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Background: Autologous conditioned serum (ACS) is used to treat osteoarthritis in horses, although its effects are not fully investigated., Objectives: To investigate the effects of equine serum and conditioned serum on chondrocytes stimulated with interleukin (IL)-1β and cartilage explants with mild osteoarthritis., Study Design: In vitro experimental study., Methods: The effect of three different serum preparations (unincubated control [PS], serum incubated 24 h [PS24h] and serum incubated 24 h in ACS containers [PCS]) pooled from lame horses were tested in two in vitro models. IL-1β and IL-1 receptor antagonist (IL-1Ra) concentrations were measured in all sera. In model 1, chondrocyte pellet cultures were stimulated with IL-1β prior to treatment with the serum preparations for 2 and 48 h. Microarray, polymerase chain reaction, and matrix metallopeptidase-13 analyses were performed. In model 2, cartilage explants from horses with structural osteoarthritis were treated with PS or PCS on days 0, 6 and 12, or left untreated, and evaluated at day 24 using the OARSI grading scale for histological evaluation of articular cartilage., Results: The IL-1Ra concentration in PS24h and PCS was significantly higher than in PS. In model 1, inflammation- and cartilage matrix degradation-related genes were upregulated after 48 h in all treatment groups versus untreated controls. Cartilage matrix molecules, aggrecan and collagens, were downregulated in PS24h- and PCS-treated pellets versus untreated controls. Growth factor signalling genes were upregulated-FGF7 in all treatment groups, BMP2 in PS24h-, and INHBA in PCS-treated-compared with untreated controls. In model 2, the OARSI score at day 24 was not significantly different between treatment groups., Main Limitations: Results from in vitro models cannot be directly translated to in vivo situations., Conclusions: In vitro treatment with conditioned serum did not alleviate IL-1β-induced responses in chondrocyte pellets or lead to morphological improvement in osteoarthritic cartilage explants., (© 2022 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Nerve growth factor (NGF) is a neurotrophin that has been implicated in pain signaling, apoptosis, inflammation and proliferation. The resultant effects depend on interaction with two different receptors; tyrosine kinase A (TrkA) and p75 NTR . NGF increases in synovial fluid from osteoarthritic joints, and monoclonal antibody therapy is trialed to treat osteoarthritis (OA)-related pain. Investigation of the complex and somewhat contradictory signaling pathways of NGF is conducted in neural research, but has not followed through to orthopaedic studies. The objectives of this study were to compare the expression of NGF receptors and the downstream regulator BAX in synovial membranes from joints in various stages of OA. The horse was used as a model. Synovial membranes were harvested from five healthy horses postmortem and from clinical cases with spontaneous OA undergoing arthroscopic surgery for lameness. Four horses with synovitis without gross cartilage changes, four horses with synovitis and cartilage damage, and four horses with synovitis and intracarpal fractures were included. Samples were investigated by immunohistochemistry and results showed that nuclear staining of TrkA, p75 NTR and BAX increases in OA-associated synovitis. TrkA expression increased in early disease stages whereas increases in p75 NTR were most prominent in later disease stages with cartilage damage and fibrosis. Clinical significance: Suppression of NGF may result in varied effects depending on different stages of the osteoarthritic disease process., (© 2022 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Clinical tools to diagnose the early changes of osteoarthritis (OA) that occur in the articular cartilage are lacking., Objectives: We 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., Methods: Articular 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., Results: Semitryptic 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 Limitations: The ELISA is based on polyclonal antisera rather than a monoclonal antibody., Conclusions: The 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., (© 2017 The Authors Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Reason for Performing Study: The glycoprotein lubricin contributes to the boundary lubrication of the articular cartilage surface. The early events of osteoarthritis involve the superficial layer where lubricin is synthesised., Objectives: To characterise the glycosylation profile of lubricin in synovial fluid from horses with osteoarthritis and study secretion and degradation of lubricin in an in vitro inflammation cartilage model., Study Design: In vitro study., Methods: Synovial fluid samples collected from horses with joints with normal articular cartilage and structural osteoarthritic lesions; with and without osteochondral fragments, were analysed for the lubricin glycosylation profiles. Articular cartilage explants were stimulated with or without interleukin-1β for 25 days. Media samples collected at 3-day intervals were analysed by quantitative proteomics, western blot and enzyme-linked immunosorbent assay., Results: O-glycosylation profiles in synovial fluid revealed both Core 1 and 2 O-glycans, with Core 1 O-glycans predominating. Synovial fluid from normal joints (49.5 ± 1.9%) contained significantly lower amounts of monosialylated Core 1 O-glycans compared with joints with osteoarthritis (53.8 ± 7.8%, P = 0.03) or joints with osteochondral fragments (57.3 ± 8.8%, P = 0.001). Additionally, synovial fluid from normal joints (26.7 ± 6.7%) showed higher amounts of disialylated Core 1 O-glycan than from joints with osteochondral fragments (21.2 ± 4.9%, P = 0.03). A C-terminal proteolytic cleavage site in lubricin was found in synovial fluid from normal and osteochondral fragment joints and in media from interleukin-1β stimulated and unstimulated articular cartilage explants., Conclusions: 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., (© 2015 EVJ Ltd.)

