All Publications

  • Effect of porosity of a functionally-graded scaffold for the treatment of corticosteroid-associated osteonecrosis of the femoral head in rabbits. Journal of orthopaedic translation Maruyama, M., Pan, C., Moeinzadeh, S., Storaci, H. W., Guzman, R. A., Lui, E., Ueno, M., Utsunomiya, T., Zhang, N., Rhee, C., Yao, Z., Takagi, M., Goodman, S. B., Yang, Y. P. 2021; 28: 90–99


    Background/Objective: Core decompression (CD) with scaffold and cell-based therapies is a promising strategy for providing both mechanical support and regeneration of the osteonecrotic area for early stage osteonecrosis of the femoral head (ONFH). We designed a new 3D printed porous functionally-graded scaffold (FGS) with a central channel to facilitate delivery of transplanted cells in a hydrogel to the osteonecrotic area. However, the optimal porous structural design for the FGS for the engineering of bone in ONFH has not been elucidated. The aim of this study was to fabricate and evaluate two different porous structures (30% or 60% porosity) of the FGSs in corticosteroid-associated ONFH in rabbits.Methods: Two different FGSs with 30% or 60% porosity containing a 1-mm central channel were 3D printed using polycaprolactone and beta-tricalcium phosphate. The FGS was 3-mm diameter and 32-mm length and was composed of three segments: 1-mm in length for the non-porous proximal segment, 22-mm in length for the porous (30% versus 60%) middle segment, and 9-mm in length for the 15% porous distal segment. Eighteen male New Zealand White rabbits were given a single dose of 20​mg/kg methylprednisolone acetate intramuscularly. Four weeks later, rabbits were divided into three groups: the CD group, the 30% porosity FGS group, and the 60% porosity FGS group. In the CD group, a 3-mm diameter drill hole was created into the left femoral head. In the FGS groups, a 30% or 60% porosity implant was inserted into the bone tunnel. Eight weeks postoperatively, femurs were harvested and microCT, mechanical, and histological analyses were performed.Results: The actual porosity and pore size of the middle segments were 26.4%​±​2.3% and 699​±​56​mum in the 30% porosity FGS, and 56.0%​±​4.5% and 999​±​71​mum in the 60% porosity FGS, respectively using microCT analysis. Bone ingrowth ratio in the 30% porosity FGS group was 73.9%​±​15.8%, which was significantly higher than 39.5%​±​13.0% in the CD group on microCT (p​<​0.05). Bone ingrowth ratio in the 60% porosity FGS group (61.3%​±​30.1%) showed no significant differences compared to the other two groups. The stiffness at the bone tunnel site in the 30% porosity FGS group was 582.4​±​192.3​N/mm3, which was significantly higher than 338.7​±​164.6​N/mm3 in the 60% porosity FGS group during push-out testing (p​<​0.05). Hematoxylin and eosin staining exhibited thick and mature trabecular bone around the porous FGS in the 30% porosity FGS group, whereas thinner, more immature trabecular bone was seen around the porous FGS in the 60% porosity FGS group.Conclusion: These findings indicate that the 30% porosity FGS may enhance bone regeneration and have superior biomechanical properties in the bone tunnel after CD in ONFH, compared to the 60% porosity FGS.Translation potential statement: The translational potential of this article: This FGS implant holds promise for improving outcomes of CD for early stage ONFH.

    View details for DOI 10.1016/

    View details for PubMedID 33816112

  • Suppression of NF-kappaB-induced chronic inflammation mitigates inflammatory osteolysis in the murine continuous polyethylene particle infusion model. Journal of biomedical materials research. Part A Utsunomiya, T., Zhang, N., Lin, T., Kohno, Y., Ueno, M., Maruyama, M., Huang, E., Rhee, C., Yao, Z., Goodman, S. B. 2021


    Wear particle-associated bone loss (periprosthetic osteolysis) constrains the longevity of total joint arthroplasty (TJA). Wear particles induce a prolonged upregulation of nuclear factor kappa B (NF-kappaB) signaling in macrophages and osteoclasts. Synthetic double-stranded oligodeoxynucleotides (ODNs) can prevent the binding of NF-kappaB to the promoter regions of targeted genes and inhibit genetic activation. We tested the hypothesis that polyethylene-particle induced chronic inflammatory bone loss could be suppressed by local delivery of NF-kappaB decoy ODNs in murine in vivo model. Polyethylene particles were continuously infused into the medullary cavity of the distal femur for 6weeks to induce chronic inflammation, and micro-computational tomography and immunohistochemical analysis were performed. Particle-induced chronic inflammation resulted in lower BMD values, an increase in osteoclastogenesis and nuclear translocation of p65, a prolonged M1 pro-inflammatory macrophage phenotype, and a decrease of M2 anti-inflammatory macrophage phenotype. Delayed timing of local infusion of NF-kappaB decoy ODN for the last 3weeks reversed polyethylene-particle associated chronic inflammatory bone loss and facilitated bone healing. This study demonstrated that polyethylene-particle associated chronic inflammatory osteolysis can be effectively modulated via interference with the NF-kappaB pathway; this minimally invasive intervention could potentially be an efficacious therapeutic strategy for periprosthetic osteolysis after TJA.

