Gross, histologic, and gene expression characteristics of osteoarthritic articular cartilage of the metacarpal condyle of horses

OBJECTIVE: To identify patterns and correlations of gross, histologic, and gene expression characteristics of articular cartilage from horses with osteoarthritis. ANIMALS: 10 clinically normal horses and 11 horses with osteoarthritis of the metacarpal condyles. PROCEDURES: Metacarpophalangeal joints were opened and digitally photographed, and gross lesions were scored and quantified. Representative cartilage specimens were stained for histologic scoring. Total RNA from dorsal and palmar articular surfaces was processed on an equine gene expression microarray. RESULTS: Histologic scores were greater in both regions of osteoarthritic joints, compared with corresponding regions in control joints. Cartilage from the palmar aspect of diseased joints had the highest histologic scores of osteoarthritic sites or of either region in control joints. A different set of genes for dorsal and palmar osteoarthritis was identified for high and low gene expression. Articular cartilage from the dorsal region had surface fraying and greater expression of genes coding for collagen matrix components and proteins with anti-apoptotic function, compared with control specimens. Articular cartilage from the palmar region had greater fraying, deep fissures, and less expression of genes coding for glycosaminoglycan matrix formation and proteins with anti-apoptotic function, compared with cartilage from disease-free joints and the dorsal aspect of affected joints. CONCLUSIONS AND CLINICAL RELEVANCE: Metacarpal condyles of horses with naturally occurring osteoarthritis had an identifiable and regional gene expression signature with typical morphologic features.     

Characterization of age- and location-associated variations in the composition of articular cartilage from the equine metacarpophalangeal joint

Objective: To characterize the impact of age, gender, location and individual animal variation on the composition of articular cartilage from the metacarpophalangeal joint of horses. Design: Cartilage specimens were obtained from the metacarpophalangeal joints of 28 male, female and castrated male horses ranging in age from one day to 27 years of age. Cartilage samples from the distal metacarpus, proximal first phalanx and proximal sesamoids were analyzed separately. Chondrocyte number, DNA content, proteoglycan concentration and total collagen content were determined for each animal and joint location. Results: Age and joint location had a significant effect on chondrocyte number and DNA content with higher cell counts and DNA content detected in cartilage from the youngest age groups and in cartilage from the metacarpus and proximal sesamoids. The influence of age on chondrocyte numbers was not significant in horses over two years of age. Both age and joint location also influenced total proteoglycan and collagen content. Lower proteoglycan and collagen concentrations were detected in younger horses, and cartilage from the metacarpus had lower proteoglycan and collagen concentrations than that from other joint locations. Gender did not appear to influence chondrocyte number or matrix content of equine articular cartilage. However, there was significant residual variation in cellularity, proteoglycan levels and collagen content between individual animals that could not be explained by the signalment factors considered in this study. Conclusions: Future studies examining equine articular cartilage should avoid direct morphologic comparisons between animals of different ages, and any comparisons made between individuals should be interpreted cautiously. In addition, in vitro tissue culture models should avoid the use of cartilage pooled from different animals or from different locations within the same joint.     

Concentration of collagen, aggrecan and cartilage oligomeric matrix protein (COMP) in synovial fluid from equine middle carpal joints

The aim of the present investigation was to study the metabolic activity of the third carpal bone and the release of COMP, aggrecan and collagen type II molecules in the synovial fluid as a result of injury. Cartilage oligomeric matrix protein (COMP), aggrecan and collagen type II or fragments of these molecules released to the synovial fluid and serum (COMP) were quantified in samples from 73 left equine middle carpal joints from 2 breeds with different activity profiles (52 Standardbred trotters [STB] and 21 Swedish Warmblood riding horses [SWH]) and different articular cartilage lesions. Synovial and serum samples were analysed using inhibition ELISA for COMP and aggrecan. An ELISA that combines features of both the competitive and capture ELISAs was used for collagen type II. COMP and aggrecan concentrations decreased in synovial fluid from the joints with moderate lesions of STB compared with the normal joints; COMP from 16.6 to 12.0 microg/ml and aggrecan from 93.0 to 68.1 microg/ml. In serum, COMP concentrations were also lowered in the STB with moderate lesions compared with the normal joints, while in the SWH, the COMP concentration in synovial fluids from joints with moderate lesions was somewhat increased at 19.6 microg/ml compared with the normal joints (17.6 microg/ml). The ratio between aggrecan/COMP in the synovial fluid from joints with moderate lesions was higher in the STB (6.2) than in the SWH (3.4). The level of collagen type II in synovial fluid was higher in the SWH (8.8 microg/ml) than the STB (1.6 microg/ml), but there was no correlation between joint damage and collagen concentrations in synovial fluids (10.0 and 1.8 microg/ml in joints with moderate lesions from SWH and STB, respectively). A marked difference in COMP synthesised upon metabolic labelling between the normal and osteoarthritic cartilage was seen and the synthesis of COMP in the articular cartilage of the third carpal bone with moderate articular lesions (from an STB) was lower than in the joint with mild lesions. This difference between breeds may reflect different load characters, in release of macromolecules in osteoarthritic and normal joints. This a novel finding that should be considered in studies of equine traumatic arthritis.     

