Osteogenesis evaluation of osteoblasts in different cell culture media: Towards the development of international standardized methods

<p>Background: In the early developmental stages of new biomaterials and therapeutic agents targeting the bone, there are cases in which in vitro reproducibility cannot be obtained or there is discrepancy with the results of in vivo experiments. Therefore, we examined the effects of different in vitro culture medium conditions, which could be a cause of such variability, on the evaluation of osteogenesis.</p><p>Methods: Mouse calvaria-derived osteoblast-like cell line MC3T3-E1 cells (MC) were cultured inαMEM with vitamin C (VC), which augments osteogenic function in osteoblasts, (α+), αMEM without VC (α-), αMEM with VC addition before culture (αVC), DMEM without VC (D-), or DMEM with VC addition before culture (DVC). Cell proliferation, alkaline phosphatase (ALP) activity, cell differentiation marker expression, and bone calcification were compared among the culture conditions.</p><p>Results: MC proliferation was fastest for αVC and slowest for D-. There were notable differences in both ALP activity and differentiation marker expression betweenαVC and DVC as well as betweenα+ andαVC. Bone calcification was observed only in theαMEM-based media 3 weeks after the induction of calcification.</p><p>Discussion: Differences in medium type and the presence and freshness of VC all exerted marked effects on MC cell proliferation, ALP activity, cell differentiation, and calcification. Future studies are needed to confirm these findings in other osteoblastic cells and clarify the optimal culture conditions to evaluate bone formation towards the development of international standardized methods.</p>