The aim of this study was to examine the response of secondary cartilage to ascorbic acid in vitro. Cells which had not attained the prechondroblast stage when cultured, did not chondrify in vitro, even when exposed to high levels (400 µg/ml) of ascorbic acid. On the other hand, prechondroblasts cultured in the presence of low levels (100 µg/ml) of ascorbic acid did chondrify. Once cytodifferentiation commenced, chondrogenesis was independent of the presence of ascorbic acid in the medium. Cartilage which differentiated in the presence of continuous high levels ( > 200 µg/ml) of ascorbic acid modulated to a tissue which consisted of hypertropic chondrocytes in a highly collagenous extracellular matrix. At the light-microscopic level, this tissue was classified as chondroid bone. At the ultrastructural level, the collagen was seen to be in the form of cross-banded fibres more typical of bone than cartilage. Prelabelling of cultures with either [3H]-thymidine or [3H]-proline showed that neither dedifferentiation of chondrocytes nor the resumption of mitotic activity was responsible for the alterations in the extracellular matrix. The differentiated chondrocytes can secrete an altered extracellular matrix without undergoing cytological dedifferentiation. The role of ascorbic acid in the attainment and maintenance of the differentiated state is briefly discussed.

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