Histochemical Visualization of the Cartilage Hyaladherins Using a Biotinylated Hyaluronan Oligosaccharide Bioaffinity Probe

Hyaluronan (HA) binding proteins (HABPs) were localized in cartilaginous ovine tissues (articular cartilage, intervertebral disc) using a biotinylated HA (bHA) oligosaccharide bioaffinity probe. The bHA oligosaccharide probe was prepared by partial digestion of HA with ovine testicular hyaluronidase, and the oligosaccharides were labeled with biotin hydrazide and purified by a combination of aggrecan G1 domain and avidin affinity chromatography. Hyaladherins were prominently visualized in tissue sections using the bHA oligosaccharide probe as pericellular components in hypertrophic epiphyseal and vertebral growth plate chondrocytes and in the enlarged cells of the cartilaginous end plate of the intervertebral disc. Weaker extracellular staining was also evident in the matrix of the ovine newborn hip and knee joint cartilages. The bHA oligosaccharide probe also visualized intracellular HABPs (IHABPs) in the hypertrophic growth plate chondrocytes of the primary ossification centers. Monolayer cultures of ovine chondrocytes rapidly internalized the bHA oligosaccharide affinity probe to discrete cytoplasmic, nuclear, and perinuclear regions, which were visualized by indirect fluorescent microscopy. This bHA oligosaccharide affinity probe may be useful in future investigations designed to characterize these novel cartilage IHABPs and the role that HA endocytosis plays in cellular regulatory processes in cartilage homeostasis.