Synergistic effects of overexpression of BMP‑2 and TGF‑β3 on osteogenic differentiation of bone marrow mesenchymal stem cells

Abstract

Bone morphogenetic protein 2 (BMP-2) and transforming growth factor β (TGF-β) isoforms are important in advancing bone regeneration. The aim of the present study was to investigate the positive and reciprocal effect of TGF‑β3, one of the three TGF‑β isoforms, on BMP‑2 in promoting osteogenic differentiation. Exogenous BMP‑2 and TGF‑β3 genes were separately, and in combination, overexpressed in rabbit bone marrow‑derived mesenchymal stem cells (rBMSCs). Expression levels of BMP‑2 and TGF‑β3 were evaluated using reverse‑transcription‑polymerase chain reaction (RT‑PCR) and Western blotting assays. Furthermore, the osteogenic differentiation capacities of BMSCs were assessed by measuring Alizarin Red S staining, an alkaline phosphatase activity assay, and quantification of the osteogenic‑specific genes, Runt‑related transcription factor 2 (Runx2) and Osterix (Osx). Using lentiviral‑mediated transfection, robust co‑transfection efficiency of >90% was achieved. RT‑PCR and immunoblotting results indicated a marked elevated expression of BMP‑2 and TGF‑β3 in rBMSCs undergoing co‑transfection, compared with transfection with BMP‑2 or TGF‑β3 alone, indicating that BMP‑2 and TGF‑β3 are synergistically expressed in rBMSCs. Furthermore, enhanced osteogenic differentiation was observed in rBMSCs co‑transfected with BMP‑2/TGF‑β3. The present study successfully delivered BMP‑2 together with TGF‑β3 into rBMSCs with high efficiency for the first time. Furthermore, TGF‑β3 overexpression was demonstrated to enhance the osteogenic efficacy of BMP‑2 in rBMSCs, and vice versa. This provides a potential clinical therapeutic approach for regenerating the function of osseous tissue, and may present a promising strategy for bone defect healing.

Figure 1.

BMP-2 and TGF-β3 were synergistically expressed in rBMSCs. (A) Strong green fluorescence intensity indicated a robust lentivirus-mediated transfection efficiency of rBMSCs (magnification, ×100). Increased suspended cells in the co-transfection group indicated decreased proliferation of rBMSCs. The expression levels of BMP-2 and TGF-β3 were increased in the rBMSCs co-transfected with BMP-2/TGF-β3 lentiviral particles, as assessed by (B) reverse transcription-polymerase chain reaction and (C) western blotting. Data are presented as the mean ± standard deviation from triplicate repeats. *P<0.05 vs. indicated groups. BMP-2, bone morphogenetic protein 2; TGF-β3, transforming growth factor β3; rBMSCs, rabbit bone marrow-derived mesenchymal stem cells; GFP, green fluorescent protein.

Figure 2.

Mutual effect of BMP-2 and TGF-β3 on osteogenic differentiation. (A) Increased expression levels of Runx2 and Osx, which are early markers for osteogenic differentiation in osteogenic cultures, were observed in cells transfected with BMP-2 and TGF-β3. (B) ALP activity of rabbit BMSCs osteogenic cultures was analyzed at the indicated time points. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. indicated groups. (C) Alizarin Red S staining was performed to visualize mineral deposition at day 21 post-transfection (magnification ×200). Marginal mineralized nodules were observed in negative control groups, however a markedly higher density of nodules were detected in BMSCs undergoing gene transfection, particularly in co-delivered cells. Control cells were stem cells normally cultured without osteogenesis induction treatment, while BMSC indicates cells undergoing induction but without exogenous gene transfection. BMP-2, bone morphogenetic protein 2; TGF-β3, transforming growth factor β; Runx-2, Runt-related transcription factor 2; Osx, Osterix; ALP, alkaline phosphatase; BMSCs, bone marrow-derived mesenchymal stem cells.