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.     

Differences in the topographical distribution of articular cartilage degeneration between equine metacarpo- and metatarsophalangeal joints

REASONS FOR PERFORMING STUDY: The equine metacarpophalangeal (MCP) and metatarsophalangeal (MTP) joints, although having virtually the same geometrical appearance, differ in the prevalence of joint pathologies, such as osteochondral fragmentation, and in biomechanical behaviour. The recently developed cartilage degeneration index (CDI) technique offers a possibility to assess quantitatively differences in cartilage degeneration between these joints and to compare these with known differences in biomechanics and clinical observations. OBJECTIVES: To compare the topographical distribution of articular cartilage degeneration across the proximal articular surface of the proximal phalanx (P1) in the equine fore- and hindlimb. METHODS: In 24 distal hindlimbs from 24 horses, articular cartilage degeneration of the proximal articular surface of P1 was quantified using the CDI. Overall CDI value (CDI(P1)) and CDI values of 6 areas of interest were determined: the medial dorsal surface (mds), lateral dorsal surface (lds), medial central fovea (mcf), lateral central fovea (lcf), medial plantar surface (mps) and lateral plantar surface (lps). The joints were divided into 4 equally sized groups of increasing CDI(P1) values. From an existing CDI database of MCP joints, 24 joints were selected with matching CDI(P1) values to the MTP joints and CDI values for the same areas of interest were determined. RESULTS: In both the MCP and MTP joints, highest CDI values were determined at the dorsal articular surfaces. Values were not significantly different between fore- and hindlimbs. In contrast to the MCP joint, CDI values at the plantar joint margin were significantly higher compared to CDI values in the central sites in the MTP joint. CDI values for the plantar surfaces of P1 were significantly higher than those for the palmar surfaces in the forelimb in joints with advanced stages of OA; and values for the central regions of P1 were significantly lower in the hindlimb compared with the forelimb in joints with severe OA. CONCLUSIONS: In both fore- and hindlimbs, initial cartilage degeneration started at the dorsal articular margin of P1. There was a major difference in the spread of cartilage degeneration; in the forelimb both the central and palmar parts are about equally involved, whereas in the hindlimb the plantar parts were significantly more and the central parts significantly less involved. These differences can be linked to differences in biomechanical loading reported elsewhere. POTENTIAL RELEVANCE: This study supports the hypothesis that differences in biokinematics between fore- and hindlimbs are associated with differences in the development of cartilage degeneration and other joint pathologies such as osteochondral fragmentation in the MCP and MTP joints. This information is indispensable for a better understanding of the dynamic nature and progression of these joint disorders and may be of help when monitoring the effects of therapeutic interventions and preventative measures.     

Cellular heterogeneity in cathepsin D distribution in equine articular cartilage

The distribution of cathepsin D in normal equine growth cartilage has been examined immunocytochemically using an antiserum raised against human cathepsin D. The cross-reactivity and specificity of the antiserum for equine cathepsin D was confirmed, and its lysosomal localisation was demonstrated in horse skin fibroblasts by confocal scanning microscopy. Cultured horse chondrocytes were heterogenous in their expression of cathepsin D. Heterogeneity of distribution of the enzyme was also seen in chondrocytes in cartilage from different anatomical sites. A high level of cathepsin D was observed in the deep layer of cartilage from the lateral trochlear ridge of the distal femur. Cathepsin D was absent in the hypertrophic zone of the distal radial growth plate.     

Functional consequences of cartilage degeneration in the equine metacarpophalangeal joint: quantitative assessment of cartilage stiffness

