal models commonly used in dentistry.

  View details for DOI 10.1177/2380084420936979

  View details for PubMedID 32660303

• Interspecies comparison of alveolar bone biology: Tooth extraction socket healing in mini pigs and mice. Journal of periodontology Pan, J., Pilawski, I., Yuan, X., Arioka, M., Ticha, P., Tian, Y., Helms, J. A. 2020

  Abstract

  BACKGROUND: in an effort to identify and validate which animal models are best suited for dental implant research, we used multiscale analyses to examine tooth extraction wound healing in a well-accepted model, the Yucatan mini pig and a more controversial model, the laboratory mouse.METHODS: first molar extractions were performed in adult, skeletally mature mini pigs and mice. Alveolar bone repair was evaluated at early, intermediate and late timepoints using quantitative micro-computed tomographic (muCT) imaging, histology, molecular, and cellular assays. Vital dye labeling was employed to quantify mineral apposition rates (MAR) in both species.RESULTS: Despite a 3000-fold difference in weight, the relative proportions of the mini pig and murine maxillae and are equivalent. Quantitative muCT demonstrated that within the posterior alveolar bone, the volume of mineralized bone was lower in mini pig than in the mice; during healing, however, the bone volume fraction was equivalent. The histologic appearance of healing sites was also comparable, and alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP) staining showed a similar temporal and spatial distribution of bone remodeling. Vital dye labeling indicated equivalent MAR between the species. The absolute duration of the healing period differed: In mice, complete healing was accomplished in 21 days. In mini pigs, the same process took four times longer.CONCLUSIONS: Extraction socket healing is histologically equivalent between mini pigs and mice, supporting the hypothesis that the underlying mechanisms of alveolar bone healing are conserved amongst species. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/JPER.19-0667

  View details for PubMedID 32347546

• Effects of condensation and compressive strain on implant primary stability A longitudinal, in vivo, multiscale study in mice BONE & JOINT RESEARCH Li, Z., Arioka, M., Liu, Y., Aghvami, M., Tulu, S., Brunski, J. B., Helms, J. A. 2020; 9 (2): 60–70

  View details for DOI 10.1302/2046-3758.92.BJR-2019-0161

  View details for Web of Science ID 000519558300002

• Bioactivating a bone substitute accelerates graft incorporation in a murine model of vertical ridge augmentation. Dental materials : official publication of the Academy of Dental Materials Chen, J. n., Yuan, X. n., Li, Z. n., Bahat, D. J., Helms, J. A. 2020

  Abstract

  Compared to autologous bone grafts, allogeneic bone grafts integrate slowly, which can adversely affect clinical outcomes. Here, our goal was to understand the molecular mechanisms underlying graft incorporation, and then test clinically feasible methods to accelerate this process.Wild-type and transgenic Wnt "reporter" mice were used in a vertical ridge augmentation procedure. The surgery consisted of tunneling procedure to elevate the maxillary edentulous ridge periosteum, followed by the insertion of bone graft. Micro-computed tomographic imaging, and molecular/cellular analyses were used to follow the bone graft over time. Sclerostin null mice, and mice carrying an activated form of β-catenin were evaluated to understand how elevated Wnt signaling impacted edentulous ridge height and based on these data, a biomimetic strategy was employed to combine bone graft particles with a formulation of recombinant WNT protein. Thereafter, the rate of graft incorporation was evaluated.Tunneling activated osteoprogenitor cell proliferation from the periosteum. If graft particles were present, then osteoprogenitor cells attached to the matrix and gave rise to new bone that augmented edentulous ridge height. Graft particles alone did not stimulate osteoprogenitor cell proliferation. Based on the thicker edentulous ridges in mice with amplified Wnt signaling, a strategy was undertaken to load bone graft particles with WNT; this combination was sufficient to accelerate the initial step of graft incorporation.Local delivery of a WNT protein therapeutic has the potential to accelerate graft incorporation, and thus shorten the time to when the graft can support a dental implant.

  View details for DOI 10.1016/j.dental.2020.06.003

  View details for PubMedID 32651017

• Optimizing autologous bone contribution to implant osseointegration. Journal of periodontology Coyac, B. R., Sun, Q. n., Leahy, B. n., Salvi, G. n., Yuan, X. n., Brunski, J. B., Helms, J. A. 2020

  Abstract

  Autologous bone can be harvested from the flutes of a conventional drill or from a bone scraper; here we compared whether autologous bone chips generated by a new slow-speed instrument were more osteogenic than the bone chips generated by conventional drills or bone scrapers. Additionally, we tested whether the osteogenic potential of bone chips could be further improved by exposure to a WNT therapeutic.Osteotomies were prepared in fresh rat maxillary first molar extraction sockets using a conventional drill or a new osseo-shaping instrument; titanium alloy implants were placed immediately thereafter. Using molecular/cellular and histologic analyses, the fates of the resulting bone chips were analyzed. To test whether increasing WNT signaling improved osteogenesis in an immediate post-extraction implant environment, a WNT therapeutic was introduced at the time of implant placement.Bone collected from a conventional drill exhibited extensive apoptosis; in contrast, bone generated by the new instrument remained in situ, which preserved their viability. Also preserved was the viability of the osteoprogenitor cells attached to the bone chips. Exogenous treatment with a WNT therapeutic increased the rate of osteogenesis around immediate post-extraction implants.Compared with conventional drills or bone scrapers, a new cutting instrument enabled concomitant site preparation with autologous bone chip collection. Histology/histomorphometric analyses revealed that the bone chips generated by this new tool were more osteogenic and could be further enhanced by exposure to a WNT therapeutic. Even though gaps still existed in L-PBS and L-WNT3A cases, the area of peri-implant bone was significantly greater in L-WNT3A treated sites. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/JPER.19-0524

  View details for PubMedID 32279310

• Root resorption and ensuing cementum repair by Wnt/β-catenin dependent mechanism. American journal of orthodontics and dentofacial orthopedics : official publication of the American Association of Orthodontists, its constituent societies, and the American Board of Orthodontics Turkkahraman, H. n., Yuan, X. n., Salmon, B. n., Chen, C. H., Brunski, J. B., Helms, J. A. 2020

  Abstract

  Physiological root resorption is a common occurrence in mammalian teeth, which suggests that there must be a corollary consisting of physiological cementum repair. The mechanism(s) responsible for this physiological repair process is unknown and was the focus of this study.Using a rat model, we explored first the prevalence of physiological root resorption and then asked whether this prevalence changed as a result of an osteoporotic phenotype. The cellular mechanisms of resorption were characterized using a combination of finite element modeling coupled with in-vivo histologic, molecular, and cellular analyses in rats. A potential molecular mechanism for cementum repair was uncovered using a strain of transgenic mice in which Wnt-responsive cells could be labeled and followed over time.In rats, most resorption lacunae were concentrated on the distal surfaces of the roots. Rat molars undergo a physiological tooth drift distally, and using finite element modeling, we calculated the magnitude of the compressive strains that accumulated on these surfaces in response to mastication. Although the overall strain magnitudes were low, they were constant and coincided with the presence of resorption lacunae. Where resorption lacunae were present, progeny from a Wnt-responsive population of stem cells, embedded in the periodontal ligament, directly contributed to the repair of the lacunae.Despite the fact that both are clastic conditions, an osteoporotic phenotype in rats was not associated with an increase in the prevalence of physiological root resorption. The location of the resorption lacunae corresponded to sites of low but constant compressive strains produced by physiological distal drift. At least 1 mechanism responsible for physiological cementum repair involved the contribution of Wnt-responsive stem or progenitor cells originating in the periodontal ligament. These data point toward a potential Wnt-based strategy to regenerate cementum in subjects with disease or damage.

  View details for DOI 10.1016/j.ajodo.2019.06.021

  View details for PubMedID 32381434

• A novel system exploits bone debris for implant osseointegration. Journal of periodontology Coyac, B. R., Salvi, G. n., Leahy, B. n., Li, Z. n., Salmon, B. n., Hoffmann, W. n., Helms, J. A. 2020

  Abstract

  Bone debris generated during site preparation is generally evacuated with irrigation; here, we evaluated whether retention of this autologous material improved the rate of peri-implant bone formation.In 25 rats, a miniatured implant system comprised of an osseo-shaping tool and a tri-oval shaped implant was compared against a conventional drill and round implant system. A split-mouth design was used, and fresh extraction sockets served as implant sites. Histology/histomorphometry, immunohistochemistry, and μCT imaging were performed immediately after implant placement, and on post-surgery days 3, 7, 14 and 28.Compared to a conventional drill design, the osseo-shaping tool produced a textured osteotomy surface and viable bone debris that was retained in the peri-implant environment. Proliferating osteoprogenitor cells, identified by PCNA and Runx2 expression, contributed to faster peri-implant bone formation. Although all implants osseointegrated, sites prepared with the osseo-shaping tool showed evidence of new peri-implant bone sooner than controls.Bone debris produced by an osseo-shaping tool directly contributed to faster peri-implant bone formation and implant osseointegration. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/JPER.20-0099

  View details for PubMedID 32829495

• Wnt responsive progenitor cells contribute to osseointegration of implants in lone bone Li, Z., Yuan, X., Helms, J. WILEY. 2019: 86–87

  View details for Web of Science ID 000508614700252

• Wnt responsive progenitor cells contribute to osseointegration of implants in lone bone. Li, Z., Yuan, X., Helms, J. WILEY. 2019: 86–87

  View details for Web of Science ID 000508356601081

• Improving intraoperative storage conditions for autologous bone grafts: an experimental investigation in mice. Journal of tissue engineering and regenerative medicine Sun, Q., Li, Z., Liu, B., Yuan, X., Guo, S., Helms, J. 2019

  Abstract

  Autologous bone grafts constitute the second most transplanted tissue in medicine today. The viability, and consequently the osteogenic capacity, of an autograft is directly impacted by the interval between harvest and transplantation, but how the temperature and the solution in which the graft is held intraoperatively affect viability is not clear. Using a syngeneic mouse model and in vivo bone-forming assays, these variables were tested for their effects on programmed cell death, osteoprogenitor cell proliferation, and ultimately the ability of the autograft to produce new bone in an ectopic site. Based on these results, the intraoperative treatment with a WNT protein therapeutic was tested for its effects on the viability and osteogenic capacity of an autograft. Viability, programmed cell death, mitotic activity, osteogenic protein expression, and bone-forming capacity were assessed. Experimental results demonstrated that the osteogenic capacity of an autograft is significantly improved by intraoperative storage in L-WNT3A at physiological temperature.

  View details for DOI 10.1002/term.2970

  View details for PubMedID 31617958

• Wnt-Responsive Stem Cell Fates in the Oral Mucosa. iScience Yuan, X., Xu, Q., Zhang, X., Van Brunt, L. A., Ticha, P., Helms, J. A. 2019; 21: 84–94

  Abstract

  Epithelia of the oral cavity exhibit variations in morphologies and turnover rates. Are these differences related to environment or to region-specific stem cell populations? A lineage-tracing strategy allowed visualization of Wnt-responsive cells, and their progeny, in the hard and soft palates. In both anatomic locations, Wnt-responsive basal cells self-renewed and gave rise to supra-basal cells. Palatal injuries triggered an enlargement of this population, and their descendants were responsible for wound re-epithelialization. Compared with the hard palate, soft palate stem cells exhibited an earlier, more robust burst in proliferation, culminating in significantly faster repair. Thereafter, excess Wnt-responsive basal cells were removed, and stem cell numbers were restored back to homeostatic level. Thus, we uncovered a stem cell population in oral mucosa, and its relative abundance is correlate with the rate of oral wound healing. Besides the activation during injury, an endogenous mechanism exists to constrain the stem cell pool after repair.

  View details for DOI 10.1016/j.isci.2019.10.016

  View details for PubMedID 31655258

• A preclinical model links osseo-densification due to misfit and osseo-destruction due to stress/strain. Clinical oral implants research Coyac, B. R., Leahy, B., Salvi, G., Hoffmann, W., Brunski, J. B., Helms, J. A. 2019

  Abstract

  OBJECTIVE: Primary stability is a prerequisite for implant osseointegration. Some degree of misfit between an implant and its osteotomy is required to ensure primary stability, and this is typically achieved by undersizing an implant osteotomy. In this preclinical study, we aimed at understanding the relationship between misfit, insertion torque, implant stability, and their cumulative short- and longer-term effects on peri-implant bone.MATERIALS AND METHODS: We placed implants in maxillary extraction sites of a rat; in the control group, these implants had minimal misfit while those in the test group had a high degree of misfit and therefore osseo-densified the peri-implant bone.RESULTS: Compared to controls, the misfit-induced stresses produced by osseo-densification led to micro-fractures in the peri-implant bone and an extensive zone of dying osteocytes. High interfacial pressures produced a pro-resorptive environment as shown by TRAP activity and Cathepsin K immunostaining. The lack of ALP activity and Collagen I IHC supported the absence of new bone formation. Collectively, CT imaging, quantification of Bone Implant Contact (BIC), Vimentin and IL1-beta IHCs demonstrated that implant failure occurred soon afterwards, which presented as a crater-like lesion filled with fibrous, inflamed granulation tissue around the test implants.CONCLUSION: By controlling every other risk indicator, we confirmed how excessive osseo-densification can lead directly to osseo-destruction.

  View details for DOI 10.1111/clr.13537

  View details for PubMedID 31520494

• A Correlation between Wnt/Beta-catenin Signaling and the Rate of Dentin Secretion. Journal of endodontics Zhao, Y., Yuan, X., Bellido, T., Helms, J. A. 2019

  Abstract

  INTRODUCTION: Odontoblasts produce dentin throughout life and in response to trauma. The purpose of this study was to identify the roles of endogenous Wnt signaling in regulating the rate of dentin accumulation.METHODS: Histology, immunohistochemistry, vital dye labeling, and histomorphometric assays were used to quantify the rate of dentin accumulation as a function of age. Two strains of Wnt reporter mice were used to identify and follow the distribution and number of Wnt-responsive odontoblasts as a function of age. To show a causal relationship between dentin secretion and Wnt signaling, dentin accumulation was monitored in a strain of mice in which Wnt signaling was aberrantly elevated.RESULTS: Dentin deposition occurs throughout life, but the rate of accumulation slows with age. This decline in dentin secretion correlates with a decrease in endogenous Wnt signaling. In a genetically modified strain of mice, instead of tubular dentin, aberrantly elevated Wnt signaling resulted in accumulation of reparative dentin or osteodentin secreted from predontoblasts.CONCLUSIONS: Wnt signaling regulates dentin secretion by odontoblasts, and the formation of reparative or osteodentin is the direct consequence of elevated Wnt signaling. These preclinical data have therapeutic implications for the development of a biologically based pulp capping medicant.

  View details for DOI 10.1016/j.joen.2019.07.014

  View details for PubMedID 31522810

• WNT3A accelerates delayed alveolar bone repair in ovariectomized mice. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA Liu, Y., Li, Z., Arioka, M., Wang, L., Bao, C., Helms, J. A. 2019

  Abstract

  Our goal was to evaluate alveolar bone healing in OVX mice, and to assess the functional utility of a WNT-based treatment to accelerate healing in mice with an osteoporotic-like bony phenotype.INTRODUCTION: Is osteoporosis a risk factor for dental procedures? This relatively simple question is exceedingly difficult to answer in a clinical setting, for two reasons. First, as an age-related disease, osteoporosis is frequently accompanied by age-related co-morbidities that can contribute to slower tissue repair. Second, the intervals at which alveolar bone repair are assessed in a clinical study are often measured in months to years. This study aimed to evaluate alveolar bone repair in ovariectomized (OVX) mice and provide preclinical evidence to support a WNT-based treatment to accelerate alveolar bone formation.METHODS: OVX was performed in young mice to produce an osteoporotic-like bone phenotype. Thereafter, the rate of extraction socket healing and osteotomy repair was assessed. A liposomal WNT3A treatment was tested for its ability to promote alveolar bone formation in this OVX-induced model of bone loss.RESULTS: Bone loss was observed throughout the murine skeleton, including the maxilla, and mirrored the pattern of bone loss observed in aged mice. Injuries to the alveolar bone, including tooth extraction and osteotomy site preparation, both healed significantly slower than the same injuries produced in young controls. Given sufficient time, however, all injuries eventually healed. In OVX mice, osteotomies healed significantly faster if they were treated with L-WNT3A.CONCLUSIONS: Alveolar bone injuries heal slower in OVX mice that exhibit an osteoporotic-like phenotype. The rate of alveolar bone repair in OVX mice can be significantly promoted with local delivery of L-WNT3A.

  View details for DOI 10.1007/s00198-019-05071-x

  View details for PubMedID 31338519

• A novel hypothesis based on clinical, radiological, and histological data to explain the dentinogenesis imperfecta type II phenotype. Connective tissue research Turkkahraman, H., Galindo, F., Tulu, U. S., Helms, J. A. 2019: 1–11

  Abstract

  Purpose/Aim: The aim of this study was to explore whether dentinogenesis imperfecta (DGI)-related aberrations are detectable in odontogenic tissues. Materials and Methods: Morphological and histological analyses were carried out on 3 teeth (two maxillary 1st molars, one maxillary central incisor) extracted from a patient with DGI Type II. A maxillary 2nd molar teeth extracted from a healthy patient was used as control. A micro-computed tomographic (muCT) data-acquisition system was used to scan and reconstruct samples. Pentachrome and picrosirius red histologic stains were used to analyze odontogenic tissues and their collagenous matrices. Results: Our findings corroborate DGI effects on molar and incisor root elongation, and the hypo-mineralized state of DGI dentin. In addition to these findings, we discovered changes to the DGI pulp cavity: Reactionary dentin formation, which we theorize is exacerbated by the early loss of enamel, nearly obliterated an acellular but still-vascularized DGI pulp cavity. We also discovered an accumulation of lamellated cellular cementum at the root apices, which we hypothesize compensates for the severe and rapid attrition of the DGI tooth. Conclusions: Based on imaging and histological data, we propose a novel hypothesis to explain the complex dental phenotypes observed in patients with DGI Type II.

  View details for DOI 10.1080/03008207.2019.1631296

  View details for PubMedID 31284784

• Mechanoadaptive Responses in the Periodontium Are Coordinated by Wnt JOURNAL OF DENTAL RESEARCH Xu, Q., Yuan, X., Zhang, X., Chen, J., Shi, Y., Brunski, J. B., Helms, J. A. 2019; 98 (6): 689–97

  View details for DOI 10.1177/0022034519839438

  View details for Web of Science ID 000470871200012

• Mechanoadaptive Responses in the Periodontium Are Coordinated by Wnt. Journal of dental research Xu, Q., Yuan, X., Zhang, X., Chen, J., Shi, Y., Brunski, J. B., Helms, J. A. 2019: 22034519839438

  Abstract

  Despite an extensive literature documenting the adaptive changes of bones and ligaments to mechanical forces, our understanding of how tissues actually mount a coordinated response to physical loading is astonishingly inadequate. Here, using finite element (FE) modeling and an in vivo murine model, we demonstrate the stress distributions within the periodontal ligament (PDL) caused by occlusal hyperloading. In direct response, a spatially restricted pattern of apoptosis is triggered in the stressed PDL, the temporal peak of which is coordinated with a spatially restricted burst in PDL cell proliferation. This culminates in increased collagen deposition and a thicker, stiffer PDL that is adapted to its new hyperloading status. Meanwhile, in the adjacent alveolar bone, hyperloading activates bone resorption, the peak of which is followed by a bone formation phase, leading ultimately to an accelerated rate of mineral apposition and an increase in alveolar bone density. All of these adaptive responses are orchestrated by a population of Wnt-responsive stem/progenitor cells residing in the PDL and bone, whose death and revival are ultimately responsible for directly giving rise to new PDL fibers and new bone.

  View details for PubMedID 30971171

• Osteoporotic Changes in the Periodontium Impair Alveolar Bone Healing JOURNAL OF DENTAL RESEARCH Arioka, M., Zhang, X., Li, Z., Tulu, U. S., Liu, Y., Wang, L., Yuan, X., Helms, J. A. 2019; 98 (4): 450–58

  View details for DOI 10.1177/0022034518818456

  View details for Web of Science ID 000462084600010

• Mechanical and Biological Advantages of a Tri-Oval Implant Design. Journal of clinical medicine Yin, X., Li, J., Hoffmann, W., Gasser, A., Brunski, J. B., Helms, J. A. 2019; 8 (4)

  Abstract

  Of all geometric shapes, a tri-oval one may be the strongest because of its capacity to bear large loads with neither rotation nor deformation. Here, we modified the external shape of a dental implant from circular to tri-oval, aiming to create a combination of high strain and low strain peri-implant environment that would ensure both primary implant stability and rapid osseointegration, respectively. Using in vivo mouse models, we tested the effects of this geometric alteration on implant survival and osseointegration over time. The maxima regions of tri-oval implants provided superior primary stability without increasing insertion torque. The minima regions of tri-oval implants presented low compressive strain and significantly less osteocyte apoptosis, which led to minimal bone resorption compared to the round implants. The rate of new bone accrual was also faster around the tri-oval implants. We further subjected both round and tri-oval implants to occlusal loading immediately after placement. In contrast to the round implants that exhibited a significant dip in stability that eventually led to their failure, the tri-oval implants maintained their stability throughout the osseointegration period. Collectively, these multiscale biomechanical analyses demonstrated the superior in vivo performance of the tri-oval implant design.

  View details for DOI 10.3390/jcm8040427

  View details for PubMedID 30925746

• Osteoporotic Changes in the Periodontium Impair Alveolar Bone Healing. Journal of dental research Arioka, M., Zhang, X., Li, Z., Tulu, U. S., Liu, Y., Wang, L., Yuan, X., Helms, J. A. 2019: 22034518818456

  Abstract

  Osteoporosis is associated with decreased bone density and increased bone fragility, but how this disease affects alveolar bone healing is not clear. The objective of this study was to determine the extent to which osteoporosis affects the jaw skeleton and then to evaluate possible mechanisms whereby an osteoporotic phenotype might affect the rate of alveolar bone healing following tooth extraction. Using an ovariectomized mouse model coupled with micro-computed tomographic imaging, histologic, molecular, and cellular assays, we first demonstrated that the appendicular and jaw skeletons both develop osteoporotic phenotypes. Next, we demonstrated that osteoporotic mice exhibit atrophy of the periodontal ligament (PDL) and that this atrophy was accompanied by a reduction in the pool of osteoprogenitor cells in the PDL. The paucity of PDL-derived osteoprogenitor cells in osteoporotic mice was associated with significantly slower extraction socket healing. Collectively, these analyses demonstrate that the jaw skeleton is susceptible to the untoward effects of osteoporosis that manifest as thinner, more porous alveolar bone, PDL thinning, and slower bone repair. These findings have potential clinical significance for older osteopenic patients undergoing reconstructive procedures.

  View details for PubMedID 30626268

• Molecular Basis for Periodontal Ligament Adaptation to In Vivo Loading. Journal of dental research Zhang, X., Yuan, X., Xu, Q., Arioka, M., Van Brunt, L. A., Shi, Y., Brunski, J., Helms, J. A. 2019: 22034518817305

  Abstract

  A soft food diet leads to changes in the periodontal ligament (PDL). These changes, which have been recognized for more than a century, are ascribed to alterations in mechanical loading. While these adaptive responses have been well characterized, the molecular, cellular, and mechanical mechanisms underlying the changes have not. Here, we implicate Wnt signaling in the pathoetiology of PDL responses to underloading. We show that Wnt-responsive cells and their progeny in the PDL space exhibit a burst in proliferation in response to mastication. If an animal is fed a soft diet from the time of weaning, then this burst in Wnt-responsive cell proliferation is quelled; as a consequence, both the PDL and the surrounding alveolar bone undergo atrophy. Returning these animals to a hard food diet restores the Wnt signaling in PDL. These data provide, for the first time, a molecular mechanism underlying the adaptive response of the PDL to loading.

  View details for PubMedID 30612508

• Systemic Immunologic Consequences of Chronic Periodontitis. Journal of dental research Gaudilliere, D. K., Culos, A. n., Djebali, K. n., Tsai, A. S., Ganio, E. A., Choi, W. M., Han, X. n., Maghaireh, A. n., Choisy, B. n., Baca, Q. n., Einhaus, J. F., Hedou, J. J., Bertrand, B. n., Ando, K. n., Fallahzadeh, R. n., Ghaemi, M. S., Okada, R. n., Stanley, N. n., Tanada, A. n., Tingle, M. n., Alpagot, T. n., Helms, J. A., Angst, M. S., Aghaeepour, N. n., Gaudilliere, B. n. 2019: 22034519857714

  Abstract

  Chronic periodontitis (ChP) is a prevalent inflammatory disease affecting 46% of the US population. ChP produces a profound local inflammatory response to dysbiotic oral microbiota that leads to destruction of alveolar bone and tooth loss. ChP is also associated with systemic illnesses, including cardiovascular diseases, malignancies, and adverse pregnancy outcomes. However, the mechanisms underlying these adverse health outcomes are poorly understood. In this prospective cohort study, we used a highly multiplex mass cytometry immunoassay to perform an in-depth analysis of the systemic consequences of ChP in patients before (n = 28) and after (n = 16) periodontal treatment. A high-dimensional analysis of intracellular signaling networks revealed immune system-wide dysfunctions differentiating patients with ChP from healthy controls. Notably, we observed exaggerated proinflammatory responses to Porphyromonas gingivalis-derived lipopolysaccharide in circulating neutrophils and monocytes from patients with ChP. Simultaneously, natural killer cell responses to inflammatory cytokines were attenuated. Importantly, the immune alterations associated with ChP were no longer detectable 3 wk after periodontal treatment. Our findings demarcate systemic and cell-specific immune dysfunctions in patients with ChP, which can be temporarily reversed by the local treatment of ChP. Future studies in larger cohorts are needed to test the boundaries of generalizability of our results.

