Vascula Endothelial Growth Factor-a Potentiates Bone Morphogenetic Protein 9-induced Osteogenic Differentiation And Bone Formation

Fractures, bone defects and non-union are the common diseases of orthopearedic. As the best method of treatment in currently also has its obvious defection(such as operation-time is too long, blood loss, limition of materials, increase the suffering of patients, etc). Thus, using the theory and techniques of tissue engineering to research and discovery one or more powerful osteogenic factor is becoming increasingly important. Mesenchymal stem cells(MSCs) are suitable seed cells for bone tissue engineering since they can self-renew and undergo differentiation into osteogenic, adipogenic, chondrogenic, or myogenic lineages. The main objective of this article is to investigate the effect of VEGF-a on the osteogenesis induced by BMP9 and the mechanism of how VEGF-a cooperates with BMP9 in the osteogenesis of MEFs. In this study, experimental methods include RT-PCR, immunocytochemistry, Western blot, alkaline phosphatase(ALP) activity assay and staining, Alizarin Red S staining, ectopic bone formation experiments, three-dimensional imaging of Micro-CT and histologic staining. Firstly, we examined the endogenous expression level of VEGF-a in MSCs(MEFs, C3H10T1 / 2, C2C12 and MC3T3E1). We found that the endogenous expression level of VEGF-a in MSCs was detectable, but was very low. Secondly, to investigate the effect of VEGF-a on BMP9-induced the activity of early and late osteogenic markers(ALP, OPN and OCN)and matrix mineralization in MEFs. We found that all the markers of osteogenesis strengthened. Nextly, to investigate the effect of VEGF-a on BMP9-induced ectopic ossification in MEFs. We found that VEGF-a can strengthen BMP9-induced ectopic bone formation activity, can strengthen increased bone volume significantly, but decreased bone density. Lastly, to investigate the mechanism of VEGF-a on BMP9-induced osteogenic differentiation in MEFs. We found that VEGF-a synergistic enhanced BMP9-induced osteogenic differentiation in MEFs would significantly activated PI3 K / Akt signaling pathway. Application of PI3 K / Akt inhibitor(LY294002) could offset this synergistic effect significantly. Our findings suggested that VEGF-a plays an important role in regulating BMP9-induced osteogenic differentiation in MEFs, which may be mediated by PI3K/Akt signaling.