Effects of oral treatment with chondroitin sulfate and glucosamine in an experimental model of metacarpophalangeal osteoarthritis in horses.

BACKGROUND: Combined chondroitin sulfate (CS) and glucosamine (GlcN) has been widely used in oral formulations to prevent and treat osteoarthritis. CS is effective for controlling pain in osteoarthritic patients, whereas GlcN can stimulate glycosaminoglycan synthesis, thus reducing extracellular matrix degradation. Although several studies have been published on this topic, the effectiveness of treatment with oral CS and GlcN remains uncertain. The objective of this study was to analyze the progression of experimentally induced osteoarthritis in horses and verify the effectiveness of an oral compound based on CS and GlcN to treat and/or modulate this disease. The study analyzed the metacarpophalangeal joint of the left thoracic limb of 16 horses divided into two groups, with eight horses treated with CS and GlcN in the treated group (GT) and eight untreated horses in the control group (GC). Chondral lesions were induced through arthroscopy, which was defined as time-point zero (T0). Physical, ultrasonographic, and radiographic examinations and synovial fluid biomarkers measurements were performed on days 0, 30, 60, 90, and 120. At the end of the experiment (T4), arthroscopy was performed again to macroscopically evaluate the joints and collect material for microscopic analysis. RESULTS: Significant differences were observed between groups in some evaluated parameters, such as visual lameness assessment, synovial concentrations of prostaglandin E2, and ultrasound examination. However, the GT still presented slightly improved results for joint flexion angle, analysis of lameness using sensors, and histopathological analysis of chondral repair tissue, however, without the statistical significance (p>0.05). CONCLUSIONS: The treatment was considered effective in the clinical modulation of experimental osteoarthritis, with improvement of some parameters in the GT. However, this type of treatment may not be entirely effective to change the catabolic process in articular cartilage and the progressive induced chondral damage.

Evaluation of platelet-rich plasma applied in the coronary band of healthy equine hooves.

Platelet-rich plasma (PRP) is a widely used hemocomponent that holds great promise in equine medicine due to its feasible production and regenerative therapy potential. Its use has been considered as a treatment for chronic laminitis, mainly in terms of its analgesic properties and because it can induce growth in affected hooves. The aim of this study was to evaluate the effect on hoof growth attributable to PRP applied in the coronary band of clinically healthy horses by comparing the responses to PRP, saline, and trimming alone. After randomization, the forelimbs of 9 horses received specific treatment at intervals of 33 days and were trimmed, measured, and radiographed at the same time. Neither hoof growth nor hoof angles were different between the treatment groups at any time point. The application of PRP in the coronary band of horses may be a safe procedure; however, it had no effect on the growth or conformation of hooves in clinically healthy horses.

Évaluation du plasma riche en plaquettes appliqué dans la bande coronaire de sabots équins sains. Le plasma riche en plaquettes (PRP) est un hémo-composant largement utilisé qui est très prometteur en médecine équine en raison de sa production réalisable et de son potentiel de thérapie régénérative. Son utilisation a été considérée comme un traitement de la fourbure chronique, principalement en termes de ses propriétés analgésiques et parce qu'elle peut induire la croissance des sabots atteints. Le but de cette étude était d'évaluer l'effet sur la croissance des sabots attribuable au PRP appliqué dans la bande coronaire de chevaux cliniquement sains en comparant les réponses au PRP, à la solution saline et à la taille seule. Après randomisation, les membres antérieurs de neuf chevaux ont reçu un traitement spécifique à des intervalles de 33 jours et ont été taillés, mesurés et radiographiés au même moment. Ni la croissance ni les angles des sabots n'étaient différents entre les groupes de traitement à aucun moment. L'application de PRP dans la bande coronaire des chevaux peut être une procédure sûre; cependant, il n'a eu aucun effet sur la croissance ou la conformation des sabots chez les chevaux cliniquement sains.(Traduit par Dr Serge Messier).

Social facilitation of trotting: Can horses perceive and adapt to the movement of another horse?

Exercise intensity is prone to be self-regulated in horses exercising freely. The main drivers include social, feeding and escape behaviors, as well as the operant conditioning. We hypothesized that self-regulated exercise intensity may increase due to the presence of another horse exercising ahead. Seven horses were assigned to a 2x2 crossover trial following treadmill familiarization. Video images of a trotting horse were displayed on the wall in front of the experimental unit (Visual), which was positioned in the treadmill. Physiological and behavioral markers were further compared with a control visual stimulus (Co), comprising a racetrack image without horses. Horses were sampled during a constant load exercise test (1) at rest (baseline), (2) after the warm-up (0 - 10th minute) and (3) after visual stimulation or control (10th- 12th minutes of the SET) to quantify plasma lactate and glucose concentration, heart rate, head angle, as well as behavioral markers. Following visual stimulation, heart rate (130.8 ± 27.8 b.p.m.) was higher than control (84.7 ± 15.1 b.p.m., P = .017), as was plasma lactate (Visual - 5.28 ± 1.48 mg/dl; Co -3.27 ± 1.24 mg/dl, P = .042) and head angle (Visual - 36.43 ± 3.69°; Co -25.14 ± 4.88°, P = .003). The prevalence of "ears forward" behavior was also higher following Visual (100% - 7/7) than Co (14% - 1/7, P = .004). These results suggest that visual stimulus (1) was safe and well tolerated and (2) prompted the anaerobic lactic pathways and shifted the behavior to a vigilant state. In conclusion, horses were able to perceive and adapt to a social environment. Our findings validate the use of social facilitation of trotting to encourage horses to move forward avoiding the use of the whip.

