Effect of toothpaste with high fluoride concentration on remineralization of dental substrate with artificial caries

Abstract Introduction fluoride toothpaste is widely used by the population. Objective dentifrices with a high concentration of sodium fluoride and the presence of tricalcium phosphate were developed, providing additional protection to patients vulnerable to the development of caries. This study aimed to evaluate the influence of different dentifrices on the surface and internal hardness and enamel wear after artificial caries, pH cycling, and toothbrushing. Material and method enamel blocks (4x4 mm) were obtained from 20 bovine teeth. The specimens were submitted to artificial caries lesion and then to pH cycling and brushing according to the dentifrice: Colgate Total 12 and Clinpro + Tricalcium Phosphate. Result after this period, they were submitted to analysis of superficial and internal hardness and profilometry. Scanning Electron Microscopy (SEM) was performed. The data obtained were evaluated by one-way Analysis of Variance (ANOVA) to evaluate the mineral recovery and profilometry, and two-way repeated measures ANOVA to evaluate the superficial and internal hardness. Conclusion analyzing the surface hardness, after caries induction, there was a significant reduction in hardness that was partially recovered regardless of the dentifrice used. For internal hardness, Clinpro obtained lower values on the control side and up to 90 micrometers on the test side. As for profilometry, Colgate Total 12 showed greater wear when compared to Clinpro. It was concluded that Clinpro promoted lower internal hardness, however, Colgate Total 12 resulted in greater surface wear of tooth enamel.


INTRODUCTION
Lack of oral hygiene care can worsen the clinical condition of hospitalized patients, as long periods of hospitalization can allow the evolution of pre-existing infections and increase the risk of developing opportunistic infections 1 .Under healthy conditions, the relationship between microorganisms in the oral cavity is symbiosis, in which different species interact beneficially with each other, without harming the oral environment 2 .However, when there are systemic changes, in the standard of oral hygiene or with the use of antimicrobials, the balance between the species is broken and the environment becomes conducive to the expression of pathogenic microorganisms.In this sense, the inclusion of a dental surgeon in the hospital team contributes to reducing the risk of dental caries, and periodontal disease and preventing the oral cavity from becoming a focus of infection and dissemination of microorganisms to the systemic circulation.
Fluoride toothpaste with 1450 ppm of fluoride is widely used by the population; dentifrices with a high concentration of sodium fluoride (5000 ppm) were recently developed for specific indications 3 .Due to their high concentration of fluoride, they are only available under professional prescription for specific cases such as a high risk of developing caries lesions.In this context, due to the ability of phosphates to stimulate the process of mineral balance and deep remineralization of caries lesions, tricalcium phosphate (PTC) 2,4 has been added to certain toothpaste to provide additional protection to patients vulnerable to the development of caries.
This study aimed to evaluate the influence of different dentifrices on the surface and internal hardness, in addition to enamel wear, after artificial caries induction, pH cycling, and brushing.The established null hypothesis was that there would be no difference between the dentifrices in superficial and internal hardness and the profilometry of the dental enamel.