Assessment of the effects of age and joint disease on hydroxyproline and glycosaminoglycan concentrations in synovial fluid from the metacarpophalangeal joint of horses

OBJECTIVE: To assess the effects of age and joint disease on hydroxyproline and glycosaminoglycan (GAG) concentrations in synovial fluid from the metacarpophalangeal joint of horses and evaluate the association of those concentrations with severity of osteoarthritis and general matrix metalloproteinase (MMP) activity. SAMPLE POPULATION: Synovial fluid was collected from the metacarpophalangeal joints of foals at birth (n = 10), 5-month-old foals (10), 11-month-old foals (5), and adult horses (73). PROCEDURE: Hydroxyproline and GAG concentrations were determined in synovial fluid samples. The severity of osteoarthritis in adult joints was quantified by use of a cartilage degeneration index (CDI) and assessment of general MMP-activity via a fluorogenic assay. RESULTS: Hydroxyproline and GAG concentrations in synovial fluid were highest in neonates and decreased with age. Concentrations reached a plateau in adults by 4 years and remained constant in healthy joints. In synovial fluid from osteoarthritic joints, hydroxyproline and GAG concentrations were not increased, compared with unaffected joints, but hydroxyproline were significantly correlated with the CDI and general MMP activity. There was no significant correlation between GAG concentration and CDI value or MMP activity. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in hydroxyproline concentration in synovial fluid appeared to indicate damage to collagen of the articular cartilage. In joints with osteoarthritis, the lack of high GAG concentration in synovial fluid and the absence of a significant correlation between GAG concentration and CDI values or MMP activity may severely limit the usefulness of this marker for monitoring equine joint disease.     

Expression of bone morphogenetic protein-6 and -2 and a bone morphogenetic protein antagonist in horses with naturally acquired osteochondrosis

OBJECTIVE: To determine the mRNA expression of bone morphogenetic protein (BMP)-6 and -2 and a BMP antagonist (Noggin) in horses with osteochondrosis. SAMPLE POPULATION: Samples of articular cartilage from affected stifle or shoulder joints of 10 immature horses with naturally acquired osteochondrosis and corresponding joints of 9 clinically normal horses of similar age; additionally, samples of distal femoral growth plate cartilage and distal femoral articular cartilage were obtained from a normal equine fetus. PROCEDURE: Cartilage specimens were snap-frozen in liquid nitrogen, and total RNA was isolated. Adjacent specimens were fixed in 4% paraformaldehyde for histologic examination. Expression of BMP-6, BMP-2, and Noggin mRNA was evaluated by real-time quantitative polymerase chain reaction (PCR) assays. Spatial tissue mRNA expression of BMP-6 was determined by in situ hybridization. RESULTS: Nucleotide sequences were obtained for portions of the BMP-6 propeptide and mature peptide region, as well as the signal and mature peptide region of Noggin. Expression of BMP-6, BMP-2, and Noggin mRNA was found to be similar in cartilage from normal and osteochondrosis-affected horses. Spatial expression of BMP-6 correlated with the middle and deep layers of articular cartilage; no differences were observed in overall expression between cartilage specimens from the 2 groups of horses. No expression of BMP-6 was found in the superficial layer, subchondral bone, or osteochondrosis-affected cleft fibrous tissue. CONCLUSIONS AND CLINICAL RELEVANCE: Although these signaling peptides may play important roles in cartilage differentiation, results did not provide evidence to suggest that they are involved in the disease process of osteochondrosis.     

