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Articular cartilage can be directly imaged using ultrasonography. The fetlock is a common site of osteochondrosis, with the sagittal ridge of the third metacarpal bone most commonly affected. In osteochondrosis, cartilage thickening may be an initial finding. This postmortem study investigated the ability of ultrasonography to accurately measure the dorsodistal articular cartilage of the third metacarpal bone in young horses, compared to computed tomographic arthrography (CTA) and histological measurements. A total of 33 metacarpophalangeal joints from 18 horses between the ages of 12 days and 10 months old were imaged ultrasonographically and with CTA and sectioned and measured using histology. Imaging measurements were made by two observers. Despite overall weak agreement between ultrasonography and histology, the best agreement was at the distal aspect of the sagittal ridge of the third metacarpal bone. Interobserver agreement at this site was also moderate. CTA showed poor agreement overall with histology. Cartilage thickness decreased with age on ultrasonography, CTA, and histology. In conclusion, ultrasonography is a more accurate imaging modality than CTA in the assessment of cartilage in young horses., (© 2024 The Author(s). Veterinary Radiology & Ultrasound published by Wiley Periodicals LLC on behalf of American College of Veterinary Radiology.)

Background: Stifle osteoarthritis (OA) lesions are most common in the medial femorotibial (MFT) compartment. Their characterisation and mapping will inform equine veterinarians towards an accurate diagnosis of OA., Objectives: Investigate and map micro-CT (μCT) changes in the hyaline articular cartilage (HAC) in the medial femoral condyle (MFC) and medial tibial plateau (MTP)., Study Design: Ex vivo cadaveric., Methods: Stifles (n = 7 OA and 17 control [CO]) were retrieved from a tissue bank. The MFC and MFT were imaged with μCT. Regions of interest (ROIs) were cranial (MFCcr; MTPcr) and caudal (MFCca; MTPca) sites. In each ROI, μCT images were scored for HAC fibrillation, surface mineralisation and for the presence of high-density mineralised protrusions (HDMP). The lesions were mapped, and site-matched histology was performed., Results: The microstructure of healthy and abnormal HAC was discernible on μCT images and confirmed with histology. HAC fibrillation was more prevalent (p = 0.019) in the MFCcr of the OA group (n = 7/7, 100%) when compared with the CO group (n = 7/17, 41%). Score 1 HAC surface mineralisation was more prevalent (p = 0.038) in the OA MFCca (n = 4/7, 57%) when compared with the CO group (n = 2/17, 12%). HDMP were heterogenous and hyperdense mineralised material protruding into the HAC and were more frequent (p = 0.033) in MFCs (n = 12/24, 50%) compared with MTPs (n = 5/24, 20%). Score 3 HDMPs were also more prevalent (p = 0.003) in the MFCcr (n = 7/24, 29%) compared with MFCca (n = 0/24, 0%) and in MFCs (n = 7/24, 29%) compared with MTPs (n = 3/24, 12.5%) (p = 0.046)., Main Limitations: Clinical history was not available for all specimens., Conclusions: Equine HDMP and HAC surface mineralisation are imaged for the first time in the MFT joint. HAC fibrillation and erosion and HDMP are more frequent in the cranial aspect of the MFC. μCT images of OA in equine stifle joints provide a novel perspective of lesions and improve understanding of OA., (© 2024 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Background: There is a knowledge gap about how equine MFC subchondral radiolucencies (SR) arise and evolve. Osteoclasts are believed to have a role but have not been studied in situ., Objectives: To measure and compare osteoclast density and the percentage of chondroclasts in healthy and MFC SR specimens from juvenile Thoroughbreds., Study Design: Cadaveric study., Methods: Medial femoral condyles (MFC) from a tissue bank of equine stifles were studied. Inclusion criteria were MFCs (≤8 months old) with a computed tomography SR lesion and histological focal failure of endochondral ossification (L group). Contralateral, lesion-free, MFCs were a control group (CC). Osteochondral slabs were cut through the lesion (L), a healthy site immediately caudal to the lesion, (internal control; IC) and the contralateral, site-matched controls (CC). Histological sections were immunostained with Cathepsin K for osteoclast counting. Osteoclasts in contact with the growth cartilage (chondroclasts) were also counted. The sections were segmented into regions of interest (ROI) at different depths in the subchondral bone: ROI 1 (0-1 mm), ROI 2 (1-3 mm) and ROI 3 (3-6 mm). Osteoclasts were counted and the bone area was measured in each ROI to calculate their density. Chondroclasts were counted in ROI 1 ., Results: Sections were studied from L and IC (n = 6) and CC sites (n = 5). Osteoclast density was significantly higher in ROI 1 when compared with ROI 3 in all groups. Although higher osteoclast density was measured in ROI 1 in the L group, no significant differences were detected when compared with control ROIs. The proportion of chondroclasts in ROI 1 was lower in the L sections when compared with controls but no significant differences were detected., Main Limitations: Limited sample size., Conclusions: Osteoclasts are important actors in MFC subchondral bone development, digesting both growth cartilage (chondroclasts) and bone, but the pathophysiology of early MFC SRs cannot be explained solely by an increased osteoclast presence in the subchondral bone., (© 2021 EVJ Ltd.)

