Bone Abstracts

The in vitro culture of calvarial osteoblasts from neonatal rodents remains an important method for studying the regulation of osteoblast function. Widespread use of transgenics has created a particular need for a reliable, simple method that allows the differentiation and bone-forming activity of mouse osteoblasts to be studied directly. We have established such a method and have identified key differences in optimal culture conditions between mouse and rat osteoblasts. Cells, isolated from neonatal rat or mouse calvariae by bacterial collagenase digestion, were cultured for 14–28 days before staining for alkaline phosphatase (TNAP) and bone mineralisation (Alizarin Red). Rat cells typically required ~14 days in culture, whilst mouse osteoblasts had to be grown for 21–28 days. We found that reliable differentiation of mouse osteoblasts, resulting in abundant TNAP expression and the formation of discretely mineralised collagenous ‘trabecular’ bone elements, occurred only in αMEM culture medium with 10% heat-inactivated FCS (HI-FCS). Dexamethasone had no effect on bone mineralisation or TNAP expression in mouse osteoblasts. In contrast, TNAP expression and bone formation by rat osteoblasts occurred in both αMEM and DMEM culture media (although ~4-fold more efficiently in αMEM), supplemented with either FCS or HI-FCS, and was strongly dependent on dexamethasone (10 nM). Both mouse and rat osteoblasts required β-glycerophosphate (2 mM) and ascorbate (50 μg/ml) for osteogenic differentiation, and both showed similar sensitivity to exogenous ATP (10 μM), a well-established inhibitor of mineralisation. The high efficiency of osteogenic differentiation observed in αMEM, compared with DMEM (which we have previously used for rat osteoblast cultures) probably reflects the much richer formulation of the former; αMEM contains many additional amino acids (including proline), vitamins and other supplements. These findings should prove useful not only to those wishing to culture mouse osteoblasts successfully but also for laboratories requiring more efficient routine culture of bone-forming rat osteoblasts.