Proteoglycan- and collagen-degrading activities of neutral proteases from fresh and cryopreserved articular cartilage explants and the chondrocytes: an in vitro biochemical study
Creators: name: Tavakol, Keramatolah
Publisher: name: University of Calgary
Year: 1989
Subjects: subject: QM 567 T38 1989
subject: Cartilage - Transplantation
subject: Cartilage - Cryopreservation

description: Bibliography: p. 134-142.
description: Cryopreservation is an established method of storing viable cells of many origins for long term. This approach has been successful in the preservation of chondrocytes isolated from articular cartilage. These cells survive the procedure and are able to grow and express their phenotype after freeze-thawing. Advances in orthopaedic medicine have made possible the successful use of fresh or cryopreserved human articular cartilage for transplantation in the repair of diseased or damaged human joints. Cryopreservation of osteoarticular segments has gained experimental and clinical support as a feasible approach for long term banking of the tissue for transplantation in humans. However, osteoarticular allografts appear to degenerate over the long term. We have hypothesized that the degeneration of articular cartilage after freeze-thawing might be due to cellular injuries which lead either to an increase in the level of proteinases, which degrade the tissue, or to decreased matrix synthesis. Our long term aim is to improve the clinical strategies of articular cartilage cryopreservation through identification of factors which affect the viability and function of the tissue. The specific objective of this project is to compare the proteolytic activity of cultured frozen-thawed cartilage explants and isolated chondrocytes with that of fresh controls in order to determine whether this parameter can be used as an index of cellular injury. This in vitro study qunatitated the protease activity released into the cultured media of fresh or frozen-thawed ovine cartilage explants and isolated chondrocytes. The proteolytic activity was also measured in the presence of interleukin-1 (IL-1) in the culture media. The presence of plasminogen activator and its inhibitor were identified in these media. The substrates were proteoglycan aggregates and collagen type II, which were purified from cartilage to homogeneity and radiolabeled. Their properties characterized by several biochemical criteria. The protease activity was shown to be synthesized de novo and was not active upon release unless incubated with aminophenylmercuric acetate. The active protease(s) had the ability to degrade both 3H-proteoglycan aggregates and 3H-collagen II. The proteoglycan-degrading and collagenolytic ac-tivities required neutral pH and calcium for full activity and were shown to be metalloproteases since their activity was abolished in the presence of EDTA and o-phenanthroline, two chelators of metal ions. Freeze-thawing of isolated chondrocytes in the culture media with DMSO (but not without) preserved the cell morphology and the synthetic and degradative functions. A 59% and 71% reduction, respectively, in the levels of proteoglycan-degrading and collagenolytic activities of cryopreserved cartilage explants correlated with a 61% loss of functional chondrocytes recovered from these samples. The surviving cells maintained their typical morphology, phenotype, degradative activities in culture, and their functionality did not appear to be dependent upon a DMSO pre-treatment of the explants. In addition, all categories of the explants and the cells which had survived the freeze-thawing, released proportion-ally increased levels of proteolytic activity into the media in response to IL-1 treatments. Our results suggest that while DMSO has an essential role in the preservation of isolated cartilage cells it may not pr otect the cells within the intact cartilage explants. These findings support the hypothesis that a reduction in the level of released pr oteolytic activity from cartilage explants in culture may reflect reduced numbers of functional chondrocytes, and hence cellular injury.