Abstract
Objective
This study compared the effect of an experimental NaF/TiF 4 mouth rinse with a commercial tin/F mouth rinse on the prevention of tooth wear in situ .
Methods
Fifteen subjects took part in this crossover and double-blind study, in which they wore a palatal appliance with 8 bovine teeth samples (4 enamel and 4 root dentine) in each of 3 phases (5 days each). Half of the samples were subjected to erosive challenges, and the other half to erosive plus abrasive challenges. The phases corresponded to the use of 1) Experimental solution containing NaF/TiF 4 (189 ppm Ti +4 , 500 ppm F − , pH 4.4); 2) commercial solution containing SnCl 2 /NaF/AmF (800 ppm Sn +2 , 500 ppm F − , pH 4.5, Elmex ® /GABA, positive control); 3) distilled water (negative control). Erosive challenges were performed using 0.1% citric acid (pH 2.5) for 90 s 4 times per day. The abrasion was done using a toothbrush and slurry of fluoride toothpaste, for 15 s 2 times per day. Thereafter, the subjects rinsed with the tested mouth rinse for 60s. Tooth wear was measured using contact profilometry (μm) and submitted to a two-way RM ANOVA/Bonferroni test (p < 0.05).
Results
No significant differences were detected between the experimental and the commercial mouth rinses, regardless of the challenge. Both fluoride mouth rinses were able to significantly reduce tooth wear compared to the negative control (p < 0.0001). No significant differences were detected with respect to tooth wear between the challenges (erosion and erosion plus abrasion).
Conclusion
The experimental NaF/TiF 4 mouth rinse has a similar protective effect to the commercial one against tooth wear in situ.
Clinical significance
The experimental NaF/TiF 4 solution protected against tooth wear in situ , regardless of the challenge (erosion or erosion plus abrasion), for both enamel and dentine, similarly to a commercial solution (tin/F-Elmex ® ) applied for this proposal. This result supports the conduction of clinical trials and a possible application of this solution in the future.
1
Introduction
Titanium tetrafluoride (TiF 4 ) has regained interest in dentistry due to the development of new formulations that have shown to be more effective against tooth demineralization (for both dental caries and erosion) compared to sodium fluoride (NaF) formulations in vitro and in situ [ ]. TiF 4 has also shown to be less cytotoxic [ ] than what was demonstrated in the past [ ]. One of the new formulations, an experimental 4% TiF 4 varnish, was developed in 2007 and first evaluated against enamel erosion in vitro . It was shown to be more effective compared to 4% TiF 4 solution and commercial NaF varnishes in reducing erosive wear [ ]. Since then, some studies have been carried out to test the effect of this experimental TiF 4 varnish against enamel and dentine erosion and abrasion in vitro and in situ , with promising results [ ].
On the other hand, patients with erosion could be benefit from a daily application of fluoride [ ]. Therefore, the professional application of a varnish, 2–4 times per year, for example, may not be the best option for patients under high daily erosive challenges. Accordingly, Magalhães et al. [ ] investigated the isolated and combined effect of a single application of TiF 4 or NaF varnish (24,500 ppm F − ) versus a daily use of a solution (mouth rinse) containing a low concentration of TiF 4 /NaF (500 ppm F − ) against tooth erosion in vitro . Both types of TiF 4 applications, isolated or combined, were effective against tooth erosion. However, the varnish lost its protective effect on enamel after 10 days of erosive challenges, whereas the protective effect of the mouth rinse was still evident until the end of the study (14 days).
To better understand the performance of this experimental solution containing both NaF and TiF 4, two in vitro studies have been conducted to compare the experimental NaF/TiF 4 solution with a known commercial fluoride mouth rinse containing tin (Elmex ® ) [ ]. Fluoride solution containing tin has shown a protective effect against tooth erosion [ ]. Its mechanism of action is due to the formation of precipitates on the dental surface (such as Ca(SnF 3 ) 2 , SnOHPO 4 , Sn 3 F 3 PO 4 ), which are more acid resistant than CaF 2 particles induced by NaF application [ ]. Furthermore, fluoride solution containing tin is able to reduce the development of dentine erosion regardless of the preservation of demineralized organic matrix (DOM) [ ].
With respect to the mechanism of action of TiF 4 , our knowledge comes from a recent study that elucidated the reaction of TiF 4 varnish with enamel. Titanium can minimize demineralization, since it tends to complex with phosphate from apatite, forming a glaze-like layer which is rich in titanium oxide and hydrated titanium phosphate, and which is also more acid resistant than CaF 2 [ ]. In addition, TiF 4 is able to produce a higher amount of CaF 2 on enamel compared to NaF, due to its low pH [ ]. TiF 4 varnish has been shown to protect against dentine erosion regardless of the presence of DOM [ ]. We assumed that a similar mechanism of action might occur if TiF 4 /NaF solution were applied daily.
