Influence of in vitro differentiation status on the in vivo bone regeneration of cell/chitosan microspheres using a rat cranial defect model.

The aim of this study was to investigate the influence of the in vitro osteogenic differentiation status on the in vivo bone regeneration of cell/chitosan microspheres qualitatively and quantitatively. To this end, rat bone-marrow-derived mesenchymal stromal cells (BMSCs) were seeded onto apatite-coated chitosan microspheres. The constructs were osteogenically differentiated for 0, 7, 14, and 21 days followed by calvarial defect implantation in vivo for up to 8 weeks. In vitro studies showed that BMSCs in the constructs proliferated from day 0 to day 7. The activity and gene expression of alkaline phosphatise increased from day 0 to day 14 and then decreased. The gene expression of collagen type I and osteocalcin peaked at day 21.In vivo, constructs retrieved from day 0 group were filled with fibrous tissues and capillaries, but no bone formation was observed. Constructs retrieved from day 7 and day 21 groups showed progressive bone formation, whereas those retrieved from day 14 group had the highest percentage of bone formation. These data suggested that to generate a substantial amount of bone in vivo, not only the in vitro osteogenic differentiation was necessary, but also the period of pre-differentiation was important for the cell-scaffold constructs. The period of pre-differentiation for 14 days was found to be the most suitable for chitosan microspheres.