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.     

Functional adaptation of articular cartilage from birth to maturity under the influence of loading: a biomechanical analysis

REASONS FOR PERFORMING STUDY: The concept of functional adapatation of articular cartilage during maturation has emerged from earlier biochemical research. However, articular cartilage has principally a biomechanical function governed by joint loading. OBJECTIVES: To verify whether the concept of functional adaptation can be confirmed by direct measurement of biomechanical properties of cartilage. HYPOTHESIS: Fetuses have homogeneous (i.e. site-independent) cartilage with regard to biomechanical properties. During growth and development to maturity, the biomechanical characteristics adapt according to functional (loading) demands, leading to distinct, site-dependent biomechanical heterogeneity of articular cartilage. METHODS: Osteochondral plugs were drilled out of the surface at 2 differently loaded sites (Site 1: intermittent impact-loading during locomotion, Site 2: low-level constant loading during weightbearing) of the proximal articular cartilage surface of the proximal phalanx in the forelimb from stillborn foals (n = 8), horses of age 5 (n = 9) and 18 months (n = 9) and mature horses (n = 13). Cartilage thickness was measured using ultrasonic, optical and needle-probe techniques. The osteochondral samples were biomechanically tested in indentation geometry. Young's modulus at equilibrium, dynamic modulus at 1 Hz and the ratios of these moduli values between Sites 1 and 2 were calculated. Age and site effects were evaluated statistically using ANOVA tests. The level of significance was set at P<0.05. RESULTS: Fetal cartilage was significantly thicker compared to the other ages with no further age-dependent differences in cartilage thickness from age 5 months onwards. Young's modulus stayed constant at Site 1, whereas at Site 2 there was a gradual, statistically significant increase in modulus during maturation. Values of dynamic modulus at both Sites 1 and 2 were significantly higher in the fetus and decreased after birth. Values for both moduli were significantly different between Sites 1 and 2 from age 18 months onwards. The ratio of values between Sites 1 and 2 for Young's modulus and dynamic modulus showed a gradual decrease from approximately 1.0 at birth to 0.5-0.6 in the mature horse. At age 18 months, all values were comparable to those in the mature horse. CONCLUSIONS: In line with the concept of functional adaptation, the neonate is born with biomechanically 'blank' or homogeneous cartilage. Functional adaptation of biomechanical properties takes place early in life, resulting in cartilage with a distinct heterogeneity in functional characteristics. At age 18 months, functional adaptation, as assessed by the biomechanical characteristics, has progressed to a level comparable to the mature horse and, after this age, no major adaptations seem to occur. POTENTIAL RELEVANCE: Throughout life, different areas of articular cartilage are subjected to different types of loading. Differences in loading can adequately be met only when the tissue is biomechanically adapted to withstand these different loading conditions without injury. This process of functional adaptation starts immediately after birth and is completed well before maturity. This makes the factor of loading at a young age a crucial variable, and emphasises the necessity to optimise joint loading during early life in order to create an optimal biomechanical quality of articular cartilage, which may well turn out to be the best prevention for joint injury later in life.     

Biochemical development of subchondral bone from birth until age eleven months and the influence of physical activity

Subchondral bone provides structural support to the overlying articular cartilage, and plays an important role in osteochondral diseases. There is growing insight that the mechanical features of bone are related to the biochemistry of the collagen network and the mineral content. In the present study, part of the normal developmental process and the influence of physical activity on biochemical composition of subchondral bone was studied. Water content, calcium content and characteristics of the collagen network (collagen, hydroxylysine, lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) crosslinking) of subchondral bone were measured in newborn foals, 5-month-old foals (pasture-grown and box-confined) and 11-month-old foals at 2 differently loaded sites of the proximal articular surface of the first phalanx. During the first 5 months postpartum, water and hydroxylysine content decreased significantly while calcium and collagen content and the amount of HP and LP crosslinks increased significantly. The withholding of physical activity during this developmental phase affected the biochemical characteristics of subchondral bone only at the site that is loaded during physical exercise. At this site, calcium content and both HP and LP crosslink levels increased significantly less than in pasture-raised animals. During development from 5-11 months, measured parameters remained essentially constant, except for water content, which decreased further. It is concluded that substantial changes, presumed to be largely exercise-driven, take place during the normal process of development in the biochemical composition of equine subchondral bone. Normal development of subchondral bone is presumably important for the normal functional adaptation of this bone to the loading conditions it is subjected to and therefore essential to resist the future biomechanical challenges the horse will encounter during its athletic career. The findings from this study and the assumed important role of subchondral bone quality in the pathogenesis of osteochondral disease merit more attention to the role of the collagen network in subchondral bone.     