  View details for DOI 10.1177/0022034519857714

  View details for PubMedID 31226001

• Relationship Between Primary/Mechanical and Secondary/Biological Implant Stability INTERNATIONAL JOURNAL OF ORAL & MAXILLOFACIAL IMPLANTS Monje, A., Ravida, A., Wang, H., Helms, J. A., Brunski, J. B. 2019; 34: S7-+

  Abstract

  This systematic review was prepared as part of the Academy of Osseointegration (AO) 2018 Summit, held August 8-10 in Oak Brook Hills, Illinois, to assess the relationship between the primary (mechanical) and secondary (biological) implant stability.Electronic and manual searches were conducted by two independent examiners in order to address the following issues. Meta-regression analyses explored the relationship between primary stability, as measured by insertion torque (IT) and implant stability quotient (ISQ), and secondary stability, by means of survival and peri-implant marginal bone loss (MBL).Overall, 37 articles were included for quantitative assessment. Of these, 17 reported on implant stability using only resonance frequncy analysis (RFA), 11 used only IT data, 7 used a combination of RFA and IT, and 2 used only the Periotest. The following findings were reached: ·Relationship between primary and secondary implant stability: Strong positive statistically significant relationship (P < .001). ·Relationship between primary stability by means of ISQ and implant survival: No statistically significant relationship (P = .4). ·Relationship between IT and implant survival: No statistically significant relationship (P = .2). ·Relationship between primary stability by means of ISQ unit and MBL: No statistically significant relationship (P = .9). ·Relationship between IT and MBL: Positive statistically significant relationship (P = .02). ·Accuracy of methods and devices to assess implant stability: Insufficient data to address this issue.Data suggest that primary/mechanical stability leads to more efficient achievement of secondary/biological stability, but the achievement of high primary stability might be detrimental for bone level stability. While current methods/devices for tracking implant stability over time can be clinically useful, a robust connection between existing stability metrics with implant survival remains inconclusive.

  View details for DOI 10.11607/jomi.19suppl.g1

  View details for Web of Science ID 000476794800002

  View details for PubMedID 31116830

• A Novel Osteotomy Preparation Technique to Preserve Implant Site Viability and Enhance Osteogenesis. Journal of clinical medicine Chen, C. H., Coyac, B. R., Arioka, M. n., Leahy, B. n., Tulu, U. S., Aghvami, M. n., Holst, S. n., Hoffmann, W. n., Quarry, A. n., Bahat, O. n., Salmon, B. n., Brunski, J. B., Helms, J. A. 2019; 8 (2)

  Abstract

  The preservation of bone viability at an osteotomy site is a critical variable for subsequent implant osseointegration. Recent biomechanical studies evaluating the consequences of site preparation led us to rethink the design of bone-cutting drills, especially those intended for implant site preparation. We present here a novel drill design that is designed to efficiently cut bone at a very low rotational velocity, obviating the need for irrigation as a coolant. The low-speed cutting produces little heat and, consequently, osteocyte viability is maintained. The lack of irrigation, coupled with the unique design of the cutting flutes, channels into the osteotomy autologous bone chips and osseous coagulum that have inherent osteogenic potential. Collectively, these features result in robust, new bone formation at rates significantly faster than those observed with conventional drilling protocols. These preclinical data have practical implications for the clinical preparation of osteotomies and alveolar bone reconstructive surgeries.

  View details for PubMedID 30717291

• Aberrantly elevated Wnt signaling is responsible for cementum overgrowth and dental ankylosis. Bone Wu, Y., Yuan, X., Perez, K. C., Hyman, S., Wang, L., Pellegrini, G., Salmon, B., Bellido, T., Helms, J. A. 2018

  Abstract

  Vertebrate teeth are attached to the jawbones using a variety of methods but in mammals, a fibrous connection is the norm. This fibrous periodontal ligament (PDL) allows teeth to move in the jawbones in response to natural eruptive forces, mastication, and orthodontic tooth movement. In some disease states the PDL either calcifies or is replaced by a mineralized tissue and the result is ankylosis, where the tooth is fused to the alveolar bone. To understand how the PDL maintains this fibrous state we examined a strain of mice in which tooth movement is arrested. DabetacatOt mice express a stabilized form of beta-catenin in DMP1-positive alveolar bone osteocytes and cementocytes, which results in elevated Wnt signaling throughout the periodontium. As a consequence, there is an accrual of massive amounts of cellular cementum and alveolar bone, the PDL itself calcifies and teeth become ankylosed. These data suggest that to maintain its fibrous nature, Wnt signaling must normally be repressed in the PDL space.

  View details for DOI 10.1016/j.bone.2018.10.023

  View details for PubMedID 30408613

• A Thermal and Biological Analysis of Bone Drilling. Journal of biomechanical engineering Aghvami, M., Brunski, J. B., Serdar Tulu, U., Chen, C., Helms, J. A. 2018; 140 (10)

  Abstract

  With the introduction of high-speed cutting tools, clinicians have recognized the potential for thermal damage to the material being cut. Here, we developed a mathematical model of heat transfer caused by drilling bones of different densities and validated it with respect to experimentally measured temperatures in bone. We then coupled these computational results with a biological assessment of cell death following osteotomy site preparation. Parameters under clinical control, e.g., drill diameter, rotational speed, and irrigation, along with patient-specific variables such as bone density were evaluated in order to understand their contributions to thermal damage. Predictions from our models provide insights into temperatures and thresholds that cause osteocyte death and that can ultimately compromise stability of an implant.

  View details for PubMedID 30029243

• An osteopenic/osteoporotic phenotype delays alveolar bone repair. Bone Chen, C., Wang, L., Serdar Tulu, U., Arioka, M., Moghim, M. M., Salmon, B., Chen, C., Hoffmann, W., Gilgenbach, J., Brunski, J. B., Helms, J. A. 2018; 112: 212–19

  Abstract

  Aging is associated with a function decline in tissue homeostasis and tissue repair. Aging is also associated with an increased incidence in osteopenia and osteoporosis, but whether these low bone mass diseases are a risk factor for delayed bone healing still remains controversial. Addressing this question is of direct clinical relevance for dental patients, since most implants are performed in older patients who are at risk of developing low bone mass conditions. The objective of this study was to assess how an osteopenic/osteoporotic phenotype affected the rate of new alveolar bone formation. Using an ovariectomized (OVX) rat model, the rates of tooth extraction socket and osteotomy healing were compared with age-matched controls. Imaging, along with molecular, cellular, and histologic analyses, demonstrated that OVX produced an overt osteoporotic phenotype in long bones, but only a subtle phenotype in alveolar bone. Nonetheless, the OVX group demonstrated significantly slower alveolar bone healing in both the extraction socket, and in the osteotomy produced in a healed extraction site. Most notably, osteotomy site preparation created a dramatically wider zone of dying and dead osteocytes in the OVX group, which was coupled with more extensive bone remodeling and a delay in the differentiation of osteoblasts. Collectively, these analyses demonstrate that the emergence of an osteoporotic phenotype delays new alveolar bone formation.

  View details for PubMedID 29704698

• WNT-activated bone grafts repair osteonecrotic lesions in aged animals (vol 7, 14254, 2017) SCIENTIFIC REPORTS Salmon, B., Liu, B., Shen, E., Chen, T., Li, J., Gillette, M., Ransom, R. C., Ezran, M., Johnson, C. A., Castillo, A. B., Shen, W. J., Kraemer, F. B., Smith, A. A., Helms, J. A. 2018; 8: 6356

  Abstract

  A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

  View details for PubMedID 29662149

• Effects of mechanical loading on cortical defect repair using a novel mechanobiological model of bone healing BONE Liu, C., Carrera, R., Flamini, V., Kenny, L., Cabahug-Zuckerman, P., George, B. M., Hunter, D., Liu, B., Singh, G., Leucht, P., Mann, K. A., Helms, J. A., Castillo, A. B. 2018; 108: 145–55

  Abstract

  Mechanical loading is an important aspect of post-surgical fracture care. The timing of load application relative to the injury event may differentially regulate repair depending on the stage of healing. Here, we used a novel mechanobiological model of cortical defect repair that offers several advantages including its technical simplicity and spatially confined repair program, making effects of both physical and biological interventions more easily assessed. Using this model, we showed that daily loading (5N peak load, 2Hz, 60 cycles, 4 consecutive days) during hematoma consolidation and inflammation disrupted the injury site and activated cartilage formation on the periosteal surface adjacent to the defect. We also showed that daily loading during the matrix deposition phase enhanced both bone and cartilage formation at the defect site, while loading during the remodeling phase resulted in an enlarged woven bone regenerate. All loading regimens resulted in abundant cellular proliferation throughout the regenerate and fibrous tissue formation directly above the defect demonstrating that all phases of cortical defect healing are sensitive to physical stimulation. Stress was concentrated at the edges of the defect during exogenous loading, and finite element (FE)-modeled longitudinal strain (εzz) values along the anterior and posterior borders of the defect (~2200με) was an order of magnitude larger than strain values on the proximal and distal borders (~50-100με). It is concluded that loading during the early stages of repair may impede stabilization of the injury site important for early bone matrix deposition, whereas loading while matrix deposition and remodeling are ongoing may enhance stabilization through the formation of additional cartilage and bone.

  View details for PubMedID 29305998

• A WNT protein therapeutic improves the bone-forming capacity of autografts from aged animals SCIENTIFIC REPORTS Chen, T., Li, J., Cordova, L. A., Liu, B., Mouraret, S., Sun, Q., Salmon, B., Helms, J. 2018; 8: 119

  Abstract

  Autografts tend to be unreliable in older patients. Some of these age-related skeletal changes appear to be attributable to a decline in endogenous WNT signaling. We used a functional in vivo transplantation assay to demonstrate that the bone-forming capacity of an autograft can be traced back to a Wnt-responsive cell population associated with the mineralized bone matrix fraction of a bone graft. Micro-CT imaging, flow cytometry and quantitative analyses demonstrate that this mineralized fraction declines with age, along with a waning in endogenous Wnt signaling; together these factors contribute to the age-related deterioration in autograft efficacy. Using a lipid formulation to stabilize the hydrophobic WNT3A protein, we demonstrate that osteogenic capacity can be restored by incubating the bone graft ex vivo with WNT3A. Compared to control bone grafts, WNT-treated bone grafts give rise to three times more bone. These preclinical results establish a pivotal role for WNT signaling in the age-related decline of autologous bone grafting efficacy, and demonstrate a means to restore that efficacy via local, transient amplification of endogenous Wnt signaling.

  View details for DOI 10.1038/s41598-017-18375-x

  View details for Web of Science ID 000419442500056

  View details for PubMedID 29311710

  View details for PubMedCentralID PMC5758817

• Wnt-Responsive Odontoblasts Secrete New Dentin after Superficial Tooth Injury. Journal of dental research Zhao, Y. n., Yuan, X. n., Liu, B. n., Tulu, U. S., Helms, J. A. 2018: 22034518763151

  Abstract

  The objective of our experiments was to identify new therapeutic strategies to stimulate dentin formation in an adult tooth. To address this objective, we evaluated dentin production in 2 acute trauma models: one involving a pulp exposure and the other involving a superficial dentin injury. Molecular, cellular, and histologic analyses revealed that in response to a severe injury, where the pulp is exposed to the oral cavity, cell death is rampant and the repair response initiates from surviving pulp cells and, to a lesser extent, surviving odontoblasts. When an injury is superficial, as in the case of a dentin injury model, then disturbances are largely confined to pulp tissue immediately underneath the damaged dentin tubules. We found that the pulp remained vital and innervated; primary odontoblasts upregulated HIF1α; and the rate of mineralization was significantly increased. A tamoxifen-inducible Axin2CreERT2/+; R26RmTmG/+reporter strain was then used to demonstrate that a population of long-lived Wnt-responsive odontoblasts, which secreted dentin throughout the life of the animal, were responsible for depositing new dentin in response to a superficial injury. Amplifying Wnt signaling in the pulp stimulates dentin secretion, and in the dentin injury model, we show that a liposomal formulation of human WNT3A protein passes through dentinal tubules and is capable of upregulating Wnt signaling in the pulp. These data provide strong proof of concept for a therapeutic pulp-capping material to stimulate Wnt signaling in odontoblasts and thus improve the pulp repair response.

  View details for DOI 10.1177/0022034518763151

  View details for PubMedID 29566345

• Biomechanics of Immediate Postextraction Implant Osseointegration. Journal of dental research Yuan, X. n., Pei, X. n., Zhao, Y. n., Li, Z. n., Chen, C. H., Tulu, U. S., Liu, B. n., Van Brunt, L. A., Brunski, J. B., Helms, J. A. 2018: 22034518765757

  Abstract

  The aim of this study was to gain insights into the biology and mechanics of immediate postextraction implant osseointegration. To mimic clinical practice, murine first molar extraction was followed by osteotomy site preparation, specifically in the palatal root socket. The osteotomy was positioned such that it removed periodontal ligament (PDL) only on the palatal aspect of the socket, leaving the buccal aspect undisturbed. This strategy created 2 distinct peri-implant environments: on the palatal aspect, the implant was in direct contact with bone, while on the buccal aspect, a PDL-filled gap existed between the implant and bone. Finite element modeling showed high strains on the palatal aspect, where bone was compressed by the implant. Osteocyte death and bone resorption predominated on the palatal aspect, leading to the loss of peri-implant bone. On the buccal aspect, where finite element modeling revealed low strains, there was minimal osteocyte death and robust peri-implant bone formation. Initially, the buccal aspect was filled with PDL remnants, which we found directly provided Wnt-responsive cells that were responsible for new bone formation and osseointegration. On the palatal aspect, which was devoid of PDL and Wnt-responsive cells, adding exogenous liposomal WNT3A created an osteogenic environment for rapid peri-implant bone formation. Thus, we conclude that low strain and high Wnt signaling favor osseointegration of immediate postextraction implants. The PDL harbors Wnt-responsive cells that are inherently osteogenic, and if the PDL tissue is healthy, it is reasonable to preserve this tissue during immediate implant placement.

  View details for DOI 10.1177/0022034518765757

  View details for PubMedID 29608868

• A Wnt-Responsive PDL Population Effectuates Extraction Socket Healing. Journal of dental research Yuan, X. n., Pei, X. n., Zhao, Y. n., Tulu, U. S., Liu, B. n., Helms, J. A. 2018: 22034518755719

  Abstract

  Stem cells residing in the periodontal ligament (PDL) support the homeostasis of the periodontium, but their in vivo identity, source(s), and function(s) remain poorly understood. Here, using a lineage-tracing mouse strain, we identified a quiescent Wnt-responsive population in the PDL that became activated in response to tooth extraction. The Wnt-responsive population expanded by proliferation, then migrated from the PDL remnants that remained attached to bundle bone, into the socket. Once there, the Wnt-responsive progeny upregulated osteogenic protein expression, differentiated into osteoblasts, and generated the new bone that healed the socket. Using a liposomal WNT3A protein therapeutic, we showed that a single application at the time of extraction was sufficient to accelerate extraction socket healing 2-fold. Collectively, these data identify a new stem cell population in the intact periodontium that is directly responsible for alveolar bone healing after tooth removal.

  View details for PubMedID 29420105

• Single-Molecule Imaging of Wnt3A Protein Diffusion on Living Cell Membranes BIOPHYSICAL JOURNAL Lippert, A., Janeczek, A. A., Furstenberg, A., Ponjavic, A., Moerner, W. E., Nusse, R., Helms, J. A., Evans, N. D., Lee, S. F. 2017; 113 (12): 2762–67

  Abstract

  Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localization at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding. We find that labeled Wnt3A transiently and dynamically associates with the membranes of Drosophila Schneider 2 cells, diffuses with Brownian kinetics on flattened membranes and on cellular protrusions, and does not transfer between cells in close contact. In S2 receptor-plus (S2R+) cells, which express Frizzled receptors, membrane diffusion rate is reduced and membrane residency time is increased. These results provide direct evidence of Wnt3A interaction with living cell membranes, and represent, to our knowledge, a new system for investigating the dynamics of Wnt transport.

  View details for PubMedID 29262368

  View details for PubMedCentralID PMC5925569

• Wnt signals control development of the periodontium. Yuan, X., Wu, Y., Zhao, Y., Perez, K., Pellegrini, G., Condon, K., McAndrews, K., Cregor, M., Bellido, T., Helms, J. WILEY. 2017: S131–S132

  View details for Web of Science ID 000418869201202

• WNT-activated bone grafts repair osteonecrotic lesions in aged animals SCIENTIFIC REPORTS Salmon, B., Liu, B., Shen, E., Chen, T., Li, J., Gillette, M., Ransom, R. C., Ezran, M., Johnson, C. A., Castillo, A. B., Shen, W. J., Kraemer, F. B., Smith, A. A., Helms, J. A. 2017; 7: 14254

  Abstract

  The Wnt pathway is a new target in bone therapeutic space. WNT proteins are potent stem cell activators and pro-osteogenic agents. Here, we gained insights into the molecular and cellular mechanisms responsible for liposome-reconstituted recombinant human WNT3A protein (L-WNT3A) efficacy to treat osteonecrotic defects. Skeletal injuries were coupled with cryoablation to create non-healing osteonecrotic defects in the diaphysis of the murine long bones. To replicate clinical therapy, osteonecrotic defects were treated with autologous bone graft, which were simulated by using bone graft material from syngeneic ACTB-eGFP-expressing mice. Control osteonecrotic defects received autografts alone; test sites received autografts treated ex vivo with L-WNT3A. In vivo µCT monitored healing over time and immunohistochemistry were used to track the fate of donor cells and assess their capacity to repair osteonecrotic defects according to age and WNT activation status. Collectively, analyses demonstrated that cells from the autograft directly contributed to repair of an osteonecrotic lesion, but this contribution diminished as the age of the donor increased. Pre-treating autografts from aged animals with L-WNT3A restored osteogenic capacity to autografts back to levels observed in autografts from young animals. A WNT therapeutic approach may therefore have utility in the treatment of osteonecrosis, especially in aged patients.

  View details for PubMedID 29079746

• Cleft Palate and Aglossia Result From Perturbations in Wnt and Hedgehog Signaling CLEFT PALATE-CRANIOFACIAL JOURNAL Yuan, G., Singh, G., Chen, S., Perez, K. C., Wu, Y., Liu, B., Helms, J. A. 2017; 54 (3): 269-280

  Abstract

  The objective of this study was to explore the molecular basis for cleft secondary palate and arrested tongue development caused by the loss of the intraflagellar transport protein, Kif3a.Kif3a mutant embryos and their littermate controls were analyzed for defects in facial development at multiple stages of embryonic development. Histology was employed to understand the effects of Kif3a deletion on palate and tongue development. Various transgenic reporter strains were used to understand how deletion of Kif3a affected Hedgehog and Wnt signaling. Immunostaining for structural elements of the tongue and for components of the Wnt pathway were performed. BrdU activity analyses were carried out to examine how the loss of Kif3a affected cell proliferation and led to palate and tongue malformations.Kif3a deletion causes cranial neural crest cells to become unresponsive to Hedgehog signals and hyper-responsive to Wnt signals. This aberrant molecular signaling causes abnormally high cell proliferation, but paradoxically outgrowths of the tongue and the palatal processes are reduced. The basis for this enigmatic effect can be traced back to a disruption in epithelial/mesenchymal signaling that governs facial development.The primary cilium is a cell surface organelle that integrates Hh and Wnt signaling, and disruptions in the function of the primary cilium cause one of the most common-of the rarest-craniofacial birth defects observed in humans. The shared molecular basis for these dysmorphologies is an abnormally high Wnt signal simultaneous with an abnormally low Hedgehog signal. These pathways are integrated in the primary cilium.

  View details for DOI 10.1597/15-178

  View details for Web of Science ID 000401016000005

• Effects of Condensation on Peri-implant Bone Density and Remodeling JOURNAL OF DENTAL RESEARCH Wang, L., Wu, Y., Perez, K. C., Hyman, S., Brunski, J. B., Tulu, U., Bao, C., Salmon, B., Helms, J. A. 2017; 96 (4): 406-413

  View details for DOI 10.1177/0022034516683932

  View details for Web of Science ID 000398161900007

• Relationships among Bone Quality, Implant Osseointegration, and Wnt Signaling. Journal of dental research Li, J., Yin, X., Huang, L., Mouraret, S., Brunski, J. B., Cordova, L., Salmon, B., Helms, J. A. 2017: 22034517700131-?

  Abstract

  A variety of clinical classification schemes have been proposed as a means to identify sites in the oral cavity where implant osseointegration is likely to be successful. Most schemes are based on structural characteristics of the bone, for example, the relative proportion of densely compact, homogenous (type I) bone versus more trabeculated, cancellous (type III) bone. None of these schemes, however, consider potential biological characteristics of the bone. Here, we employed multiscale analyses to identify and characterize type I and type III bones in murine jaws. We then combined these analytical tools with in vivo models of osteotomy healing and implant osseointegration to determine if one type of bone healed faster and supported osseointegration better than another. Collectively, these studies revealed a strong positive correlation between bone remodeling rates, mitotic activity, and osteotomy site healing in type III bone and high endogenous Wnt signaling. This positive correlation was strengthened by observations showing that the osteoid matrix that is responsible for implant osseointegration originates from Wnt-responsive cells and their progeny. The potential application of this knowledge to clinical practice is discussed, along with a theory unifying the role that biology and mechanics play in implant osseointegration.

  View details for DOI 10.1177/0022034517700131

  View details for PubMedID 28571512

• A Comparative Assessment of Implant Site Viability in Humans and Rats. Journal of dental research Chen, C. H., Pei, X. n., Tulu, U. S., Aghvami, M. n., Chen, C. T., Gaudillière, D. n., Arioka, M. n., Maghazeh Moghim, M. n., Bahat, O. n., Kolinski, M. n., Crosby, T. R., Felderhoff, A. n., Brunski, J. B., Helms, J. A. 2017: 22034517742631

  Abstract

  Our long-term objective is to devise methods to improve osteotomy site preparation and, in doing so, facilitate implant osseointegration. As a first step in this process, we developed a standardized oral osteotomy model in ovariectomized rats. There were 2 unique features to this model: first, the rats exhibited an osteopenic phenotype, reminiscent of the bone health that has been reported for the average dental implant patient population. Second, osteotomies were produced in healed tooth extraction sites and therefore represented the placement of most implants in patients. Commercially available drills were then used to produce osteotomies in a patient cohort and in the rat model. Molecular, cellular, and histologic analyses demonstrated a close alignment between the responses of human and rodent alveolar bone to osteotomy site preparation. Most notably in both patients and rats, all drilling tools created a zone of dead and dying osteocytes around the osteotomy. In rat tissues, which could be collected at multiple time points after osteotomy, the fate of the dead alveolar bone was followed. Over the course of a week, osteoclast activity was responsible for resorbing the necrotic bone, which in turn stimulated the deposition of a new bone matrix by osteoblasts. Collectively, these analyses support the use of an ovariectomy surgery rat model to gain insights into the response of human bone to osteotomy site preparation. The data also suggest that reducing the zone of osteocyte death will improve osteotomy site viability, leading to faster new bone formation around implants.

  View details for PubMedID 29202640

• Effects of Condensation on Peri-implant Bone Density and Remodeling. Journal of dental research Wang, L., Wu, Y., Perez, K. C., Hyman, S., Brunski, J. B., Tulu, U., Bao, C., Salmon, B., Helms, J. A. 2016: 22034516683932-?

  Abstract

  Bone condensation is thought to densify interfacial bone and thus improve implant primary stability, but scant data substantiate either claim. We developed a murine oral implant model to test these hypotheses. Osteotomies were created in healed maxillary extraction sites 1) by drilling or 2) by drilling followed by stepwise condensation with tapered osteotomes. Condensation increased interfacial bone density, as measured by a significant change in bone volume/total volume and trabecular spacing, but it simultaneously damaged the bone. On postimplant day 1, the condensed bone interface exhibited microfractures and osteoclast activity. Finite element modeling, mechanical testing, and immunohistochemical analyses at multiple time points throughout the osseointegration period demonstrated that condensation caused very high interfacial strains, marginal bone resorption, and no improvement in implant stability. Collectively, these multiscale analyses demonstrate that condensation does not positively contribute to implant stability.

  View details for DOI 10.1177/0022034516683932

  View details for PubMedID 28048963

  View details for PubMedCentralID PMC5384489

• Comment on "Hotair Is Dispensable for Mouse Development" PLOS GENETICS Li, L., Helms, J. A., Chang, H. Y. 2016; 12 (12): e1006406

  View details for PubMedID 27977686

• Axin2-expressing cells execute regeneration after skeletal injury SCIENTIFIC REPORTS Ransom, R. C., Hunter, D. J., Hyman, S., Singh, G., RANSOM, S. C., Shen, E. Z., Perez, K. C., Gillette, M., Li, J., Liu, B., Brunski, J. B., Helms, J. A. 2016; 6

  Abstract

  The mammalian skeleton performs a diverse range of vital functions, requiring mechanisms of regeneration that restore functional skeletal cell populations after injury. We hypothesized that the Wnt pathway specifies distinct functional subsets of skeletal cell types, and that lineage tracing of Wnt-responding cells (WRCs) using the Axin2 gene in mice identifies a population of long-lived skeletal cells on the periosteum of long bone. Ablation of these WRCs disrupts healing after injury, and three-dimensional finite element modeling of the regenerate delineates their essential role in functional bone regeneration. These progenitor cells in the periosteum are activated upon injury and give rise to both cartilage and bone. Indeed, our findings suggest that WRCs may serve as a therapeutic target in the setting of impaired skeletal regeneration.

  View details for DOI 10.1038/srep36524

  View details for Web of Science ID 000388150800001

  View details for PubMedID 27853243

  View details for PubMedCentralID PMC5113299

• From restoration to regeneration: periodontal aging and opportunities for therapeutic intervention PERIODONTOLOGY 2000 Huang, L., Salmon, B., Yin, X., Helms, J. A. 2016; 72 (1): 19-29

  Abstract

  With the march of time our bodies start to wear out: eyesight fades, skin loses its elasticity, teeth and bones become more brittle and injuries heal more slowly. These universal features of aging can be traced back to our stem cells. Aging has a profound effect on stem cells: DNA mutations naturally accumulate over time and our bodies have evolved highly specialized mechanisms to remove these damaged cells. Whilst obviously beneficial, this repair mechanism also reduces the pool of available stem cells and this, in turn, has a dramatic effect on tissue homeostasis and on our rate of healing. Simply put: fewer stem cells means a decline in tissue function and slower healing. Despite this seemingly intractable situation, research over the past decade now demonstrates that some of the effects of aging are reversible. Nobel prize-winning research demonstrates that old cells can become young again, and lessons learned from these experiments-in-a-dish are now being translated into human therapies. Scientists and clinicians around the world are identifying and characterizing methods to activate stem cells to reinvigorate the body's natural regenerative process. If this research in dental regenerative medicine pans out, the end result will be tissue homeostasis and healing back to the levels we appreciated when we were young.

