Study On Bone Formation Of Adipose Tissue Derived Stromal Cells Cotransfected By BMP-2and BFGF

BackgroundGene therapy was the most promising way to maintain effective concentration of growth factor and thus improve the efficacy of treatment to local bone defects. But there were still some deficiencies when gene engineer went to application on the treatment of bone defects, for example, limited source of seed cells, lack of coordination for single gene. Seed cells modified by BMP-2or bFGF could effectively paly a role in bone formation; ADSCs was a kind of seed cell which was easy to get and rich in content, and had important value for research.The survival rate of free fat had closely relationship with the concentration of growth factor. Free fat transplantation was a good material for tissue filler-easy to obtain and lack of tissue rejection, however, the rate of absorption was too high and the survival rate was too low. VEGF had long been known as the master regulator of angiogenesis. Chitosan microspheres could release the carried growth factor stablely with no toxicity and easy degradation. Aim1) The capacity of bone formation would be studied after the cotransfection of double gene (BMP-2/bFGF) in ADSCs mediated by chitosan nanocapsules.2) The transfection efficiency was studied through the expression of fluorescent gene in ADSCs mediated by a variety of gene vectors. The gene vector with the highest transfection efficiency was used to later experiment.3) The capacity of bone formation would be studied after the cotransfection of double gene (BMP-2/bFGF) in ADSCs mediated by lentivirus.4) The influence of the sustained-release system made of VEGF and chitosan microspheres on the survival rate of free fat transplantation was observed.Methods1) Chitosan nanocapsules was used to conduct the cotransfection of double gene (BMP-2/bFGF) in ADSCs. Four groups were made:blank group, BMP-2group (control group), bFGF group (control group), bFGF+BMP-2group (experimental group). The observations after the transfecion included the transfecion efficiency, expression of target gene, secretion of OCN and BSP. Also, the cotransfected ADSCs was implanted in the nude mouse for research on the ectopic osteogenesis in hamstrings and bone defect repairing in thighbone.2) Chitosan、TMC、TMO、X-treme GENE HP DNA Transfection Reagent、 Lipofectamine2000and lentivirus were successively used to transfect the ADSCs, the transfection efficiency was studied thereafter.3) Lentivirus was used to conduct the cotransfection of double gene (BMP-2/bFGF) in ADSCs. Four groups were made:blank virus group, BMP-2group (control group), bFGF group (control group), bFGF-BMP-2group (experimental group). The observations after the transfecion included the transfecion efficiency, expression of target gene, secretion of OCN and BSP、the influence on cell apoptosis and proliferation. Also, the cotransfected ADSCs was implanted in the nude mouse for research on the ectopic osteogenesis in subdermal and bone defect repairing in cranial bone. 4) The fat was obtained from human and four groups were made:VEGF-chitosan microspheres and fat (experimental group), blank chitosan microspheres and fat (control group), VEGF and fat (control group), fat (blank group). The ratio was3:1. Immunologically-compromised nude mice were used as free fat graft receptors, and the mixtures of the4groups was injected to the dorsa of mice subcutaneously. The grafts were harvested at different time intervals:3weeks,6weeks, and12weeks. The weight. HE slice and CD34staining of capillaries were observed after the transplantation.Results1) The transfecion efficiency of the cotransfecion mediated by chitosan nanocapsules was approximately5%to10%. The experimental group showed good coordination in the expression of target gene and was better at secretion of OCN and BSP than the other three groups, furthermore, the difference was statistically significant (p<0.01). No bone formation was detectived in the experimentation on nude mouse.2) The transfection efficiency in ADSCs was:Chitosan5%-10%; TMC1%-5%; TMO5%-10%; X-treme GENE HP DNA Transfection Reagent15%-20%; Lipofectamine20005%-10%; lentivirus90%-95%. The lentivirus won the highest transfection efficiency.3) The transfection efficiency of lentivirus was90%-95%. The experimental group showed good coordination in the expression of target gene and was better at secretion of OCN and BSP than the other three groups, furthermore, the difference was statistically significant (p<0.01). The transfected gene was apt to inhibit the cell apotosis and promote cell proliferation. No bone formation was detectived in the experimentation on nude mouse.4) The weight and vascularization of graft in experimental group (VEGF carried by chitosan microspheres was applied) was significantly higher than other control groups. No statistically significance was detectived in the other three groups.Conclusion1) The cotransfecion of the double gene (BMP-2/bFGF) in ADSCs mediated by chitosan nanocapsules was proved to be effective in the co-expression of target gene and bone formation.2) The highest transfection efficiency was obtained in lentivirus, and the ADSCs with target gene could be stably subcultured.3) The cotransfecion of the double gene (BMP-2/bFGF) in ADSCs mediated by lentivirus was proved to be effective in the co-expression of BMP-2and bFGF, and was apt to promote bone formation.4) Chitosan microspheres can improve the efficiency of VEGF application, promote the neovascularization, and thus improve the survival of free fat graft.