An In Vitro Comparison of Clinpro ™ XT and Duraphat Varnish for Protecting Teeth from Discoloration during Orthodontic Treatment

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Introduction
Aesthetics is one of the cornerstone objectives of orthodontic treatment with both fxed and clear appliances.Although orthodontic outcomes can be acceptable, several adverse efects of orthodontic treatments have been reported, such as gingivitis, loss of periodontal attachment, white spot lesions (WSLs), and tooth discoloration [1].During orthodontic treatment, a higher caries risk has been reported, so fuoride [2] and other remineralizing compounds [3] have been proposed.Many patients have complained of tooth discoloration during orthodontic treatment, which can signifcantly afect the attractiveness of a smile [4].
Te color of teeth can be infuenced by a combination of intrinsic and extrinsic staining [5].Direct extrinsic staining, such as that caused by cofee, tea, and red wine, which are commonly consumed, occurs via incorporation of the staining solution into the pellicle; the color imparted is determined by the basic color of the staining solution.Furthermore, some acidic beverages might infuence the surface microhardness [6], which increases enamel porosity and consequently increases susceptibility to further staining via adsorption [7].Te accumulation of plaque around complex appliances has been shown to increase due to the difculty in maintaining oral hygiene throughout orthodontic treatment, which leads to a potential risk of extrinsic staining.Indirect extrinsic staining has been shown to be related to cationic antiseptics and metal salts, which can cause chemical reactions on the tooth surface [8].Orthodontic metal brackets comprise various metals, and their corrosion might release large amounts of nickel and chromium ions, resulting in enamel discoloration [9].Te resin used to attach clear appliances for orthodontic treatment can preclude the problems caused by the release of metal ions.Currently, no studies have been conducted on the efects of using resin for appliance attachment on tooth discoloration compared to those of using metal brackets.
Te best strategies for the management of tooth discoloration seem to be those that prevent its occurrence.Currently, fuoride plays an important role in mitigating acid attacks [10].Te layer formed by fuoride varnish on the tooth surface can even act as a shield against tooth discoloration caused by food and drink.However, the diferent forms of fuoride agents, such as fuoride toothpaste, fuoride gel, and tooth protectors, usually are inefective.Tis inefciency is often associated with the low fuorine concentrations in the products as well as low patient cooperation [11,12].Professional fuoride varnish is the preferred method of topical application because of its simple usage and lack of patient dependency.Currently, fuoride varnish is commonly available in conventional and lightcurable forms.Conventional fuoride varnish is widely used; however, it must be reapplied every three months to maintain its efectiveness [13].Recently, a light-curableresinmodifed glass ionomer that can continuously release fuorine and calcium phosphate has attracted widespread clinical attention [11].Moreover, the manufacturer claims that this light-curable varnish can provide a protective coating for up to six months, with the potential for more controlled and sustained fuoride release [14].
According to the manufacturers, the chemical composition of the two materials was diferent, which are listed in Supplemental Table 1.To provide clinicians with better medication guidelines, it is necessary to study the efects of these two forms of fuoride varnish on protecting teeth from discoloration.Moreover, a comparison of these two forms when orthodontic appliances are attached with resin and brackets has not been performed.Terefore, the following null hypotheses were tested: ① these two forms of fuoride varnish are not efective in preventing tooth discoloration during orthodontic treatment; ② these two diferent fuoride varnishes do not show any signifcant diferences in protecting teeth from discoloration during orthodontic treatment; and ③ there are no signifcant diferences in tooth discoloration during orthodontic treatment between appliances attached with metal brackets and those attached with resin.

Materials and Methods (Supplemental
Figure 1) According to the inclusion criteria and sample size calculation (power, 0.9; α, 0.05; and efect size, 0.40), 120 teeth were included in the experiment.After extraction, the enamel of the premolars was cleaned with a rubber cup (TPC, Advanced Technology, California, USA) at slow speed for 5 seconds [1].Half of the roots of the premolars were sectioned and discarded, and after the pulp was extracted with a pulp needle, the remaining crowns were soaked in artifcial saliva in a water container at 37 °C during the entire study so that any efects of temperature and lighting were eliminated [15].Fresh artifcial saliva was prepared every day.