    View details for DOI 10.1002/jbm.a.37175

    View details for PubMedID 33779115

  • 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

  • Different Effects of Intramedullary Injection of Mesenchymal Stem Cells During the Acute vs. Chronic Inflammatory Phase on Bone Healing in the Murine Continuous Polyethylene Particle Infusion Model. Frontiers in cell and developmental biology Utsunomiya, T., Zhang, N., Lin, T., Kohno, Y., Ueno, M., Maruyama, M., Rhee, C., Huang, E., Yao, Z., Goodman, S. B. 2021; 9: 631063


    Chronic inflammation is a common feature in many diseases of different organ systems, including bone. However, there are few interventions to mitigate chronic inflammation and preserve host tissue. Previous in vitro studies demonstrated that preconditioning of mesenchymal stem cells (pMSCs) using lipopolysaccharide and tumor necrosis factor-alpha polarized macrophages from a pro-inflammatory to an anti-inflammatory phenotype and increased osteogenesis compared to unaltered MSCs. In the current study, we investigated the local injection of MSCs or pMSCs during the acute versus chronic inflammatory phase in a murine model of inflammation of bone: the continuous femoral intramedullary polyethylene particle infusion model. Chronic inflammation due to contaminated polyethylene particles decreased bone mineral density and increased osteoclast-like cells positively stained with leukocyte tartrate resistant acid phosphatase (TRAP) staining, and resulted in a sustained M1 pro-inflammatory macrophage phenotype and a decreased M2 anti-inflammatory phenotype. Local injection of MSCs or pMSCs during the chronic inflammatory phase reversed these findings. Conversely, immediate local injection of pMSCs during the acute inflammatory phase impaired bone healing, probably by mitigating the mandatory acute inflammatory reaction. These results suggest that the timing of interventions to facilitate bone healing by modulating inflammation is critical to the outcome. Interventions to facilitate bone healing by modulating acute inflammation should be prudently applied, as this phase of bone healing is temporally sensitive. Alternatively, local injection of MSCs or pMSCs during the chronic inflammatory phase may be a potential intervention to mitigate the adverse effects of contaminated particles on bone.

    View details for DOI 10.3389/fcell.2021.631063

    View details for PubMedID 33816480

  • Modulation of the Inflammatory Response and Bone Healing. Frontiers in endocrinology Maruyama, M., Rhee, C., Utsunomiya, T., Zhang, N., Ueno, M., Yao, Z., Goodman, S. B. 2020; 11: 386


    The optimal treatment for complex fractures and large bone defects is an important unsolved issue in orthopedics and related specialties. Approximately 5-10% of fractures fail to heal and develop non-unions. Bone healing can be characterized by three partially overlapping phases: the inflammatory phase, the repair phase, and the remodeling phase. Eventual healing is highly dependent on the initial inflammatory phase, which is affected by both the local and systemic responses to the injurious stimulus. Furthermore, immune cells and mesenchymal stromal cells (MSCs) participate in critical inter-cellular communication or crosstalk to modulate bone healing. Deficiencies in this inter-cellular exchange, inhibition of the natural processes of acute inflammation, and its resolution, or chronic inflammation due to a persistent adverse stimulus can lead to impaired fracture healing. Thus, an initial and optimal transient stage of acute inflammation is one of the key factors for successful, robust bone healing. Recent studies demonstrated the therapeutic potential of immunomodulation for bone healing by the preconditioning of MSCs to empower their immunosuppressive properties. Preconditioned MSCs (also known as "primed/ licensed/ activated" MSCs) are cultured first with pro-inflammatory cytokines (e.g., TNFα and IL17A) or exposed to hypoxic conditions to mimic the inflammatory environment prior to their intended application. Another approach of immunomodulation for bone healing is the resolution of inflammation with anti-inflammatory cytokines such as IL4, IL10, and IL13. In this review, we summarize the principles of inflammation and bone healing and provide an update on cellular interactions and immunomodulation for optimal bone healing.

    View details for DOI 10.3389/fendo.2020.00386

    View details for PubMedID 32655495

    View details for PubMedCentralID PMC7325942

  • Modulation of the Inflammatory Response and Bone Healing FRONTIERS IN ENDOCRINOLOGY Maruyama, M., Rhee, C., Utsunomiya, T., Zhang, N., Ueno, M., Yao, Z., Goodman, S. B. 2020; 11
  • Macrophage Effects on Mesenchymal Stem Cell Osteogenesis in a Three-Dimensional in vitro Bone Model. Tissue engineering. Part A Romero-Lopez, M., Li, Z., Rhee, C., Maruyama, M., Pajarinen, J., O'Donnell, B., Lin, T., Lo, C., Hanlon, J., Dubowitz, R., Yao, Z., Bunnell, B. A., Lin, H., Tuan, R., Goodman, S. B. 2020


