The effect of phytosphingosine associated with tooth brushing on color change, surface roughness, and microhardness of dental enamel — an in vitro and in situ study

This study evaluated the in vitro and in situ effects of phytosphingosine (PHS) associated with tooth brushing on color stability, surface roughness, and microhardness of dental enamel. Sixty-four specimens of bovine teeth (6 × 6 × 2 mm) were separated into 8 groups (n = 8): S + TB: PHS (spray) + tooth brushing; TB + S: tooth brushing + PHS (spray); I + TB: PHS (immersion) + tooth brushing; TB + I: tooth brushing + PHS (immersion); TB: tooth brushing; S: PHS spray; I: immersion in PHS solution, and Saliva: immersion in saliva. Tooth brushing simulation (Mavtec, Brazil) was performed (356 rpm on 3.8 cm area by the toothbrush — Soft Tek) for 1, 7, 15, and 30 days. PHS remained in contact with specimens for 15 min. The specimens were evaluated before and after tooth brushing for color alteration (Easy Shade, VITA), and surface roughness (Model SJ-201P Mitutoyo), and Knoop microhardness (HMV-2, Shimadzu Corporation). For the in situ analyses, 8 participants were recruited and received an intraoral device with 6 fragments of bovine enamel (6 × 6 × 2 mm). The properties evaluated were the same as those of the in vitro study. Participants were randomized following best results of in vitro tested protocols, for 15 days: TB, TB + S, I + TB. Data obtained by in vitro (two-way ANOVA, Tukey, p < .05) and in situ (one-way ANOVA, Tukey, p < .05) studies were analyzed. The in vitro study showed that greater color change was found after 30 days. The greatest differences in surface roughness occurred between the initial value and after 1 day. Regarding microhardness, the highest values occurred after 15 and 30 days, which showed similar results. The in situ study showed greater color changes for the TB and I + TB, and greater surface roughness changes for TB as well as a similar increase in microhardness for the PHS protocols, which were higher than TB. Phytosphingosine leads to an increase in performance regarding color stability, surface roughness, and microhardness when applied. In general, the application of PHS after brushing showed a positive impact on its performance. Phytosphingosine proved to be interesting for compound prevention formulations in the dentistry field.


Introduction
Dental caries is a major public and worldwide health problem. Its prevention is essential to maintain overall well-being and oral health. For example, dental caries can result in oral disorders such as pain, loss of function, infection, and loss of esthetics. If left untreated, problems may even extend across the borders of oral health resulting in anxiety, sleep disorders, and consequently leading to higher expenditure of public health resources [1,2].
Mostly, the presence of dental biofilms is directly related to the prevalence of dental caries. Dental biofilms may harbor a high number of cariogenic bacteria, which contribute to the development of caries by the production of acids from fermentable carbohydrates. As a consequence, this leads to demineralization and the subsequent dissolution of hydroxyapatite from the tooth enamel [3]. In general, dental biofilms are controlled by combined strategies, including tooth brushing, the use of disinfecting oral rinses, and reduction of sugar intake. Although these strategies have substantially contributed to the reduction of the prevalence of caries, complete prevention seems impossible.
To support the removal of the dental biofilm during oral hygiene procedures, some oral hygiene products contain antimicrobials such as phenol, aldehydes, alcohol, biguanides, and quaternary ammonia compounds [4,5]. Despite reducing the microbial load, these agents do not protect against acid-induced demineralization of the dental hard tissues. In addition, ideally, they should not affect the physical and mechanical properties of the teeth such as the color, brightness, hardness, or surface roughness [6][7][8][9]. Although, with the development of novel multifunctional oral care products, some antiseptic products even stain the dental hard tissues [10].
Thus, solutions with alternative and multiple mechanisms of action have been studied to optimize the available strategies of prevention. Accordingly, recent studies have linked the ability of phytosphingosine (PHS) to protect the dental enamel against biofilm formation and bacterial adhesion on hydroxyapatite [5,11], and also against the process of enamel mineral loss [12,13].