Background: The aetiology of equine medial femoral condyle (MFC) subchondral bone radiolucencies (SR) is unknown., Objectives: Characterise the microstructural structural features of MFC SR in juvenile Thoroughbreds with microcomputed tomography (μCT) and histology., Study Design: Cross-sectional post-mortem study., Methods: Distal femurs were collected at post-mortem. Conventional tomodensitometry was employed to scout for MFCs with and without SR lesions (SR+ and SR-, respectively). Group 1 were CT MFC SR+ and Group 2 age-matched SR- controls. Both underwent μCT and histological analysis. Group 3 CT MFC SR- foals, <6 months, were selected to search for chondronecrosis. Histological sections, processed from the lesion (Group 1) and a corresponding site in Groups 2 and 3, were assessed for chondronecrosis, fibrin, fibroplasia and osteochondral separation. Group 3 sections were surveyed for chondronecrosis alone., Results: A total of 178 femurs from 89 Thoroughbreds were harvested. Of these horses 19.1% (95% CI: 10.9%-27.3%) were CT MFC SR+ (17/23; 7.46 ± 4.36 months) and met the inclusion criteria for Group 1. Group 2 included 30 CT MFC SR- specimens (5.00 ± 2.73 months) and Group 3 had 44 CT MFC SR- s (2.68 ± 1.74 months). SR were located axially in foals <7 months of age, and centrally thereafter. All SRs had areas of thickened cartilage on histology and separation at the osteochondral junction containing fibrin (acute event) and fibroplasia (chronicity) in 73.9% (17/23; 95% CI: 56%-91.9%). In Group 1 specimens, chondronecrosis was present in 82.6% (19/23; 95% CI: 67.1%-98.1%) but four MFC SR+ had no evidence of chondronecrosis. Chondronecrosis was not detected in the Group 3 foal MFCs., Main Limitations: No longitudinal follow-up., Conclusions: The absence of chondronecrosis, pathognomic of osteochondrosis, in four MFC SR+s and in all of the CT MFC SR- foals suggests that osteochondrosis is not the cause, or the only cause, of these lesions and favours trauma as an alternate aetiological hypothesis., (© 2021 EVJ Ltd.)

Background: The administration of bisphosphonate medications, which target osteoclastic-bone remodelling, to juvenile and adult racehorses is a matter of debate owing to concerns that these molecules remain bound to the bone-mineralised matrix and may interfere with subsequent bone growth, adaptation to exercise and healing of bone microdamage in equine athletes. Osteoclasts participate in endochondral ossification, subchondral bone remodelling and bone repair. There is a knowledge gap on the role of equine osteoclast biology in the growth and maturation of joint surfaces and this information is important to inform judicious bisphosphonate use., Objectives: Measure and compare the osteoclast density in the subchondral bone of Thoroughbred (TB) distal third metacarpi (McIII) at different sites, varying depths from the articular surface and with age (0-84 months)., Study Design: Ex vivo cadaveric study., Methods: McIIIs from foals, yearlings and adults were collected, fixed in formaldehyde and stored at 4°C. Sections were cut from the lateral hemi-metacarpus, stained and scored for cartilage degeneration. Osteoclasts were counted on immunohistochemically (Cathepsin K) stained sections. Osteoclast density was compared in regions of interest (ROIs-the sagittal ridge, axial and abaxial condyle) and also at two depths (0-3 mm and 3-6 mm) into the subchondral bone below the osteochondral junction., Results: The osteoclast density was consistently highest in the subchondral cortical bone plate (0-3 mm) when compared with the deeper trabecular bone in all age groups. Furthermore, the osteoclast density was significantly higher in juvenile Thoroughbreds (foals and yearlings) within both sites in the subchondral bone when compared with adults., Main Limitations: The number of specimens available for study was restricted., Conclusions: Osteoclasts are important in normal McIII epiphyseal and articular surface maturation and have a propensity to localise at the osteochondral junction and subchondral cortical bone plate zone in juvenile Thoroughbreds., (© 2020 EVJ Ltd.)