Chondrogenic potential of mesenchymal stem cells from horses using a magnetic 3D cell culture system.

BACKGROUND: Mesenchymal stem cells (MSCs) represent a promising therapy for the treatment of equine joint diseases, studied due to their possible immunomodulatory characteristics and regenerative capacity. However, the source of most suitable MSCs for producing cartilage for regenerative processes in conjunction with biomaterials for an enhanced function is yet to be established. AIM: To compare the chondrogenicity of MSCs derived from synovial fluid, bone marrow, and adipose tissue of horses, using the aggrecan synthesis. METHODS: MSCs from ten horses were cultured, phenotypic characterization was done with antibodies CD90, CD44 and CD34 and were differentiated into chondrocytes. The 3D cell culture system in which biocompatible nanoparticles consisting of gold, iron oxide, and poly-L-lysine were added to the cells, and they were forced by magnets to form one microspheroid. The microspheroids were exposed to a commercial culture medium for 4 d, 7 d, 14 d, and 21 d. Proteoglycan extraction was performed, and aggrecan was quantified by enzyme-linked immunosorbent assay. Keratan sulfate and aggrecan in the microspheroids were identified and localized by immunofluorescence. RESULTS: All cultured cells showed fibroblast-like appearance, the ability to adhere to the plastic surface, and were positive for CD44 and CD90, thus confirming the characteristics and morphology of MSCs. The soluble protein concentrations were higher in the microspheroids derived from adipose tissue. The aggrecan concentration and the ratio of aggrecan to soluble proteins were higher in microspheroids derived from synovial fluid than in those derived from bone marrow, thereby showing chondrogenic superiority. Microspheroids from all sources expressed aggrecan and keratan sulfate when observed using confocal immunofluorescence microscopy. All sources of MSCs can synthesize aggrecan, however, MSCs from synovial fluid and adipose tissue have demonstrated better biocompatibility in a 3D environment, thus suggesting chondrogenic superiority. CONCLUSION: All sources of MSCs produce hyaline cartilage; however, the use of synovial liquid or adipose tissue should be recommended when it is intended for use with biomaterials or scaffolds.

Effects of Joint Lavage with Dimethylsulfoxide on LPS-Induced Synovitis in Horses-Clinical and Laboratorial Aspects.

Several studies in human and equine medicine have produced controversial results regarding the role of dimethylsulfoxide (DMSO) as a therapeutic agent. This study aimed to evaluate the effect of joint lavage with different DMSO concentrations on biomarkers of synovial fluid inflammation and cartilage degradation in joints with lipopolysaccharide (LPS)-induced synovitis. Twenty-six tibiotarsal joints of 13 horses were randomly distributed into four groups (lactated Ringer's solution; 5% DMSO in lactated Ringer's; 10% DMSO in lactated Ringer's; and sham). All animals were evaluated for the presence of lameness, and synovial fluid analyses were performed at 0 h, 1 h, 8 h, 24 h, and 48 h (T0, T1, T8, T24, and T48, respectively). The white blood cell counts (WBC), total protein (TP), urea, prostaglandin E2 (PGE2), interleukin (IL)-1ß, IL-6, IL-10, tumor necrosis factor-α (TNF-α), hyaluronic acid (HA), and chondroitin sulfate (CS) concentrations were measured. The WBC counts and PGE2, IL-1ß, IL-6, and TP concentrations increased in all groups at T8 compared to baseline values (p<0.05). At T48, only the 5% DMSO and 10% DMSO groups showed a significant decrease in WBC counts (p<0.05). Furthermore, the 10% DMSO group had lower concentrations of PGE2 and IL-1ß at T48 than at T8 (p<0.05) and presented lower IL-6 levels than the5% DMSO and lactated Ringer's groups at T24. All groups showed an increase in CS concentration after LPS-induced synovitis. Joint lavage with 10% DMSO in lactated Ringer´s has anti-inflammatory but not chondroprotective effects.

