Leukocyte inclusion within a platelet rich plasma-derived fibrin scaffold stimulates a more pro-inflammatory environment and alters fibrin properties

Abstract

One of the main differences among platelet-rich plasma (PRP) products is the inclusion of leukocytes that may affect the biological efficacy of these autologous preparations. The purpose of this study was to evaluate whether the addition of leukocytes modified the morphological, biomechanical and biological properties of PRP under normal and inflammatory conditions. The release of pro-inflammatory cytokines from plasma rich in growth factors (PRGF) and leukocyte-platelet rich plasma (L-PRP) scaffolds was determined by enzyme-linked immunosorbent assay (ELISA) and was significantly increased under an inflammatory condition when leukocytes were included in the PRP. Fibroblasts and osteoblasts treated with L-PRP, under an inflammatory situation, underwent a greater activation of NFĸB pathway, proliferated significantly less and secreted a higher concentration of pro-inflammatory cytokines. These cellular events were assessed through Western blot and fluorimetric and ELISA methods, respectively. Therefore, the inclusion of leukocytes induced significantly higher pro-inflammatory conditions.

Fig 1. Illustrative representation of the cell treatment experimental procedure with PRGF and L-PRP releasates under inflammatory conditions.

Fig 2. Composition and structure of fibrin net from both PRP products.

(A) Concentration of platelets in PRGF and L-PRP preparations. (B) Leukocyte content in PRGF and L-PRP preparations. (C) Macroscopic appearance and ultraestructural composition of PRGF and L-PRP scaffolds.

Fig 3. Characterization of the PRP scaffold releasates.

Concentration of different cytokines, (A) IL1-beta, (B) TNF-alpha, (C) IL-6 and (D) IL-8 in the PRGF and L-PRP releasate under both normal and inflammatory conditions. *Statistically significant differences respect to the other treatments (p < 0.05). $Statistically significant differences respect to the PRGF treatment under normal conditions (p < 0.05).

Fig 4. Characterization of the PRP scaffold releasate.

Determination of different proteins concentration. (A) Angiopoietin-1 (B) VEGF and (C) PDGF-AB in the PRGF and L-PRP releasate under both normal and inflammatory conditions. *Statistically significant differences respect to the other treatments (p < 0.05).

Fig 5. Analysis of the integrity and the mechanical properties of PRP scaffolds.

(A) Comparison of PRP scaffolds appearance after 72 hours incubations under normal and inflammatory conditions. In half of the L-PRP matrix a great degradation was observed. (B) MMP-1 activity in PRGF and L-PRP releasates was determined under both normal and inflammatory conditions. (One U = 100 pmol/min at 37°C, 100 μM thiopeptolide). (C) Mechanical testing station where the maximum elongation at failure of the PRP scaffolds was determined. (D) Maximum elongation of PRP scaffolds. *Statistically significant differences between L-PRP and PRGF under normal conditions and between L-PRP and PRGF under inflammatory conditions (p < 0.05).^# Statistically significant differences respect to the PRGF treatment under normal conditions (p < 0.05).

Fig 6. Analysis of the inflammatory mediators’ expression by western blot.

Cells were treated with PRGF+LPS and L-PRP+LPS releasates for 24 hours under inflammatory conditions and IĸB-α and p-NFĸB/NFĸB ratio were determined. (A) IĸB-α expression in gingival fibroblasts. (B) IĸB-α expression in alveolar osteoblasts. (C) p-NFĸB/NFĸB determination in gingival fibroblasts. (D) p-NFĸB/NFĸB determination in alveolar osteoblasts. *Statistically significant differences respect to the PRGF+LPS treatment (p < 0.05).

Fig 7. Effect of the releasates of PRGF+LPS and L-PRP+LPS on the proliferation of fibroblasts and osteoblast cells.

(A) Morphological alterations in cultures due to 72 hour treatment with PRP releasates under inflammatory conditions. Scale bar: 300 μm. (B) Gingival fibroblast proliferation after treatment. (C) DNA quantification of alveolar osteoblast proliferation after treatment. *Statistically significant differences respect to the L-PRP+LPS treatment (p < 0.05).

Fig 8. Pro-inflammatory cytokine synthesis due to cell treatment with PRP releasates.

Gingival and bone cells were incubated with PRGF+LPS and L-PRP+LPS releasates under inflammatory conditions for 24 and 72 hours. IL-1β, TNF-α, IL-6 and IL-8 synthesis was determined by ELISA techniques. (A) Treatment of gingival fibroblast during 24 hours. (B) Gingival fibroblast response to 72 hour treatment. (C) Treatment of alveolar osteoblasts during 24 hours. (D) Bone cells response to 72 hour treatment. *Statistically significant differences respect to the PRGF+LPS treatment (p < 0.05).