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  • Sex differences of NF-κB-targeted therapy for mitigating osteoporosis associated with chronic inflammation of bone. Bone & joint research Toya, M., Kushioka, J., Shen, H., Utsunomiya, T., Hirata, H., Tsubosaka, M., Gao, Q., Chow, S. K., Zhang, N., Goodman, S. B. 2024; 13 (1): 28-39


    Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice.We used a murine model of chronic inflammation induced by continuous intramedullary delivery of lipopolysaccharide-contaminated polyethylene particles (cPE) using an osmotic pump. Specimens were evaluated using micro-CT and histomorphometric analyses. Sex-specific osteogenic and osteoclastic differentiation potentials were also investigated in vitro, including alkaline phosphatase, Alizarin Red, tartrate-resistant acid phosphatase staining, and gene expression using reverse transcription polymerase chain reaction (RT-PCR).Local delivery of NF-κB decoy ODN in vivo increased osteogenesis in males, but not females, in the presence of chronic inflammation induced by cPE. Bone resorption activity was decreased in both sexes. In vitro osteogenic and osteoclastic differentiation assays during inflammatory conditions did not reveal differences among the groups. Receptor activator of nuclear factor kappa Β ligand (Rankl) gene expression by osteoblasts was significantly decreased only in males when treated with ODN.We demonstrated that NF-κB decoy ODN increased osteogenesis in male mice and decreased bone resorption activity in both sexes in preclinical models of chronic inflammation. NF-κB signalling could be a therapeutic target for chronic inflammatory diseases involving bone, especially in males.

    View details for DOI 10.1302/2046-3758.131.BJR-2023-0040.R3

    View details for PubMedID 38194999

  • Preclinical models for studying corticosteroid-induced osteonecrosis of the femoral head. Journal of biomedical materials research. Part B, Applied biomaterials Tsubosaka, M., Maruyama, M., Lui, E., Kushioka, J., Toya, M., Gao, Q., Shen, H., Li, X., Chow, S. K., Zhang, N., Yang, Y. P., Goodman, S. B. 2024; 112 (1): e35360


    Nontraumatic osteonecrosis of the femoral head (ONFH) is a refractory condition that commonly results in femoral head collapse and degenerative arthritis of the hip. In the early stages, surgical procedures for hip preservation, including core decompression (CD), have been developed to prevent progressive collapse of the femoral head. Optimization of bone regeneration and biological augmentation may further enhance the therapeutic efficacy of CD for ONFH. Thus, combining CD with cell-based therapy has recently been proposed. In fact, patients treated with cell-based therapy using autologous bone marrow concentrate demonstrate improved survivorship of the femoral head, compared with conventional CD alone. Preclinical research studies to investigate adjunctive therapies for CD often utilize the rabbit model of corticosteroid-induced ONFH. Mesenchymal stem cells (MSCs) are known to promote osteogenesis and angiogenesis, and decrease inflammation in bone. Local drug delivery systems have the potential to achieve targeted therapeutic effects by precisely controlling the drug release rate. Scaffolds can provide an osteoconductive structural framework to facilitate the repair of osteonecrotic bone tissue. We focused on the combination of both cell-based and scaffold-based therapies for bone tissue regeneration in ONFH. We hypothesized that combining CD and osteoconductive scaffolds would provide mechanical strength and structural cell guidance; and that combining CD and genetically modified (GM) MSCs to express relevant cytokines, chemokines, and growth factors would promote bone tissue repair. We developed GM MSCs that overexpress the anti-inflammatory, pro-reconstructive cytokines platelet-derived growth factor-BB to provide MSCs with additional benefits and investigated the efficacy of combinations of these GM MSCs and scaffolds for treatment of ONFH in skeletally mature male New Zealand white rabbits. In the future, the long-term safety, efficacy, durability, and cost-effectiveness of these and other biological and mechanical treatments must be demonstrated for the patients affected by ONFH.

    View details for DOI 10.1002/jbm.b.35360

    View details for PubMedID 38247252

  • Preclinical models for studying corticosteroid-induced osteonecrosis of the femoral head JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS Tsubosaka, M., Maruyama, M., Lui, E., Kushioka, J., Toya, M., Gao, Q., Shen, H., Li, X., Chow, S., Zhang, N., Yang, Y., Goodman, S. B. 2024; 112 (1)
  • Effects of Aging on Osteosynthesis at Bone-Implant Interfaces. Biomolecules Pius, A. K., Toya, M., Gao, Q., Ergul, Y. S., Chow, S. K., Goodman, S. B. 2023; 14 (1)


    Joint replacement is a common surgery and is predominantly utilized for treatment of osteoarthritis in the aging population. The longevity of many of these implants depends on bony ingrowth. Here, we provide an overview of current techniques in osteogenesis (inducing bone growth onto an implant), which is affected by aging and inflammation. In this review we cover the biologic underpinnings of these processes as well as the clinical applications. Overall, aging has a significant effect at the cellular and macroscopic level that impacts osteosynthesis at bone-metal interfaces after joint arthroplasty; potential solutions include targeting prolonged inflammation, preventing microbial adhesion, and enhancing osteoinductive and osteoconductive properties.

