Anionic sites in articular cartilage revealed by polyethyleneimine staining

Articular cartilage is a unique tissue that contains neither blood vessels nor nerves, and that performs mechanical loading during joint movement. The se properties are endowed by abundant glycosaminoglycans (GAGs), which are capable of retaining water-soluble substances. The GAGs attach to core prot eins and form proteoglycans. Although many studies have focused on proteogl ycans and collagen fibrils in cartilage, little is known about the nature o f the negative charge of GAGs. Recently, we investigated this subject using a cationic dye, polyethyleneimine (PEI), with several different techniques such as pre-embedding, post-embedding, and quick-freezing and deep-etching methods. In addition, we investigated whether the anionic charge is altere d at low pH, using PEI and cationic colloidal gold (CCG) labeling. The shap es of PEI-positive structures revealed by the pre-embedding method varied a t different pHs. Three-dimensional analysis using the quick-freezing and de ep-etching method demonstrated that meshwork structures composed of fine fi laments were decorated with tiny PEI granules. Additionally, the meshwork s tructure was broken down after chondroitinase ABC digestion. These data ind icate that the large PEI deposits observed in pre-embedding preparations ar e, at least in part, artificial images, and that the meshwork structure con sists of chondroitin sulfate-retaining anionic sites. Low pH conditions cha nged PEI or CCG labeling patterns, showing that negative charges of GAGs in articular cartilage are altered under environmental pH conditions. These f indings demonstrate that binding capacities of anionic sites to water-solub le or ionic substances are greatly affected by pH alterations without actua lly decreasing the number of anionic sites. Therefore, to understand cartil age dynamics and the pathogenesis of joint diseases in greater detail, alte rations of anionic charge during mechanical leading or under pathological c onditions should be examined in future studies. (C) 2001 Elsevier Science L td. All rights reserved.