Hyaluronan-mediated aggregation of limb bud mesenchyme and mesenchymal condensation during chondrogenesis.

Cell condensations are the initial structures in the formation of proper cartilage and skeletal patterning in the developing vertebrate limb. Chondrogenic differentiation is dependent upon the cell-cell and/or cell-matrix interactions which take place during the condensation process. Coincident with the onset of condensation is the expression by limb mesenchyme of specific cell surface binding sites for the extracellular matrix macromolecule hyaluronan. The association of hyaluronan with the cell surface can influence the behavior of cells, especially cell aggregation. In this study the possible involvement of hyaluronan as an extracellular linker molecule in the cell-cell adhesion event during mesenchymal condensation was investigated in the avian limb model. Hyaluronan hexasaccharides were used to prevent the multivalent interactions that occur between native hyaluronan macromolecules and the cell surface. Our studies show that hyaluronan is required for early adhesive cell-cell interactions of limb bud mesenchyme and that perturbations of hyaluronan-cell interactions with hyaluronan hexasaccharides result in a delay in the formation of condensations as well as a delay in chondrogenic differentiation of mesenchymal cells in micromass cultures.

Matrix accumulation and retention in embryonic cartilage and in vitro chondrogenesis.

Since hyaluronan anchors the proteoglycan-rich pericellular matrix to chondrocytes, hyaluronan-cell interactions may direct cartilage matrix assembly. To test this hypothesis, the competitive binding of hyaluronan hexasaccharides for native hyaluronan during matrix assembly, accumulation and retention in embryonic cartilage was studied. Chondrocytes released from explants with collagenase P retained pericellular matrices, but chondrocytes appeared "matrix-free" when released from hexasaccharide-treated explants. Decreased safranin O staining was also observed in the hexasaccharide-treated explants. This loss of proteoglycan retention was demonstrated quantitatively in the cartilage extracts and recovered in the media. The continual presence of hexasaccharides in micromass cultures resulted in decreased proteoglycan deposition. Increased proteoglycan retention, indicative of matrix repair, occurred following hexasaccharide wash-out. Thus, native hyaluronan-chondrocyte interactions are important for the assembly and maintenance of cartilage matrix.