Neuenschwander HM, et al. J Vet Sci. 2019 Oct;20:e67. https://doi.org/10.4142/jvs.2019.20.e67

Background: Bone marrow and adipose tissues are known sources of mesenchymal stem cells (MSCs) in horses; however, synovial tissues might be a promising alternative. The aim of this study was to evaluate phenotypic characteristics and differentiation potential of equine MSCs from synovial fluid (SF) and synovial membrane (SM) of healthy joints (SF-H and SM-H), joints with osteoarthritis (SF-OA and SM-OA) and joints with osteochondritis dissecans (SF-OCD and SM-OCD) to determine the most suitable synovial source for an allogeneic therapy cell bank. Methods: Expression of the markers CD90, CD105, CD44, and CD34 in SF-H, SM-H, SF-OA, SM-OA, SF-OCD and SM-OCD was verified by flow cytometry, and expression of cytokeratin, vimentin, PGP 9.5, PCNA, lysozyme, nanog, and Oct4 was verified by immunocytochemistry. MSCs were cultured and evaluated for their chondrogenic, osteogenic and adipogenic differentiation potential. Final quantification of extracellular matrix and mineralized matrix was determined using AxioVision software. A tumorigenicity test was conducted in Balb-C^nu/nu mice to verify the safety of the MSCs from these sources. Results: Cultured cells from SF and SM exhibited fibroblastoid morphology and the ability to adhere to plastic. The time elapsed between primary culture and the third passage was approximately 73 days for SF-H, 89 days for SF-OCD, 60 days for SF-OA, 68 days for SM-H, 57 days for SM-OCD and 54 days for SM-OA. The doubling time for SF-OCD was higher than that for other cells at the first passage (P < 0.05). MSCs from synovial tissues showed positive expression of the markers CD90, CD44, lysozyme, PGP 9.5, PCNA and vimentin and were able to differentiate into chondrogenic (21 days) and osteogenic (21 days) lineages, and, although poorly, into adipogenic lineages (14 days). The areas staining positive for extracellular matrix in the SF-H and SM-H groups were larger than those in the SF-OA and SM-OA groups (P < 0.05). The positive mineralized matrix area in the SF-H group was larger than those in all the other groups (P < 0.05). The studied cells exhibited no tumorigenic effects. Conclusions: SF and SM are viable sources of equine MSCs. All sources studied provide suitable MSCs for an allogeneic therapy cell bank; nevertheless, MSCs from healthy joints may be preferable for cell banking purposes because they exhibit better chondrogenic differentiation capacity.

Blood-derived autologous products are frequently used in both human and equine medicine to treat musculoskeletal disorders. These products, especially the platelet-rich plasma (PRP), may contain high concentrations of growth factors (GFs), and thus improve healing in several tissues. Nevertheless, the procedures for preparation of PRP are currently non-standardized. Several protocols, which are based on distinct centrifugation patterns (rotation speed and time), result in PRPs with different characteristics, concerning platelet and GFs concentrations, as well as platelet activation. The aim of the present study was to compare two different protocols for PRP preparation: protocol (A) that is based on a single-centrifugation step; protocol (B), which included two sequential centrifugation steps (double-centrifugation). The results here reported show that the double-centrifugation protocol resulted in higher platelet concentration, while leukocytes were not concentrated by this procedure. Although platelet activation and aggregation were increased in this protocol in comparison to the single-centrifugation one, the TGF-β1 concentration was also higher. Pearson’s correlation coefficients gave a significant, positive correlation between the platelet counts and TGF-β1 concentration. In conclusion, although the double-centrifugation protocol caused premature platelet aggregation, it seems to be an effective method for preparation of PRP with high platelet and TGF-β1 concentrations.

