Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and × collagen and matrix mineralization

In this study we describe the collagen pattern synthesized by differentiating fetal human chondrocytes in vitro and correlate type × collagen synthesis with an intracellular increase of calcium and with matrix calcification. We show that type II collagen producing fetal human epiphyseal chondrocytes differentiate in suspension culture over agarose into hyptertrophic cells in the absence of ascorbate, in contrast to chicken chondrocytes which have been shown to require ascorbate for hypertrophic differentation. Analysis of the collagen synthesis by metabolic labeling and immunoprecipitation as well as by immunofluorescence double staining with anti type I, II or × collagen antibodies revealed that type × collagen synthesis was initiated during the third week. After 4 weeks culture over agarose we identified cells staining for both type I and × collagen, indicating further differentiation of chondrocytes to a new type of ‘post-hypertrophic’ cell. This cell type, descending from a type × collagen producing chondrocyte, is different from the previously described ‘dedifferentiated’ or ‘modulated’ types I and III collagen producing cell derived from a type II collagen producing chondrocyte. The appearance of type I collagen synthesis in agarose cultures was confirmed by metabolic labeling and immunoprecipitation and challenges the current view that the chondrocyte phenotype is stable in suspension cultures. An increase in the intracellular calcium concentration from 100 to 250 nM was measured about one week after onset of type × collagen synthesis. First calcium deposits were detected by alizarine red S staining in type × collagen positive cell nodules after 4 weeks, again in the absence of ascorbate. From these observations we conclude a sequence of events ultimately leading to matrix calcification in chondrocyte nodules in vitro that begins with chondrocyte hypertrophy and the initiation of type × collagen synthesis, followed by the increase of intracellular calcium, the deposition of calcium mineral, and finally by the onset of type I collagen synthesis.