## 1 Introduction
The toothpaste Elmex Erosion Protection (Elm-EP, Colgate-Palmolive Co., New York, USA), based on the ChitoActive complex (Chitosanan/AmF/SnCl\({}_{2}\)) and the GC Tooth Mousse (GC-TM, GC Co., Tokyo, Japan) containing milk-derived casein-phospholipid and amorphous calcium phosphate (CPP-ACP), were specifically chosen because of their differences both in composition and mode of action: Elm-EP is a toothpaste based on chitosan (0.5%) and enriched with amine fluoride, sodium fluoride (1400 ppm total F\({}^{-}\)) and SnCl\({}_{2}\) (3500 ppm) [11]. Biopolymer chitosan, derived from chemical deacetylation of chitin, has been largely studied for use in preventive dentistry. Its anti-inflammatory, antimicrobial and biofilm inhibition properties have been confirmed as its ability to adhere to hydroxyapatite and to prevent dental wear [13; 14; 15]. Nevertheless, chitosan does not contribute to the remineralization of existing dental lesions [16]. On the other hand, chitosan microparticles act as carriers for the delivery of F\({}^{-}\) in the oral cavity [17]. Fluoride ions, derived from NaF and AmF, displace hydroxyapatite hydroxide groups forming fluoro-hydroxyapatite playing a double role in the surface-protection process: first, fluoride acts as a catalyst, assisting in the remineralization of enamel with phosphate ions dissolved in saliva; secondly the decreased solubility of fluoro-hydroxyapatite crystals ensures a superior resistance to acid erosion [2]. Finally, the Sn\({}^{2+}\) ions interacting with hydroxyapatite may lead to the precipitation of a protective layer of insoluble mixed tin-salts which inhibits mineral loss protecting the tooth surface [18]. Differently, CG-TM has been indicated as fluoride-free remineralizing agent, containing milk-derived multiphosphorylated casein phosphopeptides (CPP) able to form stable complexes with calcium and phosphate ions [12]. CPP is a biomimetic saliva with a significantly greater calcium-stabilizing capacity than salivary proteins, due to higher content of its phosphoseryl residues. CPP-amorphous calcium phosphate (CPP-ACP) nanocomplexes are readily soluble in saliva, acting as Ca\({}^{2+}\) and PO\({}_{4}\)\({}^{3-}\) reservoir, creating a diffusion gradient and a supersaturated level of ions in the oral cavity. Low pH conditions facilitate the release of Ca\({}^{2+}\) and PO\({}_{4}\)\({}^{3-}\) ions, inhibiting demineralization and contributing positively to the remineralization by precipitation of the released ions [5]. Published literature and systematic reviews on the CPP-ACP performances reached sometimes conflicting conclusions: some reviews suggest that CPP-ACP had significant remineralizing and caries preventive effects; conversely, other systematic reviews conclude that the evidence to support its long-term remineralizing action or synergistic effect with fluoride is limited [19; 20]. However, it has been suggested that the reason for the variability of the results obtained by using CPP-ACP, could be correlated to its remineralization mechanism, that involves an enhanced remineralization of enamel sub-surface lesions compared to predominantly surface-only remineralization produced by fluoride containing agents [21]. Given the different specific action of the two compounds and the absence of studies regarding their combined use, the present protocol for the evaluation of their potential synergic activity was designed. The treatment efficacy, in terms of remineralization potential and surface protection, was evaluated by means of profilometric and SEM analysis in an ex vivo model. Differences in surface resistance to acid attacks, before and after the treatment, were assessed by quantifying the release of Ca\({}^{2+}\) using complexometric titration. The results obtained with the combined use of Elm-EP (Elm-EP, Colgate-Palmolive Co., New York, USA) and CG-TM (GC-TM, GC Co., Tokyo, Japan) were compared with those obtained using the sole Elm-EP or only artificial remineralizing saliva.