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Osteoarthritis (OA) is a degenerative and heterogeneous disease that affects all types of joint structures. Current clinical treatments are only symptomatic and do not manage the degenerative process in animals or humans. One of the new orthobiological treatment strategies being developed to treat OA is the use of drug delivery systems (DDS) to release bioactive molecules over a long period of time directly into the joint to limit inflammation, control pain, and reduce cartilage degradation. Two vasoactive peptides, endothelin-1 and bradykinin, play important roles in OA pathogenesis. In this study, we investigated the effects of two functionalized nanogels as DDS. We assessed the effect of chitosan functionalized with a type A endothelin receptor antagonist (BQ-123-CHI) and/or hyaluronic acid functionalized with a type B1 bradykinin receptor antagonist (R-954-HA). The biocompatibility of these nanogels, alone or in combination, was first validated on equine articular chondrocytes cultured under different oxic conditions. Further, in an OA equine organoid model via induction with interleukin-1 beta (IL-1β), a combination of BQ-123-CHI and R-954-HA (BR5) triggered the greatest decrease in inflammatory and catabolic markers. In basal and OA conditions, BQ-123-CHI alone or in equimolar combinations with R-954-HA had weak pro-anabolic effects on collagens synthesis. These new nanogels, as part of a composite DDS, show promising attributes for treating OA.

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.

National audience; Osteoarthritis (OA), a common degenerative disease characterized in humans by debil- itating chronic pain and inflammation, is expected to become the fourth most important cause of disability worldwide by 2020. Horse has been considered and proved to be as one of the most relevant patient animal model to study OA based on the cellularity, structural, biochemical and mechanical properties of cartilage, but also on its ageing evolution. OA is responsible for 60% of lameness in horses and may lead to abrupt ends of racehorse’s career. No curative treatment is available and symptomatic treatments commonly used suffer from a limited efficiency and frequently adverse effects.The aim of this project is to develop a nanomedicine therapeutic approach based on func- tionalized hydrogels that could improve mechanical functions, protect the cartilage against inflammation and degradation processes, and reduce pain.Several formulations and concentrations of hydrogels were studied in vitro to evaluate their biocompatibility and phenotypic effects on equine articular chondrocytes (eAC). Dedifferen- tiated eAC were cultured in vitro as organoids in type I/III collagens sponges under hypoxia for 7 days, and interleukin-1bˆeta (IL-1β) was added into the media to mimic an inflammatory environment.First, the hydrogels formulations did not have cytotoxic effects and did not alter the prolif- eration and the viability of chondrocytes. Then, the expression of hyaline cartilage markers was downregulated by IL-1β which concomitantly induced a strong induction of inflamma- tory and catabolic markers. Interestingly, the combination of two formulations of hydrogels decreased the expression of mRNA encoding several degradation markers, such as some metalloproteinases (Mmp1, Mmp3 and Mmp13), agrecanase (Adamts5) and inflammatory markers (Ptgs2 and Inos). Finally, this combination reduced the expression of type I colla- gen protein in our cartilaginous organoids.The biocompatibility and effectiveness of these hydrogels’ formulations on inflammation and degradation allow considering in vivo study in horses first, for tolerance assessment and thereafter in horses that spontaneously suffered from osteoarthritis. These results showed that functionalized hydrogels could be an innovative therapeutic strategy in the treatment of OA.

International audience; 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.

International audience; Tibial nerve anaesthesia is often utilised in the diagnostic evaluation of hindlimb lameness, but effective analgesia is sometimes difficult to achieve using a blind injection. The objectives of this paper are to describe the ultrasonographic anatomy of the caudomedial aspect of the superficial caudal crural compartment containing the tibial nerve and to describe a technique to perform an ultrasonographic guided block of this nerve. The tibial nerve is imaged by the use of a microconvex probe on a transverse section of the caudomedial part of the crus made approximately 8–10 cm proximal to the point of the hock. The needle is first inserted caudally to the probe, through the superficial caudal crural fascia, directed to the caudal aspect of the nerve where half of the volume of anaesthetic solution is injected (5–8 mL). A second injection is made similarly, cranial to the probe. Ultrasonographic guided injection of the tibial nerve increases accuracy of the nerve block by avoiding erroneous intravascular injections or injections under the deep caudal crural fascia that reduces diffusion of the anaesthetic solution. As deposit of anaesthetic solution can be done closer to the nerve, specificity of the block increases with quicker anaesthesia of the distal part of the limb. In addition, a smaller volume of anaesthetic solution (10–12 mL) can be used, thereby reducing the risk of proximal diffusion.

