Co-culture of canine mesenchymal stem cells with primary bone-derived osteoblasts promotes osteogenic differentiation.

Tissue engineering of bone grafts with osteogenic progenitor cells such as adult mesenchymal stem cells (MSC) represents a promising strategy for the treatment of large bone defects. The aim of this experimental study was to evaluate the osteogenic potential of primary osteoblasts on MSCs in co-culture at different ratios. The co-cultures were treated with or without a specific osteogenic induction medium in monolayer and high density cultures. In monolayer co-cultures, MSCs and osteoblasts actively searched for cell-cell contact leading to cell proliferation and only in treated monolayer co-cultures osteogenesis was observed. Ultrastructural evaluation of high density co-cultures using electron microscopy demonstrated osteogenesis with no significant difference between treated or untreated co-cultures. Immunoblotting confirmed expression of collagen type I, beta1-Integrin, the osteogenic-specific transcription factor Cbfa-1 and induction of the MAPKinase pathway (Shc, Erk1/2) in both treated and untreated co-cultures. Although treatment with the induction medium enhanced osteogenesis in the co-cultures compared to untreated co-cultures, the quality of osteogenesis was proportional to the quantity of osteoblasts in the co-cultures. Fifty percent osteoblasts in the co-cultures markedly increased osteogenesis; even the presence of ten percent osteoblasts in the co-culture strongly promoted osteogenesis. This data leads us to conclude that co-culture of MSCs with osteoblasts combined with the three-dimensional environment of the high density culture strongly promotes osteogenesis and stabilizes the osteogenic potential of MSCs. This approach may prove to be of practical benefit in future tissue engineering and regenerative medicine research.