Sandrine Jacquet

Association of growth, feeding practices and exercise conditions with the severity of the osteoarticular status of limbs in French foals.

The aim of this study was to identify the risk factors for the severity of Juvenile OsteoChondral Conditions (JOCC) in limbs of French foals. Twenty-one farms in Normandy, France, were sampled and enrolled in a cohort study including 378 foals from three breeds, followed from the 8th month of pregnancy of the mares until the foals were approximately 6months old. Data on growth, feeding practices and exercise conditions were regularly collected. The carpus, the front and hind digits, the hock and the stifle of the foals were radiographed at the end of follow-up. JOCC severity in each foal was described using a global appraisal of its osteoarticular status (OAS) depending on the number and the severity of radiographic findings. Of the 378 foals, 53% had a good OAS, 34% had an intermediate OAS and 13% had a poor OAS. The breed (Selle Français and French Trotter Standardbred vs. Thoroughbred), a high girth perimeter at early age and an irregular exercise were significantly associated with a poor OAS. This study contributes to the understanding of the development of JOCC. An increased growth and reduced or irregular physical activity during the first weeks of life would be responsible for more severe lesions. Growth and exercise conditions should be carefully monitored to reduce the prevalence of severe JOCC in foals.

Radiographic findings of juvenile osteochondral conditions detected in 392 foals using a field radiographic protocol.

Radiographic examination in young horses is commonly used to detect juvenile osteochondral conditions (JOCC). The aim of this study is to present the radiographic findings (RF) detected at the age of 6months in the joints of 392 foals from three breeds: French Trotter Standardbreds (FT), Selle-Français Warmbloods (SF) and Thoroughbreds (TB). Radiographic examination included lateromedial views of the fore and hind digits, tarsi and stifles, and dorsopalmar projections of both carpi. In all areas RF indicative of JOCC were classified according to a four-grade severity scoring system. Distribution and severity of the RF varied according to breed with the SF most affected. The most frequently affected site was the hind fetlock (28.3% of foals). The second most affected site was the fore fetlock in TB and SF, while the carpus and proximal tarsus were the second and third affected sites in FT. The radiographic protocol and grading system used can serve as a basis for field research and further studies on the evolution of RF, risk factors for JOCC or the correlation between RF and the subsequent athletic career of horses.

Evolution of radiological findings detected in the limbs of 321 young horses between the ages of 6 and 18 months

The objective of this study was to evaluate the spontaneous evolution of radiological findings (RF) indicative of juvenile osteochondral conditions (JOCC) in a field study. A radiographic survey was performed at the age of 6 and 18months in 321 young horses from three breeds (French Trotter Standardbred, Selle-Français Warmblood and Thoroughbred). Each RF identified at 6months was re-evaluated at 18months, and classified as (1) disappeared, (2) improved, (3) stable, (4) deteriorated or (5) new when it was not identified at 6months. Only 32.3% of all RF identified at the age of 6months were stable, while at 18months 46.6% had disappeared and 38.7% were new radiological abnormalities. Evolution of RF varied according to the joint involved and the type of lesion. In the stifle, 85.7% of the RF of osteochondral fragmentation (OCF) of the lateral ridge of the femoral trochlea regressed (disappeared or improved). In the hock, 53.1% of the RF of OCD of the intermediate ridge of the tibial cochlea were stable. In the fetlock, dorsal and plantar OCF were frequently observed as new radiological abnormalities at 18months (51% of the dorsal RF and 45.8% of the plantar). As many lesions develop or change after 6months, a definitive radiographic assessment of the JOCC status is more reliable at 18months. Knowing the spontaneous healing capacity of some lesions is useful in helping to decide the appropriate management.

Study design for the investigation of likely aetiological factors of juvenile osteochondral conditions (JOCC) in foals and yearlings

The possible aetiology of osteochondrosis and, to a lesser extent, other developmental orthopaedic diseases or juvenile osteochondral conditions (JOCC), has been intensively investigated. However, most studies have focused on single factors of this multi-factorial disorder, or have been conducted under experimental conditions. This paper aims to present and discuss the scientific background of the BOSAC (Breeding, Osteochondral Status and Athletic Career) research program, a multi-factorial investigation on JOCC risk factors in field conditions. The epidemiology of JOCC in horses born in Normandy between 2002 and 2004 was studied. Horses were subjected to repeated body measurements, blood sampling and locomotion evaluation from birth until yearling sales. A radiographic examination, including 10 views of the limbs, was performed on each subject at approximately 6 and 17months of age. Information on nutrition and management programmes was collected by specialists from visits to the farms and the use of questionnaires. A total of 393 foals of three French breeds were monitored from birth to weaning, and 321 of these remained available for further follow-up, making the study unique as regards both the number of subjects and the variety of information collected. The study was designed to describe the evolution of JOCC, and determine possible early markers, risk factors and prognostic factors with respect to performance. Relevant data, suitable for epidemiological analyses, were collected under various field conditions that reflect current management practices in Normandy, Frances main horse breeding region.

A reliable severity scoring system for radiographic findings in the limbs of young horses.