PHS is one of the constituent bases of sphingolipids, which are lipid molecules found abundantly in tissues of fungi, plants, and animals, including humans. In the human body, small amounts of PHS are present in the epidermis [14], and also in the oral cavity, saliva, and mucous surfaces [15,16]. Salivary lipids contribute to the regulation of the immune system, the transport of fat-soluble antioxidants, and the anti-inflammatory and antimicrobial activity on the mucous membranes [16][17][18][19].
Valentijn-Benz et al. [12] showed that PHS forms a homogeneous layer on hydroxyapatite discs, and evaluated the concentration and time dependence of PHS-hydroxyapatite interaction, obtaining the best results with 0.1% in minutes. At this concentration, 90% of the adherence of Streptococcus mutans to hydroxyapatite surfaces was inhibited, under static conditions [11], and hydroxyapatite discs were protected against tissue loss [12]. Furthermore, Bikker et al. [5] evaluated the in vitro anti-adhesive and anti-biofilm properties of PHS and found that after 3 h of growing biofilm, there was a reduction of 99% of viable cells and after 6 h, the reduction was 94%, thus demonstrating effectiveness. Yönel et al. [13] evaluated the anti-erosive potential of PHS in vitro, comparing it to different fluoridated toothpastes and found that PHS protected the enamel against mineral loss compared to the control group.
Tooth brushing with toothpaste, responsible for removing surface-adhered substances, including extrinsic stains and biofilms, could also interfere in the efficiency of any previous treatment. Although strong evidence underlines the long-term protective effects of PHS coatings for at least 16 h after application [5], there is no report of the longterm sustainability of PHS on the enamel after daily tooth brushing. It is envisaged that its sustainability on the enamel surface after tooth brushing is important since the literature demonstrates changes in the surface roughness of the dental substrate by various products used in dentistry, such as toothpaste and oral hygiene solutions [20][21][22][23].
Solutions are often applied by immersion or spray to facilitate their use and decrease the amount of solution needed for antimicrobial action [24]. However, for PHS, the literature is scarce so far, and it is unknown which is the most efficient method of application. As it still lacks consistent scientific evidence and is characterized by being a long hydrophobic aliphatic molecule associated with positively charged hydrophilic radicals [19], still in the evaluation stage, it is important to understand how the PHS solution interacts with the components of the tooth enamel.
This study aimed to evaluate the effect of PHS associated with tooth brushing regarding color stability, surface roughness, and microhardness of dental enamel. The null hypothesis tested was that there would be no differences in the studied properties regardless of the submitted treatments.

Materials and methods
Protocols were first analyzed by an in vitro study and then in situ analysis was performed. They both are described below.
The equipment for color reading has 19 individual optical fibers that illuminate the dental enamel and two spectrophotometric sensors capable of reading the color numerically; in addition, the digital tip is 6 mm in diameter, the same as the sample size, ensuring the reading of the same area. The optical color measurement geometry simulates a 45/0 measurement since it is circular with the specular component excluded to avoid interference from the surface brightness [25,26]. The specimens were placed on a standard white background and inside a gray box with D65 standard lighting [27].
The color measurements were obtained in the CIEDE2000 color system, which consists of strands that represent the black-white luminosity (L *), and the color dimension of green-red (a*) and blue-yellow (b*), so that the L* axis is perpendicular to the a* and b* axes. The device emits a light source with a visible spectrum (400 to 700 nm) on the object and measured the reflection of this spectrum. The values of L*, a*, b* of each sample were measured before and after immersion.
The values of ∆E 00 were calculated by the formula [28]: where ΔL*, ΔC* and ΔH* are the differences in brightness, chroma, and hue between two specimens, and R T (rotation function) is a function that explains the interaction between chroma and hue differences in the blue region. S L , S C , and S H are the weighting functions for the luminance, chroma, and hue components, respectively. K L , K C , and K H are the parametric factors according to different visualization parameters that were defined as 1. The limits of perceptibility (0.8) and acceptability (1.8) were used to analyze the difference in color between groups [29,30].