Early exercise advances the maturation of glycosaminoglycans and collagen in the extracellular matrix of articular cartilage in the horse

REASON FOR PERFORMING STUDY: Training at a very young age may influence the characteristics of the collagen network of articular cartilage extracellular matrix (ECM) in horses. OBJECTIVES: To investigate whether increasing workload of foals results in significant changes in the biochemical composition of articular cartilage ECM. METHODS: Thoroughbred foals (n = 33) were divided into 2 different exercise groups from age 10 days-18 months. One group (PASTEX; n = 15) was reared at pasture; the other (CONDEX; n = 18) underwent a specific additional training programme that increased workload by 30%. At mean age 18 months, 6 animals from each group were subjected to euthanasia. The proximal articular surface of the proximal phalanx of the right hindlimb was examined for the presence of damage using the cartilage degeneration index (CDI). Samples were taken from 2 sites with known different loading patterns. Slices were analysed for DNA, glycosaminoglycans (GAG), collagen and post translational modifications of collagen (formation of hydroxylysylpyridinoline [HP] and pentosidine crosslinks, and hydroxylysine [Hyl]), and exercise groups and different sites compared. RESULTS: There were no differences in CDI between PASTEX and CONDEX animals, indicating the absence of extra joint damage due to the exercise regimen. There were site-related differences for most biochemical variables, corroborating earlier reports. All biochemical variables showed differences between PASTEX and CONDEX groups at one of the sites, and some at both. GAG and collagen levels were lower in the CONDEX group whereas Hyl, HP crosslinks and pentosidine crosslinks were higher. CONCLUSIONS AND POTENTIAL RELEVANCE: A measurable effect of the conditioning exercise was demonstrated. The margin between too much and too little work when training foals may be narrower than intuitively presumed.     

Influence of intensity and changes of physical activity on bone mineral density of immature equine subchondral bone

Reasons for performing study: Subchondral bone provides structural support to overlying articular cartilage and plays an important biomechanical role in osteochondral diseases. Mechanical features of bone correlate strongly with bone mineral density, which is directed by the loading conditions to which the tissue is subjected. Objective: To investigate the influence of physical activity levels on subchondral bone mineral density (sBMD) in foals during early development. Methods: Three groups of foals were subjected to different physical activity levels from birth until age 5 months. A proportion of these foals were subjected to euthanasia at 5 months while remaining foals were subjected to similar physical activity levels for 6 months until euthanasia at 11 months. Osteochondral specimens were collected for measurement of sBMD with peripheral quantitative computed tomography at 2 differently loaded anatomical sites of the proximal phalangeal bone at 1, 2, 3, 4 and 5 mm depth from the osteochondral junction. Results: Growth significantly increased sBMD but by a different amount depending on anatomical location and physical activity level. Significantly higher sBMD was found at the habitually loaded central area in comparison to the intermittently peak loaded marginal site. Exercise increased sBMD throughout the whole depth of analysed tissue, but changes were generally more obvious at a depth of 2 mm. Interestingly, foals subjected to additional sprint training preserved the exercise‐induced sBMD increase at the habitually loaded central area during the 6 months of the second phase of the study. Conclusions: Habitual low‐intensity loading elicits a greater response in sBMD in quantitative terms than high‐intensity low‐frequency loading at the sites investigated in this study. Future sBMD may be influenced by means of well‐tailored exercise regimens at young age. Potential relevance: Specific physical activity levels during early development may potentially reduce the prevalence of osteochondral injury later in life.     