    View details for DOI 10.3390/biom14010052

    View details for PubMedID 38254652

  • C-C Motif Chemokine Ligand 2 Enhances Macrophage Chemotaxis, Osteogenesis, and Angiogenesis during the Inflammatory Phase of Bone Regeneration. Biomolecules Shinohara, I., Tsubosaka, M., Toya, M., Lee, M. L., Kushioka, J., Murayama, M., Gao, Q., Li, X., Zhang, N., Chow, S. K., Matsumoto, T., Kuroda, R., Goodman, S. B. 2023; 13 (11)


    Local cell therapy has recently gained attention for the treatment of joint diseases and fractures. Mesenchymal stem cells (MSCs) are not only involved in osteogenesis and angiogenesis, but they also have immunomodulatory functions, such as inducing macrophage migration during bone regeneration via macrophage crosstalk. C-C motif chemokine ligand 2 (CCL2), a known inflammatory mediator, is associated with the migration of macrophages during inflammation. This study examined the utility of CCL2 as a therapeutic target for local cell therapy. Using lentiviral vectors for rabbit MSCs, genetically modified CCL2 overexpressing MSCs were generated. Osteogenic differentiation assays were performed using MSCs with or without macrophages in co-culture, and cell migration assays were also performed. Additionally, co-cultures were performed with endothelial cells (ECs), and angiogenesis was evaluated using a tube formation assay. Overexpression of CCL2 did not affect bone formation under monoculture conditions but promoted chemotaxis and osteogenesis when co-cultured with macrophages. Furthermore, CCL2-overexpression promoted tube formation in co-culture with ECs. These results suggest that CCL2 induces macrophage chemotaxis and osteogenesis by promoting crosstalk between MSCs and macrophages; CCL2 also stimulates ECs to induce angiogenesis. These findings indicate that CCL2 may be a useful therapeutic target for local cell therapy in areas of bone loss.

    View details for DOI 10.3390/biom13111665

    View details for PubMedID 38002347

  • Glycolytic reprogramming in macrophages and MSCs during inflammation. Frontiers in immunology Li, X., Shen, H., Zhang, M., Teissier, V., Huang, E. E., Gao, Q., Tsubosaka, M., Toya, M., Kushioka, J., Maduka, C. V., Contag, C. H., Chow, S. K., Zhang, N., Goodman, S. B. 2023; 14: 1199751


    Dysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs).We used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE.Our results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines.This study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation.

    View details for DOI 10.3389/fimmu.2023.1199751

    View details for PubMedID 37675119

    View details for PubMedCentralID PMC10477714

  • CCL2 promotes osteogenesis by facilitating macrophage migration during acute inflammation. Frontiers in cell and developmental biology Toya, M., Zhang, N., Tsubosaka, M., Kushioka, J., Gao, Q., Li, X., Chow, S. K., Goodman, S. B. 2023; 11: 1213641


    Novel minimally invasive strategies are needed to obtain robust bone healing in complex fractures and bone defects in the elderly population. Local cell therapy is one potential option for future treatment. Mesenchymal stromal cells (MSCs) are not only involved in osteogenesis but also help direct the recruitment of macrophages during bone regeneration via MSC-macrophage crosstalk. The C-C motif chemokine ligand 2 (CCL2) is an inflammatory chemokine that is associated with the migration of macrophages and MSCs during inflammation. This study investigated the use of CCL2 as a therapeutic target for local cell therapy. MSCs and macrophages were isolated from 10 to 12 week-old BALB/c male mice. Genetically modified CCL2 over-expressing MSCs were produced using murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector. Osteogenic differentiation assays were performed using MSCs with or without macrophages in co-culture. Cell migration assays were also performed. MSCs transfected with murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector showed higher levels of CCL2 secretion compared to unaltered MSCs (p < 0.05). Genetic manipulation did not affect cell proliferation. CCL2 did not affect the osteogenic ability of MSCs alone. However, acute (1 day) but not sustained (7 days) stimulation with CCL2 increased the alizarin red-positive area when MSCs were co-cultured with macrophages (p < 0.001). Both recombinant CCL2 (p < 0.05) and CCL2 released from MSCs (p < 0.05) facilitated macrophage migration. We demonstrated that acute CCL2 stimulation promoted subsequent osteogenesis in co-culture of MSCs and macrophages. Acute CCL2 stimulation potentially facilitates osteogenesis during the acute inflammatory phase of bone healing by directing local macrophage migration, fostering macrophage-MSC crosstalk, and subsequently, by activating or licensing of MSCs by macrophage pro-inflammatory cytokines. The combination of CCL2, MSCs, and macrophages could be a potential strategy for local cell therapy in compromised bone healing.

    View details for DOI 10.3389/fcell.2023.1213641

    View details for PubMedID 37457301

    View details for PubMedCentralID PMC10348816

  • Bone regeneration in inflammation with aging and cell-based immunomodulatory therapy. Inflammation and regeneration Kushioka, J., Chow, S. K., Toya, M., Tsubosaka, M., Shen, H., Gao, Q., Li, X., Zhang, N., Goodman, S. B. 2023; 43 (1): 29