Relationship between synovial fluid levels of glycosaminoglycans, hydroxyproline and general MMP activity and the presence and severity of articular cartilage change on the proximal articular surface of P1

REASONS FOR PERFORMING STUDY: Osteoarthritis (OA) is one of the most prevalent and disabling chronic conditions affecting horses and leads to degeneration of articular cartilage. Diagnosis is based on clinical signs in combination with radiography, which is relatively insensitive and provides only an indication of accumulated damage. Alternative methods, such as molecular markers, are therefore needed that can quantitatively, reliably and sensitively detect osteoarthritic changes in the joints at an early stage of the disease. If such markers are to be used reliably, it is important to know the relationship between marker concentration and cartilage composition. OBJECTIVES: To study the relationship between cartilage composition, synovial fluid levels of glycosaminoglycans (GAGs), hydroxyproline (Hyp) and general matrix metalloproteinase (MMP) activity, and the presence and severity of articular cartilage damage on the articular surface of P1. METHODS: Synovial fluid (SF) was collected from the metacarpophalangeal joints of 60 mature horses, and levels of GAGs, Hyp and general MMP activity were determined. Further, GAG and denatured collagen content of the articular cartilage were determined at the dorsal articular margin of P1 (site 1) and central cavity (site 2). The presence and severity of cartilage change was quantified using the cartilage degeneration index (CDI), measured at the same 2 sites. Correlations between SF parameters, cartilage composition and degree of cartilage degeneration were sought using correlation analysis. RESULTS: There was no correlation between GAG or Hyp content of SF and the amount of GAGs or denatured collagen, respectively, in cartilage. In joints with moderate to severe cartilage damage, the GAG content of site 1 was significantly lower than in joints with no to minimal cartilage change (P = 0.005) and there was a negative correlation between the amount of denatured collagen and GAG content at site 1 in all joints (r = -039, P = 0.002). Further, in joints with moderate to severe cartilage damage, there was a significant positive correlation between MMP activity in SF and Hyp levels in SF (r = 0.72, P < 0.001) and CDI at sites 1 (r = 0.46, P = 0.03) and 2 (r = 0.43, P = 0.04). CONCLUSIONS: General MMP activity in joints with moderate to severe cartilage damage is related to the severity of those cartilage changes and to Hyp levels in SF. Glycosaminoglycan levels in SF are not directly related to MMP activity, GAG content of articular cartilage or severity of cartilage change. POTENTIAL RELEVANCE: Glycosaminoglycan levels in SF are not helpful for the early detection of cartilage lesions. In damaged joints, Hyp levels may give an indication of the severity of cartilage change as they are strongly related to MMP activity, but do not qualify as markers for the presence or absence of cartilage lesions.     

Comparison of proteoglycan and collagen in articular cartilage of horses with naturally developing osteochondrosis and healing osteochondral fragments of experimentally induced fractures

OBJECTIVE: To compare articular cartilage from horses with naturally developing osteochondrosis (OC) with normal articular cartilage and healing cartilage obtained from horses with experimentally induced osteochondral fractures. SAMPLE POPULATION: 109 specimens of articular cartilage from 78 horses. PROCEDURE: Morphologic characteristics, proteoglycan (PG), and type II collagen were analyzed in articular cartilage of OC specimens (group 1), matched healing cartilage obtained 40 days after experimentally induced osteochondral fractures (group 2), and matched normal cartilage from the same sites (group 3). RESULTS: 79 specimens of OC cartilage were obtained from horses. Ex vivo PG synthesis was significantly greater in the femoral cartilage, compared with synthesis in the tibial cartilage, and significantly greater for groups 1 and 2, compared with group 3. For groups 1 and 2, femoral fragments had significantly greater PG content, compared with PG content in tibial fragments. Keratan sulfate content was significantly less in group 3, compared with groups 1 and 2. Cartilage from the OC specimens had loss of structural architecture. The OC tissue bed stained positive for chondroitin sulfate and type II collagen, but the fracture bed did not. CONCLUSIONS AND CLINICAL RELEVANCE: Our analyses could not distinguish articular cartilage from horses with OC and a healing fracture. Both resembled an anabolic, reparative process. Immunohistochemical analysis suggested a chondromyxoid tissue in the OC bed that was morphologically similar to fibrous tissue but phenotypically resembled hyaline cartilage. Thus, tissue in the OC bed may be degenerative cartilage, whereas tissue in the fracture bed may be reparative fibrous callus.     

