Background/Purpose: Osteoarthritis (OA) is characterized by the progressive loss of cartilage structural extracellular matrix (ECM) components. The release of these proteins from the tissue can vary according to the stage of the disease and the specific joint affected. The aim of this study was to perform a quantitative proteomics approach to identify and quantify those proteins released from normal (N) and OA human articular cartilages capable of predicting the early stage of hip and knee OA.

Methods: Tissue explants were obtained from the dissection of 4 N and 4 OA cartilages, both from 2 femoral heads and 2 tibial condyles. Among the OA samples, we differentiated the wounded zones (WZOA) from those corresponding to the area adjacent to the lesion, or unwounded zones (UWOA). Cartilage shavings from each donor were cut into 6 mm discs and three discs/donor were placed into 96-well plates and cultured during 6 days. The conditioned media from each condition (N, WZOA and UWOA) were collected and their proteins were digested with trypsin. The resulting peptides were labelled with different isobaric tags using the iTRAQ reagents (ABSciex). Then, labelled peptides from the different conditions were mixed, desalted and separated by liquid chromatography (LC). The resulting fractions were grouped and resolved by reversed-phase nano-LC coupled to mass spectrometry (MS). The identification and relative quantification of the proteins was carried out with Protein Pilot 3.0 software.

Results: Globally we were able to identify 186 proteins released from the cartilage explants. After statistical analysis we found secreted proteins showing differences in abundance (0.7≤ ratio ≥1.3, p ≤0.05) between the different OA zones (WZOA and UWOA) and N samples from the different joints. We classified them into 3 sets of proteins: a first group of proteins modulated specifically in UWOA sample (early OA biomarkers); a second group of proteins altered only in WZOA samples (late OA biomarkers), and finally a third group modified in both OA zones (progression biomarkers). Some of these modulated proteins are common early and progression biomarkers for both hip and knee OA (Table1). Furthermore, we also identified that the release of cartilage intermediate layer protein 1 (CILP1), a protein involved in cartilage scaffolding, is increased in UWOA from hip OA cartilage but not in UWOA from knee OA, being a possible early specific biomarker for hip OA. This specific modulation was confirmed by Real-time PCR and western blot in other human cartilage samples (n=3).

Conclusion : We describe a novel panel of cartilage-secreted proteins with potential biomarker value. Interestingly, we have identified a specific protein, which specifically indicates hip OA onset. This protein is now being explored in biological fluids (synovial fluid and serum) for the development of early diagnosis and/or anti-OA therapy monitoring strategies.