The quantitative evaluation of the surface roughness was performed by a roughness tester (Model SJ-201P Mitutoyo, Tokyo, Japan). Three readings were taken for each sample (4.0 mm long, 0.8 mm cut-off, and at 0.5 mm/s), one in the center and the other 1 cm from the middle on each side. The change in surface roughness (Ra) was calculated by the difference in the mean of these three readings.
The color alteration, surface roughness, and Knoop microhardness were evaluated before the application of the protocols and after 1, 7, 15, and 30 days for in vitro analyses and before the application of the protocols and after 15 days for in situ evaluation.

Preparation of PHS solution
The stock solution of PHS was prepared in ethanol with a concentration of 5 mg/mL and diluted in 20 mM Tris supplemented with 0.1% Tween 20 (pH 6.8) (Tris-Tween) at 100 µg/mL [5,11,12]. This solution was stored in an appropriate bottle, protected from light. For both methods of application, spray or immersion, 1.5 mL of a solution of PHS was dispensed on the surface of each fragment. This solution remained in contact with the sample for 15 min simulating 1, 7, 15, and 30 days, under agitation (350 rpm) [5,11,12]. After application, the specimens were stored in artificial saliva (Daterra Farmacia de Manipulacao e Industria de Cosmeticos, Ribeirao Preto, SP, Brazil) to maintain the humidity of the oral environment and keep the specimens hydrated.

In vitro assays
A pilot study estimated a priori sample size, using the data of color stability, surface roughness, and microhardness with a power of 80% and a significance level of 5% (https:// www. opene pi. com/). These parameters required 8 specimens per group, and 64 fragments of bovine teeth were prepared (6 mm high × 6 mm wide × 2 mm thick).
The fragments (6 mm high × 6 mm wide × 2 mm thick) were obtained using a low-speed diamond disc under water cooling in a metallographic cutter (Isomet 1000, Isomet, Buehler, Lake Bluff, IL, EUA). The enamel surface was flattened under refrigeration, with SiC sandpaper in decreasing granulation, 600, 1200, and 2000-grit, in a metallographic mechanical polishing machine (Polipan-U, Panambra São Paulo, SP, Brazil) for 3 min at low speed and with a standard weight of 172 g, for each sandpaper. In addition, the specimens' surface roughness was standardized with a maximum difference of 0.07 µm.
The tooth brushing simulation (Mavtec Comercio Ltda., Ribeirão Preto, SP, Brazil) was performed according to ISO/DTS 145,692 [31], applying a 200-g load at a speed of 356 rpm with a 3.8-cm path. According to Wiegand et al. [32], a healthy individual performs 14.600 cycles in 1 year of tooth brushing. The present study simulated 1, 7, 15, and 30 days of tooth brushing.
Soft toothbrushes were selected (Tek; Johnson&Johnson, Sao Jose dos Campos, SP, Brazil) and suspensions (1:1) of conventional toothpaste (Colgate Total 12, Colgate-Palmolive Ind. Ltda., Sao Bernardo do Campo, SP, Brazil) diluted in distilled water (20 mL), mixed for 90 s (A 300, Polidental Ltda., Cotia, SP, Brazil) and injected every 40 cycles. The specimens were positioned in acrylic resin plates, so that they could be adapted to the tooth brushing machine and to ensure the penetration of PHS only through the dental enamel, protecting the dentin.

In situ assays
This research project comprises an in situ, randomized, double-blind, clinical trial and was submitted to and approved by the Ethics Committee on Human Research of the Ribeirão Preto Dentistry School in the University of São Paulo, Brazil (CAAE:98,637,418.9.0000.5419/REBEC: RBR-7rmtzg) and only started after obtaining informed consent.