    Aging of the global population increases the incidence of osteoporosis and associated fragility fractures, significantly impacting patient quality of life and healthcare costs. The acute inflammatory reaction is essential to initiate healing after injury. However, aging is associated with "inflammaging", referring to the presence of systemic low-level chronic inflammation. Chronic inflammation impairs the initiation of bone regeneration in elderly patients. This review examines current knowledge of the bone regeneration process and potential immunomodulatory therapies to facilitate bone healing in inflammaging.Aged macrophages show increased sensitivity and responsiveness to inflammatory signals. While M1 macrophages are activated during the acute inflammatory response, proper resolution of the inflammatory phase involves repolarizing pro-inflammatory M1 macrophages to an anti-inflammatory M2 phenotype associated with tissue regeneration. In aging, persistent chronic inflammation resulting from the failure of M1 to M2 repolarization leads to increased osteoclast activation and decreased osteoblast formation, thus increasing bone resorption and decreasing bone formation during healing.Inflammaging can impair the ability of stem cells to support bone regeneration and contributes to the decline in bone mass and strength that occurs with aging. Therefore, modulating inflammaging is a promising approach for improving bone health in the aging population. Mesenchymal stem cells (MSCs) possess immunomodulatory properties that may benefit bone regeneration in inflammation. Preconditioning MSCs with pro-inflammatory cytokines affects MSCs' secretory profile and osteogenic ability. MSCs cultured under hypoxic conditions show increased proliferation rates and secretion of growth factors. Resolution of inflammation via local delivery of anti-inflammatory cytokines is also a potential therapy for bone regeneration in inflammaging. Scaffolds containing anti-inflammatory cytokines, unaltered MSCs, and genetically modified MSCs can also have therapeutic potential. MSC exosomes can increase the migration of MSCs to the fracture site and enhance osteogenic differentiation and angiogenesis.In conclusion, inflammaging can impair the proper initiation of bone regeneration in the elderly. Modulating inflammaging is a promising approach for improving compromised bone healing in the aging population.

    View details for DOI 10.1186/s41232-023-00279-1

    View details for PubMedID 37231450

    View details for PubMedCentralID 2880220

  • Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model. Stem cell research & therapy Teissier, V., Gao, Q., Shen, H., Li, J., Li, X., Huang, E. E., Kushioka, J., Toya, M., Tsubosaka, M., Hirata, H., Alizadeh, H. V., Maduka, C. V., Contag, C. H., Yang, Y. P., Zhang, N., Goodman, S. B. 2023; 14 (1): 99


    Continuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway.We cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species-ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs.Exposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days.This work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism.

    View details for DOI 10.1186/s13287-023-03260-4

    View details for PubMedID 37085909

    View details for PubMedCentralID PMC10122387

  • Using Microphysiological System for the Development of Treatments for Joint Inflammation and Associated Cartilage Loss-A Pilot Study. Biomolecules Makarczyk, M. J., Hines, S., Yagi, H., Li, Z. A., Aguglia, A. M., Zbikowski, J., Padget, A. M., Gao, Q., Bunnell, B. A., Goodman, S. B., Lin, H. 2023; 13 (2)


    Osteoarthritis (OA) is a painful and disabling joint disease affecting millions worldwide. The lack of clinically relevant models limits our ability to predict therapeutic outcomes prior to clinical trials, where most drugs fail. Therefore, there is a need for a model that accurately recapitulates the whole-joint disease nature of OA in humans. Emerging microphysiological systems provide a new opportunity. We recently established a miniature knee joint system, known as the miniJoint, in which human bone-marrow-derived mesenchymal stem cells (hBMSCs) were used to create an osteochondral complex, synovial-like fibrous tissue, and adipose tissue analogs. In this study, we explored the potential of the miniJoint in developing novel treatments for OA by testing the hypothesis that co-treatment with anti-inflammation and chondroinducing agents can suppress joint inflammation and associated cartilage degradation. Specifically, we created a "synovitis"-relevant OA model in the miniJoint by treating synovial-like tissues with interleukin-1β (IL-1β), and then a combined treatment of oligodeoxynucleotides (ODNs) suppressing the nuclear factor kappa beta (NF-κB) genetic pathway and bone morphogenic protein-7 (BMP-7) was introduced. The combined treatment with BMP-7 and ODNs reduced inflammation in the synovial-like fibrous tissue and showed an increase in glycosaminoglycan formation in the cartilage portion of the osteochondral complex. For the first time, this study demonstrated the potential of the miniJoint in developing disease-modifying OA drugs. The therapeutic efficacy of co-treatment with NF-κB ODNs and BMP-7 can be further validated in future clinical studies.

    View details for DOI 10.3390/biom13020384

    View details for PubMedID 36830751

  • Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs. Journal of visualized experiments : JoVE Makarcyzk, M. J., Li, Z. A., Yu, I., Yagi, H., Zhang, X., Yocum, L., Li, E., Fritch, M. R., Gao, Q., Bunnell, B. A., Goodman, S. B., Tuan, R. S., Alexander, P. G., Lin, H. 2023


    The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either "systemic administration" through all the medium streams or "intraarticular administration" by adding the drugs to only the shared "synovial fluid"-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

    View details for DOI 10.3791/64186

    View details for PubMedID 36779602

  • The efficiency of genetically modified mesenchymal stromal cells combined with a functionally graded scaffold for bone regeneration in corticosteroid-induced osteonecrosis of the femoral head in rabbits. Journal of biomedical materials research. Part A Tsubosaka, M., Maruyama, M., Lui, E., Moeinzadeh, S., Huang, E. E., Kushioka, J., Hirata, H., Jain, C., Storaci, H. W., Chan, C., Toya, M., Gao, Q., Teissier, V., Shen, H., Li, X., Zhang, N., Matsumoto, T., Kuroda, R., Goodman, S. B., Yang, Y. P. 2023


