211, P211

A translational equine approach towards development of human biomarkers of early osteoarthritis

Anders Lindahl1,*

1Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Institute of Biomedicine, Göteborg, Sweden

Introduction: Osteoarthrtitis (OA) is a chronic low-grade inflammatory disease for which the diagnosis currently relies on clinical and radiological means whereas sensitive serum biomarkers specific for an early stage of the disease are lacking. The aim of the study was to identify specific molecular alterations of articular cartilage in an in vitro inflammation model. The pro-inflammatory cytokine interleukin (IL)-1β was used to induce degradation of articular cartilage explants cultured in vitro with the purpose of mimicking OA cartilage in early and later developmental stages.

Objectives: The specific objective was to longitudinally characterize and quantify the release of cartilage matrix components to the media by using a quantitative proteomic approach and translate the findings to human early OA.

Methods: Full thickness equine articular cartilage explants were harvested from the

weight bearing part of the distal metacarpal bone III in the metacarpal-phalangeal joint of three horses. The explants were cultured in the presence or absence of interleukin IL-1β in-vitro and cell media was changed and sampled at day 3, 6, 9, 12, 15, 18 and 22. Proteins were isolated from the harvested media and following tryptic digestion the peptides were labeled with isobaric tandem mass tags (TMT) and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). The TMT reporter ion intensities in MS/MS spectra were divided by reference reporter ion intensities, giving a relative amount for each quantified peptide. The relative amount of released components in media from IL-1β stimulated and unstimulated explants were compared over time.

Results: IL-1β stimulation resulted in an abundance of proteins related to the extra cellular matrix and the proteins were released at different time points during culturing time. Release of several cartilage matrix components e.g. aggrecan, cartilage oligomeric matrix protein (COMP), chondroadherin, thrombospondin-1 and proteoglycan 4 displayed high amounts at days 3 and 6, similar to the early stage of the disease process of OA in vivo. Collagen type XII release was identified at day 9 and 12 and the release of collagen type VI increased continually from day 18-22. Several small leucine-rich proteoglycans (SLRP) such as biglycan and lumican had the highest release at day 22, together with a high amount of collagen type II in IL-1β stimulated media. The unstimulated media showed signs of new synthesis of collagen type II, by the presence of high amount of procollagen type II C-propeptide and C endopeptidase enhancers, and this collagen synthesis was not at all prominent in the IL-1β stimulated media.

Conclusion: This inflammatory in vitro model display structural changes seen at distinct developmental stages of OA in vivo. Novel findings presented are the release, at different time points after IL-1β stimulation, of cartilage components in a specific time pattern in the order as follows; COMP, collagen type XII, collagen type VI, SLRPs and finally collagen type II, indicating a degradation of the collagen network in a distinct pattern, without evidence for new synthesis of collagen type II. The results from the unstimulated explants indicate repair capacity with an increase of collagen synthesis. The identification of early biochemical changes in equine cartilage is possible to translate towards humans since both genomes are sequenced and the found cartilage components would be of great importance in both evaluating disease modifying pharmaceutical compounds as well as in the development of biomarkers for early OA.

Keywords: Bone