The Biological Effect of Enriching the Plasma Content in Platelet-Rich Plasma: An In Vitro Study

Abstract

Background: Platelet-rich plasma (PRP) formulations have become valuable therapeutic tools in regenerative medicine. In addition, these blood derivates have been successfully included in cell therapy as fetal bovine serum substitutes, due to the real need to avoid the risk of host immunologic reactions and the animal disease transmission associated with reagents from animal origin. However, the protocols for obtaining them should be optimized to improve their biological potential.

Methods: PRP-derived preparations with different concentrations of the platelet and plasma components were obtained from the blood of five donors by freeze-drying. Measurements of the pH, protein, and growth factor concentration were performed. Moreover, their biological effects on cell proliferation and migration and their angiogenic potential were assessed.

Results: An increased plasma component concentration resulted in an augmented quantity of the total protein content, a significative variation in the hepatocyte growth factor concentration, and an experimental but clinically irrelevant alteration of the pH value. No significant changes were induced in their potential to enhance proliferative and migratory responses in epithelial cells, with the latter being reduced for dermal fibroblasts. The endothelial cell capacity for tube formation was significatively reduced.

Conclusions: An increased blood plasma content did not improve the biological potential of the formulations. However, they have emerged as a promising approach for regenerative therapies where neovascularization must be avoided.

Keywords: cell therapy; lyophilization; plasma content enrichment; platelet-rich plasma; tissue regeneration.

Figure 1

Schematic illustration showing the process for obtaining the 6 supernatant preparations prepared from plasma rich in growth factors (PRGF). PRGF: Plasma Rich in Growth Factors. 1-0 formulation: preparation with 1× plasma content and no platelets. 1-2 PRP: Plasma Rich in Platelets with 1× and 2× concentration factors in plasma and platelet contents, respectively. 1-4 PRP: Plasma Rich in Platelets with 1× and 4× concentration factors in plasma and platelet contents, respectively. Snt: supernatant for cell culture medium supplementation. 1-2 Snt: supernatant with 1× and 2× plasma and platelet content, respectively. 2-2 Snt: supernatant with 2× plasma and platelet content. 3-2 Snt: supernatant with 3× and 2× plasma and platelet content, respectively. 1-4 Snt: supernatant with 1× and 4× plasma and platelet content, respectively. 2-4 Snt: supernatant with 2× and 4× plasma and platelet content, respectively. 3-4 Snt: supernatant with 3× and 4× plasma and platelet content, respectively. Created with BioRender.com.

Figure 2

Characterization of the six supernatants obtained from formulations with different plasma and platelet contents. Values of pH (A), protein concentration (B), and growth factors concentrations (C) for the six preparations are graphically represented. Moreover, the effects of both the plasmatic and the platelet factors on these parameters were individually analyzed (D–F). 1-2 Snt: supernatant with the same plasma content (one times the plasma) and twice the platelet content of the peripheral blood (two times the platelets). 2-2 Snt: supernatant with twice the platelet and plasma contents of the peripheral blood. 3-2 Snt: supernatant with three- and two-times higher concentrations of plasma and platelet contents, respectively, than the peripheral blood. 1-4 Snt: supernatant with the same plasma content and a platelet concentration four times higher than the peripheral blood. 2-4 Snt: supernatant with twice the plasma content and a four-times higher concentration of platelet-derived factors, with respect to the blood. 3-4 Snt: supernatant with three and four times the concentrations of plasma and platelets, respectively, compared to the peripheral blood. ØStatistically significant differences with respect to the 2-4 Snt, 3-2 Snt, and 3-4 Snt groups. βStatistically significant differences with respect to the supernatants derived from formulations with different plasmatic factors. ¶Statistically significant differences with respect to the 1-2 Snt and 1-4 Snt groups. γStatistically significant differences with respect to the two times and three times plasmatic factor groups. ‡Statistically significant differences with respect to the four times platelet factor group. ¥Statistically significant differences with respect to the 3-4 Snt group (p ≤ 0.05) (n = 5).

Figure 3

Cell proliferation analysis. Cells from two phenotypes were treated with supernatants (Snts) derived from six PRP-derived formulations. The code of these supernatants consisted of two numbers referring to the plasma and platelet contents, respectively, compared to the peripheral blood. A double statistical analysis was performed to determine the effect of these supernatants on the proliferation rate induced by the specific composition of both plasma and platelet-derived factors (A), and, in addition, that induced by the plasma- or platelet-derived factors considered separately (B). The results are expressed as a percentage of the proliferation achieved by the cells maintained with the routine culture medium specific for each cell phenotype (the positive control). HDFs: human dermal fibroblasts; and HCE-1: human corneal epithelial cell. $Statistically significant differences with respect to the three times plasmatic factor group (p ≤ 0.05) (n = 5).

Figure 4

Cell chemotaxis analysis. Cells from two phenotypes were treated with supernatants (Snts) derived from six PRP-derived formulations. The code of these PRPs consisted of two numbers referring to the plasma and platelet contents, respectively, compared to the peripheral blood. A double statistical analysis was performed to determine the effect of these supernatants on the migration rate induced by the specific composition of both plasma- and platelet-derived factors (A) and, in addition, that induced by plasma or platelet-derived factors considered individually (B). The results are expressed as a percentage of the proliferation achieved by the cells maintained with the routine culture medium specific for each cell phenotype (the positive control). HDFs: human dermal fibroblasts; and HCE-1: human corneal epithelial cell (n = 5).

Figure 5

Endothelial cell proliferation analysis and angiogenesis assay. The endothelial cells were treated with supernatants derived from PRP with increasing concentrations of plasmatic factors and setting the platelet content at twice that of the peripheral blood (1-2 Snt, 2-2 Snt, and 3-2 Snt). The proliferation results are expressed as a percentage of the proliferation achieved by the cells under optimal conditions, maintained with the routine culture medium (the positive control) (A). For the angiogenesis assay, the endothelial cells were seeded in a µ-plate with 96 wells for a high-throughput 3D cell culture and tube formation assays in which a solubilized basement membrane preparation was previously added. The covered area (B), the number of loops (C), the total tube length (D), the total tubes (E), and the total branching points (F), considered as the main parameters of the angiogenic process, were measured. The results are expressed as a percentage with respect to the positive control (the routine culture medium). &Statistically significant differences with respect to the 3-2 Snt group. (p ≤ 0.05). HUVECs: human umbilical vein endothelial cells (n = 5).