Extended PRF: impact of heat on gene expression in gingival fibroblasts

Extended platelet-rich fibrin (e-PRF) combines the prolonged resorption properties of heat-coagulated platelet-poor plasma (PPP), becoming an albumin gel (Alb-gel) that is mixed back with the respective native cell-rich buffy coat layer (BC), the concentrated PRF (C-PRF). E-PRF or Alb-PRF is utilized as a barrier membrane in various clinical applications, such as guided tissue regeneration. Heating of PPP might lower its biological activity, but testing this hypothesis is necessary. To this end, we exposed gingival fibroblasts to lysates of regular PPP, heated PPP (hPPP), and BC, followed by bulk RNA sequencing. Gingival fibroblasts respond to PPP lysates with a total of 153 up- and 71 down-regulated genes when considering a minimum 3.0-fold log2 expression change and a significance level 2.0 log-10. In sharp contrast, the response to hPPP was characterized by only five up-regulated and five down-regulated genes, clearly indicating that heating almost completely abolished the biological activity of PPP. As expected, BC was more potent than PPP and broadened the spectrum of regulated genes. RT-PCR and immunoassays confirmed the heat sensitivity of PPP as exemplified by IL11 and other genes. Moreover, PPP, but not hPPP, drives the phosphorylation of p65, representing NF-κB signaling. Taken together, these findings extend previous observations that PPP causes a robust response in gingival fibroblasts and also strengthen the hypothesis that this response is heat-sensitive. These operations support the clinical concept of e-PRF by mixing back the heated inactive PPP with the bioactive buffy coat C-PRF layer. We exposed gingival fibroblasts to 30% lysates of regular PPP, heated PPP (hPPP), and BC, followed by bulk RNA sequencing, to identify changes in the genetic signatures of fibroblasts. In the indicated experiments, RT-PCR and immunoassays were conducted to verify some gene expression changes relevant to the RNA-seq. Moreover, the Western blot assay was applied to confirm that hPPP loses the capacity to induce NF-κB signaling.