Evaluation of chondrocyte micromass culture for the study of cartilage degradation

Background

An understanding of the mechanisms involved in cartilage degradation that occur in rheumatoid arthritis and osteoarthritis is essential for the development of treatments to block disease progression. Culturing chondrocytes to synthesize a cartilage matrix would ideally provide a cartilage matrix of consistent composition that would reproducibly respond to different stimuli. When cultured in monolayers, chondrocytes convert to a fibroblastic morphology and gradually lose their native phenotype, no longer able to synthesize type II collagen and aggrecan. However, when chondrocytes are cultured at a high density in the presence of growth factors, the chondrocytes regain their native phenotype and native round morphology [1].

Objective

To optimise the culture conditions for chondrocyte micromass synthesis of matrix components, and to evaluate the response of the culture to cytokine stimulation by measuring the degradation of matrix components.

Methods

Chondrocytes extracted from bovine nasal cartilage were passaged several times to produce substantial cell stocks. Harvested cells were resuspended at 2 × 10⁷ cells/ml and seeded in the centre of each well of a 48-well plate (20 μl/well). Cultures were maintained in a serum-free medium with or without ITS (insulin, transferrin and selenium), ascorbic acid, and transforming growth factor beta 1 (TGFβ₁) for 14–21 days. Histological analysis of the micromass cultures examined the presence of proteoglycans and type II collagen. The effects of cytokine stimulation with IL-1, oncostatin M (OSM), and the combination of both cytokines on matrix degradation were tested by analysing proteoglycan and collagen release. Collagenase activity was measured by bioassay.

Results

Both ITS and ascorbic acid were necessary for maximum collagen incorporation into micromass cultures. Collagen and proteoglycan incorporation increased with increasing TGFβ₁ concentration and increasing culture duration, maximum incorporation detected when TGFβ₁ was used at 30 ng/ml and cultures were maintained for 21 days. Histology showed that both proteoglycans and type II collagen were present throughout the chondrocyte micromass matrix. When micromass cultures were stimulated with IL-1, OSM and IL-1/OSM for 14 days, proteoglycan release was greater compared with control, but there was no difference for collagen release (see Fig. 1). Active collagenase levels were negligible, although high levels of pro-collagenase were detected.

Figure 1

The effect on proteoglycan and collagen release when bovine nasal chondrocyte micromass cultured for 14 days in serum-free medium supplemented with 25 ng/ml transforming growth factor beta 1 and then stimulated for 14 days with IL-1 (1 ng/ml), oncostatin M (OSM) (10 ng/ml) and IL-1/OSM combination (1/10 ng/ml). A significant difference with control was identified by Student's t test, *** P < 0.005.

Conclusion

The chondrocyte micromass cultures synthesized a matrix that contained proteoglycan and type II collagen. The micromass culture may serve as a good model for studying proteoglycan but not collagen degradation.