Ce Giraldo

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.

In vitro bactericidal activity of equine platelet concentrates, platelet poor plasma, and plasma against methicillin-resistant Staphylococcus aureus

Los objetivos del estudio fueron: 1) evaluar el efecto antibacteriano de concentrados de plaquetas equinas (ePC) (activados o no con gluconato de calcio (CG)) frente a Staphylococcus aureus meticilino-resistente (MRSA) y 2) comparar su efecto antibacteriano contra plasma pobre en plaquetas (PPP) (activado con CG -PPP/GC-) y plasma (P). Los productos sanguineos fueron divididos en 4 grupos (ePC, ePC/CG, PPP/CG y P), mas un grupo control positivo (PCG) y otro control negativo. Los grupos se mezclaron con caldo Mueller-Hinton y MRSA. Las muestras fueron incubadas durante 1, 4, 8, 12 y 24 horas y se contaron las unidades formadoras de colonias. El crecimiento de las bacterias fue significativamente (P = 0,01) inhibido por el ePC, ePC/CG, PPP/CG y P en comparacion con el PCG durante las primeras 12 h. Solo a las 24 horas hubo una diferencia estadisticamente significativa (P = 0.01) y se observo un efecto antibacteriano para el ePC, ePC/CG y PPP/CG en comparacion con el PCG y P. Los ePCs y PPP equinos mostraron el mejor efecto antibacteriano in vitro contra el MRSA.

Uso de concentrados autólogos de plaquetas como terapia regenerativa de enfermedades crónicas del aparato musculoesquelético equino

SUMMARY Platelets are of pivotal importance for wound healing since they release growth factors that in turns produce chemotaxis, both cellular proliferation and differentiation, angiogenesis, and extracellular matrix deposition. The use of autologous platelet concentrates (APCs) has been proposed for accelerating wound healing, decreasing inflammation, to stimulate the regenerative capability of the injured tissues, to decrease the fibroblastic activity and to avoid the production of non functional scarring tissue. APCs could be obtained by several methods. Each method produces APCs of different both cellular and molecular quality. Recently, some basic and clinical information that justifies the usage of APCs for the treatment of degenerative musculoskeletal diseases in horses, such as osteoarthritis, tendinopathies and desmopathies has been published.

Effects of sodium citrate and acid citrate dextrose solutions on cell counts and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel

BackgroundThere is a lack information on the effects of the most commonly used anticoagulants for equine platelet rich plasmas (PRPs) elaboration on cell counts and growth factor release from platelet rich gels (PRGs). The aims of this study were 1) to compare the effects of the anticoagulants sodium citrate (SC), acid citrate dextrose solution A (ACD-A) and ACD-B on platelet (PLT), leukocyte (WBC) and on some parameters associated to platelet activation including mean platelet volume (MPV) and platelet distribution width (PDW) between whole blood, pure PRP (P-PRP) and platelet-poor plasma (PPP); 2) to compare transforming growth factor beta 1 (TGF-β1) and platelet-derived growth factor isoform BB (PDGF-BB) concentrations in supernatants from pure PRG (P-PRG), platelet-poor gel (PPG), P-PRP lysate (positive control) and plasma (negative control); 3) to establish the possible correlations between all the studied cellular and molecular parameters.ResultsIn all cases the three anticoagulants produced P-PRPs with significantly higher PLT counts compared with whole blood and PPP. The concentrations of WBCs were similar between P-PRP and whole blood, but significantly lower in PPP. The type of anticoagulant did not significantly affect the cell counts for each blood component. The anticoagulants also did not affect the MPV and PDW parameters. Independently of the anticoagulant used, all blood components presented significantly different concentrations of PDGF-BB and TGF-β1. The highest growth factor (GF) concentrations were observed from P-PRP lysates, followed by PRG supernatants, PPP lysates, PPG supernatants and plasma. Significant correlations were observed between PLT and WBC counts (ρ = 0.80), PLT count and TGF-β1 concentration (ρ = 0.85), PLT count and PDGF-BB concentration (ρ = 0.80) and PDGF-BB and TGF-β1 concentrations (ρ = 0.75). The type of anticoagulant was not correlated with any of the variables evaluated.ConclusionsThe anticoagulants did not significantly influence cell counts or GF concentrations in equine PRP. However, ACD-B was apparently the worst anticoagulant evaluated. It is necessary to perform additional research to determine the effect of anticoagulants on the kinetics of GF elution from P-PRG.

