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.

Articular cartilage (AC) has a poor innate healing capacity following significant injury. Autologous chondrocyte implantation is a repair technique which utilises in vitro-expanded chondrocytes combined with a periosteal patch. The chondrocytes are enzymatically digested from arthroscopically harvested tissue at an initial surgery and expanded in monolayer culture prior to implantation at a second procedure. Unfortunately, in vitro expanded chondrocytes appear unable to retain their fundamental phenotype resulting in dedifferentiated cells which produce a matrix of inferior quality. This study compares the matrix-component gene expression profiles of chondrocytes in their native chondrons and through multiple divisions in monolayer culture. We hypothesised that there would be a rapid decline of matrix-component gene expression within a few cell replications in monolayer culture. The goal is to understand more fully the process of chondrocyte dedifferentiation and to compare matrix-component gene expression during cellular expansion in vitro.

Human AC was obtained from tissue donors and operative patients. A portion of the AC was stored at −80°C for use as a control while the remainder was homogenised and enzymatically digested with collagenase. The released cells were plated in monolayer culture and passaged (2:1) when they approached confluence. RNA was extracted from the frozen cartilage control and the passaged chondrogenic cell lines from which cDNA was generated. Real time PCR was performed with primers specific for collagen I, collagen II, aggrecan, and GAPDH. Gene expression was quantified and profiles from the cells in their native chondron and passaged cells (p0-p9) were compared.

Cells, when removed from the extra-cellular matrix and plated in monolayer, experienced an immediate upregulation of collagen I which persisted throughout all passages. In contrast, there was a stepwise decrease in collagen II with each successive passage until p8-p9 when the expression became undetectable. Aggrecan expression only decreased minimally as the cells were passaged.

Rapid dedifferentiation of monolayer cultured chondrocytes is a persistent barrier to AC tissue engineering including ACI. This study quantified the expression of relevant genes relating to AC generation and is an important first step to understanding cellular events, as alternative expansion techniques and cellular alternatives are sought.