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Cell morphology may reflect the mechanical environment of tissues and influence tissue physiology and response to injury. Normal cruciate ligaments (CLs) from disease-free stifle joints were harvested from dog breeds with a high (Labrador retriever) and low (Greyhound) risk of cranial cruciate ligament (CCL) rupture. Antibodies against the cytoskeletal components vimentin and alpha tubulin were used to analyse cell morphology; nuclei were stained with 4′,6-diamidino-2-phenylindole, and images were collected using conventional and confocal microscopy. Both cranial and caudal CLs contained cells of heterogenous morphologies. Cells were arranged between collagen bundles and frequently had cytoplasmic processes. Some of these processes were long (type A cells), others were shorter, thicker and more branched (type B cells), and some had no processes (type C cells). Processes were frequently shown to contact other cells, extending longitudinally and transversely through the CLs. Cells with longer processes had fusiform nuclei, and those with no processes had rounded nuclei and were more frequent in the mid-substance of both CLs. Cells with long processes were more commonly noted in the CLs of the Greyhound. As contact between cells may facilitate direct communication, variances in cell morphology between breeds at a differing risk of CCL rupture may reflect differences in CL physiology.

Type IX collagen is covalently bound to the surface of type II collagen fibrils within the cartilage extracellular matrix. The N-terminal, globular noncollagenous domain (NC4) of the α1(IX) chain protrudes away from the surface of the fibrils into the surrounding matrix and is available for molecular interactions. To define these interactions, we used the NC4 domain in a yeast two-hybrid screen of a human chondrocyte cDNA library. 73% of the interacting clones encoded fibronectin. The interaction was confirmed using in vitro immunoprecipitation and was further characterized by surface plasmon resonance. Using whole and pepsin-derived preparations of type IX collagen, the interaction was shown to be specific for the NC4 domain with no interaction with the triple helical collagenous domains. The interaction was shown to be of high affinity with nanomolar K(d) values. Analysis of the fibronectin-interacting clones indicates that the constant domain is the likely site of interaction. Type IX collagen and fibronectin were shown to co-localize in cartilage. This novel interaction between the NC4 domain of type IX collagen and fibronectin may represent an in vivo interaction in cartilage that could contribute to the matrix integrity of the tissue.

Although elastin fibres and oxytalan fibres (bundles of microfibrils) have important mechanical, biochemical and cell regulatory functions, neither their distribution nor their function in cruciate ligaments has been investigated. Twelve pairs of cruciate ligaments (CLs) were obtained from 10 adult dogs with no evidence of knee osteoarthritis. Elastic fibres were identified using Verhoeff's and Miller's staining. Fibrillins 1 and 2 were immunolocalised and imaged using confocal laser scanning microscopy. Hydrated, unfixed tissue was analysed using Nomarski differential interference microscopy (NDIC), allowing structural and mechanical analysis. Microfibrils and elastin fibres were widespread in both CLs, predominantly within ligament fascicles, parallel to collagen bundles. Although elastin fibres were sparse, microfibrils were abundant. We described abundant fibres composed of both fibrillin 1 and fibrillin 2, which had a similar pattern of distribution to oxytalan fibres. NDIC demonstrated complex interfascicular and interbundle anatomy in the CL complex. The distribution of elastin fibres is suggestive of a mechanical role in bundle reorganisation following ligament deformation. The presence and location of fibrillin 2 in oxytalan fibres in ligament differs from the solely fibrillin 1-containing oxytalan fibres previously described in tendon and may demonstrate a fundamental difference between ligament and tendon.

