Professional Education


  • Master of Medicine, Suzhou University (2020)
  • Doctor of Medicine, Xuzhou Medical College (2014)
  • Master of Science, Soochow University, Orthopaedic surgery (2020)
  • Doctor of Medicine, XuZhou Medical University, Medicine (2014)

Stanford Advisors


All Publications


  • Ageing attenuates bone healing by mesenchymal stem cellsin a microribbon hydrogelwith a murine long bone critical-size defect model. Immunity & ageing : I & A Hirata, H., Zhang, N., Ueno, M., Barati, D., Kushioka, J., Shen, H., Tsubosaka, M., Toya, M., Lin, T., Huang, E., Yao, Z., Wu, J. Y., Zwingenberger, S., Yang, F., Goodman, S. B. 2022; 19 (1): 14

    Abstract

    BACKGROUND: Despite the high incidence of fractures and pseudoarthrosis in the aged population, a potential role for the use of mesenchymal stem cells (MSCs) in the treatment of bone defects in elderly patients has not been elucidated. Inflammation and the innate immune system, including macrophages, play crucial roles in the differentiation and activation of MSCs. We have developed lentivirus-transduced interleukin 4 (IL4) over-expressing MSCs (IL4-MSCs) to polarize macrophages to an M2 phenotype to promote bone healing in an established young murine critical size bone defect model. In the current study, we explore the potential of IL4-MSCs in aged mice.METHODS: A 2mm femoral diaphyseal bone defect was created and fixed with an external fixation device in 15- to 17-month-old male and female BALB/c mice. Microribbon (RB) scaffolds (Sc) with or without encapsulation of MSCs were implanted in the defect sites. Accordingly, the mice were divided into three treatment groups: Sc-only, Sc+MSCs, and Sc+IL4-MSCs. Mice were euthanized six weeks after the surgery; subsequently, MicroCT (CT), histochemical and immunohistochemical analyses were performed.RESULTS: CT analysis revealed that bone formation was markedly enhanced in the IL4-MSC group. Compared with the Sc-only, the amount of new bone increased in the Sc+MSCs and Sc+IL4-MSC groups. However, no bridging of bone was observed in all groups. H&E staining showed fibrous tissue within the defect in all groups. Alkaline phosphatase (ALP) staining was increased in the Sc+IL4-MSC group. The Sc+IL4-MSCs group showed a decrease in the number of M1 macrophages and an increase in the number of M2 macrophages, with a significant increase in the M2/M1 ratio.DISCUSSION: IL4 promotes macrophage polarization to an M2 phenotype, facilitating osteogenesis and vasculogenesis. The addition of IL4-MSCs in the RB scaffold polarized macrophages to an M2 phenotype and increased bone formation; however, complete bone bridging was not observed in any specimens. These results suggest that IL4-MSCs are insufficient to heal a critical size bone defect in aged mice, as opposed to younger animals. Additional therapeutic strategies are needed in this challenging clinical scenario.

    View details for DOI 10.1186/s12979-022-00272-1

    View details for PubMedID 35279175

  • Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice. Journal of visualized experiments : JoVE Gaffney, C. M., Muwanga, G., Shen, H., Tawfik, V. L., Shepherd, A. J. 2022

    Abstract

    Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

    View details for DOI 10.3791/63454

    View details for PubMedID 35253785

  • 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)

    Abstract

    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 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. E., Yao, Z., Bunnell, B. A., Lin, H., Tuan, R. S., Goodman, S. B. 2021; 9 (5)

    Abstract

    Wear debris generated from the bearing surfaces of joint arthroplasties leads to acute and chronic inflammation, which is strongly associated with implant failure. Macrophages derived from monocytes recruited to the local tissues have a significant impact on bone healing and regeneration. Macrophages can adopt various functional phenotypes. While M1 macrophages are pro-inflammatory, M2 macrophages express factors important for tissue repair. Here, we established a 3D co-culture system to investigate how the immune system influences the osteogenic differentiation of mesenchymal stem cells (MSCs) in the presence of micron-sized particles. This system allowed for the simulation of an inflammatory reaction via the addition of Lipopolysaccharide-contaminated polyethylene particles (cPE) and the characterization of bone formation using micro-CT and gene and protein expression. Co-cultures of MSCs with M2 macrophages in the presence of cPE in a 3D environment resulted in the increased expression of osteogenic markers, suggesting facilitation of bone formation. In this model, the upregulation of M2 macrophage expression of immune-associated genes and cytokines contributes to enhanced bone formation by MSCs. This study elucidates how the immune system modulates bone healing in response to an inflammatory stimulus using a unique 3D culture system.

    View details for DOI 10.3390/biomedicines9050499

    View details for PubMedID 34062822

  • Temporal contribution of myeloid-lineage TLR4 to the transition to chronic pain: A focus on sex differences. The Journal of neuroscience : the official journal of the Society for Neuroscience Huck, N. A., Siliezar-Doyle, J., Haight, E. S., Ishida, R., Forman, T. E., Wu, S., Shen, H., Takemura, Y., Clark, J. D., Tawfik, V. L. 2021

