Synergistic effect of Indian hedgehog and bone morphogenetic protein-2 gene transfer to increase the osteogenic potential of human mesenchymal stem cells

Abstract

Introduction: To stimulate healing of large bone defects research has concentrated on the application of mesenchymal stem cells (MSCs).

Methods: In the present study, we induced the overexpression of the growth factors bone morphogenetic protein 2 (BMP-2) and/or Indian hedgehog (IHH) in human MSCs by adenoviral transduction to increase their osteogenic potential. GFP and nontransduced MSCs served as controls. The influence of the respective genetic modification on cell metabolic activity, proliferation, alkaline phosphatase (ALP) activity, mineralization in cell culture, and osteogenic marker gene expression was investigated.

Results: Transduction had no negative influence on cell metabolic activity or proliferation. ALP activity showed a typical rise-and-fall pattern with a maximal activity at day 14 and 21 after osteogenic induction. Enzyme activity was significantly higher in groups cultured with osteogenic media. The overexpression of BMP-2 and especially IHH + BMP-2 resulted in a significantly higher mineralization after 28 days. This was in line with obtained quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses, which showed a significant increase in osteopontin and osteocalcin expression for osteogenically induced BMP-2 and IHH + BMP-2 transduced cells when compared with the other groups. Moreover, an increase in runx2 expression was observed in all osteogenic groups toward day 21. It was again more pronounced for BMP-2 and IHH + BMP-2 transduced cells cultured in osteogenic media.

Conclusions: In summary, viral transduction did not negatively influence cell metabolic activity and proliferation. The overexpression of BMP-2 in combination with or without IHH resulted in an increased deposition of mineralized extracellular matrix, and expression of osteogenic marker genes. Viral transduction therefore represents a promising means to increase the osteogenic potential of MSCs and the combination of different transgenes may result in synergistic effects.

Figure 1

Metabolic activity, proliferation, and levels of transgene products. The figure illustrates the cell metabolic activity (a) and proliferation behavior (b) of native mesenchymal progenitor cells (Ctrl) and after adenoviral transduction with GFP, BMP-2, IHH, and BMP-2 with IHH, as determined with AlamarBlue and Quibit assays. Furthermore, the levels of BMP-2 (c) and IHH transgene products (d), determined with ELISA, are demonstrated. The asterisks indicate statistically significant differences compared with control cultures.

Figure 2

ALP activity and mineralization. The figure shows the alkaline phosphatase activity of osteogenic (a) and control (b) monolayer cultures of native (Ctrl) and transduced mesenchymal progenitor cells. Moreover, the extracellular matrix mineralization of osteogenically induced (c, e) and control cultures (d, f) over time is depicted. Asterisks indicate statistical significance.

Figure 3

Monolayer mineralization. Alizarin red S stainings on days 14 and 21 of monolayer cultures transduced with GFP, BMP-2, IHH, and BMP-2 with IHH maintained with osteogenic (left panel) and control (right panel) media. Nonmodified mesenchymal progenitor cells (Ctrl) served as controls. Magnification, ×10.

Figure 4

RT-PCR analysis. Outcomes of the qRT-PCR measurements for the osteogenic marker genes alkaline phosphatase (ALP(a)), osteopontin (OP, (b)), osteocalcin (OC, (c)), and runx2(d) in osteogenic (left panel) and control cultures (right panels).