Effects of blood-derived products and sodium hyaluronate on equine synovial fluid cells and on synovial fluid from osteochondrotic joints of horses after arthroscopy and administration of treatment.

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)-1ß, 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.

Hyaluronic acid has chondroprotective and joint-preserving effects on LPS-induced synovitis in horses.

The intra-articular use of hyaluronic acid (HA) for the treatment of synovitis and osteoarthritis is still controversial. As a consequence, corticosteroids remain the most frequently employed therapeutic agents, despite their potential systemic and local deleterious effects. This study examined the anti-inflammatory, antioxidant, and chondroprotective activities of low and high molecular weight hyaluronic acid (LMW-HA and HMW-HA) on lipopolysaccharide (LPS)-induced synovitis in horses compared to triamcinolone acetonide (TA). LPS was injected in the metacarpophalangeal joints, which were treated intra-articularly with either TA (as control) or LMW-HA or HMW-HA. Joint clinical evaluation and synovial fluid (SF) analysis were performed at 0, 8, 24, and 48 h. The white blood cell counts (WBC), prostaglandin E2 (PGE2), interleukin (IL)-1, IL-6, IL-10, tumor necrosis factor-α, chondroitin sulfate (CS) and HA concentrations, oxidative burst, and HA molecular weights were measured. TA reduced the lameness, swelling, and PGE2 release but increased the SF CS concentrations enormously at 24h and 48h, and decreased the SF HA modal molecular weight. These results indicate the breakdown of articular cartilage aggrecan and SF HA. In contrast, LMW-HA and HMW-HA were less effective in reducing the inflammation symptoms, but preserved the joints because only a modest increase in CS occurred at 24 h, decreasing at 48 h, and the SF HA was maintained. The HA-treatment also had anti-inflammatory actions, and LMW-HA was the most effective in reducing the release of cytokine. In summary, the HA treatment inhibited efficiently the digestion of cartilage proteoglycans and SF HA breakdown.

Does Double Centrifugation Lead to Premature Platelet Aggregation and Decreased TGF-ß1 Concentrations in Equine Platelet-Rich Plasma?

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-ß1 concentration was also higher. Pearson's correlation coefficients gave a significant, positive correlation between the platelet counts and TGF-ß1 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-ß1 concentrations.

Effects of medical ozone upon healthy equine joints: Clinical and laboratorial aspects.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: The ozonetherapy did not cause significant inflammation process or cartilage degradation, therefore, ozonetherapy is safe at both evaluated doses.

Comparative study of equine mesenchymal stem cells from healthy and injured synovial tissues: an in vitro assessment.

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.

In vitro evaluation of hydroxyapatite, chitosan, and carbon nanotube composite biomaterial to support bone healing / Avaliação in vitro do biomaterial compósito à base de hidroxiapatita, quitosana e nanotubo de carbono como adjuvante no reparo ósseo

Hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were developed to improve bone healing. Previous studies suggested that a combination of biomaterials and mesenchymal stem cells (MSCs) can potentially help promote bone regeneration. In the present study, we first developed hydroxyapatite, chitosan, and carbon nanotube composite biomaterial. Then, the effect of different concentrations of the extract on the viability of Vero cells (ATCC CCL-81) and MSCs obtained from sheep bone marrow using methylthiazol tetrazolium (MTT) and propidium iodide (PI) assays were evaluated. The biomaterial group demonstrated an absence of cytotoxicity, similar to the control group. Samples with 50% and 10% biomaterial extract concentrations showed higher cell viability compared to samples from the control group (MTT assay). These results suggest that the presence of this composite biomaterial can be used with MSCs. This study also concluded that hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were not cytotoxic. Therefore, these could be used for performing in vivo tests.(AU)

O compósito à base de hidroxiapatita, quitosana e nanotubo de carbono foi desenvolvido com o intuito de auxiliar na consolidação óssea. Estudos anteriores sugerem que a combinação de substitutos ósseos e células-tronco mesenquimais (CTM) podem auxiliar a potencializar e promover a regeneração óssea. No presente estudo, o biomaterial foi desenvolvido e a viabilidade e a citotoxicidade de células Vero (ATCC CCL-81) e CTM obtidas de medula óssea provenientes de ovinos utilizando ensaios metil-tiazol-tetrazólio, MTT e iodeto de propídeo (PI) foram avaliadas em diferentes concentrações de extrato desse compósito. O compósito demonstrou ausência de citotoxicidade com comportamento semelhante ao grupo controle. Amostras com 50% e 10% de concentração de extrato do compósito mostraram resultados maiores comparados ao grupo controle (ensaio MTT). Esses resultados também sugerem que a presença do biomaterial pode ser utilizada em associação a CTM. Assim, esse estudo conclui que o compósito apresentado de hidroxiapatita, quitosana e nanotubo de cabono não foi considerado citotóxico e pode ser utilizado em teste in vivo.(AU)