OBJECTIVE To report history, findings from clinical examinations and diagnostic imaging, treatment, and outcomes associated with distal interphalangeal primary degenerative joint disease (DIP-PDJD) and to evaluate diagnostic usefulness and limitations of standing low-field MRI, relative to radiography and ultrasonography, for the diagnosis of DIP-PDJD in horses. DESIGN Retrospective case series with nested evaluation study. ANIMALS 12 client-owned horses. PROCEDURES Medical records were reviewed, and data were collected regarding signalment, history, results of physical and diagnostic imaging examinations, treatments, and outcomes of horses that underwent radiography, ultrasonography, and standing MRI for DIP-PDJD. Findings from radiography, ultrasonography, and MRI were recorded, and abnormal findings were graded. The diagnostic usefulness of MRI, relative to radiography and ultrasonography, in the diagnosis of DIP-PDJD in horses was evaluated. RESULTS A diagnosis of DIP-PDJD was established in 12 of 176 (6.8%) horses that underwent MRI examination of a foot for locomotor disorders. Radiography and ultrasonography enabled confirmation of DIP-PDJD in 3 of the 12 horses, and standing MRI enabled confirmation of DIP-PDJD in the remaining 9. Mean grade for thinning joint space and cartilage were significantly greater when determined with MRI, compared with radiography. Mean grade for osteophytes and periarticular bone remodeling were significantly greater when determined with radiography and ultrasonography, compared with MRI. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that DIP-PDJD can be challenging to detect with routine imaging, especially when synovial effusion and periarticular new bone formation are absent. Standing low-field MRI represents a potentially useful diagnostic tool to diagnose advanced DIP-PDJD in horses.

International audience; 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.

International audience; 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.

Summary Diagnosis of proximal suspensory ligament (PSL, third interosseous muscle) injuries in equine hindlimbs can be challenging because of the complex anatomy of the proximal metatarsal region. Ultrasonographic examination of the PSL using the angle contrast ultrasound technique in the flexed limb facilitates assessment of PSL desmopathies and enthesopathies, with subjectively improved sensitivity compared with the standard approach. The main limitation of ultrasonography is identification of deep osseous injuries, but combined with other imaging procedures, ultrasonography provides valuable diagnostic information of the injured structures. Ultrasonography also helps to specify the stage of a lesion. The repair process can be monitored, helping the clinician to adapt the rehabilitation programme and establish a prognosis for the horse to return to its previous level.

International audience; 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-β1) 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-β1 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.

ReGeneraTing Agents (RGTA) are nanopolysaccharides engineered to mimic heparan sulfates and have been shown to stimulate tissue repair and healing (skin, cornea, gingival, and muscle) in several animal models of injury and in human medicine. A preliminary study, monocentric and uncontrolled, was conducted to evaluate the efficacy of a dedicated RGTA (named Equitend) in improving the healing quality of the injured tendon after a single and ultrasound-guided intralesional injection. Improvement of ultrasonographic appearance of tendinous or ligamentous lesion was documented at least over 4 months in Equitend-treated horses and was further supported by another study on superficial digital flexor tendonitis, in 51 horses (16 French Standardbred trotters (ST), 13 Thoroughbreds (TB), and 22 eventers). Long-term follow-up of these horses showed high rates of return to racing, with 95% of eventers, 87.5% of French ST, and 77% of TB. The mean duration before the first race or competition was 6.6 and 7.4 months for ST and eventers, respectively. The group of 16 treated ST was then compared with a matched control group, showing that the Equitend-treated group was able to return to a higher level of earning after lesion (74% vs. 15% in the control group). Furthermore, the Equitend-treated group was back in racing 2.4 months earlier than the control group.

