Proteomic characterization of cartilage matrix synthesis and breakdown

Objectives: Development of neo-cartilage in vitro has important applications in tissue engineering/ cartilage repair and analysis of the response to catabolic agents and mechanical injury, two key contributors to cartilage degeneration in arthritis. However, primary chondrocyte culture is challenging due to the inter-dependence of phenotype with chondrocyte morphology, ECM and environment. We aimed to develop a high-density chondrocyte culture system and evaluate phenotype and response to catabolic agents using proteomics. Methods: Mouse P3 chondrocytes seeded at high density were maintained in DMEM/FCS/ascorbic acid. Independent 21-day cultures were then treated for 4 days (serum-free) with interleukin-1α (IL1) or all-trans-retinoic acid (RetA) to induce ECM breakdown. 7, 14 and 21-day and cytokine-treated cultures were harvested and proteins extracted sequentially using 1M NaCl then 4M GuHCl, followed by pepsin digestion of the GuHCl-insoluble fraction. Comparative analysis of media and sequential protein extracts of control and stimulated cultures was performed using SDS-PAGE, 2-DE and DIGE. Shotgun mass spectrometry was used for global profiling of the NaCl and GuHCl-soluble fractions. Results: Over 21 days, levels of GuHCl-soluble components (matrilin-1, collagen VI, SLRPs) and collagen II increased ~2-fold, while NaCl extracts showed little change. Haptoglobin, MMP3, cartilage gp-39 and lipocalin-2 were detected in the media of IL1-treated cultures, whereas RetA caused COMP release, consistent with our experiments in intact femoral head cartilage [1]. In the protein extracts of IL-1 and RetA cultures we detected loss of link protein and the appearance of aggrecan fragments. Conclusions: Our high-density chondrocyte cultures deposit a cartilage-like ECM, with further proteomic characterization of the NaCl and GuHCl-soluble fractions ongoing. Importantly, these cultures facilitate proteomic characterization of media and extracts, an improvement on previous analysis of intact cartilage degradation.