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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 35SO42-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 ofmatrix 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.Longitudinal growth of the skeleton is confined mainly to the growth plate. Basically, there are two aspects of growth processes in cartilage. The (6), and parathyroid hormone was found to induce ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis (7,8). Andersen (9, 10) and Bonucci (11) reported that earliest min eral deposits are found to be in contact with membranebounded vesicles, so-called matrix vesicles or dense globules, in the matrix around. epiphyseal chondrocytes. In this study, we developed a model system for the initial stage of endochondral ossification, composed ofGC and bone marrow cells (2, 12). With this system, we examined the relationship between the degradation of GAG and calcification associated with matrix vesicles.MATERIALS AND METHODS Cell Culture. GC were isolated from costal cartilage ofyoung male Sprague-Dawley rats weighing 80-90 g by treatment with EDTA, trypsin, and collagenase (1). The isolated cells [5 x 105 cells in 0.1 ml of Ham's F-12 medium (Nissui, Tokyo) supplemented with 10% fetal calf serum (GIBCO; previously heat treated at 560C for 30 min)] were inoculated into stainless steel Penicylinders (length, 9 mm; inside diameter, 6 mm) placed in the center of Lux Scientific plastic dishes (35 mm). These cells were incubated for about 24 hr at 370C under 5% CO2 in air until the cells became attached to the bottom ofdishes, and then the Penicylinders were removed. The cells were fed with 2 ml of the same medium and the medium was changed every other day.The bone marrow was washed out of the shafts of rat femurs and dispersed in the same medium as described above. The cell suspension was filtered through a nylon sieve (pore siz...

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The present results indicate that the systemic retinoid administration may alter the biological properties of the articular cartilage.

Multiplication-stimulating activity (MSA), a substance obtained from conditioned medium of Buffalo rat liver cells, stimulated replication of rabbit costal chondrocytes in culture and their DNA synthesis, sulfation of glycosaminoglycans, protein synthesis, and collagen synthesis. These stimulatory effects of MSA were dose-dependent in serum-free medium, indicating that MSA has intrinsic somatomedin activity. Even after several successive passages, cultured chondrocytes were more responsive to MSA than other organ- and cell-culture systems reported. Therefore, cultured rabbit costal chondrocytes proved a good in vitro system for analysis of somatomedin actions.