Sequential synthesis of cartilage and bone marker proteins during transdifferentiation of mouse Meckel's cartilage chondrocytes in vitro

Abstract

Meckel's cartilage cells cultured in vitro undergo phenotypic transformation toward osteogenic cells. We examined whether these cells synthesize type X collagen and bone morphogenetic protein-2 (BMP-2). We also examined the results of Alcian blue staining and the expression of type I and type II collagen, osteocalcin and chondroitin sulfate proteoglycan (CSPG) during this transdifferentiation. Meckel's chondrocytes, isolated from day-17 mouse embryos, were inoculated at 1 x 104/penicylinder and cultured in α-MEM for periods up to 4 weeks. Alcian blue staining and immunostaining of type II collagen and CSPG confirmed that, after cell culture for 2 weeks, the cartilaginous phenotype was expressed most intensely. Later in culture, chondrocytes underwent modification through the synthesis of bone-type proteins; nodule-forming small round cells showed ALPase activity and were immunoreactive for type 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. BMP-2 was also expressed first in similar small round cells after 3 weeks in culture, and it subsequently extended along the extracellular matrix in the calcified nodules. These results indicate that small round cells that are differentiating toward osteocyte-like cells from Meckel's chondrocytes express type X collagen and BMP-2 sequentially.