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 Platelet-rich fibrin (PRF) has been proposed as an alternative approach to the application of recombinant growth factors to enhance wound healing and bone regeneration 1. PRF is obtained by centrifugation and spontaneous coagulation of blood followed by the removal of the red corpuscle base 2. The coagulated plasma contains a complex mixture of growth factors and other bioactive molecules enmeshed within a fibrin network 3,4. This coagulated plasma can be further processed by squeezing out the serum, obtaining a PRF membrane. PRF membranes have become an attractive strategy to maximize the clinical outcomes by delivering growth factors at the surgical site, either alone or in combination with dental implants and collagen membranes 5,6. For example, PRF membranes can preserve the alveolar ridge dimension following tooth extraction 7. Furthermore, dental implants coated with PRF increase their stability during the early phases of osseointegration 5,8. Additionally, when PRF is combined with a collagen membrane in a guided bone regeneration approach it can preserve the alveolar ridge profile 9. However, and despite these promising clinical results, the underlying cellular and molecular mechanisms induced by PRF are poorly understood 10. Mesenchymal lineage cells are among the possible targets at sites where PRF is applied. In the oral cavity, mesenchymal cells are found in the gingiva 11. Consequently, it is not surprising that gingival fibroblasts are common targets for assessment of cell responses. For example, cell proliferation or osteogenic differentiation in response to PRF can be measured via changes in gene expression 12,13. This screening approach can be further refined by means of whole genome arrays. Gen...
Background Platelet‐rich fibrin (PRF) membranes can preserve alveolar ridge dimension after tooth extraction. Thus, it can be presumed that PRF suppresses the catabolic events that are caused by osteoclastic bone resorption. Methods To address this possibility, we investigated the impact of soluble extracts of PRF membranes on in vitro osteoclastogenesis in murine bone marrow cultures. Osteoclastogenesis was induced by exposing murine bone marrow cultures to receptor activator of nuclear factor kappa B ligand (RANKL), macrophage colony‐stimulating factor (M‐CSF) and transforming growth factor‐beta 1 (TGF‐β1) in the presence or absence of PRF. Osteoclastogenesis was evaluated based on histochemical, gene expression, and resorption analysis. Viability was confirmed by formation of formazan crystals, live‐dead staining and caspase‐3 activity assay. Results We report here that in vitro osteoclastogenesis is greatly suppressed by soluble extracts of PRF membranes as indicated by tartrate‐resistant acid phosphatase (TRAP) staining and pit formation. In support of the histochemical observations, soluble extracts of PRF membranes decreased expression levels of the osteoclast marker genes TRAP, Cathepsin K, dendritic cell‐specific transmembrane protein (DCSTAMP), nuclear factor of activated T‐cells (NFATc1), and osteoclast‐associated receptor (OSCAR). PRF membranes, however, cannot reverse the process once osteoclastogenesis has evolved. Conclusion These in vitro findings indicate that PRF membranes can inhibit the formation of osteoclasts from hematopoietic progenitors in bone marrow cultures. Overall, our results imply that the favorable effects of PRF membranes in alveolar ridge preservation may be attributed, at least in part, by the inhibition of osteoclastogenesis.
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.Int. J. Mol. Sci. 2020, 21, 1679 2 of 15 crevicular fluid by means of a tightly controlled expression of adhesion molecules, thereby defending microbiological antagonism in the periodontal tissue [2,3]. Thus, the increase of adhesion molecules by inflammatory mediators has to be counterbalanced by local cues to control an excessive influx of cells of the innate immune system.The influx of cells is controlled by intercellular adhesion molecule-1 (ICAM-1), allowing the transmigration of leucocytes which express the corresponding lymphocyte function-associated antigen-1 and macrophage adhesion ligand-1 [4]. ICAM-1, being induced by inflammatory cues such as interleukin-1-beta (IL1β) and tumor necrosis factor-α (TNFα) [5], is expressed by the vascular endothelium and by the junctional epithelium [6], thus, facilitating transmigration of leukocytes across vascular endothelia and the invasion of the extracellular matrix [7]. Although ICAM-1 is consistently expressed by junctional epithelial cells in healthy gingiva and in pocket epithelium, it is not detectable on the majority of keratinocytes in the external gingival epithelium [6,8]. The question then arises, how is the expression of ICAM-1 in epithelial cells controlled?The increase of ICAM-1 expression by inflammatory cues is evidently well-documented. Inflammatory mediators including IL-6 and prostaglandin E 2 increase ICAM-1 expression in human oral squamous cell carcinoma SCC4 cells in vitro [9,10]. Primary gingival epithelial cells increasingly express ICAM-1 upon inflammatory cytokines stimuli, namely, TNFα and interferon-γ [11]....
Objectives Osteoblasts lay down new bone on implant surfaces. The underlying cellular mechanism and the spatio‐temporal mode of action, however, remain unclear. It can be proposed that growth factors released upon acidification by osteoclasts adsorb to the implant surface and control the early stages of osseointegration. Methods To simulate bone lysis by osteoclasts, titanium discs were exposed to acid bone lysate (ABL) followed by vigorous washing and seeding of oral fibroblasts. The expression of TGF‐β target genes interleukin 11 (IL11) and NADPH oxidase 4 (NOX4) was evaluated by reverse transcriptase polymerase chain reaction and IL11 ELISA. TGF‐β signaling activation was assessed via Smad2/3 immunofluorescence. The impact of ABL on osteogenic differentiation was determined with murine ST2 mesenchymal stromal cells. Results We report here that ABL‐conditioned titanium discs, independent of turned or rough surface, increased the expression of IL11 and NOX4. This increase was blocked by the TGF‐β receptor 1 antagonist SB431542. Further support for the TGF‐β signaling activation came from the translocation of Smad2/3 into the nucleus of oral fibroblasts. Moreover, titanium discs exposed to ABL decreased alkaline phosphatase and osteopontin in ST2 cells. Conclusions These in vitro findings suggest that titanium can adsorb TGF‐β from ABLs. The data provide a strong impetus for studies on the protein adsorption on implant surfaces in vitro and in vivo, specifically for growth factors including bone‐derived TGF‐β during successful and failed osseointegration.
Hydrogen peroxide is a damage signal at sites of chronic inflammation. The question arises whether platelet-rich fibrin (PRF), platelet-poor plasma (PPP), and the buffy coat can neutralize hydrogen peroxide toxicity and thereby counteract local oxidative stress. In the present study, gingival fibroblasts cells were exposed to hydrogen peroxide with and without lysates obtained from PRF membranes, PPP, heated PPP (75 °C for 10 min), and the buffy coat. Cell viability was examined by trypan blue staining, live-dead staining, and formazan crystal formation. Cell apoptosis was assessed by cleaved caspase-3 Western blot analysis. Reverse transcription-quantitative polymerase chain reaction (RT-PCR) was utilized to determine the impact of PRF lysates on the expression of catalase in fibroblasts. It was reported that lysates from PRF, PPP, and the buffy coat—but not heated PPP—abolished the hydrogen peroxide-induced toxicity in gingival fibroblasts. Necrosis was confirmed by a loss of membrane integrity and apoptosis was ruled out by the lack of cleavage of caspase-3. Aminotriazole, an inhibitor of catalase, reduced the cytoprotective activity of PRF lysates yet blocking of glutathione peroxidase by mercaptosuccinate did not show the same effect. PRF lysates had no impact on the expression of catalase in gingival fibroblasts. These findings suggest that PRF, PPP, and the buffy coat can neutralize hydrogen peroxide through the release of heat-sensitive catalase.
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