In situ analyses followed the results found in the in vitro study, taking into consideration three main factors. The first is the fact that tooth brushing with toothpaste is part of an individual's daily hygiene and habits; thus, only groups with tooth brushing were included. The second and third factors were the evaluated properties, color, and microhardness. The groups that presented color change within the perceptibility limit of 0.80 [29] and with a significant increase in microhardness compared to the initial values were selected.
The in vitro study estimated a priori sample size, using the color stability, surface roughness, and microhardness data (https:// www. opene pi. com/). These parameters required at least 42 specimens per group (α = 0.05, power = 0.80). Considering that each device would house 6 specimens and the possibility of withdrawal or the need to exclude some of the participants, 8 participants were included.
Participants of both genders, with healthy general status, without oral pathologies, aged between 20 and 35 years, were included in the study. Exclusion criteria were participants wearing removable dental prostheses or braces, pregnant or nursing women, and those who had used an antibiotic or fluoride topical application within the past 2 months. Each participant was randomly assigned a computer-generated sequence, in which all volunteers used all the protocols, for 15 days: (1) TB: tooth brushing, (2) TB + S: tooth brushing + PHS (spray), and (3) I + TB: PHS (immersion) + tooth brushing.
Some attention was given to controlling biases; thus, a researcher (P1), who was not involved with the other operational phases of the project, produced a list of computergenerated random numbers, which were kept secret until the solutions were applied. Another researcher (P2) received the random numbers and distributed the PHS solutions (immersion or spray) in bottles without identification and in a separate environment from the others so that the other parties (participants and outcome evaluators) did not know how the solution was applied at each stage of the study. Researcher P3 was responsible for applying the protocols to the participants and conducting all necessary orientations. Researcher P4 was responsible for performing the color change, surface roughness, and microhardness analyses of the specimens. Researcher P2 collected the variable information and coded the protocols and forwarded them to P1, who performed the statistical analysis of the results. Thus, the participants and researchers remained blind to the solutions applied.
The salivary flow of the volunteers was measured in the morning (8 a.m.-9 a.m.), where each of the participants chewed sugarless gum for 1 min, and then poured the salivary contents into a millimeter test tube for 5 min. The pH was evaluated (pH-Indicator colorimetric strips, MERCK, Germany), and was not considered an inclusion/exclusion factor; however, no values discrepant to pH 7 were found [33].
The bovine enamel specimens were prepared as previously described and sterilized with ethylene oxide (Acecil, Campinas, SP, Brazil), for 7 h: 20 min of pre-vacuum, 3 h of cycles with ethylene oxide gas at 626 mg/L in an autoclave (45º to 47º), 2 h of aeration, and 2 h of hyperventilation. Then, the fragments were subjected to aeration for 48 h to release the toxic potential of the gas.
Each intraoral device was prepared with six bovine enamel fragments (6 × 6 × 2 mm), three on each side of the device. The devices were made with 0.3-mm-thick polyvinyl chloride plates (Bio-Art Equipamentos Odontológicos Ltda, Sao Carlos, SP, Brazil). The specimens were attached to the device by means of vacuum pressure and flow resin (Opallis-FGM Produtos Odontológicos Ltda, Joinville, SC, Brazil), which was used to protect the dentin faces of the specimens. Thus, only the dental enamel was exposed to the oral cavity.
The sequence of the in situ evaluation occurred according to the flowcharts in Fig. 1. Each participant received 1 device for each protocol evaluated, thus allowing for the evaluation of the specimens after the proposed protocols. The participants were instructed to remove the device, not only for the use of the PHS solution but also for tooth brushing it. In addition, they were instructed to rinse the device thoroughly under running water after the application of the protocol, before returning it to the oral cavity. A washout period of 7 days was established between the applications of the protocols in order to avoid any carry-over effect of the protocols evaluated. In this period, the participants did not receive intraoral devices and were instructed to maintain brushing at 3 times a day with the brush and toothpaste provided by the researchers.
The properties evaluated were the same as in the in vitro study, according to the methodology previously described. The study time of 15 days, in terms of treatment longevity, was defined according to the results obtained in the in vitro study.

