| Objective Human bone morphogenetic protein-7(hBMP-7) gene was transfected into bone marrow stromal cells (BMSCs). The transfected BMSCs with collagen membrane(BME-10X) were used to construct tissue-engineered compound in vitro to repair the canine experimental classâ…¡furcation defects. This study is to estimate the potential effects of hBMP-7 gene transfected BMSCs in periodontal regenetion and to provide a new way for restoring the periodontal defects.Methods 1. The hBMP-7 cDNA was inserted into the eukaryotic expression vector pIRES2-EGFP by genetic engineering. The recombinant plasmid was identified by restriction enzymolysis(EcoRâ… ), PCR analysis and DNA sequencing. 2. BMSCs obtained from Beagle dog were cultured and transfected with pIRES2-EGFP-hBMP-7 by liposome-mediated gene transfer method. RT-PCR, immunohistochemical straining as well as ELISA were employed to observe the transcription and expression of hBMP-7. 3. Morphological and growth feature of the transfected cells were valued. TUNEL and FCM were used to test the apoptosis. Alkaline phosphatase (ALP), osteocalcin(OC) and Von Kossa were tested to determine the phenotypes of osteoblast. 4. The hBMP-7 transfected BMSCs were cultured with BME-10X in vitro. Histological examination was performed with light microscopy, H.E stained and transmission electron microscope. The adhesion situation was analyzed by fluorescence microscope, confocal laser scanning microscope and scanning electron microscope. 5. A total of 30 esperimental classâ…¡furcation defects in five Beagle dogs were created surgically. The transfected BMSCs and non-transfected BMSCs were seeded in collagen membrane at initial concentration of 1×107/mL and transplanted into experimental classâ…¡furcation defects. The animals were sacrificed at 12 weeks postsurgery and periodontal regeneration was evaluated by histological and morphometric analysis.Results 1. The hBMP-7 gene was cloned into the vector successfully. 2. The hBMP-7 gene was transfected into BMSCs successfully. The transfected BMSCs expressing green fluorescene protein were observed under fluorescence microscope with the transfer efficiency of 17%-31%. RT-PCR, immunohistochemical straining and ELISA showed the expression of hBMP-7 gene. 3. Compared to the control group, morphological feature of the transfected cells changed a little, but there were no significant differences of cell proliferation and apoptosis rate. ALP activity and the expression of OC were increased significantly, as well as the size of calcium nodules. 4. The transfected cells adhered to BME-10X and stretched well after 24h of culture. Investigation under transmission electron microscope found that cell and material grew together, forming structure like semi-desmosome. 5. In both the experimental groups and the control group, there were different extents of regeneration. The percentage of new alveolar area in the transfected BMSCs and non-transfected BMSCs were significantly higher than the control(P<0.05), and there were significantly difference between the two experimental groups too(P<0.05). The percentage of new cementum length in the two experimental groups were significantly higher than the control too(P<0.05), but there were no significantly difference between the two BMSCs groups(P>0.05).Conclusion 1. pIRES2-EGFP-hBMP-7 eukaryotic expression plasmid, which contained the full length sequence of the hBMP-7 gene could be constructed successfully. 2. pIRES2-EGFP-hBMP-7 was transfected into BMSCs successfully. The transfected BMSCs can highly express hBMP-7. 3. 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. 4. The BME-10X has a good biocompatibility with hBMP-7 transfected BMSCs. It shows that the compound can be used in periodontal tissue engineering. 5. hBMP-7 gene enhanced tissue engineering could be a better way to repair the periodontal defects.
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