Assessment of cellular, biochemical, and histologic effects of bipolar radiofrequency treatment of canine articular cartilage

OBJECTIVE: To assess the cellular, biochemical, and histologic effects of bipolar radiofrequency-generated heat on canine articular cartilage. SAMPLE POPULATION: Articular cartilage explants (n = 72) from 6 canine cadavers and cultured articular chondrocytes from 5 canine cadavers. PROCEDURE: Cartilage explants were randomly assigned to receive no treatment or treatment with focal (3 seconds) or diffuse bipolar radiofrequency. Following treatment, methylene blue permeability assay was performed (n = 12) and remaining samples (60) were cultured. Immediately and 5, 10, and 20 days after treatment, cultured explants were assessed for glycosaminoglycan (GAG) and collagen contents, type II collagen and matrix metalloproteinase (MMP)-13 immunoreactivity, and modified Mankin histologic scores. Liquid culture media were collected every 4 days and GAG content measured. Additionally, cultured chondrocytes were exposed for 3 seconds to media preheated to 37 degrees, 45 degrees, or 55 degrees C. Cell viability was determined via 2 different assays immediately and 24 hours after treatment. RESULTS: Radiofrequency-treated cartilage had reduced permeability and considerable histologic damage, compared with control samples; most treated samples had reduced collagen II staining and increased MMP-13 immunostaining. Compared with other treatments, less GAGs were released from cartilage after diffuse radiofrequency treatment throughout the study period. Cell viability was significantly different between controls and cells treated at 55 degrees C immediately and 24 hours after heat treatment. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, bipolar radiofrequency treatment had detrimental effects on normal articular cartilage cells and extracellular matrix with probable long-term clinical consequences. The usefulness of radiofrequency for treatment of osteoarthritic articular cartilage requires further investigation.     

Morphologic and biochemical study of sternal cartilage autografts for resurfacing induced osteochondral defects in horses.

Using biodegradable pins, sternal cartilage autografts were fixed into osteochondral defects of the distal radial carpal bone in ten 2 to 3-year-old horses. The defects measured 1 cm2 at the surface and were 4 mm deep. Control osteochondral defects of contralateral carpi were not grafted. After confinement for 7 weeks, horses were walked 1 hour daily on a walker for an additional 9 weeks. Horses were euthanatized at 16 weeks. Half of the repair tissue was processed for histologic and histochemical (H&E and safranin-O fast green) examinations. The other half was used for the following biochemical analyses: type-I and type-II collagen contents, total glycosaminoglycan content, and galactosamine-to-glucosamine ratio. On histologic examination, the repair tissue in the grafted defects consisted of hyaline-like cartilage. Repair tissue in the nongrafted defects consisted of fibrocartilaginous tissue, with fibrous tissue in surface layers. On biochemical analysis, repair tissue of grafted defects was composed predominantly of type-II collagen; repair tissue of non-grafted defects was composed of type-I collagen. Total glycosaminoglycan content of repair tissue of grafted defects was similar to that of normal articular cartilage. Total glycosaminoglycan content of nongrafted defects was 62% of that of normal articular cartilage (P less than 0.05). Repair tissue of all defects was characterized by galactosamine-to-glucosamine ratio significantly (P less than 0.05) higher than that of normal articular cartilage. These results at 16 weeks after grafting indicate that sternal cartilage may potentially constitute a suitable substitute for articular cartilage in large osteochondral defects of horses.     

Influence of site and age on biochemical characteristics of the collagen network of equine articular cartilage

OBJECTIVE: To determine variations in biochemical characteristics of equine articular cartilage in relation to age and the degree of predisposition for osteochondral disease at a specific site. SAMPLE POPULATION: Articular cartilage specimens from 53 horses 4 to 30 years old. PROCEDURE: Healthy specimens were obtained from 2 locations on the proximal articular surface of the first phalanx that had different disease prevalences (site 1 at the mediodorsal margin and site 2 at the center of the medial cavity). Water, total collagen, and hydroxylysine contents and enzymatic (hydroxylysylpyridinoline [HP]) and nonenzymatic (pentosidine) crosslinking were determined at both sites. Differences between sites were analyzed by ANOVA (factors, site, and age), and age correlation was tested by Pearson's product-moment correlation analysis. Significance was set at P< 0.01. RESULTS: Correlation with age was not found for water, collagen, hydroxylysine contents, and enzymatic cross-linking. Nonenzymatic crosslinking was higher in older horses and was linearly related to age (r = 0.94). Water and collagen contents and HP and pentosidine crosslinks were significantly higher at site 1. Hydroxylysine content was significantly lower at site 1. CONCLUSIONS: Except for nonenzymatic glycation, the composition of articular cartilage collagen does not change significantly in adult horses. A significant topographic variation exists in biochemical characteristics of the articular cartilage collagen network in equine metacarpophalangeal joints. These differences may influence local biomechanical properties and, hence, susceptibility to osteochondral disease, as will greater pentosidine crosslinks in older horses that are likely to cause stiffer and more brittle cartilage.     