Objectives: Develop a species-specific ELISA for a neo-epitope generated by cathepsin K cleavage of equine type II collagen to: (1) measure cartilage type II collagen degradation by cathepsin K in vitro, (2) identify cytokines that upregulate cathepsin K expression and (3) compare cathepsin K with matrix metalloproteinase (MMP) collagenase activity in stimulated cartilage explants and freshly isolated normal and osteoarthritic (OA) articular cartilages., Design: A new ELISA (C2K77) was developed and tested by measuring the activity of exogenous cathepsin K on equine articular cartilage explants. The ELISA was then employed to measure endogenous cathepsin K activity in cultured cartilage explants with or without stimulation by interleukin-1 beta (IL-1β), tumour necrosis-alpha (TNF-α), oncostatin M (OSM) and lipopolysaccharide (LPS). Cathepsin K activity in cartilage explants (control and osteoarthritic-OA) and freshly harvested cartilage (control and OA) was compared to that of MMPs employing C2K77 and C1,2C immunoassays., Results: The addition of Cathepsin K to normal cartilage caused a significant increase (P < 0.01) in the C2K77 epitope release. Whereas the content of C1,2C, that reflects MMP collagenase activity, was increased in media by the addition to cartilage explants of TNF-α and OSM (P < 0.0001) or IL-1β and OSM (P = 0.002), no change was observed in C2K77 which also unchanged in OA cartilages compared to normal., Conclusions: The ELISA C2K77 measured the activity of cathepsin K in equine cartilage which was unchanged in OA cartilage. Cytokines that upregulate MMP collagenase activity had no effect on endogenous cathepsin K activity, suggesting a different activation mechanism that requires further study., (Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equine articular osteoarthritic cartilage., Design: Equine type II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, in vitro, and the presence of cathepsin K-derived products in equine and human osteoarthritic cartilage., Results: Six cathepsin K cleavage sites distributed throughout the triple helical region were identified in equine type II collagen. Most of the cleavages occurred following a hydroxyproline residue. The most N-terminal site was within three residues of the previously identified site in bovine type II collagen. Western blotting using anti-neoepitope antibodies showed that the initial cleavages occurred at the N-terminal sites and this was followed by more extensive degradation resulting in products too small to be resolved by SDS gel electrophoresis. Immunohistochemical staining of cartilage sections from equine or human osteoarthritic joints showed staining in lesional areas which was not observed in non-arthritic sites., Conclusions: Cathepsin K cleaves triple helical collagen by erosion from the N-terminus and with subsequent progressive cleavages. The liberated fragments can be detected in osteoarthritic cartilage and may represent useful biomarkers for disease activity., (Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

We studied changes in articular calcified cartilage (ACC) and subchondral bone (SCB) in the third carpal bones (C3) of Standardbred racehorses with naturally-occurring repetitive loading-induced osteoarthritis (OA). Two osteochondral cores were harvested from dorsal sites from each of 15 post-mortem C3 and classified as control or as showing early or advanced OA changes from visual inspection. We re-examined X-ray micro-computed tomography (µCT) image sets for the presence of high-density mineral infill (HDMI) in ACC cracks and possible high-density mineralized protrusions (HDMP) from the ACC mineralizing (tidemark) front (MF) into hyaline articular cartilage (HAC). We hypothesized and we show that 20-µm µCT resolution in 10-mm diameter samples is sufficient to detect HDMI and HDMP: these are lost upon tissue decalcification for routine paraffin wax histology owing to their predominant mineral content. The findings show that µCT is sufficient to discover HDMI and HDMP, which were seen in 2/10 controls, 6/9 early OA and 8/10 advanced OA cases. This is the first report of HDMI and HDMP in the equine carpus and in the Standardbred breed and the first to rely solely on µCT. HDMP are a candidate cause for mechanical tissue destruction in OA.

Adult articular cartilage (AC) has a well described multizonal collagen structure. Knowledge of foetal AC organisation and development may provide a prototype for cartilage repair strategies, and improve understanding of structural changes in developmental diseases such as osteochondrosis (OC). The objective of this study was to describe normal development of the spatial architecture of the collagen network of equine AC using 1.5 T magnetic resonance imaging (MRI) and polarised light microscopy (PLM), at sites employed for cartilage repair studies or susceptible to OC. T2-weighted fast-spin echo (FSE) sequences and PLM assessment were performed on distal femoral epiphyses of equine foetuses, foals and adults. Both MRI and PLM revealed an early progressive collagen network zonal organisation of the femoral epiphyses, beginning at 4 months of gestation. PLM revealed that the collagen network of equine foetal AC prior to birth was already organised into an evident anisotropic layered structure that included the appearance of a dense tangential zone in the superficial AC in the youngest specimens, with the progressive development of an underlying transitional zone. A third, increasingly birefringent, radial layer developed in the AC from 6 months of gestation. Four laminae were observed on the MR images in the last third of gestation. These included not only the AC but also the superficial growth plate of the epiphysis. These findings provide novel data on normal equine foetal cartilage collagen development, and may serve as a template for cartilage repair studies in this species or a model for developmental studies of OC.

