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Objective: The objective of this study was to characterize extracellular vesicles (EVs) in plasma and synovial fluid obtained from horses with and without naturally occurring post-traumatic osteoarthritis (PTOA)., Animals: EVs were isolated from plasma and synovial fluid from horses with (n = 6) and without (n = 6) PTOA., Methods: Plasma and synovial fluid EVs were characterized with respect to quantity, size, and surface markers. Small RNA sequencing was performed, and differentially expressed microRNAs (miRNAs) underwent bioinformatic analysis to identify putative targets and to explore potential associations with specific biological processes., Results: Plasma and synovial fluid samples from horses with PTOA had a significantly higher proportion of exosomes and a lower proportion of microvesicles compared to horses without PTOA. Small RNA sequencing revealed several differentially expressed miRNAs, including miR-144, miR-219-3p, and miR-199a-3l in plasma and miR-199a-3p, miR-214, and miR-9094 in synovial fluid EVs. Bioinformatics analysis of the differentially expressed miRNAs highlighted their potential role in fibrosis, differentiation of chondrocytes, apoptosis, and inflammation pathways in PTOA., Clinical Relevance: We have identified dynamic molecular changes in the small noncoding signatures of plasma and synovial fluid EVs in horses with naturally occurring PTOA. These findings could serve to identify promising biomarkers in the pathogenesis of PTOA, to facilitate the development of targeted therapies, and to aid in establishing appropriate translational models of PTOA.

Objective: Mesenchymal stem cell (MSC) extracellular vesicles (EVs) have emerged as a biotherapeutic for osteoarthritis; however, manufacturing large quantities is not practical using traditional monolayer (2-D) culture. We aimed to examine the effects of 3-D and 2-D culture 2 types of media: Dulbecco modified Eagle medium and a commercially available medium (CM) on EV yield., Animals: Banked bone marrow-derived MSCs (BM-MSCs) from 6 healthy, young horses were used., Methods: 4 microcarriers (collagen-coated polystyrene, uncoated polystyrene, collagen-coated dextran, and uncoated dextran) were tested in static and bioreactor cultures, and the optimal microcarrier was chosen. The BM-MSCs were inoculated into a bioreactor with collagen-coated dextran microcarriers at 5,000 cells/cm2 or onto culture dishes at 4,000 cells/cm2 in either Dulbecco modified Eagle medium or CM media. Supernatants were obtained for metabolite and pH analysis. The BM-MSCs were expanded until confluent (2-D) or for 7 days (3-D) when the 48-hour EV collection period commenced using EV-depleted media. Extracellular vesicles were isolated and characterized via nanoparticle tracking analysis, Western blot, transmission electron microscopy, and protein quantification. The BM-MSCs were harvested, quantified, and immunophenotyped., Results: The number of EVs isolated was not improved by 3-D culture or CM media, however, the CM 3-D condition improved the number of EVs produced per BM-MSC over the CM 2-D condition (mean ± SD: 306 ± 99 vs 37 ± 22, respectively). Glucose decreased and lactate and ammonium accumulated in 3-D culture. Surface markers of stemness exhibited reduced expression in 3-D culture., Clinical Relevance: Optimization of our 3-D culture methods could improve BM-MSC expansion and thus EV yield.

The use of mesenchymal stem cells (MSCs) in human and veterinary clinical applications has become a subject of increasing importance due to their roles in immunomodulation and regenerative processes. MSCs are especially relevant in equine medicine because they may have the ability to treat prevalent musculoskeletal disorders, among other conditions. However, recent evidence suggests that the components secreted by MSCs, particularly extracellular vesicles (EVs), are responsible for these properties. EVs contain proteins and nucleic acids, which possess an active role in intercellular communication and can be used as therapeutics. However, because the intersection of equine veterinary medicine with EVs remains a relatively new field, there is a demand to identify biomarkers that can discern and enrich for therapeutic EVs, progressing their clinical efficacy. In this study, we identified and characterized 84 miRNAs, between three equine donors involved in immunomodulation in cell and EV subjects. We discovered distinct groups of shared miRNAs, like miR-21-5p and miR-451a, that are abundant and enriched between the donors' EVs, respectively. By mapping and comparing the MSC-EV miRNA expression, we discovered many pathways that are involved in immunomodulation and tissue regenerative processes related to equine clinical applications. Therefore, the miRNAs highlighted in this article can be used as valuable biomarkers for screening MSC-derived EVs for potential equine therapy., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Joint injury often leads to cartilage damage and posttraumatic inflammation, which drives continued extracellular matrix degradation culminating in osteoarthritis. Mesenchymal stem cells (MSCs) have been proposed as a biotherapeutic to modulate inflammation within the joint. However, concerns have been raised regarding the immunogenicity of MSCs cultured in traditional fetal bovine serum (FBS) containing media, and the potential of xenogenic antigens to activate the immune system causing rejection and destruction of the MSCs. Xenogen-free alternatives to FBS have been proposed to decrease MSC immunogenicity, including platelet lysate (PL) and equine serum. The objective of this study was to compare the immunomodulatory properties of BM-MSCs culture-expanded in media supplemented with autologous PL (APL), pooled PL (PPL), equine serum (ES) or FBS. We hypothesized that BM-MSCs culture expanded in media with xenogen-free supplements would exhibit superior immunomodulatory properties to those cultured in FBS containing media. Bone marrow-derived MSCs (BM-MSCs) were isolated from six horses and culture expanded in each media type. Blood was collected from each horse to isolate platelet lysate. The immunomodulatory function of the BM-MSCs was assessed via a T cell proliferation assay and through multiplex immunoassay quantification of cytokines, including IL-1β, IL-6, IL-8, IL-10, and TNFα, following preconditioning of BM-MSCs with IL-1β. The concentration of platelet-derived growth factor BB (PDGF-BB), IL-10, and transforming growth factor-β (TGF-β) in each media was measured via immunoassay. BM-MSCs cultured in ES resulted in significant suppression of T cell proliferation ( p = 0.02). Cell culture supernatant from preconditioned BM-MSCs cultured in ES had significantly higher levels of IL-6. PDGF-BB was significantly higher in APL media compared to FBS media ( p = 0.016), while IL-10 was significantly higher in PPL media than ES and FBS ( p = 0.04). TGF-β was highest in APL media, with a significant difference in comparison to ES media ( p = 0.03). In conclusion, expansion of equine BM-MSCs in ES may enhance their immunomodulatory abilities, while PL containing media may have some inherent therapeutic potential associated with higher concentrations of growth factors. Further studies are needed to elucidate which xenogen-free supplement optimizes BM-MSC performance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Even, Gaesser, Ciamillo, Linardi and Ortved.)

