Pyroptosis is a caspase-dependent process relevant to the understanding of beneficial host responses and medical conditions for which inflammation is central to the pathophysiology of the disease. Pyroptosis has been recently suggested as one of the pathways of exacerbated inflammation of periodontal tissues. Hence, this focused review aims to discuss pyroptosis as a pathological mechanism in the cause of periodontitis. The included articles presented similarities regarding methods, type of cells applied, and cell stimulation, as the outcomes also point to the same direction considering the cellular events. The collected data indicate that virulence factors present in the diseased periodontal tissues initiate the inflammasome route of tissue destruction with caspase activation, cleavage of gasdermin D, and secretion of interleukins IL-1β and IL-18. Consequently, removing periopathogens’ virulence factors that trigger pyroptosis is a potential strategy to combat periodontal disease and regain tissue homeostasis.

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.

Platelet-rich fibrin (PRF) contains a broad spectrum of bioactive molecules that can trigger several cellular responses. However, these molecules along with their upstream responses remain mostly uninvestigated. By means of proteomics we revealed that PRF lysates contain more than 650 proteins, being TGF-β one of the few growth factors found. To uncover the major target genes regulated by PRF lysates, gingival fibroblasts were exposed to lysates obtained from PRF membranes followed by a whole genome array. We identified 51 genes strongly regulated by PRF including IL11, NOX4 and PRG4 which are characteristic TGF-β target genes. RT-PCR and immunoassay analysis confirmed the TGF-β receptor I kinase-dependent increased expression of IL11, NOX4 and PRG4. The PRF-derived TGF-β activity was verified by the translocation of Smad2/3 into the nucleus along with the increased phosphorylation of Smad3. Considering that PRF is clinically used in combination with dental implants and collagen membranes, we showed here that PRF-derived TGF-β activity adsorbs to titanium implants and collagen membranes indicated by the changes in gene expression and immunoassay analysis. Our study points towards TGF-β as major target of PRF and suggest that TGF-β activity released by PRF adsorbs to titanium surface and collagen membranes

Liquid platelet-rich fibrin (PRF) can be prepared by high centrifugation forces separating the blood into a platelet-poor plasma (PPP) layer and a cell-rich buffy coat layer, termed concentrated PRF (C-PRF). Heating the liquid PPP was recently introduced to prepare an albumin gel (Alb-gel) that is later mixed back with the concentrated liquid C-PRF to generate Alb-PRF. PRF is a rich source of TGF-β activity; however, the overall TGF-β activity in the PPP and the impact of heating the upper plasma layer remains unknown. Here, we investigated for the first time the in vitro TGF-β activity of all fractions of Alb-PRF. We report that exposure of oral fibroblasts with lysates of PPP and the buffy coat layer, but not with heated PPP, provoked a robust increase in the TGF-β target genes interleukin 11 and NADPH oxidase 4 by RT-PCR, and for IL11 by immunoassay. Consistent with the activation of TGF-β signaling, expression changes were blocked in the presence of the TGF-β receptor type I kinase inhibitor SB431542. Immunofluorescence and Western blot further confirmed that lysates of PPP and the buffy coat layer, but not heated PPP, induced the nuclear translocation of Smad2/3 and increased phosphorylation of Smad3. The immunoassay further revealed that PPP and particularly BC are rich in active TGF-β compared to heated PPP. These results strengthen the evidence that not only the cell-rich C-PRF but also PPP comprise a TGF-β activity that is, however, heat sensitive. It thus seems relevant to mix the heated PPP with the buffy coat C-PRF layer to regain TGF-β activity, as proposed during the preparation of Alb-PRF.

Platelet-rich fibrin (PRF) has a potent anti-inflammatory activity but the components mediating this effect remain unknown. Blood lipids have anti-inflammatory properties. The question arises whether this is also true for the lipid fraction of PRF. To answer this question, lipid fractions of solid and liquid PRF were tested for their potential to lower the inflammatory response of ST2 bone marrow stromal cells and primary bone marrow macrophages exposed to IL1β and TNFα, and LPS, respectively. Cytokine production and the underlying signalling pathway were analysed by RT-PCR, immunoassays, and Western blotting. We report here that lipids from solid and liquid PRF substantially lowered cytokine-induced expression of IL6, CCL2 and CCL5 in ST2 cells. Moreover, the inflammatory response induced by Pam3CSK4, the agonist of Toll-like receptor (TLR) TLR2, was partially reduced by the lipid extracts in ST2 cells. The PRF lipids further reduced the LPS-induced expression of IL1β, IL6 and CCL5 in macrophages at the transcriptional level. This was confirmed by showing the ability of PRF lipids to diminish IL6 at the protein level in ST2 cells and macrophages. Likewise, PRF lipid extracts reduced the phosphorylation of p38 and JNK and moderately decreased the phosphorylation of NFκB-p65 in ST2 cells. These findings suggest that the lipid fraction is at least partially responsible for the anti-inflammatory activity of PRF in vitro.

Short-chain fatty acids (SCFA) are bacterial metabolites that can be found in periodontal pockets. The expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) within the epithelium pocket is considered to be a key event for the selective transmigration of leucocytes towards the gingival sulcus. However, the impact of SCFA on ICAM-1 expression by oral epithelial cells remains unclear. We therefore exposed the oral squamous carcinoma cell line HSC-2, primary oral epithelial cells and human gingival fibroblasts to SCFA, namely acetate, propionate and butyrate, and stimulated with known inducers of ICAM-1 such as interleukin-1-beta (IL1β) and tumor necrosis factor-alfa (TNFα). We report here that butyrate but not acetate or propionate significantly suppressed the cytokine-induced ICAM-1 expression in HSC-2 epithelial cells and primary epithelial cells. The G-protein coupled receptor-43 (GPR43/ FFAR2) agonist but not the histone deacetylase inhibitor, trichostatin A, mimicked the butyrate effects. Butyrate also attenuated the nuclear translocation of p65 into the nucleus on HSC-2 cells. The decrease of ICAM-1 was independent of Nrf2/HO-1 signaling and phosphorylation of JNK and p38. Nevertheless, butyrate could not reverse an ongoing cytokine-induced ICAM-1 expression in HSC-2 cells. Overall, these observations suggest that butyrate can attenuate cytokine-induced ICAM-1 expression in cells with epithelial origin.