Objective: To explore the long-term safety and efficacy of canine allogeneic mesenchymal stromal cells (MSC) administered intra-articularly as single or repeated injections in appendicular joints of dogs affected by moderate to severe refractory osteoarthritis.Study Design: 22 pet dogs were recruited into a non-randomized, open and monocentric study initially administering one cellular injection. A second injection was offered after 6 months to owners if the first injection did not produce expected results.Materials and Methods: Anti-inflammatory treatment (if prescribed) was discontinued at last one week before the onset of treatment. Each injection consisted of at least 10 million viable neonatal allogeneic mesenchymal stromal cells obtained from fetal adnexa. Medical data was collected from veterinary clinical evaluations of joints up to 6 months post-injection and owner's assessment of their dog's mobility and well-being followed for a further 2 years when possible.Results: Mild, immediate self-limiting inflammatory joint reactions were observed in 5/22 joints after the first injection, and in almost all dogs having a subsequent injection. No other MSC-related adverse medical events were reported, neither during the 6 months follow up visits, nor during the long-term (2-years) safety follow up. Veterinary clinical evaluation showed a significant and durable clinical improvement (up to 6 months) following MSC administration. Eight dogs (11 joints) were re-injected 6 months apart, sustaining clinical benefits up to 1 year. Owner's global satisfaction reached 75% at 2 years post-treatmentConclusion: Our data suggest that a single or repeated intra-articular administration of neonatal MSC in dogs with moderate to severe OA is a safe procedure and confer clinical benefits over a 24-month period. When humoral response against MSC is investigated by flow cytometry, a positive mild and transient signal was detected in only one dog from the studied cohort, this dog having had a positive clinical outcome.

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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-β) 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-β 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.

As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine.