VIABILITY OF AN EXPLANT MODEL FOR STUDYING ARTICULAR CARTILAGE

Abstract

Current research strategies for studying articular cartilage (AC) repair include the observation of chondrocyte behaviour in monolayer cultures, the use of artificial matrices and animal models. Since AC relies on the diffusion of joint synovial fluid for nourishment, we hypothesised that it should be possible to develop a research model in which full-depth AC explants are maintained under established tissue culture conditions. Successful maintenance of explants for prolonged periods of time would represent a novel approach and provide a very powerful research tool to address a wide range of chondrocyte biology and matrix synthesis questions. The objective of the project was to examine the cell viability within an AC explant model maintained in tissue media.

AC samples were obtained from the femoral condyles of total knee arthroplasty patients. Cylindrical dowels (10mm in diameter) were harvested from these samples. The dowels consisting of full-depth AC with several mm of subchondral bone attached were placed in tissue culture flask (T-25) containing 15mls of the respective culture media and maintained at 37oC in an incubator containing 5% CO2. Dowels were cultured in a variety of different media formulations (DMEM/F12, CGM (chondrocyte growth medium)) as well as PBS (phosphate buffered saline) which served as a negative control. AC chondrocyte viability was evaluated after five weeks. After having determined the best medium for cartilage maintenance, a second study with a broader range of end-points was undertaken. All dowels collected, rated 2/4 on the Outerbridge scale for osteoarthritis, and were then grown for zero, four, eight or twelve weeks in DMEM/F12 and CDM (chondrocyte differentiating medium). At each time interval, the dowels were evaluated for viability (live/dead stain), general morphology (trichrome stain), distribution of matrix proteins and proteoglycans (aggrecan, Types I and II collagen – immunofluorescence).

After five weeks in PBS, there were no viable cells in the explant. Viability in the explants maintained in DMEM/F12 was 71% compared to 59.6% in the CGM treatment. The viability of the cells in the second study was 90% with DMEM/F12. After twelve weeks, the explant models stained well for general morphology and the distribution of proteoglycans and collagen was well maintained.

To our surprise, the DMEM/F12 medium actually demonstrated the highest cell viability. Typically, AC requires joint motion to pressurise the synovial fluid into the matrix, which augments the transport of nutrients to the cells. Given that this study did not include any form fluid pressurization, it is surprising that such high cell viability was observed. This suggests that passive diffusion alone may provide adequate nourishment in this model system. In conclusion, the explant model for studying AC damage and repair examined in this research appears to be quite promising. This novel approach may serve as the foundation for subsequent research into new treatment strategies for AC injury.