Mouse Meckel's cartilage chondrocytes evoke bone-like matrix and further transform into osteocyte-like cells in culture

Background: we reported that when Meckel's cartilage was transplanted ectopically, chondrocytes transformed into osteocyte-like cells accompanying the extracellular calcified matrix. However, we could not determine whether the osteocyte-like cells were derived from host tissues or from Meckel's cartilage itself. Therefore, we examined whether the Meckel's cartilage chondrocytes, which have a retrogressive ultimate fate, are capable of inducing the observed calcification and further transform into osteocyte- like cells in culture. Methods: Meckelian chondrocytes isolated enzymatically were plated at a low density and grown in α-MEM containing 10% FBS at 37°C under 5% CO₂ in air for up to 4 weeks. Results: Chondrocytes were fibroblast-like cells early in culture, but gradually transformed from polygonal cells into typical chondrocytes showing metachromasia with toluidine blue staining. After an additional week of culture, the chondrocytes transformed from large to small round cells accompanying nodule formations. Small round cells multiple-layered actively, and showed more intense alkaline phosphatase (ALPase) activity. Immunostaining identified type II collagen in the extracellular matrix at 2 weeks of culture, and type I collagen and osteocalcin were later synthesized by round cells. von Kossa's reaction showed extensive precipitation of calcification throughout the flocculent materials. Ultrastructural analysis showed that the cells surrounded by calcified matrix strongly resembled osteocytes. Conclusions: The present study suggested that the Meckel's cartilage chondrocytes can express the osteocyte-like phenotype in vitro during synthesis of hone-type marker proteins such as osteocalcin or type I collagen.