Biochemical analysis of the articular cartilage and subchondral and trabecular bone of the metacarpophalangeal joint of horses with early osteoarthritis

OBJECTIVE: To assess whether site-related changes in biochemical composition are present in the cartilage and subchondral and trabecular bone of the metacarpophalangeal joint of horses with early osteoarthritis. SAMPLE POPULATION: Right metacarpophalangeal joints from 59 mature warmblood horses. PROCEDURE: Biochemical data (cross-link, amino acid, DNA, and ash contents; denatured collagen and glycosaminoglycan [GAG] concentrations; bone mineral density; and mineral composition) were obtained from 2 differently loaded sites of phalanx I cartilage and subchondral and trabecular bone samples; data were compared with previously published values from nonosteoarthritic equine joints. RESULTS: Compared with findings in nonosteoarthritic joints, GAG concentration was lower in cartilage from osteoarthritic joints and there was a loss of site differences in cellularity and lysylpyridinoline (LP) cross-link content. In subchondral bone, LP cross-link content was decreased overall and there was a loss of site differences in osteoarthritic joints; ash content was higher in the osteoarthritic joints. Hydroxyproline content in trabecular bone from osteoarthritic joints was greater than that in nonosteoarthritic trabecular bone. In all 3 layers and at both sites, the linear increase of the pentosidine cross-link content with age had diminished or was not apparent in the horses with osteoarthritic joints. CONCLUSIONS AND CLINICAL RELEVANCE: In equine metacarpophalangeal joints with early osteoarthritis, distinct biochemical changes were detected in the cartilage and subchondral and trabecular bone. The dissimilarity in response of the different tissues and differences between the sites that are affected may be related to differences in biomechanical loading and transmission and dissipation of force.     

Biochemical study of repair of induced osteochondral defects of the distal portion of the radial carpal bone in horses by use of periosteal autografts

Periosteal autografts were used for repair of large osteochondral defects in 10 horses aged 2 to 3 years old. In each horse, osteochondral defects measuring 1.0 x 1.0 cm2 were induced bilaterally on the distal articular surface of each radial carpal bone. Control and experimental defects were drilled. Periosteum was harvested from the proximal portion of the tibia and was glued into the principal defects, using a fibrin adhesive. Control defects were glued, but were not grafted. Sixteen weeks after the grafting procedure, the quality of the repair tissue of control and grafted defects was assessed biochemically. Total collagen content and the proportion of type-II collagen were determined. Galactosamine and glucosamine contents also were determined. From these measurements, contents of chondroitin and keratan sulfate and total glycosaminoglycan, and galactosamine-to-glucosamine ratio were calculated. All biochemical variables were compared with those of normal equine articular cartilage taken from the same site in another group of clinically normal horses. Total collagen content was determined on the basis of 4-hydroxyproline content, using a colorimetric method. The proportions of collagen types I and II in the repair tissue were assessed by electrophoresis of their cyanogen bromide-cleaved peptides on sodium dodecyl sulfate slab gels. Peptide ratios were computed and compared with those of standard mixtures of type-I and type-II collagens. Galactosamine and glucosamine contents were determined by use of ion chromatography. In general, the biochemical composition of repair tissue of grafted and nongrafted defects was similar, but clearly differed from that of normal articular cartilage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of transforming growth factor beta1 on chondrogenic differentiation of cultured equine mesenchymal stem cells

OBJECTIVE: To determine the morphologic and phenotypic effects of transforming growth factor beta1 (TGFbeta1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes. SAMPLE POPULATION: Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8-month-old horses. PROCEDURE: After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-beta1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated from cell cultures, and expression of aggrecan, decrin, collagen type-I, and collagen type-II mRNA was assessed by means of Northern blot analyses. Cell cultures were stained with H&E or toluidine blue and examined histologically. Additional cultures were examined after immunohistochemical staining for type-I and -II collagen. RESULTS: MSC cultures exposed to TGF-beta1 had an increased cellular density with cell layering and nodule formation that was most pronounced in cultures treated with 5 ng of TGF-beta1/ml. Expression of collagen type-II mRNA in MSC cultures exposed to 5 ng of TGF-beta1/ml was 1.7 times expression in control cultures, and expression of collagen type-I mRNA was 2.8 times expression in control cultures. Treatment of MSC with TGF-beta1 led to dose-related increases in area and intensity of type-II collagen immunoreaction. CONCLUSION: Results suggest that TGF-beta1 enhances chondrogenic differentiation of bone marrow-derived MSC in a dose-dependent manner.     

