Ex vivo investigation of the use of hydrothermal energy to induce chondrocyte necrosis in articular cartilage of the metacarpophalangeal and metatarsophalangeal joints of horses

OBJECTIVE: To evaluate the use of hydrothermal ablation of articular cartilage for arthrodesis in horses through investigation of the effects of joint lavage with physiologic saline (0.9% NaCI) solution (80 degrees C) for various treatment times on chondrocyte viability in the articular cartilage of the metacarpophalangeal and metatarsophalangeal joints of cadaveric horse limbs. Sample Population-7 pairs of metacarpophalangeal and 8 pairs of metatarsophalangeal joints from 8 Thoroughbreds. PROCEDURE: The horses were euthanatized for reasons unrelated to musculoskeletal disease. On a random basis, 1 joint of each pair underwent intra-articular lavage for 5, 10, or 15 minutes with heated saline solution (80 degrees C); the other joint underwent sham treatment of similar duration with saline solution at 22 degrees C (control). Cartilage samples from the distal articular surface of metacarpus III (or metatarsus III), the proximal surface of the proximal phalanx, and the lateral and medial proximal sesamoid bones were assessed for chondrocyte viability via confocal microscopy and viability staining following enzymatic digestion. RESULTS: Compared with the control joints, findings of both viability assays indicated that the percentage of sites containing viable chondrocytes in heat-treated joints was decreased.Treatment hazard ratios of 0.048 (confocal microscopy) and 0.2 (digestion assay) were estimated. Histologically, periarticular soft tissues had minimal detrimental effects after heat treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Ex vivo intra-articular lavage with saline solution at 80 degrees C resulted in the death of almost all articular chondrocytes in the joint. This technique may be a satisfactory method for extensive cartilage ablation when performing arthrodesis by minimally invasive techniques.     

In vitro evaluation of the effect of dimethyl sulfoxide on equine articular cartilage matrix metabolism

OBJECTIVE: To evaluate the effects of dimethyl sulfoxide (DMSO) on equine articular cartilage matrix metabolism. STUDY DESIGN: Using a cartilage explant culture system, proteoglycan (PG) synthesis, PG release, lactate metabolism, chondrocyte viability, and metabolism recovery were determined after cartilage exposure to DMSO. SAMPLE POPULATION: Cartilage harvested from metacarpophalangeal and metatarsophalangeal joints of 12 horses (age range, 1 to 10 years). METHODS: Explants were exposed to concentrations of DMSO (1% to 20%) for variable times (3 to 72 hours). PG synthesis and release were determined by a radiolabel incorporation assay and dimethylmethylene blue (DMMB) dye assay, respectively. Lactate released into culture media was measured, and chondrocyte viability was assessed using the Formizan Conversion Assay and a paravital staining protocol. Metabolism recovery was assessed in explants that were allowed to recover in maintenance media after exposure to DMSO. RESULTS: PG synthesis and lactate metabolism were inhibited in a dose- and time-dependent manner after exposure to DMSO concentrations > or = 5%; there was no significant alteration in PG release. No change in chondrocyte viability was detected after incubation with DMSO. PG synthesis and lactate metabolism returned to baseline rates when allowed a recovery period after exposure to DMSO. CONCLUSIONS: DMSO concentrations > or = 5% suppress equine articular cartilage matrix metabolism. Suppression of PG synthesis and lactate metabolism is reversible and does not appear to be the result of chondrocyte death. CLINICAL RELEVANCE: Equine clinicians adding DMSO to intraarticular lavage solutions should be aware that DMSO may have deleterious effects on equine articular cartilage matrix metabolism.     