The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin-1beta (IL-1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL-1 or tumor necrosis factor-alpha (TNFalpha), promoting significant collagen degradation from equine cartilage explants within 4 days, but did not augment glycoaminoglycan (GAG) release. APC activated pro-matrix metalloproteinases (MMP)-2 but not pro-MMP-9, as assessed by gelatin zymography. APC did not directly activate pro-MMP-13. Dexamethasone, triamcinolone, and methylprednisolone acetate (MPA) were evaluated at concentrations between 10(- 5)M and 10(-10)M. High concentrations significantly increased GAG release from IL-1+APC-treated explants. With the exception of MPA at 10(-10)M, all concentrations of corticosteroids caused significant decreases in IL-1+APC-driven hydroxyproline loss. Treatment with corticosteroids suppressed expression of MMP-1, -3, and -13 mRNA. The collagenolysis associated with IL-1+APC synergy, and the inhibition of this effect by corticosteroids may involve gelatinase activation and downregulation of MMP expression, respectively.

Articular chondrocytes exist in an environment lacking in oxygen and nutrients due to the avascular nature of cartilage. The main source of metabolic energy is glucose, which is taken up by glucose transporters (GLUTs). In diseased joints, oxygen tensions and glucose availability alter as a result of inflammation and changes in vascularisation. Accordingly, in this study we examined the effects of hypoxia and the hypoxia mimetic cobalt chloride (CoCl(2)) on glucose transport in equine chondrocytes and compared expression of the hypoxia responsive GLUT1 gene in normal and diseased cartilage. Monolayers of equine chondrocytes were exposed to 20% O(2), 1% O(2), CoCl(2) (75 microM), or a combination of 1% O(2) and CoCl(2). Glucose uptake was measured using 2-deoxy-D-[2,6-(3)H] glucose. GLUT1 protein and mRNA expression were determined by FACS analysis and qPCR, respectively. GLUT1 mRNA expression in normal and diseased cartilage was analyzed using explants derived from normal, OA, and OCD cartilage. Chondrocytes under hypoxic conditions exhibited a significantly increased glucose uptake as well as upregulated GLUT1 protein expression. GLUT1 mRNA expression significantly increased in combined hypoxia-CoCl(2) treatment. Analysis of clinical samples indicated a significant reduction in GLUT1 mRNA in OA samples. In OCD samples GLUT1 expression also decreased but did not reach statistical significance. The increase in glucose uptake and GLUT1 expression under hypoxic conditions confirms that hypoxia alters the metabolic requirements of chondrocytes. The altered GLUT1 mRNA expression in diseased cartilage with significance in OA suggests that reduced GLUT1 may contribute to the failure of OA cartilage repair.

Fatigue fracture of cuboidal bones occurs in the human foot as well as the equine carpus. The racehorse provides a naturally-occurring model to study the effects of high-intensity exercise on the morphology and metabolism of cuboidal bones. We studied both the mineral and the collagenous matrix of the third (C(3)) and radial (C(r)) carpal bones of raced and non-raced Thoroughbred (TB) horses. We hypothesised that racehorses would show increases in the mineral component of these bones and post-translational modifications of the collagenous matrix alongside changes in markers of collagen remodelling and bone formation. C(3) and C(r) carpal bones were retrieved from raced TB horses (n=14) and non-raced TB horses (n=11). Standardised proximal-distal sections were taken from each bone and these were sliced transversely to study the proximal-distal differences in bone metabolism from the subchondral plate through to trabecular bone. Histomorphometry and bone mineral density measurements were performed in parallel with biochemical analyses including total collagen, collagen synthesis and cross-links, matrix metalloproteinases-2 and 9 and their inhibitors, calcium and phosphate, and bone alkaline phosphatase. The results of this study show that, while there is a net increase in bone formation in the racehorses, there is additionally an increase in bone collagen synthesis and remodelling, particularly within the trabecular regions of the bone. The increase in bone density would lead to greater stiffness, particularly in the cortical bone, and failure of this 'stiffer' cortical bone may result from its lack of support from the rapidly remodelling and structurally weakened underlying trabecular bone.

