HBMP-7 Gene Modified Bone Marrow Stromal Cells On The Healing Of Periodontal Furcation Defects In Osteoporosis Rats

Objective:This study is to assess the potential effects of human bone morphogenetic protein-7 (hBMP-7) gene transfected bone marrow stromal cells (BMSCs) in periodontal regeneration of osteoporosis Sprague Dawley rats. hBMP-7 gene was transfected into BMSCs, which was then inplanted in a collagen membrane (BME-10X) and transplanted into periodontal fenestration defects of osteoporosis Sprague Dawley rats.Methods: 1 Osteoporosis models of the female Sprague Dawley rats were induced by ovariectomy. Eight weeks after the operation, the body weight, the bone mineral density (BMD), histological examination and serum levels of biochemical indices were investigated to confirm the osteoporosis models were successful. 2. BMSCs from osteoporosis rats and the sham control were isolated and cultured in DMEM. The morphology, growth feature and alkaline phosphatase (ALP) of BMSCs were monitored. Then BMSCs were transfected with recombinant AD5-hBMP7-EGFP virus and observed by fluorescence microscope. RT-PCR, immunohistochemical straining,FCM,ELISA, as well as Western Blot were employed to observe the transcription and expression of hBMP-7. 3. The morphology and growth feature of the transfected cells were detected and FCM were used to test the cell cycle. Alkaline phosphatase (ALP) and Von Kossa were tested to determine osteogenic property of osteoblast. 4. The hBMP-7 transfected BMSCs were cultured with BME-10X in vitro. Histological examination was performed with light microscopy and H.E stained. The adhesion situation was analyzed by fluorescence microscope, confocal laser scanning microscope and scanning electron microscope. 5. BMSCs with different treatment were seeded on the collagen membranes. The complex and blank collagen membranes without cells were implanted into the acute periodontal defects in osteoporosis rats, and then the defects were covered with e-PTFE membranes. Defects only covered with e-PTFE membranes were served as empty controls. Histometric measurements with H&E staining were performed after 4 weeks, the formation of new alveolar bone area, cementum area and the width of periodontal ligament were measured and compared.Results: 1. Eight weeks after ovariectomy, osteoporosis models of the female Sprague Dawley rats were successful according to the criteria. 2. The BMSCs were transfected with recombinant AD5-hBMP7-EGFP virus showed a successful transfection. Under the inverted-phase-contrast fluorescence microscope, cells emiting green fluorescence convinced the successful transfection and the transfection efficiency was over 70% by calculating the ratio of luminescent cells. Using RT-PCR, immunocytochemistry, ELISA and Western Blot, it was convinced that effective expression of hBMP-7 in all transfected cells. 3. Compared to the control group, morphological feature of the transfected cells showed little alterations, however, there were no significant differences of cell proliferation and apoptosis rate compared with the controls. ALP activity was increased significantly, as well as the size of mineralization nodule formation in transfected cells. 4. The transfected cells adhered to BME-10X and stretched well after 24h of culture. By tomographic scanning with confocal laser scanning confocal microscope, it was showed that cell and material intergrated together, cell attached to BME-10X with different amount. 5. Animal study showed various degrees of periodontal tissue regeneration in the experimental and control groups. The area of newly formed alveolar bone was significantly larger in all experimental groups compared with the empty control group (P<0.05). The area of new alveolar bone from the transfected BMSCs was significantly higher than that of the non-transfected BMSCs and the control (P<0.05). When the defect healing was compared between the OVX rats and SHAM rats, it was noted that there was no significant difference (P>0.05) in both transfected BMSCs and non-transfected BMSCs.Conclusion: 1. Osteoporosis models of the female Sprague Dawley rats can be made by ovariectomy. 2. The transfected BMSCs can highly express hBMP-7. The transfection of hBMP-7 gene promoted the differentiation of BMSCs to osteoblasts. BMSCs transfected by hBMP-7 gene may serve as an ideal cell source for periodontal tissue engineering. 3. The transfected cells adhered to BME-10X and stretched well. hBMP-7 gene enhanced periodontal defect healing, which shows a potential in clinical application.