Leucocyte-Rich Platelet-Rich Plasma Enhances Fibroblast and Extracellular Matrix Activity: Implications in Wound Healing

Abstract

Background: Platelet-rich plasma (PRP) is an autologous blood product that contains a high concentration of platelets and leucocytes, which are fundamental fibroblast proliferation agents. Literature has emerged that offers contradictory findings about leucocytes within PRP. Herein, we elucidated the effects of highly concentrated leucocytes and platelets on human fibroblasts.

Methods: Leucocyte-rich, PRP (LR-PRP) and leucocyte-poor, platelet-poor plasma (LP-PPP) were compared to identify their effects on human fibroblasts, including cell proliferation, wound healing and extracellular matrix and adhesion molecule gene expressions.

Results: The LR-PRP exhibited 1422.00 ± 317.21 × 10³ platelets/µL and 16.36 ± 2.08 × 10³ white blood cells/µL whilst the LP-PPP demonstrated lower concentrations of 55.33 ± 10.13 × 10³ platelets/µL and 0.8 ± 0.02 × 10³ white blood cells/µL. LR-PRP enhanced fibroblast cell proliferation and cell migration, and demonstrated either upregulation or down-regulation gene expression profile of the extracellular matrix and adhesion molecules.

Conclusion: LR-PRP has a continuous stimulatory anabolic and ergogenic effect on human fibroblast cells.

Keywords: PRP; extracellular matrix; fibroblasts; leucocytes; platelet gel; platelet-rich fibrin; platelet-rich plasma; wound.

Figure 1

MTT Fibroblast Proliferation Assay. LR-PRP, LP-PPP, serum-free media (negative control) measured at 5 time points 4, 8, 12, 24, 48 h. Data were analyzed and the means for n = 3 samples (6 replicate wells for each sample) were calculated and presented as plus or minus (±) the standard error of the mean (SEM) using a two-way ANOVA and Tukey’s multiple comparison tests. Symbols represent the p value; * p < 0.024 significant difference, ** p < 0.003 very significant difference, *** p < 0.001 highly significant difference. Abbreviations: Leucocyte-rich, platelet-rich plasma (LR-PRP), leucocyte-poor, platelet poor plasma (LP-PPP).

Figure 2

Scratch wound assay. Wound closure for each time point and group is measured in percentages. Data were analyzed for n = 3 samples (6 replicate wells for each sample) using the mean ± standard error of the mean (SEM) using a two-way ANOVA and Tukey’s multiple comparison tests (* p = 0.033 statistically significant difference, *** p < 0.001 very highly statistically significant difference). Abbreviations: Leucocyte-rich-platelet-rich plasma (LR-PRP), leucocyte-poor-platelet poor plasma (LP-PPP), negative control (NC).

Figure 3

The human extracellular matrix and adhesion molecules RT² Profiler PCR gene array. Fibroblasts supplemented with (A) LR-PRP versus LP-PPP (control) at 12 h or (B) LR-PRP versus serum-free media (negative control) representing significant up- and down-regulated genes (more than two-fold) and those with no significant change (less than two-fold). Abbreviations: MMP1, matrix metallopeptidase 1 (collagenase); MMP3, matrix metallopeptidase 3 (stromelysin); MMP9, matrix metallopeptidase 9 (gelatinase); MMP11, matrix metallopeptidase 11 (stromelysin); MMP12, matrix metallopeptidase 12 (metalloelastase); ITGA2, integrin alpha 2; ITGA6, integrin, alpha 6; ITGA8, integrin alpha 8; ITGAM, integrin alpha M chain; ITGB4, integrin beta 4; TIMP3, metalloproteinase inhibitor 3; NCAM1, neural cell adhesion molecule 1; VCAM1, vascular cell adhesion molecule 1, leucocyte-rich—platelet-rich plasma (LR-PRP), leucocyte-poor—platelet poor plasma (LP-PPP). Letter A specifies that the gene’s average threshold cycle is relatively high (>30) in either the control or the test sample and is reasonably low in the other sample (<30). These data mean that the gene’s expression is relatively low in one sample and reasonably detected in the other sample, which suggests that the actual fold-change value is at least as large as the calculated and reported fold-change result. Letter B specifies that the gene’s average threshold cycle is relatively high (>30), meaning that its relative expression level is low, in both control and test samples, and the p value for the fold-change is either unavailable or relatively high (p > 0.05).