Reasons for Performing Study: Validated noninvasive detection methods for early osteoarthritis (OA) are required for OA prevention and early intervention treatment strategies., Objectives: To evaluate radiography and low-field magnetic resonance imaging (MRI) for the detection of early stage OA osteochondral lesions in equine centrodistal joints using microscopy as the reference standard., Study Design: Prospective imaging of live horses and imaging and microscopy of cadaver tarsal joints., Methods: Centrodistal (distal intertarsal) joints of 38 Icelandic research horses aged 27-29 months were radiographed. Horses were subjected to euthanasia approximately 2 months later and cadaver joints examined with low-field MRI. Osteochondral joint specimens were classified as negative or positive for OA using light microscopy histology or scanning electron microscopy. Radiographs and MRIs were evaluated for osteochondral lesions and results compared with microscopy., Results: Forty-two joints were classified OA positive with microscopy. Associations were detected between microscopic OA and the radiography lesion categories; mineralisation front defect (P<0.0001), joint margin lesion (P<0.0001), central osteophyte (P = 0.03) and the low-field MRI lesion categories; mineralisation front defect (P = 0.01), joint margin lesion (P = 0.02) and articular cartilage lesion (P = 0.0003). The most frequent lesion category detected in microscopic OA positive joints was the mineralisation front defect in radiographs (28/42 OA positive joints, specificity 97%, sensitivity 67%). No significant differences were detected between the sensitivity and specificity of radiography and low-field MRI pooled lesion categories, but radiography was often superior when individual lesion categories were compared., Conclusions: Early stage centrodistal joint OA changes may be detected with radiography and low-field MRI. Detection of mineralisation front defects in radiographs may be a useful screening method for detection of early OA in centrodistal joints of young Icelandic horses., (© 2015 EVJ Ltd.)

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.

Objective: To evaluate the use of CT and MRI for guidance of osteochondral sample collection for histologic detection of early osteoarthritic lesions in centrodistal (distal intertarsal) joints of horses., Sample: Right tarsal joints from the cadavers of 24 Icelandic horses aged 29 to 31 months., Procedures: CT and MRI were used to evaluate the extent of suspected osteoarthritic changes in centrodistal joints, which were graded with a semiquantitative system. The anatomic regions with the highest grade of change were identified, and osteochondral samples were obtained from these regions. Samples were also obtained from the same centrodistal joints at predetermined sites. Histologic examination of all samples was performed, with samples classified as negative or positive for osteoarthritis, and results were compared between sample collection methods., Results: Histologic examination revealed osteoarthritic lesions in 29% (7/24) of centrodistal joints with the predetermined method and in 63% (15/24) with the image-guided method. Significant associations were identified between histologic osteoarthritis detection and the summed image-guided sample collection site image grades, central osteophytes, articular cartilage thickness abnormalities, grade 2 articular mineralization front defects, and grade 2 marginal osteophytes., Conclusions and Clinical Relevance: CT and MRI aided the detection of focal changes suggestive of early-stage osteoarthritis in the centrodistal joints of equine cadavers and may be useful for detection of similar disease in live horses. The first morphological changes of centrodistal joint osteoarthritis were suspected to be in the articular cartilage and the articular mineralization front regions.