Sample Size
Calculation.G-power software (University of Düsseldorf, Düsseldorf, Germany) was used to estimate an appropriate sample size.Te power of the primary outcome (ΔE * ) was calculated based on our previous preexperiment with a power of 0.9, an efect size of 0.40, and an α of 0.05.Te results indicated that a minimum sample size of 19 teeth was required per group.Te fnal sample size was determined to be 20 teeth per group, which meant that 120 teeth were required for our study.

Experimental Design.
Te teeth were randomly assigned a number from 1 to 120 and randomly divided into six groups with 20 teeth each (Figure 1).Te root of each tooth was embedded in silicone rubber impression material (Supplemental Figure 2B).International Journal of Clinical Practice Two pairs of customized plastic trays with windows 6 mm (Supplemental Figure 2c2) or 3.5 mm (Supplemental Figure 2c3) in diameter and located in the middle of the buccal crown surface of each tooth were fabricated with a 1.0 mm-thicksoft-tray sheet according to the sample tooth crowns.Te 3.5 mm-diameter windows were used to limit the range of acid etching.Te 6 mm-diameter windows were used to ensure that spectrophotometric readings were obtained with a spectrophotometric probe (VITA Classical shade guide, VITA-Zahnfabrik, Bad Sackingen, Germany) at the same sites [16].Groups of resin attachments (Supplemental Figure 3A) were used to make alginate impressions (Supplemental Figure 3B) and flled with white plaster (Supplemental Figure 3C).Te resin attachment on the buccal side of the plaster model was formed with light-cured composite resin (Supplemental Figure 3E).Te size of the resin attachment was the same as the foor area (Supplemental Figure 3D) of the metal premolar bracket (MBT, Gemini ™ , 3M Unitek, São Paulo, USA), with a thickness of approximately 1.5 mm.Ten, the attachment templates (Supplemental Figure 3F) of these groups were fabricated with a 1.0 mm-thickhardtray sheet.Te Ethics Committee of the School of Stomatology at Fujian Medical University reviewed and approved the methodological protocol for this research and ensured that all the procedures met the guidelines of the Medical Ethics Committee (2019-IRB-35).

Adhesion
Procedure.Before adhesive application, all teeth were cleaned with a rubber cup at slow speed for 5 seconds, rinsed, and dried [1].Te following adhesion procedures were then performed: RCTR group: frst, the teeth were placed in a transparent retainer with 3.5 mm-diameter windows.Te exposed enamel was etched with 37% orthophosphoric acid for 15 seconds, rinsed with air-water spray for 30 seconds, and dried for 10 seconds.Ten, the resin attachment template flled with nanohybrid composite resin (Z350, 3M, São Paulo, USA) was placed on the teeth with adhesive resin (Transbond XT, 3M unit, São Paulo, USA).Te resin attachment was cured vertically for 20 seconds, with the light distance not exceeding 2 mm.Ten, the crowns were immediately soaked in artifcial saliva.
RC group: Clinpro ™ XT varnish was added to the appliance RD group: the appliance was supplemented with Duraphat varnish BCTR group: MBT metal premolar brackets were bonded with the same bonding system as in the RCTR group BC group: Clinpro ™ XT varnish was added to the appliance BD group: the appliance was supplemented with Duraphat varnish 2.5.Cofee Cycling.Te teeth were soaked in 180 ml of Nestle instant black cofee (Nestle Switzerland, Vevey, USA) for 5 min (Supplemental Figure 2D) 4 times per day between 8 a.m. and 5 p.m. Te interval between each cofee soaking was 2 hours (Supplemental Figure 4).When not soaked in cofee, these teeth were immersed in artifcial saliva, which was replaced every day.

Debonding and Resin
Removal.On days 7 and 28 of the study, 10 teeth in each group were removed from artifcial saliva separately.Te metal brackets were removed gently with a debonding plier.Te resin attachments and residual resin were removed by cleaning with a tungsten carbide bur under loupe magnifcation (3.3x) until no residual resin was observed with the naked eye.Ten, a rubber cup was applied at slow speed for 5 seconds [1].Afterward, color measurements were performed in the same environment.

