The use of large animals to facilitate the process of MSC going from laboratory to patient-'bench to bedside'.

Large animal models have been widely used to facilitate the translation of mesenchymal stem cells (MSC) from the laboratory to patient. MSC, with their multi-potent capacity, have been proposed to have therapeutic benefits in a number of pathological conditions. Laboratory studies allow the investigation of cellular and molecular interactions, while small animal models allow initial 'proof of concept' experiments. Large animals (dogs, pigs, sheep, goats and horses) are more similar physiologically and structurally to man. These models have allowed clinically relevant assessments of safety, efficacy and dosing of different MSC sources prior to clinical trials. In this review, we recapitulate the use of large animal models to facilitate the use of MSC to treat myocardial infarction-an example of one large animal model being considered the 'gold standard' for research and osteoarthritis-an example of the complexities of using different large animal models in a multifactorial disease. These examples show how large animals can provide a research platform that can be used to evaluate the value of cell-based therapies and facilitate the process of 'bench to bedside'.

The transcription factor scleraxis differentially regulates gene expression in tenocytes isolated at different developmental stages.

The transcription factor scleraxis (SCX) is expressed throughout tendon development and plays a key role in directing tendon wound healing. However, little is known regarding its role in fetal or young postnatal tendons, stages in development that are known for their enhanced regenerative capabilities. Here we used RNA-sequencing to compare the transcriptome of adult and fetal tenocytes following SCX knockdown. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes affected in adult tenocytes, indicating that scleraxis-dependent processes may differ in these two developmental stages. Gene ontology, network and pathway analysis revealed an overrepresentation of extracellular matrix (ECM) remodelling processes within both comparisons. These included several matrix metalloproteinases, proteoglycans and collagens, some of which were also investigated in SCX knockdown tenocytes from young postnatal foals. Using chromatin immunoprecipitation, we also identified novel genes that SCX differentially interacts with in adult and fetal tenocytes. These results indicate a role for SCX in modulating ECM synthesis and breakdown and provide a useful dataset for further study into SCX gene regulation.

Genome-wide transcriptome analysis reveals equine embryonic stem cell-derived tenocytes resemble fetal, not adult tenocytes.

BACKGROUND: Tendon injuries occur frequently in human and equine athletes. Treatment options are limited, and the prognosis is often poor with functionally deficient scar tissue resulting. Fetal tendon injuries in contrast are capable of healing without forming scar tissue. Embryonic stem cells (ESCs) may provide a potential cellular therapeutic to improve adult tendon regeneration; however, whether they can mimic the properties of fetal tenocytes is unknown. To this end, understanding the unique expression profile of normal adult and fetal tenocytes is crucial to allow validation of ESC-derived tenocytes as a cellular therapeutic. METHODS: Equine adult, fetal and ESC-derived tenocytes were cultured in a three-dimensional environment, with histological, morphological and transcriptomic differences compared. Additionally, the effects on gene expression of culturing adult and fetal tenocytes in either conventional two-dimensional monolayer culture or three-dimensional culture were compared using RNA sequencing. RESULTS: No qualitative differences in three-dimensional tendon constructs generated from adult, fetal and ESCs were found using histological and morphological analysis. However, genome-wide transcriptomic analysis using RNA sequencing revealed that ESC-derived tenocytes' transcriptomic profile more closely resembled fetal tenocytes as opposed to adult tenocytes. Furthermore, this study adds to the growing evidence that monolayer cultured cells' gene expression profiles converge, with adult and fetal tenocytes having only 10 significantly different genes when cultured in this manner. In contrast, when adult and fetal tenocytes were cultured in 3D, large distinctions in gene expression between these two developmental stages were found, with 542 genes being differentially expressed. CONCLUSION: The information provided in this study makes a significant contribution to the investigation into the differences between adult reparative and fetal regenerative cells and supports the concept of using ESC-derived tenocytes as a cellular therapy. Comparing two- and three-dimensional culture also indicates three-dimensional culture as being a more physiologically relevant culture system for determining transcriptomic difference between the same cell types from different developmental stages.

Radiographic retrospective study of the caudal cervical articular process joints in the horse.

