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

Abstract

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...

Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor beta and serum factors.

“…In some experiments, a cell suspension (20 x 104 cells in 0.1 ml of medium A) was transferred into stainless steel Penicylinders (inside diameter, 6 mm; Kamei, Osaka, Japan) placed in the center of 35-mm plastic tissue culture dishes. These cells were incubated for about 24 hr at 370C under 5% CO2 in air until the cells became attached to the substrate (12). The Penicylinders were then removed, and the cultures were exposed to 1 ml of medium A. Alternatively, cells were seeded at a low density (8 x 104 cells in 1 ml of medium A per 35-mm plastic tissue culture dish).…”

“…Cultures maintained for [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] days in centrifuge tubes were washed three times with methioninefree MEM and then exposed for 3 hr to 10 ,Ci of L-…”

“…However; there have been few studies demonstrating cartilage-matrix calcification in a cell culture system. It has been reported that embryonic chicken chondrocytes (9-11) and rat growth-plate chondrocytes (12) in mass cultures on plastic dishes produce an abundant extracellular matrix, although the matrix does not calcify unless high levels of phosphate (3-fold over the level in standard medium) are included (11,12). The mineral deposition with high phosphate should be regarded as a physicochemical phenomenon (12).…”

“…It has been reported that embryonic chicken chondrocytes (9)(10)(11) and rat growth-plate chondrocytes (12) in mass cultures on plastic dishes produce an abundant extracellular matrix, although the matrix does not calcify unless high levels of phosphate (3-fold over the level in standard medium) are included (11,12). The mineral deposition with high phosphate should be regarded as a physicochemical phenomenon (12). Because of the lack of a good in vitro system, no information about the regulation of cartilage calcification is currently available.…”

Molecular cloning of the mouse homologue of Rab3c

“…One of the distinguishing features of enlarging chondrocytes within the hypertrophic zone is the production of matrix vesicles (Anderson 1995). Matrix vesicles, small extracellular membranous particles containing alkaline phosphatase and ATPase (Boyan et al 1989, Matsuzawa & Anderson 1971, have been shown to dock at the side of polarised cytoplasmic membranes of chondrocytes (Glaser & Conrad 1981, Suzuki et al 1981 and osteoblasts (Boyan et al 1989, Sudo et al 1983 where they then bud from the membrane and are subsequently expelled into the extracellular matrix. The restriction of matrix vesicle deposition defines the spatial regulation of the process.…”