ULTRASTRUCTURE AND CYTOCHEMICAL DETECTION OF ALKALINE-PHOSPHATASE IN LONG-TERM CULTURES OF OSTEOBLAST-LIKE CELLS FROM RAT CALVARIA

Two methods of collecting osteoblast-like cells from newborn rat calva ria were tested, either placing individual glass fragments or tipping dense grass beads onto the endocranial surface of periosteum-free bone . Inoculated at high density, cells collected by using these two metho ds form large mineralized plates after three weeks of culture. The mai n purpose of our investigation was to analyze the progressive formatio n of this mineralized structure and to localize alkaline phosphatase a ctivity. At the beginning of the culture, flattened cells gathered int o multilayers and synthesized collagen fibers. Cells in the upper laye r became rapidly cuboidal in shape and continued to secrete collagen a t their basal pole, whereas other cells became progressively embedded in the extracellular matrix. The upper cells featured ultrastructural characters of osteoblasts, whereas the embedded cells resembled osteoc ytes. After two weeks, the matrix began to mineralize: crystals appear ed on collagen fibers, on matrix vesicles, and on cell debris. During the first days of the culture, the alkaline phosphatase activity was l ocalized on the plasma membranes and on the collagen fibers. Thereafte r, only the upper cells and collagen fibers that were juxtaposed to th ese cells showed alkaline phosphatase activity. In addition, the prese nce of mineralized matrix prevented the reaction product from being vi sualized on collagen fibers. The ultrastructural analysis reveals larg e mineralized plates with a structure resembling that of bone in vivo. This culture appears to be an appropriate model to study bone formati on and regulation.