Abstract
During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. Chondrocyte hypertrophy and matrix mineralization are associated with expression of type X collagen and the induction of high levels of the bone/liver/kidney isozyme of alkaline phosphatase. To determine what role vitamin C plays in these processes, chondrocytes derived from the cephalic portion of 14-day chick embryo sternae were grown in the absence or presence of exogenous ascorbic acid. Control untreated cells displayed low levels of type X collagen and alkaline phosphatase activity throughout the culture period. However, cells grown in the presence of ascorbic acid produced increasing levels of alkaline phosphatase activity and type X collagen mRNA and protein. Both alkaline phosphatase activity and type X collagen mRNA levels began to increase within 24 h of ascorbate treatment; by 9 days, the levels of both alkaline phosphatase activity and type X collagen mRNA were 15-20-fold higher than in non-ascorbate-treated cells. Ascorbate treatment also increased calcium deposition in the cell layer and decreased the levels of types II and IX collagen mRNAs; these effects lagged significantly behind the elevation of alkaline phosphatase and type X collagen. Addition of beta-glycerophosphate to the medium increased calcium deposition in the presence of ascorbate but had no effect on levels of collagen mRNAs or alkaline phosphatase. The results suggest that vitamin C may play an important role in endochondral bone formation by modulating gene expression in hypertrophic chondrocytes.
Highlights
During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which deposit a mineralized matrix to form calcified cartilage
Chondrocyte hypertrophy is associated with the production of a short helix collagen, type X [5,6,7,8], which far has been found only in premineralizing and mineralizing cartilage [9].The appearance of type X collagen during chondrocyte hypertrophy is associated with a decrease in levels of the major fibrillar collagen found in cartilage type I1 [10,11,12]
To clarify the roles of vitamin C in chondrocyte hypertrophy and matrix mineralization, we have studied cultures of prehypertrophic sternal chondrocytes underconditionsin which these cells mature into hypertrophic chondrocytes
Summary
Chondrocyte Culture-The isolation and culturing of sternal chondrocytes were carried out using procedures similar to those described previously for vertebral chondrocytes [15]. If cells were exposed to 50 pg/ml ascorbate at the time of initial plating of secondary cultures, a gradual increase in fibroblastic cells was observed. Cell Characterization-At each time point, cultures were rinsed with Hanks’ balanced salt solution and fixed in 2% paraformaldehyde in phosphate-buffered saline for 20 min. The cell layer matrix extract was used to determine calcium levels in each culture dish by atomic absorption spectrophotometry. The specificity of hybridization of all probes was verified by Northern blot hybridization to RNAs from cell cultures as well as samples of RNA prepared from cartilage and bone to ensure that theprobe was reacting only with mRNA of the expected size. The type I collagen probe was pGEM.821 containing 3‘ propeptide and untranslated sequences of a chick genomic a2(I) collagen gene clone [17]. Cell layers were not analyzed as theycontained minimal levels of type X collagen
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