All Publications


  • 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

    Abstract

    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., Lo, C. W., Barati, D., Conrad, B., Lin, T., Kohno, Y., Utsunomiya, T., Zhang, N., Maruyama, M., Rhee, C., Huang, E., Romero-Lopez, M., Tong, X., Yao, Z., Zwingenberger, S., Yang, F., Goodman, S. B. 2020

    Abstract

    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., Romero-Lopez, M., Maruyama, M., Kohno, Y., Rhee, C., Yao, Z., Pérez-Cruz, M., Meyer, E., Goodman, S. B. 2019

    Abstract

    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