Comparison of the Cellular Composition and Cytokine-Release Kinetics According to Different Preparations of Platelet-rich Plasma (PRP)

Abstract

Introduction: Variations in formulations used to prepare or activate platelet-rich plasmas (PRPs) result in differences in the cellular composition and biological activity of the platelets. This study evaluates the cellular composition and the cytokine release kinetics of PRP according to differences in the preparation and activation protocols. Methods: Blood samples were obtained from 14 healthy subjects, and five preparation procedures were performed for each donor including two methods that used manual procedures [single-spin (SS) at 900 g for 5 min

double-spin (DS) at 900 g for 5 min and then 1500 g for 15 min] and three methods with commercial kits (Arthrex ACP, Biomet GPS, and Prodizen Prosys). Each PRP preparation was divided into four aliquots and was incubated for 1 h, 24 h, 72 h, and 7 days. The cytokine release kinetics were evaluated by assessing the platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), interleukin-1 (IL-1), and matrix metalloproteinase-9 (MMP-9) concentrations with commercially available bead-based sandwich immunoassay kits. The SS and DS PRP preparations were tested for platelet activation with three different conditions: no activation, activation with calcium only, or calcium with a low dose of thrombin. Results: The DS PRP had a higher concentration of platelets (x 4) and leukocytes (x 9) than the SS PRP. Both PRP preparations exhibited an increase in PDGF, TGF, VEGF, and FGF release when compared to whole-blood samples, and the IL-1 and MMP-9 concentrations of the SS or DS PRP preparations were lower than those of whole-blood samples during the 7 days of the experiment. The cytokine release kinetics varied for the different cytokines, and the FGF and the TGF release occurred quickly, with maximum concentrations detected within 1 h, and then decreased over time. The PDGF and VEGF release was constant and was sustained over 7 days. The PDGF and VEGF concentrations were higher in the DS PRP than in the SS PRP, whereas the TGF and FGF concentrations were higher in the SS PRP than in the DS PRP. With respect to the commercial PRP kits, Arthrex ACP had cytokine release kinetics similar to those of SS PRP, and Prodizen Prosys had kinetics similar to those of DS PRP. Among the three commercial kits that were used, Biomet GPS had the highest platelet and leukocyte concentrations and the highest VEGF and MMP-9 concentrations but the lowest TGF concentration. Arthrex ACP had the highest FGF concentration but the lowest PDGF concentration. Ca-only activation had a significant effect on the DS PRP preparations (where the VEGF, FGF, and IL-1 concentrations were sustained) while Ca/thrombin activation had significant effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were not sustained). The IL-1 content showed a significant increase with Ca only or Ca/thrombin activation while Ca only or Ca/thrombin activation did not increase the MMP-9 concentration. Conclusions: The PRP preparations showed different qualities in terms of their cellular composition and their cytokine release kinetics, depending on the preparation and activation protocols that were used. The DS method generally led to a higher concentration of PLT relative to the SS method, and it was considered to be preferable for an efficient local cellular response of the PRP. However, the cytokine content was not necessarily proportional to the cellular composition of the PRPs since greater cytokine content could be different between the SS or the DS method, depending on the type of cytokine evaluated. In terms of the PRP activation, a low dose of thrombin/calcium activation increased the overall cytokine release of the PRP preparations over 7 days relative to that with a calcium-only supplement or non-activation.