Objective: To assess the effects of an intra-articular (IA) lidocaine or bupivacaine injection on synovial fluid (SF) biomarkers of cartilage metabolism., Study Design: Experimental., Animals: Horses (n = 6)., Methods: The study had 2 components, each with an identical design: the first arm assessed the effects of bupivacaine (0.5%) IA in the intercarpal joints and, after a 2-week washout period, the second was conducted to evaluate the effects of IA lidocaine (2%) in the tarsocrural joints. The mares were randomly assigned to receive the test local anesthetic in the target joint or the placebo (0.9% NaCl) in the contralateral joint. After a 2-week washout period, treatments were reversed, yielding 6 joints for each treatment. SF samples were collected for measurement of biomarkers of cartilage metabolism., Results: IA bupivacaine or lidocaine induced a significant increase in both markers of cartilage matrix synthesis (CS846-aggrecan and CPII-type II collagen) suggesting an anabolic effect. Bupivacaine also resulted in an unanticipated decrease in both collagen degradation markers (C2C and C1,2C)., Conclusions: These results reveal an anabolic effect of single IA injections of local anesthetics on cartilage metabolism. Although collagen degradation biomarker results were not increased, it is possible that the anabolic response is because of an undetected cartilage insult, and requires further investigation., (© Copyright 2012 by The American College of Veterinary Surgeons.)

Objective: To correlate degenerative changes in cartilage and subchondral bone in the third carpal bone (C3) of Standardbred racehorses with naturally occurring repetitive trauma-induced osteoarthritis., Design: Fifteen C3, collected from Standardbred horses postmortem, were assessed for cartilage lesions by visual inspection and divided into Control (CO), Early Osteoarthritis (EOA) and Advanced Osteoarthritis (AOA) groups. Two osteochondral cores were harvested from corresponding dorsal sites on each bone and scanned with a micro-computed tomography (CT) instrument. 2D images were assembled into 3D reconstructions that were used to quantify architectural parameters from selected regions of interest, including bone mineral density and bone volume fraction. 2D images, illustrating the most severe lesion per core, were scored for architectural appearance by blinded observers. Thin sections of paraffin-embedded decalcified cores stained with Safranin O-Fast Green, matched to the micro-CT images, were scored using a modified Mankin scoring system., Results: Subchondral bone pits with deep focal areas of porosity were seen more frequently in AOA than EOA but never in CO. Articular cartilage damage was seen in association with a reduction in bone mineral and loss of bone tissue. Histological analyses revealed significant numbers of microcracks in the calcified cartilage of EOA and AOA groups and a progressive increase in the score compared with CO bones., Conclusion: The data reveal corresponding, progressive degenerative changes in articular cartilage and subchondral bone, including striking focal resorptive lesions, in the third carpal bone of racehorses subjected to repetitive, high impact trauma., (Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Introduction: The intra-helical cleavage of type II collagen by proteases, including collagenases and cathepsin K, is increased with aging and osteoarthritis (OA) in cartilage as determined by immunochemical assays. The distinct sites of collagen cleavage generated by collagenases and cathepsin K in healthy and OA human femoral condylar cartilages were identified and compared., Methods: Fixed frozen cartilage sections were examined immunohistochemically, using antibodies that react with the collagenase-generated cleavage neoepitopes, C2C and C1,2C, and the primary cleavage neoepitope (C2K) generated in type II collagen by the action of cathepsin K and possibly by other proteases, but not by any collagenases studied to date., Results: In most cases, the staining patterns for collagen cleavage were similar for all three epitopes: weak to moderate mainly pericellular staining in non-OA cartilage from younger individuals and stronger, more widespread staining in aging and OA cartilages that often extended from the superficial to the mid/deep zone of the tissue. In very degenerate OA specimens, with significant disruption of the articular surface, staining was distributed throughout most of the cartilage matrix., Conclusions: Cleavage of collagen by proteases usually arises pericellularly around chondrocytes at and near the articular surface, subsequently becoming more intense and extending progressively deeper into the cartilage with aging and OA. The close correspondence between the distributions of these products suggests that both collagenases and cathepsin K, and other proteases that may generate this distinct cathepsin K cleavage site, are usually active in the same sites in the degradation of type II collagen.