Color Assessment.
Prior to the color measurement, the enamel was cleaned with a rubber cup at a slow speed and then thoroughly rinsed with running water.All teeth were inserted into the corresponding silicone rubber base and placed in the transparent retainer with 6 mm-diameter windows.All color measurements were obtained when the enamel surface was wet [17].A VITA spectrophotometer was used for color assessment according to the CIE (Commission Internationale de l'Eclairage) LAB color systems, where L * represents the degree of lightness, a * represents the red/green axis, and b * represents the yellow/blue axis [18].Before each measurement, the spectrophotometer was calibrated.Te color of each tooth was measured three times, and the data were averaged.Te color diferences before and after bonding (ΔE * , ΔL * , Δa * , and Δb * ) on days 7 and 28 were calculated using the following equations for color comparison [19]: where

Results
Te baseline L * , a * , and b * values of all groups and those recorded on days 7 and 28 are listed in Tables 1 and 2. Te ΔE * values of all groups recorded on days 7 and 28 are listed in Table 3.
A signifcant decrease in the L * value was observed for all groups, indicating that the teeth darkened throughout the discoloration procedure (Table 2).Tere were no diferences among the ΔL * values of the RC, RD, BC, and BD groups.On day 7, signifcant diferences were observed between the ΔL * values of the RCTR and RC groups (P = 0.001), the RCTR and RD groups (P = 0.039), the BCTR and BC groups (P = 0.009), and the BCTR and BD groups (P = 0.028) (Table 2).After an immersion time of 28 days, the ΔL * values of the RC and BC groups became signifcantly less than those of the RD and BD groups (Table 2).Tere were no signifcant diferences between the ΔL * values of the RCTR, RC, and RD groups and those of the corresponding BCTR, BC, and BD groups (Table 2).Signifcant diferences were still observed between the ΔL * values of the RCTR and RC groups (P < 0.001), the RCTR and RD groups (P = 0.011), the RC and RD groups (P < 0.001), the BCTR and BC groups (P < 0.001), the BCTR and BD groups (P < 0.001), and the BC and BD groups (P = 0.001) (Table 2).
A slight increase in the Δa * value was observed for all groups, indicating a shift toward red color components.Te Δa * values of the control groups exhibited more marked changes than those of the RC, RD, BC, and BD groups on day 7 (Table 2).Signifcant diferences were observed between the Δa * values of the RCTR and RC groups (P � 0.004), the RCTR and RD groups (P � 0.028), the BCTR and BC groups (P � 0.001), and the BCTR and BD groups (P � 0.009) (Table 2).Tere were no diferences between the Δa * values of the RC and RD groups or those of the BC and BD groups (Table 2).On day 28, the color changes indicated by the diferences in the Δa * values were similar for the control groups and the RD and BD groups (Table 2).Te Δa * values of the RC and BC groups shifted the least (Table 2).Signifcant diferences were observed between the Δa * values of the RCTR and RC groups (P � 0.009), the RC and RD groups (P � 0.026), the BCTR and BC groups (P � 0.017), and the BC and BD groups (P � 0.002) (Table 2).Tere were no diferences between the Δa * values of the RCTR and RD groups or those of the BCTR and BD groups.
A signifcant increase in the b * value was observed for all groups, indicating a shift toward yellow color components.Te Δb * values of the control groups showed signifcant changes throughout the study compared to those of the RC, RD, BC, and BD groups (Table 2).On day 7, no signifcant diferences were observed between the Δb * values of the RC and RD groups or those of the BC and BD groups (Table 2).International Journal of Clinical Practice Signifcant diferences were observed between the Δb * values of the RCTR and RC groups (P � 0.001), the RCTR and RD groups (P � 0.024), the BCTR and BC groups (P � 0.001), and the BCTR and BD groups (P < 0.001) (Table 2).However, the Δb * values of the RC and BC groups were signifcantly lower than those of the RD and BD groups on day 28 (Table 2).Signifcant diferences were observed between the △b * values of the RCTR and RC groups (P < 0.001), the RCTR and RD groups (P � 0.002), the RC and RD groups (P < 0.001), the BCTR and BC groups (P < 0.001), the BCTR and BD groups (P � 0.001), and the BC and BD groups (P < 0.001) (Table 2).
On day 7, the ΔE * values of both the RCTR and BCTR groups were higher than 2.7, which is the clinical limit of detection by the naked eye (Table 3).On day 28, the ΔE * values of all groups except for the RC group were higher than 3.7 (Table 3), which was considered unacceptable.Signifcant diferences were observed between the ΔE * values of the RCTR and RC groups (P < 0.001), the RCTR and RD groups (P < 0.001), the BCTR and BC groups (P < 0.001), and the BCTR and BD groups (P < 0.001) (Table 3).Tese diferences increased as the experiment progressed over 28 days (all P < 0.001) (Table 3).Tere were no signifcant diferences among the ΔE * values of the Clinpro ™ XT and Duraphat groups on day 7 (Table 3).However, the ΔE * values of the Duraphat groups increased with increasing immersion time (Table 3).No signifcant diferences were observed among   the ΔE * values of the resin and metal bracket groups on days 7 or 28 (Table 3).