Osteochondrosis — An international problem for the horse industry

Osteochondrosis is one of the most important skeletalproblems affecting young horses and is one of the conditions included in the syndrome of developmental orthopedic disease. It occurs worldwide in many different breeds and its incidence appears to be steadily increasing. The primary lesion of osteochondrosis is attributed to focal damage of endochondral ossification. When occurring in the articular/epiphyseal cartilage complex, there may be bone formation within the retained cartilage, resulting in “bony fragments.” These are often seen in the tarsocrural articulation on the cranial aspect of the tibia. Focal retention of necrotic cartilage into the subchondral bone may eventually lead to cleft formation resulting in osteochondrosis dissecans. Other lesions associated with osteochondrosis are subchondral cysts, fractures and osteoarthrosis.The definitive cause of osteochondrosis is unknown,but there is evidence that it is the response in growth cartilage to a number of different challenges. It appears to be multifactorial in origin, involving heredity, growth rate, body size, nutrition, mineral imbalance, endocrinological dysfunction and biomechanical trauma. Recent studies have shown cases of osteochondrosis to be preceded by radiographically detectable disturbances of endochondral ossification during the first three months of life. This early appearance of disturbed ossification necessitates further studies into the importance of nutrition/management of the pregnant and lactating mare. Whether these early disturbances will lead to fully developed osteochondrosis or if they will recede obviously depends on a number of factors such as nutrition and exercise. High energy diets have been shown in weaning foals to consistently produce lesions of osteochondrosis. If, however, foals fed a high energy diet are subjected to forced exercise, the incidence of osteochondrosis is reduced dramatically.The prevention of osteochondrosis must rate as a highpriority for the horse industry, but to achieve this more data on the mechanisms of endochondral ossification and how this process is influenced by different intrinsic and environmental factors are required. Recent research into osteochondrosis has involved studies of chondrocyte metabolism in growth cartilage, the influence of growth factors, the extent of degradation of extracellular matrix and endochondral ossification.     

The effects of copper supplementation on the prevalence of cartilage lesions in foals

The potential role of dietary copper in the development of cartilage defects in foals was investigated. Twenty-one mares were fed rations containing 13 ppm copper (CuC, control) or 32 ppm copper (CuS, supplemented) during the last three to six months of gestation and first three months of lactation. Their foals were fed pelleted concentrate containing 15 or 55 ppm Cu and were destroyed at 90 (5 CuC and 5 CuS foals) or 180 (6 CuC and 5 CuS foals) days. Focal cartilage lesions were found at multiple sites on necropsy. In foals killed at 90 days, there were over twice (9 versus 4) as many lesions of osteochondrosis and more than four times (9 versus 2) as many articular lesions of osteophyte formation or thinning in CuC foals compared with CuS foals. These differences were due predominantly to a higher number of lesions in one CuC foal. Two 90-day CuC foals had osteochondrosis of articular-epiphyseal (A-E) complex, one with thickenings and separation from subchondral bone and one with subchondral fibrosis. One 90-day CuS foal had a cartilage thickening of the A-E complex in the tibiotarsal joint with separation from subchondral bone. In foals killed at 180 days, there were seven times more articular lesions (21 versus 3) of osteophyte formation or thinning, nearly twice as many lesions of osteochondrosis (13 versus 8) [corrected] in the physis and over five times as many involving the A-E complex (11 versus 2) in six CuC foals compared with five CuS foals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of exercise on the diameter of collagen fibrils in the central core and periphery of the superficial digital flexor tendon in foals