Matrix degradation by metalloproteinases is considered a key feature in the loss of articular cartilage seen in many joint diseases. Membrane-type matrix metalloproteinase-3 (MT3-MMP) expression is elevated in human cartilage in end-stage osteoarthritis. We investigated whether MT3-MMP is similarly regulated in cartilage in two naturally occurring arthropathies in vivo and whether proinflammatory cytokines regulate its expression in vitro. MT3-MMP expression was evaluated in cartilage from horses with osteoarthritis and osteochondrosis and compared with age- and site-matched normal cartilage. MT3-MMP also was measured in normal cartilage stimulated with proinflammatory cytokines. MT3-MMP expression was not significantly altered in either osteoarthritis or osteochondrosis cartilage. However, gene expression was significantly downregulated by the addition of recombinant human interleukin-1beta, oncostatin M, or tumor necrosis factor-alpha to normal cartilage explants. The results suggest that MT3-MMP may not have a role in matrix destruction in equine cartilage diseases. Further work is required to characterize its regulation and function.

Osteochondral lesions are a major cause of pain and disability in several species including dogs, horses and human beings. The objective of this study was to assess three potential sources of canine cells for their osteochondral regenerative potential. Cartilage, synovium and adipose tissue cells were grown in pellet culture in chondrogenic or osteogenic media. Cartilage-derived pellets displayed the best chondrogenic differentiation as indicated by significantly higher COL2A1 and SOX9 mRNA expression, greater glycosaminoglycan content, and higher retention of Safranin-O stain compared to the synovium and adipose-derived cells. Following application of the osteogenic media, all three cell sources exhibited small areas of positive alizarin red staining. Poor intracellular alkaline phosphatase activity was found in all three cell types when stimulated although osteocalcin and RUNX2 expression were significantly increased. Cells isolated and cultured from canine articular cartilage retained their specific chondrocytic phenotype. Furthermore, canine adipocytes and synovial cells did not undergo chondrogenic differentiation and did not exhibit evidence of multipotency. Although osteogenic differentiation was initiated at a genomic level, phenotypic osteoblastic differentiation was not observed. The findings of this study suggest that cells isolated from canine adipose tissue and synovium are sub-optimal substitutes for chondrocytes when engineering articular cartilage in vitro.

Type II collagen is a major component of articular cartilage and its breakdown is a key feature of osteoarthritis. Products of cartilage collagen metabolism can be detected in the blood, synovial fluid and urine. Several biomarker assays have been developed which can be used to measure the synthesis and degradation of collagen, and therefore provide information regarding cartilage turnover. This is the first part of a two-part review and describes the need for accurate, reliable information regarding collagen turnover, the processes by which the biomarker epitopes are generated, their application to the study of both healthy and diseased cartilage and the results of currently published studies, with particular reference to the veterinary species. The second part of the review considers the non-collagenous biomarkers of cartilage matrix turnover.

Osteoarthritis is the most common arthropathy of mammalian species including cats. Cartilage degradation is central to the disorder and here we present, for the first time, an in vitro model of feline cartilage degradation which will be useful for further studies in this target species. Feline articular cartilage explant cultures were maintained for 28 days and in the presence of oncostatin M with and without interleukin (IL)-17, tumour necrosis factor (TNF), IL-1alpha, or IL-1beta. Media samples and digested cartilage explants were analysed for glycosaminoglycan (GAG) and collagen content. The combination of IL-1beta and OSM, both at 20 ng/ml, was able to promote GAG release to the greatest extent at 14 days. At 28 days, all groups showed relatively high release of GAG. At 14 days, only IL-1beta and OSM in combination were associated with a statistically significant increase in collagen release over and above control tissue. IL-1beta dose-response studies showed that an IL-1beta dose of 10 ng/ml promotes a statistically significant increase in GAG breakdown when used with OSM, and higher doses of IL-1beta did not result in significantly greater response. The model demonstrated both GAG and collagen degradation and will be of use for further understanding of feline cartilage metabolism and for screening of potential structure-modifying agents to be used in cats.

