Demonstration of Somatomedin Activity of “Multiplication-Stimulating Activity” in Rabbit Costal Chondrocytes in Culture1

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...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Suzuki¹,

Takase²,

Takigawa³

et al. 1981

“…On a protein basis, the purified CDF preparation was 100 times more effective than insulin and 5000 times more effective than fetal calf (Fig. 3) cells (9,10,12,16,17). (v) CDF-is a heat-stable noncollagenous peptide of Mr =12,000 (9,10).…”

Section: Methodsmentioning

confidence: 97%

“…1 and 3). Furthermore, the elution profiles were similar on repeated chromatography (at least 20 times) at high flow rates [10][11][12][13][14][15][16][17][18][19][20] (ml/cm2)/hr]. We feel that the new gel (crosslinked polymethacrylate) is promising as material for molecular sieve chromatography.…”

Section: Methodsmentioning

confidence: 98%

Somatomedin-like peptide(s) isolated from fetal bovine cartilage (cartilage-derived factor): isolation and some properties.

Kato¹,

Nomura²,

Tsuji³

et al. 1981

Communicated by H. A. Barker, August 10, 1981 ABSTRACT Fetal bovine cartilage contains a polypeptide(s) that has somatomedin-like effects on rat and rabbit costal chondrocytes in culture. This factor, named the cartilage-derived factor, was extracted from fetal bovine cartilage, fractionated with acetone, and purified by gel fitration on Toyopearl HW 55-F in 4 M guanidine hydrochloride, preparative isoelectric focusing, and subsequent gel filtration on Toyopearl HW 55-F in 1 M formic acid. The resulting preparation, which focused in the neutral pH region and eluted from a Toyopearl column in a fraction with apparent Mr 10,000-11,000, appeared homogenous by NaDodSO4 gel electrophoresis. The purified preparation markedly enhanced not only proteoglycan synthesis but also DNA synthesis in rabbit costal chondrocytes and, on a protein basis, it was 1000 times more active than insulin and 1,000,000 times more active than fetal calf serum in stimulating proteoglycan synthesis.

“…The peptides were dissolved in saline/10% polyvinylpyrrolidone and injected subcutaneously (sc) in doses of [25][26][27][28][29][30] ,ug in a volume of0.2 ml. Occasionally, the analogs were first dissolved in a small volume of propylene glycol and then diluted as above.…”

Section: Phe4]ss [D-5-f-trp8]ss and [D-5-meo-trp8]ss In Doses Ofmentioning

confidence: 99%

Inhibition of growth of the transplantable rat chondrosarcoma by analogs of hypothalamic hormones

Redding¹

1983

The Swarm chondrosarcoma is a hormone-responsive tumor whose growth is dependent on growth hormone, somatomedins, and glucocorticoids. Our previous work showed that partial functional hypophysectomy can be achieved by chronic administration of the luteinizing hormone-releasing hormone (LH-RH) analog LH-RH, which lowers blood levels ofLH and follicle-stimulating hormone. We have also demonstrated that somatostatin (SS)-28 or analogs of SS-14 depress serum prolactin, growth hormone, and corticotropin (ACTH) levels. Consequently, we investigated the effect of subcutaneous injection of these analogs on the growth of Swarm chondrosarcoma 3 days after transplanting it into male Sprague-Dawley rats. At autopsy, tumor volume was measured and tumors and various organs were weighed. In rats treated with three different analogs of (GH) and cortisone also act synergistically to stimulate the growth of this chondrosarcoma in hypophysectomized rats. In addition, they were able to show that chondrosarcomas respond, in vitro, to GH-dependent serum growth factors (somatomedins) and insulin by increases in amino acid transport and macromolecule synthesis (6). They concluded that the growth ofthe chondrosarcoma was mediated through somatomedins and by glucocorticoids. Recent biochemical data indicate that somatomedins stimulate both RNA and protein synthesis in chondrosarcoma tissue (7).Repeated administration of agonistic luteinizing hormonereleasing hormone (LH-RH) analogs leads to decreased responsiveness of the pituitary gland and inhibition of gonadal steroid secretion (8, 9). These paradoxical inhibitory effects induced by prolonged administration of LH-RH agonists have been linked with the regression of hormone-dependent mammary and prostate carcinomas in rats (10, 15).We have previously shown that chronic administration of[DTrp6]LH-RH, an agonist of LH-RH, significantly inhibited the growth of two different types of transplantable rat prostate tumors (14). In another study, we demonstrated that prolonged treatment with antagonistic analogs ofLH-RH can also suppress the growth of rat prostate tumors (15). Our group extended these findings to men and provided evidence for tumor growth inhibition in patients with prostatic carcinoma treated with LH-RH agonists (16). These studies showed that chronic administration ofLH-RH agonists or antagonists can inhibit the growth of sex steroid-dependent tumors by suppressing both pituitary and gonadal functions. The investigations with analogs of hypothalamic hormones were then extended to pituitary tumors (17). We demonstrated that chronic administration of agonistic and antagonistic LH-RH analogs or somatostatin (SS) analogs to rats bearing the estrogen-dependent prolactin-and corticotropin-secreting pituitary tumor 7315a significantly decreased growth, volume, and weight of this tumor (17).Since GH-dependent somatomedins and insulin play a vital part in growth of the Swarm chondrosarcoma, it became apparent that analogs of