Effects of enrofloxacin and magnesium deficiency on matrix metabolism in equine articular cartilage

OBJECTIVE: To investigate the effects of enrofloxacin and magnesium deficiency on explants of equine articular cartilage. SAMPLE POPULATION: Articular cartilage explants and cultured chondrocytes obtained from adult and neonatal horses. PROCEDURE: Full-thickness explants and cultured chondrocytes were incubated in complete or magnesium-deficient media containing enrofloxacin at concentrations of 0, 1, 5, 25, 100, and 500 microg/ml. Incorporation and release of sulfate 35S over 24 hours were used to assess glycosaminoglycan (GAG) synthesis and degradation. An assay that measured binding of dimethylmethylene blue dye was used to compare total GAG content between groups. Northern blots of RNA from cultured chondrocytes were probed with equine cDNA of aggrecan, type-II collagen, biglycan, decorin, link protein, matrix metalloproteinases 1, 3, and 13, and tissue inhibitor of metalloproteinase 1. RESULTS: A dose-dependent suppression of 35S incorporation was observed. In cartilage of neonates, 35S incorporation was substantially decreased at enrofloxacin concentrations of 25 mg/ml. In cartilage of adult horses, 35S incorporation was decreased only at enrofloxacin concentrations of > or =100 microg/ml. Magnesium deficiency caused suppression of 35S incorporation. Enrofloxacin or magnesium deficiency did not affect GAG degradation or endogenous GAG content. Specific effects of enrofloxacin on steady-state mRNA for the various genes were not observed. CONCLUSION AND CLINICAL RELEVANCE: Enrofloxacin may have a detrimental effect on cartilage metabolism in horses, especially in neonates.     

Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes

OBJECTIVE: To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. PROCEDURE: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related genes. Incorporation of 35S-sulfate, fluorography of 14C-proline labeled medium, zymography, and western blotting were used to confirm effects on protein synthesis. RESULTS: IL-1beta and TNF-alpha increased steady-state amounts of mRNA of matrix metalloproteinases 1, 3, and 13 by up to 100-fold. Amount of mRNA of tissue inhibitor of metalloproteinase-1 also increased but to a lesser extent (1.5- to 2-fold). Amounts of mRNA of type-II collagen and link protein were consistently decreased in a dose-dependent manner. Amount of aggrecan mRNA was decreased slightly; amounts of biglycan and decorin mRNA were minimally affected. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of cultured equine chondrocytes with IL-1beta or TNF-alpha resulted in marked alterations in expression of various matrix and matrix-related genes consistent with the implicated involvement of these genes in arthritis. Expression of matrix metalloproteinases was increased far more than expression of their putative endogenous inhibitor. Results support the suggestion that IL-1beta and TNF-alpha play a role in the degradation of articular cartilage in arthritis.     

Influence of age, site, and degenerative state on the speed of sound in equine articular cartilage

OBJECTIVE: To determine the speed of sound (SOS) in equine articular cartilage and investigate the influence of age, site in the joint, and cartilage degeneration on the SOS. SAMPLE POPULATION: Cartilage samples from 38 metacarpophalangeal joints of 38 horses (age range, 5 months to 22 years). PROCEDURE: Osteochondral plugs were collected from 2 articular sites of the proximal phalanx after the degenerative state was characterized by use of the cartilage degeneration index (CDI) technique. The SOS was calculated (ratio of needle-probe cartilage thickness to time of flight of the ultrasound pulse), and relationships between SOS value and age, site, and cartilage degeneration were evaluated. An analytical model of cartilage indentation was used to evaluate the effect of variation in true SOS on the determination of cartilage thickness and dynamic modulus with the ultrasound indentation technique. RESULTS: The mean SOS for all samples was 1,696 +/- 126 m/s. Age, site, and cartilage degeneration had no significant influence on the SOS in cartilage. The analytical model revealed that use of the mean SOS of 1,696 m/s was associated with maximum errors of 17.5% on cartilage thickness and 70% on dynamic modulus in an SOS range that covered 95% of the individual measurements. CONCLUSIONS AND CLINICAL RELEVANCE: In equine articular cartilage, use of mean SOS of 1,696 m/s in ultrasound indentation measurements introduces some inaccuracy on cartilage thickness determinations, but the dynamic modulus of cartilage can be estimated with acceptable accuracy in horses regardless of age, site in the joint, or stage of cartilage degeneration.     