Cytokine production in synovial membranes (SM) and osteochondral fragments (OCF) may influence the development of equine osteoarthritis (OA). In this study, the presence of interleukin (IL)-6 and cytoplasmic and extracellular high mobility group box protein (HMGB)-1 in SM and osteochondral tissue from healthy and diseased equine joints was investigated by immunohistochemistry. Additionally, microscopic synovitis was graded. IL-6 was commonly found in SM cells and in chondrocytes in uncalcified cartilage of OCF, whereas little staining was detected in healthy cartilage. Cytoplasmic and/or extracellular HMGB-1 was widespread only in SM from diseased joints, and also detected in OCF in areas of cartilage damage, fibrous repair tissue, and tidemark reduplication. Joints with OCF and cartilage lesions (without OCF) showed significantly higher median synovitis scores than healthy joints (p=0.013 and p=0.042, respectively). The study identifies OCF as a source of inflammatory mediators in equine OA, as shown by the presence of IL-6 and extracellular HMGB-1 in the fragment. Based upon HMGB-1 release in SM and OCF, further studies to investigate possible involvement of HMGB-1 in the pathogenesis of OA are warranted.

Reasons for Performing Study: One of the most common causes of lameness in racehorses is osteoarthritis (OA). Pathogenesis is not clear and pathological processes of the different joint tissues interact in often progressive events. The interface between cartilage and newly synthesised bone has been shown to be particularly enriched in bone sialoprotein (BSP), a cell-binding matrix protein., Objectives: To establish whether changes in the concentration of BSP may serve as a marker for early biochemical changes of the subchondral bone., Methods: Articular cartilage, cartilage/bone interface and subchondral bone of the proximal third carpal bone from 3 Standardbred trotters were analysed ultrastructurally for the presence of BSP in normal and degenerative areas., Results: A marked increase of BSP in the cartilage/bone interface with degenerative changes of the bone and cartilage compared to the morphologically intact cartilage/bone interface was noted, but levels of the protein were distinctly lower in the distal bone., Conclusions: The results indicate that BSP has the potential to be used as a marker for changes in bone metabolism in the subchondral bone., Potential Relevance: Tools to monitor early biochemical changes within the connective tissues of the joint in vivo are essential in studies of the pathogenesis of OA. These could be used to monitor and understand such changes in relation to load, exercise, training programmes, inflammation and the development of OA.

Reasons for Performing Study: Osteoarthrosis (OA) in the distal tarsal joints, bone spavin, is a well known condition which is common in Icelandic horses age 6-12 years., Objectives: To determine the nature, location and age of appearance of early radiographic and histological changes in the centrodistal tarsal joint (CD) of young Icelandic horses., Methods: Slab sections from the CD of young Icelandic horses were examined by high detail radiography (age 6 months to 6 years, n = 111) and histology (age 6 months to 4 years, n = 82) to detect and describe the early changes indicative of OA. Horses younger than 5 years were unridden., Results: Chondronecrotic lesions histologically similar to those described in the early pathogenesis of OA were seen in 33% of the joints, located both medially and laterally. Radiographic sclerosis of the subchondral bone was recorded in 60% of the specimens, most often medially. Medial location was not associated with chondronecrosis, but was strongly related to age. Sclerosis was an infrequent finding on the lateral side, and was probably secondary to chondronecrosis in the corresponding part of the joint. Small defects in the subchondral bone were considered to be the most specific radiographic sign of OA as they were strongly associated with chondronecrosis., Conclusions: The high prevalence of chondronecrosis in the young horses indicates an early onset and slow progression of the disease. The early appearance also shows that the initiation of the disease is unrelated to the use of horses for riding. As clinical manifestation of OA in the distal tarsal joints is most often described in mature or old horses, the first stages of the disease are not likely to result in clinical signs. Subchondral bone sclerosis did not appear to be a primary factor in the development of OA in the CD but was considered to reflect an uneven distribution of biomechanical forces within the joint., Potential Relevance: The development of OA in the CD of young Icelandic horses seems to be due to poor conformation or joint architecture rather than trauma or overloading. These aetiological factors are likely to be of importance for OA in the distal tarsal joints in other breeds as well. The influence of hindlimb conformation and the architecture of the distal tarsal joints on the biomechanics of joints need to be investigated, preferably by locomotion analysis in young horses.