OBJECTIVE: To determine the effects of exercise on collagen fibril diameter distribution in the superficial digital flexor tendon (SDFT) of foals. ANIMALS: 43 Dutch Warmblood foals. PROCEDURE: From 1 week until 5 months of age, group-1 foals (n = 14) were housed in stalls and not exercised, group-2 foals (14) were housed in stalls but were exercised, and group-3 foals (15) were maintained at pasture. Biopsy specimens were collected from the SDFT at 2 months, and 8 foals in each group were euthanatized at 5 months. Remaining foals were housed together in a loose stall and paddock until euthanatized at 11 months. After euthanasia, specimens were collected from the SDFT; all specimens were analyzed by use of electron microscopy. Collagen fibrillar index (CFI), mass average diameter (MAvD), and area dependent diameter (ADD) were compared among groups. RESULTS: Exercise-related differences in fibril distribution were not detected among groups at 2 months. At 5 months, ADD in peripheral specimens was significantly greater in group 1 than group 3. At 11 months, MAvD in core specimens was significantly less in group 3, compared with the other groups. However, in peripheral specimens, MAvD was significantly less in groups 2 and 3. CONCLUSIONS AND CLINICAL RELEVANCE: Collagen fibril restructuring in the SDFT of foals is in part an exercise-driven process. Withholding exercise may cause a delay in fibril development that can be partially overcome by increasing exercise at a later age. Exercise type may also affect remodeling of the SDFT in foals.     

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.     

Early lesions of articular osteochondrosis in the distal femur of foals

Failure of the cartilage canal blood supply to epiphyseal growth cartilage has been implicated in the pathogenesis of articular osteochondrosis in horses and other animal species. In a previous study of the developmental pattern of the blood supply in the tarsus of foals, early lesions of osteochondrosis were consistently found in regions where the cartilage canal vessels traversed the chondro-osseous junction. The developmental pattern of blood vessels has also been described in the distal femoral epiphysis; however, the group of foals examined in that study did not have lesions of osteochondrosis in this location. Therefore, the relationship between the occurrence of early lesions of osteochondrosis and the developmental pattern of the blood supply to epiphyseal growth cartilage in this site in foals has not been examined. Distal femora were collected from 30 fetuses and foals (up to 11 months old) submitted for postmortem examination. Sections from the lateral trochlear ridge and medial femoral condyle of both hind limbs were examined histologically. Sixteen cartilage lesions were found in 7 of the 30 fetuses and foals. All lesions contained evidence of cartilage canal necrosis and ischemic chondronecrosis. The lesions were located in regions where cartilage canal vessels traversed the chondro-osseous junction, as previously observed in the tarsus. The location and morphology of lesions indicated that a subclinical stage of ischemic chondronecrosis existed that preceded and predisposed to the development of osteochondrosis dissecans and subchondral bone cysts.     

Effects of exercise on chondrocyte viability and subchondral bone sclerosis in the distal third metacarpal and metatarsal bones of young horses

The objective was to determine the effects of early exercise on the articular cartilage and subchondral bone at specific sites of the distal third metacarpal and metatarsal bones of 12 young Thoroughbred horses allowed free choice exercise at pasture. Six of the horses had additional controlled exercise 5 days per week from mean age of 21+/-20 days of age (range: 3-83 days) until 17.1 months of age. Confocal laser scanning microscopy was used to quantify viable and non-viable chondrocytes. Proteoglycan scoring and modified Mankin scoring was performed and subchondral bone mineral density measured by computed tomography. The number of viable chondrocytes was significantly greater in the conditioned group, which also had a higher Safranin O/Fast Green (SOFG) score than did the group which could exercise only at pasture. There was no difference in mean bone mineral density between groups, nor was there relationship between subchondral bone mineral density and chondrocyte viability. The apparent beneficial effects of early conditioning exercise may support the use of exercise to optimise development of articular cartilage in young individuals.     