Analysis of cartilage oligomeric matrix protein (COMP) degradation and synthesis in equine joint disease

REASONS FOR PERFORMING STUDY: Cartilage oligomeric matrix protein (COMP) is abundant within cartilage; its turnover and/or degradation have been investigated in various equine joint diseases and it has been suggested that COMP fragmentation might be useful for monitoring such conditions. OBJECTIVES: To determine whether COMP metabolism is compromised in equine osteoarthritis (OA) and whether COMP degradation is a useful joint marker representing cartilage destruction. HYPOTHESIS: A monoclonal antibody (mAb) with a higher affinity for degraded COMP allows discrimination of diseased joints by quantifying COMP levels and fragmentation. METHODS: A mAb (clone14G4) was generated against equine cartilage COMP. The NH2-terminal sequence of enzyme-cut COMP fragments recognised by 14G4 was determined, as was the efficiency of binding to COMP (using a generated COMP peptide). COMP concentration and fragmentation were analysed in synovial fluid (SF) from normal horses and those with OA. RESULTS: The mAb 14G4 had a higher affinity for the smaller fragments of equine COMP, compared with a mAb (clone 12C4) generated against human COMP. The 14G4 epitope was identified as between C134 and F147. The COMP values in OA (mean +/- s.d. 205.8 +/- 90.9 microg/ml) were significantly higher than in the normal (133.1 +/- 31.5 microg/ml) SF. On the immunoblots of OA sample, the proportions of intact COMP were significantly lower, while smaller fragments ranging from 75 to 290 kDa were higher compared with the normal SF. CONCLUSIONS AND POTENTIAL RELEVANCE: The mAb 14G4 reliably detects COMP degradation as well as synthesis, and fragmentation analysis combined with quantification in SF could be useful to study equine OA.     

Measurement of articular cartilage stiffness of the femoropatellar, tarsocrural, and metatarsophalangeal joints in horses and comparison with biochemical data

OBJECTIVE: To determine normal cartilage stiffness values in different weight-bearing and non-weight-bearing areas of 3 different equine joints, and to evaluate the relationship between cartilage stiffness and glycosaminoglycan (GAG) and collagen content. STUDY DESIGN: Compressive stiffness of the articular cartilage was measured in 8 horse cadaver femoropatellar (FP), tarsocrural (TC), and metatarsophalangeal (MT) joints. Gross evaluation, collagen content, GAG content, and histologic appearance were assessed for each measurement location. ANIMALS: Eight equine cadavers (4 intact females, 4 castrated males; 7 Quarter Horse or Quarter Horse type, 1 Arabian; aged 4-12 years, weighing 400-550 kg). METHODS: The articular surfaces of 8 equine cadaver FP, TC, and MT joints were grossly evaluated for signs of articular cartilage pathology. Stiffness at preselected sites (FP joint-6 sites; TC joint-3 sites; MT joint-4 sites) was determined using an arthroscopic indentation instrument. Biochemical composition (collagen, GAG content) and histologic evaluation (modified Mankin score) were assessed for each measurement site. RESULTS: All cartilage from all sites evaluated was determined to be normal based on macroscopic and histologic assessments. No significant correlation between Mankin scores and cartilage stiffness values was observed. Site differences in cartilage stiffness were measured in all 3 joints (P<.001). GAG or collagen content had a significant positive correlation with stiffness values in 6 of 13 sites (P<.05, r>0.622, r2>0.387). CONCLUSION: Relative cartilage stiffness values measured in healthy equine joints are site dependent and can be measured using an indentation device intended for arthroscopic application. CLINICAL RELEVANCE: An indentation instrument provided an objective means of determining relative compressive stiffness of articular cartilage. Further research needs to be performed to confirm the site and joint differences observed in this study in clinically normal horses and to determine if the tester can be used clinically to predict articular cartilage pathology.