Effects of Platelet-Rich Fibrin Treated with No-Ozone Cold Plasma on the Alkaline Phosphatase in Rat Bone Marrow Cells: An In Vitro Study

Background/Objectives: Herein, we investigated the effect of platelet-rich fibrin (PRF) treatment combined with no-ozone cold plasma (NCP) on growth factor levels, rat bone-marrow stem cell (rBMSC) proliferation, and alkaline phosphatase (ALP) activity in the early stage of differentiation into osteoblasts. Methods: The PRF used in the experiment was prepared by collecting blood from the jugular vein of rats, followed by centrifugation. The obtained PRF was treated with NCP, and the cell culture media were conditioned with the PRF extracts alone or with NCP-treated PRF extracts. Three different experimental groups were defined: no treatment (NT); cell culture media extracted from PRF (PRF); and cell culture media extracted from PRF treated with NCP (PRF + NCP). Enzyme-linked immunosorbent assays were performed to determine the levels of transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF) AB. Water-soluble Tetrazolium-1 assay was performed to measure cell proliferation in rBMSCs. To analyze cell differentiation into osteoblasts, ALP staining and real-time PCR were performed. Results: Growth factor levels increased in response to treatment (TGF-β: p < 0.001, PDGF AB: p < 0.05), and the cell proliferation rate increased with treatment (145.29% and 150.05% for PRF and the PRF + NCP groups, respectively, relative to the NT group, p < 0.001). Evaluation of the ALP staining intensity and mRNA expression levels showed that the ALP activity was highest in the PRF + NCP group (p < 0.001). Conclusions: Our results confirmed that NCP treatment enhanced the release of several different growth factors contained in PRF to the culture media and that treatment with PRF and NCP increased the proliferation of rBMSCs and their differentiation into osteoblasts.