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.     

Study of cartilage and bone layers of the bearing surface of the equine metacarpophalangeal joint relative to different timescales of maturation

REASONS FOR PERFORMING STUDY: A detailed and comprehensive insight into the normal maturation process of the different tissues that make up functional units of the locomotor system such as joints is necessary to understand the influence of early training on musculoskeletal tissues. OBJECTIVES: To study simultaneously the maturation process in the entire composite structure that makes up the bearing surface of a joint (cartilage, subchondral and trabecular bone) in terms of biochemical changes in the tissues of juvenile horses at 2 differently loaded sites of the metacarpophalangeal joint, compared to a group of mature horses. HYPOTHESIS: In all the structures described above developmental changes may follow a different timescale. METHODS: Age-related changes in biochemical characteristics of the collagen part of the extracellular matrix (hydroxylysine, hydroxyproline, hydroxypyridinum crosslinks) of articular cartilage and of the underlying subchondral and trabecular bone were determined in a group of juvenile horses (n = 13) (Group 1, age 6 months-4 years) and compared to a group of mature horses (n = 30) (Group 2, >4 years). In both bony layers, bone mineral density, ash content and levels of individual minerals were determined. RESULTS: In cartilage, subchondral bone and trabecular bone, virtually all collagen parameters in juvenile horses were already at a similar (stable) level as in mature horses. In both bony layers, bone mineral density, ash- and calcium content were also stable in the mature horses, but continued to increase in the juvenile group. For magnesium there was a decrease in the juvenile animals, followed by a steady state in the mature horses. CONCLUSIONS: In horses age 6 months-4 years, the collagen network of all 3 layers within the joint has already attained a mature biochemical composition, but the mineral composition of both subchondral and trabecular bone continues to develop until approximately age 4 years. POTENTIAL RELEVANCE: The disparity in maturation of the various extracellular matrix components of a joint can be assumed to have consequences for the capacity to sustain load and should hence be taken into account when training or racing young animals.     

Subchondral bone density and cartilage degeneration patterns in osteoarthritic metacarpal condyles of horses

OBJECTIVE: To evaluate and correlate patterns of subchondral bone density and articular cartilage degeneration (derived by use of gross, histologic, and computed tomographic [CT] examinations) in equine third metacarpal condyles with and without osteoarthritis. SAMPLE POPULATION: 8 metacarpophalangeal (MCP) joints (n = 4 horses) without osteoarthritis and 6 osteoarthritis-affected MCP joints (4). PROCEDURES: Horses were euthanized. The third metacarpal condyles of the joints were examined grossly and via CT (3 slice images/condyle). For 6 condylar zones, mean bone density and pattern of density distribution were determined. Data for osteoarthritis-affected and control joints were compared. Histomorphometric point count analyses identified areas of bone density for comparison with CT density measurements. RESULTS: Osteoarthritis-affected condyles had heterogeneous subchondral bone with focal resorptive lesions and patterned sclerosis, whereas control condyles had symmetric bone density distribution. In osteoarthritis-affected condyles, bone density determined via gray scale image density analysis was greater (dorsal and medial pattern), compared with control condyles, and differed among zones because of resorption and sclerosis. With regard to bone density in osteoarthritis-affected condyles, histologic findings correlated with CT images, and bone lesions were significantly correlated with cartilage lesions. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, heterogeneous distribution and greater subchondral bone density were characteristic of osteoarthritis-affected condyles, compared with control condyles. Subchondral bone lesions correlated with overlying cartilage lesions in osteoarthritis-affected MCP joints. Identification of CT image characteristics appears to predict the presence of a cartilage lesion in MCP joints of horses with osteoarthritis.     

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.     

Imaging and histological features of central subchondral osteophytes in racehorses with metacarpophalangeal joint osteoarthritis