Summary The collateral ligaments (CL) of the distal interphalangeal joint (DIPJ) are important structures for DIPJ stability. Collateral ligament injuries must be considered in the differential diagnosis of foot pain. An accurate examination of the CL-DIPJ can be performed with an adequate ultrasonographic technique and equipment. This paper describes the technique and normal ultrasonographic images as reference data for the diagnosis and documentation of CL-DIPJ desmopathies and enthesopathies.

Summary Ultrasonography is an easy and rewarding technique for the assessment of collateral ligaments of the distal interphalangeal joint (CL-DIPJ) desmopathies and enthesopathies. Comparison between ligaments of both feet and oblique incidence improve sensitivity and specificity of the ultrasonographic diagnosis. The main limitation of the procedure is imaging the distal part of the CL-DIPJ and corresponding fossa of the distal phalanx. Alone, or in combination with other imaging procedures, ultrasonography is valuable in the diagnosis of the injured structures, which may help to determine the corrective shoeing and the rehabilitation programme of the horse.

OBJECTIVE To describe the clinical features, diagnostic procedures, management, and outcome of horses with peripheral neuropathy of a forelimb. DESIGN Retrospective case series. ANIMALS 27 horses. PROCEDURES Records from 2000 to 2013 were reviewed to identify horses with peripheral neuropathy of a forelimb. Horses were grouped as having predominant lesions of a suprascapular nerve, axillary nerve, or radial nerve (alone or in association with other brachial plexus nerves) on the basis of physical examination and diagnostic imaging findings. Treatments were primarily conservative. Signalment, history, lameness characteristics, diagnostic imaging features, case management, and outcomes were evaluated. RESULTS Predominant lesions of a suprascapular nerve, axillary nerve, and radial nerve were identified in 11, 2, and 14 horses, respectively. Eight horses with predominant suprascapular nerve injury and 9 with injury to a radial nerve alone or in association with other nerves returned to their previous activity level or intended use after mean recovery periods of 9.3 and 13.3 months, respectively; 2 horses with a predominant axillary nerve injury had this outcome after a mean 3.5-month recovery period. Ultrasonography was useful for evaluation of muscle atrophy and other injuries during the initial examination (in 27 horses) and the rehabilitation period (in 7 horses). CONCLUSIONS AND CLINICAL RELEVANCE Most horses with peripheral neuropathy of a forelimb returned to athletic soundness following an adequate period of rest. Horses with lesions of a radial nerve alone or in association with other nerves typically required longer recovery times than did those with predominant injuries of a suprascapular nerve.

National audience; PURPOSE: Articular cartilage is of great importance for physiological mobility. The structure and the function of this tissue which is characterized by a poor self-repair capacity are frequently disrupted or damaged upon physical trauma or in degenerative osteoarthritis (OA). Given that musculoskeletal disorders are the leading cause of poor performance or early retirement of sports and race horses treating horses for these disorders is relevant. The proposed therapeutic approach which was first developed for human has the potential of being considered as a pre-clinical step for human medicine because the horse is recognized as an excellent model for the study of articular cartilage disorders in humans. This study aims to improve the Autologous Chondrocytes Implantation (ACI) technique by using Mesenchymal Stem cells (MSCs) from bone marrow (easy to collect in horse) as the cell source. Thus MSCs were first characterized before being cultured with chondrogenic conditions in order to find the combination that best enhances and stabilizes the characteristics of the chondrocyte phenotype in order to perform clinical trials in horse. METHODS: MSCs from equine bone marrow were isolated and expanded in monolayer cultures until 4 passages. These cells were characterized by analyzing their proliferative potential their pluripotency (with microenvironmental stimuli) and their capacity to express MSCs specific phenotypic markers defining the MSCs (flow cytometry). In parallel MSCs differentiation in chondrocytes was accomplished via a combinatory approach based on the association of 3D-culture in type I collagen sponges low oxygen tension with chondrogenic factors (BMP-2 TGF-β1) and RNA interference (siRNA to down-regulate type I collagen and HtrA1 protein expression). Finally an extensive analysis at gene and protein levels corresponding to differentiated dedifferentiated and hypertrophic chondrocyte phenotypes was performed. RESULTS: Our results show a very high proliferation potential of MSCs isolated from equine bone marrow. Furthermore the isolated cells satisfy the various criteria of stem cells definition (pluripotency expression of surface markers). In addition siRNAs targeting equine Col1a1 and Htra1 have been functionally validated. Finally we show that the BMP-2 and TGF-β1 combination strongly induces the differentiation of MSCs in chondrocytes. Thus the combined use of specific culture conditions defined within the laboratory with specific growth factors and siRNAs association leads to the in vitro synthesis of a hyaline type neo-cartilage by chondrocytes which present an optimal phenotypic index comparable to the one established with induction of specific differentiation of human MSCs in chondrocytes and close to the one of differentiated/healthy chondrocytes. CONCLUSIONS: These data represent a first step in the development of equine clinical trials which are planed - to better understand the reaction of cartilage tissue after a few weeks of intra-articular implantation and - to make the proof of concept in a large animal model. This approach will allow us to explore the criteria necessary to begin to consider the development of cell therapy for cartilage repair in humans.

