Prevalence of subchondral bone pathological changes in the distal metacarpi/metatarsi of racing Thoroughbred horses.

OBJECTIVES: To investigate the prevalence of microscopic subchondral bone injury in the distal metacarpi/tarsi of Thoroughbred racehorses and associations with recent and cumulative training history. METHODS: Metacarpi/metatarsi were obtained from postmortem examination of Thoroughbred racehorses. The severity of palmar/plantar osteochondral disease (POD) was graded in forelimbs from 38 horses and in hindlimbs from a separate cohort of 45 horses. Forelimb samples were embedded in methyl methacrylate and examined using backscattered scanning electron microscopy. Microfracture density in the condylar subchondral bone was determined. Horizontal subchondral bone fractures were identified in hindlimb samples using sections of demineralised tissue. Empty osteocyte lacunae were quantified in hindlimb samples using sections of demineralised tissue. RESULTS: The prevalence of gross POD was 65.8% (95% confidence interval (CI) 48.7-80.4%) in the forelimb and 57.8% (95% CI 42.2-72.3%) in the hindlimb cohort of horses. Microfractures occurred in the forelimbs of 97.4% (95% CI 86.2-99.9%) of horses. Microfracture density in forelimbs increased with age (rs = 0.50, P = 0.001), the number of race starts (rs = 0.47, P = 0.003) and was greater in the medial condyles of horses in training than in those not in training (n = 21, median: 3.1/mm; range: 0.8-10.0 vs n = 17, 1.4/mm; 0-4.5, P = 0.008). Empty osteocyte lacunae were observed in the subchondral bone of hindlimbs in 97.7% (95% CI 88.0-99.9%) of 44 horses. CONCLUSIONS: Subchondral bone pathology occurs with a high prevalence in Thoroughbred racehorses presented for postmortem examination. The accumulation of subchondral bone damage with longer career duration is consistent with bone fatigue.

Role of subchondral bone remodelling in collapse of the articular surface of Thoroughbred racehorses with palmar osteochondral disease.

REASONS FOR PERFORMING STUDY: To gain a better understanding of the aetiology of articular surface collapse in horses with palmar osteochondral disease. OBJECTIVES: To determine whether acceleration of focal bone resorption associated with reduced physical activity contributes to articular surface collapse in racehorses with palmar osteochondral disease. STUDY DESIGN: Cross-sectional study comparing metacarpal bones from horses at varying stages of race training. METHODS: Metacarpal bones from 36 racing Thoroughbred horses were examined with high-resolution peripheral quantitative computed tomography to determine the proportion of the articular surface that had collapsed and with backscattered scanning electron microscopy to quantify porosity and eroded bone surface. Racing and training histories were obtained for comparison with imaging data. RESULTS: In 21 cases, inward collapse of the calcified cartilage layer was observed on backscattered scanning electron microscopy. An increased extent of articular surface collapse was associated with greater numbers of microfractures in the calcified cartilage and superficial subchondral bone (Spearman's correlation [rs ] = 0.62, P<0.001). In the deeper bone (6-10 mm), porosity was lower with a greater extent of articular surface collapse (rs = -0.38, P = 0.023), whereas in the superficial bone (0-4 mm) there was no association between articular surface collapse and porosity (rs = 0.19, P = 0.26). Both porosity (median 14, range 3.8-26 vs. 3.8, 1.6-17%, P = 0.008) and eroded surface (1.1, 0.74-4.5 vs. 0.64, 0.11-4.7 mm(-1) , P = 0.016) of the superficial subchondral bone were higher in resting than in training horses, and in some resting horses subchondral bone voids were highly concentrated, resulting in an apparent loss of support for the overlying calcified cartilage layer. CONCLUSIONS: Articular surface collapse is common in cases of palmar osteochondral disease and is likely to be a sequel to fatigue injury of subchondral bone. Focal subchondral bone resorption appears to contribute to collapse of the calcified cartilage and is potentiated by a reduced-intensity exercise regimen.

Thoroughbred horses in race training have lower levels of subchondral bone remodelling in highly loaded regions of the distal metacarpus compared to horses resting from training.