OBJECTIVE: To compare effects of platelet-rich plasma (PRP), interleukin-1 receptor antagonist protein (IRAP), autologous processed plasma (APP), and sodium hyaluronate treatments on synovial fluid cells in vitro and on synovial fluid obtained from osteochondrotic joints of horses. SAMPLE: Synovial fluid cells from 8 healthy equine tibiotarsal joints (in vitro experiment) and synovial fluid samples from 40 tibiotarsal joints of 25 horses with osteochondrosis dissecans (in vivo experiment). PROCEDURES: Effects of various treatments on concentrations of prostaglandin (PG) E₂, interleukin (IL)-1β, tumor necrosis factor-α, IL-10, and IL-1 receptor antagonist (IL-1ra) were analyzed in cell medium supernatant, and production of reactive oxygen species was analyzed by use of flow cytometry. In an in vivo experiment, synovial fluid samples were collected before and 48 hours after arthroscopy and treatment administration (8 joints/treatment) and evaluated to determine concentrations of hyaluronic acid, chondroitin sulfate, PGE₂, tumor necrosis factor-α, IL-1, IL-10, and IL-1ra. RESULTS: All in vitro treatments reduced reactive oxygen species production, PRP increased PGE₂ concentrations, and PRP, IRAP, and APP increased IL-1ra concentrations. Only IRAP and APP increased IL-1 concentrations. For the in vivo experiment, PRP increased and IRAP decreased PGE₂ concentrations in synovial fluid after arthroscopy. All treatments increased IL-1ra concentrations, but only sodium hyaluronate resulted in a significant increase in concentration, compared with the concentration for untreated joints. Also, IRAP reduced hyaluronic acid breakdown in synovial fluid. CONCLUSIONS AND CLINICAL RELEVANCE: PRP should be used with caution in the period immediately after arthroscopy and treatment of osteochondrotic joints of horses. All treatments had antioxidant effects. Sodium hyaluronate, APP, and IRAP might help ameliorate joint inflammation.

The aim of this study was to verify whether transient inflammatory reactions induced by intra-articular medicinal ozone administration affect joint components, by in vivo evaluation of inflammatory (prostaglandin E₂, Substance P, Interleukin-6, Interleukine-1, Tumor Necrosis Factor), anti-inflammatory (Interleukin-10) and oxidative (superoxide dismutase activity and oxidative burst) biomarkers and extracellular matrix degradation products (chondroitin sulphate and hyaluronic acid) in synovial fluid. The effects of medicinal ozone were analyzed at two ozone concentrations (groups A and B, 20 and 40 μg/ml, respectively), using oxygen-injected joints as controls (group C); each group received ten treatments (15 ml gas per treatment). Physical evaluation, evaluation of lameness, ultrasonography, and synovial fluid analysis were performed. All joints presented mild and transient effusion throughout the study. Group B exhibited the highest lameness score on day 14 (P<0.05), detected by the lameness measurement system, probably because of the higher ozone concentration. All groups exhibited increased ultrasonography scores on day 14 (P < 0.05). Groups A and B exhibited increased proteins concentrations on day 21 (P<0.05). There was no change in hyaluronic acid concentration or the percentage of high-molecular weight hyaluronic acid throughout the experiment. Chondroitin sulfate concentrations decreased in group B, and did not change in group A and C, indicating that neither treatment provoked extracellular matrix catabolism. Cytokine and eicosanoid concentrations were not significantly changed. The ozonetherapy did not cause significant inflammation process or cartilage degradation, therefore, ozonetherapy is safe at both evaluated doses.

Ozone (O3) therapy has been used for medical procedures for centuries; however, there are no extensive studies on its utilization in horses. This study aimed to evaluate the application of transrectal O3 on horses by physical and laboratorial evaluation, and production of reactive oxygen species (ROS). Sixteen healthy horses were separated in two groups: a control group (CG) and a group treated with O3 (TG). The TG animals received 1L of an oxygen and O3 mixture transrectally. The initial dose was 10µg/ml for the first two applications, 15μg/ml for the following two applications, and 20μg/ml for the next six applications. The CG animals received 1L of oxygen transrectally. In TG animals no variations in the physical examination were detected; furthermore, TG animals did not exhibit changes in biochemical evaluation results, fibrinogen concentrations, or ROS production. TG animals had increased red blood cell counts, hemoglobin concentrations, and packet cell volume values in comparison to the baseline and CG values. We could infer that O3 affected the red blood cell counts and improved rhetological properties of the blood. The transrectal application of O3 in horses is safe and can indirectly improve the oxygenation and metabolism of tissues.