  View details for DOI 10.1111/prd.12127

  View details for Web of Science ID 000385235400003

  View details for PubMedID 27501489

• Activating Hair Follicle Stem Cells via R-spondin2 to Stimulate Hair Growth. journal of investigative dermatology Smith, A. A., Li, J., Liu, B., Hunter, D., Pyles, M., Gillette, M., Dhamdhere, G. R., Abo, A., Oro, A., Helms, J. A. 2016; 136 (8): 1549-1558

  Abstract

  Wnt signaling is required for the development of the hair follicle, and for inciting the growth (anagen) phase of the hair cycle. Most strategies to enhance Wnt signaling for hair growth create a state of constitutive Wnt activation, which leads to neoplastic transformation of the epithelial hair matrix. Using Axin2(LacZ/+) and Axin2(Cre/+)R26R(mTmG/+) reporter mice and RNA analyses, we show that Wnt signaling is elevated during anagen, is reduced at the onset of catagen, and can be reamplified in the skin and surrounding hair follicles via intradermal injection of recombinant R-spondin2 protein. Using Lgr5(LacZ/+) reporter mice, we demonstrate that this amplified Wnt environment leads to activation of leucine-rich repeat-containing G-protein coupled receptor 5-positive stem cells in the hair follicle. The onset of catagen is repressed by R-spondin2 injection, and the anagen phase persists. As a consequence, hair shafts grow longer. We conclude that R-spondin2 treatment activates hair follicle stem cells and therefore may have therapeutic potential to promote hair growth.

  View details for DOI 10.1016/j.jid.2016.01.041

  View details for PubMedID 27109869

• Mechanoresponsive Properties of the Periodontal Ligament JOURNAL OF DENTAL RESEARCH Huang, L., Liu, B., Cha, J. Y., Yuan, G., Kelly, M., Singh, G., Hyman, S., Brunski, J. B., Li, J., Helms, J. A. 2016; 95 (4): 467-475

  View details for DOI 10.1177/0022034515626102

  View details for Web of Science ID 000373082000015

• Response to Letter to the Editor, "Wnt Signaling and Its Contribution to Craniofacial Tissue Homeostasis". Journal of dental research Yin, X., Li, J., Helms, J. A. 2016; 95 (3): 357-?

  View details for DOI 10.1177/0022034515627145

  View details for PubMedID 26767773

• Rescuing failed oral implants via Wnt activation. Journal of clinical periodontology Yin, X., Li, J., Chen, T., Mouraret, S., Dhamdhere, G., Brunski, J. B., Zou, S., Helms, J. A. 2016; 43 (2): 180-192

  Abstract

  Implant osseointegration is not always guaranteed and once fibrous encapsulation occurs clinicians have few options other than implant removal. Our goal was to test whether a WNT protein therapeutic could rescue such failed implants.Titanium implants were placed in over-sized murine oral osteotomies. A lack of primary stability was verified by mechanical testing. Interfacial strains were estimated by finite element modelling and histology coupled with histomorphometry confirmed the lack of peri-implant bone. After fibrous encapsulation was established peri-implant injections of a liposomal formulation of WNT3A protein (L-WNT3A) or liposomal PBS (L-PBS) were then initiated. Quantitative assays were employed to analyse the effects of L-WNT3A treatment.Implants in gap-type interfaces exhibited high interfacial strains and no primary stability. After verification of implant failure, L-WNT3A or L-PBS injections were initiated. L-WNT3A induced a rapid, significant increase in Wnt responsiveness in the peri-implant environment, cell proliferation and osteogenic protein expression. The amount of peri-implant bone and bone in contact with the implant were significantly higher in L-WNT3A cases.These data demonstrate L-WNT3A can induce peri-implant bone formation even in cases where fibrous encapsulation predominates.

  View details for DOI 10.1111/jcpe.12503

  View details for PubMedID 26718012

• Linking suckling biomechanics to the development of the palate. Scientific reports Li, J., Johnson, C. A., Smith, A. A., Hunter, D. J., Singh, G., Brunski, J. B., Helms, J. A. 2016; 6: 20419-?

  Abstract

  Skulls are amongst the most informative documents of evolutionary history but a complex geometry, coupled with composite material properties and complicated biomechanics, have made it particularly challenging to identify mechanical principles guiding the skull's morphogenesis. Despite this challenge, multiple lines of evidence, for example the relationship between masticatory function and the evolution of jaw shape, nonetheless suggest that mechanobiology plays a major role in skull morphogenesis. To begin to tackle this persistent challenge, cellular, molecular and tissue-level analyses of the developing mouse palate were coupled with finite element modeling to demonstrate that patterns of strain created by mammalian-specific oral behaviors produce complementary patterns of chondrogenic gene expression in an initially homogeneous population of cranial neural crest cells. Neural crest cells change from an osteogenic to a chondrogenic fate, leading to the materialization of cartilaginous growth plate-like structures in the palatal midline. These growth plates contribute to lateral expansion of the head but are transient structures; when the strain patterns associated with suckling dissipate at weaning, the growth plates disappear and the palate ossifies. Thus, mechanical cues such as strain appear to co-regulate cell fate specification and ultimately, help drive large-scale morphogenetic changes in head shape.

  View details for DOI 10.1038/srep20419

  View details for PubMedID 26842915

  View details for PubMedCentralID PMC4740798

• 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

• Disrupting the intrinsic growth potential of a suture contributes to midfacial hypoplasia. Bone Li, J., Johnson, C. A., Smith, A. A., Salmon, B., Shi, B., Brunski, J., Helms, J. A. 2015; 81: 186-195

  Abstract

  Children with unoperated cleft palates have nearly normal growth of their faces whereas patients who have had early surgical repair often exhibit midfacial hypoplasia. Surgical repair is responsible for the underlying bone growth arrest but the mechanisms responsible for these surgical sequelae are poorly understood. We simulated the effect of cleft palate repair by raising a mucoperiosteal flap in the murine palate. Three-dimensional micro-CT reconstructions of the palate along with histomorphometric measurements, finite element (FE) modeling, immunohistochemical analyses, and quantitative RT-PCR were employed to follow the skeletal healing process. Inflammatory bone resorption was observed during the first few days after denudation, which destroyed the midpalatal suture complex. FE modeling was used to predict and map the distribution of strains and their associated stresses in the area of denudation and the magnitude and location of hydrostatic and distortional strains corresponded to sites of skeletal tissue destruction. Once re-epithelialization was complete and wound contracture subsided, the midpalatal suture complex reformed. Despite this, growth at the midpalatal suture was reduced, which led to palatal constriction and a narrowing of the dental arch. Thus the simple act of raising a flap, here mimicked by denuding the mucoperiosteum, was sufficient to cause significant destruction to the midpalatal suture complex. Although the bone and cartilage growth plates were re-established, mediolateral skeletal growth was nonetheless compromised and the injured palate never reached its full growth potential. These data strongly suggest that disruption of suture complexes, which have intrinsic growth potential, should be avoided during surgical correction of congenital anomalies.

  View details for DOI 10.1016/j.bone.2014.04.020

  View details for PubMedID 24780877

• Response to Letter to the Editor, "Multiscale Analyses of the Bone-implant Interface". Journal of dental research Helms, J. A., Brunski, J. B. 2015; 94 (12): 1783

  View details for DOI 10.1177/0022034515603925

  View details for PubMedID 26316462

• Wnt Signaling and Its Contribution to Craniofacial Tissue Homeostasis. Journal of dental research Yin, X., Li, J., Salmon, B., Huang, L., Lim, W. H., Liu, B., Hunter, D. J., Ransom, R. C., Singh, G., Gillette, M., Zou, S., Helms, J. A. 2015; 94 (11): 1487-1494

  View details for DOI 10.1177/0022034515599772

  View details for PubMedID 26285808

• Wnt Acts as a Prosurvival Signal to Enhance Dentin Regeneration JOURNAL OF BONE AND MINERAL RESEARCH Hunter, D. J., Bardet, C., Mouraret, S., Liu, B., Singh, G., Sadoine, J., Dhamdhere, G., Smith, A., Tran, X. V., Joy, A., Rooker, S., Suzuki, S., Vuorinen, A., Miettinen, S., Chaussain, C., Helms, J. A. 2015; 30 (7): 1150-1159

  Abstract

  Wnt proteins are lipid-modified, short-range signals that control stem cell self-renewal and tissue regeneration. We identified a population of Wnt responsive cells in the pulp cavity, characterized their function, and then created a pulp injury. The repair response was evaluated over time using molecular, cellular, and quantitative assays. We tested how healing was impacted by wound environments in which Wnt signaling was amplified. We found that a Wnt-amplified environment was associated with superior pulp healing. Although cell death was still rampant, the number of cells undergoing apoptosis was significantly reduced. This resulted in significantly better survival of injured pulp cells, and resulted in the formation of more tertiary dentin. We engineered a liposome-reconstituted form of WNT3A then tested whether this biomimetic compound could activate cells in the injured tooth pulp and stimulate dentin regeneration. Pulp cells responded to the elevated Wnt stimulus by differentiating into secretory odontoblasts. Thus, transiently amplifying the body's natural Wnt response resulted in improved pulp vitality. These data have direct clinical implications for treating dental caries, the most prevalent disease affecting mankind.

  View details for DOI 10.1002/jbmr.2444

  View details for Web of Science ID 000356715900004

  View details for PubMedID 25556760

• Reengineering autologous bone grafts with the stem cell activator WNT3A. Biomaterials Jing, W., Smith, A. A., Liu, B., Li, J., Hunter, D. J., Dhamdhere, G., Salmon, B., Jiang, J., Cheng, D., Johnson, C. A., Chen, S., Lee, K., Singh, G., Helms, J. A. 2015; 47: 29-40

  Abstract

  Autologous bone grafting represents the standard of care for treating bone defects but this biomaterial is unreliable in older patients. The efficacy of an autograft can be traced back to multipotent stem cells residing within the bone graft. Aging attenuates the viability and function of these stem cells, leading to inconsistent rates of bony union. We show that age-related changes in autograft efficacy are caused by a loss in endogenous Wnt signaling. Blocking this endogenous Wnt signal using Dkk1 abrogates autograft efficacy whereas providing a Wnt signal in the form of liposome-reconstituted WNT3A protein (L-WNT3A) restores bone forming potential to autografts from aged animals. The bioengineered autograft exhibits significantly better survival in the hosting site. Mesenchymal and skeletal stem cell populations in the autograft are activated by L-WNT3A and mitotic activity and osteogenic differentiation are significantly enhanced. In a spinal fusion model, aged autografts treated with L-WNT3A demonstrate superior bone forming capacity compared to the standard of care. Thus, a brief incubation in L-WNT3A reliably improves autologous bone grafting efficacy, which has the potential to significantly improve patient care in the elderly.

  View details for DOI 10.1016/j.biomaterials.2014.12.014

  View details for PubMedID 25682158

• Multiscale Analyses of the Bone-implant Interface. Journal of dental research Cha, J. Y., Pereira, M. D., Smith, A. A., Houschyar, K. S., Yin, X., Mouraret, S., Brunski, J. B., Helms, J. A. 2015; 94 (3): 482-490

  Abstract

  Implants placed with high insertion torque (IT) typically exhibit primary stability, which enables early loading. Whether high IT has a negative impact on peri-implant bone health, however, remains to be determined. The purpose of this study was to ascertain how peri-implant bone responds to strains and stresses created when implants are placed with low and high IT. Titanium micro-implants were inserted into murine femurs with low and high IT using torque values that were scaled to approximate those used to place clinically sized implants. Torque created in peri-implant tissues a distribution and magnitude of strains, which were calculated through finite element modeling. Stiffness tests quantified primary and secondary implant stability. At multiple time points, molecular, cellular, and histomorphometric analyses were performed to quantitatively determine the effect of high and low strains on apoptosis, mineralization, resorption, and collagen matrix deposition in peri-implant bone. Preparation of an osteotomy results in a narrow zone of dead and dying osteocytes in peri-implant bone that is not significantly enlarged in response to implants placed with low IT. Placing implants with high IT more than doubles this zone of dead and dying osteocytes. As a result, peri-implant bone develops micro-fractures, bone resorption is increased, and bone formation is decreased. Using high IT to place an implant creates high interfacial stress and strain that are associated with damage to peri-implant bone and therefore should be avoided to best preserve the viability of this tissue.

  View details for DOI 10.1177/0022034514566029

  View details for PubMedID 25628271

• Alveolar Bone Turnover and Periodontal Ligament Width Are Controlled by Wnt JOURNAL OF PERIODONTOLOGY Lim, W. H., Liu, B., Mah, S., Yin, X., Helms, J. A. 2015; 86 (2): 319-326

  Abstract

  The molecular signals responsible for maintaining homeostatic control over the periodontal ligament (PDL) are unknown. The purpose of this study was to investigate the role of Wnt signaling in this process using gain- and loss-of-function approaches.The function of endogenous Wnt signal in the PDL was evaluated in Lrp5(ACT) mice in which a mutation in the low-density lipoprotein receptor-related protein 5 Wnt co-receptor causes constitutive activation of Wnt signaling, and in Ad-Dkk1 treated mice in which over-expression of the Wnt inhibitor Dkk1 causes transient Wnt signal inhibition. The PDL in both animal models was examined using histology and immunohistochemical analyses for Osteopontin, Runx2 and Fibromodulin, Osterix, ki67, RANKL and alkaline phosphatase activity.Lrp5(ACT) mice exhibited a significant narrowing of the PDL space caused by an increase in osteogenic gene expression, a reduction in RANKL expression and osteoclast activity, and an increase in alveolar bone formation. Conversely, Ad-Dkk1 treated mice showed decreased expression of osteogenic markers, coupled with an increase in osteoclast activity, which resulted in a slight widening in PDL width.The Wnt pathway is involved in the homeostatic control of the PDL and conditions that elevate or repress Wnt signaling alter the expression of osteogenic genes within the PDL space, which in turn affects its overall width.

  View details for DOI 10.1902/jop.2014.140286

  View details for Web of Science ID 000349380500018

  View details for PubMedID 25345341

• The potential for vertical bone regeneration via maxillary periosteal elevation. Journal of clinical periodontology Mouraret, S., Von Kaeppler, E., Bardet, C., Hunter, D. J., Chaussain, C., Bouchard, P., Helms, J. A. 2014; 41 (12): 1170-1177

  Abstract

  While many studies have been performed on the characteristics and regenerative capacity of long bone periosteum, the craniofacial periosteum remains poorly understood.The aim of this study was to investigate the potential for a maxillary periosteum tunnelling procedure to induce vertical alveolar bone regeneration.We employed a murine injury model that activates skeletal stem cells in the periosteum without overtly damaging the underlying cortical bone, preserving the integrity of the long bone and maxilla, and avoiding the introduction of pathological motion at the injury site. Further, we introduced a collagen sponge to serve as a scaffold, providing the necessary space for vertical bone regeneration.Periosteal elevation alone resulted in bone formation in the tibia and delayed bone resorption in the maxilla. With the presence of the collagen sponge, new bone formation occurred in the maxilla.Periosteal response to injury varies with anatomical location, so conclusions from long bone studies should not be extrapolated for craniofacial applications. Murine maxillary periosteum has the osteogenic potential to induce vertical alveolar bone regeneration.

  View details for DOI 10.1111/jcpe.12310

  View details for PubMedID 25229322

• Wnt signaling regulates homeostasis of the periodontal ligament JOURNAL OF PERIODONTAL RESEARCH Lim, W. H., Liu, B., Cheng, D., Williams, B. O., Mah, S. J., Helms, J. A. 2014; 49 (6): 751-759

  Abstract

  In health, the periodontal ligament maintains a constant width throughout an organism's lifetime. The molecular signals responsible for maintaining homeostatic control over the periodontal ligament are unknown. The purpose of this study was to investigate the role of Wnt signaling in this process by removing an essential chaperone protein, Wntless (Wls), from odontoblasts and cementoblasts, and observing the effects of Wnt depletion on cells of the periodontal complex.The Wnt responsive status of the periodontal complex was assessed using two strains of Wnt reporter mice: Axin2(LacZ/+) and Lgr5(LacZ/+) . The function of this endogenous Wnt signal was evaluated by conditionally eliminating the Wntless (Wls) gene using an osteocalcin Cre driver. The resulting OCN-Cre;Wls (fl/fl) mice were examined using micro-computed tomography and histology, immunohistochemical analyses for osteopontin, Runx2 and fibromodulin, in-situ hybridization for osterix and alkaline phosphatase activity.The adult periodontal ligament is Wnt responsive. Elimination of Wnt signaling in the periodontal complex of OCN-Cre;Wls(fl/fl) mice resulted in a wider periodontal ligament space. This pathologically increased periodontal width is caused by a reduction in the expression of osteogenic genes and proteins, which results in thinner alveolar bone. A concomitant increase in fibrous tissue occupying the periodontal space was observed, along with a disruption in the orientation of the periodontal ligament.The periodontal ligament is a Wnt-dependent tissue. Cells in the periodontal complex are Wnt responsive, and eliminating an essential component of the Wnt signaling network leads to a pathological widening of the periodontal ligament space. Osteogenic stimuli are reduced, and a disorganized fibrillary matrix results from the depletion of Wnt signaling. Collectively, these data underscore the importance of Wnt signaling in homeostasis of the periodontal ligament.

  View details for DOI 10.1111/jre.12158

  View details for Web of Science ID 000345152300010

  View details for PubMedID 24410666

• Midfacial growth and surgically induced growth arrest. Plastic and reconstructive surgery Johnson, C. A., Li, J., Smith, A. A., Brunski, J. B., Helms, J. A. 2014; 134 (4 Suppl 1): 20

  View details for DOI 10.1097/01.prs.0000455340.31525.f9

  View details for PubMedID 25254698

• The molecular and cellular effects of ageing on the periodontal ligament JOURNAL OF CLINICAL PERIODONTOLOGY Lim, W. H., Liu, B., Mah, S., Chen, S., Helms, J. A. 2014; 41 (10): 935-942

  Abstract

  Many in vitro studies have investigated age-related biological changes in cells comprising the periodontium but the basic question of whether the periodontium can maintain its integrity with age remains unanswered. Thus, the aim of this study was to understand how, in the absence of disease, advancing age impacts the structure of the periodontium.Of 4, 10, 25, and 50-week-old mice were examined using histology and immunohistochemical analyses for cell proliferation, cell turnover, collagen quantity and quality, osteogenic markers, bone turnover, and cytokine expression.The periodontal ligament (PDL) space shows a gradual decrease in width over the lifespan of the mice. Cell proliferation as well as the quantity and quality of collagen fibres decreased with age although cell density did not appear to be altered. Osteoprogenitor markers in the PDL maintained their expression with increasing age. Alkaline phosphatase (ALP) activity decreased, but osteoclast activity increased with age.Ageing is associated with a decline in the quality and quantity of collagen and an increase in bone resorption, all of which can diminish the function of the periodontium even in the absence of disease.

  View details for DOI 10.1111/jcpe.12277

  View details for Web of Science ID 000342748900001

  View details for PubMedID 24888546

• The capacity of neural crest-derived stem cells for ocular repair. Birth defects research. Part C, Embryo today : reviews Liu, B., Hunter, D. J., Smith, A. A., Chen, S., Helms, J. A. 2014; 102 (3): 299-308

  Abstract

  Whether it is due to a particular epigenetic signature, or some other component of an embryonic differentiation program, accumulating evidence indicates that the origins of a stem cell has a profound impact on the potential of a tissue to regenerate and repair. Here, we focus on Müller glia, long considered the stem cells of the retina, and their surprising derivation from the neural crest. Whether the multipotent properties of a subset of Müller glia is associated with their neural crest origin remains a tantalizing possibility.

  View details for DOI 10.1002/bdrc.21077

  View details for PubMedID 25227475

• Molecular mechanisms underlying skeletal growth arrest by cutaneous scarring. Bone Li, J., Johnson, C. A., Smith, A. A., Shi, B., Brunski, J. B., Helms, J. A. 2014; 66: 223-231

  Abstract

  In pediatric surgeries, cutaneous scarring is frequently accompanied by an arrest in skeletal growth. The molecular mechanisms responsible for this effect are not understood. Here, we investigated the relationship between scar contracture and osteogenesis. An excisional cutaneous wound was made on the tail of neonatal mice. Finite element (FE) modeling of the wound site was used to predict the distribution and magnitude of contractile forces within soft and hard tissues. Morphogenesis of the bony vertebrae was monitored by micro-CT analyses, and vertebral growth plates were interrogated throughout the healing period using assays for cell proliferation, death, differentiation, as well as matrix deposition and remodeling. Wound contracture was grossly evident on post-injury day 7 and accompanying it was a significant shortening in the tail. FE modeling indicated high compressive strains localized to the dorsal portions of the vertebral growth plates and intervertebral disks. These predicted strain distributions corresponded to sites of increased cell death, a cessation in cell proliferation, and a loss in mineralization within the growth plates and IVD. Although cutaneous contracture resolved and skeletal growth rates returned to normal, vertebrae under the cutaneous wound remained significantly shorter than controls. Thus, localized contractile forces generated by scarring led to spatial alterations in cell proliferation, death, and differentiation that inhibited bone growth in a location-dependent manner. Resolution of cutaneous scarring was not accompanied by compensatory bone growth, which left the bony elements permanently truncated. Therefore, targeting early scar reduction is critical to preserving pediatric bone growth after surgery.

  View details for DOI 10.1016/j.bone.2014.06.007

  View details for PubMedID 24933346

• A ciliopathy with hydrocephalus, isolated craniosynostosis, hypertelorism, and clefting caused by deletion of Kif3a REPRODUCTIVE TOXICOLOGY Liu, B., Chen, S., Johnson, C., Helms, J. A. 2014; 48: 88-97

  Abstract

  Malformations of the facial midline are a consistent feature among individuals with defects in primary cilia. Here, we provide a framework in which to consider how these primary cilia-dependent facial anomalies occur. We generated mice in which the intraflagellar transport protein Kif3a was deleted in cranial neural crest cells. The Kif3a phenotypes included isolated metopic craniosynostosis, delayed closure of the anterior fontanelles, and hydrocephalus, as well as midline facial anomalies including hypertelorism, cleft palate, and bifid nasal septum. Although all cranial neural crest cells had truncated primary cilia as a result of the conditional deletion, only those in the midline showed evidence of hyper-proliferation and ectopic Wnt responsiveness. Thus, cranial neural crest cells do not rely on primary cilia for their migration but once established in the facial prominences, midline cranial neural crest cells require Kif3a function in order to integrate and respond to Wnt signals from the surrounding epithelia.

  View details for DOI 10.1016/j.reprotox.2014.05.009

  View details for Web of Science ID 000341341900010

  View details for PubMedID 24887031

• The Capacity of Neural Crest-Derived Stem Cells for Ocular Repair BIRTH DEFECTS RESEARCH PART C-EMBRYO TODAY-REVIEWS Liu, B., Hunter, D. J., Smith, A. A., Chen, S., Helms, J. A. 2014; 102 (3): 299-308

  Abstract

  Whether it is due to a particular epigenetic signature, or some other component of an embryonic differentiation program, accumulating evidence indicates that the origins of a stem cell has a profound impact on the potential of a tissue to regenerate and repair. Here, we focus on Müller glia, long considered the stem cells of the retina, and their surprising derivation from the neural crest. Whether the multipotent properties of a subset of Müller glia is associated with their neural crest origin remains a tantalizing possibility.

  View details for DOI 10.1002/bdrc.21077

  View details for Web of Science ID 000342741900008

• Downregulation of Wnt causes root resorption AMERICAN JOURNAL OF ORTHODONTICS AND DENTOFACIAL ORTHOPEDICS Lim, W. H., Liu, B., Hunter, D. J., Cheng, D., Mah, S., Helms, J. A. 2014; 146 (3): 337-345

  Abstract

  There are multiple causes of external root resorption, but absent a disease state, it is most often observed when excessive physical force is used during orthodontic treatment. Even without mechanical stimulation, however, root resorption can still occur. The purpose of this study was to test whether Wnt signaling plays a role in pathologic root resorption, by conditionally deleting Wntless (Wls) from odontoblasts and osteoblasts and then evaluating the phenotypic effects on the maintenance of the root surface.Ten (age, 1 month) and 20 (age, 3 months) OCN-Cre;Wls(fl/fl) mice and their wild-type littermates were evaluated using microcomputed tomography, histology, and immunohistochemistry. Phenotypic alterations in the alveolar bone, dentin, and cementum were characterized and quantified.In a genetic model of reduced Wnt signaling, we found that RANKL expression is upregulated, and osteoprotegerin expression is downregulated. This molecular disruption results in an increase in osteoclast activity, a decrease in osteoblast activity, and extensive, spontaneous root resorption. A genetic strain of mice in which Wnt signaling is elevated exhibits thicker cementum, whereas, even in the perinatal period, OCN-Cre;Wls(fl/fl) mice exhibit thinner cementum.Taken together, these data demonstrate that Wnts regulate cementum homeostasis, and that idiopathic cases of root resorption might have as their etiology a reduction in endogenous Wnt signaling.