    Core decompression (CD) with mesenchymal stromal cells (MSCs) is an effective therapy for early-stage osteonecrosis of the femoral head (ONFH). Preconditioning of MSCs, using inflammatory mediators, is widely used in immunology and various cell therapies. We developed a three-dimensional printed functionally graded scaffold (FGS), made of beta-TCP and PCL, for cell delivery at a specific location. The present study examined the efficacy of CD treatments with genetically modified (GM) MSCs over-expressing PDGF-BB (PDGF-MSCs) or GM MSCs co-over-expressing IL-4 and PDGF-BB and preconditioned for three days of exposure to lipopolysaccharide and tumor necrosis factor-alpha (IL-4-PDGF-pMSCs) using the FGS for treating steroid-induced ONFH in rabbits. We compared CD without cell-therapy, with IL-4-PDGF-pMSCs alone, and with FGS loaded with PDGF-MSCs or IL-4-PDGF-pMSCs. For the area inside the CD, the bone volume in the CD alone was higher than in both FGS groups. The IL-4-PDGF-pMSCs alone and FGS+PDGF-MSCs reduced the occurrence of empty lacunae and improved osteoclastogenesis. There was no significant difference in angiogenesis among the four groups. The combined effect of GM MSCs or pMSCs and the FGS was not superior to the effect of each alone. To establish an important adjunctive therapy for CD for early ONFH in the future, it is necessary and essential to develop an FGS that delivers biologics appropriately and provides structural and mechanical support.

    View details for DOI 10.1002/jbm.a.37495

    View details for PubMedID 36606330

  • Experimental models to study osteoarthritis pain and develop therapeutics. Osteoarthritis and cartilage open Riewruja, K., Makarczyk, M., Alexander, P. G., Gao, Q., Goodman, S. B., Bunnell, B. A., Gold, M. S., Lin, H. 2022; 4 (4): 100306


    Pain is the predominant symptom of osteoarthritis (OA) that drives patients to seek medical care. Currently, there are no pharmacological treatments that can reverse or halt the progression of OA. Safe and efficacious medications for long-term management of OA pain are also unavailable. Understanding the mechanisms behind OA pain generation at onset and over time is critical for developing effective treatments. In this narrative review, we first summarize our current knowledge on the innervation of the knee joint, and then discuss the molecular mechanism(s) currently thought to underlie OA pain. In particular, we focus on the contribution of each joint component to the generation of pain. Next, the current experimental models for studying OA pain are summarized, and the methods to assess pain in rodents are presented. The potential application of emerging microphysiological systems in OA pain research is especially highlighted. Lastly, we discuss the current challenge in standardizing models and the selection of appropriate systems to address specific questions.

    View details for DOI 10.1016/j.ocarto.2022.100306

    View details for PubMedID 36474784

  • Differential dynamics of bone graft transplantation and mesenchymal stem cell therapy during bone defect healing in a murine critical size defect. Journal of orthopaedic translation Huang, E. E., Zhang, N., Ganio, E. A., Shen, H., Li, X., Ueno, M., Utsunomiya, T., Maruyama, M., Gao, Q., Su, N., Yao, Z., Yang, F., Gaudilliere, B., Goodman, S. B. 2022; 36: 64-74


    Background: A critical size bone defect is a clinical scenario in which bone is lost or excised due to trauma, infection, tumor, or other causes, and cannot completely heal spontaneously. The most common treatment for this condition is autologous bone grafting to the defect site. However, autologous bone graft is often insufficient in quantity or quality for transplantation to these large defects. Recently, tissue engineering methods using mesenchymal stem cells (MSCs) have been proposed as an alternative treatment. However, the underlying biological principles and optimal techniques for tissue regeneration of bone using stem cell therapy have not been completely elucidated.Methods: In this study, we compare the early cellular dynamics of healing between bone graft transplantation and MSC therapy in a murine chronic femoral critical-size bone defect. We employ high-dimensional mass cytometry to provide a comprehensive view of the differences in cell composition, stem cell functionality, and immunomodulatory activity between these two treatment methods one week after transplantation.Results: We reveal distinct cell compositions among tissues from bone defect sites compared with original bone graft, show active recruitment of MSCs to the bone defect sites, and demonstrate the phenotypic diversity of macrophages and T cells in each group that may affect the clinical outcome.Conclusion: Our results provide critical data and future directions on the use of MSCs for treating critical size defects to regenerate bone.Translational Potential of this article: This study showed systematic comparisons of the cellular and immunomodulatory profiles among different interventions to improve the healing of the critical-size bone defect. The results provided potential strategies for designing robust therapeutic interventions for the unmet clinical need of treating critical-size bone defects.

