MORPHOLOGICAL AND BIOCHEMICAL-CHARACTERIZATION OF MINERALIZING PRIMARY CULTURES OF AVIAN GROWTH-PLATE CHONDROCYTES - EVIDENCE FOR CELLULAR PROCESSING OF CA2+ AND PI PRIOR TO MATRIX MINERALIZATION

Advances in the culture of mineralizing growth plate chondrocytes prov ided an opportunity to study endochondral calcification under controll ed conditions. Here we report that these cultures synthesize large amo unts of proteins characteristically associated with mineralization: ty pe II and X collagens, sulfated proteoglycans, alkaline phosphatase, a nd the bone-related proteins, osteonectin and osteopontin. Certain cho ndrocytes appeared to accumulate large amounts of Ca2+ and Pi during t he mineralization process: laser confocal imaging revealed high levels of intracellular Ca2+ in their periphery and X-ray microanalytical ma pping revealed the presence of many Ca2+- and Pi-rich cell surface str uctures ranging from filamentous processes 0.14 +/- 0.02 mu m by 0.5-2 .0 mu m, to spherical globules 0.70 +/- 0.27 mu m in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 +/- 0.19 mu m) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca2+-rich cell surf ace blebs. The globular mineral progressively transformed into cluster s of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca2+ and Pi leads to formation of complexes o f amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These str uctures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumu lation of Ca2+ and Pi is directly involved in endochondral calcificati on. (C) 1995 Wiley-Liss, Inc.