  View details for DOI 10.1016/j.ajodo.2014.05.027

  View details for Web of Science ID 000341305800012

  View details for PubMedID 25172256

• Molecular mechanisms underlying skeletal growth arrest by cutaneous scarring. Bone Li, J., Johnson, C. A., Smith, A. A., Shi, B., Brunski, J. B., Helms, J. A. 2014; 66: 223-231

  Abstract

  In pediatric surgeries, cutaneous scarring is frequently accompanied by an arrest in skeletal growth. The molecular mechanisms responsible for this effect are not understood. Here, we investigated the relationship between scar contracture and osteogenesis. An excisional cutaneous wound was made on the tail of neonatal mice. Finite element (FE) modeling of the wound site was used to predict the distribution and magnitude of contractile forces within soft and hard tissues. Morphogenesis of the bony vertebrae was monitored by micro-CT analyses, and vertebral growth plates were interrogated throughout the healing period using assays for cell proliferation, death, differentiation, as well as matrix deposition and remodeling. Wound contracture was grossly evident on post-injury day 7 and accompanying it was a significant shortening in the tail. FE modeling indicated high compressive strains localized to the dorsal portions of the vertebral growth plates and intervertebral disks. These predicted strain distributions corresponded to sites of increased cell death, a cessation in cell proliferation, and a loss in mineralization within the growth plates and IVD. Although cutaneous contracture resolved and skeletal growth rates returned to normal, vertebrae under the cutaneous wound remained significantly shorter than controls. Thus, localized contractile forces generated by scarring led to spatial alterations in cell proliferation, death, and differentiation that inhibited bone growth in a location-dependent manner. Resolution of cutaneous scarring was not accompanied by compensatory bone growth, which left the bony elements permanently truncated. Therefore, targeting early scar reduction is critical to preserving pediatric bone growth after surgery.

  View details for DOI 10.1016/j.bone.2014.06.007

  View details for PubMedID 24933346

• Cell viability after osteotomy and bone harvesting: comparison of piezoelectric surgery and conventional bur INTERNATIONAL JOURNAL OF ORAL AND MAXILLOFACIAL SURGERY Mouraret, S., Houschyar, K. S., Hunter, D. J., Smith, A. A., Jew, O. S., Girod, S., Helms, J. A. 2014; 43 (8): 966-971

  Abstract

  The aim of this study was to evaluate and compare the influence of a piezoelectric device versus a conventional bur on osteocyte viability and osteoblast and osteoclast activity using an in vivo mouse model. Osteotomies were created and bone grafts were harvested using either a conventional bur or a piezoelectric device; the resulting injuries and bone grafts were evaluated over an extended time-course using molecular and cellular assays for cell death (TUNEL assay), cell viability (4',6-diamidino-2-phenylindole (DAPI) staining), the onset of mineralization (alkaline phosphatase activity), and bone remodelling (tartrate-resistant acid phosphatase activity). Osteotomies created with a piezoelectric device showed greater osteocyte viability and reduced cell death. Bone grafts harvested with a piezoelectric device exhibited greater short-term cell viability than those harvested with a bur, and exhibited slightly more new bone deposition and bone remodelling. The difference in response of osteocytes, osteoblasts, and osteoclasts to bone cutting via a bur and via a piezoelectric device is negligible in vivo. Given the improved visibility and the margin of safety afforded by a piezoelectric device, they are the instrument of choice when cutting or harvesting bone to preserve soft tissue.

  View details for DOI 10.1016/j.ijom.2013.11.018

  View details for Web of Science ID 000339601900009

  View details for PubMedID 24721169

• Primary Cilia Integrate Hedgehog and Wnt Signaling during Tooth Development JOURNAL OF DENTAL RESEARCH Liu, B., Chen, S., Cheng, D., Jing, W., Helms, J. A. 2014; 93 (5): 475-482

  Abstract

  Many ciliopathies have clinical features that include tooth malformations but how these defects come about is not clear. Here we show that genetic deletion of the motor protein Kif3a in dental mesenchyme results in an arrest in odontogenesis. Incisors are completely missing, and molars are enlarged in Wnt1(Cre+)Kif3a(fl/fl) embryos. Although amelogenesis and dentinogenesis initiate in the molar tooth bud, both processes terminate prematurely. We demonstrate that loss of Kif3a in dental mesenchyme results in loss of Hedgehog signaling and gain of Wnt signaling in this same tissue. The defective dental mesenchyme then aberrantly signals to the dental epithelia, which prompts an up-regulation in the Hedgehog and Wnt responses in the epithelia and leads to multiple attempts at invagination and an expanded enamel organ. Thus, the primary cilium integrates Hedgehog and Wnt signaling between dental epithelia and mesenchyme, and this cilia-dependent integration is required for proper tooth development.

  View details for DOI 10.1177/0022034514528211

  View details for Web of Science ID 000334265100007

  View details for PubMedID 24659776

• Wnt Signaling Regulates Pulp Volume and Dentin Thickness JOURNAL OF BONE AND MINERAL RESEARCH Lim, W. H., Liu, B., Cheng, D., Hunter, D. J., Zhong, Z., Ramos, D. M., Williams, B. O., Sharpe, P. T., Bardet, C., Mah, S., Helms, J. A. 2014; 29 (4): 892-901

  Abstract

  Odontoblasts, cementoblasts, ameloblasts, and osteoblasts all form mineralized tissues in the craniofacial complex, and all these cell types exhibit active Wnt signaling during postnatal life. We set out to understand the functions of this Wnt signaling, by evaluating the phenotypes of mice in which the essential Wnt chaperone protein, Wntless was eliminated. The deletion of Wls was restricted to cells expressing Osteocalcin (OCN), which in addition to osteoblasts includes odontoblasts, cementoblasts, and ameloblasts. Dentin, cementum, enamel, and bone all formed in OCN-Cre;Wls(fl/fl) mice but their homeostasis was dramatically affected. The most notable feature was a significant increase in dentin volume and density. We attribute this gain in dentin volume to a Wnt-mediated misregulation of Runx2. Normally, Wnt signaling stimulates Runx2, which in turn inhibits dentin sialoprotein (DSP); this inhibition must be relieved for odontoblasts to differentiate. In OCN-Cre;Wls(fl/fl) mice, Wnt pathway activation is reduced and Runx2 levels decline. The Runx2-mediated repression of DSP is relieved and odontoblast differentiation is accordingly enhanced. This study demonstrates the importance of Wnt signaling in the homeostasis of mineralized tissues of the craniofacial complex.

  View details for Web of Science ID 000333005100012

  View details for PubMedID 23996396

• Improving oral implant osseointegration in a murine model via Wnt signal amplification. Journal of clinical periodontology Mouraret, S., Hunter, D. J., Bardet, C., Popelut, A., Brunski, J. B., Chaussain, C., Bouchard, P., Helms, J. A. 2014; 41 (2): 172-180

  Abstract

  To determine the key biological events occurring during implant failure and then we use this knowledge to develop new biology-based strategies that improve osseointegration.Wild-type and Axin2(LacZ/LacZ) adult male mice underwent oral implant placement, with and without primary stability. Peri-implant tissues were evaluated using histology, alkaline phosphatase (ALP) activity, tartrate resistant acid phosphatase (TRAP) activity and TUNEL staining. In addition, mineralization sites, collagenous matrix organization and the expression of bone markers in the peri-implant tissues were assessed.Maxillary implants lacking primary stability show histological evidence of persistent fibrous encapsulation and mobility, which recapitulates the clinical problems of implant failure. Despite histological and molecular evidence of fibrous encapsulation, osteoblasts in the gap interface exhibit robust ALP activity. This mineralization activity is counteracted by osteoclast activity that resorbs any new bony matrix and consequently, the fibrous encapsulation remains. Using a genetic mouse model, we show that implants lacking primary stability undergo osseointegration, provided that Wnt signalling is amplified.In a mouse model of oral implant failure caused by a lack of primary stability, we find evidence of active mineralization. This mineralization, however, is outpaced by robust bone resorption, which culminates in persistent fibrous encapsulation of the implant. Fibrous encapsulation can be prevented and osseointegration assured if Wnt signalling is elevated at the time of implant placement.

  View details for DOI 10.1111/jcpe.12187

  View details for PubMedID 24164629

• A pre-clinical murine model of oral implant osseointegration. Bone Mouraret, S., Hunter, D. J., Bardet, C., Brunski, J. B., Bouchard, P., Helms, J. A. 2014; 58: 177-184

  Abstract

  Many of our assumptions concerning oral implant osseointegration are extrapolated from experimental models studying skeletal tissue repair in long bones. This disconnect between clinical practice and experimental research hampers our understanding of bone formation around oral implants and how this process can be improved. We postulated that oral implant osseointegration would be fundamentally equivalent to implant osseointegration elsewhere in the body. Mice underwent implant placement in the edentulous ridge anterior to the first molar and peri-implant tissues were evaluated at various timepoints after surgery. Our hypothesis was disproven; oral implant osseointegration is substantially different from osseointegration in long bones. For example, in the maxilla peri-implant pre-osteoblasts are derived from cranial neural crest whereas in the tibia peri-implant osteoblasts are derived from mesoderm. In the maxilla, new osteoid arises from periostea of the maxillary bone but in the tibia the new osteoid arises from the marrow space. Cellular and molecular analyses indicate that osteoblast activity and mineralization proceeds from the surfaces of the native bone and osteoclastic activity is responsible for extensive remodeling of the new peri-implant bone. In addition to histologic features of implant osseointegration, molecular and cellular assays conducted in a murine model provide new insights into the sequelae of implant placement and the process by which bone is generated around implants.

  View details for DOI 10.1016/j.bone.2013.07.021

  View details for PubMedID 23886841

• A pre-clinical murine model of oral implant osseointegration BONE Mouraret, S., Hunter, D. J., Bardet, C., Brunski, J. B., Bouchard, P., Helms, J. A. 2014; 58: 177-184

  View details for DOI 10.1016/j.bone.2013.07.021

  View details for Web of Science ID 000328304000023

  View details for PubMedID 23886841

• Drugging a stem cell compartment using Wnt3a protein as a therapeutic. PloS one Dhamdhere, G. R., Fang, M. Y., Jiang, J., Lee, K., Cheng, D., Olveda, R. C., Liu, B., Mulligan, K. A., Carlson, J. C., Ransom, R. C., Weis, W. I., Helms, J. A. 2014; 9 (1)

  Abstract

  The therapeutic potential of Wnt proteins has long been recognized but challenges associated with in vivo stability and delivery have hindered their development as drug candidates. By exploiting the hydrophobic nature of the protein we provide evidence that exogenous Wnt3a can be delivered in vivo if it is associated with a lipid vesicle. Recombinant Wnt3a associates with the external surface of the lipid membrane; this association stabilizes the protein and leads to prolonged activation of the Wnt pathway in primary cells. We demonstrate the consequences of Wnt pathway activation in vivo using a bone marrow engraftment assay. These data provide validation for the development of WNT3A as a therapeutic protein.

  View details for DOI 10.1371/journal.pone.0083650

  View details for PubMedID 24400074

  View details for PubMedCentralID PMC3882211

• CXCR4 Antagonism Attenuates Load-Induced Periosteal Bone Formation in Mice JOURNAL OF ORTHOPAEDIC RESEARCH Leucht, P., Temiyasathit, S., Russell, A., Arguello, J. F., Jacobs, C. R., Helms, J. A., Castillo, A. B. 2013; 31 (11): 1828-1838

  Abstract

  Mechanical loading is a key anabolic regulator of bone mass. Stromal cell-derived factor-1 (SDF-1) is a stem cell homing factor that is important in hematopoiesis, angiogenesis, and fracture healing, though its involvement in skeletal mechanoadaptation is virtually unknown. The objective of this study was to characterize skeletal expression patterns of SDF-1 and CXCR4, the receptor for SDF-1, and to determine the role of SDF-1 signaling in load-induced periosteal bone formation. Sixteen-week-old C57BL/6 mice were treated with PBS or AMD3100, an antagonist against CXCR4, and exposed to in vivo ulnar loading (2.8 N peak-to-peak, 2 Hz, 120 cycles). SDF-1 was expressed in cortical and trabecular osteocytes and marrow cells, and CXCR4 was primarily expressed in marrow cells. SDF-1 and CXCR4 expression was enhanced in response to mechanical stimulation. The CXCR4 receptor antagonist AMD3100 significantly attenuated load-induced bone formation and led to smaller adaptive changes in cortical geometric properties as determined by histomorphometric analysis. Our data suggest that SDF-1/CXCR4 signaling plays a critical role in skeletal mechanoadaptation, and may represent a unique therapeutic target for prevention and treatment of age-related and disuse bone loss. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res XX:XXX-XXX, 2013.

  View details for DOI 10.1002/jor.22440

  View details for Web of Science ID 000324930500022

  View details for PubMedID 23881789

• Augmenting Endogenous Wnt Signaling Improves Skin Wound Healing PLOS ONE Whyte, J. L., Smith, A. A., Liu, B., Manzano, W. R., Evans, N. D., Dhamdhere, G. R., Fang, M. Y., Chang, H. Y., Oro, A. E., Helms, J. A. 2013; 8 (10)

  Abstract

  Wnt signaling is required for both the development and homeostasis of the skin, yet its contribution to skin wound repair remains controversial. By employing Axin2(LacZ/+) reporter mice we evaluated the spatial and temporal distribution patterns of Wnt responsive cells, and found that the pattern of Wnt responsiveness varies with the hair cycle, and correlates with wound healing potential. Using Axin2(LacZ/LacZ) mice and an ear wound model, we demonstrate that amplified Wnt signaling leads to improved healing. Utilizing a biochemical approach that mimics the amplified Wnt response of Axin2(LacZ/LacZ) mice, we show that topical application of liposomal Wnt3a to a non-healing wound enhances endogenous Wnt signaling, and results in better skin wound healing. Given the importance of Wnt signaling in the maintenance and repair of skin, liposomal Wnt3a may have widespread application in clinical practice.

  View details for DOI 10.1371/journal.pone.0076883

  View details for Web of Science ID 000326029300061

  View details for PubMedID 24204695

  View details for PubMedCentralID PMC3799989

• Targeted disruption of hotair leads to homeotic transformation and gene derepression. Cell reports Li, L., Liu, B., Wapinski, O. L., Tsai, M., Qu, K., Zhang, J., Carlson, J. C., Lin, M., Fang, F., Gupta, R. A., Helms, J. A., Chang, H. Y. 2013; 5 (1): 3-12

  Abstract

  Long noncoding RNAs (lncRNAs) are thought to be prevalent regulators of gene expression, but the consequences of lncRNA inactivation in vivo are mostly unknown. Here, we show that targeted deletion of mouse Hotair lncRNA leads to derepression of hundreds of genes, resulting in homeotic transformation of the spine and malformation of metacarpal-carpal bones. RNA sequencing and conditional inactivation reveal an ongoing requirement of Hotair to repress HoxD genes and several imprinted loci such as Dlk1-Meg3 and Igf2-H19 without affecting imprinting choice. Hotair binds to both Polycomb repressive complex 2, which methylates histone H3 at lysine 27 (H3K27), and Lsd1 complex, which demethylates histone H3 at lysine 4 (H3K4) in vivo. Hotair inactivation causes H3K4me3 gain and, to a lesser extent, H3K27me3 loss at target genes. These results reveal the function and mechanisms of Hotair lncRNA in enforcing a silent chromatin state at Hox and additional genes.

  View details for DOI 10.1016/j.celrep.2013.09.003

  View details for PubMedID 24075995

• Rhinoplasty: Congenital Deficiencies of the Alar Cartilage AESTHETIC SURGERY JOURNAL Kosins, A. M., Daniel, R. K., Sajjadian, A., Helms, J. 2013; 33 (6): 799-808

  Abstract

  Congenital deficiencies of the alar cartilages are rare and often visible at birth but can occasionally present later.The authors review the anatomical development and discuss the incidence and treatment of congenital defects within the alar cartilages seen in rhinoplasty cases.The charts of 869 consecutive patients who underwent open rhinoplasty were retrospectively reviewed, and 8 cases of congenital defects of the alar cartilage within the middle crura were identified. Intraoperative photographs were taken of the alar deformities, and each patient underwent surgical correction. To simplify analysis, a classification of the defects was developed. A division was a cleft in the continuity of the alar cartilage with the 2 ends separate. A gap was a true absence of cartilage ranging from 1 to 4 mm, which can be accurately assessed in unilateral cases. A segmental loss was a defect greater than 4 mm.The 8 cases of deformity could be classified as 4 divisions, 3 gaps, and 1 segmental loss. None of the patients had a history of prior nasal trauma or nasal surgery. Six patients were women and 2 patients were men. In all cases, adequate projection and stability were achieved with a columellar strut. Asymmetry was minimized through concealer or tip grafts. There were no complications.Surgeons performing rhinoplasty surgery will encounter and should be prepared to deal with unexpected congenital defects of the alar cartilage. These defects within the middle crura will require stabilization with a columellar strut and, often, coverage with a concealer tip graft. We speculate that the cause of these defects is a disruption of the hedgehog signals that may arrest the condensation or block the differentiation of the underlying neural crest cells.

  View details for DOI 10.1177/1090820X13495692

  View details for Web of Science ID 000322590500006

  View details for PubMedID 23838255

• 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

• Micromotion-induced strain fields influence early stages of repair at bone-implant interfaces. Acta biomaterialia Wazen, R. M., Currey, J. A., Guo, H., Brunski, J. B., Helms, J. A., Nanci, A. 2013; 9 (5): 6663-6674

  Abstract

  Implant loading can create micromotion at the bone-implant interface. The interfacial strain associated with implant micromotion could contribute to regulating the tissue healing response. Excessive micromotion can lead to fibrous encapsulation and implant loosening. Our objective was to characterize the influence of interfacial strain on bone regeneration around implants in mouse tibiae. A micromotion system was used to create strain under conditions of (1) no initial contact between implant and bone and (2) direct bone-implant contact. Pin- and screw-shaped implants were subjected to displacements of 150 or 300 μm for 60 cycles per day for 7 days. Pin-shaped implants placed in five animals were subjected to three sessions of 150 μm displacement per day, with 60 cycles per session. Control implants in both types of interfaces were stabilized throughout the healing period. Experimental strain analyses, microtomography, image-based displacement mapping, and finite element simulations were used to characterize interfacial strain fields. Calcified tissue sections were prepared and Goldner trichrome stained to evaluate the tissue reactions in higher and lower strain regions. In stable implants bone formation occurred consistently around the implants. In implants subjected to micromotion bone regeneration was disrupted in areas of high strain concentrations (e.g. >30%), whereas lower strain values were permissive of bone formation. Increasing implant displacement or number of cycles per day also changed the strain distribution and disturbed bone healing. These results indicate that not only implant micromotion but also the associated interfacial strain field contributes to regulating the interfacial mechanobiology at healing bone-implant interfaces.

  View details for DOI 10.1016/j.actbio.2013.01.014

  View details for PubMedID 23337705

• 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

• Wnt Signaling Promotes Muller Cell Proliferation and Survival after Injury INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Liu, B., Hunter, D. J., Rooker, S., Chan, A., Paulus, Y. M., Leucht, P., Nusse, Y., Nomoto, H., Helms, J. A. 2013; 54 (1): 444-453

  Abstract

  Müller glia respond to retinal injury by a reactive gliosis, but only rarely do mammalian glial cells re-enter the cell cycle and generate new neurons. In the nonmammalian retina, however, Müller glia act as stem/progenitor cells. Here, we tested the function of Wnt signaling in the postinjury retina, focusing on its ability to influence mammalian Müller cell dedifferentiation, proliferation, and neurogenesis.A 532 nm frequency doubled neodymium-doped yttrium aluminum garnet (ND:YAG) laser was used to create light burns on the retina of Axin2(LacZ/+) Wnt reporter mice. At various time points after injury, retinas were analyzed for evidence of Wnt signaling as well as glial cell response, proliferation, and apoptosis. Laser injuries also were created in Axin2(LacZ/LacZ) mice, and the effect of potentiated Wnt signaling on retinal repair was assessed.A subpopulation of mammalian Müller cells are Wnt responsive and, when Wnt signaling is increased, these cells showed enhanced proliferation in response to injury. In an environment of heightened Wnt signaling, caused by the loss of the Wnt negative regulator Axin2, Müller cells proliferated after injury and adopted the expression patterns of retinal progenitor cells (RPCs). The Wnt-responsive Müller cells also exhibited long-term survival and, in some cases, expressed the rod photoreceptor marker, rhodopsin.The Wnt pathway is activated by retinal injury, and prolonging the endogenous Wnt signal causes a subset of Müller cells to proliferate and dedifferentiate into RPCs. These data raised the possibility that transient amplification of Wnt signaling after retinal damage may unlock the latent regenerative capacity long speculated to reside in mammalian neural tissues.

  View details for DOI 10.1167/iovs.12-10774

  View details for Web of Science ID 000314338400055

  View details for PubMedID 23154457

• Augmenting endogenous Wnt signaling improves skin wound healing. PloS one Whyte, J. L., Smith, A. A., Liu, B., Manzano, W. R., Evans, N. D., Dhamdhere, G. R., Fang, M. Y., Chang, H. Y., Oro, A. E., Helms, J. A. 2013; 8 (10)

  Abstract

  Wnt signaling is required for both the development and homeostasis of the skin, yet its contribution to skin wound repair remains controversial. By employing Axin2(LacZ/+) reporter mice we evaluated the spatial and temporal distribution patterns of Wnt responsive cells, and found that the pattern of Wnt responsiveness varies with the hair cycle, and correlates with wound healing potential. Using Axin2(LacZ/LacZ) mice and an ear wound model, we demonstrate that amplified Wnt signaling leads to improved healing. Utilizing a biochemical approach that mimics the amplified Wnt response of Axin2(LacZ/LacZ) mice, we show that topical application of liposomal Wnt3a to a non-healing wound enhances endogenous Wnt signaling, and results in better skin wound healing. Given the importance of Wnt signaling in the maintenance and repair of skin, liposomal Wnt3a may have widespread application in clinical practice.

  View details for DOI 10.1371/journal.pone.0076883

  View details for PubMedID 24204695

• Wntless functions in mature osteoblasts to regulate bone mass PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhong, Z., Zylstra-Diegel, C. R., Schumacher, C. A., Baker, J. J., Carpenter, A. C., Rao, S., Yao, W., Guan, M., Helms, J. A., Lane, N. E., Lang, R. A., Williams, B. O. 2012; 109 (33): E2197-E2204

  Abstract

  Recent genome-wide association studies of individuals of Asian and European descent have found that SNPs located within the genomic region (1p31.3) encoding the Wntless (Wls)/Gpr177 protein are associated significantly with reduced bone mineral density. Wls/Gpr177 is a newly identified chaperone protein that specifically escorts Wnt ligands for secretion. Given the strong functional association between the Wnt signaling pathways and bone development and homeostasis, we generated osteoblast-specific Wls-deficient (Ocn-Cre;Wls-flox) mice. Homozygous conditional knockout animals were born at a normal Mendelian frequency. Whole-body dual-energy X-ray absorptiometry scanning revealed that bone-mass accrual was significantly inhibited in homozygotes as early as 20 d of age. These homozygotes had spontaneous fractures and a high frequency of premature lethality at around 2 mo of age. Microcomputed tomography analysis and histomorphometric data revealed a dramatic reduction of both trabecular and cortical bone mass in homozygous mutants. Bone formation in homozygotes was severely impaired, but no obvious phenotypic change was observed in mice heterozygous for the conditional deletion. In vitro studies showed that Wls-deficient osteoblasts had a defect in differentiation and mineralization, with significant reductions in the expression of key osteoblast differentiation regulators. In summary, these results reveal a surprising and crucial role of osteoblast-secreted Wnt ligands in bone-mass accrual.

  View details for DOI 10.1073/pnas.1120407109

  View details for Web of Science ID 000307807000005

  View details for PubMedID 22745162

  View details for PubMedCentralID PMC3421196

• Wnt Signaling and Injury Repair COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Whyte, J. L., Smith, A. A., Helms, J. A. 2012; 4 (8)

  Abstract

  Wnt signaling is activated by wounding and participates in every subsequent stage of the healing process from the control of inflammation and programmed cell death, to the mobilization of stem cell reservoirs within the wound site. In this review we summarize recent data elucidating the roles that the Wnt pathway plays in the injury repair process. These data provide a foundation for potential Wnt-based therapeutic strategies aimed at stimulating tissue regeneration.

  View details for DOI 10.1101/cshperspect.a008078

  View details for Web of Science ID 000308030500007

  View details for PubMedID 22723493

• Differentiation of multipotent vascular stem cells contributes to vascular diseases NATURE COMMUNICATIONS Tang, Z., Wang, A., Yuan, F., Yan, Z., Liu, B., Chu, J. S., Helms, J. A., Li, S. 2012; 3

  Abstract

  It is generally accepted that the de-differentiation of smooth muscle cells, from the contractile to the proliferative/synthetic phenotype, has an important role during vascular remodelling and diseases. Here we provide evidence that challenges this theory. We identify a new type of stem cell in the blood vessel wall, named multipotent vascular stem cells. Multipotent vascular stem cells express markers, including Sox17, Sox10 and S100β, are cloneable, have telomerase activity, and can differentiate into neural cells and mesenchymal stem cell-like cells that subsequently differentiate into smooth muscle cells. On the other hand, we perform lineage tracing with smooth muscle myosin heavy chain as a marker and find that multipotent vascular stem cells and proliferative or synthetic smooth muscle cells do not arise from the de-differentiation of mature smooth muscle cells. In response to vascular injuries, multipotent vascular stem cells, instead of smooth muscle cells, become proliferative, and differentiate into smooth muscle cells and chondrogenic cells, thus contributing to vascular remodelling and neointimal hyperplasia. These findings support a new hypothesis that the differentiation of multipotent vascular stem cells, rather than the de-differentiation of smooth muscle cells, contributes to vascular remodelling and diseases.