    View details for DOI 10.1016/

    View details for PubMedID 35979174

  • Therapeutic effects of MSCs, genetically modified MSCs, and NFkB-inhibitor on chronic inflammatory osteolysis in aged mice. Journal of orthopaedic research : official publication of the Orthopaedic Research Society Kushioka, J., Toya, M., Shen, H., Hirata, H., Zhang, N., Huang, E., Tsubosaka, M., Gao, Q., Teissier, V., Li, X., Utsunomiya, T., Goodman, S. B. 2022


    The number of total joint replacements is increasing, especially in elderly patients, and so too are implant-related complications such as prosthesis loosening. Wear particles from the prosthesis induce a chronic inflammatory reaction and subsequent osteolysis, leading to the need for revision surgery. This study investigated the therapeutic effect of NF-kB decoy oligodeoxynucleotides (ODN), mesenchymal stem cells (MSCs), and genetically-modified NF-kB sensing interleukin-4 over-secreting MSCs (IL4-MSCs) on chronic inflammation in aged mice. The model was generated by continuous infusion of contaminated polyethylene particles into the intramedullary space of the distal femur of aged mice (15-17-month-old) for six weeks. Local delivery of ODN showed increased bone mineral density (BMD), decreased osteoclast-like cells, increased alkaline phosphatase (ALP)-positive area, and increased M2/M1 macrophage ratio. Local injection of MSCs and IL4-MSCs significantly decreased osteoclast-like cells and increased the M2/M1 ratio, with a greater trend for IL4-MSCs than MSCs. MSCs significantly increased ALP-positive area and BMD values compared to the control. The IL4-MSCs demonstrated higher values for both ALP-positive area and BMD. These findings demonstrated the therapeutic effects of ODN, MSCs, and IL4-MSCs on chronic inflammatory osteolysis in aged mice. The two MSC-based therapies were more effective than ODN in increasing the M2/M1 macrophage ratio, reducing bone resorption, and increasing bone formation. Specifically, MSCs were more effective in increasing bone formation, and IL4-MSCs were more effective in mitigating inflammation. This study suggests potential therapeutic strategies for treating wear particle-associated inflammatory osteolysis after arthroplasty in the elderly. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/jor.25434

    View details for PubMedID 36031590

  • Human Mesenchymal Stem Cell-Derived Miniature Joint System for Disease Modeling and Drug Testing. Advanced science (Weinheim, Baden-Wurttemberg, Germany) Li, Z., Lin, Z., Liu, S., Yagi, H., Zhang, X., Yocum, L., Romero-Lopez, M., Rhee, C., Makarcyzk, M. J., Yu, I., Li, E. N., Fritch, M. R., Gao, Q., Goh, K. B., O'Donnell, B., Hao, T., Alexander, P. G., Mahadik, B., Fisher, J. P., Goodman, S. B., Bunnell, B. A., Tuan, R. S., Lin, H. 2022: e2105909


    Diseases of the knee joint such as osteoarthritis (OA) affect all joint elements. An in vitro human cell-derived microphysiologica system capable of simulating intraarticular tissue crosstalk is desirable for studying etiologies/pathogenesis of joint diseases and testing potential therapeutics. Herein, a human mesenchymal stem cell-derived miniature joint system (miniJoint) is generated, in which engineered osteochondral complex, synovial-like fibrous tissue, and adipose tissue are integrated into a microfluidics-enabled bioreactor. This novel design facilitates different tissues communicating while still maintaining their respective phenotypes. The miniJoint exhibits physiologically relevant changes when exposed to interleukin-1beta mediated inflammation, which are similar to observations in joint diseases in humans. The potential of the miniJoint in predicting in vivo efficacy of drug treatment is confirmed by testing the "therapeutic effect" of the nonsteroidal anti-inflammatory drug, naproxen, as well as four other potential disease-modifying OA drugs. The data demonstrate that the miniJoint recapitulates complex tissue interactions, thus providing a robust organ chip model for the study of joint pathology and the development of novel therapeutic interventions.

    View details for DOI 10.1002/advs.202105909

    View details for PubMedID 35436042

  • Novel Techniques and Future Perspective for Investigating Critical-Size Bone Defects. Bioengineering (Basel, Switzerland) Huang, E. E., Zhang, N., Shen, H., Li, X., Maruyama, M., Utsunomiya, T., Gao, Q., Guzman, R. A., Goodman, S. B. 2022; 9 (4)


    A critical-size bone defect is a challenging clinical problem in which a gap between bone ends will not heal and will become a nonunion. The current treatment is to harvest and transplant an autologous bone graft to facilitate bone bridging. To develop less invasive but equally effective treatment options, one needs to first have a comprehensive understanding of the bone healing process. Therefore, it is imperative to leverage the most advanced technologies to elucidate the fundamental concepts of the bone healing process and develop innovative therapeutic strategies to bridge the nonunion gap. In this review, we first discuss the current animal models to study critical-size bone defects. Then, we focus on four novel analytic techniques and discuss their strengths and limitations. These four technologies are mass cytometry (CyTOF) for enhanced cellular analysis, imaging mass cytometry (IMC) for enhanced tissue special imaging, single-cell RNA sequencing (scRNA-seq) for detailed transcriptome analysis, and Luminex assays for comprehensive protein secretome analysis. With this new understanding of the healing of critical-size bone defects, novel methods of diagnosis and treatment will emerge.

