Hypertrophy and physiological death of equine chondrocytes in vitro

REASON FOR PERFORMING STUDY: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. OBJECTIVE: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. METHODS: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. RESULTS: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. CONCLUSIONS: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. POTENTIAL RELEVANCE: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.     

Immunolocalization of cathepsin B in equinedyschondroplastic articular cartilage

A polyclonal antiserum raised in sheep against human cathepsin B was tested for specificity and cross-reactivity with the horse homologue by SDS-PAGE and Western blotting, prior to being used for immunolocalization of the enzyme in equine articular cartilage. In Western blots, the antiserum recognized the 30 kDa single chain and 25 kDa heavy chain of the mature enzyme in purified bovine cathepsin B, and corresponding bands at 32 and 27 kDa in equine chondrocyte and fibroblast lysates. This antiserum was then used to compare the expression and distribution of cathepsin B in normal and dyschondroplastic cartilage of young horses.In normal articular cartilage (n=6 animals), significant amounts of enzyme were detected only in hypertrophicchondrocytes in the deep zone. The enzyme was intracellular, located in the lysosomal granules. No extracellular matrix staining was observed. Levels of cathepsin B were increased slightly above normal in the deep zone in age-matched dyschondroplastic cartilage (n=5 animals). The most striking finding, however, was the abundance of the enzyme in chondrocyte clonal clusters associated with the lesions. Cathepsin B levels were low in chondrocytes isolated from normal cartilage (n=6), but increased progressively during serial subculture, reaching a maximum at passage 5–6. In contrast, primary cultures of dyschondroplastic chondrocytes (n=3) expressed abundant cathepsin B.     

Localisation and activity of cathepsins K and B in equine osteoclasts

Cathepsin K and cathepsin B were immunolocalised in equine osteoclasts (OC s) present in ex vivo cartilage/subchondral bone samples. Samples were obtained post mortem from the lateral trochlear ridge (LTR) of six horses and ponies aged between 303 days gestation to 8 months. Strong expression of cathepsin K was detected in OC s, particularly those located at the osteochondral junction, apparently involved in the resorption of calcified cartilage. Cathepsin K expression was also detected in hypertrophic chondrocytes and in the endothelial cells of some blood vessels penetrating the hypertrophic zone of cartilage. By contrast, cathepsin B was either absent or present at very low levels in OC s.Osteoclast-like cells (OCL s) were generated in vitro from bone marrow (BM), obtained from the femurs of one horse and two ponies. High levels of cathepsin K activity but only very low levels of cathepsin B activity were demonstrated in OCL s using fluorogenic substrates for these enzymes. The cathepsin K activity could be blocked by the general cysteine proteinase inhibitor, E-64, but not by the cathepsin B inhibitor, CA-074Me. The cathepsin B activity was completely blocked by both CA-074Me and E-64. Taken together, these results suggest that cathepsin K is more important than cathepsin B in the osteoclastic resorption of bone and calcified cartilage of developing equine long bones. Given the apparent importance of cathepsin K in equine endochondral ossification further investigation into the possibility that abnormal expression of this enzyme is involved in the pathogenesis of equine developmental orthopaedic disease is warranted.     

Studies on growth cartilage in the horse and their application to aetiopathogenesis of dyschondroplasia (osteochondrosis)

