M. Prades

Autologous platelet concentrates as a treatment for musculoskeletal lesions in five horses

Two horses with acute tendinopathy of a superficial digital flexor tendon (sdft) and three horses with chronic proximal desmitis of the suspensory ligament (pdsl) were treated by injecting autologous concentrates of their platelets into the lesions. The lesions were monitored ultrasonographically and clinically. There were significant ultrasonographic and clinical improvements in the two horses with sdft, but no ultrasonographic improvements in the horses with pdsl; however, they improved clinically and became less lame. All the horses had returned to their pre-injury level of performance by six months after the completion of the treatment, and none of them had suffered a recurrence after 20 months.

Effects of the breed, sex and age on cellular content and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel

BackgroundThere is no information on the effects of the breed, gender and age on the cellular content and growth factor (GF) release from equine pure-platelet rich plasma (P-PRP) and pure-platelet rich gel (P-PRG). The objectives of this study were: 1) to compare the cellular composition of P-PRP with whole blood and platelet poor plasma (PPP); 2) to compare the concentration of transforming GF beta 1 (TGF-β1) and platelet derived GF isoform BB (PDGF-BB) between P-PRP treated with non-ionic detergent (P-PRP+NID), P-PRG (activated with calcium gluconate -CG-), PPP+NID, PPP gel (PPG), and plasma and; 3) to evaluate and to correlate the effect of the breed, gender and age on the cellular and GF concentration for each blood component. Forty adult horses, 20 Argentinean Creole Horses (ACH) and, 20 Colombian Creole Horses (CCH) were included. Data were analyzed by parametric (i.e.: t-test, one way ANOVA) and non parametric (Kruskal-Wallis test, Wilcoxon test) tests. Correlation analysis was also performed by using the Spearman and Pearson tests. A p ≤ 0.05 was set as significant for all tests. All the blood components were compared for platelet (PLT), leukocyte (WBC), TGF-β1 and PDGF-BB concentrations. The effect of the breed, gender and age on these variables was analyzed. A P ≤ 0.05 was accepted as significant for all the tests.ResultsPLT counts were 1.8 and 0.6 times higher in P-PRP than in whole blood and PPP, respectively; WBC counts were 0.5 and 0.1 times lower in P-PRP, in comparison with whole blood and PPP, respectively. TGF-β1 and PDGF-BB concentrations were 2.3 and 262 times higher, respectively, in P-PRG than in plasma, and 0.59 and 0.48 times higher, respectively, in P-PRG than in PPG. P-PRG derived from CCH females or young horses presented significantly (P < 0.001) higher PDGF-BB concentrations than P-PRG derived from ACH males or older horses.ConclusionsOur results indicated that P-PRP obtained by a manual method was affected by intrinsic factors such as the breed, gender and age. Equine practitioners should be aware that cellular and GF release from P-PRP/P-PRG could change according with the intrinsic variables associated with a patient in particular.

Uso de concentrados autólogos de plaquetas obtenidos mediante el método del tubo como tratamiento de artropatías en caballos

The clinical effect of the intra-articular injection of an autologous platelet concentrate (APC) in 7 horses with severe joint disease (4 with osteoarthritis...

Inflammatory response to the administration of mesenchymal stem cells in an equine experimental model: effect of autologous, and single and repeat doses of pooled allogeneic cells in healthy joints

BackgroundMesenchymal 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.ResultsA 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.ConclusionsIntra-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.