Influence of exercise and joint topography on depth‐related spatial distribution of proteoglycan and collagen content in immature equine articular cartilage

Reasons for performing study: There is ample evidence on topographical heterogeneity of the principal biochemical components of articular cartilage over the surface of the joint and the influence of loading thereon, but no information on depth‐related zonal variation in horses. Objectives: To study depth‐related zonal variation in proteoglycan (PG) and collagen content in equine articular cartilage. Methods: Two techniques (safranin‐O densitometry and Fourier transform infrared spectroscopy) were applied to sections of articular cartilage from the proximal phalangeal bone of the metacarpophalangeal joint of 18‐month‐old Thoroughbreds that had been raised at pasture from age 0–18 months without (PASTEX) and with (CONDEX) additional exercise. Two sites were investigated: site 1 at the joint margin that is unloaded at rest or at slow gaits, but subjected to high‐intensity loading during athletic activity; and site 2, a continuously, but less intensively, loaded site in the centre of the joint. Results: Proteoglycan values increased from the surface to the deep layers of the cartilage, collagen content showed a reverse pattern. PG content was significantly higher at site 2 in both PASTEX and CONDEX animals without an effect of exercise. In the PASTEX animals collagen content was significantly higher at site 1, but in the CONDEX group the situation was reversed, due to a significant exercise effect on site 1, leading to a reduced collagen content. Conclusions: Collagen and PG content gradients agree with findings in other species. The observations on PG levels suggest that the exercise level was not strenuous. The collagen results in the PASTEX group confirmed earlier findings, the lower levels at site 1 in the CONDEX group being possibly due to an advancement of the physiological maturation process of collagen remodelling. Potential relevance: This study confirms earlier observations that even moderate variations in exercise level in early age may have significant effects on the collagen network of articular cartilage.     

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.     

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.     

Functional adaptation of equine articular cartilage: the formation of regional biochemical characteristics up to age one year

Biochemical heterogeneity of cartilage within a joint is well known in mature individuals. It has recently been reported that heterogeneity for proteoglycan content and chondrocyte metabolism in sheep develops postnatally under the influence of loading. No data exist on the collagen network in general or on the specific situation in the horse. The objective of this study was to investigate the alterations in equine articular cartilage biochemistry that occur from birth up to age one year, testing the hypothesis that the molecular composition of equine cartilage matrix is uniform at birth and biochemical heterogeneity is formed postnatally. Water content, DNA content, glycosaminoglycan content (GAG) and biochemical characteristics of the collagen network (collagen content, hydroxylysine content and hydroxylysylpyridinoline [HP] crosslinks) were measured in immature articular cartilage of neonatal (n = 16), 5-month-old foals (n = 16) and yearlings (n = 16) at 2 predefined differently loaded sites within the metacarpophalangeal joint. Statistical differences between sites were analysed by ANOVA (P<0.01), and age correlation was tested by Pearson's product moment correlation analysis (P<0.01). In neonatal cartilage no significant site differences were found for any of the measured biochemical parameters. This revealed that the horse has a biochemically uniform joint (i.e. the cartilage) at birth. In the 5-month-old foals and yearlings, significant site differences, comparable to those in the mature horse, were found for DNA, GAG, collagen content and hydroxylysine content. This indicates that functional adaptation of articular cartilage to weight bearing for these biochemical parameters takes place during the first months postpartum. Water content and HP crosslinks showed no difference between the 2 sites from neonatal horses, 5-month-old animals and yearlings. At both sites water, DNA and GAG decreased during maturation while collagen content, hydroxylysine content and HP crosslinks increased. We propose that a foal is born with a uniform biochemical composition of cartilage in which the functional adaptation to weight bearing takes place early in life. This adaptation results in biochemical and therefore biomechanical heterogeneity and is thought to be essential to resist the different loading conditions to which articular cartilage is subjected during later life. As collagen turnover is extremely low at mature age, an undisturbed functional adaptation of the collagen network of articular cartilage at a young age may be of significant importance for future strength and resistance to injury.     

Use of an antineoepitope antibody for identification of type-II collagen degradation in equine articular cartilage.

