| Objective To investigate the effects of bFGF gene modified GFs alone or together with BMSCs modified by hBMP-7 gene on periodontal regeneration and to provide a new approach to repair the periodontal defects.Methods①Complete sequence of human bFGF gene was cloned into pIRES2-EGFP with recombination DNA technique, named pIRES2-EGFP-bFGF. The recombinant plasmid was identified by restriction enzymolysis(HindⅢ) and DNA sequencing.②Recombinant plasmid pIRES2-EGFP-bFGF and vector pIRES2-EGFP were transfected into GFs of Beagle dogs respectively. Expression of bFGF in GFs was identified by RT–PCR, ELISA and immunohistochemistry.③Proliferation and apoptosis feature of the transfected cells were evaluated by MTT and AO/EB.The activity of cell alkaline phosphatase(ALP) was assayed to evaluated the ability of bone formation.④GFs expressed hbFGF gene were cultured with ADM in vitro. Histological examination was performed with light microscopy. Adhesion situation was analyzed by fluorescence microscope, confocal laser scanning microscope and transmission electron microscope.⑤A total 36 experimental classⅡfurcation defects were created surgically in six Beagle dogs, and randomly divided into two groups under therapy of six weeks and twelve weeks. Each group was divided into three subgroups, named BMP-7, BMP-7/GFs and BMP-7/hbFGF. 6wk or 12wk later, CAL(clinical attachment level), KG(width of keratinized gingival) and content of NO in gingival crevicular fluid were detected. After animals were sacrificed, periodontal regeneration was evaluated by histological and morphmetric analysis to calculate the percentage of new alveolar area and new cementum length.Results①Human bFGF gene was inserted into vector pIRES2-EGFP correctly.②24hr after transfection, green fluorescence can be detected in GFs, the efficiency of transfection was nearly 15-25%. RT-PCR, ELISA and immunohistochemistry showed that bFGF was only expressed in GFs which transfected pIRES2-EGFP-bFGF. Transfected GFs had some morphological changes, and the proliferation was promoted, while apoptosis was impressed (P< 0.05). ALP activity remained unchanged (P>0.05).④The transfected cells adhered to ADM and stretched well after 24h of culture. Investigation under transmission electron microscope found that cells and material grew together, forming semi-desmosome like structure.⑤Improvement of CAL was observed in each group. Although there was no difference between each subgroup on 6wk (P>0.05), CAL were significantly improved in BMP-7/GFs and BMP-7/hbFGF on 12wk, comparing with BMP-7 subgroup (P<0.05). In all groups, there were different extent of KG recoveries. BMP-7/hbFGF subgroup surpassed the other subgroup on 6wk (P<0.05); meanwhile, both of BMP-7/GFs and BMP-7/hbFGF subgroups showed more recoveries than BMP-7 on 12wk (P<0.05). NO level in every subgroup was elevated after therapy. However, no difference was observed among groups on 6wk as well as 12wk (P>0.05). In both the experimental groups and the control group, there were different extents of regeneration. There was no significant difference on percentage of new alveolar area and new cementum length between three subgroups on 6wk(P>0.05),BMP-7/hbFGF subgroup increased apparently compared with others(P<0.05).Conclusion①Human bFGF gene recombinant eukaryotic expression plasmid was constructed successfully.②bFGF gene can express in GFs of Beagle dogs by transfeting recombinant plasmid into cells.③The introduction of bFGF gene to GFs promoted the proliferation of cells as well as depressed the apoptosis. GFs expressed bFGF gene may be served as an ideal cell source for periodontal tissue engineering.④The AMD has a good biocompatibility with hbFGF transfected GFs. It shows that the compound caould be used in periodontal tissue engineering.⑤Introduction of bFGF gene to GFs promoted the regeneration of periodontium, especially the regeneration of alveolar bone and cementum. hbFGF gene enhanced tissue engineering could successfully repair the periodontal defects.
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