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.

Osteoarthrosis (OA) of the antebrachiocarpal joint from 7 riding horses is described. The horses were old mares and developed severe OA, with ankylosis in some of the joints. The lesions were bilateral, and the owners noticed the lameness in a late event. The cause of severe OA in these mares is not clear. The fact that OA was bilateral indicates that a single traumatic injury is unlikely as an etiologic factor. Considering the severe joint lesions it took long time before the horse-owners noticed the lameness. It is discussed if the threshold of pain is higher in the antebrachiocarpal joint compared with the middle carpal joint.

Objective: Native biglycan (BGN), which can undergo proteolytic cleavage in pathological conditions, is well known to be involved in bone formation and mineralization. This study aimed to delineate the specific cleavage fragment, a neo-epitope for BGN (BGN262), in synovial fluid (SF) from young racehorses in training, osteoarthritic (OA) joints with subchondral bone sclerosis (SCBS), and chip fracture joints.Design: A custom-made inhibition ELISA was developed to quantify BGN262 in SF. Cohort 1: A longitudinal study comprising 10 racehorses undergoing long-term training. Cohort 2: A cross-sectional study comprising joints from horses (N = 69) with different stages of OA and radiographically classified SCBS. Cohort 3: A cross-sectional study comprising horses (N = 9) with chip fractures. Receiver operating characteristic (ROC) curve analysis was performed (healthy joints vs chip joints) to evaluate BGN262 robustness.Results: Cohort 1: SF BGN262 levels from racehorses showed a statistical increase during the first 6 months of the training period. Cohort 2: BGN262 levels were significantly higher in the SF from severe SCBS joints. Cohort 3: SF BGN262 levels in chip fracture joints showed a significant increase compared to normal joints. The ROC analysis showed an AUC of 0.957 (95% C.I 0.868-1.046), indicating good separation between the groups.Conclusions: The data presented show that BGN262 levels increase in SF in correlation with the initiation of training, severity of SCBS, and presence of chip fractures. This suggests that BGN262 is a potential predictor and a novel biomarker for early changes in subchondral bone (SCB), aiming to prevent catastrophic injuries in racehorses. [ABSTRACT FROM AUTHOR]

The aim of the present study was to correlate the levels of COMP and aggrecan as indicators of tissue damage, in synovial fluid (sf) from carpal joints of acutely lame racehorses, with macroscopical lesions of articular cartilage (OA), osteochondral fractures and ligament tears found at arthroscopy.Sixty-three lame horses [49 Standardbred trotters (STB) and 14 Thoroughbreds (TB)] in conventional training and racing that underwent arthroscopy of their middle carpal or radiocarpal joints were included in the study. Intact as well as fragmented COMP and aggrecan released into the synovial fluid were quantified by western blot analyses and ELISA. The expression of COMP in tissues was estimated by mRNA in situ hybridisation and protein immunolocalisation in cartilage and osteochondral fractures.The concentration of sf-COMP was higher in TB with an osteochondral fracture than in STB with osteochondral fractures and TB and STB with OA. The chondrocytes in middle and deep zones of the articular cartilage of the osteochondral fragments (from a TB) expressed COMP mRNA, in contrast to the cartilage on the opposite side of the fracture where no expression was detected. In the synovial fluid from a joint (TB) with osteochondral fractures only intact COMP was present, whereas, fragmented COMP was more prominent in synovial fluid from a joint with OA. The concentration of sf-aggrecan did not differ between the two breeds, or between different lesions. The increased concentration of sf-COMP in TB with osteochondral fractures, but not in synovial fluid from equine joints with OA, is a novel finding. The results from this study indicate that elevated sf-COMP concentration in the joints of Thoroughbreds may be a useful marker for carpal joint osteochondral fragments. [Copyright &y& Elsevier]

