SEQUENTIAL SYNTHESIS OF CARTILAGE AND BONE MARKER PROTEINS DURING TRANSDIFFERENTIATION OF MOUSE MECKELS CARTILAGE CHONDROCYTES IN-VITRO

Meckel's cartilage cells cultured in vitro undergo phenotypic transfor mation toward osteogenic cells. We examined whether these cells synthe size type X collagen and bone morphogenetic protein-2 (BMP-2). We also examined the results of Alcian blue staining and the expression of ty pe I and type II collagen, osteocalcin and chondroitin sulfate proteog lycan (CSPG) during this transdifferentiation. Meckel's chondrocytes, isolated from day-17 mouse embryos, were inoculated at 1x10(4)/penicyl inder and cultured in alpha-MEM for periods up to 4 weeks. Alcian blue staining and immunostaining of type II collagen and CSPG confirmed th at, after cell culture for 2 weeks, the cartilaginous phenotype was ex pressed most intensely. Later in culture, chondrocytes underwent modif ication through the synthesis of bone-type proteins; nodule-forming sm all round cells showed ALPase activity and were immunoreactive for typ e I collagen and osteocalcin. Immunoreactivity for type X collagen was detected in the small round cells at the top of the nodules prior to calcification of the matrix, as well as in large hypertrophic cells. B MP-2 was also expressed first in similar small round cells after 3 wee ks in culture, and it subsequently extended along the extracellular ma trix in the calcified nodules. These results indicate that small round cells that are differentiating toward osteocyte-like cells from Mecke l's chondrocytes express type X collagen and BMP-2 sequentially.