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

Abstract

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.

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

“…Csaki et al demonstrated that MSCs and osteoblasts actively search for cell-cell contact, leading to cell proliferation and osteogenic differentiation, only in monolayer co-cultures with osteoinductive treatment [14]. The quality of osteogenesis, as evidenced by protein expression, is proportional to the quantity of osteoblasts in the co-cultures, a finding that is consistent with our results.…”

“…However, it remains unclear as to whether and how osteoblasts affect the self-renewal and differentiation of another major stem cell population in bone marrow MSCs. There is little in vivo data available on this, and in vitro studies using conditioned medium or co-culture models have produced inconsistent results [13][14][15][16][17]. In experiments using conditioned medium, the real-time dynamic cell-cell interactions are missing and osteogenic inducers such as dexamethasone were used.…”

Effect of diabetic osteoblasts on osteogenic differentiation of human umbilical cord mesenchymal stem cells

“…These results are similar to the view that osteoblasts can promote MSC or hematopoietic progenitor cell proliferation [22][23] . Osteoblasts can also regulate osteogenic differentiation of MSCs in stimulating osteogenesis 24 .…”

Indirect co-culture with tenocytes promotes proliferation and mRNA expression of tendon/ligament related genes in rat bone marrow mesenchymal stem cells

“…Co-culture system allowed the contact of different cell types in vitro which ideally simulated the coexisting microenvironment of cells in vivo, thereby made it possible to investigate the molecular events during the crosstalk and made cell fate determination more controllable than directly injecting MSCs into tissues. The differentiation of MSCs affected by full differentiated cells in direct or indirect co-culture systems were investigated by a number of groups and differentiation of MSCs were achieved or promoted (Lange et al 1999;Lee et al 2007;Csaki et al 2009;Chen et al 2009;Wang et al 2006;Ball et al 2004). However, little is known about the proliferation and commitment of MSCs subjected to co-culture conditions with primary tenocytes.…”

“…Moreover, this in vitro culture system made MSCs fate more controllable than in vivo and easier for understanding the molecular events within the crosstalk of MSCs and the local environment. Co-culturing with appropriate cell types, MSCs have been elucidated to commit to corresponding lineages, such as osteoblasts (Csaki et al 2009), chondrocytes (Chen et al 2009), ligament cells (Lee et al 2007), cardiomyocytes , hepatocytes (Lange et al 1999) and smooth muscle cells Ball et al 2004). Interestingly, direct cell-to-cell contact has been proven to be indispensable for the commitment of a sort of cell types, such as smooth muscle cells and cardiomyocytes Ball et al 2004).…”