REASONS FOR PERFORMING STUDY: No quantitative data currently exist on the relationship of the occurrence of cartilage degeneration and changes in site-specific biomechanical properties in the metacarpophalangeal (MCP) joint in the horse. OBJECTIVES: To gain insight into the biomechanical consequences of cartilage deterioration at 2 differently loaded sites on the proximal articular surface of the proximal phalanx (P1). HYPOTHESIS: Static and dynamic stiffness of articular cartilage decreases significantly in degenerated cartilage. METHODS: Cartilage degeneration index (CDI) values were measured at the lateral dorsal margin (Site 1), lateral central fovea (Site 2) and entire joint surface of P1 (CDIP1) in 30 horses. Group 1 contained joints without (CDIP1 values <25 %, n = 22) and Group 2 joints with (CDIP1 values >25 %, n = 8) signs of cartilage degeneration. Cartilage thickness at Sites 1 and 2 was measured using ultrasonic and needle-probe techniques. Osteochondral plugs were drilled out from Sites 1 and 2 and subsequently tested biomechanically in indentation geometry. Young's modulus at equilibrium and dynamic modulus were determined. RESULTS: Cartilage thickness values were not significantly different between the 2 groups and sites. Young's modulus at Site 1 was significantly higher in Group 1 than in Group 2; at Site 2, the difference was not significant. Dynamic modulus values were significantly higher in Group 1 than in Group 2 at both sites. CONCLUSIONS: Degenerative cartilage changes are clearly related to loss of stiffness of the tissue. Absolute changes in cartilage integrity in terms of CDI are greatest at the joint margin, but concomitant changes are also present at the centre, with a comparable decrease of the biomechanical moduli at the 2 sites. Therefore, significant cartilage degradation at the joint margin not only reflects local deterioration of biomechanical properties, but is also indicative of the functional quality in the centre. POTENTIAL RELEVANCE: These findings may be important for improving prognostication and developing preventative measures.     

Intra-articular pressure profiles of the cadaveric equine fetlock joint in motion

The study of the influence of motion and initial intra-articular pressure (IAP) on intra-articular pressure profiles in equine cadaver metatarsophalangeal (MTP) joints was undertaken as a prelude to in vivo studies. Eleven equine cadaver MTP joints were submitted to 2 motion frequencies of 5 and 10 cycles/min of flexion and extension, simulating the condition of lower and higher (double) rates of passive motion. These frequencies were applied and pressure profiles generated with initial normal intra-articular pressure (-5 mmHg) and subsequently 30 mmHg intra-articular pressure obtained by injection of previously harvested synovial fluid. The 4 trials performed were 1) normal IAP; 5 cyles/min; 2) normal IAP; 10 cycles/min; 3) IAP at 30 mmHg; 5 cycles/min and 4) IAP at 30 mmHg; 10 cycles/min. The range of joint motion applied (mean +/- s.e.) was 67.6+/-1.61 degrees with an excursion from 12.2+/-1.2 degrees in extension to 56.2+/-2.6 degrees in flexion. Mean pressure recorded in mmHg for the first and last min of each trial, respectively, were 1) -5.7+/-0.9 and -6.3+/-1.1; 2) -5.3+/-1.1 and -6.2+/-1.1; 3) 58.8+/-8.0 and 42.3+/-7.2; 4) 56.6+/-3.7 and 40.3+/-4.6. Statistical analyses showed a trend for difference between the values for the first and last minute in trial 3 (0.05>P<0.1) with P = 0.1 and significant difference (P = 0.02) between the mean IAP of the first and last min in trial 4. The loss of intra-articular pressure associated with time and motion was 10.5, 16.9, 28.1 and 28.9% for trials 1-4, respectively. As initial intraarticular pressure and motion increased, the percent loss of intra-articular pressure increased. The angle of lowest pressure was 12.2+/-1.2 degrees (mean +/- s.e.) in extension in trials 1 and 2. In trials 3 and 4, the lowest pressures were obtained in flexion with the joints at 18.5+/-2.0 degrees (mean +/- s.e.). This demonstrated that the joint angle of least pressure changed as the initial intra-articular pressure changed and there would not be a single angle of least pressure for a given joint. The volume of synovial fluid recovered from the MTP joints in trial 3 compared to 4 (trials in which fluid was injected to attain IAP of 30 mmHg) was not significantly different, supporting a soft tissue compliance change as a cause for the significant loss of intra-articular pressure during the 15 min of trial 4. The pressure profiles generated correlate well with in vivo values and demonstrated consistent pressure profiles. Our conclusions are summarised as follows: 1. Clinically normal equine MTP joints which were frozen and then later thawed were found to have mostly negative baseline intra-articular pressures, as would be expected in living subjects. 2. Alternate pressure profiles of the dorsal and plantar pouch at baseline intra-articular pressure document the presence of pressure forces that would support 'back and forth' fluid movement between joint compartments. This should result in movement of joint fluid during motion, assisting in lubrication and nutrition of articular cartilage. 3. If joint pressure was initially greater than normal (30 mmHg), as occurs in diseased equine MTP joints, joint motion further increased joint capsule relaxation (compliance) and, therefore, reduced intra-articular pressure. 4. Peak intra-articular pressures reached extremely high values (often >100 mmHg) in flexion when initial pressure was 30 mmHg. Joint effusion pressures recorded for clinical MCP joints are frequently 30 mmHg. These IAP values are expected to produce intermittent synovial ischaemia in clinical cases during joint flexion. 5. Additional in vivo studies are necessary to confirm our conclusions from this study and to identify the contributions of fluid absorption and the presence of ischaemia in a vascularised joint.     

