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.

Osteoarthritis (OA) is a significant cause of pain in both humans and horses with a high socio-economic impact. The horse is recognized as a pertinent model for human OA. In both species, regenerative therapy with allogeneic mesenchymal stem cells (MSCs) appears to be a promising treatment but, to date, no in vivo studies have attempted to compare the effects of different cell sources on the same individuals. The objective of this study is to evaluate the ability of a single blinded intra-articular injection of allogeneic bone-marrow (BM) derived MSCs and umbilical cord blood (UCB) derived MSC to limit the development of OA-associated pathological changes compared to placebo in a post-traumatic OA model applied to all four fetlock joints of eight horses. The effect of the tissue source (BM vs. UCB) is also assessed on the same individuals. Observations were carried out using clinical, radiographic, ultrasonographic, and magnetic resonance imaging methods as well as biochemical analysis of synovial fluid and postmortem microscopic and macroscopic evaluations of the joints until Week 12. A significant reduction in the progression of OA-associated changes measured with imaging techniques, especially radiography, was observed after injection of bone-marrow derived mesenchymal stem cells (BM-MSCs) compared to contralateral placebo injections. These results indicate that allogeneic BM-MSCs are a promising treatment for OA in horses and reinforce the importance of continuing research to validate these results and find innovative strategies that will optimize the therapeutic potential of these cells. However, they should be considered with caution given the low number of units per group.

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.

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.

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.

A randomized controlled trial was performed on racing horses, to evaluate the efficacy of a new class of therapeutic agents in regenerative medicine—ReGeneraTing Agents^® (RGTA^®), to treat tendinopathies. Preliminary uncontrolled studies on tendon healing in racing horses with RGTA^® (OTR4131)—Equitend^® showed encouraging results, justifying performing a randomized, controlled, multicenter study with a two-year racing performance follow up. The objective of this study was to evaluate the effect of Equitend^® versus placebo on acute superficial digital flexor tendonitis in racing French Standardbred Trotters (ST). Twenty-two ST were randomly and blindly assigned to receive with a ratio of 2 to 1, a single Equitend^® (n = 14) or placebo (n = 8) intralesional injection under ultrasonographic guidance. Horses were evaluated over 4 months, by clinical and ultrasonographic evaluations (day 0, months 1, 2, 4), and their racing performances followed up over the 2 years after treatment. During the first month of treatment, a significant decrease in the cross-sectional area (CSA) was found in the Equitend^® group (p = 0.04). After 4 months, the number of Equitend^® treated horses with an improved CSA was significantly higher than the placebo-treated horses (p = 0.03571). The Equitend^® group returned to their pre-injury performance level, racing in, and winning, significantly more races than the placebo group (p = 0.01399 and 0.0421, respectively). Furthermore, recurrence was significantly higher in the placebo group than in the Equitend^® group (71.4% vs 16.6%, p = 0.02442). In conclusion, we measured a significant, short-term, reduction effect on CSA and demonstrated a long-term beneficial effect of intralesional injection of Equitend^® for the treatment of superficial digital flexor tendonitis on racing ST, racing 2. 3 times more often than placebo, with 3.3 times fewer recurrences maintaining pre-injury performance level. This study may open the way for the development of a human treatment of tendonitis.