The use of arginine (Arg) for the ecological management of dental caries has been extensively studied due to its ability to modulate microbial composition and biofilm metabolism, besides inhibiting dentin and enamel demineralization [1]. Arg effectively inhibit virulence factors of biofilms, including growth modulation, gene expression and Streptococcus mutans metabolism [2]. Additionally, it contributes to reducing the biofilms thickness and the concentration of extracellular polysaccharides in both S. mutans and polymicrobial biofilms [1,3]. Arginine's effect on oral biofilms originates from its metabolism by oral microorganisms, leading to the generation of alkalis [4]. This process assists in maintaining a less cariogenic oral flora [5].

Moreover, it has been demonstrated that, when associated to fluoride (F), there is a potentiation of Arg effect in dental biofilms, with an increased amount of arginolytic species and a decreased amount of cariogenic species [1,6,7]. The precise mechanism of action of this association is not fully understood. Nonetheless, it is known that this association increases F absorption by dental enamel compared to F alone, thereby enhancing protection against demineralization [8].

Besides complexing with F, Arg also form complexes with metallic ions, such as calcium [8,9]. Arg affinity for metallic ions could promote its reaction with Ca ions from saliva, biofilm fluid and within dental formulations. The ability to form complexes can further facilitate arginine affinity with demineralized dental structure, potentially enhancing arginine concentration in sites where its presence is needed [10].

Marketed dentifrices that associate Arg to F also have calcium in their composition. However, the calcium originates from an insoluble source with an abrasive function, such as calcium carbonate, and is not added as an active ingredient [[11], [12], [13], [14]]. This insoluble source can limit both the amount of calcium available for complex formation with arginine and its action in de- and remineralization process. Moreover, it is possible that this effect is observed only in the presence of phosphate and thus would be self-limited by the amount of phosphate present in saliva. Based on the above, it is interesting to study if a source of soluble calcium associated with F can increase the anticaries effect of Arg. The hypothesis raised is based on the fact that Arg acts as a ligand in monoprotonated form, but does not form chelates with calcium, as observed by the low stability constant values described by Antonilli et al. [15]. These data indicate that the binding of calcium to the Arg molecule is reversible and that there is no sequestration of calcium present in the medium. Thus, the formation of these complexes would not decrease calcium availability but could increase even more the anticaries effect of dental formulations.

Among the sources of soluble calcium available as active ingredients in dental formulation, calcium glycerophosphate (CaGP), an organic polyphosphate with anticariogenic properties is particularly notable [[16], [17], [18]].]. Studies have demonstrated many mechanisms by which CaGP express its anticariogenic properties, including the ability to 1) increase enamel resistance by its binding with phosphate radicals in CaGP molecule; 2) reduce mineral loss [19,20]; 3) increase calcium levels in biofilm [21]; 4) buffer the pH [16,22] 5) reduce biofilm biomass [23] and 6) increased remineralization of initial caries lesions [24].

Despite the potential application of Arg in preventing dental caries, systematic reviews and meta-analysis highlight the lack of evidence supporting its clinical use [[25], [26], [27]]. They also demonstrate the need for a deeper comprehension of its mechanism of action and behavior when combined with other substances [28]. Given the known mechanism of action of Arg and considering that its action is reinforced by the presence of Ca and F ions, the association of these compounds could promote a beneficial synergistic effect in maintaining biofilm homeostasis and enamel mineral balance, representing a promising approach for controlling dental caries. Therefore, the objective of this work was to evaluate the antibiofilm and anticaries properties of the association of Arg with CaGP and F. The primary outcome assessed was the inhibition of enamel demineralization, measured by surface hardness loss and enamel subsurface mineral content. Secondary outcomes included the analysis of (I) chemical composition of enamel; (II) bacterial viability of biofilm; and (III) biofilm virulence factors (pH of the culture medium, biomass and extracellular insoluble polysaccharides). The null hypothesis tested were that the association of Arg with CaGP and F in aqueous solution does not decrease enamel demineralization (H0), does not reduce bacterial viability (H0’), does not affect biofilm virulence factors (H0’’) in comparison to Control group.