    View details for DOI 10.3390/bioengineering9040171

    View details for PubMedID 35447731

  • AN INNERVATED SYNOVIUM-CARTILAGE CHIP FOR MODELING JOINT INFLAMMATION AND ASSOCIATED PAIN Li, Z., Makarcyzk, M. J., Moy, J. K., Yu, I., Liu, F., Gao, Q., Cho, S., Weber, D. J., Bunnell, B. A., Goodman, S. B., Gold, M. S., Lin, H. MARY ANN LIEBERT, INC. 2022: S505
  • Sex differences in the therapeutic effect of unaltered versus NFkappaB sensing IL-4 over-expressing mesenchymal stromal cells in a murine model of chronic inflammatory bone loss. Frontiers in bioengineering and biotechnology Shen, H., Kushioka, J., Toya, M., Utsunomiya, T., Hirata, H., Huang, E. E., Tsubosaka, M., Gao, Q., Li, X., Teissier, V., Zhang, N., Goodman, S. B. 2022; 10: 962114


    Wear particles from joint arthroplasties induce chronic inflammation associated with prolonged upregulation of nuclear factor kappa-B (NF-kappaB) signaling in macrophages and osteoclasts, which leads to osteolysis and implant loosening. Mesenchymal stromal cell (MSC)-based therapy showed great potential for immunomodulation and mitigation of osteolysis in vivo, especially in the chronic phase of inflammation. We previously generated genetically modified MSCs that secrete the anti-inflammatory cytokine interleukin 4 (IL-4) in response to NF-kappaB activation (NFkappaB-IL-4 MSCs). However, whether the impact of sexual difference in the internal environment can alter the therapeutic effects of IL-4 over-secreting MSCs that simultaneously mitigate prolonged inflammation and enhance bone formation remains unknown. This study investigated the therapeutic effects of unaltered MSCs versus NFkappaB-IL-4 MSCs in mitigating chronic inflammation and enhancing bone formation in male and female mice. The murine model was established by continuous infusion of polyethylene particles contaminated with lipopolysaccharide (cPE) into the medullary cavity of the distal femur for 6 weeks to induce chronic inflammation. Unaltered MSCs or NFkappaB-IL-4 MSCs were infused into the femoral intramedullary cavity in sex-matched groups beginning 3 weeks after primary surgery. Femurs were harvested at 6 weeks, and bone marrow density was measured with micro-computational tomography. Numbers of osteoclast-like cells, osteoblasts, and macrophages were evaluated with histochemical and immunofluorescence staining. cPE infusion resulted in severe bone loss at the surgery site, increased tartrate-resistant acid phosphatase positive osteoclasts and M1 pro-inflammatory macrophages, and decreased alkaline phosphatase expression. MSC-based therapy effectively decreased local bone loss and polarized M1 macrophages into an M2 anti-inflammatory phenotype. In females, unaltered MSCs demonstrated a larger impact in enhancing the osteogenesis, but they demonstrated similar anti-inflammatory effects compared to NFkappaB-IL-4 MSCs. These results demonstrated that local inflammatory bone loss can be effectively modulated via MSC-based treatments in a sexually dimorphic manner, which could be an efficacious therapeutic strategy for treatment of periprosthetic osteolysis in both genders.

    View details for DOI 10.3389/fbioe.2022.962114

    View details for PubMedID 36046680

  • Macrophages Modulate the Function of MSC- and iPSC-Derived Fibroblasts in the Presence of Polyethylene Particles. International journal of molecular sciences Gao, Q., Li, Z., Rhee, C., Xiang, S., Maruyama, M., Huang, E. E., Yao, Z., Bunnell, B. A., Tuan, R. S., Lin, H., Gold, M. S., Goodman, S. B. 2021; 22 (23)


    Fibroblasts in the synovial membrane secrete molecules essential to forming the extracellular matrix (ECM) and supporting joint homeostasis. While evidence suggests that fibroblasts contribute to the response to joint injury, the outcomes appear to be patient-specific and dependent on interactions between resident immune cells, particularly macrophages (Mphis). On the other hand, the response of Mphis to injury depends on their functional phenotype. The goal of these studies was to further explore these issues in an in vitro 3D microtissue model that simulates a pathophysiological disease-specific microenvironment. Two sources of fibroblasts were used to assess patient-specific influences: mesenchymal stem cell (MSC)- and induced pluripotent stem cell (iPSC)-derived fibroblasts. These were co-cultured with either M1 or M2 Mphis, and the cultures were challenged with polyethylene particles coated with lipopolysaccharide (cPE) to model wear debris generated from total joint arthroplasties. Our results indicated that the fibroblast response to cPE was dependent on the source of the fibroblasts and the presence of M1 or M2 Mphis: the fibroblast response as measured by gene expression changes was amplified by the presence of M2 Mphis. These results demonstrate that the immune system modulates the function of fibroblasts; furthermore, different sources of differentiated fibroblasts may lead to divergent results. Overall, our research suggests that M2 Mphis may be a critical target for the clinical treatment of cPE induced fibrosis.

    View details for DOI 10.3390/ijms222312837

    View details for PubMedID 34884641

  • Effect on Osteogenic Differentiation of Genetically Modified IL4 or PDGF-BB Over-Expressing and IL4-PDGF-BB Co-Over-Expressing Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro. Bioengineering (Basel, Switzerland) Tsubosaka, M., Maruyama, M., Huang, E. E., Zhang, N., Utsunomiya, T., Gao, Q., Shen, H., Li, X., Kushioka, J., Hirata, H., Yao, Z., Yang, Y. P., Goodman, S. B. 2021; 8 (11)


