Lee, David Alan; (1991) The effect of matrix depletion on articular cartilage. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The object of the present study was to investigate the effect of matrix depletion on the morphology, ultrastructure and metabolism of adult bovine cartilage in explant culture. Matrix depletion was achieved by incubating the tissue in medium containing either Streptomyces hyaluronidase (used at 10 unit.ml-1) or Clostidium collagenase (used at either 20 or 100 unit.ml-1) for 24 hr at 31°C. The tissue was subsequently cultured for a further two weeks in medium without enzymes. Control explants were cultured in medium without enzymes throughout the culture period. The morphology and ultrastructure of matrix-depleted tissue has been investigated using light and both transmission and scanning electron microscopy. DNA synthesis has been assessed using tritiated thymidine labelling, whilst proteoglycan (PG) synthesis has been investigated using labelling and immunolo-calisation. Proteoglycans and hyaluronan were lost from control tissue at a steady rate, equating to about 9% per day for PG and 5% per day for hyaluronan. An increased loss of PG and hyaluronan was detected from both hyaluronidase and collagenase-treated tissue during enzyme incubation although the rate of loss rapidly returned to normal once the enzymes were removed. The loss of collagen from control and hyaluronidase-treated cultures was minimal, whilst collagenase-treated cultures lost up to 50% of their collagen during treatment. The rate of PG synthesis in control and hyaluronidase-treated explants showed an initial rise followed by a reduction throughout the rest of the culture period. Collagenase-treated explants exhibited an increased rate of PG synthesis. We have reported that PG depletion in hyaluronidase-treated cultures is primarily from the surface zones and that the overall ultrastructure and architecture remains intact. Collagenase-induced disruption of tissue architecture has been reported and is most noted in the surface zones. Cellular changes and the formation of abnormal outgrowths have been described in collagenase-treated tissue. The investigation of DNA synthesis, as assessed by tritiated thymidine labelling, revealed that few chondrocytes had undergone DNA replication during the culture period in control and hyaluronidase-treated cartilage (about 4% in two weeks). We have detected an increase in the number of labelled cells in collagenase-treated tissue. The increase was dose-dependent and most marked in area of the tissue which showed the greatest morphological tissue disruption. A link between cell flattening and the onset of DNA synthesis is proposed, based on observations of explant cultures and cultures of isolated chondrocytes. We have reported the presence of novel chondroitin sulphate epitopes, recognised by the antibodies 3B3 (without chondroitinase) and 7D4 in both control and enzyme-treated cartilage from day 3 onwards. These epitopes are not normally found in normal adult cartilage and showed differential distribution in cultured cartilage.

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