Reasons for performing study: Marginal osteophytes represent a well known component of osteoarthritis in man and animals. Conversely, central subchondral osteophytes (COs), which are commonly present in human knees with osteoarthritis, have not been reported in horses. Objectives: To describe and compare computed radiography (CR), single‐slice computed tomography (CT), 1.5 Tesla magnetic resonance imaging (MRI), and histological features of COs in equine metacarpophalangeal joints with macroscopic evidence of naturally‐occurring osteoarthritis. Methods: MRI sequences (sagittal spoiled gradient recalled echo [SPGR] with fat saturation, sagittal T2‐weighted fast spin echo with fat saturation [T2‐FS], dorsal and transverse T1‐weighted gradient‐recalled echo [GRE], and sagittal T2*‐weighted gradient echo with fast imaging employing steady state acquisition [FIESTA]), as well as transverse and reformatted sagittal CT, and 4 computed radiographic (CR) views of 20 paired metacarpophalangeal joints were acquired ex vivo. Following macroscopic evaluation, samples were harvested in predetermined sites of the metacarpal condyle for subsequent histology. The prevalence and detection level of COs was determined for each imaging modality. Results: Abnormalities consistent with COs were clearly depicted on MRI, using the SPGR sequence, in 7/20 (35%) joints. They were identified as a focal hypointense protuberance from the subchondral plate into the cartilage, at the palmarodistal aspect (n = 7) and/or at the very dorsal aspect (n = 2) of the metacarpal condyle. COs were visible but less obvious in 5 of the 7 joints using FIESTA and reformatted sagittal CT, and were not identifiable on T2‐FS, T1‐GRE or CR. Microscopically, they consisted of dense bone protruding into the calcified cartilage and disrupting the tidemarks, and they were consistently associated with overlying cartilage defects. Conclusions: Subchondral osteophytes are a feature of osteoarthritis of equine metacarpophalangeal joints and they may be diagnosed using 1.5 Tesla MRI and CT. Potential relevance: Central subchondral osteophytes on MRI represent indirect evidence of cartilage damage in horses.     

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.     

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.     

Reliability of high‐ and low‐field magnetic resonance imaging systems for detection of cartilage and bone lesions in the equine cadaver fetlock

Reasons for performing study: To determine the reliability of 2 magnetic resonance imaging (MRI) systems for detection of cartilage and bone lesions of the equine fetlock. Objectives: To test the hypotheses that lesions in cartilage, subchondral and trabecular bone of the equine fetlock verified using histopathology can be detected on high‐ and low‐field MR images with a low incidence of false positive or negative results; that low‐field images are less reliable than high‐field images for detection of cartilage lesions; and that combining results of interpretation from different pulse sequences increases detection of cartilage lesions. Methods: High‐ and low‐field MRI was performed on 19 limbs from horses identified with fetlock lameness prior to euthanasia. Grading systems were used to score cartilage, subchondral and trabecular bone on MR images and histopathology. Sensitivity and specificity were calculated for images. Results: High‐field T2*‐weighted gradient echo (T2*W‐GRE) and low‐field T2‐weighted fast spin echo (T2W‐FSE) images had high sensitivity but low specificity for detection of cartilage lesions. All pulse sequences had high sensitivity and low–moderate specificity for detection of subchondral bone lesions and moderate sensitivity and moderate–high specificity for detection of trabecular bone lesions (histopathology as gold standard). For detection of lesions of trabecular bone low‐field T2*W‐GRE images had higher sensitivity and specificity than T2W‐FSE images. Conclusions: There is high likelihood of false positive results using high‐ or low‐field MRI for detection of cartilage lesions and moderate–high likelihood of false positive results for detection of subchondral bone lesions compared with histopathology. Combining results of interpretation from different pulse sequences did not increase detection of cartilage lesions. MRI interpretation of trabecular bone was more reliable than cartilage or subchondral bone in both MR systems. Potential relevance: Independent interpretation of a variety of pulse sequences may maximise detection of cartilage and bone lesions in the fetlock. Clinicians should be aware of potential false positive and negative results.     

Comparison of mean bone densities of three preparations of the distal portion of the equine third metacarpal bone measured by use of quantitative computed tomography

OBJECTIVE: To evaluate whether cutting equine subchondral bone to demarcate specific regions of interest (ROIs) influences the mean density for that bone as measured via quantitative computed tomography (QCT). Sample population-2 metacarpophalangeal joints from equine cadavers. PROCEDURES: The distal portion of the third metacarpal bone of each intact metacarpophalangeal joint was scanned via CT to simulate in vivo conditions. Each joint was subsequently disarticulated and dissected, and the distal portion of the dissected third metacarpal bone in air was scanned. Then, six 1-cm(2) areas representing ROIs were cut into the distal condylar surfaces to depths of approximately 1 cm, and the bone was scanned again. Three-dimensional CT models of the 3 bone preparations were generated for each third metacarpal bone on the basis of data from each set of scan images, and densities of the 6 ROIs were measured. Mean bone densities for the 6 ROIs were compared among models of intact, dissected, and cut third metacarpal bone scans. RESULTS: Mean bone density was significantly lower in cut bone preparations, compared with that in intact or dissected bone. Differences between mean bone densities for intact and dissected bone preparations were not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Cutting subchondral bone to demarcate specific ROIs prior to CT imaging significantly lowered mean bone density as measured via QCT and thus introduced substantial artifacts. These findings have direct implications on techniques for CT modeling of equine subchondral bone in the characterization of joint diseases in horses.     