The importance of osteochondrosis (dyschondroplasia) to the horse industry has been well documentedsince it was first recorded 50 years ago. The condition is known to be multifactorial in origin, arising from focal failure of endochondral ossification at predilection sites in articular/epiphyseal growth cartilage, but specific information on its aetiopathogenesis is sparse. This paper reviews the current knowledge of growth cartilate metabolism and the process of normal endochondral ossification in the horse. It highlights the localization of various protein products of chondrocytes and the differences in the zones of articular cartilage. In the early focal lesions (referred to as dyschondroplasia) there are alterations in the chondrocytes, extracellular matrix and some of the local protein products. The most obvious feature is an alteration in matrix metabolism which may be responsible for triggering a range of other factors leading to the development of a retained core of cartilage and a primary lesion of dyschondroplasia.Based on available evidence, a preliminary hypothesis for pathogenesis is presented. This suggests thatthere are a number of factors capable of initiating the condition. One of these involves high circulating insulin levels from high energy feeding which may affect chondrocyte maturation leading to altered matrix metabolism and faulty mineralization resulting in the formation of cartilage cores which characterize the condition. Further research to test this hypothesis is needed before there can be a rational basis for prophylaxis.     

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.     

Chondrodysplasia of Texel sheep - a new disease of suspected genetic aetiology (abstract)

A chondrodysplasia characterised by dwarfism and varus deformity of the forelimbs has been recognised over four seasons in Texel and Texel-cross lambs on three related properties. Affected lambs appear normal at birth, but by 2 weeks of age have reduced growth rate, shortened neck and legs, varus forelimb deformities and a progressive reluctance to walk. Most die within the first 4 months of life. Some affected lambs have had normal co-twins. Post mortem examination of two severely affected 3-month-old lambs revealed extensive loss of articular cartilage and exposure of subchondral bone on the weight-bearing surfaces of the proximal humeri and femurs. Articular and physeal cartilages were thicker than normal, suggesting delayed or impaired endochondral ossification. Histopathologically, there was disorganisation of chondrocytes in articular and physeal cartilage of all bones examined. In addition, there were multiple foci of chondrolysis, which coalesced in some areas to form large clefts. The chondroid matrix surrounding chondrocytes had an abnormal fibrillar appearance. One lamb that exhibited early signs of the disease died of an unrelated cause at 3 weeks of age, and although there was no erosion of articular cartilage, characteristic microscopic lesions were present in articular and physeal cartilage. Although most of the affected lambs were born on a commercial property with a mix of different sheep breeds, all had Texel genes on both sire and dam sides of the pedigree. The farmer had been purchasing his Texel rams from the same flock for several seasons. Circumstantial evidence favours a genetic aetiology, characterised by either autosomal recessive inheritance or over dominance. Studies on the genetic and biochemical basis of the disease are continuing.     

Consequences of capital femoral dyschondroplasia in young adult and skeletally mature broilers

Radiographs revealed defective endochondral ossification in proximal femora of 52 lame broilers. Cartilage retention either involved epiphyseal cartilage close to the articular surface or growth plate cartilage (dyschondroplasia). In at least seven birds proximal femoral lesions were the only abnormalities detected which could have caused lameness. In cases of moderate or severe cartilage retention, fracture lines traversed necrotic cartilage to undermine part or all of the femoral head. Evidence of reparative fibrosis was seen with augmentation of metaphyseal trabeculae by woven bone apposition. Foveal blood vessels aided repair by re-establishing a new ossification centre but in many cases attempts at repair were inadequate.     

Epiphyseal cartilage canal blood supply to the tarsus of foals and relationship to osteochondrosis

