RNAseq of peripheral blood mononucleated cells exposed to platelet-rich fibrin and enamel matrix derivatives.

Platelet-rich fibrin (PRF) and Enamel Matrix Derivatives (EMD) can support the local regenerative events in periodontal defects. There is reason to suggest that PRF and EMD exert part of their activity by targeting the blood-derived cells accumulating in the early wound healing blastema. However, the impact of PRF and EMD on blood cell response remains to be discovered. To this aim, we have exposed human peripheral blood mononucleated cells (PBMCs) to PRF lysates and EMD, followed by bulk RNA sequencing. A total of 111 and 8 genes are up- and down-regulated by PRF under the premise of an at least log2 two-fold change and a minus log10 significance level of two, respectively. Representative is a characteristic IFN response indicated by various human leukocyte antigens (HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DRA, HLA-DRB1, HLA-DRB5), gamma Fc receptors (FCGR1A, FCGR1B, FCGR3B), chemokines (CXCL9-11), and calprotectin (S100A8/9 and S100A12), complement (C1QA/B, C2) and interferon-induced guanylate-binding proteins (GBP1, GBP5). With EMD, 67 and 29 genes are up- and down-regulated, respectively. Characteristics of the upregulated genes are tensins (TNS1 and TNS3). Among the genes downregulated by EMD were epsilon Fc receptors (FCER1A; FCER2) and Fc receptor-like proteins (FCRL1, FCRL3) and CX3CR1. Genes commonly upregulated by PRF and EMD were most noticeably NXPH4 and MN1, but also FN1, MMP14, MERTK, and AXL. Our findings suggest that PRF provokes an inflammatory response, while EMD dampens IgE signaling in peripheral mononucleated blood cells. Overall design: Preparation and stimulation of PBMCs: This isolation of PBMCs was approved by the ethics committee of the Medical University of Vienna and conducted according to the principles of the Helsinki Declaration and Good Clinical Practice. Written informed consent was obtained from all donors. Human peripheral blood mononuclear cells (PBMCs) were obtained from three healthy male volunteers by venous blood draw. Cells were separated by Ficoll-Paque (G.E. Healthcare Bio-Sciences AB, Sweden) density gradient centrifugation. Heparinized anticoagulated blood specimens were processed immediately after venipuncture, diluted 1:2 in Hanks balanced salt solution (HBBS, Lonza, Basel, Switzerland), and transferred carefully to 50 ml tubes containing Ficoll?Paque solution (G.E. Healthcare Bio-Sciences AB, Sweden). The tubes were centrifuged for 15 minutes at 800 g at room temperature without braking, and buffy coats with mononuclear cells were obtained. Cells were washed in HBSS and resuspended in a CellGro serum-free medium (CellGenix, Freiburg, Germany; 25?×?106 cells/ml). Cell concentrations were determined on a Sysmex automated cell counter (Sysmex Inc., USA). For each experiment, PBMCs from the three donors were incubated for 20 hours without or with 300 µg/mL of enamel derivative matrix (EMD; Straumann AG, Basel, Switzerland) or a 30% solution of a PRF lysate.