Discussion
Information regarding the efcacy of Clinpro ™ XT and   Duraphat varnish in protecting teeth from discoloration is limited.Te current study can be considered the frst investigation in this feld.Based on the fndings of this study, the following null hypotheses were rejected: ① Clinpro ™ XT and Duraphat varnish are not efective in preventing tooth discoloration during orthodontic treatment; ② Clinpro ™ XT and Duraphat varnish do not show any signifcant diferences in protecting teeth from discoloration during orthodontic treatment.However, the following null hypothesis was accepted: ③ there are no signifcant diferences in tooth discoloration during orthodontic treatment between appliances attached with metal brackets and those attached with resin.Tis study was performed in vitro not only for ethical reasons but also to standardize the study conditions to exclude diferences in diet as a confounding factor and to facilitate identical testing conditions for Clinpro ™ XT and Duraphat.Artifcial saliva was also used to better refect the oral environment due to its ability to deposit a pellicle layer on teeth [21].Generally, tooth discoloration is infuenced by not only food colorants or dietary pigments but also low pH media [22].As the most popular beverage worldwide, cofee has the most potential for staining among common daily drinks because it is acidic and contains a variety of chemical compounds [23].Te cofee in this study had an acidic pH value, similar to that in previous studies [24,25].Te low pH value of cofee might induce enamel dissolution and increase surface porosity, thus promoting tooth discoloration.Te 28-day immersion time was chosen based on many previous and similar studies having a maximum immersion time of 30 days [26,27].Ertas et al. reported that 28 days were equivalent to approximately 2.5 years of clinical aging, which is approximately the duration of routine orthodontic treatment [28].
Usually, cofee causes an acidic and low-polarity stain via the demineralization of the enamel surface and the adsorption of cofee onto the tooth.Furthermore, demineralization changes the refractive index of enamel, altering its color properties [20].Fluoride varnish forms a layer of calcium fuoride material on the enamel surface that can protect against acid attack.Tus, it inhibits the demineralization of the enamel surface [29].Te layer formed by fuoride varnish on the enamel surface can even act as a shield, reducing the adsorption of cofee [30].
According to our results, the Clinpro ™ XT and Duraphat varnish groups both showed less discoloration than the control groups.
On day 7 of the study, no signifcant diferences were found between the Clinpro ™ XT and Duraphat groups, whereas these two groups exhibited signifcant diferences on day 28.Due to the inherent diference in the fuoride carrier among commercially available fuoride varnishes, all fuoride varnishes release fuoride, but the method and duration of release vary [31].Clinpro ™ XT is a resin- modifed glass ionomer cement that contains fuoroaluminosilicate glass particles.Te remineralization potential of this material is attributed to its manufacturing process, which creates functionalized calcium and free phosphate.Te fuoride at the surface is released immediately, while the internal fuoride serves as a storage pool for sustained fuoride release during orthodontic treatment [11].
Shah et al. reported that a single application of Clinpro ™ XT prevented enamel demineralization for up to 120 days, while the conventional fuoridated varnish prevented enamel demineralization for just 40 days; this diference was attributed to the functionalized calcium and free phosphate in Clinpro ™ XT [13].On the other hand, Duraphat is a varnish containing 5% sodium particles that adhere to the tooth surface after encountering saliva and detach after 24 hours, which difers from the behavior of light-curable varnish.Tus, due to the short retention time of Duraphat on the tooth surface, it needs to be applied frequently (every three months) to maintain its efectiveness [32].Te lower remineralization capacity of Duraphat leads to less protection and less tooth discoloration prevention.
In clinical situations, the corrosion of orthodontic alloys can occur, followed by the release of large amounts of nickel and chromium ions [9], resulting in tooth discoloration [1].Gölz et al. observed high metal ion levels after 2 weeks of metal appliance exposure [33].Nevertheless, no diferences were observed between metallic and nonmetallic braces in the present in situ experiment.Tis diference is most likely due to the variations in the conditions of metal corrosion.Mashallah et al. and Antonija et al. also reported the release of orthodontic metal ions from orthodontic metal brackets [34,35], which was evaluated 6 months after the beginning of their studies.Te limited experimental time of the present study may also have been a factor contributing to this diference.Another possible explanation could be that cofee has a much greater efect on tooth discoloration than metal alloys, thus preventing the observation of the efects of metal ions.Reduced or no friction between brackets and archwires might reduce the release of metal ions, which could also serve as a possible explanation for this diference [33].
In this study, we observed increases in both the a * and b * values with cofee exposure, suggesting that the color of the teeth became dark and shifted toward more reddishyellowish colors, which should be related to the adsorption of cofee onto the enamel [7].Te direction of tooth color change was consistent with the color of the staining agent [4].In this study, the changes in the a * and b * values of the fuoride varnish groups were signifcantly diferent from those of the control groups.Fluoride varnish not only acts as a barrier on the tooth surface, reducing the direct adsorption of cofee onto the enamel, but also prevents pigments from entering the internal structure of the tooth by facilitating remineralization in micropores, gaps between the glaze columns, and places with poor mineralization.
If the average orthodontic treatment duration is assumed to be 2 years, traditional fuoride varnish would need to be applied up to 24 times throughout the course of treatment to 6 International Journal of Clinical Practice achieve almost complete protection, whereas Clinpro ™ XT would only need to be applied 4 to 6 times [36].Te frequency of Clinpro ™ XT application would be nearly three times less, which is a considerable reduction that merits its recommendation for protecting teeth from discoloration.
Although the results of the study further the understanding of the potential diferences in fuoride release characteristics, they must be interpreted with caution since the study period was limited.In vitro experimental conditions cannot simulate all complex factors afecting the human mouth in vivo, including aging and plaque, and the fnal results regarding the efciency of these forms of fuoride varnish should be obtained through controlled clinical trials in the future.Additionally, the protective efects of these fuoride varnishes were evaluated without mechanical action, which can be considered another limitation of this study.Notably, a recent study showed that the use of a toothpaste containing hydroxyapatite could be a more reliable method for the domiciliary management of WSLs than the use of conventional fuoride toothpaste [37].It might be interesting to compare the efcacy of toothpaste containing biomimetic hydroxyapatite with that of the two varnishes investigated in this study.

