Combined effects of cartilage-derived factor with epidermal growth factor in bone and tooth developments

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We have succeeded in purifyin gvarious molecular species of cartilage-derived factor (CDF) including neutral 11k, basic 16k, and 26k CDFs. The purified CDF enhanced the synthesis of proteoglycans of rabbit costal chondrocytes in culture, but had relatively smaller effect on DNA synthesis and cell division. In contrast, epidermal growth factor (EGF) stimulated DNA synthesis and cell division of chondrocytes, but did not stimulate nor inhibit proteoglycan synthesis. CDF affected events occurring in the early stage of the G_1 phase of the cell cycle of chondrocytes to stimulate DNA synthesis. CDF had synergistic effects with EGF in stimulating DNA synthesis of the cells. We found that the culured chondrocytes become activated to interact with EGF for commitment to DNA synthesis when they are exposed to a somatomedin-like growth factor at an early stage of G_1 phase. This means that CDF activates cells at this regulatory stage to respond synergistically to the non-somatomedin growth factor … More EGF. Therefore, sutocrine factors in cartilage, such as CDFs, fibroblast growth factor, and transforming growth factor-<beta>, must We found that vanadate (0.6-6<micrn>M), an inibitor of phosphotyrosine phosphatase, stimulates the synthesis of cartilage-specific proteoglycans in rabbit costal chondrocytes and induced morphologic differentiation. In contrast, vanadate (20-60 <micrn>M) decreased cartilage proteoglycan synthesis by chondrocytes and induced morphologic transformation. Therefore, moderate increases in the level of phosphotyrosine are required for the expression of the chondrocyte phoenotype, excess increases cause cells to express the transformed phenotype.Rabbit chondrocytes were grown successfully in suspension in Eagle's medium supplemented with 10 % fetal calf serum. The cells proliferated and formed cartilage-like nodules, Where hypertrophic chondrocytes were embedded in matrix, composed of 'cartilage- specific' proteoglycans, type II and X-like collagens. The calcification occurred only after marked increases of alkaline phosphatase activity and 1,25(OH)_2D_3 receptor levels in this system. Therefore, sequential events occurring at the growth-plate of rabbit rib in vivo were simulated by this high-density suspension culture. Less