The Study On Bmscs Mediated By HIF-1α Combined With PLGA To Repairing The Critical-sized Calvarial Defect Of Rat

Objective HIF-1α gene was used to modify BMSCs by gene transfection technique and to detect the role of HIF-1α gene in regulating the differentiation of BMSCs into osteogenesis and angiogenesis in vitro,and construct the vascular tissue engineering bone of HIF-1α mediated BMSCs combined with PLGA scaffold,and observe the effect of repairing the critical-sized calvarial bone defect of SD rat,which is explored the bidirectional regulation of target genes in vivo.In the future,this study will provide the experimental basis and theoretical basis of using HIF-1α to repair clinical bone defect.Methods BMSCs of SD rat was isolated and cultured in vitro,and then transduced by the lentiviral vector carrying HIF-1α gene(Lenti-HIF-1α).The transfection efficiency was observed in inverted fluorescence microscope on day 1~7 after transduced with Lenti-HIF-1α,and best MOI was determined.The effect of HIF-1αon proliferation of BMSCs was tested by the MTT method.Expressions of VEGF and OCN m RNA were detected by RT-PCR on day 1,4,7,14 and 21 after the transfection of Lenti-HIF-1α gene.The target gene HIF-1α was transfected into BMSCs,and combined with PLGA scaffold,then implanted to repair bilateral calvarial defect(5mm diameter)of SD rat(n=18).The rats were randomly divided into three groups:HIF-1α-BMSCs/-PLGA group(n=6),BMSCs/PLGA group(n=6)and only PLGA scaffold group(n=6).The new bone formation was observed by gross inspection,X-ray examination and HE staining respectively at 8 weeks after surgery.The software SPSS was used to analysis the data,which were collected from this study and described as mean±standard deviation,Analysis of Variance was used to compare the differences between groups and with P<0.05 as significant statistical differences.Results Observed by the microscope,the BMSCs were uniformly distributed in the bottom of wall and grew in the shape of polygon,spindle or spiral.When MOI was 12,transduction efficiency was the best(>80%),and MTT colorimetric assay showed that HIF-1α had no significant effect on cell proliferation(P>0.05).After the HIF-1α gene was transduced,expressions of VEGF and OCN at the m RNA levels were significantly increased compared with only BMSCs by RT-PCR(P<0.05).Scanning electron microscopy showed that BMSCs and BMSCs transfected with the target gene HIF-1α could be attached to the surface of the PLGA scaffold,and the PLGA scaffold showed a 3D porous structure.The gross inspection,X-ray examination and HE staining showed that the amount of new bone formation in the experimental group was significantly higher than that in the BMSCs/PLGA group and the only PLGA group,and the difference was statistically significant(P<0.05).Conclusion Lenti-HIF-1α could successfully transfected into BMSCs,and the expression of osteogenic and angiogenic factors in BMSCs were up-regulated,this showed that HIF-1α could significantly promote the differentiation of BMSCs into osteogenesis and angiogenesis in vitro.The in vivo study showed that HIF-1α can mediate BMSCs to promote bone tissue formation,PLGA is an ideal scaffold material and the bone defect can be repaired effectively by using this vascular tissue engineering bone.