Statistical analysis
The values obtained from the analyses were submitted to appropriate statistical tests with the help of a statistical program (GraphPad Prism 8.0.1). Kolmogorov-Smirnov normality test was applied, and adherence to the normal distribution of data was identified for in vitro (two-way repeated measures ANOVA, Tukey, p < 0.05) and in situ (one-way ANOVA, Tukey, p < 0.05) evaluations. The properties of color change, surface roughness, and microhardness were statistically analyzed at the different proposed application times and protocols.

In vitro assays
The comparison of the mean values (two-way repeated measures ANOVA, Tukey's test, p < 0.05) regarding color alteration is shown in Fig. 2. The results of ΔE 00 showed differences in the periods tested for the S + TB, TB + S, I + TB, and saliva groups, which revealed higher changes after 30 days. In addition, the only groups that presented ΔE 00 within the limits of perceptibility (0.80) after 1 day were the S + TB; after 7 days, TB + S, I + TB, spray, and saliva groups; after 15 days, the TB + S, I + TB, and spray groups; and after 30 days, only the spray group. Table 1 shows the in vitro results for surface roughness and Knoop microhardness. Changes in surface roughness occurred in the S + TB, and TB + S groups after 7 days; I + TB after 15 days; and for the TB group, after 1 day Regarding microhardness, the results showed that after 15 and 30 days, all groups using PHS associated with tooth brushing were different from the group immersed in artificial saliva and TB, showing that the solution of PHS was able to increase the microhardness of the specimens when compared to the initial microhardness. The TB, spray, and saliva groups maintained their microhardness values, regardless of the evaluated times. The groups that were brushed with toothpaste achieved a significant increase in microhardness in a shorter time compared to groups brushed with saliva. Regardless of the protocol used, the microhardness values were similar after 15 and 30 days (p < 0.05).

In situ assays
Ten participants were invited to the study (screening). One participant was excluded from the sample because he was undergoing orthodontic treatment with a removable appliance, and one refused to participate in the research. Thus, eight healthy adults (6 females, 2 males, age 27.5 ± 4.11, salivary pH = 6.86 ± 0.35) participated in this study. None of the participants included in the randomization were removed from the research. Thus, this study had a total of 8 participants (Fig. 3). Considering that each participant used a device with 6 specimens for each group, this research had in the end a total of 48 specimens per group evaluated.
The results of the in situ study for ΔE 00 , ΔL, Δa, Δb, and KHN are described in Table 2. The results showed higher ΔE 00 changes for the TB and I + TB groups than for TB + S. All ΔE 00 values were higher than the perceptibility level (0.80), but lower than the acceptability level (1.80) [29].
By observing the results obtained by the coordinates L*, a*, and b*, it is possible to determine that color change occurred from the decrease of these three coordinates, leading to the decrease of the white, red, and yellow chromas with the greatest decrease being in the b* coordinate, moving the color of the sample in the direction of blue or against yellow.
The highest values of surface roughness were found for the TB group, which were similar to the I + TB group. The TB + S group showed the lowest values for this property and were also similar to the I + TB group.
Regarding the difference between final and initial microhardness (ΔKHN), the two groups that used PHS solutions, regardless of the form of application, showed increased dental enamel microhardness when compared to the TB group. The highest microhardness values after the application of the protocols were found in I + TB. Taking into consideration the relative microhardness (KHN%), Table 1 Means and standard deviation for Ra and KHN. Different uppercase letters in the column, indicate significant difference (p < .05) between the times. Different lowercase letters in line indicate significant difference (p < .05) between treatments for the same time  where the increase in microhardness is observed from the initial values of each specimen for each group, it was observed that the protocols with PHS were similar and superior to the TB group.

