Synergistic Effects of Recombinant Lentiviral-mediated BMP2 and TGF-beta3 on the Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells in Vitro.

BACKGROUND:Bone marrow mesenchymal stem cells (BMSCs) are considered good candidates for seed cells in bone engineering. The study aim to investigate the synergistic effects of human bone morphogenetic protein 2 (hBMP2) and transforming growth factor beta3 (hTGF-beta3) modified BMSCs on inducing osteogenic differentiation in vitro.METHODS:Lentivirus (LV) carrying hBMP2 and/or hTGF-beta3 genes were constructed and used to transduce rat BMSCs. The expression of osteogenic molecules was detected by qRT-PCR and western blotting.RESULTS:Targeted genes were PCR-amplified and confirmed by DNA sequencing and BLAST analysis. BMSCs infected by vectors effectively resulted in the overexpressions of hBMP2 and hTGF-beta3 and higher levels of hBMP2 and hTGF-beta3 in the culture supernatant. The co-transduction of hBMP2 and hTGF-beta3 induced BMSCs osteogenic differentiation more effectively than the transduction of hBMP2 or hTGF-beta3 individually. The expression levels of osteopontin (OPN), osteocalcin (OCN), and osteoprotegerin (OPG) in LV-hBMP2 + LV-hTGF-beta3 group (BMSCs transfected by vectors respectively carrying hBMP-2 gene and hTGF-beta3 gene) and LV-hBMP2-hTGF-beta3 group (BMSCs transfected by vector carrying hBMP2 and hTGF-beta3 fusion gene) were significantly higher than in LV-BMP2 (BMSCs transfected by vector carrying hBMP2 gene) and LV-TGF-beta3 (BMSCs transfected by vector carrying hTGF-beta3 gene) groups (P < 0.05). The hBMP2 and/or hTGF-beta3 overexpression upregulated alkaline phosphatase (ALP) activity.CONCLUSION:The present study showed that hBMP2 and/or hTGF-beta3 genes can be successfully overexpressed in BMSCs. Our study proved that the two cytokines (hBMP2 and hTGF-beta3) could induce bone differentiation synergistically, which foresees the use of the combination of these two cytokines as a therapeutic strategy in the future.