MATERIAL AND METHOD
The project was submitted and approved by the animal research ethics committee (Process 00513-2019).Twenty bovine teeth were selected and stored in 0.1% thymol solution for 30 days.Organic debris was removed with a periodontal curette.Enamel blocks (4x4mm) were obtained through cuts in the mesiodistal and inciso-cervical directions in the central region of the crown with a diamond disk (Buehler, Illinois, USA) mounted on a precision cutter (Isomet 1000, Buehler, Illinois, USA).
Enamel blocks were standardized according to surface hardness, and specimens with values 10% below and above average were excluded 5 .Initial hardness was analyzed at the center of the specimens on a micrometer (Micromet 5114 and OmniMet Software -Buehler, Lake Bluff, Ill, USA), performing 5 indentations with a pyramidal Knoop tip, 50 cm apart, under a static load of 25 g for 10 seconds 5 .Stratified randomization was performed to distribute samples with similar hardness values among the groups, ensuring that the values were statistically similar after the caries challenge.
Then, for pH cycling, the specimens were immersed in 35 ml of demineralizing solution (2.0 mmol L-1 Ca, 2.0 mmol L-1 P, 0.075 mol L-1 acetate buffer, 0.04 g F/mL, with pH 4.7) for 6 hours, and in remineralizing solution (1.5 mmol/L Ca, 0.9 mmol/LP, 0.15 mmol/L KCl, 0.02 mmol/L cacodylate buffer of sodium, 0.05 g of F/mL, with pH 7.0) for 18 hours 7 .For brushing, Colgate Total 12 (CT) and Clinpro + Tricalcium Phosphate (CL) toothpaste were used, described in Table 1.The dentifrices were weighed and diluted in distilled water (1:3); 2 ml was pipetted onto the specimens of each group and brushed manually with an electric brush (Oral B Professional Care 5000 Oral B Schwalbacham Taunus, DE) equipped with a pressure sensor whose red light turned on when the pressure exerted reached a value of 2N.Brushing was performed twice a day at an interval of 12 hours, with 15 seconds of simulated brushing and immersion in the toothpaste for up to 2 minutes 8 .For surface hardness, 5 indentations were made 100 µm apart in the control and test areas 9 , to assess whether the dentifrices altered the final surface hardness (SH2) 9 .The percentage recovery of surface hardness (% SH) was calculated by the formula [% RSH = 100 (SH2-SH1) / SH1] 9 .For the internal hardness analysis, the specimens were longitudinally sectioned in the central region and embedded in acrylic resin, sequentially polished with silicon carbide sandpaper (#600, #800, and #1200) until the entire enamel surface was exposed.Seven indentations were performed with depth distances of 10, 30, 50, 70, 90, 110, and 220 µm, both on the control side and on the test side, with a Knoop indenter under a load of 5g for 10 seconds 9 .The averages were calculated for each distance and values converted to mineral content (vol% min = 3.66 * (√KHN) + 21.19) 9 .The integrated mineral area of the lesion and healthy enamel was calculated using a trapezoidal rule.The values obtained were subtracted from the integrated area of the post-demineralized enamel, resulting in the integrated mineral recovery (IMR).
Mineral tissue loss was determined using an optical profilometer (Surftest SJ 401, Mitutoyo American Corp) 8 .In the center of the enamel samples, 3 scans (2 mm in length) were performed with an interval of 0.5 mm between them.The scans were interpreted with specific software coupled to the profilometer based on the construction of regression lines between the reference and experimental areas.Tissue loss (µm) was defined from the vertical distance between the regression lines of the reference area (previously protected by varnish) and the area submitted to pH cycling and brushing.The average between the three scans was calculated 8 .Three representative specimens from each group (control and test) were analyzed under a Scanning Electron Microscope (SEM) (EVO LS-15, Carl Zeiss, Oberkochen, DE) at 3000x magnification, to observe the differences structures in healthy and caries-affected enamel after pH cycling and toothbrushing.
The data obtained in this study were submitted to the normality and homogeneity test.Oneway Analysis of Variance (ANOVA) was used to assess mineral recovery and profilometry, twoway repeated measures ANOVA to assess surface and internal hardness, and Tukey's test posttest for multiple comparisons.All analyzes were performed with a significance level at 5%.

RESULT
According to the data obtained in the surface hardness analysis (Table 2), when comparing the dentifrices, there was no statistically significant difference in any treatment period.When comparing the surfaces, there was a difference between sound enamel, decayed enamel, and after treatment with the respective dentifrices, with lower values for decayed enamel.Through the calculation of the mineral recovery, we observed that both groups did not present statistically significant differences after the treatments.The results of the internal hardness (Table 3) showed that the CL dentifrice obtained lower values on the control side and up to 90 micrometers on the test side.When evaluating the different depths of each dentifrice on the control side, the CT group showed less hardness for the distance of 10 and 30 micrometers, with a statistical difference for the other measures, stabilizing from 50 micrometers.Likewise, the CL group presented the lowest hardness observed for the distance of 10 micrometers and a gradual increase up to 220 micrometers.When analyzing the side submitted to the challenges, there were statistical differences of up to 50 micrometers for the CT group and of up to 70 micrometers for the CL group.From 70 to 90 micrometers, the CT group approached the deepest distances and upon reaching 110 micrometers, it reached hardness stability, while the CL group no longer showed statistical differences at 110 micrometers.Finally, there was a difference between the control and test sides at the same depth, and toothpaste was only up to 50 micrometers.As for profilometry, CT toothpaste showed a higher enamel wear profile (-47.9 ± 4.9) when compared to CL (-17.5 ± 2.2). Figure 1 represents micrographs obtained by SEM with a magnification of 3000x illustrating the surface appearance of the samples on the control side (A), treated with CT (B), and treated with CL (C).In Figure 1B, it is possible to observe greater disorganization of the surface in Figure 1A and 1C, while Figure 1C is characterized by greater homogeneity of the surface after treatment.

