Ultrastructural immunolocalization of type-VI collagen and chondroitin sulphate in ligament

Immunological methods were used to determine the identity of the major components comprising a network of electron-dense seams (described by the authors in a previous work) within the extracellular matrix of medial collateral ligament (MCL) from humans and rabbits. Tissue obtained from MCL midsubstance was subjected to pre-embedding labelling with colloidal gold at the electron microscopic level with monoclonal antibodies (MAbs) against type-VI collagen and chondroitin sulphate (CS), before and after digestion with chondroitinase ABC and testicular hyaluronidase. Tissue labelled with anti-type-VI MAbs showed gold conjugates attached to the microfilamentous component of the seams both before and after enzyme digestion, which confirmed the identity of the beaded microfilaments as type-VI collagen. Treatment of the tissue with anti-CS MAbs resulted in labelling of undigested tissue only. In these treatments, gold particles were found attached to granules that were interspersed throughout the network of type-VI microfilaments. Both the granules and gold labels were absent from the network following enzyme digestion. Thin nonbeaded microfilaments that did not label with anti-type-VI MAbs also were present within the seams. The loss of these nonbeaded microfilaments following enzyme digestion suggested that they might represent strands of hyaluronan. The codistribution and sequestering of type-VI collagen and CS within discrete seams or channels suggests that these regions of the MCL midsubstance may contain higher concentrations of water than the surrounding dense fibrillar matrix.