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.     

Equine carpal articular cartilage fibronectin distribution associated with training, joint location and cartilage deterioration

Processes involved in equine carpal osteochondral injury have not been established. In other species, fibronectin appears important in chondrocyte-matrix interactions, and levels are increased in osteoarthritis. This investigation aimed to (a) describe fibronectin immunoreactivity in the middle carpal joint of 2-year-old Thoroughbreds, (b) determine topographical variations, (c) compare strenuously trained (Group 1) or gently exercised horses (Group 2) and (d) describe sites with early osteoarthritis. Group 1 (n = 6) underwent a 19 week high intensity treadmill training programme. Group 2 (n = 6) underwent 40 min walking until euthanasia. Dorsal and palmar sites on radial, intermediate and third carpal articular surfaces were prepared. Immunohistochemistry was performed using a biotin-streptavidin/peroxidase method. Cross-reactivity of rabbit antihuman fibronectin antiserum with equine fibronectin was confirmed using Western blotting. Results showed: (a) fibronectin was present primarily in pericellular and interterritorial matrix locations, (b) dorsal sites had zonal immunoreactivity compared to palmar sites, (c) Group 1 dorsal radial carpal cartilage had increased superficial staining compared to Group 2 and (d) fibrillated cartilage showed increased intracellular and local matrical immunoreactivity (superficial zone). These findings suggest topographical and exercise-related variations in fibronectin distribution, and indicate equine fibronectin is localised at sites of cartilage degeneration and released into the matrix by chondrocytes in the local area.     

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.     

The distribution of cartilage oligomeric matrix protein (COMP) in equine carpal articular cartilage and its variation with exercise and cartilage deterioration

Based on previous studies where tendons receiving the most load have been shown to have the highest levels of cartilage oligomeric matrix protein (COMP), we hypothesized that COMP distribution in articular cartilage may be influenced by mechanical loading. This investigation aimed (a) to describe the pattern of COMP immunoreactivity in middle carpal joint cartilage of two-year-old Thoroughbred horses; (b) to determine topographical variations; (c) to compare high (group 1) and low (group 2) intensity training and (d) to describe COMP immunoreactivity at sites with early osteoarthritis.Group 1 (n =6) underwent a 19 week high-intensity treadmill training programme and group 2 (n =6) were given daily walking until euthanasia. Dorsal and palmar sites on radial and third carpal articular surfaces were prepared. Immunohistochemistry was performed with polyclonal rabbit anti-equine COMP antiserum using a biotin-streptavidin/peroxidase method. Results showed: (a) intracellular immunoreactivity was present in all cartilage zones, but the distribution of COMP staining within the matrix varied between cartilage zones; (b) differences in distribution between sites were not observed, but total COMP levels in exercised horses (n =2) did vary between sites with dorsal sites containing less COMP than palmar sites on the radial, intermediate and third carpal lateral facet; (c) group 1 cartilage showed marked interterritorial distribution in the deep layer compared to group 2 where staining was more generalized throughout the matrix and (d) fibrillated cartilage showed increased local immunoreactivity in the matrix. These findings demonstrate zonal variations in equine COMP distribution which may be influenced by loading.     