Niveles de factor de crecimiento transformante beta-3 y óxido nítrico en cuatro concentrados autólogos de plaquetas y plasma derivados de sangre equina

El objetivo de este estudio fue evaluar el metodo de centrifugado simple y doble en tubo para concentrar plaquetas, leucocitos, factor de crecimiento transformante beta-3 (TGF-s3) y oxido nitrico (NO). Se recogieron muestras de sangre entera de caballos clinicamente normales, las cuales fueron procesadas por medio del metodo de centrifugado simple y doble en tubo para obtener 4 concentrados de plaquetas (PCs): PC-A, PC-B, PC-C y PC-D. Los PCs y la sangre entera fueron analizados para hemograma mediante un sistema hematologico de citometria de flujo. Las concentraciones de TGF-s3 fueron analizadas mediante ELISA y el NO mediante la reaccion de Greiss. Las concentraciones de plaquetas y leucocitos fueron estadisticamente diferentes (P < 0,05) para cada PC y sangre entera. Los niveles de TGF-s3 y NO fueron similares para cada PC y sangre entera. En conclusion, el metodo de centrifugado simple y doble en tubo es un procedimiento de confianza para concentrar plaquetas en el caballo. Sin embargo, los niveles de TGF-s3 y NO no dependen del numero de plaquetas o de leucocitos concentrados con esta tecnica.

Concentrados autólogos de plaquetas como tratamiento de lesiones de tejidos blandos del aparato locomotor en caballos

The efficacy of an autologous platelet concentrate (APC) in 5 horses with soft tissue musculoskeletal injuries namely: superficial digital flexor tendon (SDF...

Does intraoperative low arterial partial pressure of oxygen increase the risk of surgical site infection following emergency exploratory laparotomy in horses

Decreased tissue oxygenation is a critical factor in the development of wound infection as neutrophil mediated oxidative killing is an essential mechanism against surgical pathogens. The objective of this prospective case series was to assess the impact of intraoperative arterial partial pressure of oxygen (PaO2) on surgical site infection (SSI) in horses undergoing emergency exploratory laparotomy for acute gastrointestinal disease. The anaesthetic and antibiotic protocol was standardised. Demographic data, surgical potential risk factors and PaO2, obtained 1h after induction of anaesthesia were recorded. Surgical wounds were assessed daily for infection during hospitalisation and follow up information was obtained after discharge. A total of 84 adult horses were included. SSI developed in 34 (40.4%) horses. Multivariate logistic regression showed that PaO2, anaesthetic time and subcutaneous suture material were predictors of SSI (AUC=0.76, sensitivity=71%, specificity=65%). The use of polyglycolic acid sutures increased the risk and horses with a PaO2 value < 80 mm Hg [10.6 kPa] and anaesthetic time >2h had the highest risk of developing SSI (OR=9.01; 95% CI 2.28-35.64). The results of this study confirm the hypothesis that low intraoperative PaO2 contributes to the development of SSI following colic surgery.

Synovial Fluid D-Dimer Concentration in Foals with Septic Joint Disease

BACKGROUND Increased synovial fibrinolytic activity (detected by increases in synovial D-Dimer concentrations) has been observed in different joint diseases in humans and adult horses, presumably in order to minimize fibrin deposition within the joint and thus avoid its detrimental effects. OBJECTIVE To investigate fibrinolytic pathway activation in joint sepsis in foals by measuring synovial D-Dimer concentrations. ANIMALS Eighteen septic foals with septic joints, 9 septic foals without septic joints, 9 systemically healthy foals with septic joint, and 3 controls are included. METHODS Prospective observational clinical study of foals admitted for septic arthritis. Synovial D-Dimer concentration and routine synovial fluid analysis were performed. Diagnosis of joint sepsis was made whenever synovial total nucleated cell count was >30,000 cells/μL, synovial total protein >4 g/dL, and neutrophil percentage of >80%, or synovial fluid culture resulted positive. Results were compared among groups by general lineal models. RESULTS Synovial D-Dimer concentration was significantly (P < .001) higher in the foals with septic joints compared with foals without joint disease (P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE Septic joint disease is associated with a marked increase of synovial D-Dimer concentration (marked activation of the fibrinolytic activity) within the affected joint. Although further studies are needed, the measurement of synovial D-Dimer concentration may be considered a complementary diagnostic marker of septic joint disease.

