Cytochemical evidence for a proteoglycan-associated filamentous network in ligament extracellular matrix

The purpose of this investigation was to examine the extracellular matrix of rabbit ligament before and after digestion with glycosaminoglycan degrading enzymes. In order to preserve and enhance the visibility of negatively charged tissue components, particularly the glycosaminoglycan-containing proteoglycans, the cationic stains ruthenium red (RR) and ruthenium hexamine trichloride (RHT) were used. Cross-sections of the midsubstance of 10-month-old (mature) rabbit medial collateral ligaments fixed using conventional procedures revealed a sparse population of stellate-shaped cells that did not appear to be interconnected. Similar tissue fixed in either RR or RHT showed an extensive network of thin, electron-dense "seams" that interconnected cells and appeared to irregularly subdivide the extracellular matrix (ECM). These seams mainly consisted of a meshwork of microfilaments throughout which small granules were dispersed. Numerous 14-nm microfibrils, as well as mature elastic fibers were also present within the seams. The size and shape of the microfilaments, together with their threadlike, beaded appearance suggested that they could be Type VI collagen. The seam granules were easily removed with chondroitinase ABC, chondroitinase AC II, and mild (0.18 M) salt treatment. Only chondroitinase ABC succeeded in removing additional granules, tentatively identified as proteodermatan sulphate molecules, that were periodically located at d band sites along the Type I collagen fibrils. These results suggest that the seam granules are not dermatan sulphate containing proteoglycans, and further, that these proteoglycans may be sequestered into specific zones within the ECM through loose association with the seam microfilaments. While the functional significance of the seams remains unknown and their specific composition clearly requires further study, it is likely that they represent important functional (e.g., viscoelastic) or biological (e.g., nutritional) subdivisions of ligament substance.