Haematological and blood biochemical characteristics of Dutch warmblood foals managed under three different rearing conditions from birth to 5 months of age

Reference values for haematological and blood biochemical variables may vary per breed and are influenced by age and, to a certain extent, by rearing conditions. To investigate the influence of age and rearing conditions, these variables were measured in Dutch Warmblood foals from birth to 5 months of age. The foals were divided into three groups with different exercise regimens: 14 foals got boxrest with no exercise; 14 foals were kept in comparable boxes, but received daily exercise; and 15 foals were maintained on pasture with free exercise. Blood samples were collected each month and 36 haematological and biochemical variables were measured. The influence of age and rearing conditions was statistically evaluated. Significant age effects were found for all variables with the exception of band-shaped leucocytes, eosinophilic leucocytes, basophilic leucocytes, monocytes, platelets, pCO2, and sodium, potassium, calcium, magnesium, creatinine and creatine phosphokinase levels. Rearing conditions appeared to influence the haemoglobin, packed cell volume, pH, base excess, bicarbonate, chloride, urea, and alkaline phosphatase values. Most age-related differences can be explained by growth and differentiation-related processes that are specific for young animals. Differences due to the different rearing conditions can partly be explained by the higher metabolism and greater maturation of tissues in foals maintained on pasture with free exercise. Some other differences were minor and were probably of no clinical relevance. The conclusion is that haematological and blood biochemical variables in the Dutch Warmblood foal mainly depend on age, thus warranting the use of specific age-related reference values for foals of this breed.     

The influence of strenuous exercise on collagen characteristics of articular cartilage in Thoroughbreds age 2 years

In order to assess the influence of strenuous exercise on collagen characteristics of articular cartilage, the response of the collagen network was studied in seven 2-year-old Thoroughbreds subjected to strenuous exercise compared to 7 nontrained individuals. After 13 weeks, the animals were subjected to euthanasia, fetlock joints of the forelimbs were scored macroscopically after Indian Ink staining, and articular cartilage from different locations of the articular surface of the proximal first phalanx was sampled and analysed for water content, collagen content, hydroxylysine content and amount of hydroxylysylpyridinoline (HP) crosslinks. Gross lesions were significantly more severe in the exercised than in the nonexercised group. In the control animals, the characteristic site-specific differences in collagen parameters were found as described earlier, but in the strenuously exercised animals this physiological biochemical heterogeneity had disappeared. In the exercised animals, an increase in water content and a sharp decrease in HP crosslinking was found that was correlated with the presence of wear lines. It is concluded that the strenuous exercise provoked significant alterations in the characteristics of the collagen network of the articular cartilage of the fetlock joint which were suggestive of microdamage and loosening of the collagen network. The collagen component of cartilage, in contrast to the proteoglycan component, is known to have a very limited capacity for repair and remodelling due to an extremely low turnover rate. Therefore, alterations within the articular collagen network might be expected to play an important role in the pathophysiology of degenerative joint disorders.     

The Effects of Fixation of the Ulna to the Radius in Young Foals

The effects of radioulnar fixation were studied in 21 Quarter horse foals by applying a bone plate to the caudal aspect of the proximal part of the ulna, with screws engaging both the radius and the ulna. The plates were applied at 1 month of age in six foals (group I), 5 months of age in six foals (group II), and 7 months of age in three foals (group III). Six foals underwent sham operations at 1 month of age to serve as controls (group IV). Ulnar dysplasia and elbow subluxation developed in all treated foals. The magnitude of ulnar dysplasia was inversely related to the patient's age at fixation and was accompanied by degenerative joint disease and lameness in foals undergoing fixation at 1 and 5 months of age. Removal of the fixation appliances 16 weeks after implantation in three foals from each of groups I and II failed to reverse the degree of ulnar dysplasia. Although foals undergoing fixation at 7 months of age (group III) were not lame, radiographic evidence of subluxation and subtle degenerative changes in the articular cartilage of the treated elbow did develop. Recommendations for avoidance of radioulnar fixation were developed from these observations.     

