# Effect of Three Chlorhexidine-Based Mouthwashes on Human Gingival Fibroblastsets: An In Vitro Study
## 1 Introduction  
Oral biofilm is considered the principal etiologic factor responsible for the onset, the development and the recurrence of periodontitis and peri-implantiis [1, 2, 3, 4, 5, 6]. Furthermore, tissue healing can be impaired by the presence and accumulation of oral biofilm after the surgical treatment of periodontal and peri-implant diseases, when effective mechanical self-care cannot be adequately performed [7]. Thus, plaque control is deemed to be essential for both the recovery and the maintenance of healthy tissue conditions [7, 8].  
In adjunction to professional mechanical debridement, adequate self-administered daily home care is fundamental for the long-term success of the treatments [8, 9, 10]. At-home measures frequently include the use of antiseptic mouthwashes. In addition to adequate antibacterial activity, these products should not trigger any allergic reactions or provoke tissue damage [11, 12].  
Concerns may arise in cases of prolonged usage or when the antimicrobial agent comes in direct contact with the connective tissues, for instance during postoperative wound healing [11, 13]. In vitro assays are frequently utilized for analyzing the cytotoxicity of antiseptic agents as well as of some filling resin frequently used dental materials, owingto their reduced costs and their high repeatability and reproducibility [14]. In particular, human gingival fibroblasts are commonly used to mimic connective tissue exposure to mouthwashes and to investigate cell-induced stress [15; 16; 17; 18; 19].  
Chloriexidine (CHX), a bisbiguanide broad-spectrum antiseptic, has been widely used for chemical plaque control [20]. However, it is well documented that the prolonged use of mouthwashes with CHX at high concentration can lead to several undesired side effects, including tooth staining, taste alteration and burning sensation [20; 21; 22; 23]. CHX-based mouthwashes on the market are generally at a concentration of 0.1%, 0.12% or 0.2% CHX digluconate, or they present a low concentration equal to or below 0.06% [20]. Research has tended towards the formulation of mouthwashes presenting lower cytotoxicity, while maintaining high antibacterial properties. To overcome the aforementioned drawbacks, mouthwashes containing low concentrations of CHX, alone or in combination with additional compounds, have been proposed [20]. Among them, cetylpyridinium chloride (CPC) seems to be particularly promising [24; 25; 26; 27; 28]. CPC is an amphiphilic cationic quaternary ammonium compound, whose antimicrobial activity is mainly related to its capability to bind to and destroy the bacterial cell membrane. Whereas, at low concentrations, it indirectly promotes cell autolysis through the activation of intracellular latent ribonucleases [29]. Several mouthwash formulations containing both CHX and CPC have been investigated, including solutions with CHX at low concentration, such as CHX 0.05 % + CPC 0.05 % [26; 30; 31] or CHX 0.03 % + CPC 0.05 % [24; 25], but also at higher concentration (e.g., CHX 0.12 % + CPC 0.05 %) [32].  
In a recent study by our group, a CHX 0.05 % + CPC 0.05 % was found to be effective against oral bacteria in vitro; however, limited data on its cytotoxicity are available [30]. Therefore, the aim of the present in vitro study was to investigate the effects of three mouthwashes containing CHX at different dilutions, alone or combined with CPC, on human gingival fibroblasts (HGFs) by examining cell viability, cytotoxicity, and apoptosis after 0, 3 and 6 days from the exposure. The null hypotheses were that, at the three time points, there would be no significant difference among groups in cell viability, cytotoxicity and apoptosis.