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-ß3 alone showed promising result but the previously tested association of BMP-2 and TGF-ß1 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-ß1, 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.

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-ß1 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.

Water depth and speed may have an opposite effect on the trunk vertical displacement in horses trotting on a water treadmill.

OBJECTIVE: To measure the trunk vertical displacement (VD) in horses trotting on a water treadmill (WT) at different water depths (WDs) and speeds. ANIMALS: 6 sound Standardbred horses (median age 12 years [IQR:10.5-12]). METHODS: The horses were trotted on a WT at 2 speeds (3.5 m/s and 5 m/s) and during 4 conditions: dry treadmill (DT), WD at mid-cannon (WD-CAN), mid-radius (WD-RAD), and shoulder (WD-SHOUL). The dorsoventral movement was obtained with accelerometers placed over the withers, thoracolumbar junction (T18), tuber sacrale (TS), and sacrum (S5). The VD was defined with the median value of the upward (Up) and downward (Down) amplitudes of the vertical excursion during each stride. The difference of VD at each sensor location was compared between the DT and the 3 WDs, and between the 2 trotting speeds for the same condition. RESULTS: The VD amplitudes were significantly increased at any sensor location when trotting in water at WD-CAN and WD-RAD compared to DT (P < .05 for all), with the highest increase at WD-RAD and T18. When the speed increased from 3.5 to 5 m/s, the VD amplitudes were significantly decreased at T18 at each water level (P = .03), and at WD-RAD only for the withers and TS (P = .03). CLINICAL RELEVANCE: Both water depth and speed affect the trunk VD in horses at trot on a WT with an opposite effect. The VD increases when increasing the WD up to mid-radius, while the VD decreases when increasing the trotting speed, with the main effects observed at the thoracolumbar junction.

Asymmetry Thresholds Reflecting the Visual Assessment of Forelimb Lameness on Circles on a Hard Surface.

The assessment of lameness in horses can be aided by objective gait analysis tools. Despite their key role of evaluating a horse at trot on a circle, asymmetry thresholds have not been determined for differentiating between sound and lame gait during this exercise. These thresholds are essential to distinguish physiological asymmetry linked to the circle from pathological asymmetry linked to lameness. This study aims to determine the Asymmetry Indices (AIs) with the highest power to discriminate between a group of sound horses and a group of horses with consistent unilateral lameness across both circle directions, as categorized by visual lameness assessment conducted by specialist veterinarians. Then, thresholds were defined for the best performing AIs, based on the optimal sensitivity and specificity. AIs were calculated as the relative comparison between left and right minima, maxima, time between maxima and upward amplitudes of the vertical displacement of the head and the withers. Except the AI of maxima difference, the head AI showed the highest sensitivity (≥69%) and the highest specificity (≥81%) for inside forelimb lameness detection and the withers AI showed the highest sensitivity (≥72%) and the highest specificity (≥77%) for outside forelimb lameness detection on circles.

Investigation of Thresholds for Asymmetry Indices to Represent the Visual Assessment of Single Limb Lameness by Expert Veterinarians on Horses Trotting in a Straight Line.