Evaluation of serum concentrations of biomarkers of skeletal metabolism and results of radiography as indicators of severity of osteochondrosis in foals

OBJECTIVE: To determine whether serum concentrations of biomarkers of skeletal metabolism can, in conjunction with radiographic evaluation, indicate severity of osteochondrosis in developing horses. ANIMALS: 43 Dutch Warmblood foals with varying severity of osteochondrosis. PROCEDURE: 24 foals were monitored for 5 months and 19 foals were monitored for 11 months. Monthly radiographs of femoropatellar-femorotibial and tibio-tarsal joints were graded for osteochondral abnormalities. Serial blood samples were assayed for 8 cartilage and bone biomarkers. At the end of the monitoring period, foals were examined for macroscopic osteochondrosis lesions. RESULTS: Temporal relationships were evident between certain serum biomarkers and osteochondrosis severity in foals during their first year. Biomarkers of collagen degradation (collagenase-generated neoepitopes of type-II collagen fragments, type-I and -II collagen fragments [COL2-3/4C(short)], and cross-linked telopeptide fragments of type-I collagen) and bone mineralization (osteocalcin) were positive indicators of osteochondrosis severity at 5 months of age. In foals with lesions at 11 months of age, osteochondrosis severity correlated negatively with COL2-3/4C(short) and osteocalcin and positively with C-propeptide of type-II procollagen (CPII), a collagen synthesis marker. Radiographic grading of osteochondrosis lesions significantly correlated with macroscopic osteochondrosis severity score at both ages and was strongest when combined with osteocalcin at 5 months and CPII at 11 months. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of serum biomarkers to indicate osteochondrosis severity appears to depend on stage of disease and is strengthened with radiography. In older foals with more permanent lesions, osteochondrosis severity is significantly related to biomarker concentrations of decreased bone formation and increased cartilage synthesis.     

Osteochondrosis and copper: histology of articular cartilage from foals out of copper supplemented and non-supplemented dams

Copper (Cu) supplementation of dams in late gestation may be protective against articular cartilage abnormalities in foals. Articular cartilage was harvested from 22 Thoroughbred foals at 160 days of age, at sites predisposed to osteochondrosis (OC), and examined for evidence of early cartilage abnormalities and established dyschondroplastic (DCP) lesions to determine if there were any significant differences due to mare Cu supplementation by injection during late gestation, or foal liver Cu concentration. Cu supplemented mares received calcium Cu edetate injections in late gestation (250 mg at around 220, 248, 276 and 304 days gestation, then every two weeks until foaling). Foals were euthanased at 160 days of age and articular cartilage was harvested from four defined sites. Samples were examined for histological appearance of chondrocytes after staining with haematoxylin and eosin, and were also stained with toluidine blue to indicate proteoglycan content. Alkaline phosphatase (ALP) activity was detected by histochemistry, and histocytochemical techniques were used to determine the expression of cathepsin B. Cu supplementation of the dam, or liver Cu concentration of the foal at birth or 160 days of age had no statistically significant effect on the frequency of cartilage irregularities observed grossly, or abnormalities detected histologically at four defined sites. ALP expression was similar in all samples. Cathepsin B expression varied between sites, and was seen in chondrocyte clusters. The intensity of toludine blue staining varied between sites. Minor histological cartilage abnormalities were observed in cartilage from clinically normal animals. These abnormalities might be 'early' dyschondroplastic lesions, which could resolve or progress. The role of Cu in the development, resolution or progression of dyschondroplastic lesions is poorly understood.     

The influence of exercise during growth on ultrasonographic parameters of the superficial digital flexor tendon of young Thoroughbred horses