REASONS FOR PERFORMING STUDY: Arthropathy of the caudal cervical articular process joints (APJs) in the horse is documented as a cause of ataxia and paresis secondary to spinal cord compression. Enlargement of the caudal APJs is reported to increase with age, but there are no known associations of any other factors. No association of the degree of APJ enlargement with neurological signs seen has been documented. This study investigated the associations of cervical APJ enlargement at the C5-C6 and C6-C7 articulations with case subject details (breed, age, sex, usage) and clinical signs. OBJECTIVES: To ascertain if there are of any associations between: the subject details and enlargement of the caudal cervical APJs; and the degree of APJ enlargement and the presence and type of clinical signs. HYPOTHESES: There would be an effect of age, breed and usage on APJ grade, with no effect of sex. Association between grade and clinical signs seen was also investigated. MATERIALS AND METHODS: The radiographs of 122 horses qualified for inclusion. Horses were excluded if they were known to have a neck lesion cranial to C5-C6, or if the radiographs were rotated or of poor quality. In order to standardise the interpretation of APJ enlargement, a novel grading system was developed and used. RESULTS: An association was found between age and APJ grade at C5-C6 but not C6-C7. There was no association between grade, breed, sex and usage, or clinical signs seen. Data also showed a trend for increasing enlargement the more caudal the APJ. CONCLUSION AND POTENTIAL RELEVANCE: The data in this study support that the size of the caudal cervical APJ at the level of C5-C6, appear to increase with age, but this enlargement may not be significant. Enlargement cannot be associated with breed, sex or discipline of the horse at present, and specific grades and therefore degree of enlargement, cannot necessarily be assumed to be the cause of neurological deficits.

Treatment of periocular and non-ocular sarcoids in 18 horses by interstitial brachytherapy with iridium-192.

Treatment of the equine sarcoid has posed a significant challenge to clinicians for years and many different methods have been tried with varying success, including ionising radiation. The aim of this study was to review the efficacy of iridium-192 interstitial brachytherapy for the treatment of eight periocular sarcoids and 15 non-ocular sarcoids on 18 horses. All the periocular sarcoids and 13 of the 15 non-ocular sarcoids were treated successfully.

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 documented since 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 cartilage 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 that there 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.

Reliability and repeatability of thermographic examination and the normal thermographic image of the thoracolumbar region in the horse.

REASONS FOR PERFORMING STUDY: Thermographic imaging is an increasingly used diagnostic tool. When performing thermography, guidelines suggest that horses should be left for 10-20 mins to 'acclimatise' to the thermographic imaging environment, with no experimental data to substantiate this recommendation. In addition, little objective work has been published on the repeatability and reliability of the data obtained. Thermography has been widely used to identify areas of abnormal body surface temperature in horses with back pathology; however, no normal data is available on the thermographic 'map' of the thoracolumbar region with which to compare horses with suspected pathology. OBJECTIVES: To i) investigate whether equilibration of the thermographic subject was required and, if so, how long it should take, ii) investigate what factors affect time to equilibration, iii) investigate the repeatability and reliability of the technique and iv) generate a topographic thermographic 'map' of the thoracolumbar region. METHODS: A total of 52 horses were used. The following investigations were undertaken: thermal imaging validation, i.e. detection of movement around the baseline of an object of constant temperature; factors affecting equilibration; pattern reproducibility during equilibration and over time (n = 25); and imaging of the thoracolumbar region (n = 27). RESULTS: A 1 degrees C change was detected in an object of stable temperature using this detection system, i.e the 'noise' in the system. The average time taken to equilibrate, ie. reach a plateau temperature, was 39 mins (40.2 in the gluteal region, 36.2 in lateral thoracic region and 40.4 in metacarpophalangeal region). Only 19% of horses reached plateau within 10-20 mins. Of the factors analysed hair length and difference between the external environment and the internal environment where the measurements were being taken both significantly affected time to plateau (P<0.05). However, during equilibration, the thermographic patterns obtained did not change, nor when assessed over a 7 day period. A 'normal' map of the surface temperature of the thoracolumbar region has been produced, demonstrating that the midline is the hottest, with a fall off of 3 degrees C either side of the midline. CONCLUSIONS: This study demonstrates that horses may not need time to equilibrate prior to taking thermographic images and that thermographic patterns are reproducible over periods up to 7 days. A topographical thermographic 'map' of the thoracolumbar region has been obtained. POTENTIAL RELEVANCE: Clinicians can obtain relevant thermographic images without the need for prior equilibration and can compare cases with thoracolumbar pathology to a normal topographic thermographic map.