The purpose was to evaluate the capacity of 1.5 T magnetic resonance (MR) imaging to assess articular cartilage in racehorses with naturally occurring metacarpophalangeal joint osteoarthritis. A sagittal, three-dimensional spoiled gradient-recalled echo (SPGR) with fat saturation (FS) sequence was acquired ex vivo on 20 joints. Following joint dissection, specific areas on the third metacarpal condyle were designated for subsequent sampling for histologic cartilage thickness measurement and modified Mankin scoring. Cartilage thickness was measured and cartilage signal intensity was also graded (0-3) on MR images at these selected metacarpal sites. Cartilage structure was graded (0-3) macroscopically and on MR images by two examiners in defined subregions of the proximal phalanx, third metacarpal, and proximal sesamoid bones. There was good precision (mean error 0.11 mm) and moderate correlation (r = 0.44; P < 0.0001) of cartilage thickness measurements between MR images (0.90 +/- 0.17mm) and histology (0.79 +/- 0.16 mm). There was moderate correlation between modified Mankin histologic score and signal intensity of cartilage (r = 0.36; P < 0.01) or MR cartilage structure assessment (r = 0.49, P > 0.001) on SPGR-FS. The sensitivity to detect full-thickness cartilage erosion on MR was only moderate (0.56), and these lesions were often underestimated, particularly when linear in nature. However, the specificity to detect such lesions on MR was high (0.92). While few limitations were identified, the use of a clinically applicable SPGR-FS sequence allows a reasonably accurate method to assess structural changes affecting the articular cartilage of the equine metacarpophalangeal joint.

Objective: The mechanisms leading to degeneration of articular cartilage in osteoarthritis (OA) are complex and not yet fully understood. Cathepsin K (CK) is a cysteine protease which can also cleave the triple helix of type II collagen. This exposes a neoepitope that can now be identified by specific antibodies. The aim of this study was to obtain evidence suggesting a role for CK in naturally occurring equine OA in both lesional and peri-lesional regions., Methods: Articular cartilages (n=12 horses; 5 healthy, 7 OA) were harvested from animals postmortem. A gross macroscopic examination, histologic (Safranin O-Fast Green and Picrosirius red staining) and immunohistochemical evaluation were performed. Samples were divided into normal appearing cartilage, peri-lesional and lesional cartilage. Cartilage degradation in the samples was graded histologically and immunohistochemically. CK and possible CK cleavage were detected immunohistochemically with specific anti-protein and anti-neoepitope antibodies, respectively. A comparison of CK neoepitope (C2K) production with the collagenase-generated neoepitope produced by matrix metalloproteinases (MMP)-1, 8 and 13 (C2C) was also assessed immunohistochemically., Results: CK and CK cleavage were significantly more abundant in OA cartilage (both peri-lesional and lesional) when compared to remote cartilage within the sample joint or cartilage from healthy joints. The immunohistochemical pattern observed for CK degradation (C2K) was similar to that of collagenase degradation (C2C). Macroscopic cartilage changes and histologic findings were significantly correlated with immunohistochemistry results., Conclusion: The data generated suggests that CK may be involved in cartilage collagen degradation in naturally occurring osteoarthritis.

Cathepsin K is a cysteine protease of the papain family that cleaves triple-helical type II collagen, the major structural component of the extracellular matrix of articular cartilage. In osteoarthritis (OA), the anabolic/catabolic balance of articular cartilage is disrupted with the excessive cleavage of collagen II by collagenases or matrix metalloproteinases. A polyclonal antibody against a C-terminal neoepitope (C2K) generated in triple-helical type II collagen by the proteolytic action of cathepsin K was prepared and used to develop an enzyme-linked immunosorbent assay to study the generation of this epitope and the effects of its presence in normal adult and osteoarthritic femoral condylar articular cartilage. The generation of the C2K epitope in explant culture and the effect of a specific cathepsin K inhibitor were studied. The neoepitope, which is not generated by the collagenase matrix metalloproteinase-13, increased with age in articular cartilage and was significantly elevated in osteoarthritic cartilage compared with adult nonarthritic cartilage. Moreover, in explants from three of eight OA patients, the generation of the neoepitope in culture was significantly reduced by a specific, nontoxic inhibitor of cathepsin K. These data suggest that cathepsin K is involved in the cleavage of type II collagen in human articular cartilage in certain OA patients and that it may play a role in both OA pathophysiology and the aging process.

Reasons for Performing Study: The earliest osteochondrosis (OC) microscopic lesion reported in the literature was present in the femorotibial joint of a 2-day-old foal suggesting that OC lesions and factors initiating them may arise prior to birth., Objective: To examine the developing equine epiphysis to detect histological changes that could be precursors to OC lesions., Methods: Osteochondral samples from 21 equine fetuses and 13 foals were harvested from selected sites in the scapulohumeral, humeroradial, metacarpophalangeal, femoropatellar, femorotibial, tarsocrural and metatarsophalangeal joints. Sections were stained with safranin O and picrosiruis red to assess cartilage changes and structural arrangement of the collagen matrix., Results: Extracellular matrix changes observed included perivascular areas of paleness of the proteoglycan matrix associated with hypocellularity and, sometimes, necrotic chondrocytes. These changes were most abundant in the youngest fetuses and in the femoropatellar/femorotibial (FP/FT) joints. Indentations of the ossification front were also observed in most specimens, but, most frequently, in scapulohumeral and FP/FT joints. A cartilage canal was almost always present in these indentations. The vascular density of the cartilage was higher in the youngest fetuses. In these fetuses, the most vascularised joints were the metacarpo- and metatarsophalangeal joints but their cartilage canals regressed quickly. After birth, the most vascularised cartilage was present in the FP/FT joint. Articular cartilage differentiated into 4 zones early in fetal life and the epiphyseal cartilage also had a distinct zonal cartilage structure. A striking difference was observed in the collagen structure at the junction of the proliferative and hypertrophic zones where OCD lesions occur., Conclusion: Matrix and ossification front changes were frequently observed and significantly associated with cartilage canals suggesting that they may be physiological changes associated with matrix remodelling and development. The collagen structure was variable through the growing epiphysis and a differential in biomechanical properties at focal sites may predispose them to injury.

