## 4 Discussion
gingivalis_ biofilm with CAPP, although the virulence of literature's strains and biofilm maturation stages were different [12; 35], which prevents comparison. As _P. gingivalis_ HW24D-1 is a strain associated with deep pockets in chronic periodontitis, and in a translational study, more than one application may be necessary in an unknown application period, in vivo, the safety of our setup in oral cells became necessary. Epithelial gingival cells (OBA-9) and human oral fibroblasts (HGF) were then chosen to be tested in 1 to 7 min of CAPP exposure to evaluate the safety of the treatment to the cell DNA content using the static cytometry assay. Static cytometry, also known as DNA image cytometry in Feulgen-stained cells, is an internationally accepted method for oral cell dysplasia and cervical cancer diagnosis for more than a decade [15; 16]. Image analysis systems classify a cell population integrated optical density (IOD) through calibrated filters that help with cell nucleus selection and capture the optical density proportional to the DNA content [17]. Optical density is selected and automatically calculated in each field through a specific algorithmic function. Commonly, 100 to 300 cell nuclei are selected, and inflammatory or stroma cells are used as a diploid control [17]. In this study, we selected 90 HGF and 100 OBA-9 cells, based on the cell type yield in culture. The diploid control cell was the lymphocyte based on studies in dentistry [15; 20; 21], although the IOD mean from oral keratinocytes was 0.64, and lymphocytes was 0.65. Our results showed slight aneuploidy for OBA-9 cells in 5 and 7 min of He-CAPP treatment (DI mean = 1.2 \(\pm\) 0.05) and HGF in 3 min. There was no significance in the ID value distribution among the groups for both cell types (OBA-9 \(p\) = 0.993; HGF \(p\) = 0.996). These results cannot be solely considered as discussed in [PERSON] et al. [21]. Based on the cut edge DI between euploid (DI = 0.9-1.1) and slightly aneuploid (DI = 1.2-1.4), and in the results previously presented for our group regarding cytotoxicity and histological safety of the plasma jet in the used parameters [13], we can conclude that the plasma device and conditions used in this study are safe for oral human cells. Analyzing the dispersion histograms, we can observe that in all groups, there is a small percentage of cells classified as slightly aneuploid, which is in accordance with clinical trials of studying smokers [20; 21], in which inside the healthy control, there was always a small percentage of aneuploid cells. Plasma genotoxic evaluation has been previously evaluated in different cell types and devices, and different assays, such as micronucleus (MN), comet assay, and hypoxanthine phosphoribosyltransferase (HPRT1) assay, indicate no mutagenicity of the treated cells [4; 36; 37; 38]. Unfortunately, the static cytometry has been used for CAPP safety evaluation in only one study [14] in which plasma treatment was not genotoxic to fibroblast-like cells from mice. In the study of [PERSON] et al. [14], the same periods of treatment as the present study, but different setup parameters, were adopted. DI obtained from treated cells corresponded to slight aneuploid scores for all treatment times, including the non-treated group, with a mean of 1.27, the same obtained after 7 min-treatment, which led authors to conclude that CAPP was non-cytotoxic, as well as in the present study. Although static cytometry is an internationally accepted method for oral cell dysplasia and cervical cancer diagnosis, the limited number of studies using this methodology for genotoxicity evaluation of CAPP application makes it difficult to compare our study with others in the literature. Future assays using the same conditions and periods of treatments will confirm our results. In addition, the use of culture cells limits the number of analyzed cells. Clinical trials in which exfoliative cytology could be applied would improve the translation of results. Our study shows that CAPP is effective against a _P.