Bone is repaired by remodelling, a process influenced by its loading environment. The aim of this study was to investigate the effect of a change in loading environment on bone remodelling by quantifying bone resorption and formation activity in the metacarpal subchondral bone in Thoroughbred racehorses. Sections of the palmar metacarpal condyles of horses in race training (n = 24) or resting from training (n = 24) were examined with light microscopy and back scattered scanning electron microscopy (BSEM). Bone area fraction, osteoid perimeter and eroded bone surface were measured within two regions of interest: (1) the lateral parasagittal groove (PS); (2) the lateral condylar subchondral bone (LC). BSEM variables were analysed for the effect of group, region and interaction with time since change in work status. The means ± SE are reported. For both regions of interest in the training compared to the resting group, eroded bone surface was lower (PS: 0.39 ± 0.06 vs. 0.65 ± 0.07 per mm, P = 0.010; LC: 0.24 ± 0.04 vs. 0.85 ± 0.10 per mm, P < 0.001) and in the parasagittal groove osteoid perimeter was higher (0.23 ± 0.04% vs. 0.12 ± 0.02%). Lower porosity was observed in the subchondral bone, reflected by a higher bone area fraction in the LC of the training group (90.8 ± 0.6%) compared to the resting group (85.3 ± 1.4%, P = 0.0010). Race training was associated with less bone resorption and more bone formation in the subchondral bone of highly loaded areas of the distal metacarpus limiting the replacement of fatigued bone. Periods of reduced intensity loading are important for facilitating subchondral bone repair in Thoroughbred racehorses.

Exercise-induced inhibition of remodelling is focally offset with fatigue fracture in racehorses.

UNLABELLED: Bone remodelling is inhibited by high repetitive loading. However, in subchondral bone of racehorses in training, eroded surface doubled in association with fatigue fracture and there was greater surrounding trabecular bone volume suggesting trabecular modelling unloads the bone focally, allowing damage repair by remodelling. INTRODUCTION: Remodelling replaces damaged bone with new bone but is suppressed during high magnitude repetitive loading when damage is most likely. However, in cortical bone of racehorses, at sites of fatigue fracture, focal porosity, consistent with remodelling, is observed in proportion to the extent of surrounding callus. Focal areas of porosity are also observed at sites of fatigue damage in subchondral bone. We hypothesised that fatigued subchondral bone, like damaged cortical bone, is remodelled focally in proportion to the modelling of surrounding trabecular bone. METHODS: Eroded and mineralizing surfaces and bone area were measured using backscattered scanning electron microscopy of post-mortem specimens of the distal third metacarpal bone in 11 racehorses with condylar fractures (cases) and eight racehorses in training without fractures (controls). RESULTS: Cases had a two-fold greater eroded surface per unit area at the fracture site than controls (0.81 ± 0.10 vs. 0.40 ± 0.12 mm(-1), P = 0.021) but not at an adjacent site (0.22 ± 0.09 vs. 0.30 ± 0.11 mm(-1), P = 0.59). Area fraction of surrounding trabecular bone was higher in cases than controls (81 ± 2 vs. 72 ± 2 %, P = 0.0020) and the eroded surface at the fracture site correlated with the surrounding trabecular area (adjusted R (2) = 0.63, P = 0.0010). CONCLUSION: In conclusion, exercise-induced inhibition of remodelling is offset at sites of fatigue fracture. Modelling of trabecular bone may contribute to unloading these regions, allowing repair by remodelling.

The skeleton: a multi-functional complex organ: the growth plate chondrocyte and endochondral ossification.

Endochondral ossification is the process that results in both the replacement of the embryonic cartilaginous skeleton during organogenesis and the growth of long bones until adult height is achieved. Chondrocytes play a central role in this process, contributing to longitudinal growth through a combination of proliferation, extracellular matrix (ECM) secretion and hypertrophy. Terminally differentiated hypertrophic chondrocytes then die, allowing the invasion of a mixture of cells that collectively replace the cartilage tissue with bone tissue. The behaviour of growth plate chondrocytes is tightly regulated at all stages of endochondral ossification by a complex network of interactions between circulating hormones (including GH and thyroid hormone), locally produced growth factors (including Indian hedgehog, WNTs, bone morphogenetic proteins and fibroblast growth factors) and the components of the ECM secreted by the chondrocytes (including collagens, proteoglycans, thrombospondins and matrilins). In turn, chondrocytes secrete factors that regulate the behaviour of the invading bone cells, including vascular endothelial growth factor and receptor activator of NFκB ligand. This review discusses how the growth plate chondrocyte contributes to endochondral ossification, with some emphasis on recent advances.

Identification of light and dark hypertrophic chondrocytes in mouse and rat chondrocyte pellet cultures.

Hypertrophic "light" and "dark" chondrocytes have been reported as morphologically distinct cell types in growth cartilage during endochondral ossification in many species, but functional differences between the two cell types have not been described. The aim of the current study was to develop a pellet culture system using chondrocytes isolated from epiphyseal cartilage of neonatal mice and rats, for the study of functional differences between these two cell types. Hypertrophic chondrocytes resembling those described in vivo were observed by light and electron microscopy in sections of pellets treated with triiodothyronine, 1% fetal calf or mouse serum, 10% fetal calf serum or 1.7MPa centrifugal pressure at day 14, and in pellets cultured with insulin or 0.1% fetal calf or mouse serum at day 21. A mixed population of light and dark chondrocytes was found in all conditions leading to induction of chondrocyte hypertrophy. This rodent culture system allows the differentiation of light and dark chondrocytes under various conditions in vitro and will be useful for future studies on tissue engineering and mechanisms of chondrocyte hypertrophy.