Conclusion
Within the limitations of this study, the following conclusions can be drawn: (i) Te application of both tested fuoride varnishes during orthodontic treatment demonstrated signifcant and benefcial efects in preventing tooth discoloration (ii) Clinpro ™ XT varnish was more efective in pro- tecting teeth from discoloration than Duraphat varnish over time (iii) No signifcant diference was observed in tooth discoloration between the appliances attached with metal brackets and those attached with resin.
[6]determine the normality and homogeneity of the data, respectively[6].Diferences in the changes in the L * , a * , b * , and ΔE * values among all groups were compared using one-way ANOVA.Bonferroni's test was performed for post hoc comparisons.Te statistical signifcance level was 0.05.

Table 2 :
Comparison of the means and standard deviations (SDs) of the ΔL * , Δa * , and Δb * values of each group on days 7 and 28.
① RCTR: resin control group; RC: resin Clinpro group; RD: resin Duraphat group; BCTR: bracket control group; BC: bracket Clinpro group; BD: bracket Duraphat group.② Diferent uppercase letters in the same column indicate signifcant diferences for the diferent groups on day 7 or 28 (P < 0.05).Diferent lowercase letters in the same row indicate signifcant diferences among the diferent time points in the same groups (P < 0.05), whereas the same letter in a column or in a row indicates no signifcant diferences (P > 0.05).

Table 3 :
Comparison of the means and standard deviations (SDs) of the ΔE * values of each group on days 7 and 28.

Table 1 :
Means and standard deviations (SDs) of the baseline L * , a * , and b * values of each group.