# Effect of Three Chlorhexidine-Based Mouthwashes on Human Gingival Fibroblastsets: An In Vitro Study
## 4 Discussion  
The purpose of the present in vitro study was to assess the possible effects of three commercially available CHX-based mouthwashes on human gingival fibroblasts (HGFs). The null hypotheses were that there would be no significant difference among the groups in cell viability, cytotoxicity and apoptosis. They were partially rejected, and the corresponding alternatives were accepted. Cell viability, as expected, was generally higher in the saline control group (NaCl) as compared to the use of mouthwashes, with similar values among the latter at all time points. Furthermore, cell viability was found not to be influenced by mouthwash application time in the large majority of cases. Insights into the cellular death mechanisms provoked by the different treatment procedures were also uncovered. Despite the modifications of the plasma membrane in the final stages of apoptotic cell death, the rupture and the integrity of the cell membrane are generally considered as main features of necrosis and apoptosis, respectively [35; 36]. Cytotoxicity was analyzed by measuring a proteolytic biomarker dependent on cell membrane disruption. In agreement with cell viability findings, at day 0, all the mouthwashes presented higher values as compared to the control. Interestingly, at this time point, CHX 0.2 exhibited significantly higher cytotoxicity compared to the CHX + CPC group after the long application time (120 s) and was significantly more cytotoxic once applied for 120 s than for the short treatment time (30 s). Moreover, at day 0, no statistically significant differences could be observed between CHX + CPC and the control group for all the application times. By contrast, at day 3, NaCl showed the highest cytotoxicity; significant differences were also observed between CHX + CPC and CHX 0.1 groups after both 60 s and 120 s of treatment. It can be speculated that CHX-based mouthwashes induced HGF death immediately after exposure, when the phenomenon could clearly be observed. Since most of the cells were likely to be dead in the early phases after the contact with the mouthwashes, in particular at higher CHX percentages, their cytotoxicity values drastically decreased already at day 3. Whereas, in the NaCl group, a balance between living and dead cells was maintained up to day 6 of culture. Caspases are deemed to be responsible for the proteolytic cleavages leading to cell disassembly, which is typical of apoptosis [37]. Therefore, a luminescent assay measuring the activity of two effector caspases, which are expressed and activated in apoptotic cells, was here utilized. In accordance with a previous investigation of our team [16], the control group exhibited significantly higher values of apoptosis as compared to the test groups. The predominant cytotoxic action exerted by the mouthwashes could be an explanation for the low apoptosis values. Although optimal culture conditions were provided through constant cell coverage by culture medium, in the NaCl group, apoptosis values tended to increase over time; this might be ascribed to environmental stress, which can result from changes in cell density, nutrient depletion, or waste product accumulation [38]. For all the selected parameters, changes were mainly observed between day 0 and day 3, while the values remained almost unmodified from day 3 to day 6. As the majority of the events took place in the early phases after mouthwash application, it would be interesting to map and quantify these dynamic processes in real time and over time by means of live cell imaging [39; 40]. Mouthwashes are widely used concomitant with periodontal and peri-implantitis treatments [8; 41; 42]. As these pathological conditions are associated with bacterial biofilm formation, antimicrobial properties are of major importance for supragingival plaque control [11; 43]. Owing to its well-documented antibacterial activity, CHX is frequently employed to reduce oral bacterial load [20].