The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin-1beta (IL-1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL-1 or tumor necrosis factor-alpha (TNFalpha), promoting significant collagen degradation from equine cartilage explants within 4 days, but did not augment glycoaminoglycan (GAG) release. APC activated pro-matrix metalloproteinases (MMP)-2 but not pro-MMP-9, as assessed by gelatin zymography. APC did not directly activate pro-MMP-13. Dexamethasone, triamcinolone, and methylprednisolone acetate (MPA) were evaluated at concentrations between 10(- 5)M and 10(-10)M. High concentrations significantly increased GAG release from IL-1+APC-treated explants. With the exception of MPA at 10(-10)M, all concentrations of corticosteroids caused significant decreases in IL-1+APC-driven hydroxyproline loss. Treatment with corticosteroids suppressed expression of MMP-1, -3, and -13 mRNA. The collagenolysis associated with IL-1+APC synergy, and the inhibition of this effect by corticosteroids may involve gelatinase activation and downregulation of MMP expression, respectively.

Recent studies have identified a range of interactions between type IX collagen and other cartilage matrix components. To determine the extent to which these interactions are important in maintaining the integrity of ageing articular cartilage, we analyzed an age range of normal healthy articular cartilage samples by Western blotting, immunohistochemical, and PCR analyses. Reduced levels of type IX collagen were detected in post adolescence cartilage. Type IX collagen epitopes were evident throughout the matrix in all cartilage samples up to 19 years of age. Post adolescence, however, the pattern of immunoreactivity revealed territorial staining only. Type IX collagen expression at the transcriptional level is maintained at all ages. Type IX collagen fragments were extracted from young tissue, supporting the hypothesis that young cartilage is continually remodelled, while mature cartilage maintains relatively low levels of collagen turnover. Clearly the age changes we observed may have significant effects on the integrity of the tissue as the chondrocytes in ageing articular cartilage have limited capacity to turnover the interterritorial matrix. However, this study provides evidence that even in old age, the chondrocyte attempts to maintain its pericellular environment and hence its mechanical role. Therefore, the potential of type IX collagen to interact with other matrix components continues to be of importance in the territorial environment, and these interactions may have significant roles in mechanotransduction.

Objectives: To evaluate the hypothesis that the concentration of the 1/20/5D4 epitope of keratan sulphate, cartilage oligomeric matrix protein and total sulphated glycosaminoglycans in synovial fluids from dogs with cranial cruciate ligament disease would be affected by tibial plateau levelling osteotomy. In addition, to evaluate the hypothesis that medial meniscal release or meniscal injury would alter the expression of these candidate biomarkers. Methods: Forty-one dogs with naturally occurring cranial cruciate ligament disease were recruited prospectively. Synovial fluids were collected from the index joint before surgery and six weeks and six months postsurgery. Following tibial plateau levelling osteotomy, synovial fluids were assayed for 1/20/5D4 epitope of keratan sulphate and cartilage oligomeric matrix protein concentration using an inhibition ELISA and for sulphated glycosaminoglycans using a direct dye-binding assay. Results: The sulphated glycosaminoglycans ratio did not change significantly during the study. Medial meniscal injury at entry was associated with lower concentrations of synovial fluid cartilage oligomeric matrix protein (P

Objective—To assess 2 methods of RNA purification by use of different quality metrics and identify the most useful metric for quality assessment of RNA extracted from articular cartilage from dogs with osteoarthritis. Sample Population—40 articular cartilage specimens from the femoral heads of 3 clinically normal dogs and 37 dogs with osteoarthritis. Procedures—RNA was extracted from articular cartilage by 2 purification methods. Quality metrics of each sample were determined and recorded by use of a UV spectrophotometer (Spec I; to determine the 260 to 280 nm absorbance ratio [A260:A280 ratio]), a second UV spectrophotometer (Spec II; to determine A260:A280 and A260:A230 absorbance ratios), and a microfluidic capillary electrophoresis analyzer (to determine the ribosomal peak ratio [RR], degradation factor [DF], and RNA integrity number [RIN]). The RNA was extracted from affected (osteoarthritic) articular cartilage and assessed with the same quality metrics. Metric results were compared with visual analysis of the electropherogram to determine the most useful RNA quality metric. Results—No differences in methods of RNA purification were determined by use of quality metrics. The RNA extracted from unaffected (normal) cartilage was of higher quality than that extracted from affected (osteoarthritic) cartilage, as determined by the RIN and Spec II A260:A230 ratio. The RIN and RR were the most sensitive metrics for determining RNA quality, whereas the DF was most specific. A significant proportion (32%) of RNA extracted from osteoarthritic articular cartilage specimens was determined as being of low quality. Conclusions and Clinical Relevance—No single metric provided a completely sensitive and specific assessment of the quality of RNA recovered from articular cartilage.