In the present study, right hind limb bones from 46 12-month-old bulls with no clinical signs were examined to identify and describe lesions that could predispose the stifle and tarsal joints to osteoarthritis. The bulls came from a performance testing station and were slaughtered due to a low index at the end of the testing period 1996–97. Differences in frequency of lesions among breeds as well as the relationship between lesions and growth rate were evaluated. Forty-five (97.8 %) of the 46 bulls had lesions in the joints and/or growth plates. Prevalence of lesions was 100 % in the Charolais (22/22), the Hereford (8/8), and the Limousin (4/4) breeds, and 85.7 % (6/7) in the Simmental breed. The stifle was affected in 37, the tarsus in 33, and the growth plates in 34, of the 46 bulls. Lesions found in the stifle joint were: osteochondrosis of the articular-epiphyseal cartilage complex (AECC) (25), subchondral bone cyst of the distal femur (1), fragmentation of the medial intercondylar eminence of the tibia (20), cleft in the distal part of the patellar groove (28), and an avulsion fracture of the lateral condyle together with a partial tear of the cranial cruciate ligament (1). Lesions found in the tarsal joint were: osteochondrosis of the AECC (23), ulcerative lesions of the articular cartilage of the talus (25), and fracture of the medial malleolus (4). Twenty-eight bulls had lesions of osteochondrosis at the AECC and 37 at the growth plates. When osteochondrosis at the AECC and thickening of the growth plates were combined, 44 of the 46 bulls had at least one lesion at the AECC and/or the growth plate. Prevalence of bulls with at least one lesion was similar between breeds, but the number of lesions per bull was significantly higher in Charolais followed by Simmental, Hereford, and Limousin. Number of lesions per bull was significantly correlated with daily weight gain, carcase weight, and the width of the proximal tibial epiphysis. Lesions were... [ABSTRACT FROM AUTHOR]

• Nerve Growth Factor (NGF) and its receptors are present in equine joints. • NGF is increased in acutely inflamed joints and joints with chronic osteoarthritis (OA). • Receptor expression was variable and affected by the degree of OA. Nerve growth factor (NGF) is a neurotrophin with many functions. In humans, it is involved in inflammation, nerve growth, apoptosis and pain signalling. Increased concentrations of NGF in synovial fluid has been shown in humans and dogs with osteoarthritis. Despite osteoarthritis being a common problem in horses, no studies have previously been published on NGF in the equine joint. The aim of this study was to quantify NGF in equine synovial fluid from healthy joints, acutely inflamed septic joints and joints with structural changes associated with osteoarthritis. A secondary aim was to identify the localisation of NGF and its two receptors, TrkA and p75NTR, in healthy and osteoarthritic articular cartilage. NGF concentrations in synovial fluid from osteoarthritic joints (n = 27), septic joints (n = 9) and healthy joints (n = 16) were determined by ELISA. In addition, articular cartilage from osteoarthritic and healthy joints was examined for NGF, TrkA and p75NTR using immunohistochemistry staining. NGF was present in equine synovial fluid and articular cartilage. Compared to synovial fluid from healthy joints, NGF concentration was higher in synovial fluid from joints with structural osteoarthritic changes (P = 0.032) or acute septic inflammation (P = 0.006). In articular cartilage with severe osteoarthritic changes, there was more abundant positive immunohistochemistry staining for NGF and its receptors than in normal articular cartilage. Further studies should focus on identifying precursor forms of NGF, and on receptor expression and downstream signalling of TrkA and P75NTR in health and disease. [ABSTRACT FROM AUTHOR]