Discussion
According to recent studies, the use of the solution of PHS results in less microbial adhesion and lower demineralization of the hydroxyapatite crystals [5,11,12,[16][17][18][19]. This solution has demonstrated antimicrobial action against microorganisms such as Streptococcus mutans and Candida albicans [5,11,12,[16][17][18][19]. However, there is no evidence about the performance of PHS associated with tooth brushing, which is a common oral hygiene habit, especially regarding its immediate and long-lasting effects. The objective of this study was to analyze the effect of PHS associated with tooth brushing in two types of studies: in vitro and in situ. For this reason, treatments were selected with different PHS applications at different times. In the in situ study, since it included participants, fewer groups were tested. However, the groups tested needed to be compared to control groups. Thus, for the in vitro group, the control groups were tooth brushing and saliva,  while in the in situ study, the control group used only tooth brushing, as saliva was present in all participants who had normal salivary flows, as established in the methodology. The results showed that the null hypothesis was rejected since the PHS associated with tooth brushing, in different forms of application, presented different effects from the control group.
Regarding the in vitro evaluation, the results demonstrated that the PHS solution seems not to influence the color stability (ΔE 00 ) as groups treated with PHS, at different times of use, presented ΔE 00 values within the limit of perceptibility (0.80) established by the literature [30], different from the tooth brushing group, which showed values above this limit.
In addition, all the tested groups, regardless of the period of use, revealed values below the limit of acceptability (1.80) [30]. This fact is important, as it shows that the PHS was able to protect the dental enamel from the color change, and therefore can be considered safe to be tested in future stages, such as in situ and in vivo assessments.
To date, there is only one report in the literature that involves the color stability of dental enamel using PHS solution [34]; this study demonstrated that it was not able to protect the enamel against staining with coffee and tea after 15 days of simulation of daily consumption, compared to distilled water and cigarette smoke. It is important to highlight that for the simulation of the cigarette smoke, after each staining cycle, the authors performed manual tooth brushing with smooth movements. Thus, adding to the knowledge acquired through the present study, we now infer that tooth brushing seems to be an important factor in helping to maintain the color of dental enamel after the use of PHS. Furthermore, in general, the results of the present study showed no difference for ΔE 00 when the PHS was used alone, associated with tooth brushing, or between the different times evaluated.
Regarding surface roughness (ΔRa), less change was observed when PHS was associated with tooth brushing, regardless of the form of application. Furthermore, the highest values for the surface roughness were found after a 30-day simulation protocol, which is in accordance with the literature that shows that tooth brushing can promote loss of tooth structure due to abrasion, which increases proportionally with the time of brushing and can be enhanced with the inappropriate brushing technique and type of brush [35].
In general terms, the abrasiveness of toothpaste depends on the amount, particle size, surface structure of the abrasive particle, and on chemical influence of other types of ingredients in the products [36]. The toothpaste chosen for this study is composed of silica and sodium fluoride (NaF; 1450 ppm) and has a relative dentin abrasion (RDA) of 70, which is considered low. According to the American Dental Association, all toothpaste with RDA value equal to or less than 250 is considered safe and effective [37]. In addition, a previous in vitro study [38] suggested that the presence of NaF in the toothpaste was able to guarantee lower surface roughness compared to other toothpastes with lower values of RDA and sodium monofluorophosphate (MFP) in their composition, suggesting that the ability of toothpaste with NaF to release more fluorides offsets the fact of having higher values of RDA than other toothpastes.
Regarding microhardness, the results showed that after 7, 15, and 30 days, all the groups using PHS were different from the group immersed in artificial saliva, showing that the PHS solution was able to increase the microhardness of the specimens when compared to the initial microhardness. In the oral environment, fluoride can decrease the solubility of the tooth enamel, making it more resistant to acid-induced demineralization, decreasing the enamel mineral loss, and accelerating the natural remineralization process [39].
A concern of the researchers for the present study was related to the choice of the toothpaste used, composed of NaF and silica. This is important since some components of the toothpaste, such as sodium MFP, require prior enzymatic action to become ionic and present an anti-caries effect. Thus, the use of a toothpaste with NaF and silica provides confidence that any difference related to the microhardness of the enamel would not be caused by the toothpaste, but by the protocol performed.