  View details for DOI 10.1038/ncomms1867

  View details for Web of Science ID 000306099900002

  View details for PubMedID 22673902

  View details for PubMedCentralID PMC3538044

• A Cross-Species Analysis of MicroRNAs in the Developing Avian Face PLOS ONE Powder, K. E., Ku, Y., Brugmann, S. A., Veile, R. A., Renaud, N. A., Helms, J. A., Lovett, M. 2012; 7 (4)

  Abstract

  Higher vertebrates use similar genetic tools to derive very different facial features. This diversity is believed to occur through temporal, spatial and species-specific changes in gene expression within cranial neural crest (NC) cells. These contribute to the facial skeleton and contain species-specific information that drives morphological variation. A few signaling molecules and transcription factors are known to play important roles in these processes, but little is known regarding the role of micro-RNAs (miRNAs). We have identified and compared all miRNAs expressed in cranial NC cells from three avian species (chicken, duck, and quail) before and after species-specific facial distinctions occur. We identified 170 differentially expressed miRNAs. These include thirty-five novel chicken orthologs of previously described miRNAs, and six avian-specific miRNAs. Five of these avian-specific miRNAs are conserved over 120 million years of avian evolution, from ratites to galliforms, and their predicted target mRNAs include many components of Wnt signaling. Previous work indicates that mRNA gene expression in NC cells is relatively static during stages when the beak acquires species-specific morphologies. However, miRNA expression is remarkably dynamic within this timeframe, suggesting that the timing of specific developmental transitions is altered in birds with different beak shapes. We evaluated one miRNA:mRNA target pair and found that the cell cycle regulator p27(KIP1) is a likely target of miR-222 in frontonasal NC cells, and that the timing of this interaction correlates with the onset of phenotypic variation. Our comparative genomic approach is the first comprehensive analysis of miRNAs in the developing facial primordial, and in species-specific facial development.

  View details for DOI 10.1371/journal.pone.0035111

  View details for Web of Science ID 000305345000043

  View details for PubMedID 22523571

• Mechanosensing by the Primary Cilium: Deletion of Kif3A Reduces Bone Formation Due to Loading PLOS ONE Temiyasathit, S., Tang, W. J., Leucht, P., Anderson, C. T., Monica, S. D., Castillo, A. B., Helms, J. A., Stearns, T., Jacobs, C. R. 2012; 7 (3)

  Abstract

  Primary cilia, solitary microtubule-based structures that grow from the centriole and extend into the extracellular space, have increasingly been implicated as sensors of a variety of biochemical and biophysical signals. Mutations in primary cilium-related genes have been linked to a number of rare developmental disorders as well as dysregulation of cell proliferation. We propose that primary cilia are also important in mechanically regulated bone formation in adults and that their malfunction could play a role in complex multi-factorial bone diseases, such as osteoporosis. In this study, we generated mice with an osteoblast- and osteocyte-specific knockout of Kif3a, a subunit of the kinesin II intraflagellar transport (IFT) protein; IFT is required for primary cilia formation, maintenance, and function. These Colα1(I) 2.3-Cre;Kif3a(fl/fl) mice exhibited no obvious morphological skeletal abnormalities. Skeletally mature Colα1(I) 2.3-Cre;Kif3a(fl/fl) and control mice were exposed to 3 consecutive days of cyclic axial ulna loading, which resulted in a significant increase in bone formation in both the conditional knockouts and controls. However, Colα1(I) 2.3-Cre;Kif3a(fl/fl) mice did exhibit decreased formation of new bone in response to mechanical ulnar loading compared to control mice. These results suggest that primary cilia act as cellular mechanosensors in bone and that their function may be critical for the regulation of bone physiology due to mechanical loading in adults.

  View details for DOI 10.1371/journal.pone.0033368

  View details for Web of Science ID 000302381500135

  View details for PubMedID 22428034

  View details for PubMedCentralID PMC3299788

• Vascular endothelial growth factor improves bone repair in a murine nonunion model. The Iowa orthopaedic journal Ogilvie, C. M., Lu, C., Marcucio, R., Lee, M., Thompson, Z., Hu, D., Helms, J. A., Miclau, T. 2012; 32: 90-94

  Abstract

  Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that plays an important role during skeletal development and fracture healing. Previous experimental studies have shown that VEGF applied immediately after injury can stimulate bone repair in animal fracture nonunion models. However, the effectiveness of VEGF on an established fracture non-union has not been determined. the goal of this work was to test the ability of VEGF applied at a later stage on the healing of fracture nonunions.In this study, a murine non-union model was induced by rapid distraction of a tibia osteotomy. this model exhibits radiological and histological evidence of impaired fracture healing at 7 days after the completion of distraction. VEGF (10 µg in 20 µl Pbs/day, n=10) or control (20 µl Pbs/day, n=10) was injected directly into the distraction gap through the posterior musculature on three consecutive days (7, 8, and 9 days after completing distraction). A third group of animals (n=10) with rapid distraction, but no injections, served as non-treated controls. Fracture healing was analyzed by x-ray, histology, and histomorphometry at 27 days after the last round of distraction.radiographs showed that half of the VEGF treated animals (5/10) achieved bony healing whereas the majority of Pbs treated (7/10) and non-treated controls (8/10) did not exhibit bone bridging. Histological and histomorphometric analyses demonstrated that VEGF increased, but not significantly, the amount of bone formed in the distraction gap (1.35 ± 0.35 mm(3)), compared to the saline treated (0.77 ± 0.25 mm(3), p=0.19) and non-treated animals (0.79 ± 0.23mm(3), p=0.12).Results from this study demonstrate that VEGF potentially promotes bone repair, warranting further research in this direction.

  View details for PubMedID 23576927

• Wnt/beta-catenin signaling and Msx1 promote outgrowth of the maxillary prominences FRONTIERS IN PHYSIOLOGY Medio, M., Yeh, E., Popelut, A., Babajko, S., Berdal, A., Helms, J. A. 2012; 3

  View details for DOI 10.3389/fphys.2012.00375

  View details for Web of Science ID 000209173000368

• Wnt/ß-catenin signaling and Msx1 promote outgrowth of the maxillary prominences. Frontiers in physiology Medio, M., Yeh, E., Popelut, A., Babajko, S., Berdal, A., Helms, J. A. 2012; 3: 375-?

  Abstract

  Facial morphogenesis requires a series of precisely orchestrated molecular events to promote the growth and fusion of the facial prominences. Cleft palate (CP) results from perturbations in this process. The transcriptional repressor Msx1 is a key participant in these molecular events, as demonstrated by the palatal clefting phenotype observed in Msx1(-/-) embryos. Here, we exploited the high degree of conservation that exists in the gene regulatory networks that shape the faces of birds and mice, to gain a deeper understanding of Msx1 function in CP. Histomorphometric analyses indicated that facial development was disrupted as early as E12.5 in Msx1(-/-) embryos, long before the palatal shelves have formed. By mapping the expression domain of Msx1 in E11.5 and E12.5 embryos, we found the structures most affected by loss of Msx1 function were the maxillary prominences. Maxillary growth retardation was accompanied by perturbations in angiogenesis that preceded the CP phenotype. Experimental chick manipulations and in vitro assays showed that the regulation of Msx1 expression by the Wnt/β-catenin pathway is highly specific. Our data in mice and chicks indicate a conserved role for Msx1 in regulating the outgrowth of the maxillary prominences, and underscore how imbalances in Msx1 function can lead of growth disruptions that manifest as CP.

  View details for DOI 10.3389/fphys.2012.00375

  View details for PubMedID 23055979

• 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

• A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression NATURE Wang, K. C., Yang, Y. W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B. R., Protacio, A., Flynn, R. A., Gupta, R. A., Wysocka, J., Lei, M., Dekker, J., Helms, J. A., Chang, H. Y. 2011; 472 (7341): 120-U158

  Abstract

  The genome is extensively transcribed into long intergenic noncoding RNAs (lincRNAs), many of which are implicated in gene silencing. Potential roles of lincRNAs in gene activation are much less understood. Development and homeostasis require coordinate regulation of neighbouring genes through a process termed locus control. Some locus control elements and enhancers transcribe lincRNAs, hinting at possible roles in long-range control. In vertebrates, 39 Hox genes, encoding homeodomain transcription factors critical for positional identity, are clustered in four chromosomal loci; the Hox genes are expressed in nested anterior-posterior and proximal-distal patterns colinear with their genomic position from 3' to 5'of the cluster. Here we identify HOTTIP, a lincRNA transcribed from the 5' tip of the HOXA locus that coordinates the activation of several 5' HOXA genes in vivo. Chromosomal looping brings HOTTIP into close proximity to its target genes. HOTTIP RNA binds the adaptor protein WDR5 directly and targets WDR5/MLL complexes across HOXA, driving histone H3 lysine 4 trimethylation and gene transcription. Induced proximity is necessary and sufficient for HOTTIP RNA activation of its target genes. Thus, by serving as key intermediates that transmit information from higher order chromosomal looping into chromatin modifications, lincRNAs may organize chromatin domains to coordinate long-range gene activation.

  View details for DOI 10.1038/nature09819

  View details for PubMedID 21423168

• Long noncoding RNA programs active chromatin domain to coordinate homeotic gene expression 71st Annual Meeting of the Society-for-Investigative-Dermatology Wang, K. C., Yang, Y. W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B. R., Protacio, A., Flynn, R. A., Gupta, R. A., Wysocka, J., Lei, M., Dekker, J., Helms, J. A., Chang, H. Y. NATURE PUBLISHING GROUP. 2011: S63–S63

  View details for Web of Science ID 000289035600373

• The acceleration of implant osseointegration by liposomal Wnt3a BIOMATERIALS Popelut, A., Rooker, S. M., Leucht, P., Medio, M., Brunski, J. B., Helms, J. A. 2010; 31 (35): 9173-9181

  Abstract

  The strength of a Wnt-based strategy for tissue regeneration lies in the central role that Wnts play in healing. Tissue injury triggers local Wnt activation at the site of damage, and this Wnt signal is required for the repair and/or regeneration of almost all tissues including bone, neural tissues, myocardium, and epidermis. We developed a biologically based approach to create a transient elevation in Wnt signaling in peri-implant tissues, and in doing so, accelerated bone formation around the implant. Our subsequent molecular and cellular analyses provide mechanistic insights into the basis for this pro-osteogenic effect. Given the essential role of Wnt signaling in bone formation, this protein-based approach may have widespread application in implant osseointegration.

  View details for DOI 10.1016/j.biomaterials.2010.08.045

  View details for Web of Science ID 000284393300004

  View details for PubMedID 20864159

• Craniofacial Ciliopathies: A New Classification for Craniofacial Disorders AMERICAN JOURNAL OF MEDICAL GENETICS PART A Brugmann, S. A., Cordero, D. R., Helms, J. A. 2010; 152A (12): 2995-3006

  Abstract

  Craniofacial anomalies are some of the most variable and common defects affecting the population. Herein, we examine a group of craniofacial disorders that are the result of defects in primary cilia; ubiquitous, microtubule-based organelles that transduce molecular signals and facilitate the interactions between the cell and its environment. Based on the frequent appearance of craniofacial phenotypes in diseases born from defective primary cilia (ciliopathies) we propose a new class of craniofacial disorders referred to as craniofacial ciliopathies. We explore the most frequent phenotypes associated with ciliopathic conditions and the ciliary gene mutations responsible for craniofacial defects. Finally, we propose that some non-classified disorders may now be classified as craniofacial ciliopathies.

  View details for DOI 10.1002/ajmg.a.33727

  View details for Web of Science ID 000285251800008

  View details for PubMedID 21108387

  View details for PubMedCentralID PMC3121325

• 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

• rBMP Represses Wnt Signaling and Influences Skeletal Progenitor Cell Fate Specification During Bone Repair JOURNAL OF BONE AND MINERAL RESEARCH Minear, S., Leucht, P., Miller, S., Helms, J. A. 2010; 25 (6): 1196-1207

  Abstract

  Bone morphogenetic proteins (BMPs) participate in multiple stages of the fetal skeletogenic program from promoting cell condensation to regulating chondrogenesis and bone formation through endochondral ossification. Here, we show that these pleiotropic functions are recapitulated when recombinant BMPs are used to augment skeletal tissue repair. In addition to their well-documented ability to stimulate chondrogenesis in a skeletal injury, we show that recombinant BMPs (rBMPs) simultaneously suppress the differentiation of skeletal progenitor cells in the endosteum and bone marrow cavity to an osteoblast lineage. Both the prochondrogenic and antiosteogenic effects are achieved because rBMP inhibits endogenous beta-catenin-dependent Wnt signaling. In the injured periosteum, this repression of Wnt activity results in sox9 upregulation; consequently, cells in the injured periosteum adopt a chondrogenic fate. In the injured endosteum, rBMP also inhibits Wnt signaling, which results in the runx2 and collagen type I downregulation; consequently, cells in this region fail to differentiate into osteoblasts. In muscle surrounding the skeletal injury site, rBMP treatment induces Smad phosphorylation followed by exuberant cell proliferation, an increase in alkaline phosphatase activity, and chondrogenic differentiation. Thus different populations of adult skeletal progenitor cells interpret the same rBMP stimulus in unique ways, and these responses mirror the pleiotropic effects of BMPs during fetal skeletogenesis. These mechanistic insights may be particularly useful for optimizing the reparative potential of rBMPs while simultaneously minimizing their adverse outcomes.

  View details for DOI 10.1002/jbmr.29

  View details for Web of Science ID 000279441300002

  View details for PubMedID 20200943

• Molecular control of facial morphology SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Liu, B., Rooker, S. M., Helms, J. A. 2010; 21 (3): 309-313

  Abstract

  We present a developmental perspective on the concept of phylotypic and phenotypic stages of craniofacial development. Within orders of avians and mammals, a phylotypic period exists when the morphology of the facial prominences is minimally divergent. We postulate that species-specific facial variations arise as a result of subtle shifts in the timing and the duration of molecular pathway activity (e.g., heterochrony), and present evidence demonstrating a critical role for Wnt and FGF signaling in this process. The same molecular pathways that shape the vertebrate face are also implicated in craniofacial deformities, indicating that comparisons between and among animal species may represent a novel method for the identification of human craniofacial disease genes.

  View details for DOI 10.1016/j.semcdb.2009.09.002

  View details for Web of Science ID 000275805500011

  View details for PubMedID 19747977

  View details for PubMedCentralID PMC3410822

• Wnt Proteins Promote Bone Regeneration SCIENCE TRANSLATIONAL MEDICINE Minear, S., Leucht, P., Jiang, J., Liu, B., Zeng, A., Fuerer, C., Nusse, R., Helms, J. A. 2010; 2 (29)

  Abstract

  The Wnt signaling pathway plays a central role in bone development and homeostasis. In most cases, Wnt ligands promote bone growth, which has led to speculation that Wnt factors could be used to stimulate bone healing. We gained insights into the mechanism by which Wnt signaling regulates adult bone repair through the use of the mouse strain Axin2(LacZ/LacZ) in which the cellular response to Wnt is increased. We found that bone healing after injury is accelerated in Axin2(LacZ/LacZ) mice, a consequence of more robust proliferation and earlier differentiation of skeletal stem and progenitor cells. In parallel, we devised a biochemical strategy to increase the duration and strength of Wnt signaling at the sites of skeletal injury. Purified Wnt3a was packaged in liposomal vesicles and delivered to skeletal defects, where it stimulated the proliferation of skeletal progenitor cells and accelerated their differentiation into osteoblasts, cells responsible for bone growth. The end result was faster bone regeneration. Because Wnt signaling is conserved in mammalian tissue repair, this protein-based approach may have widespread applications in regenerative medicine.

  View details for DOI 10.1126/scitranslmed.3000231

  View details for Web of Science ID 000277304700001

  View details for PubMedID 20427820

• A primary cilia-dependent etiology for midline facial disorders HUMAN MOLECULAR GENETICS Brugmann, S. A., Allen, N. C., James, A. W., Mekonnen, Z., Madan, E., Helms, J. A. 2010; 19 (8): 1577-1592

  Abstract

  Human faces exhibit enormous variation. When pathological conditions are superimposed on normal variation, a nearly unbroken series of facial morphologies is produced. When viewed in full, this spectrum ranges from cyclopia and hypotelorism to hypertelorism and facial duplications. Decreased Hedgehog pathway activity causes holoprosencephaly and hypotelorism. Here, we show that excessive Hedgehog activity, caused by truncating the primary cilia on cranial neural crest cells, causes hypertelorism and frontonasal dysplasia (FND). Elimination of the intraflagellar transport protein Kif3a leads to excessive Hedgehog responsiveness in facial mesenchyme, which is accompanied by broader expression domains of Gli1, Ptc and Shh, and reduced expression domains of Gli3. Furthermore, broader domains of Gli1 expression correspond to areas of enhanced neural crest cell proliferation in the facial prominences of Kif3a conditional knockouts. Avian Talpid embryos that lack primary cilia exhibit similar molecular changes and similar facial phenotypes. Collectively, these data support our hypothesis that a severe narrowing of the facial midline and excessive expansion of the facial midline are both attributable to disruptions in Hedgehog pathway activity. These data also raise the possibility that genes encoding ciliary proteins are candidates for human conditions of hypertelorism and FNDs.

  View details for DOI 10.1093/hmg/ddq030

  View details for Web of Science ID 000276544000017

  View details for PubMedID 20106874

  View details for PubMedCentralID PMC2846163

• Comparative gene expression analysis of avian embryonic facial structures reveals new candidates for human craniofacial disorders HUMAN MOLECULAR GENETICS Brugmann, S. A., Powder, K. E., Young, N. M., Goodnough, L. H., Hahn, S. M., James, A. W., Helms, J. A., Lovett, M. 2010; 19 (5): 920-930

  Abstract

  Mammals and birds have common embryological facial structures, and appear to employ the same molecular genetic developmental toolkit. We utilized natural variation found in bird beaks to investigate what genes drive vertebrate facial morphogenesis. We employed cross-species microarrays to describe the molecular genetic signatures, developmental signaling pathways and the spectrum of transcription factor (TF) gene expression changes that differ between cranial neural crest cells in the developing beaks of ducks, quails and chickens. Surprisingly, we observed that the neural crest cells established a species-specific TF gene expression profile that predates morphological differences between the species. A total of 232 genes were differentially expressed between the three species. Twenty-two of these genes, including Fgfr2, Jagged2, Msx2, Satb2 and Tgfb3, have been previously implicated in a variety of mammalian craniofacial defects. Seventy-two of the differentially expressed genes overlap with un-cloned loci for human craniofacial disorders, suggesting that our data will provide a valuable candidate gene resource for human craniofacial genetics. The most dramatic changes between species were in the Wnt signaling pathway, including a 20-fold up-regulation of Dkk2, Fzd1 and Wnt1 in the duck compared with the other two species. We functionally validated these changes by demonstrating that spatial domains of Wnt activity differ in avian beaks, and that Wnt signals regulate Bmp pathway activity and promote regional growth in facial prominences. This study is the first of its kind, extending on previous work in Darwin's finches and provides the first large-scale insights into cross-species facial morphogenesis.

  View details for DOI 10.1093/hmg/ddp559

  View details for Web of Science ID 000274341400015

  View details for PubMedID 20015954

  View details for PubMedCentralID PMC2816616

• Role of Wnt Signaling in the Biology of the Periodontium DEVELOPMENTAL DYNAMICS Rooker, S. M., Liu, B., Helms, J. A. 2010; 239 (1): 140-147

  Abstract

  Continuously erupting teeth have associated with them a continuously regenerating periodontal ligament, but the factors that control this amazing regenerative potential are unknown. We used genetic strategies to show that the periodontal ligament arises from the cranial neural crest. Despite their histological similarity, the periodontal ligament of continuously erupting incisor teeth differs dramatically from the periodontal ligament of molar teeth. The most notable difference was in the distribution of Wnt responsive cells in the incisor periodontal ligament, which coincided with regions of periodontal ligament cell proliferation. We discuss these findings in the context of dental tissue regeneration.

  View details for DOI 10.1002/dvdy.22003

  View details for Web of Science ID 000273703900012

  View details for PubMedID 19530172

  View details for PubMedCentralID PMC3280952

• Regeneration, repair and remembering identity: the three Rs of Hox gene expression TRENDS IN CELL BIOLOGY Wang, K. C., Helms, J. A., Chang, H. Y. 2009; 19 (6): 268-275

  Abstract

  Hox genes encode transcription factors that specify embryonic positional identity in cells and guide tissue differentiation. Recent advances have greatly increased our understanding of the epigenetic mechanisms that ensure the faithful expression of Hox genes in adult cells and which involve the interplay of histone methylation, demethylation and intergenic transcription of long non-coding RNAs. The transcriptional memory of Hox genes poses both an opportunity and a challenge for regenerative medicine. Matching the positional identity of transplanted stem cells with that of the host environment, as reflected by their respective Hox profiles, is likely to be required to achieve regenerative healing. Strategies to manipulate the plasticity of Hox gene expression will probably become a major focus in regenerative medicine.

  View details for DOI 10.1016/j.tcb.2009.03.007

  View details for PubMedID 19428253

• Endochondral ossification is required for haematopoietic stem-cell niche formation NATURE Chan, C. K., Chen, C., Luppen, C. A., Kim, J., DeBoer, A. T., Wei, K., Helms, J. A., Kuo, C. J., Kraft, D. L., Weissman, I. L. 2009; 457 (7228): 490-U9

  Abstract

  Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.

  View details for DOI 10.1038/nature07547

  View details for PubMedID 19078959

• CONTROLLING THE IN VIVO ACTIVITY OF WNT LIPOSOMES METHODS IN ENZYMOLOGY LIPOSOMES, PT G Zhao, L., Rooker, S. M., Morrell, N., Leucht, P., Simanovskii, D., Helms, J. A. 2009; 465: 331-347

  Abstract

  Liposomes offer a method of delivering small molecules, nucleic acids, and proteins to sites within the body. Typically, bioactive materials are encapsulated within the liposomal aqueous core and liposomal phase transition is elicited by pH or temperature changes. We developed a new class of liposomes for the in vivo delivery of lipid-modified proteins. First, we show that the inclusion of a chromophore into the liposomal or vesosomal membrane renders these lipid vesicles extremely sensitive to very small (muJ) changes in energy. Next, we demonstrate that the lipid-modified Wnt protein is not encapsulated within a liposome but rather is tethered to the exoliposomal surface in an active configuration. When applied to intact skin, chromophore-modified liposomes do not penetrate past the corneal layer of the epidermis, but remain localized to the site of application. Injury to the epidermis allows rapid penetration of liposomes into the dermis, which suggests that mild forms of dermabrasion will greatly enhance transdermal delivery of liposome-packaged molecules. Finally, we demonstrate that topical application of Wnt3a liposomes rapidly stimulates proliferation of cells in the corneal layer, resulting in a thicker, more fibrillous epidermis.

  View details for DOI 10.1016/S0076-6879(09)65017-5

  View details for Web of Science ID 000272400200018

  View details for PubMedID 19913175

• Translating insights from development into regenerative medicine: The function of Wnts in bone biology SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Leucht, P., Minear, S., Ten Berge, D., Nusse, R., Helms, J. A. 2008; 19 (5): 434-443

  Abstract

  The Wnt pathway constitutes one of the most attractive candidates for modulating skeletal tissue regeneration based on its functions during skeletal development and homeostasis. Wnts participate in every stage of skeletogenesis, from the self-renewal and proliferation of skeletal stem cells to the specification of osteochondroprogenitor cells and the maturation of chondrocytes and osteoblasts. We propose that the function of Wnts depend upon a skeletogenic cell's state of differentiation. In this review we summarize recent data with a focus on the roles of Wnt signaling in mesenchymal stem cell fate, osteoprogenitor cell differentiation, chondrocyte maturation, bone remodeling, and bone regeneration.

  View details for DOI 10.1016/j.semcdb.2008.09.002

  View details for Web of Science ID 000262670500003

  View details for PubMedID 18824114

• Wnt and FGF signals interact to coordinate growth with cell fate specification during limb development DEVELOPMENT ten Berge, D., Brugmann, S. A., Helms, J. A., Nusse, R. 2008; 135 (19): 3247-3257

  Abstract

  A fundamental question in developmental biology is how does an undifferentiated field of cells acquire spatial pattern and undergo coordinated differentiation? The development of the vertebrate limb is an important paradigm for understanding these processes. The skeletal and connective tissues of the developing limb all derive from a population of multipotent progenitor cells located in its distal tip. During limb outgrowth, these progenitors segregate into a chondrogenic lineage, located in the center of the limb bud, and soft connective tissue lineages located in its periphery. We report that the interplay of two families of signaling proteins, fibroblast growth factors (FGFs) and Wnts, coordinate the growth of the multipotent progenitor cells with their simultaneous segregation into these lineages. FGF and Wnt signals act together to synergistically promote proliferation while maintaining the cells in an undifferentiated, multipotent state, but act separately to determine cell lineage specification. Withdrawal of both signals results in cell cycle withdrawal and chondrogenic differentiation. Continued exposure to Wnt, however, maintains proliferation and re-specifies the cells towards the soft connective tissue lineages. We have identified target genes that are synergistically regulated by Wnts and FGFs, and show how these factors actively suppress differentiation and promote growth. Finally, we show how the spatial restriction of Wnt and FGF signals to the limb ectoderm, and to a specialized region of it, the apical ectodermal ridge, controls the distribution of cell behaviors within the growing limb, and guides the proper spatial organization of the differentiating tissues.

  View details for DOI 10.1242/dev.023176

  View details for Web of Science ID 000258989400009

  View details for PubMedID 18776145

  View details for PubMedCentralID PMC2756806

• Primary cilia: Cellular sensors for the skeleton 37th International Sun Valley Workshop on Skeletal Tissue Biology Anderson, C. T., Castillo, A. B., Brugmann, S. A., Helms, J. A., Jacobs, C. R., Stearns, T. WILEY-BLACKWELL. 2008: 1074–78

  Abstract

  The primary cilium is a solitary, immotile cilium that is present in almost every mammalian cell type. Primary cilia are thought to function as chemosensors, mechanosensors, or both, depending on cell type, and have been linked to several developmental signaling pathways. Primary cilium malfunction has been implicated in several human diseases, the symptoms of which include vision and hearing loss, polydactyly, and polycystic kidneys. Recently, primary cilia have also been implicated in the development and homeostasis of the skeleton. In this review, we discuss the structure and formation of the primary cilium and some of the mechanical and chemical signals to which it could be sensitive, with a focus on skeletal biology. We also raise several unanswered questions regarding the role of primary cilia as mechanosensors and chemosensors and identify potential research avenues to address these questions.

