Simulation of the initial stage of endochondral ossification: in vitro sequential culture of growth cartilage cells and bone marrow cells.

Growth cartilage cells were isolated from the ribs of young rats and cultured at high cell density in Ham's F-12 medium supplemented with 10% fetal calf serum. During 7 days, glycosaminoglycans and proteoglycans were actively synthesized and secreted, forming a metachromatic matrix. When cultured together with growth cartilage cells precultured and biosynthetically prelabeled with 35SO4(2-) in their glycosaminoglycans, bone marrow cells caused release of 35S-labeled material into the culture medium. Glycosaminoglycan was also released by addition of conditioned medium obtained from cultures of bone marrow cells or peritoneal macrophages to the growth cartilage cell cultures. Electron microscopic studies of the extracellular matrix of growth cartilage cells cocultured with bone marrow cells showed that needles of apatite mineral were deposited within and in close apposition to the surfaces of matrix vesicles. These findings suggest that enzymes released from bone marrow cells or macrophages removed glycosaminoglycan or proteoglycans, which may be inhibitors of mineral growth, and consequently mineralization was initiated. From these findings, sequential culture of growth cartilage cells and bone marrow cells is promising as an experimental system for investigating the mechanism of the initial stage of endochondral ossification.