Endochondral ossification: how cartilage is converted into bone in the developing skeleton.

Endochondral ossification is the process by which the embryonic cartilaginous model of most bones contributes to longitudinal growth and is gradually replaced by bone. During endochondral ossification, chondrocytes proliferate, undergo hypertrophy and die; the cartilage extracellular matrix they construct is then invaded by blood vessels, osteoclasts, bone marrow cells and osteoblasts, the last of which deposit bone on remnants of cartilage matrix. The sequential changes in chondrocyte behaviour are tightly regulated by both systemic factors and locally secreted factors, which act on receptors to effect intracellular signalling and activation of chondrocyte-selective transcription factors. Systemic factors that regulate the behaviour of chondrocytes in growth cartilage include growth hormone and thyroid hormone, and the local secreted factors include Indian hedgehog, parathyroid hormone-related peptide, fibroblast growth factors and components of the cartilage extracellular matrix. Transcription factors that play critical roles in regulation of chondrocyte gene expression under the control of these extracellular factors include Runx2, Sox9 and MEF2C. The invasion of cartilage matrix by the ossification front is dependent on its resorption by members of the matrix metalloproteinase family, as well as the presence of blood vessels and bone-resorbing osteoclasts. This review, which places an emphasis on recent advances and current areas of debate, discusses the complex interactions between cell types and signalling pathways that govern endochondral ossification.

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.

Physiological death of hypertrophic chondrocytes.

OBJECTIVE: Post-proliferative chondrocytes in growth cartilage are present in two forms, light and dark cells. These cells undergo hypertrophy and die by a mechanism that is morphologically distinct from apoptosis, but has not been characterized. The aims of the current study were to document the ultrastructural appearance of dying hypertrophic chondrocytes, and to establish a culture system in which the mechanism of their death can be examined. DESIGN: Growth cartilage from fetal and growing postnatal horses was examined by electron microscopy. Chondrocytes were isolated from epiphyseal cartilage from fetal horses and grown in pellet culture, then examined by light and electron microscopy, and quantitative polymerase chain reaction. RESULTS: In tissue specimens, it was observed that dying dark chondrocytes underwent progressive extrusion of cytoplasm into the extracellular space, whereas light chondrocytes appeared to disintegrate within the cellular membrane. Pellets cultured in 0.1% fetal calf serum (FCS) contained dying light and dark chondrocytes similar to those seen in vivo. Transforming growth factor-beta1 or 10% FCS increased the proportion of dark cells and induced cell death. Triiodothyronine increased the differentiation of dark and light cells and induced their death. Dark cells were associated with higher levels of matrix metalloproteinase-13 expression than light cells, and light cells were associated with higher levels of type II collagen expression. CONCLUSIONS: Light and dark hypertrophic chondrocytes each undergo a distinctive series of non-apoptotic morphological changes as they die. Pellet culture can be used as a model of the two forms of physiological death of hypertrophic chondrocytes.

Molecular epidemiology of 'Norwalk-like viruses' associated with gastroenteritis outbreaks in New Zealand.

Outbreaks of gastroenteritis are a major public health problem in New Zealand. The introduction of molecular detection methods has now shown that the 'Norwalk-like viruses' (NLVs) are the major cause of food and waterborne nonbacterial gastroenteritis. Reverse transcription and polymerase chain reaction (RT-PCR) were used to determine the presence of NLVs in faecal specimens from 83 nonbacterial gastroenteritis outbreaks occurring in New Zealand between August 1995 and July 1999. Further characterisation of the NLVs for epidemiological purposes was carried out by dot blot DNA hybridisation and DNA sequencing of representative outbreak strains. The majority of NLV strains occurring in New Zealand since August 1995 are similar to those occurring overseas. The predominant New Zealand strain is genetically similar to the Bristol/Lordsdale virus group. Several New Zealand outbreaks were attributed to Auckland virus, a Mexico-like NLV strain identified as the most likely cause of gastroenteritis after consumption of contaminated oysters in 1994. A new strain, designated Napier virus, has been identified in six outbreaks since 1996. A number of strains closely resembling internationally recognised strains, including Southampton virus, Saratoga virus; Desert Shield virus and Melksham virus have been associated with gastroenteritis outbreaks across New Zealand. Application of these typing methods has provided information on disease transmission for epidemiological investigations of public health significance.