The effect of intra-articular methylprednisolone acetate and exercise on equine carpal subchondral and cancellous bone microhardness

Dorsal carpal osteochondral injury is a major cause of lameness in horses undergoing high intensity training. Intra-articular corticosteroid treatment is used commonly to manage exercise-associated articular pain, but its use remains highly controversial in the equine athlete. This project, therefore, aimed to compare the mechanical properties of intra-articular MPA and diluent-treated middle carpal subchondral and cancellous bone in horses undergoing a short-term treadmill exercise programme. It was hypothesised that subchondral and cancellous bone mechanical properties are influenced by intra-articular administration of methylprednisolone acetate (MPA). Eight 2-year-old female horses had MPA or diluent administered into contralateral middle carpal joints at 14 day intervals, for a total of 4 treatments per horse. Horses underwent a standard treadmill exercise protocol until euthanasia (Day 70). Standard sites were located on the dorsal aspect of third, radial and intermediate carpal bones. Osteochondral samples from each test site were divided into subchondral bone and cancellous bone portions. These were dried, resin-embedded and gold-coated. Microhardness measurements were obtained at each test site. No significant effect of intra-articular treatment was detected. At each site, cancellous bone trabecular struts had an 18-19% higher microhardness value than the overlying subchondral bone. These findings indicate that intra-articular administration of MPA at this dose has no effect on subchondral or cancellous bone adaptation to short-term exercise and, therefore, on the propensity of carpal bones to injury. Further investigation into the calcified cartilage layer, effect of different corticosteroid preparations and diffusion of medication are required.     

Glycosaminoglycans in horses with osteoarthritis

Horse articular cartilage glycosaminoglycans (GAGs) were measured in synovial fluids from 48 joints affected with osteoarthritis (OA), 22 normal joints, four joints with osteochondritis, three joints with traumatic arthritis and seven joints infected with bacteria. Serum and urine from individual horses were also examined for the presence of GAGs. High levels of GAGs were found in synovial fluids (SF) from horses with OA. In each case, the level was higher in the synovial fluid than in the serum or urine from the same horse. Horses with OA showed high GAG levels in SF, serum and urine compared to horses with normal and infected joints. High levels were also found in horses with osteochondritis and traumatic arthritis. Levels of synovial fluid GAG reflect cartilage destruction in arthritis and may be useful for monitoring disease progression in the equine species.     

Validation of magnetic resonance imaging for measurement of equine articular cartilage and subchondral bone thickness

OBJECTIVE: To validate use of magnetic resonance images (MRIs) for measurement of equine articular cartilage and subchondral bone thickness by comparison with measurements in histologic specimens. SAMPLE POPULATION: 32 cadaveric carpal joints from 16 horses. PROCEDURE: Magnetic resonance imaging was performed by use of 3-dimensional fast spoiled gradient echo (SPGR) and T2* 3-dimensional fast gradient echo (GRE) pulse sequences with and without fat saturation. Standard sites on the medial and lateral facets of the intermediate, radial, and third carpal bones were used for subchondral bone and articular cartilage thickness measurements. Digital image analysis software was used for MRI measurements 10 mm from the dorsal extent and perpendicular to the articular surface. Histomorphometric measurements of hyaline, calcified cartilage, and subchondral bone thickness were obtained at selected sites. Comparisons between histomorphometric and MRI measurements and between magnetic resonance pulse sequences were evaluated. RESULTS: There were significant correlations between GRE and SPGR and SPGR and histologic measurements of articular cartilage, with no significant difference between measurements and good agreement. When calcified cartilage was excluded from the histologic measurement, MRI measurements were significantly greater than histologic measurements. For subchondral bone thickness, there was significant correlation between GRE and SPGR but GRE was significantly greater than SPGR measurements. Histomorphometric and MRI measurements were strongly correlated and not significantly different. CONCLUSIONS AND CLINICAL RELEVANCE: Magnetic resonance imaging provides a good representation of cartilage and subchondral bone thickness, supporting its use in the study and clinical diagnosis of osteochondral structure and alteration.     

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.     

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.     

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.     

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.     

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.     