The validity of methods used to score the severity of radiographic findings (RFs) in horses is uncertain since only one or two joints are usually studied, classification criteria are heterogeneous and the internal validity is not assessed. The aim of this study was to assess the internal validity of a severity scoring system (SSS) of RFs by repeated scoring of a sample of radiographs. This SSS of RFs is based on four criteria that can be applied to every type of RF observed in limb joints. It consists of five weighted severity indexes (0, 1, 2, 4, 8) and was used to assess RFs found on the limbs of 392 young horses. The internal validity of the SSS was assessed using Kappa coefficients calculated on a subsample of 137 horses whose radiographs were interpreted twice. The final RF severity indices from these radiographs were obtained after three experienced veterinarians had reached a consensus, similar to the procedure used at foal and yearling sales. The majority of RFs from the 392 horses were scored either 1 or 2, while scores of 8 were only observed in the stifle and tarsus. Among the subsample, the overall agreement on the presence or absence of RFs was good (κ=0.63; 95% Confidence Interval [CI], 0.56-0.69), and was excellent for the severity of RFs (weighted κ=0.82; 95% CI, 0.75-0.87). Most disagreements involved RFs that scored 1. The fore fetlock and the carpus experienced the lowest agreement. The SSS was a stable and reliable procedure applicable to any RF on any limb joint of the horse. It will be of potential interest in clinical practice and in the pre-purchase evaluation of young horses and could also be used in additional studies on the evolution or risk factors of RFs.

Developmental orthopaedic disease in limbs of foals: between-breed variations in the prevalence, location and severity at weaning.

Developmental orthopaedic disease (DOD) affects all breeds and is a common cause of pain and lameness for horses in sports. A thorough knowledge of between-breed variations for the prevalence of DOD, for its distribution among the various joints and for its severity at earlier stages in the disease process is needed in order to improve the relevance and the cost-effectiveness of DOD screening protocols. However, no prevalence study for DOD simultaneously performed on several breeds with similar farming systems and based on radiographic findings (RF) on quite a large number of joints and views, has been reported earlier. The objective of this study was to describe variations in the prevalence, location and severity of DOD in foals at weaning among Warmbloods (Wb), Standardbreds (St) and Thoroughbreds (Tb) with similar farming systems. DOD assessment was based on RF on the limb joints. A total of 392 foals from 21 volunteer stud farmers were included. To determine the statuses of foals regarding DOD, they were X-rayed on the front- and hind-limb digit, carpus, hock and stifle joints. X-ray data were analysed by three experienced equine veterinarians who gave a common assessment about the entity and the severity of RF. Between-breed variations were analysed in two steps: the first implemented for each anatomical site; the second considered only foals affected by DOD to explore RF association patterns on the affected sites, at foal level. The three breeds were represented by 25.0% of Wb, 41.1% of St and 33.9% of Tb. DOD was present in 66.3% of the foals (95% confidence interval (CI) = 61.6% to 71.0%). Prevalence of foals affected by DOD and distribution of the RF severity score on the anatomical sites differed depending on the breed: Wb foals seemed to be the most affected by DOD. Cluster analyses showed no clear association among sites. However, Wb and Tb foals were preferentially classified together because they were affected on the same sites, whereas St foals were distributed in other classes. The most severely affected sites were the proximal part of the hock and the femoro-patellar joint for Wb and St foals, and the fore fetlock and the distal part of the hock for Tb foals. This is the first epidemiological study reporting between-breed variations in DOD distribution and severity, for the limb joints of foals. These results contribute to broaden the knowledge on DOD and are of great interest to improve detection of DOD within a particular breed.

Influence of juvenile osteochondral conditions on racing performance in Thoroughbreds born in Normandy

The relationship between osteoarticular status and future athletic capacity is commonly accepted in equine practice, but there is little to support this belief in Thoroughbreds. The objective of this study was to assess the prevalence of juvenile osteochondral conditions (JOCC) in Thoroughbred yearlings and to investigate the significance of these with regard to subsequent racing performance. The radiographic files from 328 Thoroughbred yearlings born in Normandy were assessed in a consistent manner and entered into a database together with racing records. Logistic regression models were used to quantify the association between each radiographic parameter and racing performance (raced/not raced, placed/not placed, performer/not performer) at 2, 3, 4 and 5years of age. The front fetlock (30.2% of horses), the dorsal aspect of the hind fetlock (18%), the carpus (15.9%) and the distal part of the hock (15.5%) were the most commonly affected joints. Most horses (87.5%) raced either in turf flat races or in hurdle races. Starting a race at 2years old was more frequent for yearlings without radiographic findings (RF) on the carpus or with less than one RF of moderate severity. The proportions of horses placed at 3years old decreased with increasing number or severity of RF. In racing horses, there was no association between the presence of RF and earnings. The radiographic score, calculated as the sum of all the severity indices found on the radiographic file of the horse appeared well correlated with performance. Using this synthetic index might help veterinarians to evaluate radiographs of Thoroughbred yearlings for potential buyers.

