Mineralization in vitro of matrix formed by osteoblasts isolated by collagenase digestion

Osteoblasts from calvaria of 18-day-old fetal Sprague-Dawley rats were isolated using a dissecting procedure followed by collagenase digestion. Freshly isolated or previously frozen cells were cultured for up to 4 weeks in a Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 μg/ml ascorbic acid, with or without 10 m M β-glycerophosphate. Most of the cells were alkaline phosphatase positive throughout the culture period and expressed a type-I collagen as assessed by immunofluorescence. Cells cultured in the presence of β-glycerophosphate formed a matrix with type-I collagen in 7 days. The matrix underwent mineralization in less than 2 weeks. In the absence of β-glycerophosphate, only the formation of a nonmineralized matrix was observed. Electron-microscopic examination revealed osteoblasts embedded in a dense network of collagen fibers, with a well-defined mineralization process in association with matrix vesicles. Scanning electron-microscopy showed that the matrix composed of layers of irregularly shaped spread cells with smooth surfaces trapped in a fiber matrix. No mineralization process was observed when rat skin fibroblasts were cultured under similar conditions. These data demonstrate the ability of enzymatically isolated osteoblasts cultured in the presence of β-glycerophosphate to form bone in vitro, and that this process is similar to bone formation in vivo.