IN-VITRO DIFFERENTIATION AND MINERALIZATION OF CARTILAGINOUS NODULES FROM ENZYMATICALLY RELEASED RAT NASAL CARTILAGE CELLS

Nasal cartilage cells from 21-day-old rat fetuses were cultured at hig h density in the presence of ascorbic acid and beta-glycerophosphate o ver a 12-day period. Immediately after plating, the cells exhibited a fibroblastic morphology, lost their chondrocyte phenotype and expresse d type I collagen. On day 3, clusters of enlarged polygonal cells were found. These cell clusters synthetised type II collagen and formed an alcian-blue-positive matrix. The following days, a progressive increa se in the number of cells positive for type II collagen was noted and, on day 8, typical cartilaginous nodules were formed. These nodules in creased in size and number, spreading outward, laying down a dense mat rix which mineralized. Light and electron microscopy observations of c ross-sections of nodules confirmed the cartilaginous nature of this ti ssue formed in vitro with typical chondrocytes embedded in a hyaline m atrix. Furthermore, at the electron microscopic level, matrix vesicles were seen in extracellular matrix associated with the initiation of m ineralization. Typical rod-like crystals were present in the intercell ular spaces along the collagen fibers. These results indicated that in a specific environment, dedifferentiated chondrocytes were able to re differentiate and to form nodular structures with morphological ultras tructure of calcified cartilage observed in vivo.