Congenital bilateral aplasia of the metacarpophalangeal joints in a foal

A 2‐day‐old Warmblood filly was presented for examination of an angular limb deformity of the left front limb and an upright conformation of both metacarpophalangeal joints. Radiological examination revealed bilateral absence of the metacarpophalangeal joint space with fusion of the third metacarpal bone and first phalanx (synostosis). No treatment was undertaken. The filly was readmitted to the clinic 10 weeks later for bilateral front limb lameness. On radiological examination, the synostosis of the front metacarpophalangeal joints was still present. Physitis of the distal growth plate of the right third metacarpal bone and proximal growth plate of the right proximal phalanx, and an avulsion fracture of the palmaromedial and proximal aspect of the left middle phalanx, with a cystic like lesion on the medial aspect of distal first phalanx and proximal middle phalanx were diagnosed. Given the poor prognosis, the foal was subjected to euthanasia. Post mortem examination confirmed the absence of the metacarpophalangeal joint space with a trabecular bony union between the third metacarpal bone and the first phalanx. A rudimentary joint capsule was present at the level of the absent joints as well as a small zone of articular cartilage, which invaginated over a short distance into the dorsal trabecular bone on the right front limb. On the medial aspect of the left proximal interphalangeal joint, a focal defect of articular cartilage with exposure of subchondral bone was observed. This is the first case report of a foal born with congenital aplasia of both metacarpophalangeal joints. Congenital malformations should be considered as differential diagnosis in lame foals or foals born with angular or flexural limb deformities.     

The effects of radial shock waves on the metabolism of equine cartilage explants in vitro

AIM: To investigate, in vitro, the effects of radial shock waves on the release of nitric oxide (NO) and synthesis of prostaglandin E2 (PGE2) and glycosaminoglycan (GAG), and liberation of GAG, from equine articular cartilage explants. METHODS: Equine cartilage from normal metacarpophalangeal and metatarsophalangeal joints was exposed to radial shock waves at various impulse doses and then maintained as explants in culture for 48 h. Shock waves were delivered at 1,876 Torr pressure and a frequency of 10 Hz. Treatment groups consisted of a negative control group, or application of 500, 2,000, or 4,000 impulses by use of either a convex handpiece (Group A) or concave handpiece (Group B). Synthesis of GAG was measured using incorporation of 35S-labelled sodium sulphate. Additionally, the synthesis of NO and PGE2, and content of GAG of the explants and media were determined. RESULTS: No significant effects (p>0.05) of radial shock-wave treatment were evident on the synthesis of NO or PGE2, or release of GAG by cartilage explants. However, radial shock waves decreased synthesis of GAG measured 48 h after exposure for all treatment groups other than the 500-impulse Group-A explants (p<0.05). CONCLUSIONS: Radial shock waves impact the metabolism of GAG in chondrocytes in equine articular cartilage. Further studies will be required to fully investigate the impact of this effect on the health of joints, and to elucidate the clinical impact.     

COMPARISON OF MAGNETIC RESONANCE IMAGING,COMPUTED TOMOGRAPHY,AND RADIOGRAPHY FOR ASSESSMENT OF NONCARTILAGINOUS CHANGES IN EQUINE METACARPOPHALANGEAL OSTEOARTHRITIS

We compared the ability of 1.5 T magnetic resonance imaging (MRI), computed tomography (CT), and computed radiography (CR) to evaluate noncartilaginous structures of the equine metacarpophalangeal joint (MCP), and the association of imaging changes with gross cartilage damage in the context of osteoarthritis. Four CR projections, helical single‐slice CT, and MRI (T1‐weighted gradient recalled echo [GRE], T2^*‐weighted GRE with fast imaging employing steady‐state acquisition [FIESTA], T2‐weighted fast spin echo with fat saturation, and spoiled gradient recalled echo with fat saturation [SPGR‐FS]) were performed on 20 racehorse cadaver forelimbs. Osteophytosis, synovial effusion, subchondral bone lysis and sclerosis, supracondylar lysis, joint fragments, bone marrow lesions, and collateral desmopathy were assessed with each modality. Interexaminer agreement was inferior to intraexaminer agreement and was generally moderate (i.e., 0.4<κ<0.6). Subchondral bone sclerosis scores using CT or MRI were correlated significantly with the reference quantitative CT technique used to assess bone mineral density (P<0.0001). Scores for subchondral lysis and osteophytosis were higher with MRI or CT vs. CR (P<0.0001). Although differences between modalities were noted, osteophytosis, subchondral sclerosis, and lysis as well as synovial effusion were all associated with the degree of cartilage damage and should be further evaluated as potential criteria to be included in a whole‐organ scoring system. This study highlights the capacity of MRI to evaluate noncartilaginous changes in the osteoarthritic equine MCP joint.