SRA

Platelet-rich fibrin (PRF) is prepared from the coagulated plasma of fractionated blood. When squeezing between two plates, PRF is separated into the solid PRF membranes and a liquid exudate, the PRF serum. The question arises regarding the extent to which the overall PRF activity remains in the membranes and what is lost in the serum. To this aim, we have exposed gingival fibroblasts to lysates prepared from PRF membranes and PRF serum, followed by bulk RNA sequencing. A total of 268 up- and 136 down-regulated genes in gingival fibroblasts exposed to PRF lysates are significantly regulated under the premise of a minimum log2 2.5-fold change and a minus log10 significance level of two, respectively. PRF serum caused 62 up- and 32 down-regulated genes when gingival fibroblasts were exposed to PRF serum, respectively. Among the 61 genes commonly up-regulated by PRF lysate and serum were CXCL1, CXCL5, CXCL6, CXCL8, IL33, and IL6 and PTGS2, STC1. PRF lysate further increased the chemokines CCL2, CCL7, CXCL2, CXCL3, and the IL1R1, IL1RL1, and IL1RN – as well as the paracrine factors IL11, LIF, IGF1, BMP2, BMP6, FGF2, CCN2/CTGF and HAS1, HAS2, HAS3. The 16 up-regulated genes by PRF serum included DKK1. Among the 122 down-regulated genes by PRF lysate were IFIT1, IFIT2, IFIT3, OSR1, OSR2. Among the 32 down-regulated genes by PRF serum were FGF18 and GDF15. Taken together, PRF lysates, compared to PRF serum, cause a more complex response of gingival fibroblasts with a chemokine with an obvious increase in chemokine expression and spectrum of paracrine factors. Overall design: The preparation of PRF was approved by the Ethical Committee of the Medical University of Vienna (NR. 1644/2018). Glass tubes (Bio-PRF, Venice, FL, USA) supporting blood coagulation were subjected to 700 g for 8 min (Z306 Hermle, Universal Centrifuge, Wehingen, Germany). The yellow PRF clot was removed and squeezed between two metal plates. The PRF serum fraction was collected and stored frozen. One cm of the PRF membrane was used per mL of serum-free medium to prepare PRF lysates. PRF membranes underwent two freeze-thaw cycles at -80 °C, followed by 30 s sonication at room temperature (Sonopuls 2000.2, Bandelin electronic, Berlin, Germany). After centrifugation at 15,000× g for 10 min, aliquots of the PRF lysates were frozen for less than one month. PRF lysates were exposed to 72° and 95°C for 10 minutes for indicated experiments. Gingival fibroblasts of human origin were derived from small gingival tissue specimens procured during wisdom teeth extraction from three healthy individuals who provided informed consent. The protocol was endorsed by the Ethical Committee of the Medical University of Vienna (EK Nr. 631/2007). Fibroblasts obtained by explant cultures exhibited the typical spindle-shaped morphology. Gingival fibroblasts were exposed to 30% PRF lysate and 10% PRF serum for 6 hours under standard conditions at 37 °C, 5% CO2, and 95% humidity.