OBJECTIVE: To develop an antibody that specifically recognizes collagenase-cleaved type-II collagen in equine articular cartilage. SAMPLE POPULATION: Cartilage specimens from horses euthanatized for problems unrelated to the musculoskeletal system. PROCEDURE: A peptide was synthesized representing the carboxy- (C-) terminus (neoepitope) of the equine type-II collagen fragment created by mammalian collagenases. This peptide was used to produce a polyclonal antibody, characterized by western analysis for reactivity to native and collagenase-cleaved equine collagens. The antibody was evaluated as an antineoepitope antibody by ELISA, using peptides +/- an amino acid at the C-terminus of the immunizing peptide. Collagen cleavage was assayed from equine articular cartilage cultured with interleukin-1 (IL-1), +/- a synthetic MMP inhibitor, BAY 12-9566. Cartilage specimens from osteoarthritic and nonarthritic joints were compared for antibody staining. RESULTS: An antibody, 234CEQ, recognized only collagenase-generated 3/4-length fragments of equine type-II collagen. This was a true antineoepitope antibody, as altering the C-terminus of the immunizing peptide significantly decreased competition for binding in an inhibition ELISA. The IL-1-induced release of type-II collagen fragments from articular cartilage was prevented with the MMP inhibitor. Cartilage from an osteoarthritic joint of a horse had increased staining with the 234CEQ antibody, compared with normal articular cartilage. CONCLUSIONS AND CLINICAL RELEVANCE: We generated an antineoepitope antibody recognizing collagenase-cleaved type-II collagen of horses. This antibody detects increases in type-II collagen cleavage in diseased equine articular cartilage. The 234CEQ antibody has the potential to aid in the early diagnosis of arthritis and to monitor treatment responses.     

FAT‐SUPPRESSED SPOILED GRADIENT‐RECALLED IMAGING OF EQUINE METACARPOPHALANGEAL ARTICULAR CARTILAGE

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±0.17 mm) 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.     

CORRELATION OF ARTICULAR CARTILAGE THICKNESS MEASUREMENTS MADE WITH MAGNETIC RESONANCE IMAGING,MAGNETIC RESONANCE ARTHROGRAPHY,AND COMPUTED TOMOGRAPHIC ARTHROGRAPHY WITH GROSS ARTICULAR CARTILAGE THICKNESS IN THE EQUINE METACARPOPHALANGEAL JOINT

Osteoarthritis of the metacarpophalangeal joint is common cause of lameness in equine athletes, and is hallmarked by articular cartilage damage. An accurate, noninvasive method for measuring cartilage thickness would be beneficial to screen for cartilage injury and allow for prompt initiation of interventional therapy. The objective of this methods comparison study was to compare computed tomographic arthrography (CTA), magnetic resonance imaging (MRI), and magnetic resonance arthrography (MRA) measurements of articular cartilage thickness with gross measurements in the metacarpophalangeal joint of Thoroughbred horses. Fourteen cadaveric, equine thoracic limbs were included. Limbs were excluded from the study if pathology of the metacarpophalangeal articular cartilage was observed with any imaging modality. Articular cartilage thickness was measured in nine regions of the third metacarpal bone and proximal phalanx on sagittal plane MRI sequences. After intra‐articular contrast administration, the measurements were repeated on sagittal plane MRA and sagittal CTA reformations. In an effort to increase cartilage conspicuity, the volume of intra‐articular contrast was increased from 14.5 ml, to maximal distention for the second set of seven limbs. Mean and standard deviation values were calculated, and linear regression analysis was used to determine correlations between gross and imaging measurements of cartilage thickness. This study failed to identify one imaging test that consistently yielded measurements correlating with gross cartilage thickness. Even with the use of intra‐articular contrast, cartilage surfaces were difficult to differentiate in regions where the cartilage surfaces of the proximal phalanx and third metacarpal bone were in close contact with each other.     

