Protein Levels and Microstructural Changes in Localized Regions of Early Cartilage Degeneration Compared with Adjacent Intact Cartilage

Abstract

Objective It was hypothesized that the respective protein profiles of bovine cartilage from sites of localized mild to moderate (GI to GII) degeneration versus adjacent sites of intact tissue would vary in accordance with the tissue microstructural changes associated with a pre-osteoarthritic state. Methods A total of 15 bovine patellae were obtained for this study. Paired samples of tissue were collected from the lateral region of each patella. If the patella contained a site of degeneration, a paired tissue set involved taking one sample each from the degenerated site and the intact tissue adjacent to it. Sufficient tissue was collected to facilitate 2 arms of investigation: microstructural imaging and proteome analysis. The microstructural analysis used a bespoke tissue preparation technique imaged with differential interference contrast optical microscopy to assess fibrillar scale destructuring and underlying bone spicule formation. An iTRAQ-based proteome analysis was performed using liquid chromatography–tandem mass spectrometry to identify the differential levels of proteins across the intact and degenerated cartilage and further, the results were validated with multiple reaction monitoring assay. Results In the healthy cartilage pairs, there was no significant variation in protein profiles between 2 adjacent sample sites. In pairs of tissue that contained a sample of GI/GII tissue, there were both significant microstructural changes as well as the difference in abundance levels of 24 proteins. Conclusions From the known functions of the 24 proteins, found to be strongly aligned with the specific microstructural changes observed, a unique “proteins ensemble” involved in the initiation and progression of early cartilage degeneration is proposed.