María E. Álvarez

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.

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.

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.

Temporal Bacteriostatic Effect and Growth Factor Loss in Equine Platelet Components and Plasma Cultured with Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus: A Comparative In Vitro Study

The aims were (1) to evaluate the bacteriostatic effect of platelet-rich plasma (PRP), platelet-rich gel (PRG), leukocyte-poor plasma (LPP), leukocyte-poor gel (LPG), plasma, and heat-inactivated plasma (IP) on both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) over a period of 24 h; (2) to determine and to compare the concentrations and degradation over time of platelet factor 4 (PF-4), transforming growth factor beta 1 (TGF-β 1), and platelet-derived growth factor isoform BB (PDGF-BB); and (3) to identify any correlations between MSSA and MRSA growth and either the cellular, PF-4, TGF-β 1, or PDGF-BB concentrations in the blood components. PRP and its byproducts from 18 horses were obtained by the tube method. All blood components were cultured with either MSSA or MRSA. Bacterial growth, PF-4, TGF-β 1, and PDGF-BB were determined at 6 h and 24 h. At six hours, bacterial growth was significantly inhibited by all blood components, with the exception of IP. MSSA was more sensitive to the treatments than MRSA. At 24 hours, bacterial growth was significantly higher in IP. MRSA bacterial growth was significantly higher in PRP, LPP, and plasma when compared to MSSA. Growth factor concentrations were not significantly affected by bacteria.

Evaluation of the effect of calcium gluconate and bovine thrombin on the temporal release of transforming growth factor beta 1 and platelet-derived growth factor isoform BB from feline platelet concentrates.

BackgroundThere are not reported regarding the protocols for obtaining platelet concentrates (PC) in cats for medical purposes. The objectives of this study were: 1) to describe a manual method for producing two kinds of PC in cats (PC-A and PC-B), 2) to describe the cellular population of the PC, 3) to measure and compare the effect of calcium gluconate (CG) and bovine thrombin (BT) on the temporal release of transforming growth factor beta 1 (TGF-β1) and platelet-derived growth factor type BB (PDGF-BB) at 3 and 12 hours post-activation and 4) to establish correlations between the cellular population of both PCs and the concentration of growth factors (GF). Blood samples were taken from 16 cats for complete blood count, plasma collection and PC preparation. The PC were arbitrarily divided into two fractions, specifically, PC-A (lower fraction) and PC-B (upper fraction).ResultsThe platelet counts were significantly different (P<0.05) between the PC and whole blood but not between the PC fractions. The TGF-β1 concentration efficiencies for PC-A and PC-B activated with CG were 42.86% and 46.54%, and activated with BT were 42.88% and 54.64%, respectively. The PDGF-BB concentration efficiencies for PC-A and PC-B activated with CG were 61.36% and 60.61%, and activated with BT were 65.64% and 72.12%, respectively. The temporal release of GFs showed no statistically significant difference (P>0.05) between the activating substances at the time or for any PC fraction.ConclusionsWhatever the activation means, these preparations of cat PC provide significant concentrations of platelets and GFs for possible clinical or experimental use.

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.

Proinflammatory and Anabolic Gene Expression Effects of Platelet-Rich Gel Supernatants on Equine Synovial Membrane Explants Challenged with Lipopolysaccharide

Platelet-rich plasma (PRP) preparations are used in horses with osteoarthritis (OA). However, some controversies remain regarding the ideal concentration of platelets and leukocytes to produce an adequate anti-inflammatory and anabolic response in the synovial membrane. The aims of this study were to study the influence of leukoconcentrated platelet-rich gel (Lc-PRG) and leukoreduced platelet-rich gel (Lr-PRG) supernatants on the quantitative expression of some proinflammatory and anabolic genes in equine synovial membrane explants (SMEs) challenged with lipopolysaccharide (LPS). SMEs from six horses were cultured over 96 h. Then, SMEs were harvested for RNA extraction and quantitative gene expression analysis by RT-qPCR for nuclear factor kappa B (NFκB), matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), collagen type I alpha 1 (COL1A1), collagen type II alpha 1 (COL2A1), and cartilage oligomeric matrix protein (COMP). The 25% and 50% Lc-PRG supernatants led to downregulation of NFκB, MMP-13, ADAMTS-4, COL1A1, COL2A1, and COMP in SMEs. Lr-PRG supernatants (particularly at the 50% concentration) induced downregulation of NFκB, MMP-13, ADAMTS-4, and COL1A1 and upregulation of COL2A1 and COMP. Lr-PRG supernatants should be used for the treatment of inflammatory arthropathies in horses because they have anti-inflammatory and anabolic effects in the synovial membrane.

Long-term cytokine and growth factor release from equine platelet-rich fibrin clots obtained with two different centrifugation protocols

Objectives: To compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF‐&agr;), interleukin 4 (IL‐4), IL‐1 receptor antagonist (IL‐1ra), platelet‐derived growth factor BB (PDGF‐BB) and transforming growth factor beta‐1 (TGF‐&bgr;1) from two platelet‐rich fibrin (PRF) preparations from equine blood obtained at either 240 g/8 min or 416 g/10 min. Methods: Whole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1 h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6 h, 24 h and then every 48 h to 21 days. Cytokines and GFs were measured by ELISA at 1 h (serum supernatants from PRF clots) and all time points of culture medium change. A negative control (plasma) and a positive control (blood lysate) were also included. Results: There were no relevant differences between the two protocols for the temporal release of proteins. However, a significant (p = 0.01) effect of time was noted. All cytokines were detected after 6 h of PRF clot culture until day 21. GF were detected at 1 h until day 21. The concentrations for these proteins diminished gradually over time. A highly significant (p = 0.01) correlation was noticed between all the proteins evaluated. Conclusions: Leukocytes enmeshed in PRF clots were able to produce cytokines, TGF‐&bgr;1 and PDGF‐BB. These findings demonstrate a paramount role of leukocytes in wound healing induced or modified by PRF clots in mammals. HighlightsPRF is a technology for the treatment of wounds in man and horses.Equine PRF retained and released cytokines and anabolic GFs over three weeks.Leukocytes are of paramount importance to produce cytokines in PRF.GFs are initially released od platelets from PRF and then released from leukocytes.TNF‐&agr; acts more as a regulatory protein than a proinflammatory cytokine in PRF.