The combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF), and bone morphogenetic protein (BMP) to sites of bone injury results in enhanced repair compared to the administration of a single factor or a combination of two factors. Based on these findings, we hypothesized that coexpression of VEGF and BMP-6 genes would enhance the osteoblastic differentiation of rat bone-marrow-derived stem cells (rMSCs) and osteogenesis by comparison to rMSCs that do not express VEGF and BMP-6. We prepared a GFP tagged adenovirus vector (Ad-VEGF+BMP-6) that contained DNA encoding the hVEGF and hBMP-6 genes. rMSCs were transduced with the virus, and the successful transduction was confirmed by green fluorescence and by production of VEGF and BMP-6 proteins. The cells were cultured to assess osteoblastic differentiation or administered in the Fischer 344 rats to assess bone formation. Mineralization of rMSCs transduced with Ad-VEGF+BMP-6 was significantly enhanced over the nontransduced rMSCs. Only transduced rMSCs could induce osteogenesis in vivo, whereas Ad-VEGF+BMP-6 or nontransduced rMSCs alone did not induce osteogenesis. The data suggests that the combined delivery of MSCs, VEGF, and BMP-6 is an attractive option for bone repair therapy. 1. Introduction Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily that possess a number of physiologic activities including the maintenance and stimulation of osteoblast differentiation [1]. The BMPs exert their effects on target tissues by binding to two types of serine/threonine kinase receptors forming a complex that phosphorylates transcription factors referred to as SMADs [2]. SMADs then act at the genomic level to alter the expression of proteins by target cells [1–3]. While BMPs have been shown to have a wide range of physiologic activities, the primary focus in the orthopaedic literature has been on their osteogenic properties that relate to bone formation in vitro and in vivo, as well as the ability of BMPs to promote skeletal repair and healing of critical sized bone defects [2, 4–28]. Several studies have attempted to determine the osteogenic potential of individual BMPs in comparison to one another [2, 12, 14, 17–19, 29]. Studies by Kang et al. [14], Li et al. [18], and Luu et al. [19] have shown that BMP-6 and BMP-9 possess superior osteogenic potential compared to BMP-4 and BMP-7 and at least equal to the osteogenic potential of BMP-2. Investigations by Vukicevic and Grgurevic [26] and Ebisawa et al. [2] have shown that BMP-6 is a more potent inducer of
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