Bacteriostatic effect of equine pure platelet-rich plasma and other blood products against methicillin-sensitive Staphylococcus aureus. An in vitro study.

OBJECTIVES 1) To evaluate the bacteriostatic in vitro effect of pure platelet-rich plasma (P-PRP), pure platelet-rich gel (P-PRG), leukocyte-poor gel (LPG), platelet-poor plasma (PPP), and heat inactivated plasma (IP) against methicillin-sensitive Staphylococcus aureus (MSSA) over a period of 24 hours. 2) To determine the degradation of platelet factor-4 (PF-4), transforming growth factor beta 1 (TGF-β1), and platelet-derived growth factor isoform BB (PDGF-BB) in these equine blood components. 3) To establish correlations between platelet and leukocyte counts, PF-4 concentrations, and MSSA growth. METHODS Fourteen horses were used. Blood components were obtained by a manual protocol. Every blood component was mixed with MSSA and Müller-Hinton Broth and cultured at 37°C for 24 hours. Samples for the determination of bacterial growth (colony-forming units) and PF-4, TGF-β1 and PDGF-BB concentrations were taken at one, four, eight, 12 and 24 hours. RESULTS The bacterial growth was significantly (p = 0.01) inhibited for P-PRP, P-PRG, LPG and PPP in comparison with IP and, the positive control group during the first 12 hours. The P-PRG had higher and sustained TGF-β1 and PDGF-BB concentrations over time in comparison with the other blood components. CLINICAL SIGNIFICANCE The plasma complement could be one of the most responsible components of the in vitro bacteriostatic effect of P-PRP, P-PRG, LPG and PPP against MSSA. Additionally, P-PRG was the better biomaterial because it had an acceptable bacteriostatic effect and the highest concentration of growth factors.

Monitoring bacterial contamination in equine platelet concentrates obtained by the tube method in a clean laboratory environment under three different technical conditions

REASONS FOR PERFORMING STUDY There is a growing interest in the use of autologous platelet concentrates (PCs) as treatment for chronic musculoskeletal diseases in horses. However, there is no information on the risk of bacterial contamination during their preparation. OBJECTIVES To: 1) assess the risk of bacterial contamination in equine PCs obtained by the tube method under 3 technical conditions: a) in a laminar flow cabinet, in a clean laboratory environment both with (b) and without (c) Bunsen burner; 2) identify the critical points of the process of PCs preparation with risk of bacterial contamination; and 3) identify the potential bacterial contaminants in the process and their antibiotic susceptibility. METHODS Bacteriological samples were taken from: the skin (shaved or unshaved) of the venipuncture site in 15 horses, both before and after being disinfected; hands and throat of the operator; caps of the tubes where the blood was processed; environment where the equine blood samples were collected; laboratory environment; laminar flow cabinet; bacteriological stove; and PCs obtained under 3 technical conditions. RESULTS Bacteria were isolated from nonaseptically prepared equine skin, hands and throat of the operator, and the place where the blood samples were taken. Bacteria were not isolated from tube caps, laboratory environment, laminar flow cabinet or PCs. The isolated bacteria were normal biota from equine skin, human skin and throat, and environmental contaminants. Of the isolated bacteria, 23% were resistant to penicillin, 19% to ampicillin, 2.12% to ceftiofur, 3.2% to sulphamethoxazole/trimethoprim and 1.1% to enrofloxacin. Resistance to amikacin and gentamicin was not seen. CONCLUSIONS AND POTENTIAL RELEVANCE Uncontaminated PCs can be obtained by the tube method in a clean laboratory environment without the need for either a laminar flow cabinet or a Bunsen burner. It is mandatory to perform the procedure following strict aseptic technique.