    The use of genetically modified (GM) mesenchymal stromal cells (MSCs) and preconditioned MSCs (pMSCs) may provide further opportunities to improve the outcome of core decompression (CD) for the treatment of early-stage osteonecrosis of the femoral head (ONFH). GM interleukin-4 (IL4) over-expressing MSCs (IL4-MSCs), platelet-derived growth factor (PDGF)-BB over-expressing MSCs (PDGF-BB-MSCs), and IL4-PDGF-BB co-over-expressing MSCs (IL4-PDGF-BB-MSCs) and their respective pMSCs were used in this in vitro study and compared with respect to cell proliferation and osteogenic differentiation. IL4-MSCs, PDGF-BB-MSCs, IL4-PDGF-BB-MSCs, and each pMSC treatment significantly increased cell proliferation compared to the MSC group alone. The percentage of Alizarin red-stained area in the IL4-MSC and IL4-pMSC groups was significantly lower than in the MSC group. However, the percentage of Alizarin red-stained area in the PDGF-BB-MSC group was significantly higher than in the MSC and PDGF-BB-pMSC groups. The percentage of Alizarin red-stained area in the IL4-PDGF-BB-pMSC was significantly higher than in the IL4-PDGF-BB-MSC group. There were no significant differences in the percentage of Alizarin red-stained area between the MSC and IL4-PDGF-BB-pMSC groups. The use of PDGF-BB-MSCs or IL4-PDGF-BB-pMSCs increased cell proliferation. Furthermore, PDGF-BB-MSCs promoted osteogenic differentiation. The addition of GM MSCs may provide a useful supplementary cell-based therapy to CD for treatment of ONFH.

    View details for DOI 10.3390/bioengineering8110165

    View details for PubMedID 34821731

  • The effect of genetically modified platelet-derived growth factor-BB over-expressing mesenchymal stromal cells during core decompression for steroid-associated osteonecrosis of the femoral head in rabbits. Stem cell research & therapy Guzman, R. A., Maruyama, M., Moeinzadeh, S., Lui, E., Zhang, N., Storaci, H. W., Tam, K., Huang, E. E., Utsunomiya, T., Rhee, C., Gao, Q., Yao, Z., Yang, Y. P., Goodman, S. B. 2021; 12 (1): 503


    BACKGROUND: Approximately one third of patients undergoing core decompression (CD) for early-stage osteonecrosis of the femoral head (ONFH) experience progression of the disease, and subsequently require total hip arthroplasty (THA). Thus, identifying adjunctive treatments to optimize bone regeneration during CD is an unmet clinical need. Platelet-derived growth factor (PDGF)-BB plays a central role in cell growth and differentiation. The aim of this study was to characterize mesenchymal stromal cells (MSCs) that were genetically modified to overexpress PDGF-BB (PDGF-BB-MSCs) in vitro and evaluate their therapeutic effect when injected into the bone tunnel at the time of CD in an in vivo rabbit model of steroid-associated ONFH.METHODS: In vitro studies: Rabbit MSCs were transduced with a lentivirus vector carrying the human PDGF-BB gene under the control of either the cytomegalovirus (CMV) or phosphoglycerate (PGK) promoter. The proliferative rate, PDGF-BB expression level, and osteogenic differentiation capacity of unmodified MSCs, CMV-PDGF-BB-MSCs, and PGK-PDGF-BB-MSCs were assessed. In vivo studies: Twenty-four male New Zealand white rabbits received an intramuscular (IM) injection of methylprednisolone 20mg/kg. Four weeks later, the rabbits were divided into four groups: the CD group, the hydrogel [HG, (a collagen-alginate mixture)] group, the MSC group, and the PGK-PDGF-BB-MSC group. Eight weeks later, the rabbits were sacrificed, their femurs were harvested, and microCT, mechanical testing, and histological analyses were performed.RESULTS: In vitro studies: PGK-PDGF-BB-MSCs proliferated more rapidly than unmodified MSCs (P<0.001) and CMV-PDGF-BB-MSCs (P<0.05) at days 3 and 7. CMV-PDGF-BB-MSCs demonstrated greater PDGF-BB expression than PGK-PDGF-BB-MSCs (P<0.01). However, PGK-PDGF-BB-MSCs exhibited greater alkaline phosphatase staining at 14days (P<0.01), and osteogenic differentiation at 28days (P=0.07) than CMV-PDGF-BB-MSCs. In vivo: The PGK-PDGF-BB-MSC group had a trend towards greater bone mineral density (BMD) than the CD group (P=0.074). The PGK-PDGF-BB-MSC group demonstrated significantly lower numbers of empty lacunae (P<0.001), greater osteoclast density (P<0.01), and greater angiogenesis (P<0.01) than the other treatment groups.CONCLUSION: The use of PGK-PDGF-BB-MSCs as an adjunctive treatment with CD may reduce progression of osteonecrosis and enhance bone regeneration and angiogenesis in the treatment of early-stage ONFH.

    View details for DOI 10.1186/s13287-021-02572-7

    View details for PubMedID 34526115

  • The efficacy of lapine preconditioned or genetically modified IL4 over-expressing bone marrow-derived mesenchymal stromal cells in corticosteroid-associated osteonecrosis of the femoral head in rabbits. Biomaterials Maruyama, M., Moeinzadeh, S., Guzman, R. A., Zhang, N., Storaci, H. W., Utsunomiya, T., Lui, E., Huang, E. E., Rhee, C., Gao, Q., Yao, Z., Takagi, M., Yang, Y. P., Goodman, S. B. 2021; 275: 120972


