Protection of calcium silicate/sodium phosphate/fluoride toothpaste with serum on enamel and dentin erosive wear

Abstract Objective To evaluate the effect of a toothpaste containing calcium silicate/sodium phosphate/fluoride associated or not to the boost serum (BS) against erosive tooth wear (ETW) on enamel and dentin. Methodology Bovine enamel and dentin specimens were subjected to an erosion-abrasion cycling model (1% citric acid - pH 3.6 -2 min / artificial saliva – 60 min, 4×/day, 5 days). Toothbrushing was performed for 15 s (2 min exposed to slurry), 2×/day, with the toothpastes (n=10): control without fluoride (Weleda), Arg/Ca/MFP (Colgate Pro-Relief), Si/PO4/MFP (Regenerate-Unilever), and Si/PO4/MFP/BS (Si/PO4/MFP with dual BS – Advanced Enamel Serum-Unilever). The effect of treatments on the eroded tissues was assessed by surface microhardness in the first day, and surface loss (SL) resulting from ETW was evaluated by profilometry (μm) after three and five days. Additional dentin specimens (n=5/group) were subjected to 20,000 brushing cycles to verify the abrasivity of the toothpastes. Data were subjected to ANOVA and correlation tests (5%). Results For enamel, no difference in microhardness was observed among the treated groups, and similar SL was obtained after 5 days. For dentin, Si/PO4/MFP/BS resulted in higher microhardness values, but none of the groups presented significantly lower SL than the control. There was no significant correlation between SL and abrasiveness. Conclusion The calcium silicate/sodium phosphate toothpaste and serum increased microhardness of eroded dentin, but they did not significantly reduce enamel and dentin loss compared to the non-fluoride control toothpaste. The abrasiveness of the toothpastes could not predict their effect on ETW.


Introduction
The initial stage of enamel dissolution (early erosion) is associated to the weakening of the surface when it contacts an acidic solution. This softened layer presents reduced hardness and is more prone to abrasive wear. 1 The maintenance of the erosive/ abrasive events induces to the more advanced stages of the process, with loss of the dental hard tissues, which is known as erosive tooth wear (ETW). 2 This is an increasing condition, which affects populations worldwide, especially children, adolescents, and young adults. 3,4 It has been associated with high consumption of acidic foodstuff, changes in lifestyles and some medical conditions, such as gastroesophageal reflux and frequent vomiting. 3,5 Although the main etiological factors associated with ETW are known, controlling the exposure to these factors is challenging, because it involves the individual's compliance. 6 Thus, different strategies have been investigated to prevent and control ETW. The use of conventional fluoride toothpastes has shown some protection, although it seems limited. [7][8][9][10] Noteworthy, even with the wide use of these products, the global prevalence of erosive tooth wear is high, being estimated in 20-45% in permanent teeth. 5 Therefore, agents intended to increase the protective potential of toothpastes against ETW are relevant.
Some agents added to the toothpastes may modulate their anti-erosive effect on enamel (presence of Sn 2+ , higher concentration of Ca 2+ and PO 4 -) 11 and dentin (concentration of F -). 12 Furthermore, dentifrices containing desensitizing agents, such as arginine/ calcium carbonate, associated with fluoride were found to protect enamel against erosive attacks in vitro and in situ, 13 whereas for dentin, the evidence is still scarce. Also, there are some previous investigations about the protective effect of different toothpastes against ETW, but the evidence is not robust enough. 7,10 Another aspect that should be considered is that the dentifrices are used during brushing, thus the abrasive potential is an important factor that can influence their protection against ETW. 7 A dentifrice containing calcium silicate and sodium phosphate salts (monosodium phosphate and trisodium phosphate) with 1,450 ppm of sodium monofluorophosphate presented promising in vitro and in situ results regarding the control of initial erosion on enamel, because it lead to the rehardening of the softening layer. 14,15 Furthermore, a dual-phase boost serum (BS) gel containing calcium silicate salts and sodium phosphate plus sodium fluoride was developed to complement the dentifrice action in the treatment of early erosive lesions. 15 Their mechanism of action is based on the deposition of calcium silicate over the enamel surface, protecting it from demineralization, whereas the dual-phase gel acts by promoting the remineralization of eroded enamel. [14][15][16][17] However, the ability of this system (dentifrice and boost serum gel) to control the ETW on enamel is not fully established, with variable results regarding efficacy, especially when abrasion is present in the model. [18][19][20][21] Moreover, there is not much data about the effect of these products on ETW in dentin.
Thus, our study aimed to evaluate the effect of the toothpaste containing calcium silicate/sodium phosphate and fluoride, associated or not to the dual phase boost serum (BS) against erosive wear of enamel and dentin. The null hypotheses tested were; 1. There is no difference among the microhardness of eroded tissues treated with the tested products; 2. For both substrates, surface loss is not different among groups after 3 and 5-days erosion-abrasion cycling; 3. There is no significant correlation between dentin surface loss after cycling and dentin abrasiveness.