The effects of methylprednisolone on normal and monocyte-conditioned medium-treated articular cartilage from dogs and horses

OBJECTIVE: To study in vitro (1) the dose-response relationships between proteoglycan metabolism in normal and corticosteroid-treated articular cartilage; (2) long-term proteoglycan metabolism after treatment of articular cartilage with corticosteroids; and (3) the effect of corticosteroids on proteoglycan metabolism in articular cartilage treated with monocyte-conditioned medium (MCM). STUDY DESIGN: Equine and canine articular cartilage explants were treated with corticosteroids and MCM. Proteoglycan synthesis and degradation were measured by radioactive labeling in short-term culture, and the long-term effect of corticosteroid treatment on proteoglycan metabolism was studied in normal explants. ANIMALS: Two young cross-breed horses and 3 young Labrador retrievers. METHODS: Equine articular cartilage explants were incubated in medium containing methylprednisolone sodium succinate (MPS) at 0, .001, .01, .1, 1, and 10 mg/mL (final concentration) for 1 day and then in fresh medium without MPS. Proteoglycan synthesis was measured by incorporation of sodium [35S]sulfate at 1, 3, 7, 10, and 13 days after initial treatment with MPS. Proteoglycan release was measured from separate explants prelabeled with sodium [35S]sulfate and treated similarly. Equine articular cartilage explants were treated with equine MCM simultaneously with, and 24 hours before MPS, at 0, 0.01, 0.1, 1, or 5 mg/mL for 72 hours. Proteoglycan synthesis and degradation in these explants was compared. Proteoglycan synthesis and degradation were measured similarly in canine articular cartilage explants treated simultaneously with canine MCM and MPS at 0, 0.001, 0.01, 0.1, 1 and 10 mg/mL for 72 hours. Equine articular cartilage explants treated with 0, 0.01, 0.1, 1, and 5 mg/mL of MPS for 72 hours were evaluated histologically. RESULTS: Proteoglycan synthesis in normal equine articular cartilage was severely depressed by 10 mg/mL MPS for 24 hours, and proteoglycan synthesis failed to recover after 13 days of culture in medium without MPS. Cartilage treated with 5 mg/mL MPS had pyknotic chondrocyte nuclei and empty lacunae. Concentrations of 1 and 0.1 mg/mL MPS depressed proteoglycan synthesis in normal equine cartilage explants. For these 2 concentrations, proteoglycan synthesis recovered 2 days after MPS removal and increased significantly (P < .05) 7 days after treatment with MPS compared with controls without MPS. Concentrations of 0.001 and 0.01 mg/mL MPS did not significantly affect proteoglycan synthesis in normal equine cartilage explants. Cumulative proteoglycan loss over 13 days in culture from normal equine explants treated for 24 hours with different concentrations of MPS was not significantly different between treatment groups at any time point. MCM significantly depressed proteoglycan synthesis in both canine and equine articular cartilage explants and significantly increased proteoglycan release. These effects were prevented in the canine explants by simultaneous treatment with MPS at 1 and 0.1 mg/mL, and proteoglycan release induced by MCM in equine articular cartilage was inhibited by 1 mg/mL MPS. CONCLUSIONS: Concentrations of 1.0 and 0.1 mg/mL MPS alleviated articular cartilage degradation in MCM-treated articular cartilage in vitro. These concentrations of MPS in contact with normal cartilage explants for 24 hours are unlikely to be detrimental in the long term to proteoglycan synthesis. The response of articular cartilage to MPS was affected by treatment with MCM so that results of experiments with normal articular cartilage explants may not reflect results obtained with abnormal cartilage. CLINICAL RELEVANCE: It may be possible to find an intraarticular concentration of corticosteroid that protects articular cartilage against cytokine-induced matrix degradation yet not have prolonged or permanent detrimental effects on chondrocyte matrix synthesis.     

Proteoglycan synthesis and content in articular cartilage and cartilage repair tissue in horses

Hexosamine concentration, DNA concentration, and [35S]sulfate incorporation for articular cartilage obtained from various sites in the metacarpophalangeal and carpal joints of horses were measured. The same measurements were made on the repair tissue filling full-thickness articular defects in the intermediate carpal bone and on cartilage surrounding partial-thickness defects 6 weeks after the defects were created arthroscopically. Cellularity (measured as DNA concentration), proteoglycan content (measured as hexosamine concentration), and proteoglycan synthesis (measured as [35S]sulfate incorporation) varied according to the site sampled. Cartilage from the transverse ridge of the head of the third metacarpal bone and the radial facet of the third carpal bone had the lowest hexosamine concentration, whereas rate of proteoglycan synthesis was lowest in cartilage from the transverse ridge of the head of the third metacarpal bone and the distal articular surface of the radial carpal bone. Repair tissue filling a full-thickness cartilage defect at 6 weeks was highly cellular. It was low in proteoglycan content, but was actively synthesizing these macromolecules. In contrast, the cartilage surrounding a partial-thickness defect was unchanged 6 weeks after the original defect was made.     