REASON FOR PERFORMING STUDY: Pathological changes in the blood supply to growth cartilage have been implicated in the pathogenesis of osteochondrosis (OC) in horses, but have not been reported using vascular perfusion techniques. OBJECTIVE: To describe the developmental pattern of cartilage canal vessels in the distal tibial epiphysis and talar growth cartilage of foals. METHODS: Nine foals bred from parents with OC were sacrificed between the ages of 0 and 7 weeks to undergo a barium perfusion procedure. The distal end of the tibia and the entire talus were cleared in methyl salicylate and perfused vessels studied in the intact bones. Slabs with a thickness of 4-5 mm from 3 predilection sites for OC were examined in the stereomicroscope and with light microscopy. RESULTS: Cartilage canals were present for a limited period of growth. Perfused vessels initially entered canals from the perichondrium. Vessels in the proximal portion of canals retained their perichondrial arterial source throughout. With time, the ossification front advanced to incorporate the mid-portion of canals; and anastomoses formed between canal vessels and subchondral vessels. A shift occurred and vessels in the distal terminus of canals came to use subchondral vessels as their arterial source. Twelve histological lesions were found in 7 foals. All contained necrotic vessels surrounded by necrotic growth cartilage and 3 caused macroscopically visible delay in endochondral ossification. Lesions were located where vessels traversed the ossification front to enter the distal terminus of canals. CONCLUSION: Cartilage canal vessels are particularly susceptible to failure at the point where they cross the ossification front, with consequences for the viability of those chondrocytes that depend on them. POTENTIAL RELEVANCE: A better understanding of how lesions of OC arise may improve the ability to identify, monitor, prevent and treat this disorder. Involvement of cartilage canals in the pathogenesis of equine tarsal OC plausibly explains several clinical features of this disease.     

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.     

Cytokine and chemokine gene expression of IL-1beta stimulated equine articular chondrocytes

OBJECTIVE: To evaluate mRNA expression of several proinflammatory and anti-inflammatory cytokines and chemokines in equine unstimulated and interleukin-1beta (IL-1beta)-stimulated chondrocytes. STUDY DESIGN: In vitro experiment using equine chondrocyte cultures. SAMPLE POPULATION: Whole articular cartilage from metacarpophalangeal joints (n=5 horses; 10 fetlocks). METHODS: Chondrocyte monolayer cultures were established from digested adult equine articular cartilage and stimulated with 5 ng/mL of recombinant human IL-1beta. RNA was extracted from the cells 24 hours after stimulation. IL-1beta, IL-4, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and ubiquitin (house keeping gene) mRNA expression were investigated by real-time RT-PCR. RESULTS: IL-1beta, IL-6, and IL-8 mRNA were expressed in unstimulated chondrocytes from macroscopically normal joints and were significantly up-regulated after stimulation (5/5 horses). IL-4 mRNA was not detected in any samples (0/5 horses). TNF-alpha mRNA, by comparison, was expressed in 2/5 unstimulated samples and in all stimulated samples but a considerable sample variation in response to IL-1beta stimulation was observed. CONCLUSIONS: Equine chondrocytes express mRNA for several proinflammatory cytokines and chemokines and IL-1beta modulates their expression. CLINICAL RELEVANCE: Chondrocytes express proinflammatory cytokines and chemokines capable of modulating a local inflammatory cascade in articular cartilage, which could potentially lead to focal degradation and osteoarthritis.     

Distribution of type VI collagen in the cartilaginous tissue of the proximal tibia in the domestic cat

To investigate the distribution of the early stage chondrocytes during the formation and closure of epiphyseal growth plate (EGP) of the domestic cat, we examined the EGP of proximal tibiae by immunohistochemistry for type VI collagen. In the epiphyseal cartilage without the secondary ossification center (SOC) and EGP in newborn cats aged 1 and 10 days, type VI collagen-positive chondrocytes were located around the cartilage canals and articular surface. In the epiphyseal cartilage with the SOC and EGP in young cats aged 1 to 3 months, type VI collagen-positive chondrocytes were located in the upper resting zone of the EGP, and then increased throughout the resting zone along with maturation. In the adult cats with the partially closed EGP, type VI collagen-positive chondrocytes were distributed throughout the remaining EGP. These findings indicate that the early stage chondrocytes characterized with type VI collagen are continuously located in the EGP during maturation. In addition, the increase of the early stage chondrocytes and the decrease of the reserve chondrocytes in the EGP along with maturation may cause the cessation of the longitudinal growth of the EGP, and finally bring about the EGP closure.     