    As musculoskeletal disorders continue to increase globally, there is an increased need for novel, in vitro models to efficiently study human bone physiology in the context of both healthy and diseased conditions. For these models, the inclusion of innate immune cells is critical. Specifically, signaling factors generated from macrophages play key roles in the pathogenesis of many musculoskeletal processes and diseases, including fracture, osteoarthritis, infection, etc. In this study, we aim to engineer three-dimensional (3D) and macrophage-encapsulated bone tissues in vitro, to model cell behavior, signaling, and other biological activities in vivo, in comparison to current two-dimensional (2D) models. We first investigated and optimized 3D culture conditions for macrophages, and then co-cultured macrophages with mesenchymal stem cells (MSCs) which were induced to undergo osteogenic differentiation to examine the effect of macrophage on new bone formation. Seeded within a 3D hydrogel scaffold fabricated from photocrosslinked methacrylated gelatin, macrophages maintained high viability and were polarized toward an M1 or M2 phenotype. In co-cultures of macrophages and human MSCs, MSCs displayed immunomodulatory activities by suppressing M1 and enhancing M2 macrophage phenotypes. Lastly, addition of macrophages, regardless of polarization state, increased MSC osteogenic differentiation, compared to MSCs alone, with pro-inflammatory M1 macrophages enhancing new bone formation most effectively. In summary, this study illustrates the important roles that macrophage signaling and inflammation play in bone tissue formation.

    View details for DOI 10.1089/ten.TEA.2020.0041

    View details for PubMedID 32312178

  • IL-4 Overexpressing Mesenchymal Stem Cells within Gelatin-Based Microribbon Hydrogels Enhance Bone Healing in a Murine Long Bone Critical-size Defect Model. Journal of biomedical materials research. Part A Ueno, M. n., Lo, C. W., Barati, D. n., Conrad, B. n., Lin, T. n., Kohno, Y. n., Utsunomiya, T. n., Zhang, N. n., Maruyama, M. n., Rhee, C. n., Huang, E. n., Romero-Lopez, M. n., Tong, X. n., Yao, Z. n., Zwingenberger, S. n., Yang, F. n., Goodman, S. B. 2020


    Mesenchymal stem cell (MSC)-based therapy is a promising strategy for bone repair. Furthermore, the innate immune system, and specifically macrophages, play a crucial role in the differentiation and activation of MSCs. The anti-inflammatory cytokine IL-4 converts pro-inflammatory M1 macrophages into a tissue regenerative M2 phenotype, which enhances MSC differentiation and function. We developed lentivirus-transduced IL-4 over-expressing MSCs (IL-4 MSCs) that continuously produce IL-4 and polarize macrophages toward an M2 phenotype. In the current study, we investigated the potential of IL-4 MSCs delivered using a macroporous gelatin-based microribbon (μRB) scaffold for healing of critical size long bone defects in Mice. IL-4 MSCs within μRBs enhanced M2 marker expression without inhibiting M1 marker expression in the early phase, and increased macrophage migration into the scaffold. Six weeks after establishing the bone defect, IL-4 MSCs within μRBs enhanced bone formation and helped bridge the long bone defect. IL-4 MSCs delivered using macroporous μRB scaffold is potentially a valuable strategy for the treatment of critical size long bone defects. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/jbm.a.36982

    View details for PubMedID 32363683

  • Optimization and characterization of calcium phosphate transfection in mesenchymal stem cells. Tissue engineering. Part C, Methods Lo, C. W., Lin, T. H., Ueno, M. n., Romero-Lopez, M. n., Maruyama, M. n., Kohno, Y. n., Rhee, C. n., Yao, Z. n., Pérez-Cruz, M. n., Meyer, E. n., Goodman, S. B. 2019


    Mesenchymal stem cells (MSCs) have been used as a therapy to modulate diverse biological processes. To fulfill the requirements for different MSC therapies, safe and effective gene transfer methods for MSCs are critical. Calcium phosphate transfection is an inexpensive and well-described method without discernible biosafety issues; however, an optimal protocol has not been developed for MSCs. In this report, we optimized the protocol of calcium phosphate transfection for murine MSCs, and compared this protocol with other gene transfer methods in different strains of mice and in human cells. We found that transfection efficiency and cell viability showed an inverse relationship depending on serum concentration during the process of calcium phosphate transfection, in which 2% serum was chosen in the optimized protocol. The optimized protocol of calcium phosphate transfection showed a fine balance between efficiency (about 70-80%) and viability (doubling original cell number) compared to other methods. Human MSCs were more resistant to this protocol (about 30% efficiency) compared with murine MSCs. Moreover, MSC potential for osteogenesis, adipogenesis, and chondrogenesis was not affected by calcium phosphate transfection. Finally, MSCs transfected with the luciferase gene were injected into the murine distal femoral bone marrow cavity to monitor gene expression overtime in vivo. MSCs in the bone marrow environment showed extended expression of the luciferase that was transfected by calcium phosphate. This report provides an optimized protocol for calcium phosphate transfection for murine MSCs and characterizes gene over-expression in MSCs in the in vitro and in vivo environments.

    View details for DOI 10.1089/ten.TEC.2019.0147

    View details for PubMedID 31441373