Objective: To evaluate mRNA expression of several proinflammatory and anti-inflammatory cytokines and chemokines in equine unstimulated and interleukin-1beta (IL-1beta)-stimulated chondrocytes., Study Design: In vitro experiment using equine chondrocyte cultures., Sample Population: Whole articular cartilage from metacarpophalangeal joints (n=5 horses; 10 fetlocks)., Methods: Chondrocyte monolayer cultures were established from digested adult equine articular cartilage and stimulated with 5 ng/mL of recombinant human IL-1beta. RNA was extracted from the cells 24 hours after stimulation. IL-1beta, IL-4, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and ubiquitin (house keeping gene) mRNA expression were investigated by real-time RT-PCR., Results: IL-1beta, IL-6, and IL-8 mRNA were expressed in unstimulated chondrocytes from macroscopically normal joints and were significantly up-regulated after stimulation (5/5 horses). IL-4 mRNA was not detected in any samples (0/5 horses). TNF-alpha mRNA, by comparison, was expressed in 2/5 unstimulated samples and in all stimulated samples but a considerable sample variation in response to IL-1beta stimulation was observed., Conclusions: Equine chondrocytes express mRNA for several proinflammatory cytokines and chemokines and IL-1beta modulates their expression., Clinical Relevance: Chondrocytes express proinflammatory cytokines and chemokines capable of modulating a local inflammatory cascade in articular cartilage, which could potentially lead to focal degradation and osteoarthritis.

Objective: To characterize expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) and regulation of prostaglandin E2 (PGE2) production by equine articular chondrocytes., Sample Population: Articular cartilage from the metacarpophalangeal joints of 7 adult horses., Procedure: Equine chondrocyte monolayer cultures were stimulated with different concentrations (2.5, 5, 10, and 20 ng/mL) of recombinant human interleukin-1beta (rhIL-1beta) for 24 hours and then with rhIL-1beta (5 ng/mL) for 3, 6, 9, 12, and 24 hours. Concentration of PGE2 in the media was measured via radioimmunoassay. Total RNA was extracted from harvested chondrocytes, and regulation of COX-2 and mPGES-1 mRNA was studied via reverse transcriptase-polymerase chain reaction assay and Southern blot analysis with equine-specific probes. Western blot analyses were performed on cellular extracts to characterize expression of COX-2 and mPGES-1 protein., Results: Stimulation with 5, 10, and 20 ng of rhIL-1beta/mL caused a significant increase in PGE2 concentrations in the culture media, and incubation of cells with rhIL-1beta (5 ng/mL) for 6 to 24 hours increased PGE2 production significantly. The increase in prostaglandin production was associated with an induction of COX-2 and mPGES-1 transcripts. There also was an rhIL-1beta-dependent induction in COX-2 and mPGES-1 protein expression., Conclusions and Clinical Relevance: Collectively, results indicated that the rhIL-1beta-dependent increase in PGE2 production in equine chondrocytes in monolayer culture was associated with coordinated upregulation of COX-2 and mPGES-1 expression. The pathophysiologic consequences of upregulated COX-2 and mPGES-1 expression and of PGE2 synthesis in rhIL-1beta-stimulated equine chondrocytes remain to be elucidated.

