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

BMC Veterinary Research 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 Carlos E Giraldo 0 Catalina Lpez 0 Mara E lvarez 0 Ismael J Samudio Marta Prades Jorge U Carmona 0 0 Grupo de Investigacion Terapia Regenerativa, Departamento de Salud Animal, Universidad de Caldas , Manizales , Colombia Background: There 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-1) 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-1 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. Results: PLT 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-1 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. Conclusions: Our 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. Horse; Platelet concentrate; Transforming growth factor beta 1; Platelet derived growth factor isoform BB; Regenerative therapy - Background The clinical use of platelet concentrates (PC) is currently a common biological therapy for musculoskeletal diseases [1-4] and wounds in horses [5]. PC intended for regenerative medicine are classified as: pure-platelet rich plasma (P-PRP), leukocyte-platelet rich plasma (L-PRP) and platelet rich fibrin (PRF). P-PRP and L-PRP are obtained in a liquid form by using anticoagulants. PRF is as a second generation PC that does not require anticoagulant for its elaboration [6]. Equine P-PRP displays slightly higher platelet counts (1.3-4 fold) and leukocyte (WBC) counts (0.5-2 fold) than whole blood. Equine L-PRP has increased platelet (5 fold) and leukocyte (3 fold or more) counts when compared to whole blood. When PC are activated by adding thrombin or a calcium salt, they form a fibrin polymer and are known as platelet gels (PG). Thus, PG from P-PRP is denominated as pure-platelet rich gel (P-PRG) and PG from L-PRP is termed leukocyteplatelet rich gel (L-PRG) [7]. Platelet gels release several growth factors (GF), such as transforming growth factor beta 1 (TGF-1) [8,9] and platelet derived growth factor type BB (PDGF-BB) [10]. These proteins have anti-inflammatory, anabolic and angiogenic effects [11]. Both GF are mainly stored in platelet alpha granules and their release correlates with the degree of platelet activation. The GF profile released from P-PRP or L-PRP will be determined by the cellular components concentrated in each substance [12]. Many questions emerge when a particular PC (either P-PRP or L-PRP) is proposed for regenerative therapy in horses. First, it is essential to know the cellular and GF profile of this substance before its (experimental or clinical) use. Several intrinsic and extrinsic aspects could influence the cellular and molecular features of a PC. Intrinsic factors such as breed, gender and, age or, extrinsic factors associated to the technique used for the PC preparation and activation could potentially influence on the final composition of these substances. This study evaluates the effect of intrinsic factors such as, the breed, gender and age on the cellular characteristics and release of TGF-1 and PDGF-BB from equine P-PRP/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-1) 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. The hypothesis from our study was that intrinsic factors, such as breed, gender and age could influence the final concentration of cells and growth factors in equine P-PRP/P-PRG. Results General hematological results There were statistically significant (P < 0.001) differences for all the general hematological variables evaluated between whole blood, P-PRP and PPP (Figure 1A), except MPV and PDW. Mean platelet volume values were similar between whole blood and PPP and both parameters differed (P < 0.001) from P-PRP. Platelet distribution width values were similar for whole blood and P-PRP; however these PDW values differed (P < 0.001) from same parameter in PPP (Table 1). General results for total protein, transforming growth factor beta 1 and platelet derived growth factor isoform BB Total protein concentration was significantly (P < 0.001) higher in P-PRP+NID when compared to plasma, P-PRG, PPP+NID and, PPG. Both TGF-1 and PDGFBB concentrations (either in pg/mL or pg/mg of TP) were significantly lower for plasma in comparison with the other blood components. P-PRP+NID and P-PRG showed the highest significant concentrations for these GF in comparison with plasma, PPP+NID and, PPG (Table 2 and Figure 1B). Breeds effect on hematological results Platelet and WBC counts for whole blood and PPP were statistically (P < 0.001) higher for CCH in comparison with ACH. Lymphocyte relative count was significantly (P < 0.001) higher in whole blood from CCH in comparison with ACH. GRA% was significantly (P < 0.001) (...truncated)