BMP2 and VEGF165 transfection to bone marrow stromal stem cells regulate osteogenic potential in vitro.

Abstract

An exogenous supply of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factors 165 (VEGF165) will synergize to promote bone regeneration in vivo. The aim of this study was to confirm the role of VEGF165 on the osteogenesis potential of bone mesenchymal stem cells (BMSCs) transduced by adenovirus vector containing BMP2 gene in vitro.Rabbit BMSCs were isolated and transfected with various adenovirus vectors: Ad-BMP2-VEGF165 (BMP2+VEGF165 group), Ad-BMP2 (BMP2 group), Ad-VEGF165 (VEGF165 group), and Ad-green fluorescent protein (GFP group). The multiplicity of infection was detected by GFP expression. Expression of BMP2 and VEGF165 was detected by Western blot and ELISA, and the osteogenic biological activity of BMP2 and VEGF165 by osteogenic assay. Meanwhile, the osteogenic biological activity of BMP2 and VEGF165 was evaluated by detection of Col I (collagen type I), OC (osteocalcin), and ALP (alkaline phosphatase) activity using OC staining, ALP activity assay, and real-time PCR assay.Expression of target genes and proteins reached peak values at 5 days and then gradually declined. The OC staining, ALP activity, and real-time PCR assay of ColI, OC, and ALP were all increased in cells transfected with Ad-BMP2-VEGF165, Ad-BMP2, Ad-VEGF165, and Ad-GFP. However, the osteogenic biological activity in cells transfected with Ad-BMP2 was higher compared to cells transfected with other vectors after transfection at 14 and 21 days. We also found that BMP2 +VEGF165 group showed more osteogenic activity effect than the VEGF165 or control group. Furthermore, osteogenic assays in VEGF165 showed that a slightly lower osteogenic effect when compared to controls at 21 days.VEGF165 might be a potent inhibitor of BMSCs differentiation into osteoblasts. The strategies to use BMP2 and VEGF165 in bone regeneration and the molecular mechanism of their interaction require further investigation.