Although intraarticular (IA) corticosteroids are frequently used to treat joint disease, the effects of their repeated use on articular cartilage remains controversial. The aim of our study was to determine the effects of a clinically recommended dose of IA triamcinolone acetonide (TA), on synovial fluid (SF) biomarkers of cartilage metabolism. Ten adult horses, free of osteoarthritis (OA) in their radiocarpal joints, were studied. One radiocarpal joint of each horse was randomly chosen for treatment and the contralateral anatomically paired joint acted as the control. Aseptic arthrocentesis was performed weekly on both joints for 13 weeks. The initial results from the first 3 weeks of the experimental period established baseline untreated control marker levels for each joint, each being its own control. On weeks 3, 5, and 7, a sterile suspension of 12 mg of TA was injected into the treated joint and an equivalent volume of sterile saline solution (0.9%) was injected into the control joint. SF was immunoassayed for biomarkers of aggrecan turnover (CS 846 & KS), types I and II collagen cleavage (C1,2C) and type II collagen synthesis (CPII). In treated joints, there was a significant increase in CS 846, KS, C1,2C and CPII epitope concentrations following IA TA injections when compared to baseline levels. There was also a significant increase in C1,2C and CPII epitope concentrations in the contralateral control joints following IA TA injections in the treated joint. Significant differences were observed between treated and control joints for all markers except CPII. These findings indicate that TA alters articular cartilage and collagen metabolism in treated and, interestingly, also in control joints, suggesting a systemic effect of the drug. Though intuitively the observed findings would favor the hypothesis that long-term IA TA treatment changes joint metabolism and this may have detrimental effects; further studies would be necessary to confirm this.

Objective: To determine whether oral glucosamine alleviates cartilage degradation in an animal model of osteoarthritis (OA)., Methods: The effect of 8 weeks of daily oral glucosamine hydrochloride on degeneration of articular cartilage was evaluated in rabbits in which anterior cruciate ligament transection (ACLT) was performed to induce OA. Animals were treated with glucosamine (n = 16) or a placebo (n = 16) and necropsied at 11 weeks. Seven unoperated rabbits served as controls. The articular cartilage was evaluated macroscopically and histologically and analyzed for total type II collagen and glycosaminoglycan (GAG) content., Results: Histologic analysis revealed that loss of proteoglycan, based on Safranin O-fast green staining, was significantly reduced in the lateral tibial plateau cartilage of ACL-transected limbs in the glucosamine group compared with ACL-transected limbs in the placebo group, with a similar, but not significant, trend for the lateral femoral condylar cartilage. Likewise, macroscopic analysis of cartilage showed that the lateral tibial plateau alone had a significantly lower rate of disease in the glucosamine group, which was consistent with the results of the independent histologic assessment. However, no significant treatment effect was detected when composite histologic scores were analyzed. A significant reduction in GAG content was observed in the femoral condyles of placebo-treated ACL-transected joints, but not in the same region of glucosamine-treated ACL-transected joints, compared with their respective contralateral unoperated joints., Conclusion: Oral administration of glucosamine had a detectable, site-specific, partial disease-modifying effect in this model of OA. From a clinical perspective, the administration of glucosamine did not prevent fibrillation and/or erosions of the articular cartilage in all of the treated animals, and no effects were detected in the medial joint compartments.

Objective: To evaluate high frequency (40 MHz) B-mode ultrasound for the detection of osteoarthritis (OA) lesions of varying severity in an animal model of OA., Design: Ultrasound biomicroscopy (UBM) was performed on the femoral articular surface of adult rabbits with unilateral transection of the anterior cruciate ligament at 4, 8 and 12 weeks post-surgery and on control rabbits. The articular cartilage was examined and graded macroscopically and histologically for OA lesions. Histological examination was used as a reference to determine sensitivity and specificity of ultrasonographic and macroscopic examination regarding fibrillation and ulceration of articular cartilage., Results: Identification of slight surface irregularities was made possible with UBM. The sensitivity and specificity of UBM were 92.3% and 96.4%, respectively, to detect histological fibrillation and 90.9% and 97.6%, respectively, to identify histological ulceration. Macroscopic examination using India Ink had a sensitivity and specificity of 80% and 96.4%, respectively, for fibrillation and 90.9% and 90.5%, respectively, for ulceration when compared to histology. A high correlation (rsp=0.90) was found between ultrasonographic and histological scores., Conclusions: UBM of articular cartilage reflects histological structure and can accurately detect early changes such as fibrillation. UBM has the potential to be a valuable tool for the in vivo identification of early lesions of OA and for monitoring the disease or efficacy of novel therapy if it can be packaged in a minimally invasive format suitable for intra-articular imaging.

Objective: To characterize the rabbit anterior cruciate ligament transection (ACLT) model of osteoarthritis (OA) at various stages of disease using high-resolution 3-D medical imaging systems, which, in turn, will facilitate future longitudinal studies evaluating disease progression and response to therapy in live animals., Methods: Degenerative changes in femorotibial cartilage, volumetric bone mineral density (vBMD), bone volume fraction (BV/TV), and osteophyte volume were characterized ex vivo using 4-T magnetic resonance imaging (MRI) and micro-computed tomography (micro-CT) at 4, 8, and 12 weeks post-ACLT. These changes were subsequently correlated to macroscopic joint evaluation., Results: Macroscopic assessment demonstrated progressive cartilage degeneration post-surgery, which was significantly correlated to MRI evaluation (r=0.82, P<0.0001). Linear regression analysis indicated that vBMD and BV/TV are linearly related such that as vBMD increases, BV/TV increases (P<0.0001). Micro-CT revealed bone loss at 4 and 8 weeks post-ACLT, but recovery to control values at 12 weeks post-ACLT. Volumetric BMD was not strongly correlated with macroscopic assessment of articular cartilage degeneration (r=-0.35, P<0.0001). Quantitative measurement of osteophyte volume demonstrated a statistically significant difference (with respect to control groups) at both 8 and 12 weeks post-ACLT, but not at 4 weeks post-ACLT., Conclusions: The rabbit ACLT model of OA demonstrates progressive cartilage degeneration and intermediate bone changes at 4, 8, and 12 weeks post-surgery. Cartilage and bone lesions were characterized ex vivo using 4-T MRI and micro-CT, and MRI assessment of cartilage degeneration was correlated to macroscopic grading.