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

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

Equine dyschondroplasia (osteochondrosis)--histological findings and type VI collagen localization.

This study describes (1) the histological appearance of dyschondroplasia, the primary lesion of osteochondrosis, in articular cartilage of the horse and (2) the localization of type VI collagen which is an important constituent of the extracellular matrix (ECM). Dyschondroplastic cartilage was identified on the basis of the presence of cartilage cores (i.e., cartilage extending into the subchondral bone) and confirmed with subsequent histological examination. Full-thickness cartilage samples from 57 horses were collected and paraffin embedded. Histological examination was used to examine the normal architecture of equine growth cartilage and to determine the presence of various pathological changes in dyschondroplastic lesions. Immunolocalization was used to identify type VI collagen in normal and dyschondroplastic lesions. The abnormalities observed in the dyschondroplastic cartilage fell into two groups. In Group A (n = 18) the lesions were associated with a disruption in the normal sequential transition of the chondrocytes through proliferation and maturation resulting in an accumulation of large numbers of small, rounded chondrocytes. A decrease in type VI collagen immunoreactivity compared with normal animals was detected except around chondrocyte clusters. Group B lesions (n = 9) were characterized by an alteration in the staining pattern of the mineralized cartilage and underlying bone. In these lesions type VI collagen immunoreactivity was increased. In both groups the presence of retained blood vessels, chondrocyte clusters, chondronecrosis and fissure formation was detected. These two histologically-distinct groups suggest that equine dyschondroplasia may be comprised of different pathological entities and that it is associated with alterations in the pattern of distribution of an ECM protein.

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

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

Expression of transforming growth factor-beta 1 in normal and dyschondroplastic articular growth cartilage of the young horse.

This study describes the distribution pattern of transforming growth factor-beta 1 (TGF-beta 1) mRNA and protein in normal pre- and post natal growth cartilage and alterations present in lesions of dyschondroplasia (osteochondrosis). TGF-beta 1 expression and immunoreactivity have been investigated by in situ hybridisation and immunolocalisation in the articular/epiphyseal growth cartilage of the lateral trochlear ridge of the distal femur. Cartilage was obtained from 19 normal Thoroughbred horses (5 prenatal and 14 post natal horses) and 15 post natal horses with dyschondroplasia (DCP). TGF-beta 1 mRNA expression and immunoreactivity were detected in the proliferative and upper hypertrophic zones in both pre- and post natal normal articular/epiphyseal cartilage. However, mRNA itself was only detected in the mid- and lower hypertrophic zones. Immunoreactivity was identified intracellularly with some nuclear staining observed. In focal lesions of DCP mRNA expression and immunoreactivity were reduced compared to normal cartilage, but strong mRNA expression was observed in the chondrocyte clusters immediately surrounding a lesion of DCP. The results described in this study demonstrate alterations in TGF-beta 1 dyschondroplastic lesions and indicate that it could be involved in the pathogenesis of this condition in the horse.

Effects of insulin and insulin-like growth factors I and II on the growth of equine fetal and neonatal chondrocytes.

The effects of insulin and insulin-like growth factors (IGFs) I and II on fetal and foal chondrocytes were investigated in vitro. Chondrocytes from the lateral trochlear ridge of the distal femur were obtained from 2 fetuses (280 and 320 days gestation) and one 4-day-old foal and cultured. Membrane proteins consistent with type 1 and type 2 IGF receptors were demonstrated by radioligand cross linking and equilibrium binding analysis. It was demonstrated that both IGF-I and IGF-II acted as mitogens for isolated equine chondrocytes when present as the sole mitogenic factor in monolayer culture. It was further shown that whereas insulin was able to promote the survival and expansion of cell populations of chondrocytes in culture there was significantly reduced mitogenic stimulation compared to the IGFs. These results suggest that the role of insulin in growth cartilage may be to promote chondrocyte survival, or to suppress differentiation/apoptosis. This supports the hypothesis that relative hyperinsulinaemia may be a contributory factor to equine dyschondroplasia (osteochondrosis). Understanding of contributory, and possibly triggering factors such as this may allow the development of modified methods of husbandry which minimise the risk of disease in populations with a known predisposition.