Evaluation of subchondral bone mineral density associated with articular cartilage structure and integrity in healthy equine joints with different functional demands

OBJECTIVE: To determine and correlate subchondral bone mineral density and overlying cartilage structure and tensile integrity in mature healthy equine stifle (low magnitude loading) and metacarpophalangeal (high magnitude loading) joints. ANIMALS: 8 healthy horses, 2 to 3 years of age. PROCEDURE: Osteochondral samples were acquired from the medial femoral condyle (FC) and medial trochlear ridge (TR) of the stifle joint and from the dorsal (MC3D) and palmar (MC3P) aspects of the distal medial third metacarpal condyles of the metacarpophalangeal joint. Articular cartilage surface fibrillation (evaluated via India ink staining) and tensile biomechanical properties were determined. The volumetric bone mineral density (vBMD) of the underlying subchondral plate was assessed via dual-energy x-ray absorptiometry. RESULTS: Cartilage staining (fibrillation), tensile moduli, tensile strength, and vBMD were greater in the MC3D and MC3P locations, compared with the FC and TR locations, whereas tensile strain at failure was less in MC3D and MC3P locations than FC and TR locations. Cartilage tensile moduli correlated positively with vBMD, whereas cartilage staining and tensile strain at failure correlated negatively with vBMD. CONCLUSIONS AND CLINICAL RELEVANCE: In areas of high joint loading, the subchondral bone had high vBMD and the articular cartilage surface layer had high tensile stiffness but signs of structural wear (fibrillation and low failure strain). The site-dependent variations and relationships in this study support the concept that articular cartilage and subchondral bone normally adapt to physiologic loading in a coordinated way.     

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.     

Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint

The aim of this study was to evaluate topographical differences in the biochemical composition of the extracellular matrix of articular cartilage of the normal equine fetlock joint. Water content, DNA content, glycosaminoglycan (GAG) content and a number of characteristics of the collagen network (total collagen content, levels of hydroxylysine- (Hyl) and the crosslink hydroxylysylpyridinoline, (HP) of articular cartilage in the proximal 1st phalanx (P1), distal 3rd metacarpal bone (MC), and proximal sesamoid bones (PSB) were determined in the left and right fetlock joint of 6 mature horses (age 5-9 years). Twenty-eight sites were sampled per joint, which included the clinically important areas often associated with pathology. Biochemical differences were evaluated between sampling sites and related with the predisposition for osteochondral injury and type of loading. Significant regional differences in the composition of the extracellular matrix existed within the joint. Furthermore, left and right joints exhibited biochemical differences. Typical topographic distribution patterns were observed for each parameter. In P1 the dorsal and palmar articular margin showed a significantly lower GAG content than the more centrally located sites. Collagen content and HP crosslinks were higher at the joint margins than in the central area. Also, in the MC, GAG content was significantly lower at the (dorsal) articular margin compared with the central area. Consistent with findings in P1, collagen and HP crosslinks were significantly lower in the central area compared to the (dorsal) articular margin. Biochemical and biomechanical heterogeneity of articular cartilage is supposed to reflect the different functional demands made at different sites. In the present study, GAG content was highest in the constantly loaded central areas of the joint surfaces. In contrast, collagen content and HP crosslinks were higher in areas intermittently subjected to peak loading which suggests that the response to a certain type of loading of the various components of the extracellular matrix of articular cartilage are different. The differences in biochemical characteristics between the various sites may help to explain the site specificity of osteochondral lesions commonly found in the equine fetlock joint. Finally, these findings emphasise that the choice of sampling sites may profoundly influence the outcome of biochemical studies of articular cartilage.     