Defining whether a gait asymmetry should be considered as lameness is challenging. Gait analysis systems now provide relatively accurate objective data, but their interpretation remains complex. Thresholds for discriminating between horses that are visually assessed as being lame or sound, as well as thresholds for locating the lame limb with precise sensitivity and specificity are essential for accurate interpretation of asymmetry measures. The goal of this study was to establish the thresholds of asymmetry indices having the best sensitivity and specificity to represent the visual single-limb lameness assessment made by expert veterinarians as part of their routine practice. Horses included in this study were evaluated for locomotor disorders at a clinic and equipped with the EQUISYM® system using inertial measurement unit (IMU) sensors. Visual evaluation by expert clinicians allocated horses into five groups: 49 sound, 62 left forelimb lame, 67 right forelimb lame, 23 left hindlimb lame, and 23 right hindlimb lame horses. 1/10 grade lame horses were excluded. Sensors placed on the head (_H), the withers (_W), and the pelvis (_P) provided vertical displacement. Relative difference of minimal (AI-min) and maximal (AI-max) altitudes, and of upward (AI-up) and downward (AI-down) amplitudes between right and left stance phases were calculated. Receiver operating characteristic (ROC) curves discriminating the sound horses from each lame limb group revealed the threshold of asymmetry indice associated with the best sensitivity and specificity. AI-up_W had the best ability to discriminate forelimb lame horses from sound horses with thresholds (left: -7%; right: +10%) whose sensitivity was greater than 84% and specificity greater than 88%. AI-up_P and AI-max_P discriminated hindlimb lame horses from sound horses with thresholds (left: -7%; right: +18% and left: -10%; right: +6%) whose sensitivity was greater than 78%, and specificity greater than 82%. Identified thresholds will enable the interpretation of quantitative data from lameness quantification systems. This study is mainly limited by the number of included horses and deserves further investigation with additional data, and similar studies on circles are warranted.

Low field magnetic resonance imaging of the equine distal interphalangeal joint: Comparison between weight-bearing and non-weight-bearing conditions.

This descriptive study aimed to compare the magnetic resonance appearance of the distal interphalangeal joint articular cartilage between standing weight-bearing and non-weight-bearing conditions. Ten forefeet of live horses were scanned in a standing low-field magnetic resonance system (0.27 T). After euthanasia for reasons unrelated to the study, the non-weight-bearing isolated feet were scanned in a vertical positioning reproducing limb orientation in live horses. The same acquisition settings as during the weight-bearing examination were used. Thickness and cross-sectional area of the distal interphalangeal articular cartilage and joint space were measured on tridimensional T1-weighted gradient echo high resolution frontal and sagittal images at predetermined landmarks in both conditions and were compared using a linear mixed-effects model. Frontal images were randomized and submitted to 9 blinded readers with 3 different experience levels for identification of weight-bearing versus non-weight-bearing acquisitions based on cartilage appearance. Weight-bearing limbs had significantly thinner distal interphalangeal cartilage (p = 0.0001) than non-weight-bearing limbs. This change was greater in the distal phalanx cartilage than that of the middle phalanx. Blinded readers correctly identified 83% (range 65 to 95%) of the images as weight-bearing or non-weight-bearing acquisitions, with significantly different results observed among the different readers (p < 0.001) and groups (p < 0.001). These results indicate that distal interphalangeal articular cartilage and particularly cartilage of the distal phalanx thins when weight-bearing compared to the non-weight-bearing standing postmortem conditions and suggest that cartilage abnormalities may be more difficult to identify on weight-bearing standing magnetic resonance imaging.

Intra-Articular Injection of 2 Different Dosages of Autologous and Allogeneic Bone Marrow- and Umbilical Cord-Derived Mesenchymal Stem Cells Triggers a Variable Inflammatory Response of the Fetlock Joint on 12 Sound Experimental Horses.

Osteoarthritis is a significant and costly cause of pain for both humans and horses. The horse has been identified as a suitable model for human osteoarthritis. Regenerative therapy with allogeneic mesenchymal stem cells (MSCs) is a promising treatment, but the safety of this procedure continues to be debated. The aim of this study is to evaluate the safety of intra-articular injections of allogeneic MSCs on healthy joints by comparing two different dosages and two different tissue sources, namely, bone marrow and umbilical cord blood, with a placebo treatment on the same individuals. We also assessed the influence of autologous versus allogeneic cells for bone marrow-derived MSC treatment. Twelve clinically sound horses were subjected to injections in their 4 fetlock joints. Each of the three fetlocks was administered a different MSC type, and the remaining fetlock was injected with phosphate-buffered saline as a control. Six horses received 10 million cells per joint, and the 6 other horses received 20 million cells per joint. Clinical and ultrasound monitoring revealed that allogeneic bone marrow-derived MSCs induced significantly more synovial effusion compared to umbilical cord blood-derived MSCs but no significant difference was noted within the synovial fluid parameters. The administration of 10 million cells in horses triggered significantly more inflammatory signs than the administration of 20 million cells. Mesenchymal stem cell injections induced mild to moderate local inflammatory signs compared to the placebo, with individual variability in the sensitivity to the same line of MSCs. Understanding the behavior of stem cells when injected alone is a step towards the safer use of new strategies in stem cell therapy, where the use of either MSC secretome or MSCs combined with biomaterials could enhance their viability and metabolic activity.