Methodology Study Design
Enamel and dentin polished specimens, obtained from bovine incisors (n=10 / group), were exposed to 5 days of an erosion-abrasion cycling. Four different treatments were tested; dentifrice without fluoride; dentifrice with arginine, calcium carbonate and sodium monofluorophosphate; dentifrice with calcium silicate, sodium phosphate and sodium monofluorophosphate; and the association of the previous one with dual phase boost serum gel, containing calcium silicate/sodium phosphate/sodium monofluorophosphate and sodium fluoride. The variables were surface microhardness (SMH), measured at baseline (B) and at the first day of cycling, after first acid challenge (E) and treatment (T), and surface loss (SL) measured by contact profilometry after the 3 rd and 5 th days of cycling.
Furthermore, the abrasiveness of the dentifrices was assessed by profilometry after 5, 10, 15 and 20 thousand toothbrushing cycles on dentin.

Sample Preparation
Freshly extracted and intact bovine incisors were selected, cleaned, and stored in 0.1% thymol solution at 4ºC, until required. Crowns were separated from roots using a diamond disk, and one hundred cylindrical specimens were obtained from their labial surface using a custom-made diamond trephine mill with 3 mm internal diameter. 22 Specimens were ground flat with water-cooled silicon carbide (SiC) paper discs (#1200 / Fepa-P, Struers, Ballerup, Denmark) to standardize a height in 2 mm with the aid of a metallic device, and then allocated into two groups (n=50) according with tooth substrate (enamel or root dentin).

Profilometry
To maintain the reference surfaces for lesiondepth determination (profilometry) and to allow exact replacement, two parallel grooves were marked on the sides of the acrylic resin surface to serve as guides.
Before the erosive-abrasive challenge, profiles of each specimen were obtained from the enamel and dentin surfaces with a contact profilometer (MaxSurf XT 20, Mahr, Goettingen, Germany). The diamond stylus moved from the first reference area in acrylic resin into the second one (4.2 mm long). Three profile measurements were performed for each specimen at intervals of 0.25 mm.

Erosion-abrasion challenge
An erosion-abrasion cycling model was performed for 5 days. The daily treatment consisted of immersing specimens in 1% citric acid (2 min -4 times / day, pH adjusted to 3.6 with KOH) 23 , followed by immersion in artificial saliva (6 times / day) for 30 minutes before treatments and 60 minutes between exposure to citric acid. Abrasion plus immersion in the toothpaste slurry was performed twice a day simulating two daily brushings. Figure 1 shows a chart of the erosive/ abrasive cycling.
Toothpaste slurries were prepared immediately before each treatment (1:3 -dentifrice : artificial saliva), to use fresh solutions on the specimens. 15 Artificial saliva used in our study was composed by

Toothpastes abrasivity analysis
To check the differences in the abrasiveness of the toothpastes used in this study, additional dentin specimens were prepared (n=5, each group) as previous described and subjected to 20,000 abrasion cycles. Profilometry was assessed 5 times (initial, after 5,000, 10,000, 15,000 and 20,000 abrasion cycles) to create the surface loss pattern of each dentifrice.

Statistical analysis
Data were checked for normality and homogeneity assumptions (Kolmogorov Smirnov and Levene tests), and then one-way ANOVA test was applied for microhardness and profilometry values, separated for enamel and dentin data, followed by Tukey's test.
Dentin loss data after 20,000 abrasive cycles was analyzed by one-way ANOVA, followed by Tukey's test. The correlation between surface loss data at the end of the cycling and dentin abrasiveness was made using the Pearson's correlation test. Statistica for Windows Software (StatSoft, Tulsa, OK, USA) was used and a 5% level of significance was considered in all the analyses.

