Liquid PRF Reduces the Inflammatory Response and Osteoclastogenesis in Murine Macrophages

Abstract

Macrophage activation and osteoclastogenesis are hallmarks of inflammatory osteolysis and may be targeted by the local application of liquid platelet-rich fibrin (PRF). Liquid PRF is produced by a hard spin of blood in the absence of clot activators and anticoagulants, thereby generating an upper platelet-poor plasma (PPP) layer, a cell-rich buffy coat layer (BC; termed concentrated-PRF or C-PRF), and the remaining red clot (RC) layer. Heating PPP has been shown to generate an albumin gel (Alb-gel) that when mixed back with C-PRF generates Alb-PRF having extended working properties when implanted in vivo. Evidence has demonstrated that traditional solid PRF holds a potent anti-inflammatory capacity and reduces osteoclastogenesis. Whether liquid PRF is capable of also suppressing an inflammatory response and the formation of osteoclasts remains open. In the present study, RAW 264.7 and primary macrophages were exposed to lipopolysaccharides (LPS), lactoferrin, and agonists of Toll-like receptors (TLR3 and TLR7) in the presence or absence of lysates prepared by freeze-thawing of liquid PPP, BC, Alb-gel, and RC. For osteoclastogenesis, primary macrophages were exposed to receptor activator of nuclear factor kappa B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and human transforming growth factor-β1 (TGF-β1) in the presence or absence of PPP, BC, Alb-gel, RC lysates and hemoglobin. We show here that it is mainly the lysates prepared from PPP and BC that consistently reduced the agonist-induced expression of interleukin 6 (IL6) and cyclooxygenase-2 (COX2) in macrophages, as determined by RT-PCR and immunoassay. With respect to osteoclastogenesis, lysates from PPP and BC but also from RC, similar to hemoglobin, reduced the expression of osteoclast marker genes tartrate-resistant acid phosphatase (TRAP) and cathepsin K, as well as TRAP histochemical staining. These findings suggest that liquid PRF holds a potent in vitro heat-sensitive anti-inflammatory activity in macrophages that goes along with an inhibition of osteoclastogenesis.

Platelet-Rich Fibrin Decreases the Inflammatory Response of Mesenchymal Cells

“…To assess the anti-inflammatory effects of different fractions of PRF, we exposed murine ST2, 3T3-L1, and calvaria cells along with human gingival fibroblast and HSC2 cells to TNFα and IL1β, either with or without 10% of PPP, BC, Alb-gel, RC, and 30% of PRF lysates [ 25 , 26 ]. Gene expression analysis showed that PPP, BC, and PRF lysates notably reduced the inflammatory response in ST2 and 3T3-L1 cells.…”

“…Today’s basic research mainly focuses on how PRF affects the proliferation and migration of cells of the mesenchymal lineage [ 47 ] and the impact of PRF on gene expression driven by TGF-β [ 21 , 27 , 45 , 47 ]. Studies investigating the impact of PRF on inflammation have been restricted to cells of the hematopoietic lineage, e.g., macrophages [ 25 , 26 , 28 ] and dendritic cells [ 28 ]. This evidence suggests that PRF is capable of shifting the pro-inflammatory M1 towards a resolving M2 phenotype [ 25 , 26 , 28 ].…”

“…Studies investigating the impact of PRF on inflammation have been restricted to cells of the hematopoietic lineage, e.g., macrophages [ 25 , 26 , 28 ] and dendritic cells [ 28 ]. This evidence suggests that PRF is capable of shifting the pro-inflammatory M1 towards a resolving M2 phenotype [ 25 , 26 , 28 ]. Recently, L-PRF was reported to exert anti-inflammatory properties in LPS-treated gingival fibroblasts [ 30 ].…”

“…The clinical effect of PRF can partially be explained by its growth factors, which can be identified by proteomics analysis [ 21 , 22 ] and immunoassays [ 23 , 24 ]. In vitro, PRF causes an M1-to-M2 transition in macrophage polarization [ 25 , 26 ] and reduces osteoclastogenesis [ 26 , 27 ]. Injectable PRF was shown to have an anti-inflammatory effect on macrophages and dendritic cells [ 28 ].…”

“…Moreover, the platelet-poor plasma (PPP) layer above the buffy coat can be subjected to heat treatment so that the albumin fraction is coagulated, thereby forming an “albumin gel” that maintains stability for months in vivo [ 35 , 36 ]. We have recently shown that it is in particular the PPP, BC, and PRF lysates that greatly suppress the inflammatory response of macrophages in vitro [ 26 ]. However, their respective effects on cells of the mesenchymal lineage are not fully understood.…”