Biochemical and histological analysis of proteoglycans systhsized by cartilaginous cell line, ATDC5.

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I cultured ATDC 5 cells with insulin, and made these cells differentiate into chondrocytes. After reaching confluency, these cells grew further and made cartilage-like nodules stained with alcian blue. The nodules were detected on 2 weeks after reaching confluency and gradually increased until 4 weeks after confluent. However, even on 4 weeks after confluent, non-cartilage tissue still remained internodullar area. While, on 2 weeks after reaching confluency, I changed the medium into alpha-MEM with Vitamine C and further cultured for 2 weeks, then calcified tissue was induced. By adding beta-glycerophosphate (2-5mM), the calcification was stably induced.Next, I tried matabolic labeling with ^<35>S04 of ^3H-Glucosamine on 0w, 1w, 2w, 3w after reaching confluency, and analyzed the synthesized proteoglycan structure with gel filtration and ion exchange chromatography. The results indicate that large cartilage type proteoglycan (aggrecan type) and small non-cartilage type proteoglycan (Decorin, Biglycan type) were synthesized by this cell. Especially, even on 3 weeks after reaching confluency, considerable amount of non-cartilage type proteoglycan existed in culture medium, while in cell layr, cartilage type proteoglycan exclusively existed. These results suggested that cartilage tissue is formed inner layr of cartilage nodules and non-cartilage tissue still remains on the surface layr of cartilage nodules, and this concept was conssisted with histological observations in this study.As a conclude, ATDC5 cell is appropriate as a model of chondrogenesis, but some attentions are needed when analyzing medium component.