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 distal row of carpal bones (C2, C3, and C4) from eight left intercarpal joints--four from Standardbred Trotters and four from Swedish Warmblood horses--were used to assess the potential of magnetic resonance (MR) imaging to detect cartilage and bone lesions. The joints used in the study were classified by macroscopic and radiographic examinations as having normal, mild, moderate, or severe articular cartilage lesions and bone sclerosis. Those classifications correlated well with the appearance of the MR images. Bone sclerosis in the MR images was observed as regions of decreased signal intensity. Upon quantitative analysis of the MR images there was a significant difference (p < 0.0001) in the MR signal intensity from areas where radiographic bone sclerosis was observed compared to areas of radiographic nonsclerotic bone. In addition, the MR images were used to pilot the location of histology slices through areas of interest that were then examined microscopically; hence, the lesions found from the MR imaging examination were verified microscopically. It was concluded that cartilage lesions and cartilage loss are related to the sclerotic state of the underlying bone. The MR protocols developed in this study were applied on five intact cadaveric carpal joints, and it was concluded that MR imaging could successfully be used in the intact joint to detect minor cartilage and bone lesions not visualized by either radiography or macroscopic examination. Hence, MR imaging can be used to delineate interactions between articular cartilage and subchondral bone over time and in vivo.

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.

BackgroundOsteoarthritis (OA) is a destructive joint disease and there are no known biomarkers available for an early diagnosis. To identify potential disease biomarkers and gain further insight into the disease mechanisms of OA we applied quantitative proteomics with SILAC technology on the secretomes from chondrocytes of OA knees, designated as high Mankin (HM) scored secretome. A quantitative comparison was made between the secretomes of the medial and lateral femur condyle chondrocytes in the same knee since the medial femur condyle is usually more affected in OA than the lateral condyle, which was confirmed by Mankin scoring. The medial/lateral comparison was also made on the secretomes from chondrocytes taken from one individual with no clinically apparent joint-disease, designated as low Mankin (LM) scored secretome.ResultsWe identified 825 proteins in the HM secretome and 69 of these showed differential expression when comparing the medial and lateral femoral compartment. The LM scored femoral condyle showed early signs of OA in the medial compartment as assessed by Mankin score. We here report the identification and relative quantification of several proteins of interest for the OA disease mechanism e.g. CYTL1, DMD and STAB1 together with putative early disease markers e.g. TIMP1, PPP2CA and B2M.ConclusionsThe present study reveals differences in protein abundance between medial/lateral femur condyles in OA patients. These regulatory differences expand the knowledge regarding OA disease markers and mechanisms.

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.

Several organs in the body comprise cells coupled into networks. These cells have in common that they are excitable but do not express action potentials. Furthermore, they are equipped with Ca2+ signaling systems, which can be intercellular and/or extracellular. The transport of small molecules between the cells occurs through gap junctions comprising connexin 43. Examples of cells coupled into networks include astrocytes, keratinocytes, chondrocytes, synovial fibroblasts, osteoblasts, connective tissue cells, cardiac and corneal fibroblasts, myofibroblasts, hepatocytes, and different types of glandular cells. These cells are targets for inflammation, which can be initiated after injury or in disease. If the inflammation reaches the CNS, it develops into neuroinflammation and can be of importance in the development of systemic chronic inflammation, which can manifest as pain and result in changes in the expression and structure of cellular components. Biochemical parameters of importance for cellular functions are described in this review.

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.