Platelet-Rich Fibrin Decreases the Inflammatory Response of Mesenchymal Cells

Abstract

Chronic inflammation is a pathological process where cells of the mesenchymal lineage become a major source of inflammatory mediators. Platelet-rich fibrin (PRF) has been shown to possess potent anti-inflammatory activity in macrophages, but its impact on mesenchymal cells has not been investigated. The aim of this study was, therefore, to expose mesenchymal cells to inflammatory cytokines together with lysates generated from liquid platelet-poor plasma (PPP), the cell-rich buffy coat layer (BC; concentrated-PRF or C-PRF), and the remaining red clot layer (RC), following centrifugation of blood. Heating PPP generates an albumin gel (Alb-gel) that when mixed back with C-PRF produces Alb-PRF. Membranes prepared from solid PRF were also subjected to lysis. We report here that lysates of PPP, BC, and PRF decreased the cytokine-induced expression of interleukin 6 (IL6) and nitric oxide synthase (iNOS) in the bone marrow-derived ST2 cells. Consistently, PPP, BC, and PRF greatly decreased the phosphorylation and nuclear translocation of p65 in ST2 cells. The inflammatory response caused by Pam3CSK4 was reduced accordingly. Moreover, PPP, BC, and PRF reduced the enhanced expression of inflammatory mediators IL6 and iNOS in 3T3-L1 pre-adipocyte mesenchymal cells, and iNOS and CCL5 in murine calvarial cells. Surprisingly, PRF lysates were not effective in reducing the inflammatory response of human gingival fibroblasts and HSC2 epithelial cells. The data from the present study suggest that both liquid PRF and solid PRF exert potent anti-inflammatory activity in murine mesenchymal cells.

Keywords: Toll-like receptor; cytokine; inflammation; mesenchymal cells; platelet-rich fibrin.

Figure 1

PPP, BC, and PRF lysates reduced cytokine-induced inflammation in murine ST2 cells. ST2 cells were treated with 10% of PPP, BC, Alb-gel, RC, and 30% of PRF in the presence of the inflammatory cytokines TNFα and IL1β. (A,B) Data indicate the x-fold changes of IL6 and iNOS gene expression (C) and the IL6 levels in the cell supernatant, n = 4. WO means without and represents unstimulated cells.

Figure 2

PPP, BC, and PRF lysates reduced cytokine-induced inflammation in murine 3T3-L1 cells. 3T3-L1 cells were treated with 10% of PPP, BC, Alb-gel, RC, and 30% of PRF in the presence of the inflammatory cytokines TNFα and IL1β. (A,B) Data indicate the x-fold changes of IL6 and iNOS expression (C) and the IL6 levels in the cell supernatant, n = 4.

Figure 3

PPP, BC, and PRF lysates reduced inflammation in murine ST2 cells preincubated by BMP2. ST2 cells were treated with BMP2 for 48 h, followed by addition of 10% PPP, BC, and 30% PRF in the presence of the inflammatory cytokines TNFα and IL1β. (A,B) Data indicate the x-fold changes of IL6 and iNOS gene expression, n = 4.

Figure 4

PPP, BC, and PRF lysates reduced inflammation in murine calvaria-derived cells. Calvaria cells were exposed to 10% of PPP, BC, and 30% of PRF in the presence of the inflammatory cytokines TNFα and IL1β. (A,B) Data indicate the x-fold changes of iNOS and CCL5 gene expression, n = 4.

Figure 5

None of the fractions reduced inflammation in gingival fibroblasts and HSC2. Gingival fibroblasts and HSC2 were incubated with 10% of PPP, BC, and 30% of PRF in the presence of TNFα and IL1β. In gingival fibroblasts, 10% of Alb-gel and RC were also added. (A,B) Data indicate the x-fold changes of IL6 gene expression, n = 4.

Figure 6

PPP, BC, and PRF could weaken phosphorylation of p65 in ST2 cells. Western blot analysis was carried out for phospho-p65 and total p65. (A) ST2 cells were treated with TNFα and IL1β in the presence or absence of 10% of PPP, BC, Alb-gel, RC, and 30% of PRF lysates. (B) Data indicate the relative changes normalized to p65.

Figure 7

PPP, BC, and PRF attenuated the translocation of NF-κB from the cytoplasm into the nucleus. ST2 cells were exposed to TNFα and IL1β with or without PPP, BC, Alb-gel, RC, and PRF. Immunofluorescence analysis of intracellular translocation of NF-κB p65 into the nucleus. Blue nuclei indicate the unstained cells and green nuclei are positive stained cells. WO means without and represents unstimulated cells.

Figure 8

PPP, BC, and PRF can reduce inflammation provoked by TLR2 agonist in ST2 cells. The ST2 cells were exposed to 10% PPP, BC, and 30% PRF lysates in the presence of 5 μg/mL Pam3CSK4, agonists of TLR2. (A,B) Data show the x-fold changes of IL6 and iNOS gene expression, and (C) the concentration of IL6 in the supernatant of ST2 cells. n = 4.