The results suggest a cellular MR from the IVD stem cell niche toward the annulus fibrosus and the inner parts of the IVD. These findings may be of importance for understanding IVD regenerative mechanisms and for future development of biological treatment strategies.

The molecular aspects of inflammation were investigated in equine articular cartilage explants using quantitative proteomics. Articular cartilage explants were stimulated with interleukin (IL)-1β in vitro for 25 days, and proteins released into cell culture media were chemically labeled with isobaric mass tags and analyzed by liquid chromatography-tandem mass spectrometry. A total of 127 proteins were identified and quantified in media from explants. IL-1β-stimulation resulted in an abundance of proteins related to inflammation, including matrix metalloproteinases, acute phase proteins, complement components and IL-6. Extracellular matrix (ECM) molecules were released at different time points, and fragmentation of aggrecan and cartilage oligomeric matrix protein was observed at days 3 and 6, similar to early-stage OA in vivo. Degradation products of the collagenous network were observed at days 18 and 22, similar to late-stage OA. This model displays a longitudinal quantification of released molecules from the ECM of articular cartilage. Identification of dynamic changes of extracellular matrix molecules in the secretome of equine explants stimulated with IL-1β over time may be useful for identifying components released at different time points during the spontaneous OA process.

The effects of high mobility group box protein (HMGB)-1, interleukin (IL)-1β, and IL-6 on equine articular chondrocytes were investigated, with emphasis on detecting differences between anatomical sites exposed to different loading in vivo, using three-dimensional (3D) cell cultures established with chondrocytes from dorsal radial facet (DRF, highly loaded) and palmar condyle (PC, less loaded) of the third carpal bone (C3). Expression of important genes involved in cartilage metabolism, presence of glycosaminoglycans and cartilage oligomeric matrix protein (COMP) in pellets, and concentrations of matrix metalloproteinase (MMP)-13 and aggrecan epitope CS 846 were evaluated. Compared to controls, IL-1β treatment increased gene expression of versican, matrix-degrading enzymes, and tissue inhibitor of metalloproteinase (TIMP)-1, and decreased aggrecan and collagen type I and type II expression. In addition, IL-1β-treated pellets showed decreased safranin O staining and increased COMP immunostaining and MMP-13 concentrations in culture supernatants. Effects of IL-6 and HMGB-1 on gene expression were variable, although upregulation of Sry-related high-mobility group box 9 (Sox9) was often present and statistically increased in HMGB-1-treated pellets. Response to cytokines rarely differed between DRF and PC pellets. Thus, site-associated cartilage deterioration in equine carpal osteoarthritis (OA) is not explained by topographically different responses to inflammatory mediators. Differences in gene expressions of structural matrix proteins in untreated DRF and PC pellets were noted in the youngest horses, which may indicate differences in the chondrocytes potential to produce matrix in vivo. Overall, a strong catabolic response was induced by IL-1β, whereas slight anabolic effects were induced by IL-6 and HMGB-1.

This is the first demonstration of a change in the glycosylation profile of lubricin in synovial fluid from diseased equine joints compared with that from normal joints. We demonstrate an identical proteolytic cleavage site of lubricin both in vitro and in vivo. The reduced sialation of lubricin in synovial fluid from diseased joints may affect the boundary lubricating ability of the superficial layer of articular cartilage and could be one of the early events in the progression of osteoarthritis.

SummaryBackgroundClinical tools to diagnose the early changes of osteoarthritis (OA) that occur in the articular cartilage are lacking.ObjectivesWe sought to identify and quantify a novel cartilage oligomeric matrix protein (COMP) neoepitope in the synovial fluid from the joints of healthy horses and those with different stages of OA.Study design In vitro quantitative proteomics and assay development with application in synovial fluids samples obtained from biobanks of well‐characterised horses.MethodsArticular cartilage explants were incubated with or without interleukin‐1β for 25 days. Media were analysed via quantitative proteomics. Synovial fluid was obtained from either normal joints (n = 15) or joints causing lameness (n = 17) or with structural OA lesions (n = 7) and analysed for concentrations of the COMP neoepitope using a custom‐developed inhibition enzyme‐linked immunosorbent assay (ELISA). Explants were immunostained with polyclonal antibodies against COMP and the COMP neoepitopes.ResultsSemitryptic COMP peptides were identified and quantified in cell culture media from cartilage explants. A rabbit polyclonal antibody was raised against the neoepitope of the N‐terminal portion of one COMP fragment (sequence SGPTHEGVC). An inhibition ELISA was developed to quantify the COMP neoepitope in synovial fluid. The mean concentration of the COMP neoepitope significantly increased in the synovial fluid from the joints responsible for acute lameness compared with normal joints and the joints of chronically lame horses and in joints with chronic structural OA. Immunolabelling for the COMP neoepitope revealed a pericellular staining in the interleukin‐1β‐stimulated explants.Main limitationsThe ELISA is based on polyclonal antisera rather than a monoclonal antibody.ConclusionsThe increase in the COMP neoepitope in the synovial fluid from horses with acute lameness suggests that this neoepitope has the potential to be a unique candidate biomarker for the early molecular changes in articular cartilage associated with OA.

Objective Chondrocytes are responsible for remodeling and maintaining the structural and functional integrity of the cartilage extracellular matrix. Because of the absence of a vascular supply, chondrocytes survive in a relatively hypoxic environment and thus have limited regenerative capacity during conditions of cellular stress associated with inflammation and matrix degradation, such as osteoarthritis (OA). Glucose is essential to sustain chondrocyte metabolism and is a precursor for key matrix components. In this study, we investigated the importance of glucose as a fuel source for matrix repair during inflammation as well as the effect of glucose on inflammatory mediators associated with osteoarthritis. Design To create an OA model, we used equine chondrocytes from 4 individual horses that were differentiated into cartilage pellets in vitro followed by interleukin-1β (IL-1β) stimulation for 72 hours. The cells were kept at either normoglycemic conditions (5 mM glucose) or supraphysiological glucose concentrations (25 mM glucose) during the stimulation with IL-1β. Results We found that elevated glucose levels preserve glucose uptake, hyaluronan synthesis, and matrix integrity, as well as induce anti-inflammatory actions by maintaining low expression of Toll-like receptor-4 and low secretion of glutamate. Conclusions Adequate supply of glucose to chondrocytes during conditions of inflammation and matrix degradation interrupts the detrimental inflammatory cycle and induces synthesis of hyaluronan, thereby promoting cartilage repair.

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Results indicated IL-6 stimulation decreased chondrocyte expression of the canonical Wnt signaling pathway transactivator β-catenin, induced expression of inhibitors of the Wnt pathway, and increased expression of GDF-5. This suggested IL-6 may inhibit the Wnt signaling pathway with subsequent upregulation of GDF-5 expression. Anabolic extracellular matrix metabolism in OCFs may be attributable to GDF-5 expression. This information could be useful for development of cartilage repair methods.