Effect of the administration of an oral hyaluronan formulation on clinical and biochemical parameters in young horses with osteochondrosis

The aim of this study was to evaluate the clinical and biochemical effects of the administration of oral hyaluronan (Hyal-Joint [HJ]) on young horses with osteochondrosis (OC). Our hypotheses were that HJ administration is safe, would decrease the degree of synovial effusion and the concentration of nitric oxide (NO) and prostaglandin E2 (PGE2) in synovial fluid, and would increase the concentration of hyaluronic acid (HA) in plasma and synovial fluid. Eleven young horses with tarsocrural OC were included in a randomised, double-blinded, placebo-controlled pilot clinical trial. Six horses received 250 mg/day HJ for 60 days (T60) and five horses received a placebo. The initial values of the degree of synovial effusion, NO, PGE2 and HA concentrations in synovial fluid and HA concentration in plasma were obtained. The horses were evaluated in terms of the same parameters at the end of treatment (T60) and 30 days thereafter (T90). The differences between the groups for each of the parameters evaluated at T0, T60 and T90 were not significant. Nevertheless, the horses treated with HJ tended to show a lower score for synovial effusion as well as higher HA, NO and PGE2 concentrations in synovial fluid, but these differences were non-significant. At a dose of 250 mg/day, HJ did not produce any adverse clinical effects and was well tolerated by the horses.

Effect of inflammatory environment on equine bone marrow derived mesenchymal stem cells immunogenicity and immunomodulatory properties.

Mesenchymal stem cells (MSCs) are being investigated for the treatment of equine joint diseases because of their regenerative potential. Recently, the focus mainly has addressed to their immunomodulatory capacities. Inflammation plays a central role in joint pathologies, since the release of proinflammatory mediators to the synovial fluid (SF) leads to the activation of enzymatic degradation of the cartilage. MSCs can modulate the local immune environment through direct or paracrine interaction with immune cells, suppressing their proliferation and re-addressing their functions. Proinflammatory molecules can induce MSC immunoregulatory potential, but they could also increase the expression of immunogenic molecules. Studying the effect of inflammatory environment on MSC immunomodulation and immunogenicity profiles is mandatory to improve cellular therapies. The aim of this study was to analyse the response of equine bone marrow MSCs (eBM-MSCs) to three inflammatory conditions. Equine BM-MSCs from three animals were exposed to: (a) 20% allogeneic inflammatory SF (SF); (b) 50 ng/ml of TNFα and IFNγ (CK50) and (c) 20 ng/ml of TNFα and IFNγ (CK20). After 72 h of exposure, expression of immunogenic and immunomodulation-related molecules, including cell-to-cell contact and paracrine signalling molecules, were analysed by RT-qPCR and flow cytometry. The gene expression of adhesion molecules was upregulated whereas MSC migration-related genes were downregulated by all inflammatory conditions tested. CK culture conditions significantly upregulated the expression of COX-2, iNOS, IDO and IL-6. MHC-I gene expression was upregulated by all conditions, whereas MHC-II was upregulated only after CK priming. The expression of CD40 did not significantly change, whereas the ligand, CD40L, was downregulated in CK conditions. Flow cytometry showed an increase in the percentage of positive cells and mean fluorescence intensity (MFI) of the MHC-I and MHC-II molecules at CK50 conditions, supporting the gene expression results. These outcomes reinforce the change of the immunophenotype of the eBM-MSCs according to the surrounding conditions. Inflammatory synovial environment did not lead to significant changes, so the environment found by eBM-MSCs when they are intraarticular administered may not be enough to activate their immunomodulatory potential. CK priming at tested doses enhances the immunoregulatory profile of eBM-MSCs, which may promote a therapeutic benefit. Even if CK priming induced an upregulation of MHC expression, costimulatory molecule expression however was not upregulated, suggesting that immunogenicity might not be increased. This study provides a better understanding about the behaviour of eBM-MSCs inside the inflamed joint and constitutes a first step to improve MSC-based therapies for equine joint diseases.