While intervertebral disc (IVD) degeneration is associated with the majority of cases of low back pain, current treatments are symptomatic rather than curative. Tissue engineering offers a treatment that both cures the problem of disc degeneration and restores normal disc function. One of the major problems for any tissue engineering strategy, however, is ensuring that both the cells and matrices used are suitable for the target tissue. In this study, we have developed and studied a potential system for tissue engineering of the nucleus pulposus (NP) of the severely degenerate IVD. While cells from degenerate discs are not suitable for tissue engineering, bone-marrow-derived mesenchymal stem cells, which are capable of differentiating into chondrocyte-like cells such as those found within the NP of the disc, offer a potential source of cells. We have used transfection with adenoviral SOX-9, a transcription factor involved in differentiation of MSCs along the chondrogenic lineage, combined with culture in a specialised medium, to differentiate monolayer MSCs to NP-like (chondrocyte-like) cells, as shown by real-time quantitative polymerase chain reaction for NP-marker genes. We have also replicated these findings on porous, biodegradable three-dimensional (3D) poly-l-lactic acid scaffolds and shown expression and deposition of NP matrix markers such as type II collagen and aggrecan. We are therefore proposing predifferentiation of human MSCs and seeding on porous, biodegradable 3D synthetic polymer scaffolds as a realistic tissue engineering strategy for regeneration of the degenerate human IVD. © 2006 Elsevier Ltd. All rights reserved.

Felid herpesvirus 1 (FeHV-1) is an important feline pathogen of the upper respiratory tract which can be identified in clinical cases by virus isolation and PCR. Studies on the effect and mode of spread of FeHV-1 in the respiratory epithelium, however, have previously only been performed in infected cats. Feline tracheal organ cultures which were inoculated with FeHV-1 at varying multiplicity of infection (MOI) were established. A dose-dependent response was observed. Low MOIs induced multifocal infection in the otherwise viable respiratory epithelium, which allowed monitoring of viral growth over several days. Therefore, tracheal organ cultures represent a suitable model for further study of the morphological and functional effects of FeHV-1 on respiratory epithelium, mimicking the in vivo situation.

There is growing recognition of the importance of nutritional factors in the maintenance of bone and joint health, and that nutritional imbalance combined with endocrine abnormalities may be involved in the pathogenesis of osteoarthritis (OA) and osteochondritis dissecans (OCD). Despite this, dietary programs have played a secondary role in the management of these connective tissue disorders. Articular cartilage is critically dependent upon the regular provision of nutrients (glucose and amino acids), vitamins (particularly vitamin C), and essential trace elements (zinc, magnesium, and copper). Therefore, dietary supplementation programs and nutraceuticals used in conjunction with non-steroidal, anti-inflammatory drugs (NSAIDs) may offer significant benefits to patients with joint disorders, such as OA and OCD. This article examines the available clinical evidence for the efficacy of nutraceuticals, antioxidant vitamin C, polyphenols, essential fatty acids, and mineral cofactors in the treatment of OA and related joint disorders in humans and veterinary species. This article also attempts to clarify the current state of knowledge. It also highlights the need for additional targeted research to elucidate the changes in nutritional status and potential alterations to the expression of plasma membrane transport systems in synovial structures in pathophysiological states, so that current therapy and future treatments may be better focused.

