Confocal analysis of the molecular heterogeneity in the pericellular microenvironment produced by adult canine chondrocytes cultured in agarose gel.

Adult articular chondrocytes are each surrounded by a heterogeneous microenvironment and together form the chondron. Since little is known of chondron development, agarose gel culture, confocal immunohistochemistry and image analysis have been used to characterize the molecular anatomy and temporal development of the chondrocyte pericellular microenvironment in vitro. Two structurally distinct domains were identified during the 12-week culture period. The first comprised a narrow glycocalyx, 1-3 microns in width, which consolidated over time and was rich in collagen types II, VI, IX and XI, fibronectin, decorin and the aggrecan epitopes, 5D4 and HABR. The second region emerged after 4-6 weeks in culture and progressively developed a broad territorial region up to 12 microns wide around the chondrocyte and pericellular glycocalyx. Co-localization studies confirmed the dominance of aggrecan epitopes 2B6, EFG-4, 5D4 and HABR in the territorial domain, whereas surface density mapping with NIH image revealed two patterns of staining, one punctate and stippled, the other more uniform in distribution. The pericellular differentiation identified appeared analogous to the chondrons of adult articular cartilage, and provides an appropriate in vitro model for further studies of cell surface receptor function in the orchestration of pericellular matrix assembly.