Osteochondrosis lesions of the lateral trochlear ridge of the distal femur in four ponies.

Lesions of the lateral trochlear ridge (LTR) of the distal femur were investigated in four pony or pony cross horses. The animals were all geldings and were six to 15 months of age. Lesions were bilateral in three ponies and unilateral in one. Femoropatellar joint effusion and lameness were present in two ponies; clinical signs were absent in the others. The proximal LTR was affected in all four animals. The radiographic appearance of the lesions was a subchondral defect containing mineralised bodies. Arthroscopic and postmortem examination findings included an osteochondral flap, a fissured or irregular articular surface and a smooth surface overlying focally thickened cartilage that extended into subchondral bone. Thickened articular cartilage was a histological feature of all the lesions. Among the other histological features, the most common were chondronecrosis, chondrocyte clusters, phenotypically abnormal chondrocytes, horizontal fissures at the osteochondral junction and retained blood vessels. The signalment of the four ponies, their clinical signs and the pathological features of their lesions were consistent with osteochondrosis of the LTR in horses. The use of multiple criteria was considered to be important in making a specific diagnosis.

Promotion of the intrinsic damage-repair response in articular cartilage by fibroblastic growth factor-2.

OBJECTIVE: To identify the effect of fibroblastic growth factor-2 (FGF-2) on the intrinsic damage-repair response in articular cartilage in vitro. METHODS: Articular equine cartilage explants, without subchondral bone, had a single impact load of 500 g applied from a height of 2.5 cm. Explants were then cultured in 0, 12, 25, 50 or 100 ng/ml FGF-2 for up to 28 days. Unimpacted discs served as controls for each time-point. Histological and immunohistochemical techniques were used to quantify and characterise the response of putative chondrocyte progenitor cells (CPC) to damage and FGF-2 treatment. RESULTS: FGF-2 significantly accelerated the appearance and increased the numbers of de novo repair cells identified histologically at the cartilage surface. The response was affected by the dose of FGF-2. The repair cells were shown to be chondrocytes by their expression of collagen types II, IX/XI, but not of type I collagen. In addition, these cells, and those underlying the articular surface, were shown to be immunopositive for Notch-1 and PCNA, markers for proliferating cartilage progenitor cells. CONCLUSIONS: The results of this study indicate that, following single impact load, CPC can be stimulated in mature articular cartilage in vitro. These CPC and the cells arising from them appear to represent the cartilage's response to damage. The timing of the appearance of CPC and their overall numbers can be significantly increased by FGF-2, providing further evidence for an important role for FGF-2 in modulating cartilage repair. These results indicate that further study into the mechanisms of repair in mature cartilage using this in vitro model are vital in understanding the repair capacity of mature cartilage.

Localisation of alkaline phosphatase in equine growth cartilage.

The aim of this study was to localise alkaline phosphatase (ALP) activity in equine growth cartilage both histochemically and ultrastructurally. For histochemical studies, full thickness growth cartilage samples were obtained from 6 anatomical sites from 16 horses and ponies ranging in age from 90 d postconception to 12 years of age. For ultrastructural studies, samples were obtained from the lateral trochlear ridge of the distal femur of 3 animals ranging in age from 157 d postconception to 12 months of age. Alkaline phosphatase in histological sections was localised using a substituted naphthol reaction which revealed enzyme activity around the cell surfaces of hypertrophic chondrocytes. ALP activity was quantified by determining the labelling index of ALP positive chondrocytes. The labelling index depended on (1) the age of the animal (activity being highest in animals < 6 months old), and (2) the anatomical site (activity being lowest in the proximal phalanges and highest in the growth plate, reflecting the degree of long bone growth at each site). In order to demonstrate ALP activity ultrastructurally, two capturing agents were used: cerium and lead. This enzyme was visualised by the precipitation of electron-dense salts at the site of activity. ALP activity was localised at low levels on the surface membranes of chondrocytes in the midproliferative zone. The enzyme activity increased throughout the proliferative zone and was most intense on chondrocytes of the hypertrophic zone. ALP activity was seen to be concentrated on the matrix vesicles, the putative sites of mineralisation. This work demonstrates, for the first time in the horse, that ALP is a useful marker of incipient bone formation.