The Experimental Study Of Tissue Engineering In BMP2 Gene Transfected Into Rat Buccal Mucosa Fibroblasts Curluredwith Xenogeneic Bone Ceramice

Objective: Rat Buccal mucosa fibroblasts(RBMF) are easier to be obtained and cultured.To establish the bone morphogenetic proteins 2-rat Buccal mucosa fibroblasts (BMP2-RBMF) as seed cells for tissue engineered bone and dentin, RBMF were transfected by human BMP2.To evaluate the feasibility of using BMP2-RBMF as seed cells for tissue engineered bone and dentin, the biological function of BMP2-RBMF and the expression of alkaline phosphatase was detected.BMP2-RBMF cells which were established as seed cells were seeded onto Xenogeneic Bone Ceramice (XBC) scaffold.In order to evaluate the feasibility of using XBC as scaffold for tissue engineered jaw and dentin,the biocompatibility between the seed cells(BMP2-RBMF)and scaffold was detected by scanning electron microscopy and MTT analysis.Methods: 1 Rat buccal mucosa fibroblasts(RBMF) primary culture and source identification 2 Construction of pEGFP-N1-BMP2 plasmid 3 Bone morphogenetic proteins 2 transfection 4 The biological influence on fibroblasts of gene transfection 4.1 RT-PCR 4.2 Cell proliferation 4.3 TEM observation of cell ultrastructure 4.4 Immunohistochemical observation of the expression of BMP2 4.5 Alkaline phosphatase activity detection 5 Cell combined with xenogeneic bone ceramice scaffolds in vitro 5.1 Cell proliferation 5.2 Scanning electron microscopy deteced cell and xenogeneic bone ceramice (XBC) scaffold Culture.Results:1Rat buccal mucosa fibroblasts in vitro morphological observationInverted microscope observation reveled that the original generation of rat buccal mucosa fibroblasts climbing from rat buccal mucosa tissue, adherenting extending, showing fusiform, triangular and polygon.Just subcultured fibroblasts spherical.During 2h, living cells has been the settlement adherent, During 24h , completely start tringle,spindle,nuclear significantly increased.2 Identification of fibroblast originFibroblast vimentin immunofluorescence staining, the cell cytoplasm (green fluorescence), nuclei are not colored. Keratin fluorescence immunostaining negative, cytoplasm nucleus no green fluorescence. Description cultured cells as the source of mesoderm fibroblasts.3 Plasmid IdentificationThe constructed pEGFP-BMP2 could produced 1.2kb and 4.7kb fragments by an enzyme cutting technique at Xho I and HindIII sites and theirsize were same as those of BMP2 and pEGFP -Nl. This indicated that BMP2 cDNA was successfully subcloned into pEGFP-Nl.4 pEGFP-N1-BMP2 plasmid fibroblasts were observed under fluorescence microscopeThe fluorescence microscopy showed that fluorescence light was spread over a broader cellular distribution both in the nucleus and cytoplasm in all of RBMF transfected with pEGFP-Nl, but fluorescence light was focused on the cellular membrane and cytoplasm of RBMF transfected with pEGFP-BMP2.5 Cell proliferationRBMF, N1-RBMF, BMP2-RBMF changes in proliferative activity, Results showed that BMP2-RBMF cells proliferation was no significant difference with RBMF, N1-RBMF. pEGFP-N1-BMP2 plasmid transfection does not affect the proliferative activity of buccal mucosa fibroblasts.6 Immunohistochemical detection the expression of BMP2 BMP2-RBMF can be seen with positive expression of BMP2 in the cell cytoplasm and membrane was stained brown, Nucleus are not coloring.7 The variation of fibroblasts by gene transfectionRT-PCR analysis demonstrated that transfection of BMP2 triggered the differentiation of BMP2-RBMF into osteoblasts-like cells.8 The results of Alkaline phosphatase (ALP) activity pEGFP-N1-BMP2 plasmid transfected fibroblasts to promote the activity of alkaline phosphatase.9 The results of Expression of BMP2 in cell supernatantspEGFP-N1-BMP2 plasmid transfection to promote secretion of BMP2 in fibroblasts10 The Result of Transmission Electron Microscope(TEM)DiscoveryLarge amount of cytoplasm and incremental myelin sheath-like figures showed concentric lamellar arrangement .The cisternae were dilated to varying degrees and were fulled of abundant proteinoid substances. The collagenous fibrillae was distributed widely intercellular.11 The effect of BMP2-RBMF curlured with xenogeneic bone ceramiceMTT detected the proliferative activity of BMP2-RBMF with XBC,XBC were not affect the proliferative activity of BMP2-RBMF. BMP2-RBMF attached on the surface of XBC and stretch well.Conclusion:1 Bone morphogenetic protein 2 (BMP2) gene transfer in rat buccal mucosa fibroblasts (RBMF), can be stable expression of BMP2, had no effect on cell proliferation.2 Transfected fibroblasts had osteoblast phenotype and can be used as seed cells for tissue engineering.3 BMP2-RBMF can be secreted BMP2, involved in inducing osteogenesis.4 BMP2-RBMF could be attached on the surface of XBC and proliferated actively, XBC could be used as scaffold material for bone and dentin formation. Anyway, RBMF and XBC composite sintering can be used as the material of jaws and dentin repair tissue engineering , the next step to be carried out animal experiments and clinical application .