Objective—To determine history; clinical, radiographic, ultrasonographic, and scintigraphic features; management; and outcome associated with third trochanter fractures in horses. Design—Retrospective case series. Animals—8 horses. Procedures—Records from 2000 to 2012 were reviewed, and signalment, case history, severity and duration of lameness, results of physical and lameness examinations, imaging findings, management, and outcome were evaluated. Results—All horses had a history of acute onset of severe lameness. Four of the 8 horses had localizing physical signs of fracture. No specific gait characteristics were identified. Ultrasonographically, there was a single bony fragment displaced cranially in 7 of 8 horses and multiple bony fragments in 1. Concurrent gluteus superficialis muscle enthesopathy was identified in 7 horses. A standing craniolateral-caudomedial 25° oblique radiographic view was obtained in 3 horses to document the lesion and revealed in all 3 horses a simple complete longitudinal fracture between the midlevel and the base of the third trochanter. Nuclear scintigraphy was used to identify the affected area of the limb for further examination in 2 horses. Follow-up revealed that fractures healed with a fibrous union, with persistence of cranial displacement of the fragment. Lameness resolved after nonsurgical management for all horses. Conclusions and Clinical Relevance—Fracture of the third trochanter should be considered as a cause of hind limb lameness in horses when the proximal portion of the limb is affected. Diagnosis can easily be made with ultrasonography, but nuclear scintigraphy may help in identifying the lesion. Prognosis for return to athletic activity is good after an appropriate period of rest and restricted exercise.

Objective—To characterize the clinical features, diagnostic procedures, treatment, and outcome for horses with an exostosis on the palmar or plantar cortex of the third metacarpal bone (MC3) or third metatarsal bone (MT3). Design—Retrospective case series. Animals—16 horses. Procedures—Records from 2001 through 2010 were evaluated to identify horses with radiographic and ultrasonographic evidence of an exostosis on an MC3 or MT3. Signalment, history, lameness examination results, diagnostic imaging results, surgical and histopathologic findings, treatment, and outcome were evaluated. Results—9 horses (group A) had unilateral lameness of the exostosis-affected limb that was alleviated with local or perineural analgesia. Seven horses (group B) had inconsistent lameness of the affected limb. The exostosis was located between the middle and distal third of the MC3 or MT3 in all horses. Desmopathy or peritendinous fibrosis (or both) of the suspensory ligament at the exostosis site was identified in 6 horses. All horses in group A returned to full function after conservative or surgical management. Lameness did not recur in any of the horses in group B despite no treatment or only conservative management. Conclusions and Clinical Relevance—Exostosis of the palmar cortex of an MC3 or plantar cortex of an MT3 should be considered as a cause of lameness in horses. The diagnosis can be made by the use of radiography and ultrasonography combined with specific diagnostic analgesia. Prognosis for return to athletic activity can be good but should be modified contingent on concurrent desmopathy of the suspensory ligament.