Objective To examine the collagens in cruciate ligaments of young Dunkin-Hartley guinea pigs, to determine whether a change in specific collagen types is an early feature of the spontaneous osteoarthritis (OA), which consistently develops in the medial compartment of the knee in this strain.Design Collagen types I, II, III, IX, and XI were detected by immunofluorescence microscopy in the anterior and posterior cruciate ligaments of animals at 3, 4–5 and 12 weeks of age. Type II collagen in PCL was further analysed by confocal microscopy or biochemical assay after cyanogen bromide digestion, SDS-PAGE and immunoblotting. Interfibrillar proteoglycans were visualized by transmission electron microscopy.Results Collagen types I and III formed the bulk of fibrous mid-ligament tissue in all animals. Typical cartilage collagens, types II, IX and XI, were identified by immunolabeling where ligaments attached to tibial bone. Type II collagen, normally restricted to the fibrocartilage attachment sites, was also found at separate foci in anterior fiber bundles of the posterior cruciate ligament in 12-week-old animals. Biochemical data confirmed these observations which, together with electron microscopy showing large atypical proteoglycan structures, suggested the deposition of fibrocartilage within the fibrous mid-ligament.Conclusions Cruciate ligaments, especially posterior cruciate ligament in Dunkin-Hartley guinea pigs synthesize cartilage-like matrix in mid-ligament prior to the appearance of classical signs of OA. Copyright 2002 Published by Elsevier Science Ltd on behalf of OsteoArthritis Research Society International.

Previously we have reported expression of an mRNA with homology to the Na+-dependent glutamate transporter, GLAST-1, in bone. Here we demonstrate that the complete open reading frame of GLAST-1 mRNA and corresponding 69 kDa protein are expressed in rat bone in vivo. We have also discovered a novel splice variant (GLAST-1a), lacking exon 3, expressed in rat bone and brain. A 55 kDa protein detected by anti-GLAST antibody in rat cerebellum corresponds to the molecular weight of unglycosylated GLAST-1a. This has led us to propose that GLAST-1a has an opposite orientation in the cell membrane to GLAST-1.

Using competitive binding experiments, it was found that native type XI collagen binds heparin, heparan sulfate, and dermatan sulfate. However, interactions were not evident with hyaluronic acid, keratan sulfate, or chondroitin sulfate chains over the concentration range studied. Chondrocyte-matrix interactions were investigated using cell attachment to solid phase type XI collagen. Pretreatment of chondrocytes with either heparin or heparinase significantly reduced attachment to type XI collagen. Incubation of denatured and cyanogen bromide-cleaved type XI collagen with radiolabeled heparin identified sites of interaction on the alpha1(XI) and alpha2(XI) chains. NH(2)-terminal sequence data confirmed that the predominant heparin-binding peptide contained the sequence GKPGPRGQRGPTGPRGSRGAR from the alpha1(XI) chain. Using rotary shadowing electron microscopy of native type XI collagen molecules and heparin-bovine serum albumin conjugate, an additional binding site was identified at one end of the triple helical region of the collagen molecule. This coincides with consensus heparin binding motifs present at the amino-terminal ends of both the alpha1(XI) and the alpha2(XI) chains. The contribution of glycosaminoglycan-type XI collagen interactions to cartilage matrix stabilization is discussed.