Regarding the in situ evaluation, to minimize the occurrence of biases, all participants were instructed to employ the protocols in a standardized way, so they were employed following the crossover configuration and in randomized sequence (randomized). The washout period was established in order to avoid the occurrence of a possible residual effect from one protocol to the other (carry-over effect). In addition, the study sought to "blind" the parties involved (researchers and participants), so that there was no knowledge, by any of the parties, of the allocation of the protocols. Furthermore, the fact that there were no losses or drop-outs during the study indicates the motivation of the participants in relation to attending return visits and participating in the research. It is important to report that throughout the research, there were no complaints from the patients about the protocols used.
The results showed greater color change for the B and I + TB groups. However, all groups showed color change within the limit of acceptability [29]. The TB + S group was the only group in which brushing was not the last procedure to be performed, but rather the use of the PHS solution. The literature shows that the hydrophobic characteristic of lipids allows for the formation of a homogeneous PHS layer on hydroxyapatite disks [5,12,19]. Apparently, this PHS layer formed on the tooth enamel was able to maintain itself when this solution was applied after tooth brushing and consequently was able to protect the tooth enamel and thus minimize the color change caused by tooth brushing. However, when the PHS solution is applied prior to brushing the teeth, even by immersion, which provides greater availability of solution to the specimens than spray, it appears that this film cannot be consistently maintained, and thus is not able to protect the tooth enamel in the same way as when it is applied after tooth brushing.
The results for surface roughness change provide further substantiation for this finding. The highest change in surface roughness was found for the TB group, while the I + TB group showed intermediate results and the TB + S group demonstrated the lowest values of change in surface roughness. According to Vieira-Júnior et al. [41], the increase in enamel surface roughness variation was able to influence the decrease in L* values, related to the luminosity of the tooth, and also decrease the a* coordinate, moving the color of the sample in the direction of red or against green. In the present study, the color change was due to the decrease in the L*, a*, and b* coordinates; perhaps, as in the study of Vieira-Júnior et al. [40], brushing may have influenced the decrease in the coordinates, especially the L* coordinate.
The microhardness change values compared the raw microhardness values and showed that the I + TB group showed higher microhardness values, followed by the TB + S group and a slight mineral loss of the enamel of the TB group specimens. However, when evaluating the relative microhardness, which considers the initial and final microhardness values for the same sample, it was found that the increase in microhardness was similar for the two forms of PHS application, immersion and spray, and higher than the results found by the TB group. Yonel et al. [13] found that dentifrices containing Sn2 + and F − were more effective in protecting tooth enamel from a mineral loss than PHS solution when the anti-erosive potential of PHS was evaluated.
One limitation of the present research was that the change in the mineral content was only measured by indirect methods, such as hardness and roughness. Other tests would contribute to better understanding of the mechanism of action of the PHS solution, such as the chemistry characterization of dental enamel. In addition, the lack of studies about the action and efficacy of PHS in dentistry makes it difficult to properly discuss the data.
In the present study, the PHS solutions associated with the use of dentifrices showed better results than the dentifrice used alone, suggesting that perhaps the interaction of PHS with fluorine can improve the performance in the protection of dental enamel, rather than when these two solutions are used alone, which may impact the development of future studies on the addition of this lipid as a component of fluoride formulations.

Conclusions
According to the methodology used in this study, it is plausible to conclude that the presence of PHS in solution improves oral health maintenance as it leads to an increase in performance regarding color stability, surface roughness, and microhardness when applied, for 15 days, as a spray after tooth brushing, or in the form of immersion before tooth brushing.
Author contribution All the authors contributed substantially throughout the drafting, data interpretation, and critical revision of the paper. Moreover, they approved the final version of the paper and agreed with all aspects of the work. Additionally, CNFA worked in all stages of the research; RGV helped with the methodology and writing process; AAA, ACF, and RTT helped with the methodology; FJB conceived the PHS solution and worked in all stages of the writing process; FCPPS worked in all stages of the research.