Contaminación bacteriana en concentrados de plaquetas de caballos

SUMMARY The aims of the study were to: 1) assess the risk of bacterial contamination in equine platelet concentrates (PCs) obtained by the tube method under three technical conditions (laminar flow cabinet or in a clean laboratory environment both with burner and without burner) 2) identify the critical points of the process of PCs preparation with risk of bacterial contamination; and 3) identify the potential bacterial contaminants in the process. Bacteriological samples were taken from the skin (shaved or unshaved) of the venipuncture site in 15 horses, both before and after being disinfected; hands and throat of the operator; caps of the tubes where the blood was processed; environment where the equine blood samples were collected; laboratory environment; laminar flow cabinet; bacteriological stove; and PCs obtained under 3 technical conditions. Bacteria were isolated from non-aseptically prepared equine skin, hands and throat of the operator, and the place where the blood samples were taken. Bacteria were not isolated from tube caps, laboratory environment, laminar flow cabinet, or PCs. The isolated bacteria were normal biota from equine skin, human skin and throat, and environmental contaminants. Uncontaminated PCs can be obtained by the tube method in a clean laboratory environment.

Evaluación de un método de doble centrifugación en tubo para concentrar plaquetas bovinas: estudio celular

Resumen es: El objetivo de este estudio fue evaluar un metodo de centrifugado doble en tubo para concentrar plaquetas bovinas. Muestras de sangre fueron recolect...

Conjuntivectomía periglandular: Una nueva alternativa para el tratamiento quirúrgico del prolapso de la glándula del tercer párpado en caninos

El prolapso de la glandula del tercer parpado se presenta con frecuencia en perros braquiocefalicos, aunque algunas razas meso o dolicocefalicas como Cocker Spaniel y Beagle, entre otras, pueden padecer esta patologia. En este articulo se describe una nueva y sencilla tecnica quirurgica para reubicar la glandula del tercer parpado prolapsada mediante la creacion de un bolsillo subconjutival acompanado de eliminacion parcial del tejido conjuntival que recubre la glandula prolapsada. La ventaja de este procedimiento quirurgico radica en que los componentes anatomico y fisiologico son ampliamente conservados

Influence of calcium salts and bovine thrombin on growth factor release from equine platelet-rich gel supernatants

OBJECTIVE To compare five activation methods in equine platelet-rich plasma (PRP) by determination of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) concentrations in platelet-rich gel (PRG) supernatants. METHODS Platelet-rich plasma from 20 horses was activated by calcium chloride (CC), calcium gluconate (CG), bovine thrombin (BT), and their combinations, BTCC and BTCG. Both growth factor concentrations in PRG supernatants were measured by ELISA and compared with plasma and platelet lysates (PL) over time. RESULTS Growth factor concentrations were significantly lower in plasma and higher for all PRG supernatants. Platelet lysates contained a significantly lower concentration of PDGF-BB than PRG supernatants and a significantly higher concentration of TGF-β1 than PRG supernatants. Clots from PRP activated with sodium salts were more stable over time and had significant growth factor release, whereas CC produced gross salt deposition. Significant correlations were noticed for platelet with leukocyte concentrations in PRP (rs: 0.76), platelet counts in PRP with TGF-β1 concentrations in PRG supernatants (rs: 0.86), platelet counts in PRP with PDGF-BB concentrations in PRG supernatants (rs: 0.78), leukocyte counts in PRP with TGF-β1 concentrations in PRG supernatants (rs: 0.76), and PDGF-BB concentrations with activating substances (rs: 0.72). CLINICAL SIGNIFICANCE Calcium gluconate was the better substance to induce PRP activation. It induced growth factor release free from calcium precipitates in the clots. Use of BT alone or combined with calcium salts was not advantageous for growth factor release.