Autologous protein solution (APS) has been used anecdotally for intralesional treatment of tendon and ligament injuries, however, its use in these injuries has never been studied in vivo . Our objective was to evaluate the effect of APS on tendon healing in an equine superficial digital flexor (SDF) tendonitis model. We hypothesized intralesional injection of APS would result in superior structural and biomechanical healing. SDF tendonitis was induced in both forelimbs of eight horses using collagenase injection. One forelimb was randomly assigned to receive an intralesional injection of APS, while the other was injected with saline. Ultrasonographic examinations were performed at weeks -1, 0, 2, 4, 8, and 12 following treatment. At 12 weeks, horses were euthanized and SDF samples harvested. Histologic evaluation, biomechanical testing, gene expression analysis, total glycosaminoglycan (GAG) and total DNA quantification were performed. Collagen type III ( COL3A1 ) expression was significantly higher ( p = 0.028) in saline treated tendon than in normal tendon. Otherwise, there were no significant differences in gene expression. There were no significant differences in histologic or ultrasonographic scores between groups. Mean total DNA content was significantly higher ( p = 0.024) in saline treated tendons than normal tendons, whereas total DNA content was not significantly different between APS treated tendon and normal tendon. Elastic modulus was higher in APS treated than saline treated tendon, but the difference was not significant. Reduced expression of COL3A1 in APS treated tendon may indicate superior healing. Increased total DNA content in saline treated tendon may indicate ongoing healing processes, vs. APS treated tendons which may be in the later stages of healing. Limitations include a relatively short study period and inconsistency in size and severity of induced lesions. Intralesional injection of APS resulted in some improvements in healing characteristics., Competing Interests: WK is the Director of Research and Development at Owl Manor, who's product was investigated in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gaesser, Underwood, Linardi, Even, Reef, Shetye, Mauck, King, Engiles and Ortved.)

Objective: To evaluate the effects of intra-articular (IA) mepivacaine administration prior to carpal arthroscopy on anesthetic drug requirements, blood pressure support, hemodynamic variables, and quality of recovery in horses., Study Design: Experimental, analytical, cohort study., Sample Population: Twenty-two horses (n = 11 horses/group)., Methods: Horses were anesthetized by using the same protocol, but an IA injection of mepivacaine or saline was performed before carpal arthroscopy. End-tidal isoflurane concentration, heart rate, and mean arterial pressure were recorded at specific time points. Quality of recovery was scored by the anesthetist, who was unaware of group assignment. Data were analyzed by using two-way repeated-measures analysis of variance., Results: Mean arterial pressure was higher during joint distension in the control group compared with baseline (7% higher, P = .02) and with the treatment group (10% higher, P = .04). Heart rate was higher in the control group compared with the treatment group during joint distension (8% higher, P = .04) and chip removal (11% higher, P = .03). Heart rate was higher in the control group compared with baseline during chip removal (5.5% higher, P = .04). Two horses in the control group required additional ketamine vs none in the treatment group. Quality of recovery was not different between groups., Conclusion: Intra-articular mepivacaine resulted in fewer detectable reactions to surgical stimulation, with similar recovery scores and blood pressure support requirements., Clinical Significance: Intra-articular anesthesia prior to arthroscopy can be used safely in the horse and should be considered as a part of balanced anesthetic protocols., (© 2020 American College of Veterinary Surgeons.)