Evaluation of Allogeneic Bone-Marrow-Derived and Umbilical Cord Blood-Derived Mesenchymal Stem Cells to Prevent the Development of Osteoarthritis in An Equine Model.

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.

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.

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.

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.

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.

Vascular abnormalities of the distal deep digital flexor tendon in 8 draught horses identified on histological examination.

The purpose of this study was to provide a detailed description of the vascular changes in the distal part of deep digital flexor tendon (DDFT). Eight isolated forelimbs were collected from 8 horses with DDF tendinopathy diagnosed post-mortem by ultrasound and gross anatomopathological examination. The samples were fixed in 10% neutral buffered formalin, softened in 4% phenol and dehydrated with ethylic alcohol. Goldner's Trichrome staining method was used. The histopathological examination revealed vascular proliferation associated with structural disorders of blood vessels. Angiogenesis, fibroplasia and consecutive hypertrophy of the vascular wall with or without vascular occlusion were the most common findings. Other histopathological findings were: endothelial cell edema, progressive metaplasia from squamous to cubic cells, vascular wall hyalinization, endothelial cells apoptosis/necrosis and endothelial desquamation. These results demonstrated damage of the distal deep digital flexor tendon vasculature which may progressively alter the structural integrity of the tendon and contribute to degenerative lesions.

Exostoses on the palmar or plantar aspect of the diaphysis of the third metacarpal or metatarsal bone in horses: 16 cases (2001-2010).

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.