ULTRASTRUCTURAL IMMUNOLOCALIZATION OF TYPE-VI COLLAGEN AND CHONDROITIN SULFATE 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 me dial collateral ligament (MCL) from humans and rabbits. Tissue obtaine d from MCL midsubstance was subjected to pre-embedding labelling with colloidal gold at the electron microscopic level with monoclonal antib odies (MAbs) against type-VI collagen and chondroitin sulphate (CS), b efore and after digestion with chondroitinase ABC and testicular hyalu ronidase. Tissue labelled with anti-type-VI MAbs showed gold conjugate s 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 tre atments, gold particles were found attached to granules that were inte rspersed throughout the network of type-VI microfilaments. Both the gr anules and gold labels were absent from the network following enzyme d igestion. Thin nonbeaded microfilaments that did not label with anti-t ype-VI MAbs also were present within the seams. The loss of these nonb eaded microfilaments following enzyme digestion suggested that they mi ght represent strands of hyaluronan. The codistribution and sequesteri ng of type-VI collagen and CS within discrete seams or channels sugges ts that these regions of the MCL midsubstance may contain higher conce ntrations of water than the surrounding dense fibrillar matrix.