    Abstract

    Complex regional pain syndrome (CRPS) is a chronic pain disorder with a clear acute-to-chronic transition. Preclinical studies demonstrate that toll-like receptor 4 (TLR4), expressed by myeloid-lineage cells, astrocytes, and neurons, mediates a sex-dependent transition to chronic pain; however, evidence is lacking on which exact TLR4-expressing cells are responsible. We used complementary pharmacologic and transgenic approaches in mice to more specifically manipulate myeloid-lineage TLR4 and outline its contribution to the transition from acute-to-chronic CRPS based on three key variables: location (peripheral vs. central), timing (prevention vs. treatment), and sex (male vs. female). We demonstrate that systemic TLR4 antagonism is more effective at improving chronic allodynia trajectory when administered at the time of injury (early) in the tibial fracture model of CRPS in both sexes. In order to clarify the contribution of myeloid-lineage cells peripherally (macrophages) or centrally (microglia), we rigorously characterize a novel spatiotemporal transgenic mouse line, Cx3CR1-CreERT2-eYFP;TLR4fl/fl (TLR4 cKO) to specifically knock-out TLR4 only in microglia and no other myeloid-lineage cells. Using this transgenic mouse, we find that early TLR4 cKO results in profound improvement in chronic, but not acute, allodynia in males, with a significant but less robust effect in females. In contrast, late TLR4 cKO results in partial improvement in allodynia in both sexes, suggesting that downstream cellular or molecular TLR4-independent events may have already been triggered. Overall, we find that the contribution of TLR4 is time- and microglia-dependent in both sexes; however, females also rely on peripheral myeloid-lineage (or other TLR4 expressing) cells to trigger chronic pain.Significance statementThe contribution of myeloid cell TLR4 to sex-specific pain progression remains controversial. We used complementary pharmacologic and transgenic approaches to specifically manipulate TLR4 based on three key variables: location (peripheral vs. central), timing (prevention vs. treatment), and sex (male vs. female). We discovered that microglial TLR4 contributes to early pain progression in males, and to a lesser extent in females. We further found that maintenance of chronic pain likely occurs through myeloid TLR4-independent mechanisms in both sexes. Altogether we define a more nuanced contribution of this receptor to the acute-to-chronic pain transition in a mouse model of complex regional pain syndrome.

    View details for DOI 10.1523/JNEUROSCI.1940-20.2021

    View details for PubMedID 33846230

  • Sex Differences in Mesenchymal Stem Cell Therapy With Gelatin-Based Microribbon Hydrogels in a Murine Long Bone Critical-Size Defect Model. Frontiers in bioengineering and biotechnology Ueno, M., Zhang, N., Hirata, H., Barati, D., Utsunomiya, T., Shen, H., Lin, T., Maruyama, M., Huang, E., Yao, Z., Wu, J. Y., Zwingenberger, S., Yang, F., Goodman, S. B. 2021; 9: 755964

    Abstract

    Mesenchymal stem cell (MSC)-based therapy and novel biomaterials are promising strategies for healing of long bone critical size defects. Interleukin-4 (IL-4) over-expressing MSCs within a gelatin microribbon (RB) scaffold was previously shown to enhance the bridging of bone within a critical size femoral bone defect in male Balb/c mice. Whether sex differences affect the healing of this bone defect in conjunction with different treatments is unknown. In this study, we generated 2-mm critical-sized femoral diaphyseal bone defects in 10-12-week-old female and male Balb/c mice. Scaffolds without cells and with unmodified MSCs were implanted immediately after the primary surgery that created the bone defect; scaffolds with IL-4 over-expressing MSCs were implanted 3days after the primary surgery, to avoid the adverse effects of IL-4 on the initial inflammatory phase of fracture healing. Mice were euthanized 6weeks after the primary surgery and femurs were collected. MicroCT (CT), histochemical and immunohistochemical analyses were subsequently performed of the defect site. RB scaffolds with IL-4 over-expressing MSCs enhanced bone healing in both female and male mice. Male mice showed higher measures of bone bridging and increased alkaline phosphatase (ALP) positive areas, total macrophages and M2 macrophages compared with female mice after receiving scaffolds with IL-4 over-expressing MSCs. Female mice showed higher Tartrate-Resistant Acid Phosphatase (TRAP) positive osteoclast numbers compared with male mice. These results demonstrated that sex differences should be considered during the application of MSC-based studies of bone healing.

    View details for DOI 10.3389/fbioe.2021.755964

    View details for PubMedID 34738008

  • Modeling Complex Orthopedic Trauma in Rodents: Bone, Muscle and Nerve Injury and Healing. Frontiers in pharmacology Shen, H., Gardner, A. M., Vyas, J., Ishida, R., Tawfik, V. L. 2020; 11: 620485

    Abstract

    Orthopedic injury can occur from a variety of causes including motor vehicle collision, battlefield injuries or even falls from standing. Persistent limb pain is common after orthopedic injury or surgery and presents a unique challenge, as the initiating event may result in polytrauma to bone, muscle, and peripheral nerves. It is imperative that we understand the tissue-specific and multicellular response to this unique type of injury in order to best develop targeted treatments that improve healing and regeneration. In this Mini Review we will first discuss current rodent models of orthopedic trauma/complex orthotrauma. In the second section, we will focus on bone-specific outcomes including imaging modalities, biomechanical testing and immunostaining for markers of bone healing/turnover. In the third section, we will discuss muscle-related pathology including outcome measures of fibrosis, muscle regeneration and tensile strength measurements. In the fourth section, we will discuss nervous system-related pathology including outcome measures of pain-like responses, both reflexive and non-reflexive. In all sections we will consider parallels between preclinical outcome measures and the functional and mechanistic findings of the human condition.

    View details for DOI 10.3389/fphar.2020.620485

    View details for PubMedID 33597884