Characterization and use of Equine Bone Marrow Mesenchymal Stem Cells in Equine Cartilage Engineering. Study of their Hyaline Cartilage Forming Potential when Cultured under Hypoxia within a Biomaterial in the Presence of BMP-2 and TGF-ß1.

Articular cartilage presents a poor capacity for self-repair. Its structure-function are frequently disrupted or damaged upon physical trauma or osteoarthritis in humans. Similar musculoskeletal disorders also affect horses and are the leading cause of poor performance or early retirement of sport- and racehorses. To develop a therapeutic solution for horses, we tested the autologous chondrocyte implantation technique developed on human bone marrow (BM) mesenchymal stem cells (MSCs) on horse BM-MSCs. This technique involves BM-MSC chondrogenesis using a combinatory approach based on the association of 3D-culture in collagen sponges, under hypoxia in the presence of chondrogenic factors (BMP-2 + TGF-ß1) and siRNA to knockdown collagen I and HtrA1. Horse BM-MSCs were characterized before being cultured in chondrogenic conditions to find the best combination to enhance, stabilize, the chondrocyte phenotype. Our results show a very high proliferation of MSCs and these cells satisfy the criteria defining stem cells (pluripotency-surface markers expression). The combination of BMP-2 + TGF-ß1 strongly induces the chondrogenic differentiation of MSCs and prevents HtrA1 expression. siRNAs targeting Col1a1 and Htra1 were functionally validated. Ultimately, the combined use of specific culture conditions defined here with specific growth factors and a Col1a1 siRNAs (50 nM) association leads to the in vitro synthesis of a hyaline-type neocartilage whose chondrocytes present an optimal phenotypic index similar to that of healthy, differentiated chondrocytes. Our results lead the way to setting up pre-clinical trials in horses to better understand the reaction of neocartilage substitute and to carry out a proof-of-concept of this therapeutic strategy on a large animal model.

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.

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, France's 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.

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.

Axial speed of sound for the monitoring of injured equine tendons: a preliminary study.

Equine superficial digital flexor tendons (SDFT) are often injured, and they represent an excellent model for human sport tendinopathies. While lesions can be precisely diagnosed by clinical evaluation and ultrasonography, a prognosis is often difficult to establish; the knowledge of the injured tendon's mechanical properties would help in anticipating the outcome. The objectives of the present study were to compare the axial speed of sound (SOS) measured in vivo in normal and injured tendons and to investigate their relationship with the tendons' mechanical parameters, in order to assess the potential of quantitative axial ultrasound to monitor the healing of the injured tendons. SOS was measured in vivo in the right fore SDFTs of 12 horses during walk, before and 3.5 months after the surgical induction of a bilateral core lesion. The 12 horses were then euthanized, their SDFTs isolated and tested in tension to measure their elastic modulus and maximal load (and corresponding stress). SOS significantly decreased from 2179.4 ± 31.4 m/s in normal tendons to 2065.8 ± 67.1 m/s 3.5 months after the surgical induction, and the tendons' elastic modulus (0.90 ± 0.17 GPa) was found lower than what has been reported in normal tendons. While SOS was not correlated to tendon maximal load and corresponding stress, the SOS normalized on its value in normal tendons was correlated to the tendons' elastic modulus. These preliminary results confirm the potential of axial SOS in helping the functional assessment of injured tendon.