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 zonal distributions of alkaline phosphatase, adenosine triphosphatase, laminin, fibronectin and chondroitin 4-sulphate in growing rat humerus proximal epiphyseal cartilage: a histochemical and an immunohistochemical study

Although there are many studies about epiphyseal cartilage extracellular matrix (ECM) macromolecules in bone formation, studies of their distribution and role in the mineralization of these components in growing rat humerus proximal epiphyseal cartilage have not been sufficiently detailed. The aim of this study was to determine the distributions of alkaline phosphatase (ALP), adenosine triphosphatase (ATPase), laminin, fibronectin and chondroitin 4-sulphate in growing rat humerus proximal epiphyseal cartilage. The rats were killed by cervical dislocation, and the humeri were removed, sectioned (6 and 10 microm) on a cryotome or paraffin microtome, and stained using histochemical and immunohistochemical methods. ALP and ATPase were markedly observed in the hypertrophy and calcifying cartilage. In addition, ATPase was found to be very strongly positive in the tangential zone of articular cartilage. Results of immunohistochemical staining for laminin, fibronectin and chondroitin 4-sulphate showed that the immunostaining was the heaviest in the tangential zone of articular cartilage. In growing epiphyseal plates, there were differences in the density of these macromolecules of chondrocytes as a function of the maturation process. In conclusion, these ECM macromolecules of epiphyseal cartilage may regulate the cell-cell and cell-matrix interactions as well as the matrix calcification during the ossification of epiphyseal cartilage.     

Osteochondrosis in the horse--searching for the key to pathogenesis

This paper reviews current developments in equine osteochondrosis complex and the clinical syndromes associated with it. Although the primary lesion has been defined as a failure of endochondral ossification, its definitive cause is unknown and appears to involve heredity, growth rate, nutrition, mineral imbalance, endocrinological dysfunction and biomechanical trauma. Despite the international importance of osteochondrosis in horses, surprisingly few controlled investigations have been performed on its pathogenesis. The studies that have been conducted suggest that local effects on differentiating growth cartilage are the key to a more complete understanding of the problem. Gaps in the current knowledge include in-depth understanding of the life cycle of chondrocytes in growth cartilage, the process of mineralisation and the use of a standard experimental model for the induction of osteochondrosis. The ultimate goal of osteochondrosis research must be to prevent or reduce the incidence of the condition in horses.     

肉鸡骨骼发育的调控机制

肉鸡骨骼发育主要是通过软骨内骨化完成的。在软骨内骨化的进程中,生长板软骨细胞经历增殖、肥大、转分化和软骨基质矿化等,最终成骨逐渐取代软骨原基,实现骨骼的线性延长。软骨内成骨是一个复杂精密的过程,由SOX9、RUNX2、MEF2C、OSX、TGF-β、BMP2、FGFs、IHH和PTHrP等多种信号因子和转录因子协调调控,这些调控因子由生长板不同区的软骨细胞表达或特异性的调控软骨细胞的增殖、分化及血管侵入等过程。在家禽养殖中,肉鸡常发腿病且治疗难度大,而有关肉鸡腿病发病机制的研究报道相对较少。本文综述了骨形成过程及具体的分子调控机制,为了解肉鸡腿病的发生以及提供有效治疗方案提供参考。     

Effect of diet on longitudinal bone growth and osteochondrosis in swine

Weanling gilts were fed either a 12% or 16% protein diet for 10 weeks. Animals fed the 12% protein diet had reduced body weights and reduced longitudinal bone growth as measured in the distal radial growth plate. There was no difference in the growth plate widths between the two animal groups, but there was a significant reduction in the daily rate of cell production in the proliferative zone of animals fed the 12% protein diet. No effect of diet on the rate of expansion of the epiphysis at the articular-epiphyseal junction of the distal femur or humerus could be detected. All animals in both groups had morphologic cartilage lesions consistent with early changes associated with osteochondrosis (OCD), and there was no difference in the lesion morphology between the dietary groups. Areas of disorderly endochondral ossification in the radial growth plate were associated with perpendicular growth cartilage infractions. Growth plate lesions were characterized by increased widths of the maturing cartilage zone without increased width of the proliferative zone or an increase in the daily rate of cell production. Focal growth plate lesions developed because of a transitory inhibition of cartilage mineralization and resorption. Disorderly foci of endochondral ossification beneath articular cartilage were characterized by an area of chondrocyte necrosis which prevented normal cartilage matrix mineralization. Lamellae of cartilage necrosis were also present within the reserve zone of the articular cartilage. These were associated with abnormalities of the cartilage canal vessels, and chondrocyte necrosis was considered to precede degenerative changes in articular cartilage matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cartilage matrix changes in the developing epiphysis: early events on the pathway to equine osteochondrosis?