  View details for DOI 10.1002/ar.20754

  View details for Web of Science ID 000259324900004

  View details for PubMedID 18727074

  View details for PubMedCentralID PMC2879613

• SDF-1 is Expressed in Osteocytes and Periosteal Cells in Response to Mechanical Loading. 30th Annual Meeting of the American-Society-for-Bone-and-Mineral-Research Castillo, A. B., Leucht, P., Tang, J., Helms, J. A., Jacobs, C. R. WILEY-BLACKWELL. 2008: S399–S399

  View details for Web of Science ID 000259411002242

• Wingless protein (Wnt) expression in fetal wounds 94th Annual Clinical Congress of the American-College-of-Surgeons/63rd Annual Sessions of the Owen H Wangensteen Forum on Fundamental Surgical Problems Carre, A. L., Joseph, K., Chang, E., Longaker, M. T., Helms, J., Lorenz, H. P. ELSEVIER SCIENCE INC. 2008: S60–S61

  View details for Web of Science ID 000259288500125

• Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration DEVELOPMENT Leucht, P., Kim, J., Amasha, R., James, A. W., Girod, S., Helms, J. A. 2008; 135 (17): 2845-2854

  Abstract

  The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.

  View details for DOI 10.1242/dev.023788

  View details for Web of Science ID 000258395500003

  View details for PubMedID 18653558

• Liposomal Packaging Generates Wnt Protein with In Vivo Biological Activity PLOS ONE Morrell, N. T., Leucht, P., Zhao, L., Kim, J., Ten Berge, D., Ponnusamy, K., Carre, A. L., Dudek, H., Zachlederova, M., McElhaney, M., Brunton, S., Gunzner, J., Callow, M., Polakis, P., Costa, M., Zhang, X. M., Helms, J. A., Nusse, R. 2008; 3 (8)

  Abstract

  Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.

  View details for DOI 10.1371/journal.pone.0002930

  View details for Web of Science ID 000264412600016

  View details for PubMedID 18698373

  View details for PubMedCentralID PMC2515347

• Beta-catenin-dependent Wnt signaling in mandibular bone regeneration. journal of bone and joint surgery. American volume Leucht, P., Kim, J., Helms, J. A. 2008; 90: 3-8

  Abstract

  Osteoblasts are derived from two distinct embryonic lineages: cranial neural crest, and mesoderm. Both populations of cells are capable of forming bone and cartilage during fetal development and during adult bone repair, but whether they use equivalent molecular pathways to achieve osteoblast differentiation is unknown. We addressed this question in the context of cranial repair and focused on the role of Wnt signaling in mandibular skeletal healing. Transgenic Wnt reporter mice were used to pinpoint Wnt-responsive cells in the injury callus, and in situ hybridization was used to identify some of the Wnt ligands expressed by cells during the repair process. A gene transfer technique was employed to abrogate Wnt signaling during mandibular healing, and we found that reparative intramembranous ossification requires a functional Wnt pathway. Finally, we evaluated how constitutive activation of the Wnt pathway, caused by mutation of the LRP5 receptor, affected bone repair in the mandible. Taken together, these data underscore the functional requirement for Wnt signaling in cranial skeletal healing.

  View details for DOI 10.2106/JBJS.G.01136

  View details for PubMedID 18292349

• Beta-catenin-dependent Wnt signaling in mandibular bone regeneration 74th Annual Meeting of the American-Academy-of-Orthopaedic-Surgeons Leucht, P., Kim, J., Helms, J. A. JOURNAL BONE JOINT SURGERY INC. 2008: 3–8

  Abstract

  Osteoblasts are derived from two distinct embryonic lineages: cranial neural crest, and mesoderm. Both populations of cells are capable of forming bone and cartilage during fetal development and during adult bone repair, but whether they use equivalent molecular pathways to achieve osteoblast differentiation is unknown. We addressed this question in the context of cranial repair and focused on the role of Wnt signaling in mandibular skeletal healing. Transgenic Wnt reporter mice were used to pinpoint Wnt-responsive cells in the injury callus, and in situ hybridization was used to identify some of the Wnt ligands expressed by cells during the repair process. A gene transfer technique was employed to abrogate Wnt signaling during mandibular healing, and we found that reparative intramembranous ossification requires a functional Wnt pathway. Finally, we evaluated how constitutive activation of the Wnt pathway, caused by mutation of the LRP5 receptor, affected bone repair in the mandible. Taken together, these data underscore the functional requirement for Wnt signaling in cranial skeletal healing.

  View details for DOI 10.2106/JBJS.G.01136

  View details for Web of Science ID 000252981300002

• A dermal HOX transcriptional program regulates site-specific epidermal fate GENES & DEVELOPMENT Rinn, J. L., Wang, J. K., Allen, N., Brugmann, S. A., Mikels, A. J., Liu, H., Ridky, T. W., Stadler, H. S., Nusse, R., Helms, J. A., Chang, H. Y. 2008; 22 (3): 303-307

  Abstract

  Reciprocal epithelial-mesenchymal interactions shape site-specific development of skin. Here we show that site-specific HOX expression in fibroblasts is cell-autonomous and epigenetically maintained. The distal-specific gene HOXA13 is continually required to maintain the distal-specific transcriptional program in adult fibroblasts, including expression of WNT5A, a morphogen required for distal development. The ability of distal fibroblasts to induce epidermal keratin 9, a distal-specific gene, is abrogated by depletion of HOXA13, but rescued by addition of WNT5A. Thus, maintenance of appropriate HOX transcriptional program in adult fibroblasts may serve as a source of positional memory to differentially pattern the epithelia during homeostasis and regeneration.

  View details for DOI 10.1101/gad.1610508

  View details for PubMedID 18245445

• 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

• Bone regeneration is regulated by Wnt signaling JOURNAL OF BONE AND MINERAL RESEARCH Kim, J., Leucht, P., Lam, K., Luppen, C., Ten Berge, D., Nusse, R., Helms, J. A. 2007; 22 (12): 1913-1923

  Abstract

  Tissue regeneration is increasingly viewed as reactivation of a developmental process that, when misappropriated, can lead to malignant growth. Therefore, understanding the molecular and cellular pathways that govern tissue regeneration provides a glimpse into normal development as well as insights into pathological conditions such as cancer. Herein, we studied the role of Wnt signaling in skeletal tissue regeneration.Some adult tissues have the ability to regenerate, and among these, bone is one of the most remarkable. Bone exhibits a persistent, lifelong capacity to reform after injury, and continual bone regeneration is a prerequisite to maintaining bone mass and density. Even slight perturbations in bone regeneration can have profound consequences, as exemplified by conditions such as osteoporosis and delayed skeletal repair. Here, our goal was to determine the role of Wnts in adult bone regeneration.Using TOPgal reporter mice, we found that damage to the skeleton instigated Wnt reporter activity, specifically at the site of injury. We used a skeletal injury model to show that Wnt inhibition, achieved through adenoviral expression of Dkk1 in the adult skeleton, prevented the differentiation of osteoprogenitor cells.As a result, injury-induced bone regeneration was reduced by 84% compared with controls. Constitutive activation of the Wnt pathway resulting from a mutation in the Lrp5 Wnt co-receptor results in high bone mass, but our experiments showed that this same point mutation caused a delay in bone regeneration. In these transgenic mice, osteoprogenitor cells in the injury site were maintained in a proliferative state and differentiation into osteoblasts was delayed.When considered together, these data provide a framework for understanding the roles of Wnt signaling in adult bone regeneration and suggest a feasible approach to treating clinical conditions where enhanced bone formation is desired.

  View details for DOI 10.1359/JBMR.070802

  View details for Web of Science ID 000251292400012

  View details for PubMedID 17696762

• Cross-regulatory interactions between Fgf8 and Shh in the avian frontonasal prominence. Congenital anomalies Abzhanov, A., Cordero, D. R., Sen, J., Tabin, C. J., Helms, J. A. 2007; 47 (4): 136-148

  Abstract

  The frontonasal prominence of the developing avian embryo contains an organizing center, defined by juxtaposition of the Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) expression domains. This molecular interface presages any detectable growth of the frontonasal prominence, and experiments involving transplantation of this boundary epithelium have demonstrated it is a source of dorsal-ventral and rostral-caudal patterning information for the neural crest-derived mesenchyme of the upper beak. We explored the ontogeny of this organizing center by mapping the expression domains of both genes and their receptors and downstream targets. We tested the extent to which Shh and Fgf8 regulate each other's expression in this frontonasal organizer by either blocking or ectopically activating these pathways. Our experiments revealed mutual antagonism between the two molecules, which aids in establishing and maintaining a molecular boundary that subsequently influences patterning and growth of the middle and upper face.

  View details for PubMedID 17988255

• Bone voyage: An expedition into the molecular and cellular parameters affecting bone graft fate BONE Helms, J. A., Amasha, R. R., Leucht, P. 2007; 41 (4): 479-485

  Abstract

  The demand for bone grafts in orthopaedic and craniofacial surgery is steadily increasing. Estimations suggest that about 500,000 are performed annually in the United States that include bone grafting as a component of the surgery, and the majority of these surgeries employ autografts. This perspective focuses on the biological events that occur during osseointegration of such bone grafts. Here, three key factors of graft osseointegration--the embryonic origin, the inclusion of skeletal progenitor cells, and the integrity of the recipient site--are discussed. Altogether, they form the foundation for survival of the bone graft and eventually for a positive clinical outcome of the procedure.

  View details for DOI 10.1016/j.bone.2007.06.023

  View details for Web of Science ID 000249800800001

  View details for PubMedID 17692586

• Shaping up and shipping out: the role of cilia in growth and patterning. Journal of musculoskeletal & neuronal interactions Brugmann, S., Helms, J. 2007; 7 (4): 300-?

  View details for PubMedID 18094481

• Wnt signaling mediates regional specification in the vertebrate face DEVELOPMENT Brugmann, S. A., Goodnough, L. H., Gregorieff, A., Leucht, P., Ten Berge, D., Fuerer, C., Clevers, H., Nusse, R., Helms, J. A. 2007; 134 (18): 3283-3295

  Abstract

  At early stages of development, the faces of vertebrate embryos look remarkably similar, yet within a very short timeframe they adopt species-specific facial characteristics. What are the mechanisms underlying this regional specification of the vertebrate face? Using transgenic Wnt reporter embryos we found a highly conserved pattern of Wnt responsiveness in the developing mouse face that later corresponded to derivatives of the frontonasal and maxillary prominences. We explored the consequences of disrupting Wnt signaling, first using a genetic approach. Mice carrying compound null mutations in the nuclear mediators Lef1 and Tcf4 exhibited radically altered facial features that culminated in a hyperteloric appearance and a foreshortened midface. We also used a biochemical approach to perturb Wnt signaling and found that in utero delivery of a Wnt antagonist, Dkk1, produced similar midfacial malformations. We tested the hypothesis that Wnt signaling is an evolutionarily conserved mechanism controlling facial morphogenesis by determining the pattern of Wnt responsiveness in avian faces, and then by evaluating the consequences of Wnt inhibition in the chick face. Collectively, these data elucidate a new role for Wnt signaling in regional specification of the vertebrate face, and suggest possible mechanisms whereby species-specific facial features are generated.

  View details for DOI 10.1242/dev.005132

  View details for Web of Science ID 000249013700006

  View details for PubMedID 17699607

• The origins of species-specific facial morphology: the proof is in the pigeon Symposium on Linking Genes and Morphology in Vertebrates Helms, J. A., Brugmann, S. A. OXFORD UNIV PRESS INC. 2007: 338–42

  Abstract

  One of the principal objectives of developmental research is to understand morphogenesis and in doing so, gain insights into the genetic basis of variation observed throughout the Animal Kingdom. In this review we take an approach, first popularized by Darwin, to understanding how diversity is created by using the domesticated pigeon as a model organism. Nearly 3000 years of selective breeding has produced an astonishing array of feather patterns, behaviors, skeletal shapes, and body sizes. Cumulatively, these features make the pigeon an exemplar of morphological variation. Our research interests center around exploiting the unique properties of domesticated pigeons to gain critical insights into the molecular and cellular basis for craniofacial variation.

  View details for DOI 10.1093/icb/icm051

  View details for Web of Science ID 000249765100002

  View details for PubMedID 21672843

• 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

• Molecular analysis of healing at a bone-implant interface JOURNAL OF DENTAL RESEARCH Colnot, C., Romero, D. M., Huang, S., Rahman, J., Currey, J. A., Nanci, A., Brunski, J. B., Helms, J. A. 2007; 86 (9): 862-867

  Abstract

  While bone healing occurs around implants, the extent to which this differs from healing at sites without implants remains unknown. We tested the hypothesis that an implant surface may affect the early stages of healing. In a new mouse model, we made cellular and molecular evaluations of healing at bone-implant interfaces vs. empty cortical defects. We assessed healing around Ti-6Al-4V, poly(L-lactide-co-D,L,-lactide), and 303 stainless steel implants with surface characteristics comparable with those of commercial implants. Our qualitative cellular and molecular evaluations showed that osteoblast differentiation and new bone deposition began sooner around the implants, suggesting that the implant surface and microenvironment around implants favored osteogenesis. The general stages of healing in this mouse model resembled those in larger animal models, and supported the use of this new model as a test bed for studying cellular and molecular responses to biomaterial and biomechanical conditions.

  View details for Web of Science ID 000249013200011

  View details for PubMedID 17720856

• Stage-dependent craniofacial defects resulting from Sprouty2 overexpression DEVELOPMENTAL DYNAMICS Goodnough, L. H., Brugmann, S. A., Hu, D., Helms, J. A. 2007; 236 (7): 1918-1928

  Abstract

  Sprouty genes encode intracellular regulators of receptor tyrosine kinases that function in a variety of developmental events. Although mice carrying null mutations in Sprouty genes exhibit craniofacial anomalies, the precise role of these regulatory proteins in facial development remains unclear. Here, we show that overexpression of spry2 at the initiation of craniofacial development results in a dramatic arrest in outgrowth of the facial prominences. Although endogenous spry2 and fibroblast growth factor 8 (fgf8) are coexpressed throughout much of craniofacial development, overexpression of spry2 did not alter the spatiotemporal patterns of fgf target gene expression. The morphological consequences of spry2 overexpression were specific: all of the facial prominences were truncated, but despite this gross malformation, the programs of osteogenesis and chondrogenesis were not impaired. Collectively, these data suggest that Sprouty2 plays a role in the outgrowth of facial prominences independent of canonical Fgf signaling.

  View details for DOI 10.1002/dvdy.21195

  View details for Web of Science ID 000248041000018

  View details for PubMedID 17576140

• 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

• Reconciling the roles of FAK in osteoblast differentiation, osteoclast remodeling, and bone regeneration BONE Kim, J., Leuch, P., Luppen, C. A., Park, Y. J., Beggs, H. E., Damsky, C. H., Helms, J. A. 2007; 41 (1): 39-51

  Abstract

  Integrins link the inside of a cell with its outside environment and in doing so regulate a wide variety of cell behaviors. Integrins are well known for their roles in angiogenesis and cell migration but their functions in bone formation are less clear. The majority of integrin signaling proceeds through focal adhesion kinase (FAK), an essential component of the focal adhesion complex. We generated transgenic mice in which FAK was deleted in osteoblasts and uncovered a previously unknown role in osteoblast differentiation associated with bone healing. FAK mutant cells migrated to the site of skeletal injury and angiogenesis was unaffected yet the transgenic mice still exhibited numerous defects in reparative bone formation. Osteoblast differentiation itself was unperturbed by the loss of FAK, whereas the attachment of osteoclasts to the bone matrix was disrupted in vivo. We postulate that defective bi-directional integrin signaling affects the organization of the collagen matrix. Finally, we present a compensatory candidate molecule, Pyk2, which localized to the focal adhesions in osteoblasts that were lacking FAK.

  View details for DOI 10.1016/j.bone.2007.01.024

  View details for Web of Science ID 000247854900006

  View details for PubMedID 17459803

  View details for PubMedCentralID PMC2699353

• Functional demarcation of active and silent chromatin domains in human HOX loci by Noncoding RNAs CELL Rinn, J. L., Kertesz, M., Wang, J. K., Squazzo, S. L., Xu, X., Brugmann, S. A., Goodnough, L. H., Helms, J. A., Farnham, P. J., Segal, E., Chang, H. Y. 2007; 129 (7): 1311-1323

  Abstract

  Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. Here we characterize the transcriptional landscape of the four human HOX loci at five base pair resolution in 11 anatomic sites and identify 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes and possess unique sequence motifs, and their expression demarcates broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. We identified a 2.2 kilobase ncRNA residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states.

  View details for DOI 10.1016/j.cell.2007.05.022

  View details for PubMedID 17604720

• FAK-Mediated Mechanotransduction in Skeletal Regeneration PLOS ONE Leucht, P., Kim, J., Currey, J. A., Brunski, J., Helms, J. A. 2007; 2 (4)

  Abstract

  The majority of cells are equipped to detect and decipher physical stimuli, and then react to these stimuli in a cell type-specific manner. Ultimately, these cellular behaviors are synchronized to produce a tissue response, but how this is achieved remains enigmatic. Here, we investigated the genetic basis for mechanotransduction using the bone marrow as a model system. We found that physical stimuli produced a pattern of principal strain that precisely corresponded to the site-specific expression of sox9 and runx2, two transcription factors required for the commitment of stem cells to a skeletogenic lineage, and the arrangement and orientation of newly deposited type I collagen fibrils. To gain insights into the genetic basis for skeletal mechanotransduction we conditionally inactivated focal adhesion kinase (FAK), an intracellular component of the integrin signaling pathway. By doing so we abolished the mechanically induced osteogenic response and thus identified a critical genetic component of the molecular machinery required for mechanotransduction. Our data provide a new framework in which to consider how physical forces and molecular signals are synchronized during the program of skeletal regeneration.

  View details for DOI 10.1371/journal.pone.0000390

  View details for Web of Science ID 000207445600004

  View details for PubMedID 17460757

  View details for PubMedCentralID PMC1849965

• Effect of mechanical stimuli on skeletal regeneration around implants BONE Leucht, P., Kim, J., Wazen, R., Currey, J. A., Nanci, A., Brunski, J. B., Heims, J. A. 2007; 40 (4): 919-930

  Abstract

  Due to the aging population and the increasing need for total joint replacements, osseointegration is of a great interest for various clinical disciplines. Our objective was to investigate the molecular and cellular foundation that underlies this process. Here, we used an in vivo mouse model to study the cellular and molecular response in three distinct areas of unloaded implants: the periosteum, the gap between implant and cortical bone, and the marrow space. Our analyses began with the early phases of healing, and continued until the implants were completely osseointegrated. We investigated aspects of osseointegration ranging from vascularization, cell proliferation, differentiation, and bone remodeling. In doing so, we gained an understanding of the healing mechanisms of different skeletal tissues during unloaded implant osseointegration. To continue our analysis, we used a micromotion device to apply a defined physical stimulus to the implants, and in doing so, we dramatically enhanced bone formation in the peri-implant tissue. By comparing strain measurements with cellular and molecular analyses, we developed an understanding of the correlation between strain magnitudes and fate decisions of cells shaping the skeletal regenerate.

  View details for DOI 10.1016/j.bone.2006.10.027

  View details for Web of Science ID 000245419800015

  View details for PubMedID 17175211

  View details for PubMedCentralID PMC1987325

• Sonic hedgehog in the pharyngeal endoderm controls arch pattern via regulation of Fgf8 in head ectoderm DEVELOPMENTAL BIOLOGY Haworth, K. E., Wilson, J. M., Grevellee, A., Cobourne, M. T., Healy, C., Helms, J. A., Sharpe, P. T., Tucker, A. S. 2007; 303 (1): 244-258

  Abstract

  Fgf8 signalling is known to play an important role during patterning of the first pharyngeal arch, setting up the oral region of the head and then defining the rostral and proximal domains of the arch. The mechanisms that regulate the restricted expression of Fgf8 in the ectoderm of the developing first arch, however, are not well understood. It has become apparent that pharyngeal endoderm plays an important role in regulating craniofacial morphogenesis. Endoderm ablation in the developing chick embryo results in a loss of Fgf8 expression in presumptive first pharyngeal arch ectoderm. Shh is locally expressed in pharyngeal endoderm, adjacent to the Fgf8-expressing ectoderm, and is thus a candidate signal regulating ectodermal Fgf8 expression. We show that in cultured explants of presumptive first pharyngeal arch, loss of Shh signalling results in loss of Fgf8 expression, both at early stages before formation of the first arch, and during arch formation. Moreover, following removal of the endoderm, Shh protein can replace this tissue and restore Fgf8 expression. Overexpression of Shh in the non-oral ectoderm leads to an expansion of Fgf8, affecting the rostral-caudal axis of the developing first arch, and resulting in the formation of ectopic cartilage. Shh from the pharyngeal endoderm thus regulates Fgf8 in the ectoderm and the role of the endoderm in pharyngeal arch patterning may thus be indirectly mediated by the ectoderm.

  View details for DOI 10.1016/j.ydbio.2006.11.009

  View details for Web of Science ID 000244542800021

  View details for PubMedID 17187772

• Visualizing in vivo liposomal drug delivery in real-time JOURNAL OF DRUG TARGETING Kim, J., Leucht, P., Morrell, N. T., Schwettman, H. A., Helms, J. A. 2007; 15 (9): 632-639

  Abstract

  Liposomes have tremendous potential for efficient small molecule delivery. Previous studies, however, have been hampered by an inability to monitor their distribution and release of contents. Here, the authors demonstrate the real time monitoring of small molecule delivery using luciferin as a model. To monitor the release of luciferin in vivo, luciferin was packaged in thermosensitive liposomes and delivered into transgenic mice that constitutively express luciferase. Their experiments show the thermally induced release of the liposomal content in real time. In addition, the model provides evidence that the thermosensitive liposomes are stable over a long period of time ( approximately 3 weeks), and still release their content upon heating. These data present a strategy to monitor liposomal drug delivery in vivo with luciferin.

  View details for DOI 10.1080/10611860701538651

  View details for Web of Science ID 000251334600007

  View details for PubMedID 17968717

• 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

• Rapid growth of cartilage rudiments may generate perichondrial structures by mechanical induction BIOMECHANICS AND MODELING IN MECHANOBIOLOGY Henderson, J. H., De la Fuente, L., Romero, D., Colnot, C. I., Huang, S., Carter, D. R., Helms, J. A. 2007; 6 (1-2): 127-137

  Abstract

  Experimental and theoretical research suggest that mechanical stimuli may play a role in morphogenesis. We investigated whether theoretically predicted patterns of stress and strain generated during the growth of a skeletal condensation are similar to in vivo expression patterns of chondrogenic and osteogenic genes. The analysis showed that predicted patterns of compressive hydrostatic stress (pressure) correspond to the expression patterns of chondrogenic genes, and predicted patterns of tensile strain correspond to the expression patterns of osteogenic genes. Furthermore, the results of iterative application of the analysis suggest that stresses and strains generated by the growing condensation could promote the formation and refinement of stiff tissue surrounding the condensation, a prediction that is in agreement with an observed increase in collagen bundling surrounding the cartilage condensation, as indicated by picro-sirius red staining. These results are consistent with mechanical stimuli playing an inductive or maintenance role in the developing cartilage and associated perichondrium and bone collar. This theoretical analysis provides insight into the potential importance of mechanical stimuli during the growth of skeletogenic condensations.

  View details for DOI 10.1007/s10237-006-0038-x

  View details for PubMedID 16691413

• Looking different: understanding diversity in facial form. American journal of medical genetics. Part A Brugmann, S. A., Kim, J., Helms, J. A. 2006; 140 (23): 2521-2529

  Abstract

  The face is perhaps the most distinguishing feature of the vertebrate body. Six billion human faces decorate the earth, each of them unique and exceptional in their own way. Likewise, facial variation is the cornerstone of species-specific diversity within the animal kingdom. Yet despite this multiplicity in form, the underlying architecture of the vertebrate face is remarkably conserved. If early embryos of different species first resemble one another, then how is this facial diversity generated? Our primary goal is to elucidate the molecular origins of species-specific craniofacial morphogenesis. We examined one facial primordia, the frontonasal prominence, of phylogenetically related (chick vs. quail vs. duck) and distant (mouse vs. chick) embryos and asked how such drastically different forms (e.g., beak, bill, or muzzle) could be generated from a once-similar entity. We examined the morphological ontogeny and a number of molecular expression patterns in an attempt to shed light on when species-specific variations occur and what molecules (BMPs, FGFs, etc.) are implicated in its differential growth. We hypothesize that subtle changes in the signaling of these morphogens can reproducibly alter the morphology of the frontonasal prominence. Taken together, these data facilitate our fledgling understanding of the process by which facial morphogenesis is regulated.

  View details for PubMedID 16838331

• Looking different: Understanding diversity in facial form AMERICAN JOURNAL OF MEDICAL GENETICS PART A Brugmann, S. A., Kim, J., Helms, J. A. 2006; 140A (23): 2521-2529

  Abstract

  The face is perhaps the most distinguishing feature of the vertebrate body. Six billion human faces decorate the earth, each of them unique and exceptional in their own way. Likewise, facial variation is the cornerstone of species-specific diversity within the animal kingdom. Yet despite this multiplicity in form, the underlying architecture of the vertebrate face is remarkably conserved. If early embryos of different species first resemble one another, then how is this facial diversity generated? Our primary goal is to elucidate the molecular origins of species-specific craniofacial morphogenesis. We examined one facial primordia, the frontonasal prominence, of phylogenetically related (chick vs. quail vs. duck) and distant (mouse vs. chick) embryos and asked how such drastically different forms (e.g., beak, bill, or muzzle) could be generated from a once-similar entity. We examined the morphological ontogeny and a number of molecular expression patterns in an attempt to shed light on when species-specific variations occur and what molecules (BMPs, FGFs, etc.) are implicated in its differential growth. We hypothesize that subtle changes in the signaling of these morphogens can reproducibly alter the morphology of the frontonasal prominence. Taken together, these data facilitate our fledgling understanding of the process by which facial morphogenesis is regulated.