DISCUSSION
In this study, the enamel surface hardness test was used at three different times (initial, after artificial caries induction, and after treatments) to identify the achieved mineral reduction.Despite having a 96% of inorganic load, the mineral balance of enamel is compromised due to constant exposure to acids, which promote the dissolution of ions and create subsurface lesions into the interprismatic spaces 10 .Through salivary action and adjuvant remineralizing therapies, there is a tendency to recover lost ions; however, recovery may not occur in its entirety and leave unevenness in the tooth structure.For this reason, profilometry was used to identify the enamel wear between the healthy side and the side subjected to artificial caries after brushing with the dentifrices to be analyzed.
The hypothesis of this study was denied, as the dentifrices showed differences in internal hardness and profilometry.After inducing the formation of artificial caries, the enamel showed a significantly reduced hardness in the initial reading, confirming the methodology used to induce artificial caries 6 .In general, the mechanism of action of dentifrices occurs through the inhibition of the demineralization process and increased remineralization, promoting preventive action on healthy substrates and therapy against caries 11 .Also, dentifrices with sodium fluoride act by forming fluorapatite, which is the union of fluoride with hydroxyapatite crystals present in tooth enamel 12 .According to the manufacturer, during the remineralization process, CL promotes a neutral or slightly alkaline pH 13 .With the addition of TCP, the action on subsurface areas is expected to be more effective than toothpaste with sodium fluoride alone.In the toothpaste production process, the ions are protected by a modified carboxylic acid (fumaric acid) that produces β-TCP 14,15 .When this substance came into contact with the aqueous medium, the interface between the acid that protects the ions was disrupted, releasing calcium and phosphate into the medium.
In contrast to this study, there are some in vitro and in situ evidences on the effects of β-TCP present in solutions, mouthwashes, dentifrices, and varnishes, showing that the association with fluoride improves enamel hardness, fluoride absorption, remineralization of white spot lesions, and release of calcium in saliva 13,[16][17][18][19][20] .Some factors may explain the difference between the results of this study in the literature, such as the presence of biofilm, brushing time, and type of storage.In the present study, a chemical process was used to induce artificial caries, unlike models with biofilm where the dentifrice can bind to the acquired film, acting as an additional reservoir to enhance the action of the dentifrice 21 .In addition, the total time of contact of the samples with the dentifrice was 2 minutes, based on the time it takes an individual to brush the teeth 22 .Featherstone et al. * indicated that 5 minutes of contact of the dentifrice with the surface is adequate to promote greater fluoride retention 2,* .It is also known that storage in artificial saliva plays a fundamental role in the remineralization process, which may influence the results of in vitro studies that evaluate toothpaste 23 .
The advantages of the presence of a high concentration of fluoride and TCP in the CL toothpaste could be evidenced in the profilometry results, as it presented better resistance to degreasing the CT.It is known that in addition to compositional characteristics, abrasiveness can affect the action of dentifrices on enamel wear.In addition to containing active principles, dentifrices have characteristics related to abrasiveness.In this context, measures such as RDA (relative dentine abrasion) and REA (relative enamel abrasion) are present in studies that investigate the effect of toothpaste abrasiveness, showing that dentin is more susceptible to abrasion than enamel.However, not all companies provide RDA and REA values, as well as the relationship between RDA and REA is not necessarily direct, as a toothpaste that has a high RDA value may have a low REA value and vice versa 24 .The CL toothpaste used in the present study had an RDA of 62.15 and REA of 3.49, while the CT toothpaste had an RDA value between 70 and 78 and an unspecified REA value 25 .It is noteworthy that representative SEM images demonstrated the superior roughness promoted by CT toothpaste compared to CL (Figure 1A and 1B).
Correlating the results of internal hardness and profiling, it is speculated that the superiority of the CT results about the CL is due to the removal of enamel weakened by the artificial caries process.The effect of artificial caries associated with the treatments promoted a porous surface, mainly in the CT group.There is a condition called lamination, in which the acid diffuses through the porosities without plugging them, resulting in the demineralization of the previously treated area 26 .It is noteworthy that this condition varies according to the pH cycling model and the fluorine concentration in the dentifrice.Although the surface was rougher on the CT dentifrice, as seen in Figure 1B, the interaction between sodium fluoride and the available minerals was able to neutralize the acids and create an effective barrier against demineralization.The initial distances are fundamental to detect the differences found in the hardness values, and in this study, it was evident that the CL toothpaste traveled a greater distance to achieve stability in the internal hardness.Other reported factors also contribute to the surface appearance, such as the presence of abrasive silica, incorporated to increase the cleaning potential, daily contact time with acids, strength, and abrasiveness promoted by brushing, and relative abrasiveness of the dentifrice.As this is an in vitro study where some variables are subject to control, the total time of contact with the acid was followed according to the protocol, and the strength and abrasiveness during brushing were also considered adequate since the electric toothbrush used has a pressure sensor that is activated in the face of excessive force 23 .On the other hand, it was not the objective of the study to quantify abrasives and other components in the substrates.
As limitations of this study, the artificial caries model, brushing time and type of storage may have enabled us to find results that are different from those found in the literature and that use protocols with biofilm or longer brushing time.Future studies that combine β-TCP with titanium dioxide or other metallic oxides may make it more stable [14][15][16] .An alternative is to functionalize it with organic and/or inorganic molecules, allowing the creation of barriers that prevent premature interactions of fluorine-calcium and facilitating the availability of ions when applied to teeth through dentifrices, varnishes, and other means.

Figure 1 .
Figure 1.Scanning electron microscopy of the tooth enamel surface on the control side (A), treated with Colgate Total 12 toothpaste (B) and Clinpro toothpaste (C).3000x magnification.

Table 1 .
Trade name, fluoride concentration, composition, and manufacturer of the dentifrices used in this study

Table 2 .
Mean and standard deviation of surface hardness (KH) and mineral recovery (%) analysis Capital letters compare toothpaste.Lowercase letters compare the surfaces.

Table 3 .
Mean and standard deviation of internal hardness (KH) analysis Capital letters compare toothpaste.Lowercase letters compare depths.# Statistical differences within the same dentifrice and depth after challenges.