Immunocytochemical expression of type I and type II collagens by rat Meckel’s chondrocytes in culture during phenotypic transformation

In culture, chondrocytes of Meckel’s cartilage can differentiate further to become bone-type collagen-synthesizing cells. Here, the replacement of type II collagen by type I collagen, accompanying expression of the osteocytic phenotype, was analysed by double immunofluorescence staining, histochemistry and electron microscopy. After 1 week in culture, formation of a toluidine blue-positive matrix, demonstrating the synthesis of cartilaginous proteoglycans, and the expression of type II collagen were detected. After 2 weeks, immunoreactivity specific for type II collagen was detected along the cartilaginous areas of the nodules, and type I collagen appeared in association with the immunopositive extracellular matrix around spindle-shaped cells. Electron microscopy revealed that the extracellular matrix at this stage was composed of homogeneous fine fibrils of type II collagen and thick cross-banded bundles of type I collagen; there was also continuity between the type I and II collagens. Double immunofluorescence staining of 3 week-old cultures revealed that type II collagen had been replaced by type I, which was synthesized by small round cells that appeared at the top of the nodules. With further passage of time in culture, the distribution of type I collagen expanded further towards the peripheral areas from the central areas of the nodules. The present combination of ultrastructural analysis and double immunofluorescence staining shows that the transition from synthesis of cartilage-specific type II collagen to expression of type I collagen occurred sequentially in spindle-shaped cells located at the top of nodules in conjunction with the further differentiation of Meckel’s cartilage cells.