Variations in cartilage catabolism in different equine joints in response to interleukin-1 in vitro

An explant system was used to investigate the hypothesis that cartilage from different equine joints might respond differently to challenge with interleukin-1alpha (IL-1alpha). Pairs of normal cartilage samples were taken from the metacarpophalangeal, proximal interphalangeal and distal interphalangeal joints of six horses. One of each pair was stimulated with 10 ng/ml human recombinant IL-1alpha for three days, and the supernatants and remaining cartilage explants were analysed for their total content of glycosaminoglycans. A significantly higher percentage of glycosaminoglycans was released from the cartilage of the proximal and distal interphalangeal joints than from the metacarpophalangeal joint.     

Effects of methylprednisolone acetate and glucosamine on proteoglycan production by equine chondrocytes in vitro

OBJECTIVE: To evaluate the effects of methylprednisolone acetate (MPA) on proteoglycan production by equine chondrocytes and to investigate whether glucosamine hydrochloride modulates these effects at clinically relevant concentrations. SAMPLE POPULATION: Articular cartilage with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints of 8 horses (1 to 10 years of age). PROCEDURES: In vitro chondrocyte pellets were pretreated with glucosamine (0, 1, 10, and 100 microg/mL) for 48 hours and exposed to MPA (0, 0.05, and 0.5 mg/mL) for 24 hours. Pellets and media were assayed for proteoglycan production (Alcian blue precipitation) and proteoglycan content (dimethylmethylene blue assay), and pellets were assayed for DNA content. RESULTS: Methylprednisolone decreased production of proteoglycan by equine chondrocytes at both concentrations studied. Glucosamine protected proteoglycan production at all 3 concentrations studied. CONCLUSIONS AND CLINICAL RELEVANCE: Methylprednisolone, under noninflammatory conditions present in this study, decreased production of proteoglycan by equine chondrocytes. Glucosamine had a protective effect against inhibition of proteoglycan production at all 3 concentrations studied. This suggested that glucosamine may be useful as an adjunct treatment when an intra-articular injection of a corticosteroid is indicated and that it may be efficacious at concentrations relevant to clinical use.     

In vitro dose-dependent effects of enrofloxacin on equine articular cartilage.

OBJECTIVE: To determine whether enrofloxacin has detrimental, dose-dependent effects on equine articular cartilage in vitro. ANIMALS: Cartilage explants were developed from 6 healthy horses between 0 and 96 months old. PROCEDURE: Patellar cartilage explants were incubated in 5 concentrations of enrofloxacin (2 microg/ml, 10 microg/ml, 1,000 microg/ml, 10,000 microg/ml, and 50,000 microg/ml) for 72 hours. Proteoglycan synthesis (Na35SO4 incorporation for 24 hours), proteoglycan degradation (Na35SO4 release for 72 hours), endogenous proteoglycan content (dimethylmethlene blue assay), and total protein content were determined. Cartilage explants were evaluated by use of histomorphologic and histomorphometric techniques (toluidine blue stain) for cytologic and matrix characteristics. Quantitative data were analyzed with a one-way ANOVA to compare results among various enrofloxacin concentration groups and the control group. A general linear model was used to determine whether age had an effect. RESULT: Proteoglycan synthesis was excellent in control specimens and in specimens incubated in low concentrations of enrofloxacin (2 microg/ml and 10 microg/ml). High concentrations of enrofloxacin (> 1,000 microg/ml) effectively eliminated proteoglycan synthesis regardless of horse age. Proteoglycan degradation at low concentrations (2 microg/ml and 10 microg/ml) was not different than control. High concentrations of enrofloxacin (> 1,000 microg/ml) caused significant degradation. Different concentrations of enrofloxacin did not affect endogenous proteoglycan. High concentrations of enrofloxacin were associated with a significant increase in number of pyknotic nuclei. CONCLUSION: Concentrations of enrofloxacin that might be achieved following systemic administration did not suppress chondrocyte metabolism in vitro. High concentrations of enrofloxacin (> 1,000 microg/ml) were toxic to chondrocytes.     

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.     

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.