  View details for DOI 10.1002/ajmg.a.31361

  View details for Web of Science ID 000242466100005

• Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Colnot, C., Huang, S., Helms, J. 2006; 350 (3): 557-561

  Abstract

  The bone marrow is believed to play important roles during fracture healing such as providing progenitor cells for inflammation, matrix remodeling, and cartilage and bone formation. Given the complex nature of bone repair, it remains difficult to distinguish the contributions of various cell types. Here we describe a mouse model based on bone marrow transplantation and genetic labeling to track cells originating from bone marrow during fracture healing. Following lethal irradiation and engraftment of bone marrow expressing the LacZ transgene constitutively, wild type mice underwent tibial fracture. Donor bone marrow-derived cells, which originated from the hematopoietic compartment, did not participate in the chondrogenic and osteogenic lineages during fracture healing. Instead, the donor bone marrow contributed to inflammatory and bone resorbing cells. This model can be exploited in the future to investigate the role of inflammation and matrix remodeling during bone repair, independent from osteogenesis and chondrogenesis.

  View details for DOI 10.1016/j.bbrc.2006.09.079

  View details for Web of Science ID 000241584300009

  View details for PubMedID 17022937

• 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

• The molecular origins of species-specific facial pattern CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY, VOL 73 Brugmann, S. A., Tapadia, M. D., Helms, J. A. 2006; 73: 1-?

  Abstract

  The prevailing approach within the field of craniofacial development is focused on finding a balance between tissues (e.g., facial epithelia, neuroectoderm, and neural crest) and molecules (e.g., bone morphogenetic proteins, fibroblast growth factors, Wnts) that play a role in sculpting the face. We are rapidly learning that neither these tissues nor molecular signals are able to act in isolation; in fact, molecular cues are constantly reciprocating signals between the epithelia and the neural crest in order to pattern and mold facial structures. More recently, it has been proposed that this crosstalk is often mediated and organized by discrete organizing centers within the tissues that are able to act as a self-contained unit of developmental potential (e.g., the rhombomere and perhaps the ectomere). Whatever the molecules are and however they are interpreted by these tissues, it appears that there is a remarkably conserved mechanism for setting up the initial organization of the facial prominences between species. Regardless of species, all vertebrates appear to have the same basic bauplan. However, sometime during mid-gestation, the vertebrate face begins to exhibit species-specific variations, in large part due to differences in the rates of growth and differentiation of cells comprising the facial prominences. How do these differences arise? Are they due to late changes in molecular signaling within the facial prominences themselves? Or are these late changes a reflection of earlier, more subtle alterations in boundaries and fields that are established at the earliest stages of head formation? We do not have clear answers to these questions yet, but in this chapter we present new studies that shed light on this age-old question. This chapter aims to present the known signals, both on a molecular and cellular level, responsible for craniofacial development while bringing to light the events that may serve to create difference in facial morphology seen from species to species.

  View details for DOI 10.1016/S0070-2153(05)73001-5

  View details for Web of Science ID 000238776000001

  View details for PubMedID 16782454

• It's all in your head: new insights into craniofacial development and deformation JOURNAL OF ANATOMY Tapadia, M. D., Cordero, D. R., Helms, J. A. 2005; 207 (5): 461-477

  View details for Web of Science ID 000232997900004

  View details for PubMedID 16313388

• Molecular interactions coordinating the development of the forebrain and face DEVELOPMENTAL BIOLOGY Marcucio, R. S., Cordero, D. R., Hu, D., Helms, J. A. 2005; 284 (1): 48-61

  Abstract

  From an architectural point of view, the forebrain acts as a framework upon which the middle and upper face develops and grows. In addition to serving a structural role, we present evidence that the forebrain is a source of signals that shape the facial skeleton. In this study, we inhibited Sonic hedgehog (Shh) signaling from the neuroectoderm then examined the molecular changes and the skeletal alterations resulting from the treatment. One of the first changes we noted was that the dorsoventral polarity of the forebrain was disturbed, which manifested as a loss of Shh in the ventral telencephalon, a reduction in expression of the ventral markers Nkx2.1 and Dlx2, and a concomitant expansion of the dorsal marker Pax6. In addition to changes in the forebrain neuroectoderm, we observed altered gene expression patterns in the facial ectoderm. For example, Shh was not induced in the frontonasal ectoderm, and Ptc and Gli1 were reduced in both the ectoderm and adjacent mesenchyme. As a consequence, a signaling center in the frontonasal prominence was disrupted and the prominence failed to undergo proximodistal and mediolateral expansion. After 15 days of development, the upper beaks of the treated embryos were truncated, and the skeletal elements were located in more medial and proximal locations in relation to the skeletal elements of the lower jaw elements. These data indicate that a role of Shh in the forebrain is to regulate Shh expression in the face, and that together, these Shh domains mediate patterning within the frontonasal prominence and proximodistal outgrowth of the middle and upper face.

  View details for DOI 10.1016/j.ydbio.2005.04.030

  View details for Web of Science ID 000231352800005

  View details for PubMedID 15979605

• Indian hedgehog regulates skeletal angiogenesis in association with cartilage and bone development. 64th Annual Meeting of the Society-for-Development-Biology Colnot, C., De la Fuente, L., Huang, S., Hu, D., Lu, C. Y., St-Jacques, B., Helms, J. A. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2005: 695–95

  View details for Web of Science ID 000230683800600

• Head, shoulders, knees, and toes DEVELOPMENTAL BIOLOGY De la Fuente, L., Helms, J. A. 2005; 282 (2): 294-306

  View details for DOI 10.1016/j.ydbio.2005.03.036

  View details for Web of Science ID 000230005900002

  View details for PubMedID 15950599

• The fickle finger of fate JOURNAL OF CLINICAL INVESTIGATION De la Fuente, L., Helms, J. A. 2005; 115 (4): 833-836

  Abstract

  In this issue of the JCI, Niedermaier and colleagues demonstrate that a chromosomal inversion in mice results in dysregulation of Sonic hedgehog (Shh), such that Shh is ectopically expressed in a skeletogenic domain typically occupied by Indian hedgehog (Ihh). This molecular reversal eliminates phalangeal joint spaces, and consequently, Short digits (Dsh) heterozygotes (Dsh/+) have brachydactyly (shortened digits). Ihh is normally downregulated in regions that will become the joint space, but in Dsh/+ mice, Shh bypasses this regulatory control and persists; accordingly, cells maintain their chondrogenic fate and the developed digits are shorter than normal. The significance of these data extends far beyond the field of skeletal biology: they hint at the very real possibility that the endogenous Shh regulatory region contains a repressor designed to segregate the activity of Shh from Ihh. The existence of such a repressor provides a window into the distant past, revealing that Shh and Ihh must once have shared responsibilities in establishing tissue boundaries and orchestrating vertebrate tissue morphogenesis.

  View details for DOI 10.1172/JCI200524840

  View details for Web of Science ID 000228145700012

  View details for PubMedID 15841172

  View details for PubMedCentralID PMC1070437

• New insights into craniofacial morphogenesis DEVELOPMENT Helms, J. A., Cordero, D., Tapadia, M. D. 2005; 132 (5): 851-861

  Abstract

  No region of our anatomy more powerfully conveys our emotions nor elicits more profound reactions when disease or genetic disorders disfigure it than the face. Recent progress has been made towards defining the tissue interactions and molecular mechanisms that control craniofacial morphogenesis. Some insights have come from genetic manipulations and others from tissue recombinations and biochemical approaches, which have revealed the molecular underpinnings of facial morphogenesis. Changes in craniofacial architecture also lie at the heart of evolutionary adaptation, as new studies in fish and fowl attest. Together, these findings reveal much about molecular and tissue interactions behind craniofacial development.

  View details for DOI 10.1242/dev.01705

  View details for Web of Science ID 000228093300001

  View details for PubMedID 15705856

• Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development DEVELOPMENT Colnot, C., De la Fuente, L., Huang, S., Hu, D., Lu, C. Y., St-Jacques, B., Helms, J. A. 2005; 132 (5): 1057-1067

  Abstract

  A null mutation in the morphogen Indian hedgehog (IHH) results in an embryonic lethal phenotype characterized by the conspicuous absence of bony tissue in the extremities. We show that this ossification defect is not attributable to a permanent arrest in cartilage differentiation, since Ihh(-/-) chondrocytes undergo hypertrophy and terminal differentiation, express angiogenic markers such as Vegf, and are invaded, albeit aberrantly, by blood vessels. Subsequent steps, including vessel expansion and persistence, are impaired, and the net result is degraded cartilage matrix that is devoid of blood vessels. The absence of blood vessels is not because the Ihh(-/-) skeleton is anti-angiogenic; in fact, in an ex vivo environment, both wild-type and Ihh mutant vessels invade the Ihh(-/-) cartilage, though only wild-type vessels expand to create the marrow cavity. In the ex vivo setting, Ihh(-/-) cells differentiate into osteoblasts and deposit a bony matrix, without benefit of exogenous hedgehog in the new environment. Even more surprising is our finding that the earliest IHH-dependent skeletal defect is obvious by the time the limb mesenchyme segregates into chondrogenic and perichondrogenic condensations. Although Ihh(-/-) cells organize into chondrogenic condensations similar in size and shape to wild-type condensations, perichondrial cells surrounding the mutant condensations are clearly faulty. They fail to aggregate, elongate and flatten into a definitive, endothelial cell-rich perichondrium like their wild-type counterparts. Normally, these cells surrounding the chondrogenic condensation are exposed to IHH, as evidenced by their expression of the hedgehog target genes, patched (Ptch) and Gli1. In the mutant environment, the milieu surrounding the cartilage -- comprising osteoblast precursors and endothelial cells -- as well as the cartilage itself, develop in the absence of this important morphogen. In conclusion, the skeletal phenotype of Ihh(-/-) embryos represents the sum of disturbances in three separate cell populations, the chondrocytes, the osteoblasts and the vasculature, each of which is a direct target of hedgehog signaling.

  View details for DOI 10.1242/dev.01649

  View details for Web of Science ID 000228093300019

  View details for PubMedID 15689378

• Mechanisms of osteogenic differentiation of mouse adipose-derived mesenchymal cells 11th International Congress of the International-Society-of-Craniofacial-Surgery Shi, Y. Y., Nacamuli, R. P., Salim, A., Quarto, N., LYONS, K. M., Wan, D. C., Cowan, C. M., Helms, J. A., Longaker, M. T. MEDIMOND S R L. 2005: 63–65

  View details for Web of Science ID 000243381600016

• Fgfr1 gain-of-function results in premaxillomaxillary suture fusion and mid-face retrusion 11th International Congress of the International-Society-of-Craniofacial-Surgery Lenton, K. A., Helms, J. A., Longaker, M. T. MEDIMOND S R L. 2005: 271–272

  View details for Web of Science ID 000243381600068

• Analysis of cartilages in the developing skull vault 11th International Congress of the International-Society-of-Craniofacial-Surgery Lenton, K. A., Helms, J. A., Longaker, M. T. MEDIMOND S R L. 2005: 267–269

  View details for Web of Science ID 000243381600067

• 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

• 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

• Temporal perturbations in sonic hedgehog signaling elicit the spectrum of holoprosencephaly phenotypes JOURNAL OF CLINICAL INVESTIGATION Cordero, D., Marcucio, R., Hu, D., Gaffield, W., Tapadia, M., Helms, J. A. 2004; 114 (4): 485-494

  Abstract

  One of the most perplexing questions in clinical genetics is why patients with identical gene mutations oftentimes exhibit radically different clinical features. This inconsistency between genotype and phenotype is illustrated in the malformation spectrum of holoprosencephaly (HPE). Family members carrying identical mutations in sonic hedgehog (SHH) can exhibit a variety of facial features ranging from cyclopia to subtle midline asymmetries. Such intrafamilial variability may arise from environmental factors acting in conjunction with gene mutations that collectively reduce SHH activity below a critical threshold. We undertook a series of experiments to test the hypothesis that modifying the activity of the SHH signaling pathway at discrete periods of embryonic development could account for the phenotypic spectrum of HPE. Exposing avian embryos to cyclopamine during critical periods of craniofacial development recreated a continuum of HPE-related defects. The craniofacial malformations included hypotelorism, midfacial hypoplasia, and facial clefting and were not the result of excessive crest cell apoptosis. Rather, they resulted from molecular reprogramming of an organizing center whose activity controls outgrowth and patterning of the mid and upper face. Collectively, these data reveal one mechanism by which the variable expressivity of a disorder such as HPE can be produced through temporal disruption of a single molecular pathway.

  View details for DOI 10.1172/JCI200419596

  View details for Web of Science ID 000223287500008

  View details for PubMedID 15314685

• Distinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development DEVELOPMENTAL BIOLOGY Colnot, C., Lu, C. Y., Hu, D., Helms, J. A. 2004; 269 (1): 55-69

  Abstract

  During the initiation of endochondral ossification three events occur that are inextricably linked in time and space: chondrocytes undergo terminal differentiation and cell death, the skeletal vascular endothelium invades the hypertrophic cartilage matrix, and osteoblasts differentiate and begin to deposit a bony matrix. These developmental programs implicate three tissues, the cartilage, the perichondrium, and the vascular endothelium. Due to their intimate associations, the interactions among these three tissues are exceedingly difficult to distinguish and elucidate. We developed an ex vivo system to unlink the processes initiating endochondral ossification and establish more precisely the cellular and molecular contributions of the three tissues involved. In this ex vivo system, the renal capsule of adult mice was used as a host environment to grow skeletal elements. We first used a genetic strategy to follow the fate of cells derived from the perichondrium and from the vasculature. We found that the perichondrium, but not the host vasculature, is the source of both trabecular and cortical osteoblasts. Endothelial cells residing within the perichondrium are the first cells to participate in the invasion of the hypertrophic cartilage matrix, followed by endothelial cells derived from the host environment. We then combined these lineage analyses with a series of tissue manipulations to address how the absence of the perichondrium or the vascular endothelium affected skeletal development. We show that although the perichondrium influences the rate of chondrocytes maturation and hypertrophy, it is not essential for chondrocytes to undergo late hypertrophy. The perichondrium is crucial for the proper invasion of blood vessels into the hypertrophic cartilage and both the perichondrium and the vasculature are essential for endochondral ossification. Collectively, these studies clarify further the contributions of the cartilage, perichondrium, and vascular endothelium to long bone development.

  View details for DOI 10.1016/j.ydbio.2004.01.011

  View details for Web of Science ID 000221034400005

  View details for PubMedID 15081357

• Altered fracture repair in the absence of MMP9 DEVELOPMENT Colnot, C., Thompson, Z., Miclau, T., WERB, Z., Helms, J. A. 2003; 130 (17): 4123-4133

  Abstract

  The regeneration of adult skeletal tissues requires the timely recruitment of skeletal progenitor cells to an injury site, the differentiation of these cells into bone or cartilage, and the re-establishment of a vascular network to maintain cell viability. Disturbances in any of these cellular events can have a detrimental effect on the process of skeletal repair. Although fracture repair has been compared with fetal skeletal development, the extent to which the reparative process actually recapitulates the fetal program remains uncertain. Here, we provide the first genetic evidence that matrix metalloproteinase 9 (MMP9) regulates crucial events during adult fracture repair. We demonstrate that MMP9 mediates vascular invasion of the hypertrophic cartilage callus, and that Mmp9(-/-) mice have non-unions and delayed unions of their fractures caused by persistent cartilage at the injury site. This MMP9- dependent delay in skeletal healing is not due to a lack of vascular endothelial growth factor (VEGF) or VEGF receptor expression, but may instead be due to the lack of VEGF bioavailability in the mutant because recombinant VEGF can rescue Mmp9(-/-) non-unions. We also found that Mmp9(-/-) mice generate a large cartilage callus even when fractured bones are stabilized, which implicates MMP9 in the regulation of chondrogenic and osteogenic cell differentiation during early stages of repair. In conclusion, the resemblance between Mmp9(-/-) fetal skeletal defects and those that emerge during Mmp9(-/-) adult repair offer the strongest evidence to date that similar mechanisms are employed to achieve bone formation, regardless of age.

  View details for DOI 10.1242/dev.00559

  View details for Web of Science ID 000187398500019

  View details for PubMedID 12874132

• Cranial skeletal biology NATURE Helms, J. A., SCHNEIDER, R. A. 2003; 423 (6937): 326-331

  Abstract

  To artists, the face is a mirror of the soul. To biologists, the face reflects remarkable structural diversity--think of bulldogs and wolfhounds or galapagos finches. How do such variations in skeletal form arise? Do the same mechanisms control skeletogenesis elsewhere in the body? The answers lie in the molecular machinery that generates neural crest cells, controls their migration, and guides their differentiation to cartilage and bone.

  View details for DOI 10.1038/nature01656

  View details for Web of Science ID 000182853100052

  View details for PubMedID 12748650

• A zone of frontonasal ectoderm regulates patterning and growth in the face DEVELOPMENT Hu, D., Marcucio, R. S., Helms, J. A. 2003; 130 (9): 1749-1758

  Abstract

  A fundamental set of patterning genes may define the global organization of the craniofacial region. One of our goals has been to identify these basic patterning genes and understand how they regulate outgrowth of the frontonasal process, which gives rise to the mid and upper face. We identified a molecular boundary in the frontonasal process ectoderm, defined by the juxtaposed domains of Fibroblast growth factor 8 and Sonic hedgehog, which presaged the initial site of frontonasal process outgrowth. Fate maps confirmed that this boundary region later demarcated the dorsoventral axis of the upper beak. Ectopic transplantation of the ectodermal boundary region activated a cascade of molecular events that reprogrammed the developmental fate of neural crest-derived mesenchyme, which resulted in duplications of upper and lower beak structures. We discuss these data in the context of boundary/morphogen models of patterning, and in view of the recent controversy regarding neural crest pre-patterning versus neural crest plasticity.

  View details for DOI 10.1242/dev.00397

  View details for Web of Science ID 000182811600003

  View details for PubMedID 12642481

• Molecular ontogeny of the skeleton. Birth defects research. Part C, Embryo today : reviews Eames, B. F., de la Fuente, L., Helms, J. A. 2003; 69 (2): 93-101

  Abstract

  From a traditional viewpoint, skeletal elements form by two distinct processes: endochondral ossification, during which a cartilage template is replaced by bone, and intramembranous ossification, whereby mesenchymal cells differentiate directly into osteoblasts. There are inherent difficulties with this historical classification scheme, not the least of which is that bones typically described as endochondral actually form bone through an intramembranous process, and that some membranous bones may have a transient chondrogenic phase. These innate contradictions can be circumvented if molecular and cellular, rather than histogenic, criteria are used to describe the process of skeletal tissue formation. Within the past decade, clinical examinations of human skeletal syndromes have led to the identification and subsequent characterization of regulatory molecules that direct chondrogenesis and osteogenesis in every skeletal element of the body. In this review, we survey these molecules and the tissue interactions that may regulate their expression. What emerges is a new paradigm, by which we can explain and understand the process of normal- and abnormal-skeletal development.

  View details for PubMedID 12955855

• The cellular and molecular origins of beak morphology SCIENCE SCHNEIDER, R. A., Helms, J. A. 2003; 299 (5606): 565-568

  Abstract

  Cellular and molecular mechanisms underlying differences in beak morphology likely involve interactions among multiple embryonic populations. We exchanged neural crest cells destined to participate in beak morphogenesis between two anatomically distinct species. Quail neural crest cells produced quail beaks in duck hosts and duck neural crest produced duck bills in quail hosts. These transformations involved morphological changes to non-neural crest host beak tissues. To achieve these changes, donor neural crest cells executed autonomous molecular programs and regulated gene expression in adjacent host tissues. Thus, neural crest cells are a source of molecular information that generates interspecific variation in beak morphology.

  View details for Web of Science ID 000180559800050

  View details for PubMedID 12543976

• In situ analysis of osteogenic differentiation in mouse cranial sutures 10th International Congress of the International-Society-of-Craniofacial-Surgery Nacamuli, R. P., Fong, K. D., Warren, S. M., Fang, T. D., Song, H. M., Salim, A., Helms, J. A., Longaker, M. T. MEDIMOND PUBLISHING CO. 2003: 455–457

  View details for Web of Science ID 000227470500093

• Prenatal morphogenesis of the human mental foramen EUROPEAN JOURNAL OF ORAL SCIENCES Radlanski, R. J., Renz, H., Muller, U., Schneider, R. S., Marcucio, R. S., Helms, J. A. 2002; 110 (6): 452-459

  Abstract

  The mental foramen, at first glance, merely looks like a hole where the mental nerve and the vascular bundle runs through. From a morphogenetic point of view, however, the mental foramen is a suitable model to study the development of a structure where different components are involved. To understand this developmental process, a three-dimensional description at different developmental stages first has to be given. From histological serial sections of human embryos and fetuses, ranging in size from 19 to 117 mm crown rump length (CRL), three-dimensional reconstructions of the foraminar regions were made. Outline and form of the developing foramen, size, course of the mental nerve and the adjacent blood vessels could be shown in detail. In this way, the formation of these structures became concrete in three dimensions. In the future, to understand the mechanisms regulating this complex system, where a nerve and blood vessels became successively surrounded by bone, molecular biological data have to be correlated with morphological findings.

  View details for Web of Science ID 000179381400008

  View details for PubMedID 12507219

• A model for intramembranous ossification during fracture healing JOURNAL OF ORTHOPAEDIC RESEARCH Thompson, Z., Miclau, T., Hu, D., Helms, J. A. 2002; 20 (5): 1091-1098

  Abstract

  We have developed a method to study the molecular basis of intramembranous fracture healing. Unlike intramedullary rods that permit rotation of the fractured bone segments, our murine model relies on an external fixation device to provide stabilization. In this study we compare stabilized fracture callus tissues with callus tissues from non-stabilized fractures during the inflammatory, soft callus, hard callus, and remodeling stages of healing. Histological analyses indicate that stabilized fractures heal with virtually no evidence of cartilage whereas non-stabilized fractures produce abundant cartilage at the fracture site. Expression patterns of collagen type IIa (colIIa) and osteocalcin (oc) reveal that mesenchymal cells at the fracture site commit to either a chondrogenic or an osteogenic lineage during the earliest stages of healing. The mechanical environment influences this cell fate decision, since mesenchymal cells in a stabilized fracture express oc and fail to express colIIa. Future studies will use this murine model of intramembranous fracture healing to explore, at a molecular level, how the mechanical environment exerts its influence on healing of a fracture.

  View details for Web of Science ID 000178383400028

  View details for PubMedID 12382977

• Local retinoid signaling coordinates forebrain and facial morphogenesis by maintaining FGF8 and SHH DEVELOPMENT SCHNEIDER, R. A., Hu, D., Rubenstein, J. L., Maden, M., Helms, J. A. 2001; 128 (14): 2755-2767

  Abstract

  Correlations between facial anomalies and brain defects are well characterized throughout the clinical literature, yet a developmental basis for this association has not been identified. We demonstrate that the frontonasal process, which gives rise to the mid- and upper face, and the forebrain are linked early in their morphogenesis by a local retinoid signaling event that maintains the expression of key regulatory molecules. First, we show that aldehyde dehydrogenase 6, which synthesizes the ligand, retinoic acid, is localized to the ventral epithelium of the presumptive frontonasal process of chick embryos. At least two retinoid receptors are expressed in adjacent populations of mesenchyme. Second, using synthetic pan-specific retinoid antagonists, we transiently inhibit the ability of retinoid receptors to bind retinoic acid in the rostral head and we generate embryos with a hypoplastic forebrain, fused eyes, and no frontonasal process-derived structures such as the upper beak. These defects are not due to eliminating mesenchymal progenitors, as neural crest cells still migrate into the frontonasal process, despite disruptions to retinoid signaling. Rather, these malformations result from loss of fibroblast growth factor 8 and sonic hedgehog expression, which leads to increased programmed cell death and decreased proliferation in the forebrain and frontonasal process. Most significantly, we can rescue the morphological defects by re-introducing retinoic acid, or fibroblast growth factor and sonic hedgehog proteins into antagonist-treated embryos. We propose that the local source of retinoic acid in the rostral head initiates a regulatory cascade that coordinates forebrain and frontonasal process morphogenesis.

  View details for Web of Science ID 000170209900012

  View details for PubMedID 11526081

• A molecular analysis of matrix remodeling and angiogenesis during long bone development MECHANISMS OF DEVELOPMENT Colnot, C. I., Helms, J. A. 2001; 100 (2): 245-250

  Abstract

  The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a 'molecular map' of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias.

  View details for Web of Science ID 000166837600008

  View details for PubMedID 11165481

• Molecular aspects of healing in stabilized and non-stabilized fractures 43rd Annual Meeting of the Orthopaedic-Research-Society Le, A. X., Miclau, T., Hu, D., Helms, J. A. JOHN WILEY & SONS INC. 2001: 78–84

  Abstract

  Bone formation is a continuous process that is initiated during fetal development and persists in adults in the form of bone regeneration and remodeling. These latter two aspects of bone formation are clearly influenced by the mechanical environment. In this study we tested the hypothesis that alterations in the mechanical environment regulate the program of mesenchymal cell differentiation, and thus the formation of a cartilage or bony callus, at the site of injury. As a first step in testing this hypothesis we produced stabilized and non-stabilized tibial fractures in a mouse model, then used molecular and cellular methods to examine the stage of healing. Using the "molecular map" of the fracture callus, we divided our analyzes into three phases of fracture healing: the inflammatory or initial phase of healing, the soft callus or intermediate stage, and the hard callus stage. Our results show that indian hedgehog(ihh), which regulates aspects of chondrocyte maturation during fetal and early postnatal skeletogenesis, was expressed earlier in an non-stabilized fracture callus as compared to a stabilized callus. ihh persisted in the non-stabilized fracture whereas its expression was down-regulated in the stabilized bone. IHH exerts its effects on chondrocyte maturation through a feedback loop that may involve bone morphogenetic protein 6 [bmp6; (S. Pathi, J.B. Rutenberg, R.L. Johnson, A. Vortkamp, Developmental Biology 209 (1999) 239-253)] and the transcription factor gli3. bmp6 and gli3 were re-induced in domain adjacent to the ihh-positive cells during the soft and hard callus stages of healing. Thus, stabilizing the fracture, which circumvents or decreases the cartilaginous phase of bone repair, correlates with a decrease in ihh signaling in the fracture callus. Collectively, our results illustrate that the ihh signaling pathway participates in fracture repair, and that the mechanical environment affects the temporal induction of ihh, bmp6 and gli3. These data support the hypothesis that mechanical influences affect mesenchymal cell differentiation to bone.