    Cell-based therapy for augmentation of core decompression (CD) using mesenchymal stromal cells (MSCs) is a promising treatment for early stage osteonecrosis of the femoral head (ONFH). Recently, the therapeutic potential for immunomodulation of osteogenesis using preconditioned (with pro-inflammatory cytokines) MSCs (pMSCs), or by the timely resolution of inflammation using MSCs that over-express anti-inflammatory cytokines has been described. Here, pMSCs exposed to tumor necrosis factor-alpha and lipopolysaccharide for 3 days accelerated osteogenic differentiation in vitro. Furthermore, injection of pMSCs encapsulated with injectable hydrogels into the bone tunnel facilitated angiogenesis and osteogenesis in the femoral head in vivo, using rabbit bone marrow-derived MSCs and a model of corticosteroid-associated ONFH in rabbits. In contrast, in vitro and in vivo studies demonstrated that genetically-modified MSCs that over-express IL4 (IL4-MSCs), established by using a lentiviral vector carrying the rabbit IL4 gene under the cytomegalovirus promoter, accelerated proliferation of MSCs and decreased the percentage of empty lacunae in the femoral head. Therefore, adjunctive cell-based therapy of CD using pMSCs and IL4-MSCs may hold promise to heal osteonecrotic lesions in the early stage ONFH. These interventions must be applied in a temporally sensitive fashion, without interfering with the mandatory acute inflammatory phase of bone healing.

    View details for DOI 10.1016/j.biomaterials.2021.120972

    View details for PubMedID 34186237

  • The Effects of Macrophage Phenotype on Osteogenic Differentiation of MSCs in the Presence of Polyethylene Particles BIOMEDICINES Gao, Q., Rhee, C., Maruyama, M., Li, Z., Shen, H., Zhang, N., Utsunomiya, T., Huang, E., Yao, Z., Bunnell, B. A., Lin, H., Tuan, R. S., Goodman, S. B. 2021; 9 (5)
  • Current Models for Development of Disease-Modifying Osteoarthritis Drugs. Tissue engineering. Part C, Methods Makarczyk, M. J., Gao, Q., He, Y., Li, Z., Gold, M. S., Hochberg, M., Bunnell, B., Tuan, R. S., Goodman, S. B., Lin, H. 2021


    Osteoarthritis (OA) is a painful and disabling disease that affects millions of people worldwide. Symptom-alleviating treatments exist, although none with long-term efficacy. Furthermore, there are currently no disease-modifying OA drugs (DMOADs) with demonstrated efficacy in OA patients, which is, in part, attributed to a lack of full understanding of the pathogenesis of OA. The inability to translate findings from basic research to clinical applications also highlights the deficiencies in the available OA models at simulating the clinically relevant pathologies and responses to treatments in humans. In this review, the current status in the development of DMOADs will be first presented, with special attention to those in Phase II-IV clinical trials. Next, current in vitro, ex vivo, and in vivo OA models are summarized and the respective advantages and disadvantages of each are highlighted. Notably, the development and application of microphysiological or tissue-on-a-chip systems for modeling OA in humans are presented and the issues that need to be addressed in the future are discussed. Microphysiological systems should be given serious consideration for their inclusion in the DMOAD development pipeline, both for their ability to predict drug safety and efficacy in human clinical trials at present, as well as for their potential to serve as a test platform for personalized medicine.

    View details for DOI 10.1089/ten.TEC.2020.0309

    View details for PubMedID 33403944

  • PDGF-BB and IL-4 co-overexpression is a potential strategy to enhance mesenchymal stem cell-based bone regeneration. Stem cell research & therapy Zhang, N. n., Lo, C. W., Utsunomiya, T. n., Maruyama, M. n., Huang, E. n., Rhee, C. n., Gao, Q. n., Yao, Z. n., Goodman, S. B. 2021; 12 (1): 40


    Mesenchymal stem cell (MSC)-based therapy has the potential for immunomodulation and enhancement of tissue regeneration. Genetically modified MSCs that over-express specific cytokines, growth factors, or chemokines have shown great promise in pre-clinical studies. In this regard, the anti-inflammatory cytokine interleukin (IL)-4 converts pro-inflammatory M1 macrophages into an anti-inflammatory M2 phenotype; M2 macrophages mitigate chronic inflammation and enhance osteogenesis by MSC lineage cells. However, exposure to IL-4 prematurely inhibits osteogenesis of MSCs in vitro; furthermore, IL-4 overexpressing MSCs inhibit osteogenesis in vivo during the acute inflammatory period. Platelet-derived growth factor (PDGF)-BB has been shown to enhance osteogenesis of MSCs with a dose-dependent effect.In this study, we generated a lentiviral vector that produces PDGF-BB under a weak promoter (phosphoglycerate kinase, PGK) and lentiviral vector producing IL-4 under a strong promoter (cytomegalovirus, CMV). We infected MSCs with PDGF-BB and IL-4-producing lentiviral vectors separately or in combination to investigate cell proliferation and viability, protein expression, and the capability for osteogenesis.PDGF-BB and IL-4 co-overexpression was observed in the co-infected MSCs and shown to enhance cell proliferation and viability, and osteogenesis compared to IL-4 overexpressing MSCs alone.Overexpression of PDGF-BB together with IL-4 mitigates the inhibitory effect of IL-4 on osteogenesis by IL-4 overexpressing MSCS. PDGF-BB and IL-4 overexpressing MSCs may be a potential strategy to facilitate osteogenesis in scenarios of both acute and chronic inflammation.

    View details for DOI 10.1186/s13287-020-02086-8

    View details for PubMedID 33413614