Introduction Cruciate ligament disease or injury is common in humans and dogs. There is some evidence to indicate that both its incidence and the degree of tissue pathology may vary between breeds and in relation to body weight, age, gender and body condition. In order to investigate a direct role of endocrine influences on pathology, we have attempted to establish a model system using isolated canine cranial cruciate ligament (CCL) cells in culture. In this preliminary study, we isolated CCL cells and characterized the matrix metalloproteinase (MMP) activities expressed by these cells in culture. MMPs contribute to the extracellular matrix turnover of connective tissue, and an imbalance between matrix synthesis and degradation leads to changes in the matrix, which may compromise ligament integrity and strength. Materials and methods CCLs were collected from animals following euthanasia for reasons other than joint disease. Ligament cells were isolated using collagenase digestion and maintained in culture in DMEM/F12 containing 10%(v/v) fetal bovine serum. Early passage number cells were incubated in medium containing reduced serum concentrations [1% (v/v)] in order to determine levels of MMP production. Gelatinase activities (MMP-2 and MMP-9) in the media of cultured cells were determined using gelatin zymography. The identification of MMP was confirmed by using EDTA to inhibit gelatinolytic activity. In order to determine whether the cells express collagenase activities, a fluorogenic substrate assay was used in which cell-conditioned medium was treated with APMA prior to incubation with the substrate (MCA-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2). Hormones (relaxin, leptin, growth hormone and oestrogen) were added at near physiological serum levels, where known, to 12-well plates after cells were 100% confluent, and medium samples were analysed 72 h after treatment. Results Pro-MMP-2 was the predominant gelatinase activity expressed by canine cruciate ligament cells in culture. The active forms of both MMP-2 and MMP-9 were observed at very low levels or not at all, differences being observed between animals. The volumes of medium samples analysed were adjusted such that the levels of MMP detected were within the range of activities that yielded a linear densitometric response curve. Our preliminary results show no change in levels of pro and active forms of MMP-2 and MMP-9 in cell culture after treatment with the hormones, oestradiol, relaxin, growth hormone and leptin. Collagenase activity was detected in the ligament cell-conditioned medium using the fluorogenic substrate, and the volume of medium used for assay was adjusted to lie within the standard curve. Our preliminary data suggest that hormonal treatment may modulate the production of collagenase activity. However, these findings require confirmation. Discussion These results show that pro-MMP-2 is the predominant gelatinase activity synthesized by cultured ligament cells. Previous studies have shown that this is also the predominant gelatinase activity present in extracts of canine cruciate tissue. We have also shown that CCL cells express a collagenase activity in culture. Therefore, we intend to use the CCL cell-culture system as a model to investigate the effects of systemic and local endocrine influences on the matrix degradative cascades. This model will allow us to obtain information on the roles of hormones in pathological processes prior to ligament rupture in vivo.

Introduction Injuries to the superficial digital flexor tendon (SDFT) are very common in the horse. The macroscopic degeneration of the tendon that is often apparent is associated with an altered matrix composition indicating substantial tissue-remodelling activity. The matrix metalloproteinases (MMPs) are responsible for degrading matrix components during tissue remodelling or turnover. There is evidence that mediators of angiogenesis, particularly vascular endothelial growth factor (VEGF), regulate the activities of gelatinases, and it is likely that tendon overuse or injury leads to disruption of tissue homeostasis, leading to hypoxia and VEGF up-regulation. In this study, we investigated the presence and levels of MMP-2 and MMP-9 and VEGF within both grossly normal and pathological tendon tissue. Furthermore, we investigated the presence of cartilage oligomeric matrix protein (COMP) within tendon. Materials and methods SDFT samples were collected post euthanasia. From tendons showing no gross pathology, samples were taken along the length of the SDFT and pulverized in a liquid nitrogen-cooled mill. Tendon tissue in which a central degenerate core lesion was observed was sampled and processed identically. Extracts of soluble protein in SDS-PAGE sample buffer were analysed by gelatin zymography for the presence of MMP-2 and MMP-9 and also by Western blot analysis for the presence of VEGF isoforms and COMP. SDS-PAGE followed by coomassie-blue staining was used to obtain a profile of the proteins extracted, and casein substrate gel zymography was used for anaylsis of serum-associated proteolytic activities. Results In the nonpathological tendon samples, no significant differences were observed along the length of the tendon. Basically, protein profiles, MMP-2 activity, activities co-migrating with serum-derived plasminogen and VEGF isoforms were constant. Pro-MMP-2 was the predominant gelatinase activity but active MMP-2 was also present. The main immunoreactive band detected using a polyclonal antiserum to VEGF had an apparent Mr of 16.5 kDa. Analysis of the pathological tendon samples revealed less consistent expression of these molecules and activities. The protein profile showed an apparent absence of some serum-derived soluble proteins in some areas, with raised levels of both pro and active MMP-2. An activity co-migrating with an active MMP-9 standard was also observed. Intact subunits of COMP were observed in all samples of pathological tissue. The presence of VEGF within the pathological tissue is being investigated. Discussion These results indicate that mediators of tissue remodelling are present both within normal and pathological SDFT. However, it is apparent that there may be imbalance between some protein components and enzyme activities within the pathological tendon. We will undertake further studies to determine whether our findings are common to further samples. This study should aid our understanding of the mechanisms that underlie SDFT pathology in the horse.

