Early Cartilage Degeneration: Correlation of Micro-Structural and Proteomic Analysis

Abstract

Pre-osteoarthritis in the joint remains elusive to the cartilage researcher. To address this, a bovine model of early cartilage degeneration has been used in recent years. In this study the state of early tissue degeneration in bovine cartilage was investigated, correlating subtle microstructural changes in the tissue with its protein profiles. A total of 44 bovine patellae showing a range of tissue states from completely intact (healthy) to those with localised mild-to-moderate degeneration of the cartilage surface (non-healthy) were used. The degeneration was highly localised to the distal-lateral quarter of the patella and this tissue state has been validated in previous studies to be an early state of degeneration in osteoarthritis. From each patella showing mild-to-moderate tissue degeneration, two samples were used for the analyses, one from the localised site of degeneration and the other from an adjacent intact region. The healthy patellae provided control samples, where two intact cartilage samples were obtained from the distal lateral quarter, and a region adjacent to it. In the first part of this study, the cartilage samples were systematically characterised based on their microstructural changes using Differential Interference Contrast optical microscopy and Mankin histological scoring. The main changes in the microstructure in early cartilage degeneration were found to be as follows: (1) cartilage surface irregularity with a progressive diminishing of the tangential zone, where in severe degeneration there were clefts and fissures, (2) chondrocyte clustering, (3) fibrillar matrix ‘destructuring’ and re-aggregation, (4) thickening of the zone of calcified cartilage and (5) presence of bone spicules and signs of an advancing cement line. These microstructural changes are consistent to being analogous to a pre-osteoarthritic state in human joints. Based on the above microstructural variations from early degeneration, the second part of the study reports on the associated protein profile variation. Comprehensive iTRAQ (Isobaric tags for relative and absolute quantitation) labelled LC-MS/MS were performed on whole cartilage tissue samples. A total of 191 proteins were identified. Correlating microstructural variations to known protein functions, 15 proteins, further validated using MRM assays, were identified as potential markers for the early degenerative process. These proteins, including a novel protein WFDC18-like protein, and their specific role in the subtle structural changes in cartilage are discussed in this work. Of interest also is that in the range of tissues with early degeneration, the differences in protein profiles were more pronounced in those from the mildly degenerated tissue group. The third part of this study involved analysing secretome profiles (unstimulated) of the cartilage explants from the different examined groups. The explants from cartilage with moderate levels of degeneration were found to have enhanced secretory action than samples from mildly degenerated tissues, contrasting to the abundance levels noted in the earlier part of the study (see above). An explanation for this reduction in protein abundance levels in the cartilage tissue with moderate level of degeneration, in which the secretome levels were higher than cartilage with mild degeneration, may be due to the increasingly destructured fibrillar matrix that facilitates protein loss. Also seen in the secretome profile was the novel protein WFDC18-like, with potential anti-protease activity, which was found to be significantly upregulated in the intact cartilage regions of the non-healthy patellae. Finally, in the last part of the study, a relatively new technique for studying cartilage protein profiles, MALDI-IMS, was explored. This technique allowed for easy quantification of region-to-region peptide variation of localised degenerated sites and adjacent intact tissue.