Experimental study of the synergistic effect and network regulation mechanisms of an applied combination of BMP‐2, VEGF, and TGF‐β1 on osteogenic differentiation

The study aimed to explore the osteogenic effect induced by the combined use of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1), attain the best combination for osteogenic quality and efficiency, and explore the network regulation mechanisms of induced osteogenesis. MC3T3-E1 cells were cultured in vitro, and BMP-2, VEGF, and TGF beta1 were added to osteogenic induction mediums in different combinations to conduct experiments. At 7 and 14 days, the alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining of the applied BMP-2 and VEGF combination were deeper and the quantitative analysis were higher than those of the other groups. After optimizing the time-effect relationship of the combined application, with BMP-2, VEGF, and TGF-beta1 adding in the early stage and BMP-2 and VEGF adding in the late, the ALP and ARS staining of these groups were deeper and the quantitative analyses were meaningfully higher than the BMP-2 and VEGF combination group at 7 and 14 days. The expression of the RUNX2 gene and the Smad1 signaling pathway in the optimized combination group was also significantly higher. The results demonstrate that the combination of BMP-2, VEGF, and TGF-beta1 applied according to the time-effect relationship can significantly promote osteogenic differentiation mainly through the classical BMP-receptor-Smad signal pathway.