Allogeneic Bone Marrow Mesenchymal Stem Cell Therapy For Bisphosphonate-related Osteonecrosis Of The Jaw In Miniature Pigs

Bisphosphonates (BPs), analogues of naturally occurring pyrophosphate compound, are widely used in skeletal-related diseases, including osteoporosis, Paget disease and bone complications in cancer patients. Increasing evidence indicates that BP treatment results in jawbone necrosis (Bisphosphonate-Related Osteonecrosis of the Jaws, BRONJ). Although there are many clinical and basic studies performed on BRONJ, the pathogenesis still remains unknown, and neither is there an effective therapeutic method. In present study, we first generated a larger animal model of BRONJ in miniature pigs, during which we observed the dynamic biological and pathological changes to approach the possible pathogensis of BRONJ. We next transplanted allogeneic bone marrow mesenchymal stem cells into the diseased animals for treatment, which may have potential clinical applications for treating BRONJ.Part1Generation of BRONJ animal model in miniature pigsObjective:To develop a reliable animal model of BRONJ in miniature pigs for further study in possible pathogenesis and effective treatment for the disease.Methods:Eight minipigs were intravenously injected with4mg zolendronic acid (ZA) every2weeks. A total of10doses of ZA were administered. The first mandibular premolars were extracted after ZA injection. The BRONJ was diagnosed by gross observation, CT scanning and histopathological examination.Results:Four weeks after tooth extraction, the clinical examination revealed incomplete mucosal healing in BP-injected pigs. Eight weeks after tooth extraction, BPs treated minipigs had exposed necrotic bone in the oral cavity that persisted until treatment was performed. CT scan as well as histopathological analysis showed the presence of BRONJ.Conclusion:A stable BRONJ animal model can be induced by ZA intravenous injection and tooth extraction in miniature pigs.Part2Biological and immunological properties of BMMSCs and immune functions were impaired in BP-treated minipigsObjective:To investigate the functional significance of BPs on biological and immunological properties of BMMSCs and immune function in minipigs so as to approach the possible pathogenesis of BRONJ.Methods:Porcine BMMSCs were isolated and cultured. Proliferation, immunoregulatory activities and multi-lineage differentiation of BMMSCs were investigated. Blood samples from the precaval vein were obtained and flow cytometric staining analysis was performed to detect levels of CD4+CD25+T cells and γδT cells. Foxp3and IL-17expression were detected by Q-PCR.Results:BMMSCs from only2of the8animals in BP-treated group were successfully isolated and cultured. The results revealed a decreased proliferation of cells, impaired immunoregulatory activity and suppressed adipose and osteogenic potential. Pigs treated with ZA showed a significant decrease level in CD4+CD25+T cells and Foxp3mRNA in peripheral blood. In contrast, the levels of IL-17mRNA and γδT cells in peripheral blood increased.Conclusion:The biological and immunological properties of BMMSCs were impaired in the BP-treated minipigs. Moreover, the ratio of Foxp3-positive regulatory T cells (Tregs) in peripheral blood decreased, and IL-17increased in the serum of BP-treated minipigs. Thus, the impaired BMMSCs function and immune function may contribute to the pathogenesis of BRONJ.Part3BMMSCs transplantation for treating BRONJ in minipigsObjective:To investigate the therapeutic effect of BMMSCs on BRONJ in minipigs.Methods:8weeks after necrotic bone exposure, the BRONJ animals were divided into BMMSC group treated with allogeneic BMMSCs intravenous injection and control group treated with normal saline. Gross and histological observation, CT examination were used to evaluate the therapeutic effect on BRONJ. Impaired immune functions of BRONJ minipigs were also evaluated after BMMSCs treatment.Results: Twelve weeks after the BMMSC treatment, the open alveolar sockets of all five minipigs with BRONJ healed with complete soft tissue coverage. CT examination revealed healing of the bony defects. Histological analysis showed new bone formation in previously necrotic area.After BMMSC transplantation, the level of CD4+CD25+T cells and Foxp3expression increased in the BMMSC group. IL-17and γδT cell levels were declined after BMMSC treatment. Moreover, the expression of ALP and TRAP increased12months after BMMSC treatment, whereas the level of IFN-γ and IL-6decreased.Conclusion:BRONJ in minipigs was clinically cured by treatment with BMMSC infusions, which may have potential clinical applications for treating BRONJ.