This study was designed to investigate the morphological features of osteogenic cultures that were established from canine marrow derived-mesenchymal stem cells (MSCs). Tripotent canine MSCs were plated in osteogenic conditions for 3 weeks, at the end of which the cultures were observed by light and transmission electron microscopy. Alkaline phosphatase (ALP) activity of the culture was determined during the differentiation period. To assess whether endochondral or intramembranous ossification was involved in MSC bone differentiation, the cultures were explored for cartilage-related gene expression. Multiple nodule-like cell aggregates appeared to form in the osteogenic cultures. These nodules were covered by a periosteum-like layer and osteocyte-like cells of varying morphology were located in lacuna-like cavities within the nodule mass. Furthermore, the bone nodules possessed an abundant matrix in which clearly striated collagen I fibres were arranged in perpendicular bundles. Matrix vesicles involving in matrix mineralization were evident in the nodules. This was in accordance with increased ALP activity in the culture. No expression of cartilage-related genes was observed, which suggested that osteogenesis might occur by intramembranous ossification. In conclusion, canine MSCs could be an appropriate model for studying in vitro bone development.
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