Background To study the ability of tepoxalin, a dual inhibitor of cyclooxygenase (COX) and lipoxygenase (LOX) and its active metabolite to reduce the catabolic response of cartilage to cytokine stimulation in an in vitro model of canine osteoarthritis (OA). Grossly normal cartilage was collected post-mortem from seven dogs that had no evidence of joint disease. Cartilage explants were cultured in media containing the recombinant canine interleukin-1β (IL-1β) at 100 ng/ml and recombinant human oncostatin-M (OSM) at 50 ng/ml. The effects of tepoxalin and its metabolite were studied at three concentrations (1 × 10-5, 1 × 10-6 and 1 × 10-7 M). Total glycosaminoglycan (GAG) and collagen (hydroxyproline) release from cartilage explants were used as outcome measures of proteoglycan and collagen depletion respectively. PGE2 and LTB4 assays were performed to study the effects of the drug on COX and LOX activity. Results Treatment with IL-1β and OSM significantly upregulated both collagen (p = 0.004) and proteoglycan (p = 0.001) release from the explants. Tepoxalin at 10-5 M and 10-6 M caused a decrease in collagen release from the explants (p = 0.047 and p = 0.075). Drug treatment showed no effect on GAG release. PGE2 concentration in culture media at day 7 was significantly increased by IL-1β and OSM and treatment with both tepoxalin and its metabolite showed a trend towards dose-dependent reduction of PGE2 production. LTB4 concentrations were too low to be quantified. Cytotoxicity assays suggested that neither tepoxalin nor its metabolite had a toxic effect on the cartilage chondrocytes at the concentrations and used in this study. Conclusion This study provides evidence that tepoxalin exerts inhibition of COX and can reduce in vitro collagen loss from canine cartilage explants at a concentration of 10-5 M. We can conclude that, in this model, tepoxalin can partially inhibit the development of cartilage degeneration when it is available locally to the tissue.

Background There is a paucity of data regarding molecular markers that identify the phenotype of the tendon cell. This study aims to quantify gene expression markers that distinguish between tendon fibroblasts and other mesenchymal cells which may be used to investigate tenogenesis. Methods Expression levels for 12 genes representative of musculoskeletal tissues, including the proposed tendon progenitor marker scleraxis, relative to validated reference genes, were evaluated in matched samples of equine tendon (harvested from the superficial digital flexor tendon), cartilage and bone using quantitative PCR (qPCR). Expression levels of genes associated with tendon phenotype were then evaluated in healthy, including developmental, and diseased equine tendon tissue and in tendon fibroblasts maintained in both monolayer culture and in three dimensional (3D) collagen gels. Results Significantly increased expression of scleraxis was found in tendon compared with bone (P = 0.002) but not compared to cartilage. High levels of COL1A2 and scleraxis and low levels of tenascin-C were found to be most representative of adult tensional tendon phenotype. While, relative expression of scleraxis in developing mid-gestational tendon or in acute or chronically diseased tendon did not differ significantly from normal adult tendon, tenascin-C message was significantly upregulated in acutely injured equine tendon (P = 0.001). Relative scleraxis gene expression levels in tendon cell monolayer and 3D cultures were significantly lower than in normal adult tendon (P = 0.002, P = 0.02 respectively). Conclusion The findings of this study indicate that high expression of both COL1A2 and scleraxis, and low expression of tenascin-C is representative of a tensional tendon phenotype. The in vitro culture methods used in these experiments however, may not recapitulate the phenotype of normal tensional tendon fibroblasts in tissues as evidenced by gene expression.