# Effect of Three Chlorhexidine-Based Mouthwashes on Human Gingival Fibroblastsets: An In Vitro Study
## 4 Discussion
However, mouthwashes containing a high percentage of CHX have been associated with cytotoxic effects in vitro [12; 16; 44; 45]. Therefore, usage of lower concentrations of CHX in combination with CPC have been proposed [24; 25; 26; 27; 28]. In a recent in vitro study by our group, a CHX 0.05% + CPC 0.05% mouthwash was revealed to be effective against oral living bacteria after in situ plaque accumulation, showing similar properties as comparted to CHX 0.1% solution [30]. Furthermore, utilizing the same studydesign of the present work, the authors found the highest cytotoxicity on osteoblast-like cells at day 0 in the CHX 0.2% group, which also presented significantly higher values compared to CHX 0.05% + CPC 0.05% for all the application times [31]. Due to the limited data available on the cytotoxicity on fibroblasts of the former, the current work was conceived as a complementary study in support of our recent investigations [30,31]. In addition to the NaCl and CHX 0.1 groups, it was decided to add the CHX 0.2 group, as it still represents a commonly used solution, in particular after periodontal and peri-implantitis surgeries. In such cases, the protective epithelial barriers would no longer inhibit the direct contact between the connective tissue and the mouthwash; therefore, the cytotoxicity of the antiseptic agent should be carefully considered [11,13]. A limitation of this study is that it was based on monolayer cultures. Two-dimensional (2D) cell cultures were chosen due to the relatively easy environmental control and cell observation, which allow for the minimizing of measuring errors. Flat cultures are, indeed, considered particularly suitable for preliminary toxicity tests, but three-dimensional (3D) human oral mucosal models might be considered for further studies, due to their closer resemblance to the complex in vivo tissue microenvironment [46, 47, 48, 49]. As the oral mucosa is characterized by multiple layers, in 3D models the direct contact of the fibroblasts with the mouthwashes can be avoided, better mimicking the tissue permeability of the agents through the epithelial outer layer [47]. This would be particularly relevant for translating the data to the chronic usage of mouthwashes, which might affect oral mucosa health in the long term [50]. Our results mark out a starting point for future clinical investigations, aiming at understanding not only the impact of different mouthwashes, but also the influence of different rinsing regimens on oral mucosa health and periodontal and peri-implant disease control. In summary, the results obtained in the present study showed that the three tested CHX-based mouthwashes had similar effects on in vitro HGF viability, thus rejecting the null hypotheses that assumed no significant differences per time point and variable. At day 0, the CHX + CPC group presented milder cytotoxic effects as compared to the CHX 0.2 group after an application time of 120 s. In addition, at this time point, no significant differences could be identified between the CHX + CPC and the NaCl control group for all the application times, thus confirming the relatively moderate cytotoxicity of the CHX 0.05% + CPC 0.05% mouthwash. Although extrapolating in vitro data to predict side effects in patients remains difficult, rinsing regimens should be carefully considered by the clinicians balancing the risks of cytotoxicity and the required antimicrobial effect in specific clinical circumstances. High concentration of CHX might have detrimental effects on oral mucosa, not only in the case of prolonged usage, but also when applied directly in contact with connective tissues during wound healing. Conceptualization, K.B. and G.J. ; formal analysis, G.B. and K.B. ; investigation, L.S. and G.J. ; data curation, K.B. and G.J. ; writing--original draft preparation, G.B. and K.B. ; writing--review and editing, G.B., K.B., D.D., J.B. and G.J. ; supervision, D.D., J.B. and G.J. ; project administration, K.B. and G.J. ; funding acquisition, K.B. (mouthwashes).