This study aimed at assessing the effectiveness and safety of repeated administrations of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) primed with tumor necrosis factor (TNF)-α and interferon-γ in an equine model of chemically-induced osteoarthritis. Arthritis was induced in both radio-carpal (RC)-joints by amphotericin-B in 18 ponies, divided into three groups depending on the treatment injected: MSC-naïve (n = 7), MSC-primed (n = 7) and control (n = 4). The study consisted of two phases and used one RC-joint of each animal in each phase, with four months time-lapse, in order to assess two end-points. Clinical, synovial, radiological and ultrasonographic follow-up was performed. At six months, animals were euthanized and both carpi were assessed by magnetic resonance imaging (MRI), gross anatomy, histopathology, histochemistry and gene expression. Clinical and synovial inflammatory signs were quicker reduced in MSC-treated groups and repeated allogeneic administration did not produce adverse reactions, but MSC-primed group showed slight and transient local inflammation after second injection. Radiology and MRI did not show significant differences between treated and control groups, whereas ultrasonography suggested reduced synovial effusion in MSC-treated groups. Both MSC-treated groups showed enhanced cartilage gross appearance at two compared to six months (MSC-naïve, p < 0.05). Cartilage histopathology did not reveal differences but histochemistry suggested delayed progression of proteoglycan loss in MSC-treated groups. Synovium histopathology indicated decreased inflammation (p < 0.01) in MSC-primed and MSC-naïve at two and six months, respectively. At two months, cartilage from MSC-primed group significantly (p < 0.05) upregulated collagen type II (COL2A1) and transforming growth factor (TGF)-β1 and downregulated cyclooxygenase-2 and interleukin (IL)-1β. At six months, MSC-treatments significantly downregulated TNFα (p < 0.05), plus MSC-primed upregulated (p < 0.05) COL2A1, aggrecan, cartilage oligomeric protein, tissue inhibitor of metalloproteinases-2 and TGF-β1. In synovium, both MSC-treatments decreased (p < 0.01) matrix metalloproteinase-13 expression at two months and MSC-primed also downregulated TNFα (p < 0.05) and IL-1β (p < 0.01). Both MSC-treatments provided beneficial effects, mostly observed at short-term. Despite no huge differences between MSC-treatments, the findings suggested enhanced anti-inflammatory and regulatory potential of MSC-primed. While further research is needed to better understand these effects and clarify immunogenicity implications, these findings contribute to enlarge the knowledge about MSC therapeutics and how they could be influenced.

Mesenchymal stem cells (MSCs) transplantation has become a promising therapeutic choice for musculoskeletal injuries. Joint-related disorders are highly prevalent in horses. Therefore, these animals are considered as suitable models for testing MSC-based therapies for these diseases. The aim of this study was to investigate the clinical and inflammatory responses to intra-articular single and repeat dose administration of autologous or of pooled allogeneic MSCs in healthy equine healthy joints. Six horses were intra-articularly injected with a single autologous dose of bone marrow derived MSCs (BM-MSCs) and two separate doses of allogeneic BM-MSCs pooled from several donors. All contralateral joints were injected with Lactated Ringer’s Solution (LRS) as the control vehicle. Signs of synovitis and lameness were evaluated at days 0, 1, 2, 3, 5 and 10 after injection. Total protein (TP), white blood cell count (WBC) and neutrophil count (NC) in synovial fluid were also measured at the same time-points. A mild synovial effusion without associated lameness was observed after all BM-MSCs injections. The second allogeneic injection caused the lowest signs of synovitis. Local temperature slightly increased after all BM-MSCs treatments compared to the controls. TP, WBC and NC in synovial fluids also increased during days 1 to 5 after all BM-MSCs injections. Both, clinical and synovial parameters were progressively normalized and by day 10 post-inoculation appeared indistinguishable from controls. Intra-articular administration of an allogeneic pool of BM-MSCs represents a safe therapeutic strategy to enhance MSCs availability. Importantly, the absence of hypersensitivity response to the second allogeneic BM-MSCs injection validates the use of repeat dose treatments to potentiate the therapeutic benefit of these cells. These results notably contribute to the development of stem cell based therapies for equine and human joint diseases.

Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O₂). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O₂. At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O₂ for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state. Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.

Introduction: Patellar tendon overuse injuries are common in athletes. Imaging may show a change in tissue structure with tendon thickening and disruption of the intratendinous substance. We wish to test the hypothesis that both autologous bone marrow expanded mesenchymal stem cells and autologous leukocyte-poor platelet-rich plasma (LP-PRP) implanted into the area of the disrupted tendinopathic patellar tendon will restore function, but tendon regeneration tissue will only be observed in the subjects treated with autologous bone marrow expanded mesenchymal stem cells. Methods and analysis: This is a single-centre, pilot phase I/II, double-blinded clinical trial with randomisation with active control. Twenty patients with a diagnosis of patellar tendinopathy with imaging changes (tendon thickening and disruption of the intratendinous substance at the proximal portion of the patellar tendon) will be randomised in a 1:1 ratio to receive a local injection of either bone-marrow autologous mesenchymal stem cells (MSC), isolated and cultured under GMP at The Institute of Biology and Molecular Genetics (IBGM) (Spain) or P-PRP. The study will have two aims: first, to ascertain whether a clinically relevant improvement after 3, 6 and 12 months according to the visual analogue scale (VAS), Victorian Institute of Sport Assessment for patellar tendons (VISA-P) and dynamometry scales (DYN) will be achieved; and second, to ascertain whether the proposed intervention will restore tendon structure as determined by ultrasonography (US), Doppler ultrasonography (DUS), and innovative MRI and ultrasound techniques: Magnetic Resonance T2 FAT SAT (UTE, Ultrashort Echo TE) sequence and Ultrasound Tissue Characterization (UTC). Patients who are randomised to the P-PRP treatment group but do not achieve a satisfactory primary endpoint after 6 months will be offered treatment with MSC. Trial registration: NCT03454737.

First cannulation is a critical manoeuvre in equine laparoscopy. This retrospective study aimed at the comparison of the frequency and type of complications detected when using different human laparoscopy devices for laparoscopic access in standing horses, and the influence of body condition in such complications. Forty-four procedures were included, and retrieved data comprised cannula insertion technique, body condition, and type and frequency of complications. Laparoscopic access techniques were classified into five groups: P: pneumoperitoneum created using Veress needle prior to cannulation; T: sharp trocar; D: direct access via surgical incision; V: Visiport optical trocar and H: optical helical cannula (OHC). In groups T, D, V and H, access was achieved without prior induction of pneumoperitoneum. Complications were registered in 13/44 procedures, of which retroperitoneal insufflation was the most common (6/13). Statistically significant association was found between the complication incidence and the type of access, with group D showing the highest complication frequency (80%) and group H the lowest frequency (0%). The majority of complications (9/13) were observed in overweight horses. We conclude that devices designed for human patients can be used for laparoscopic access in standing horses, with the use of OHC minimizing the appearance of complications, especially in overweight horses with OW.