Articular osteochondrosis (OCD) occurs in both man and animals. The etiology remains to be determined. Studies of OCD lesions in animals may provide clues as to its pathogenesis. The aim of our study was to determine whether there was evidence for increased degradation namely proteoglycan (PG) release and type II collagen cleavage in articular cartilage harvested from OCD lesions. We examined ex vivo explants at post-mortem from equine OCD lesions and macroscopically normal site and age matched cartilage. These were cultured over a 10 day period in serum-free medium. Type II collagen cleavage was measured in articular cartilage and media using an Elisa assay to detect the COL2-3/4C(short) epitope, which is generated on cleavage of the triple helix of type II collagen by collagenases. PG release was measured by a dye-binding assay. Cumulative release of PG and COL2-3/4C(short) and their contents in cartilage at the end of the culture period were determined. In OCD lesions there was a significant increase in type II collagen cleavage by collagenase but no evidence for increase of PG degradation. These findings point to a selective increase in type II collagen cleavage by collagenases, in OCD lesions of the kind observed in osteoarthritis. Further work is needed to determine whether changes represent primary or secondary events in the pathogenesis of OCD.

The authors review the structure and composition of articular cartilage. This tissue is composed of an extensive extracellular matrix synthesized by chondrocytes. It contains different zones with respect to depth from the articular surface and has a regional organization around the chondrocytes. Its composition varies regionally and zonally in its collagen and proteoglycan contents and those of other matrix molecules. There is a macrofibrillar collagen network and a microfilamentous network about which other noncollagenous molecules are organized. Its structure and composition are reflective of its special mechanical properties that primarily reflect its tensile strength (collagens) and compressive stiffness (proteoglycan aggrecan) and cell-matrix interactions (noncollagenous proteins).

In vivo the effects of intra-articular (IA) corticosteroids on articular cartilage remain controversial. This study was designed to examine this issue using synovial fluid (SF) markers of cartilage metabolism. Paired radiocarpal joints, without clinical or radiographic signs of joint disease, were studied in 10 adult horses. Aseptic arthrocentesis was performed weekly for 13 weeks. IA injections of methylprednisolone acetate (MPA) into the treatment joint and the vehicle into the control joint were performed at weeks 3, 5 and 7. We used radioimmunoassays on SF samples which measure a keratan sulfate epitope (KS) and the 846 epitope on cartilage aggrecan (PG) and the C-propeptide (CPII) of cartilage type II procollagen which is released following synthesis of this molecule. Gel chromatography was performed on selected SF samples to evaluate the sizes of SF PG molecules. The total joint KS and the 846 epitopes were both present on a heterogeneous population of mainly molecules which, from chromotographic analysis, appeared to be mainly fragments of the articular cartilage aggrecan. They were significantly elevated in MPA joints whereas CPII was significantly reduced compared to the control during the treatment period. These results indicate that the repeated use of IA MPA leads to a potentially harmful inhibition of procollagen II synthesis and an increased release of degradation products of the PG aggrecan from articular cartilage.

The etiology and pathophysiology of osteochondrosis remain poorly understood because it is difficult to obtain material from lesions in the early stage of this disease and because there is no satisfactory experimental animal model. We wished to determine whether there are changes in articular cartilage turnover in equine osteochondrosis, which closely resembles the human disease, by assaying cartilage matrix molecules in synovial fluids. We used immunoassays that measure a keratan sulfate epitope and the epitope 846 on the cartilage proteoglycan aggrecan and the C-propeptide of cartilage type-II procollagen, which is released following the synthesis of this molecule, to analyse synovial fluids from equine tarsocrural joints with and without osteochondrosis. In young horses with osteochondrosis, there was a significant increase of C-propeptide of type-II procollagen accompanied by a decrease in the 846 and keratan sulfate epitopes. The results identify differential alterations in aggrecan and type-II collagen turnover in the cartilage matrix in young animals with osteochondrosis that may contribute to the pathological degeneration of articular cartilage in this disease.