REASONS FOR PERFORMING STUDY: The earliest osteochondrosis (OC) microscopic lesion reported in the literature was present in the femorotibial joint of a 2-day-old foal suggesting that OC lesions and factors initiating them may arise prior to birth. OBJECTIVE: To examine the developing equine epiphysis to detect histological changes that could be precursors to OC lesions. METHODS: Osteochondral samples from 21 equine fetuses and 13 foals were harvested from selected sites in the scapulohumeral, humeroradial, metacarpophalangeal, femoropatellar, femorotibial, tarsocrural and metatarsophalangeal joints. Sections were stained with safranin O and picrosiruis red to assess cartilage changes and structural arrangement of the collagen matrix. RESULTS: Extracellular matrix changes observed included perivascular areas of paleness of the proteoglycan matrix associated with hypocellularity and, sometimes, necrotic chondrocytes. These changes were most abundant in the youngest fetuses and in the femoropatellar/femorotibial (FP/FT) joints. Indentations of the ossification front were also observed in most specimens, but, most frequently, in scapulohumeral and FP/FT joints. A cartilage canal was almost always present in these indentations. The vascular density of the cartilage was higher in the youngest fetuses. In these fetuses, the most vascularised joints were the metacarpo- and metatarsophalangeal joints but their cartilage canals regressed quickly. After birth, the most vascularised cartilage was present in the FP/FT joint. Articular cartilage differentiated into 4 zones early in fetal life and the epiphyseal cartilage also had a distinct zonal cartilage structure. A striking difference was observed in the collagen structure at the junction of the proliferative and hypertrophic zones where OCD lesions occur. CONCLUSION: Matrix and ossification front changes were frequently observed and significantly associated with cartilage canals suggesting that they may be physiological changes associated with matrix remodelling and development. The collagen structure was variable through the growing epiphysis and a differential in biomechanical properties at focal sites may predispose them to injury.     

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.     

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.     

Expression of matrix metalloproteinases during vascularization and ossification of normal and impaired avian growth plate

Enzymes of the matrix metalloproteinase (MMP) family regulate angiogenesis and are involved in the endochondral ossification process. Tibial dyschondroplasia (TD) and rickets are 2 disorders associated with impairments in this process, mainly in the vascularization of the avian growth plate. In this paper, we induced TD and rickets and studied the expression patterns of 4 members of the MMP family known to be important for endochondral ossification, MMP-2, 3, 9, and 13, in normal and impaired avian growth plates. The expression of MMP-3, 9, and 13 was reduced in the lesions and lined up parallel to the expulsion of blood vessels, which was extended up to the border of the lesion, but did not penetrate into it. Matrix metallopro-teinase-2 was not expressed in the TD lesion but was overexpressed in the rachitic lesion. We also studied the differentiation stage of the chondrocytes populating the lesions and found that the rachitic lesions were populated with proliferative chondrocytes, whereas the TD lesions were filled with chondrocytes that presented both proliferative and hypertrophic markers. These results suggest that MMP-3, 9, and 13 play a role in the vascularization and ossification processes, whereas MMP-2 is related to chondrocyte differentiation and may be involved in cartilage remodeling in the avian growth plate.