Comparative morphological and biochemical analysis of hypertrophic, non-hypertrophic and 1,25(OH)2D3 treated non-hypertrophic chondrocytes.

A comparative study of Type X collagen expression, chondrocyte morphology, and the expression of two genes controlling chondrocyte morphology (beta-actin and fibronectin) was carried out on chondrocytes derived from a tissue that remains as permanent hyaline cartilage in vivo (embryonic chicken caudal sterna), from a tissue that undergoes endochondral replacement (embryonic chicken ventral vertebrae) and caudal sternal chondrocytes treated with 1,25(OH)2D3. Under identical in vitro growth conditions and times, sternal chondrocytes grew as rounded non-adherent cells, and vertebral chondrocytes grew as adherent polygonal cells. Upon treatment with 10(-8) M 1,25(OH)2D3 over a twelve day period the sternal chondrocytes showed complete adherence and took on an identical appearance as the vertebral chondrocytes. Cellular adherence of both vertebral and 1,25(OH)2D3 treated sternal chondrocytes was associated with 10 X increased beta-actin, fibronectin and their corresponding mRNA's. Changes in connective tissue expression were observed with altered cellular morphology. Total collagen synthesis was 35-50% lower in both hormone treated and vertebral chondrocytes. Type II collagen was the major collagen type produced by all chondrocyte cultures; however, in both vertebral and 1,25(OH)2D3 treated sternal chondrocytes, a 60 kD collagenous protein was identified. This short chain collagen was determined to be Type X collagen based on its molecular weight and its CNBr peptide maps. Analysis of Type X mRNA levels using a 33 base pair anti sense oligonucleotide sequence to Type X, demonstrated that vertebral cells showed six to seven times more mRNA than sternal chondrocytes. However, the low mRNA levels of type X mRNA in sternal chondrocytes were increased two to three times by 1,25(OH)2D3 treatment. These studies demonstrate that the steroid hormone 1,25(OH)2 vitamin D3 induced morphological, biochemical and molecular changes indicative of chondrocyte maturation from a hyaline to a more hypertrophic phenotype.