Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture.

Abstract

A method is presented for isolating osteoblasts from newborn mouse calvaria without the use of digestive enzymes. The procedure is based on the ability of osteoblasts to migrate from bone onto small glass fragments. The isolated cells were cultured for up to 14 d [days] in Dulbecco''s modified Eagle''s medium supplemented wiht 10% fetal calf serum and 50 .mu.g/ml of ascorbic acid. Cultures (7-d) were incubated for 24 h with [3H]proline. High levels of collagen synthesis relative to total protein were found, as measured by collagenase digestion of medium and cell layer proteins. Analysis of pepsin-digested proteins from the same cultures by SDS-PAGE [sodium dodecyl sulfate-polyacrylamide gel electrophoresis] showed that type I collagen was predominantly produced with small amounts of type III and V (.alpha.1 chains) collagens. Osteoblasts grown in the presence of .beta.-glycerophosphate were able to initiate mineral deposition in culture. EM analysis of the cultures revealed the presence of needle-shaped apatite-like crystals associated with collagen fibrils and vesicles in the extracelluar space. Mouse skin fibroblasts cultured under identical conditions failed to initiate mineralization. Electron histochemical studies revealed the presence of alkaline phosphatase activity, associated with osteoblast membranes, matrix vesicles and on or near collagen fibrils. Thus, these isolated osteoblasts retained in culture their unique property of initiating mineralization and, therefore, represent a model of value for studying the mineralization process in vitro.