Microhardness
For enamel, the percentage of surface microhardness alteration after treatment (%SMH alt ) showed no significant differences (p=0.4894) among the groups (Table 2). For dentin, there were significant differences (p<0.0001) among the treatments, and Tukey's test revealed that Si/PO4/MFP and Si/PO4/MFP/BS resulted in significantly increased microhardness compared to non-fluoride control toothpaste (Table 3).

Profilometry
Profilometric analysis was performed after the 3 rd and 5 th days of the cycle to assess surface loss. RM ANOVA test showed differences among the dentifrices for enamel and for dentin both after 3 and 5 days.  showed that after 20,000 cycles the control dentifrice, without fluoride, was the less abrasive one. Arg/Ca/ MFP and Si/PO 4 /MFP presented similar intermediate abrasivity potential. The graph at Figure 2 shows the surface loss after 5,000, 10,000, 15,000 and 20,000 abrasive cycles. There was no significant correlation between enamel and dentin surface loss at the end of the cycling and dentin abrasivity after 20,000 abrasive cycles (r enamel loss × dentin abrasivity = 0.87; r dentin loss × dentin abrasivity = 0.57; all p>0.05).

Discussion
Toothpastes play an important role in oral hygiene, since they are affordable, easy to obtain, and have been traditionally incorporated into the dental hygiene routine of individuals. Many products offer multiple benefits due to the addition different active ingredients. 26 Therefore, the toothpaste stands out as an interesting vehicle for providing agents to control ETW 26,27 and its undesirable consequences, such as tooth sensitivity. The products tested in this study exhibited no significant differences on eroded enamel   Regarding the Si/PO 4 /MFP toothpaste, in vitro and in situ studies showed that its formulation based on calcium silicate and sodium phosphate salts (monosodium phosphate and trisodium phosphate), and MFP presented efficacy against enamel demineralization and was also able to improve its rehardening 14,15,21 . The presence of calcium silicate is expected to release calcium ions into the oral fluids under erosive conditions, increasing their saturation, thus reducing enamel dissolution. 16,33 Furthermore, calcium silicate may act as a chemical and physical barrier against acids due to its ability to cause pH buffering and the formation of a hydroxyapatitenucleated layer. 16,33 Our results showed that the Si/PO 4 / MFP toothpaste alone was not able to promote higher values of enamel microhardness after treatments compared to the control toothpaste. Moreover, the system did not significantly protect the enamel against advanced tissue loss. This suggests that the phosphate and calcium-based salts, that promotes the deposition of calcium silicate particles onto the softened enamel, were not able to effectively resist the toothbrush abrasion. 18 The favorable results reported previously with this toothpaste 14,15,20 are usually related to its protective effect against acid challenges, since abrasion was not considered in many studies.
The presence of abrasion modulates the process and increases the complexity of choosing a control group.
A previous study showed favorable results when Si/ PO 4 /MFP toothpaste was compared to experimental products with similar composition and abrasiveness potential. 21 When applied to dentin, the Si/PO 4  The application of the boost serum for three days followed the manufacturer recommendation, therefore profilometry was assessed after the 3 rd day, to verify its immediate protective effect, and after five days, to quantify the evolution of tissue loss promoted by different treatments. For dentin, the highest protection against erosive wear was obtained for Si/PO 4 /MFP/ BS group with three days. However, this improved efficacy provided by the serum was not significantly different from the control group after 5 days, although it promoted lower dentin loss than the Si/PO 4 /MFP and Arg/Ca/MFP toothpastes, this might suggest the necessity of regular application of the serum for a sustained effect.
The presence of arginine in Arg/Ca/MFP did not show improved efficacy on protecting the enamel and dentin against erosion. Arginine and calcium carbonate acts by deposition, physically sealing the exposed dentin tubules and forming a mass composed by calcium, phosphate and arginine that reduces acid solubility. 35 Although this dentifrice has shown the ability to reharden enamel softened by an previously erosive challenge, 36

Conclusions
The calcium silicate/sodium phosphate/fluoride toothpaste associated to the boost serum showed favorable effect on dentin microhardness, however this was not maintained with the persistence of the erosive-abrasive challenges. Similar enamel and dentin loss was observed when this system was compared to the non-fluoride control toothpaste. The abrasivity potential of the toothpastes could not predict their effect on erosive tooth wear.

Conflict of interests
The authors do not have any financial interest in the companies whose materials are included in this article.