Background Platelet‐rich fibrin (PRF) serves as a reservoir of bioactive molecules to support wound healing and bone regeneration. The beneficial action of PRF might involve macrophage polarization from proinflammatory M1 toward pro‐resolving M2 phenotypes. This study aims to evaluate the effect of PRF on macrophage polarization. Methods Murine primary macrophages and RAW 264.7 cells were exposed to saliva and lipopolysaccharides (LPS) with and without PRF lysates obtained by repeated freeze‐thawing or the secretome of PRF membranes, termed PRF conditioned medium. The expression of the M1 marker genes interleukin 1β (IL1β) and interleukin 6 (IL6) along with the M2 markers arginase‐1 and chitinase‐like 3 (Chil3 or YM1) were evaluated by real time polymerase chain reaction. Immunoassay and immunofluorescence staining were performed for IL6 and p65 translocation, a subunit nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐kB), respectively. Results We report here that PRF lysates and PRF conditioned medium, the latter containing the secretome, greatly decreased the proinflammatory response of primary macrophages and RAW 264.7 cells as indicated by the expression of IL1β and IL6. The anti‐inflammatory activity of PRF lysates was further confirmed by IL6 immunoassay. Moreover, PRF lysates suppressed the translocation of p65 from the cytoplasm into the nucleus after incubation with saliva. In support of M2 polarization, PRF lysates and PRF conditioned medium enhanced the expression of arginase‐1 and YM1 in primary macrophages. Conclusion Our results indicate that PRF holds an anti‐inflammatory activity and shifts the macrophage polarization from an M1 toward an M2 phenotype.

Background: Organic solvents are widely used in cell biology experiments. Despite increasing the solubility, they have some moderate toxic effects. Therefore, selecting the appropriate solvent along with the use of suitable concentration insures the accuracy and reliability of experimental results. Objectives: The current study aimed to examine the cytotoxic effects of some organic solvents on various cell models including MCF-7, RAW-264.7 and human umbilical vein endothelial cells (HUVEC). Materials and Methods:To evaluate the cytotoxicity effect of common organic solvents on the MCF-7, RAW-264.7 and HUVEC cells, multi-table tournament (MTT) colorimetric assay, the widely used and validated cytotoxicity test was applied. For this purpose, the selected cells were treated with different concentrations (0, 0.1%, 0.5%, 1%, 1.5%, 2%, 3% and 5% v/v) of four most commonly used organic solvents (acetone, ethanol, dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) and then subjected to MTT experiment. Results: According to the obtained results, the cytotoxicity increased significantly with increasing the concentration of all four solvents compared to that of the control group. Studies with MCF-7, RAW-264.7 and HUVEC suggested that acetone, ethanol and DMSO at concentrations of 0.1% and 0.5%, had little or no toxicity, whereas higher concentrations inhibited the growth of all three cells. Compared with other three solvents, DMF displayed rather greater toxicity. Based on the results, proliferation of MCF-7, RAW-264.7 and HUVEC cells were inhibited by all used organic solvents, dose dependently. Conclusions: Thus, the background experimental error can be reduced remarkably by maximal concentration of 0.5% ethanol, acetone and DMSO and 0.1% DMF in the final treatment medium.

Objective To systematically assess the effects of platelet-rich fibrin (PRF) on in vitro cellular behavior. Methods A systematic electronic search using MEDLINE database was performed. In vitro studies using PRF were considered and articles published up to June 31, 2018 were screened. Eligible studies were selected based on the use of human PRF. Results In total, 1746 titles were identified with the search terms, from these 37 met the inclusion criteria and were chosen for data extraction. In addition, 16 new studies, mainly published in 2019, were also included in the analysis resulting in 53 studies. No meta-analysis could be performed due to the heterogeneity of study designs. Included studies show that PRF enhances proliferation, migration, adhesion, and osteogenic differentiation on a variety of cell types along with cell signaling activation. Furthermore, PRF reduces inflammation, suppresses osteoclastogenesis, and increases the expression of various growth factors in mesenchymal cells. Summary and conclusions Despite some notable differences of the studies, the overall findings suggest a positive effect of PRF on cell proliferation, migration, adhesion, differentiation, and inflammation pointing towards a therapeutic potential in regenerative dentistry. Clinical relevance PRF serves as a reservoir of bioactive molecules to support wound healing and bone regeneration. Although the cellular mechanisms by which PRF supports the clinical outcomes remain unclear, in vitro research provides possible explanations. This systematic review aims to provide an update of the existing research on how PRF affects basic physiological processes in vitro. The overall findings suggest that PRF induces cell proliferation, migration, adhesion, and differentiation along with possessing anti-inflammatory properties further supporting its therapeutic potential in wound healing and bone regeneration.

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...