The null hypothesis of this work was accepted, since the inclusion of chitosan did not improve the protective effect of TiF4 toothpaste, which in fact was even better than the positive control at least for reducing enamel wear. The experimental toothpastes containing TiF4 and chitosan demonstrated a protective effect of 89% for enamel and 78% for dentin compared to placebo, whereas the commercial Elmex® presented 43% and 71% of protective fraction, respectively.
According to previous studies, the protective capacity of TiF4 on the enamel is justified by the action not only of fluoride, but also of titanium [29, 30]. TiF4 has been added to several formulations (such as varnish and solution), proving to be an effective compound against tooth demineralization (both caries and dental erosion) when compared to formulations containing NaF in vitro and in situ [13, 15, 30–32] Titanium minimizes tooth demineralization, since it tends to complex with apatite, forming a “glaze” layer rich in titanium oxide and hydrated titanium phosphate, which is more acid-resistant than the layer of CaF2 induced by the application of NaF . In addition, TiF4 induces greater CaF2 precipitation than NaF due to its low pH .
Chitosan was added into the toothpaste containing TiF4, to improve its protective effect, since the literature has shown that the addition of chitosan to fluoridated solutions and toothpastes leads to a reduction in ETW [23, 24, 33, 34]. A recent work has shown benefit of the association between TiF4/NaF and 0.5% chitosan on the protection of enamel wear in vitro . This biopolymer is able to adsorb to enamel, creating a positively charged and more hydrophobic surface [20, 35], providing a mechanical barrier against acids . This mechanism justifies its role as a mechanical barrier against the penetration of acids, contributing to the inhibition of demineralization . However, when applied isolated (without fluoride), its protective effect is reduced by brushing forces, as shown by our study and others [19, 20]. Our study also showed that the addition of chitosan to TiF4 toothpaste did not improve the protection, regardless the tooth substrate, indicating that chitosan, under the tested conditions, may not interact with the tooth surface producing an organic layer as expected.
An interesting result found in this study is that the experimental TiF4 toothpaste was superior to the commercial version indicated for controlling ETW in case of enamel. The commercial Elmex® Erosion Protection toothpaste has in its formulation: F (as AmF and NaF, 1400 ppm), Sn2+ (as SnCl2, 3500 ppm), and chitosan (0.5%) [21, 22, 37]. Ganss et al.  observed, in an in vitro study, applying more frequent erosive challenges and a longer treatment time with toothpaste, 68% reduction in enamel wear with the use of Elmex® Erosion Protection toothpaste compared to placebo. Schlueter et al. [21, 37], using similar methodology, but in situ, reported a reduction of approximately 50% of enamel wear by the use of Elmex® Erosion Protection compared to placebo, which is in agreement with our work.
Tin, like titanium, has an interaction with the tooth structure, being incorporated into the tooth surface and creating a mechanical barrier together with fluoride precipitates. When tin is combined with chitosan, there is a synergistic effect due to the formation of tightly connected multilayers [12, 22, 37] acting as a shield for the deposition of Sn2+ and increasing the preventive effect of this complex structure against ETW. This scenario was not observed in the case of TiF4.
Differently from enamel, studies involving dentin are scarce. Elmex® Erosion Protection toothpaste reduces dentine wear, but not at superior level compared to a conventional fluoride toothpaste . Tin may react with the dentin surface regardless of the presence of the demineralized collagen layer. In cases in which the organic matrix is preserved, phosphoproteins might attract the tin ion, which is then retained in the organic matrix to some extent but also accumulates in the underlying mineralized tissue. Under the absence of the demineralized organic matrix layer, the reaction is by precipitation .
One factor that could have influenced the lack of synergic effect of chitosan and TiF4 is the erosive challenge, since the benefit of metal fluorides has been more evident when erosive challenges are longer [12, 37]. Another important aspect is that the presence of abrasive silica in toothpaste can have limited the protective effect of chitosan associated with TiF4, when compared to fluoridated gels that do not have abrasive that could interact with chitosan [20, 40]. It is also relevant to discuss that the effect of chitosan is also dependent of the low pH of the vehicle. In case of our study, the final pH value of toothpaste containing chitosan alone was close to neutral, which can reduce the protonation of the molecule and its protective effect, which should be considered. Previous works testing solution containing chitosan, at low pH, showed better effect on ETW [23, 24] than the tested toothpaste.
It is known that abrasive wear of eroded hard tissues is considered an adverse side effect of aggressive tooth brushing, which is determined mainly by the abrasiveness of toothpaste rather than by the toothbrush [41, 42]. Based on this, the beneficial effect of TiF4 toothpaste, regardless of chitosan, may be not only due to the fluoride and titanium, but also to its low abrasivity. Thus, the need for further studies in the area is irrefutable, to analyze RDA/REA values of the experimental toothpastes and the effect of aggressive erosive challenges on their protection capacity. Other important point for the future is to buffer the toothpastes in order to have similar final pH (around 4.5) for all.
A limitation of the present study was the absence of human saliva, which is justified by the difficult to collect the amount needed for a pH cycling model of 7 days. Chitosan seems to have a great affinity to salivary proteins, reacting better with the tooth surface in the presence of those proteins [40, 43, 44]. Despite Luka et al.  showed no improvement of the protective effect of Sn2+/F−/chitosan toothpastes under the presence of mucin in vitro, the present result shall be confirmed under in situ model, a condition closer to in vivo situation, with the presence of human saliva that may interplay the action of active compounds on the tooth [46, 47]. Although chitosan did not have any protective effect when included into TiF4 toothpaste, it increased the toothpaste pH closer to the pH of the commercial toothpaste, value more suitable for a daily use.