  View details for Web of Science ID 000168179000011

  View details for PubMedID 11332624

• Genetic and teratogenic approaches to craniofacial development CRITICAL REVIEWS IN ORAL BIOLOGY & MEDICINE Young, D. L., SCHNEIDER, R. A., Hu, D., Helms, J. A. 2000; 11 (3): 304-317

  Abstract

  Craniofacial malformations are the most common birth defects that occur in humans, with facial clefting representing the majority of these defects. Facial clefts can arise at any stage of development due to perturbations that alter the extracellular matrix as well as affect the patterning, migration, proliferation, and differentiation of cells. In this review, we focus on recent advances in the understanding of the developmental basis for facial clefting through the analysis of the effects of gene disruption experiments and treatments with teratogens in both chickens and mice. Specifically, we analyze the results of disruptions to genes such as Sonic hedgehog (Shh), epidermal growth factor receptor (EGFR), Distal-less (Dlx), and transforming growth factor beta 3 (TGFbeta3). We also describe the effects that teratogens such as retinoic acid, jervine, and cyclopamine have on facial clefting and discuss mechanisms for their action. In addition to providing insight into the bases for abnormal craniofacial growth, genetic and teratogenic techniques are powerful tools for understanding the normal developmental processes that generate and pattern the face.

  View details for Web of Science ID 000175180100002

  View details for PubMedID 11021632

• The role of Sonic hedgehog in normal and abnormal craniofacial morphogenesis DEVELOPMENT Hu, D., Helms, J. A. 1999; 126 (21): 4873-4884

  Abstract

  There is growing evidence that implicates a role for Sonic hedgehog (SHH) in morphogenesis of the craniofacial complex. Mutations in human and murine SHH cause midline patterning defects that are manifested in the head as holoprosencephaly and cyclopia. In addition, teratogens such as jervine, which inhibit the response of tissues to SHH, also produce cyclopia. Thus, the loss of SHH signaling during early stages of neural plate patterning has a profound influence of craniofacial morphogenesis. However, the severity of these defects precludes analyses of SHH function during later stages of craniofacial development. We have used an embryonic chick system to study the role of SHH during these later stages of craniofacial development. Using a combination of surgical and molecular experiments, we show here that SHH is essential for morphogenesis of the frontonasal and maxillary processes (FNP and MXPs), which give rise to the mid- and upper face. Transient loss of SHH signaling in the embryonic face inhibits growth of the primordia and results in defects analogous to hypotelorism and cleft lip/palate, characteristics of the mild forms of holoprosencephaly. In contrast, excess SHH leads to a mediolateral widening of the FNP and a widening between the eyes, a condition known as hypertelorism. In severe cases, this widening is accompanied by facial duplications. Collectively, these experiments demonstrate that SHH has multiple and profound effects on the entire spectrum of craniofacial development, and perturbations in SHH signaling are likely to underlie a number of human craniofacial anomalies.

  View details for Web of Science ID 000083881600018

  View details for PubMedID 10518503

• Does adult fracture repair recapitulate embryonic skeletal formation? MECHANISMS OF DEVELOPMENT Ferguson, C., Alpern, E., Miclau, T., Helms, J. A. 1999; 87 (1-2): 57-66

  Abstract

  Bone formation is a continuous process that begins during fetal development and persists throughout life as a remodeling process. In the event of injury, bones heal by generating new bone rather than scar tissue; thus, it can accurately be described as a regenerative process. To elucidate the extent to which fetal skeletal development and skeletal regeneration are similar, we performed a series of detailed expression analyses using a number of genes that regulate key stages of endochondral ossification. They included genes in the indian hedgehog (ihh) and core binding factor 1 (cbfa1) pathways, and genes associated with extracellular matrix remodeling and vascular invasion including vascular endothelial growth factor (VEGF) and matrix metalloproteinase 13 (mmp13). Our analyses suggested that even at the earliest stages of mesenchymal cell condensation, chondrocyte (ihh, cbfa1 and collagen type II-positive) and perichondrial (gli1 and osteocalcin-positive) cell populations were already specified. As chondrocytes matured, they continued to express cbfa1 and ihh whereas cbfa1, osteocalcin and gli1 persisted in presumptive periosteal cells. Later, VEGF and mmp13 transcripts were abundant in chondrocytes as they underwent hypertrophy and terminal differentiation. Based on these expression patterns and available genetic data, we propose a model where Ihh and Cbfa1, together with Gli1 and Osteocalcin participate in establishing reciprocal signal site of injury. The persistence of cbfa1 and ihh, and their targets osteocalcin and gli1, in the callus suggests comparable processes of chondrocyte maturation and specification of a neo-perichondrium occur following injury. VEGF and mmp13 are expressed during the later stages of healing, coincident with the onset of vascularization of the callus and subsequent ossification. Taken together, these data suggest the genetic mechanisms regulating fetal skeletogenesis also regulate adult skeletal regeneration, and point to important regulators of angiogenesis and ossification in bone regeneration.

  View details for Web of Science ID 000082828000006

  View details for PubMedID 10495271

• From head to toe: conservation of molecular signals regulating limb and craniofacial morphogenesis CELL AND TISSUE RESEARCH SCHNEIDER, R. A., Hu, D., Helms, J. A. 1999; 296 (1): 103-109

  Abstract

  Recent evidence indicates that many molecules involved in generating and patterning the limbs also play a role during craniofacial morphogenesis. On the surface, this is an unexpected finding given that these regions of the body have separate evolutionary origins, are composed of different embryonic tissues, and are quite dissimilar in their anatomy. Results from several experiments involving Sonic hedgehog and retinoic acid point to a remarkable conservation of the signaling pathways mediated by these morphogens across multiple organ systems. Moreover, mutants such as the extra-toes and doublefoot mouse, and the talpid chicken also provide insights on common developmental processes that underlie the formation of the limbs and face. The identification of highly conserved aspects of morphogenesis is important for understanding fundamental mechanisms of development, as well as for revealing the common denominator of countless birth defects and providing new strategies for their prevention and cure.

  View details for Web of Science ID 000079824500012

  View details for PubMedID 10199970

• LMP-1, a LIM-domain protein, mediates BMP-6 effects on bone formation ENDOCRINOLOGY Boden, S. D., Liu, Y. S., Hair, G. A., Helms, J. A., Hu, D., Racine, M., Nanes, M. S., Titus, L. 1998; 139 (12): 5125-5134

  Abstract

  Glucocorticoids can promote osteoblast differentiation from fetal calvarial cells and bone marrow stromal cells. We recently reported that glucocorticoid specifically induced bone morphogenetic protein-6 (BMP-6), a glycoprotein signaling molecule that is a multifunctional regulator of vertebrate development. In the present study, we used fetal rat secondary calvarial cultures to determine genes induced during early osteoblast differentiation as initiated by glucocorticoid treatment. Glucocorticoid, and subsequently BMP-6, was found to induce a novel rat intracellular protein, LIM mineralization protein-1 (LMP-1), that in turn resulted in synthesis of one or more soluble factors that could induce de novo bone formation. Blocking expression of LMP-1 using antisense oligonucleotide prevented osteoblast differentiation in vitro. Overexpression of LMP-1 using a mammalian expression vector was sufficient to initiate de novo bone nodule formation in vitro and in sc implants in vivo. These data demonstrate that LMP-1 is an essential positive regulator of the osteoblast differentiation program as well as an important intermediate step in the BMP-6 signaling pathway.

  View details for Web of Science ID 000077104000043

  View details for PubMedID 9832452

• Mechanobiology of skeletal regeneration Workshop on Fracture Healing Enhancement Carter, D. R., Beaupre, G. S., Giori, N. J., Helms, J. A. SPRINGER. 1998: S41–S55

  Abstract

  Skeletal regeneration is accomplished by a cascade of biologic processes that may include differentiation of pluripotential tissue, endochondral ossification, and bone remodeling. It has been shown that all these processes are influenced strongly by the local tissue mechanical loading history. This article reviews some of the mechanobiologic principles that are thought to guide the differentiation of mesenchymal tissue into bone, cartilage, or fibrous tissue during the initial phase of regeneration. Cyclic motion and the associated shear stresses cause cell proliferation and the production of a large callus in the early phases of fracture healing. For intermittently imposed loading in the regenerating tissue: (1) direct intramembranous bone formation is permitted in areas of low stress and strain; (2) low to moderate magnitudes of tensile strain and hydrostatic tensile stress may stimulate intramembranous ossification; (3) poor vascularity can promote chondrogenesis in an otherwise osteogenic environment; (4) hydrostatic compressive stress is a stimulus for chondrogenesis; (5) high tensile strain is a stimulus for the net production of fibrous tissue; and (6) tensile strain with a superimposed hydrostatic compressive stress will stimulate the development of fibrocartilage. Finite element models are used to show that the patterns of tissue differentiation observed in fracture healing and distraction osteogenesis can be predicted from these fundamental mechanobiologic concepts. In areas of cartilage formation, subsequent endochondral ossification normally will proceed, but it can be inhibited by intermittent hydrostatic compressive stress and accelerated by octahedral shear stress (or strain). Later, bone remodeling at these sites can be expected to follow the same mechanobiologic adaptation rules as normal bone.

  View details for Web of Science ID 000077173200007

  View details for PubMedID 9917625

• Histochemical and molecular analyses of distraction osteogenesis in a mouse model JOURNAL OF ORTHOPAEDIC RESEARCH Tay, B. K., Le, A. X., Gould, S. E., Helms, J. A. 1998; 16 (5): 636-642

  Abstract

  A tibial lengthening scheme in the mouse was used to study the molecular and cellular events regulating tissue regeneration during distraction osteogenesis. Here, we report on the surgical technique and frame design and describe the histochemical and molecular aspects of distraction during different phases of treatment. A total of 26 mice were used in this study. The treatment protocol was divided into a latency period of 7 days, a phase of active distraction that lasted 10 days with a distraction rate of 0.42 mm/day, and a maturation phase of 9 days. During latency, the distraction site resembled a stabilized fracture callus on both a histochemical and a molecular level. During active distraction, the gap was characterized by a central fibrous interzone bordered by primary matrix fronts, regenerate bone aligned with the distraction force, parallel columns of vascular sinusoids, and a medullary cavity. Alkaline phosphatase activity was detected in the endosteal and periosteal surfaces of the bone ends. Tartrate resistant acid phosphatase staining revealed that osteoclasts remodeled the bone regenerate as it formed. Collagen type I was expressed in the periosteum and the primary matrix front during distraction, whereas collagen type-II transcripts were localized to discrete regions on the periosteal surfaces, immediately adjacent to the osteotomy ends. Collagen type-II transcripts were not detected in the fibrous interzone. During the maturation phase, cells within the fibrous interzone expressed collagen type I and exhibited abundant alkaline phosphatase activity, suggesting that they had begun to terminally differentiate. Collectively, these data demonstrate the utility of a mouse model to study the molecular and cellular bases for the regeneration and remodeling of tissue.

  View details for Web of Science ID 000076861700017

  View details for PubMedID 9820290

• MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes CELL Vu, T. H., Shipley, J. M., Bergers, G., Berger, J. E., Helms, J. A., Hanahan, D., Shapiro, S. D., Senior, R. M., WERB, Z. 1998; 93 (3): 411-422

  Abstract

  Homozygous mice with a null mutation in the MMP-9/gelatinase B gene exhibit an abnormal pattern of skeletal growth plate vascularization and ossification. Although hypertrophic chondrocytes develop normally, apoptosis, vascularization, and ossification are delayed, resulting in progressive lengthening of the growth plate to about eight times normal. After 3 weeks postnatal, aberrant apoptosis, vascularization, and ossification compensate to remodel the enlarged growth plate and ultimately produce an axial skeleton of normal appearance. Transplantation of wild-type bone marrow cells rescues vascularization and ossification in gelatinase B-null growth plates, indicating that these processes are mediated by gelatinase B-expressing cells of bone marrow origin, designated chondroclasts. Growth plates from gelatinase B-null mice in culture show a delayed release of an angiogenic activator, establishing a role for this proteinase in controlling angiogenesis.

  View details for Web of Science ID 000073471500016

  View details for PubMedID 9590175

• Common molecular pathways in skeletal morphogenesis and repair Conference on Morphogenesis - Cellular Interactions Ferguson, C. M., Miclau, T., Hu, D., Alpern, E., Helms, J. A. NEW YORK ACAD SCIENCES. 1998: 33–42

  Abstract

  The formation of bone is a continual process in vertebrate development, initiated during fetal development and persisting in adulthood in the form of remodeling and repair. The remarkable capacity of skeletal tissues to regenerate has led to the hypothesis that the molecular signaling pathways regulating skeletogenesis are shared during fetal development and adult wound healing. A number of key regulatory pathways that are required for endochondral ossification during fetal development are described, and their reintroduction in fracture repair demonstrated. Secreted proteins such as Sonic and Indian hedgehog exert their effect on pattern formation and chondrogenesis in the appendicular skeleton, partly through regulation of molecules such as bone morphogenic proteins (Bmps) and parathyroid hormone-related peptide (PTHrP). Once chondrocytes have matured and hypertrophied, they undergo apoptosis and are replaced by bone; the transcription factor Cbfal plays a critical role in this process of chondrocyte differentiation and ossification. Analyses of the expression patterns of these genes during fracture healing strongly suggest that they play equivalent roles in adult wound repair. Knowledge acquired through the study of fetal skeletogenesis will undoubtedly contribute to an understanding of fracture repair, and subsequently guide the development of biologically based therapeutic interventions.

  View details for Web of Science ID 000077211500004

  View details for PubMedID 9917830

• The role of Hedgehog genes in skeletogenesis and repair Workshop on Skeletal Growth and Development - Clinical Issues and Basic Science Advances Helms, J. A., Le, A. X., Iwasaki, M. AMER ACAD ORTHOPAEDIC SURGEONS. 1998: 131–145

  View details for Web of Science ID 000076319100008

• Expression of Indian hedgehog, bone morphogenetic protein 6 and gli during skeletal morphogenesis 43rd Annual Meeting of the Orthopaedic-Research-Society Iwasaki, M., Le, A. X., Helms, J. A. ELSEVIER SCIENCE BV. 1997: 197–202

  Abstract

  A complex signaling pathway involving members of the Hedgehog, Bone morphogenetic protein (Bmp) and Gli families regulates early patterning events in fetal skeletogenesis (Hui and Joyner, 1993. A mouse model of Greig cephalopolysyndactyly syndrome: the extra-toes mutation contains an intragenic deletion of the Gli3 gene. Nat. Genet. 3, 241-246; Bitgood and McMahon, 1995. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev. Biol. 172, 126-138; Lanske et al., 1996. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273, 663-666; Vortkamp et al., 1996. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273, 613-622). Hedgehog genes encode secreted proteins that mediate patterning and growth through the induction of secondary signals (reviewed in Hammerschmidt et al., 1997. The world according to hedgehog. Trends Genet. 13, 14-21). Two potential targets of Ihh are bmp6 and gli (Johnson et al., 1995. Patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets. Development 121, 4161-4170; Dominguez et al., 1996. Sending and receiving the hedgehog signal: control by the Drosophila Gli protein Cubitus interruptus. Science 272, 1621-1625; Marigo et al., 1996. Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development. Dev. Biol. 180, 273-283). We investigated the molecular similarities and differences between fetal and postnatal skeletal development by analyzing the coincident and complimentary expression domains of indian hedgehog (ihh), bmp6 and gli in adjacent sections throughout the process of skeletogenesis. In almost all of the skeletal tissues examined, the expression domains of ihh and bmp6 were adjacent to one another and this region was surrounded by gli-expressing cells. These observations are in keeping with the proposed function of gli as a negative regulator of Ihh signaling and the induction of Bmps by Hedgehog proteins (Roberts et al., 1995. Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development 121, 3163-3174; Kawakami et al., 1996. BMP signaling during bone pattern determination in the developing limb. Development 122, 3557-3566). By puberty, ihh, bmp6 and gli transcripts were no longer detected in the growth plate, despite the fact that physeal chondrocytes continued to hypertrophy and differentiate. Although bmp6 was expressed, ihh transcripts were not found in primordia of intramembranous bones, nor in cells lining the future articular surfaces. Collectively our findings suggest that ihh participates in, but is not required for chondrocyte hypertrophy.

  View details for Web of Science ID 000071716400015

  View details for PubMedID 9486541

• Neuronal pentraxin receptor, a novel putative integral membrane pentraxin that interacts with neuronal pentraxin 1 and 2 and taipoxin-associated calcium-binding protein 49 JOURNAL OF BIOLOGICAL CHEMISTRY Dodds, D. C., Omeis, I. A., Cushman, S. J., Helms, J. A., Perin, M. S. 1997; 272 (34): 21488-21494

  Abstract

  We have identified the first putative integral membrane pentraxin and named it neuronal pentraxin receptor (NPR). NPR is enriched by affinity chromatography on columns of a snake venom toxin, taipoxin, and columns of the taipoxin-binding proteins neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and taipoxin-associated calcium-binding protein 49 (TCBP49). The predominant form of NPR contains an putative NH2-terminal transmembrane domain and all forms of NPR are glycosylated. NPR has 49 and 48% amino acid identity to NP1 and NP2, respectively, and NPR message is expressed in neuronal regions that express NP1 and NP2. We suggest that NPR, NP1, NP2, and TCBP49 are involved in a pathway responsible for the transport of taipoxin into synapses and that this may represent a novel neuronal uptake pathway involved in the clearance of synaptic debris.

  View details for Web of Science ID A1997XR78900077

  View details for PubMedID 9261167

• Sonic hedgehog participates in craniofacial morphogenesis and is down-regulated by teratogenic doses of retinoic acid DEVELOPMENTAL BIOLOGY Helms, J. A., Kim, C. H., Hu, D., Minkoff, R., Thaller, C., Eichele, G. 1997; 187 (1): 25-35

  Abstract

  The face is one of the most intricately patterned structures in human and yet little is known of the mechanisms by which the tissues are instructed to grow, fuse, and differentiate. We undertook a study to determine if the craniofacial primordia used the same molecular cues that mediate growth and patterning in other embryonic tissues such as the neural tube and the limb. Here we provide evidence for the presence of organizer-like tissues in the craniofacial primordia. These candidate organizers express the polarizing signal sonic hedghog (shh) and its putative receptor, patched, as well as fibroblast growth factor 8 and bone morphogeneic protein 2. Shh-expressing epithelial grafts functioned as organizing tissues in a limb bud assay system, where they evoked duplications of the digit pattern. High doses of retinoic acid, which are known to truncate the growth of the frontonasal and maxillary processes and thus produce bilateral clefting of the lip and palate, inhibited the expression of shh and patched but not fgf8, in the craniofacial primordia, and abolished polarizing activity of these tissues. From these studies we conclude that the embryonic face contains signaling centers in the epithelium that participate in craniofacial growth and patterning. In addition, we discuss a novel mechanism whereby retinoids can exert a teratogenic effect on craniofacial morphogenesis independent of its effects on Hox gene expression or neural crest cell migration.

  View details for Web of Science ID A1997XL41900003

  View details for PubMedID 9224671

• Retinoic acid signaling is required during early chick limb development DEVELOPMENT Helms, J. A., Kim, C. H., Eichele, G., Thaller, C. 1996; 122 (5): 1385-1394

  Abstract

  In the chick limb bud, the zone of polarizing activity controls limb patterning along the anteroposterior and proximodistal axes. Since retinoic acid can induce ectopic polarizing activity, we examined whether this molecule plays a role in the establishment of the endogenous zone of polarizing activity. Grafts of wing bud mesenchyme treated with physiologic doses of retinoic acid had weak polarizing activity but inclusion of a retinoic acid-exposed apical ectodermal ridge or of prospective wing bud ectoderm evoked strong polarizing activity. Likewise, polarizing activity of prospective wing mesenchyme was markedly enhanced by co-grafting either a retinoic acid-exposed apical ectodermal ridge or ectoderm from the wing region. This equivalence of ectoderm-mesenchyme interactions required for the establishment of polarizing activity in retinoic acid-treated wing buds and in prospective wing tissue, suggests a role of retinoic acid in the establishment of the zone of polarizing activity. We found that prospective wing bud tissue is a high-point of retinoic acid synthesis. Furthermore, retinoid receptor-specific antagonists blocked limb morphogenesis and down-regulated a polarizing signal, sonic hedgehog. Limb agenesis was reversed when antagonist-exposed wing buds were treated with retinoic acid. Our results demonstrate a role of retinoic acid in the establishment of the endogenous zone of polarizing activity.

  View details for Web of Science ID A1996UM55800005

  View details for PubMedID 8625827

• EFFECTS OF CHLORHEXIDINE ON HUMAN TASTE PERCEPTION ARCHIVES OF ORAL BIOLOGY Helms, J. A., DELLAFERA, M. A., Mott, A. E., Frank, M. E. 1995; 40 (10): 913-920

  Abstract

  Chlorhexidine gluconate at a dose used to control bacteria in the mouth has a reversible effect on taste perception. Taste-intensity ratings and taste-quality identification for concentration series of sucrose, sodium chloride, citric acid and quinine hydrochloride were obtained from 15 healthy humans. The participants rinsed with 0.12% chlorhexidine for 3 min twice a day. Each individual was tested 3 times: before the 4-day rinse period, 30 min after the final rinse, and 4 days after the rinse period. Chlorhexidine rinses reduced the perceptual intensity of sodium chloride and quinine hydrochloride, not sucrose or citric acid. No effects on taste perception were detected 4 days after the rinse period. The identification of sodium chloride as salty was seriously impaired by chlorhexidine but the identification of quinine hydrochloride as bitter was not affected. Specific sites of action of chlorhexidine on the taste epithelium are not known but its effects on salty taste may be related to its strong positive charge and its effect on bitter taste may be related to its amphiphilicity. Chlorhexidine has promise as a probe of taste transduction, as well as for the management of salty/bitter dysgeusias in humans.

  View details for Web of Science ID A1995TB53700004

  View details for PubMedID 8526801

• NEURONAL PENTRAXIN, A SECRETED PROTEIN WITH HOMOLOGY TO ACUTE-PHASE PROTEINS OF THE IMMUNE-SYSTEM NEURON SCHLIMGEN, A. K., Helms, J. A., Vogel, H., Perin, M. S. 1995; 14 (3): 519-526

  Abstract

  We have identified, by affinity chromatography, a binding protein for the snake venom toxin taipoxin. The sequence of this 47 kDa protein is unique, is characteristic of a secreted protein, and has homology to the acute phase proteins serum amyloid P protein and C-reactive protein of the pentraxin family. We have named this protein neuronal pentraxin (NP), as Northern analysis and in situ hybridization demonstrate high message levels in neurons of cerebellum, hippocampus, and cerebral cortex. Because NP may be released synaptically and has homology to immune proteins potentially involved in uptake of lipidic, toxic, or other antigenic material, we suggest that NP may be involved in a general uptake of synaptic macromolecules.

  View details for Web of Science ID A1995QP23000005

  View details for PubMedID 7695898

• CLONING AND ANALYSIS OF A NEW DEVELOPMENTALLY-REGULATED MEMBER OF THE BASIC HELIX-LOOP-HELIX FAMILY MECHANISMS OF DEVELOPMENT Helms, J. A., Kuratani, S., Maxwell, G. D. 1994; 48 (2): 93-108

  Abstract

  We have isolated a basic helix-loop-helix (bHLH) family member from an embryonic chick-brain cDNA library. This 3.86-Kb cDNA, GbHLH1.4, exhibits extensive sequence similarity in the bHLH domain with Drosophila daughterless and the vertebrate cDNAs E12 and HTF4. Outside of the bHLH region the similarity is significantly reduced. GbHLH1.4 recognizes a 4.0-Kb mRNA and in situ hybridization analysis shows that GbHLH1.4 mRNA is widely expressed at early stages of development but becomes progressively restricted as embryogenesis proceeds. At later stages of embryonic development, mRNA transcripts are localized to several structures including the ventricular layers of the spinal cord and brain, the facial primordia, dorsal root ganglia and heart muscle and cardiac valves. Strikingly, GbHLH1.4 expression in chick embryos exhibits significant overlap with that reported for the murine negative HLH regulator, Id.

  View details for Web of Science ID A1994PU06200003

  View details for PubMedID 7873406

• RELATIONSHIP BETWEEN RETINOIC ACID AND SONIC HEDGEHOG, 2 POLARIZING SIGNALS IN THE CHICK WING BUD DEVELOPMENT Helms, J., Thaller, C., Eichele, G. 1994; 120 (11): 3267-3274

  Abstract

  Local application of all-trans-retinoic acid (RA) to the anterior margin of chick limb buds results in pattern duplications reminescent of those that develop after grafting cells from the zone of polarizing activity (ZPA). RA may act directly by conferring positional information to limb bud cells, or it may act indirectly by creating a polarizing region in the tissue distal to the RA source. Here we demonstrate that tissue distal to an RA-releasing bead acquires polarizing activity in a dose-dependent manner. Treatments with pharmacological (beads soaked in 330 micrograms/ml) and physiological (beads soaked in 10 micrograms/ml) doses of RA are equally capable of inducing digit pattern duplication. Additionally, both treatments induce sonic hedgehog (shh; also known as vertebrate hedgehog-1, vhh-1), a putative ZPA morphogen and Hoxd-11, a gene induced by the polarizing signal. However, tissue transplantation assays reveal that pharmacological, but not physiological, doses create a polarizing region. This differential response could be explained if physiological doses induced less shh than pharmacological doses. However, our in situ hybridization analyses demonstrate that both treatments result in similar amounts of mRNA encoding this candidate ZPA morphogen. We outline a model describing the apparently disparate effects of pharmacologic and physiological doses RA on limb bud tissue.

  View details for Web of Science ID A1994PV97100020

  View details for PubMedID 7720566