New Formulation of Platelet-Rich Plasma Enriched in Platelet and Extraplatelet Biomolecules Using Hydrogels

Abstract

Platelet-rich plasma (PRP) is an autologous biologic product used in several fields of medicine for tissue repair due to the regenerative capacity of the biomolecules of its formulation. PRP consists of a plasma with a platelet concentration higher than basal levels but with basal levels of any biomolecules present out of the platelets. Plasma contains extraplatelet biomolecules known to enhance its regenerative properties. Therefore, a PRP containing not only a higher concentration of platelets but also a higher concentration of extraplatelet biomolecules that could have a stronger regenerative performance than a standard PRP. Considering this, the aim of this work is to develop a new method to obtain PRP enriched in both platelet and extraplatelet molecules. The method is based on the absorption of the water of the plasma using hydroxyethyl acrylamide (HEAA)-based hydrogels. A plasma fraction obtained from blood, containing the basal levels of platelets and proteins, was placed in contact with the HEAA hydrogel powder to absorb half the volume of the water. The resulting plasma was characterized, and its bioactivity was analyzed in vitro. The novel PRP (nPRP) showed a platelet concentration and platelet derived growth factor (PDGF) levels similar to the standard PRP (sPRP), but the concentration of the extraplatelet growth factors IGF-1 (p < 0.0001) and HGF (p < 0.001) were significantly increased. Additionally, the cells exposed to the nPRP showed increased cell viability than those exposed to a sPRP in human dermal fibroblasts (p < 0.001) and primary chondrocytes (p < 0.01). In conclusion, this novel absorption-based method produces a PRP with novel characteristics compared to the standard PRPs, with promising in vitro results that could potentially trigger improved tissue regeneration capacity.

Keywords: biomolecules; growth factors; hydrogel; platelet-rich plasma; platelets; water absorption.

Figure 1

Water absorption and platelet concentration capacity of hydroxyethyl acrylamide (HEAA) hydrogel. (A) Plot of water % absorption and platelet concentration upon exposure of 4 mL of plasma samples to different weights of hydrogels ranging from 0 to 0.6 g in a specific time set in 5 min. (B) Water absorption capacity (%) of 0.4 g of HEAA hydrogel in relation to time. (C) Comparison of blood, the plasma obtained after the centrifugation and the novel PRP (nPRP) obtained after hydrogel exposure, in terms of platelet content. Error bars = standard deviation (n = 8). Statistically significant differences were calculated using one-way ANOVA (**** p < 0.0001).

Figure 2

Platelet and total protein concentration levels in standard and novel PRP. Mean values of platelet (A) and protein (B) in blood and standard and novel PRP are shown. Error bars = standard deviation (n = 8). Statistically significant differences were calculated using Kruskal–Wallis for the platelet count and one-way ANOVA for the total protein levels (*** p < 0.001; **** p < 0.0001).

Figure 3

Platelet and extraplatelet growth factor levels. Mean values of platelet growth factor PDGF (A), the extra-platelet IGF-1 growth factor (B), and the intra- and extra-platelet growth factor HGF (C) of blood and standard and novel PRP are shown. Error bars = standard deviation (n = 8). Statistically significant differences were calculated using one-way ANOVA (** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 4

Ion concentration of the standard and novel PRP. Mean values of sodium (A), potassium (B), chlorine (C), calcium (D), phosphorous (E), and magnesium (F) are shown. Error bars = standard deviation (n = 8). Statistically significant differences were calculated using one-way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 5

Platelet activation in standard and novel PRP. The graph represents the CD62-positive cells that are indicative of activated platelets. Error bars = standard deviation (n = 8). Statistically significant differences were calculated using t-test (*** p < 0.001).

Figure 6

Fibrinogen levels and clot properties of the standard and novel PRP. Mean values of fibrinogen levels (A) and Young’s module of the fibrin clot (B) of the standard and novel PRP are shown. Error bars = standard deviation (n = 8). Statistically significant differences were calculated using t-test (* p < 0.05; **** p < 0.0001).

Figure 7

Cellular viability in NHDF cells and human primary chondrocytes. The viability levels of (A) NHDF cells and (B) human primary chondrocytes incubated with standard and novel PRP are expressed as relative light units (RLUs), and each point represents a different donor (n = 8). Serum-free conditions served as negative control. Statistical analysis was calculated using one-way ANOVA (**** p < 0.0001).

Figure 8

PRP obtaining process. (A) Schematic representation of the process to obtain sPRP: 2 mL plasma above the red blood cell fraction is collected from an initial volume of 9 mL of whole blood, with expected doubled concentration of platelets and total proteins. (B) Schematic representation of the process to obtain nPRP using 0.5–1 mm size hydrogel particles. A basal plasma fraction is obtained with expected similar levels of platelets and total protein as in blood (1×). After water absorption and separation of the hydrogel from the concentrated plasma, the final product obtained is a PRP expected to double its concentration of extraplatelet biomolecules and platelets (2×).