Osteogenic differentiation is synergistically influenced by osteoinductive treatment and direct cell-cell contact between murine osteoblasts and mesenchymal stem cells

The present study was designed to address whether osteoblasts play a synergistic role in promoting mesenchymal stem cell (MSC) osteogenesis in a direct cell-cell contact co-culture model. Murine C3H10T1/2 and MC3T3-E1 cell lines were mixed and plated onto 12-well culture plates and co-cultured at various ratios of initial cell densities. To compare the possible improvement on osteogenic differentiation, co-culture cells were served with or without osteogenic supplements in culture medium. Weak osteogenesis was induced in MSCs co-cultured in an untreated medium with different ratios of osteoblasts. An osteoblast-dependent increase in osteogenic gene expression of Runx2, type I collagen, and osteocalcin was observed over time. Moreover, both alkaline phosphatase (ALP) activity and calcium deposition were distinctly enhanced at levels that were proportional to the quantity of osteoblasts in the culture. The increases in mRNA expression and ALP activity were greater in co-cultures treated with osteogenic supplements than in untreated cultures. However, the production of ALP activity followed by a distinct matrix mineralization was lower in osteogenic-treated cultures containing greater numbers of osteoblasts. This suggests that a higher density of osteoblasts may lead to weak osteogenesis of MSCs by direct cell-cell contact co-culture in an untreated environment. Furthermore, additional osteogenic supplements may act synergistically with osteoblasts to accelerate matrix mineralization by reducing the process of osteogenic differentiation in osteogenic treated co-cultures. The present work may improve the understanding of MSC osteogenesis and may provide benefits for regenerative medicine.