A Novel Fibrin Matrix Derived from Platelet-Rich Plasma: Protocol and Characterization

Abstract

Although fibrin matrices derived from Platelet-Rich Plasma (PRP) are widely used in regenerative medicine, they have some limitations that can hinder their application. Modifying the composition of the PRP-derived fibrin matrix may improve its properties, making it suitable for certain medical uses. Three types of fibrin matrices were obtained: a PRP-derived fibrin matrix (FM), a PRP-derived fibrin matrix with a high fibrinogen content and platelets (FM-HFP) and a PRP-derived fibrin matrix with a high fibrinogen content (FM-HF). The fibrinogen levels, biomechanical properties and cell behavior were analyzed. The presence of platelets in the FM-HFP generated an inconsistent fibrin matrix that was discarded for the rest of the analysis. The fibrinogen levels in the FM-FH were higher than those in the FM (p < 0.0001), with a concentration factor of 6.86 ± 1.81. The values of clotting and swelling achieved using the FM-HF were higher (p < 0.0001), with less clot shrinkage (p < 0.0001). The FM had a significantly higher stiffness and turned out to be the most adherent composition (p = 0.027). In terms of cell viability, the FM-HF showed less cell proliferation but higher live/dead ratio values (p < 0.01). The increased fibrinogen and platelet removal in the FM-HF improved its adhesion and other biomechanical properties without affecting cell viability.

Keywords: fibrin; fibrinogen; growth factors; matrix; platelet-rich plasma; platelets; scaffold.

Figure 1

Fibrinogen concentration for matrix preparation. The graph represents the concentration of fibrinogen in blood (basal levels), in the PRP used to prepare the FM and in the fibrinogen concentrate (FC) used to create FM-HF. Error bars = standard deviation (n = 8). Statistically significant differences were calculated using one-way ANOVA (**** p < 0.0001).

Figure 2

Retraction of the matrices. Macroscopic images show the initial volume of FM (A) and FM-HF (B) and the volume after 24 h, showing retraction in FM (C) and not in FM-HF (D).

Figure 3

Surface of the matrices. SEM images show that the FM surface is rougher (A), with more contracted fibrin fibers (B). In contrast, the FM-HF surface is smoother (C), with more evenly arranged fibers (D).

Figure 4

Mechanical properties of the matrices. The graphs represent Young’s modulus (stiffness) (A), the dissipation energy (cushioning) (B) and the adhesion capacity (C) of the FM and the FM-HF. Error bars = standard deviation (n = 8). Statistically significant differences were calculated using Student’s t-test (* p < 0.05; **** p < 0.0001).

Figure 5

Images of live/dead assay. Fluorescence microscope images show live (green) and dead (red) cells at 24 h in the FM (A) and the FM-HF (C) and their evolution after 120 h of culture, with a greater increase in the number of live and dead cells in the FM (B) than in the FM-HF (D). Scale bar = 500 µm (5× objective).

Figure 6

Cell viability. Data show a greater increase in both live and dead cells in the FM (A) than in the FM-HF (B). In both matrices, there is proliferation over time; however, the live/dead ratio is higher in the FM-HF (C). Error bars = standard deviation (n = 4). Statistically significant differences were calculated using Student’s t-test (* p < 0.05; ** p < 0.01).