Chondrogenic Differentiation of Defined Equine Mesenchymal Stem Cells Derived from Umbilical Cord Blood for Use in Cartilage Repair Therapy

Cartilage engineering is a new strategy for the treatment of cartilage damage due to osteoarthritis or trauma in humans. Racehorses are exposed to the same type of cartilage damage and the anatomical, cellular, and biochemical properties of their cartilage are comparable to those of human cartilage, making the horse an excellent model for the development of cartilage engineering. Human mesenchymal stem cells (MSCs) differentiated into chondrocytes with chondrogenic factors in a biomaterial appears to be a promising therapeutic approach for direct implantation and cartilage repair. Here, we characterized equine umbilical cord blood-derived MSCs (eUCB-MSCs) and evaluated their potential for chondrocyte differentiation for use in cartilage repair therapy. Our results show that isolated eUCB-MSCs had high proliferative capacity and differentiated easily into osteoblasts and chondrocytes, but not into adipocytes. A three-dimensional (3D) culture approach with the chondrogenic factors BMP-2 and TGF-β1 potentiated chondrogenic differentiation with a significant increase in cartilage-specific markers at the mRNA level (Col2a1, Acan, Snorc) and the protein level (type II and IIB collagen) without an increase in hypertrophic chondrocyte markers (Col10a1 and Mmp13) in normoxia and in hypoxia. However, these chondrogenic factors caused an increase in type I collagen, which can be reduced using small interfering RNA targeting Col1a2. This study provides robust data on MSCs characterization and demonstrates that eUCB-MSCs have a great potential for cartilage tissue engineering.

Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index

Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-β3 alone showed promising result but the previously tested association of BMP-2 and TGF-β1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-β1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine.

16 Matrix Therapy With Regenerating Agent Improves Horse Tendonitis’ Recovery

Introduction Matrix therapy was introduced as a new branch in regenerative medicine led by the concept that reorganisation of the extracellular matrix (ECM) and restoration of the cellular micro-environment can trigger a regeneration process. Robust demonstration were provided in many publications [1] describing the efficacy of ReGeneraTing Agents (RGTA®) in improving speed and quality of healing of various injured tissues (skin, cornea, muscle….) both in preclinical models and in clinic without any report of toxicity. RGTA® are nano-biodegradable polymers engineered to mimic and replace heparan sulfates both as a storage and protector site for growth factors as well as a scaffold element in the reconstruction and organisation of the ECM of injured tissues. Horse tendonitis represents a big challenge for trainers and veterinarians and currently, no treatment has clearly proven its effectiveness in improving tendon healing. We now present several studies demonstrating that one dedicated RGTA® named EQUITEND® presents an efficient alternative for the treatment of tendonitis and recovery of horses’ performances. Methods Three studies were conducted by the CIRALE’s team. EQUITEND® has been tested in tendon healing after a single intra-lesional injection and under ultrasound guidance. The first one is a retrospective study included 16 French trotters with Superficial Digital Flexor Tendinopathy (SDFT) treated once with EQUITEND® and compared with a matching group. The second one is an experimental efficacy study on 12 experimental French Trotters after surgical induced SDFT [2]. The last one is a controlled randomised clinical trial conducted on 22 French Trotters (FT) race horses with spontaneous cases of tendonitis. Results The retrospective study demonstrates that horses treated with EQUITEND® went back in racing 2.4 months earlier and earned more in races than control group (74%, versus 15% in the control group). In the experimental efficacy study, improvement was suspected during the follow up of healing by echography but confirmed and quantified by measuring and comparing the mechanical resistance to rupture on isolated tendons at 3 months after injury. Taking into account of the horses’ age in the statistical analysis, (as it is a determining factor in the SDFT incidence), results revealed a significant positive effect of the EQUITEND® treatment on maximal force (p = 0.0038) (Figure 1) and on the stress at maximal force (p = 0.058). Abstract 16 Figure 1 Maximal Force (N) evaluated on isolated tendons of horses’ tendons correlated to age of horses Finally, clinical trial has shown that EQUITEND® treated horses went back to racing in a shorter time, their performance as earning per race (compared to earnings per race before injury) was higher and they tend to have fewer tendonitis recurrence than placebo horses. Discussion All these studies demonstrated a good tolerance of EQUITEND®, a quick improvement of healing assessed by the ultrasound examination and a better recovery of horse’s performances in EQUITEND® treated group. The experimental study detailed the improvement of quality of healing evaluated by mechanical resistance properties of tendon to rupture, significantly higher for EQUITEND® horses. We conclude that EQUITEND® offers a real advantage upon existing therapies for tendonitis in racing horses. References 1 Johan van Neck, Bastiaan Tuk, Denis Barritault and Miao Tong Heparan Sulfate Proteoglycan Mimetics Promote Tissue Regeneration: An Overview chapter 4 in Tissue Regeneration – From Basic Biology to Clinical Application ISBN 978–953-51–0387-5, edited by Jamie Davies 2 Schramme et al. Vet Comp Orthop Traumatol. 2010;23(4):231–9