Effect Of Different Ratios Of BMP2 And VEGFA On The Osteogenic Differentiation Of Bone Marrow-derived Mesenchymal Stem Cells

Background:Bone defects repair is one of the common clinical problems,and it is necessary to explore effective methods to accelerate bone healing and bone regeneration.Bone marrow-derived mesenchymal stem cells(BMSCs)are dominant seed cell sources for bone regeneration.Bone morphogenetic proteins(BMPs)initiate cartilage and bone formation in a sequential cascade.Vascular endothelial growth factor(VEGF)is an essential coordinator of extracellular matrix remodeling,angiogenesis and bone formation.The combined application of the two factors is beneficial to optimize the realization of osteogenesis.At present,the method of transferring gene of interest into cells by gene therapy to conduct bone regeneration research has been widely applied.Objectives:The bone marrow mesenchymal stem cells(BMSCs)were cultured in vitro,and the cytological behaviors of BMSCs after transfecting the BMP-2 and VEGFA were analyzed.The optimal ratio of BMP-2 and VEGFA gene-modified cells were explored to regulate the suitable expression of two growth factors.So as to achieve the best effect of promoting microvascular and new bone formation.Method:BMSCs were isolated,cultured and purified in vitro by adherence method;BMSCs and VEGFA gene-modified BMSCs were constructed with adenovirus vector,and transfection efficiency was determined by fluorescence monitoring.On days 3,7,14,and 21,the expressions of two transduced genes were verified by RT-qPCR and ELISA,and the cell proliferation ability after transfection was detected by CCK-8,respectively.Then,the two genetically modified cells were mixed and cultured in the following proportions:BMP-2:VEGFA=1:3,1:1,3:1,BMP-2 alone group,VEGFA alone group,control group;the expressions of osteogenic related factors(Runx2 and OCN)were detected by RT-qPCR and ELISA on 14,21 days,respectively.Qualitative and quantitative detection of alkaline phosphatase(ALP)and alizarin red staining analysis were used to compare the osteogenesis effects of six groups.Results:The BMSCs were successfully isolated and cultured in vitro.When MOI=50,the transfection efficiency of Ad-BMP-2 and Ad-VEGFA was up to 95%.The results of RT-qPCR and ELISA confirmed the successful expression of the two genes.The expression of the target gene showed a downward trend after prolonged culture time.The cell proliferation ability after transfection had no significant difference compared with the control group(p>0.05).As for the expression of osteogenic related factors,Runx2 and OCN,qualitative and quantitative analysis of alkaline phosphatase(ALP)and alizarin red staining analysis showed that there was no significant difference between the VEGFA group and the low proportion BMP-2:VEGFA(1:3)group and the control group(p>0.05),while the high proportion of BMP-2:VEGFA(3:1)group was significantly higher than the BMP2 group and other groups(p<0.05).The results indicatied that excess VEGFA had no benefit in promoting osteogenic differentiation,while the high proportion group had the best osteogenesis effect.Conclusion:Adenoviral vector-mediated gene therapy can achieve efficient transfection of BMSCs and has no significant effect on cell proliferation.Excessive VEGFA may inhibit osteogenic differentiation of BMSCs.High proportion of BMP-2:VEGFA modified BMSCs has the best osteogenic effect,and it is a good choice for optimizing bone regeneration.