REASON FOR PERFORMING STUDY: Conditioning by early training may influence the composition of certain musculoskeletal tissues, but very few data exist on its effect during growth on tendon structure and function. OBJECTIVES: To investigate whether conditioning exercise in young foals would lead to any ultrasonographically detectable damage to the superficial digital flexor tendon or an increase in cross-sectional area (CSA). METHODS: Thirty-three Thoroughbred foals reared at pasture were allocated to 2 groups: control (PASTEX) allowed exercise freely at pasture; and CONDEX, also at pasture, began conditioning exercise from mean age 21 days over 1030 m on a purpose-built oval grass track, for 5 days/week until mean age 18 months. Foals were observed daily, and underwent orthopaedic examination monthly. Ultrasonographic images of the superficial digital flexor tendon (SDFT) at the mid-metacarpal level of both forelimbs were obtained in all foals at ages 5, 8, 12, 15 and 18 months. CSA was validated (r(2) = 0.89) by determining CSA from digital photographs of the transected SDFT surface from 12 of the horses necropsied at age 17.1 months. RESULTS: here was no clinical or ultrasonographic evidence of tendonopathy in either group and the greatest increase in mean CSA in both groups occurred between age 5 and 8 months. Across all age categories, there was no significant difference in mean CSA between the left and right limbs, or colts and fillies; there was a trend towards a larger CSA in the CONDEX group (P = 0.058). CONCLUSIONS: There was no conclusive evidence for a structural adaptive hypertrophy of the SDFT, probably because the regimen was insufficiently rigorous or because spontaneous pasture exercise may induce maximal development of energy storing tendons. POTENTIAL RELEVANCE: A moderate amount of early conditioning exercise against a background of constant exercise at pasture is not harmful to the development of the flexor tendons.     

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.     

Iron deficiency in stabled Dutch warmblood foals

Forty-three Dutch Warmblood foals were divided at random into 3 rearing groups immediately after birth: a box-rest group, a box-rest with exercise group, and a pasture group. All stabled foals (box-rest and exercise groups) were fed freshly cut grass harvested from the same pastures on which the pasture group foals were grazing. Blood samples were obtained monthly for CBC and biochemical analyses. At 1-3 months of age, the foals at pasture were active but the foals in the box-rest and exercise groups were listless. Mean hemoglobin concentrations, PCVs, blood iron concentrations, and saturation of total iron binding capacity were significantly lower (P < .05) in the box-rest group (11.3+/-1.2 g/dL, 33+/-3%, 123+/-67 microg/dL, and 18+/-9%) and the exercise group (11.6+/-1.5 g/dL, 34+/-4%, 101+/-61 microg/dL, and 15+/-10%) compared with the pasture group (14.0+/-0.8 g/dL, 42+/-3%, 212+/-67 microg/dL, and 32+/-10%). Oral supplementation of iron to all foals resulted in significant increases in the values of these variables in the box-rest group and exercise group at 4-5 months of age, and the stabled foals were as active as the foals at pasture. In the pasture group, no significant changes occurred. Management practices clearly influence the iron state in young growing foals from 1 to 3 months of age, and although not a frequent cause of anemia in the horse, an absolute deficit of body iron may occur in stabled foals fed freshly cut grass.     

Evaluation of the role of keratan sulphate as a molecular marker to monitor cartilage metabolism in horses

The role of keratan sulphate (KS) as a metabolic marker of cartilage was evaluated using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n = 3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor-I alpha (IGF-I alpha) or interleukin-1 alpha (IL-1 alpha) for 2 weeks. The concentrations of sulphated glycosaminoglycans (GAG) and KS in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition enzyme-linked immunosorbent assay using a 1/20/5D4 antibody, respectively. The concentration of GAG was significantly increased both in the media of pellets stimulated by IGF-I alpha and in those stimulated by IL-1 alpha. KS concentration was significantly increased in those stimulated by IL-1 alpha, while no significant change was found in those stimulated by IGF-I alpha. A high correlation between GAG and KS concentrations was found in the media of pellets stimulated by IL-1 alpha (r = 0.84), but not in those stimulated by IGF-I alpha (r = 0.59). The results suggest that the concentration of KS reacting to 1/20/5D4 mirrors the GAG concentration during the stage of cartilage catabolism, but not during the cartilage anabolic stage. The KS concentration in biological fluids could therefore be a useful marker to understand further the cartilage catabolic process. It may also represent some aspects of the cartilage anabolic process.