Technique for equine cervical articular process joint injection

Degenerative changes and osteochondrosis of articular processes are common sources of stiffness or pain in the equine cervical spine. Temporary relief of the clinical signs related to these maladies may be achieved by injecting corticosteroids into the joint. This is routinely done by ultrasound-guided needle placement. The cervical articular processes and joint form an easily identifiable sonographic landmark, a step-like echogenic surface described as having the appearance of a "chair."     

The effect of sodium heparin on equine articular cartilage.

This study compared the effect of sodium heparin and gentamicin sulphate on equine articular cartilage (AC) explants in order to investigate the possible use of sodium heparin in the treatment of infectious arthritis. Six concentrations of sodium heparin and gentamicin sulphate were tested. The supernatant and explant digest were assayed for glycosaminoglycan (GAG) content with the dimethyl-methylene blue assay and the per cent loss of GAG was calculated. A significant (P< 0.001) increase in percentage GAG loss was noted for the sodium heparin groups when compared to the control, whilst no significant increase was found among the treatment groups (P =0.782). For gentamicin, no significant difference in percentage GAG loss was found between the control and three of the five treatment groups (P =0.667). The percentage GAG loss in the sodium heparin treated AC explants was greater than for any of the gentamicin-treated AC explants. It can be concluded that sodium heparin sulphate stimulates an increase in GAG release from equine articular cartilage explants, though no firm conclusions can be drawn on its use in treating equine infectious arthritis. Copyright Harcourt Publishers Ltd.     

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.     

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.     

Influence of polysulfated glycosaminoglycan on equine articular cartilage in explant culture.

Articular cartilage explants from 3 horses were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan on proteoglycan biosynthesis. Cultures were exposed to concentrations of 0, 50, or 200 micrograms of the drug/ml for either 2 days or 6 days, and labeled with 35S, before measuring the content of sulfated proteoglycan in the culture media and in extracts of cartilage. In a second experiment, the explants were incubated with the isotope and subsequently exposed to the same concentrations of the polysulfated glycosaminoglycan for 4 days. Subsequently, the amount of remaining labeled proteoglycan was determined. Gel filtration chromatography was used to compare the hydrodynamic size of proteoglycans from the cartilage explants in each experiment. Polysulfated glycosaminoglycan caused a dose-dependent depression of sulfated proteoglycan synthesis, which was statistically significant after 6 days of exposure. Radioactive proteoglycan content in explants was similar in the experiment involving isotopic labeling prior to exposure to the drug. Proteoglycan monomer size was similar in all treatment groups. It was concluded that polysulfated glycosaminoglycan caused a modest depression in proteoglycan synthesis, had little effect on endogenous proteoglycan degradation, and did not influence the size of sulfated proteoglycans synthesized by normal equine chondrocytes in explant culture.     

Effects of polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage

The effect of intra-articular polysulfated glycosaminoglycan (PSG) on repair of chemical and physical articular cartilage injuries was evaluated in 8 horses. In each horse, a partial- and a full-thickness articular cartilage defect was made on the distal articular surface of the radial carpal bone. In the contralateral middle carpal joint, a chemical articular cartilage injury was induced by injecting 50 mg of Na monoiodoacetate (MIA). Four of the 8 horses were not treated (controls), and 4 horses were treated by intra-articular injection of 250 mg of PSG into both middle carpal joints once a week for 5 treatments starting 1 week after cartilage injury. Horses were maintained for 8 weeks. There was less joint circumference enlargement in PSG-treated horses in MIA-injected and physical defect carpi, compared with that in controls. In MIA-injected joints, there was less articular cartilage fibrillation and erosion, less chondrocyte death, and greater safranin-O staining for glycosaminoglycans in PSG-treated horses. Evaluation of joints in which physical defects were made revealed no differences between control and PSG-injected joints. None of the partial-thickness defects had healed. Full-thickness defects were repaired with fibrous tissue (which was more vascular and cellular in PSG-injected joints) and occasionally small amounts of fibrocartilage. Seemingly, PSG had chondroprotective properties in a model of chemically induced articular cartilage damage, whereas PSG had no obvious effect in a physical articular cartilage-defect model.