In both cited studies, the daily application of TiF 4 /NaF solution was demonstrated to be inferior or similar to that of a fluoride solution containing tin in reducing erosive wear of enamel and dentine respectively [ ]. However, we do not know if the results would be the same if erosion were combined with abrasion. Furthermore, it is essential to confirm the findings under models closer to the in vivo condition.
Therefore, the aim of this study was to compare the protective effect of an experimental solution containing TiF 4 and NaF with that of a commercial fluoride and tin solution (Elmex ® , GABA) on enamel and dentine erosion and erosion plus abrasion in situ . The tested null hypotheses were: 1) there is no significant difference between the fluoride solutions in reducing enamel and dentine wear, regardless of the type of challenge (erosion vs. erosion plus abrasion); and 2) there is no significant difference between the type of challenge (erosion vs. erosion plus abrasion) with respect to enamel and dentine wear, regardless of the fluoride mouth rinse.
2
Material and methods
2.1
Ethical aspects and subject selection
The study followed a double blind, randomized, crossover in situ protocol, comprising 3 phases of 5 days each, with an interval of 10 days among them. Firstly, the study protocol was submitted and approved by the local Ethics Committee (no. 67610417.3.0000.5417; Ethics Committee of the Bauru School of Dentistry, University of São Paulo, SP, Brazil).
Fifteen healthy adults (11 women and four men, 18–27 years old) were then selected according to the study inclusion and exclusion criteria. The inclusion criteria were good general and oral health (no caries or significant gingivitis/periodontitis), stimulated salivary flow rate > 1 ml/min and unstimulated salivary flow rate > 0.25 ml/min. The exclusion criteria were the presence of systemic illness, pregnancy or breastfeeding, use of orthodontic appliances, professional fluoride application within 2 months prior to the study and smoking habit. The subjects received written instructions and the schedules, and they were trained for all procedures required during the study (e.g., how to brush the samples). Informed consent was obtained from all subjects before the beginning of the study.
2.2
Preparation of samples
One hundred eighty enamel and 180 dentine samples (4 mm × 4 mm × 3 mm) were prepared from the labial surfaces of bovine crowns and labial/lingual surfaces of the cervical portion of bovine roots respectively. The teeth were stored in 0.1% buffered thymol solution (pH 7.0). The samples were cut using an ISOMET Low Speed Saw cutting machine (Buehler Ltd., Lake Bluff, USA) separated by a 4-mm-thick spacer. The surfaces were ground flat with water-cooled silicon-carbide discs (320, 600 and 1200 grades of Al 2 O 3 papers; Buehler, Lake Bluff, USA). Thereafter, the baseline profile was measured using a contact profilometer (Mahr Perthometer, Göttingen, Germany), and two thirds of the outer samples’ surfaces were protected with red nail polish (Risque, São Paulo, Brazil) to obtain two control areas for the determination of tooth wear. Before the in situ protocol, the samples were sterilized using ethylene oxide [Gas exposure time (30% ETO/70% CO 2 ) for 4 h under a pressure of 0.5 ± 0.1 kgF/cm 2 ].
2.3
In situ protocol and experimental groups
Acrylic palatal appliances were prepared individually for each subject and for each in situ phase. They were made with eight cavities (5 mm × 5 mm × 4 mm), which were distributed on the left and right sides. Enamel and dentine samples were randomly accommodated into the cavities and fixed in place with wax, resulting in a total of four samples subjected to erosion only (2 enamel and 2 dentine samples) and four samples subjected to erosion plus abrasion (2 enamel and 2 dentine samples). All samples were placed at the same level of the acrylic resin and were replaced for each in situ phase.
The study comprised 3 crossover phases of 5 days each (12 h of use a day, from 8:00 a.m. to 8:00 p.m. During the night, the appliance was stored in gauze moistened with tap water), with an interval period of 10 days between each phase. In each phase, five subjects were assigned to one of the three treatments, as follows: the experimental solution containing 0.042% NaF and 0.049% TiF 4 (NaF: 200 ppm F − , TiF 4 : 189 ppm Ti +4 and 300 ppm F − , pH 4.4); commercial solution containing SnCl 2 /NaF/AmF (800 ppm Sn +2 , 500 ppm F − , pH 4.5, Elmex ® − GABA International AG, Therwil, Switzerland, positive control); and distilled water (negative control). The experimental fluoride solution was prepared a day before the beginning of each phase. Fig. 1 summarizes the study design.
The erosive challenges were done at pre-established times of day (10:00 a.m., 2:00 p.m., 4:00 p.m., 6:00 p.m.). The subjects were instructed to immerse the appliance in 150 ml of 0.1% citric acid solution (pH 2.5) at room temperature for 90 s. Afterward, the subjects washed the appliances with tap water for 5 s and replaced them in their mouths. Thirty minutes after the first and the last erosive challenges, the subjects brushed half of the samples using a soft toothbrush (5460 ultrasoft, Curaprox ® , Kriens, Switzerland) and one drop of fluoride toothpaste slurry (1 g toothpaste: 3 ml deionized water, 1100 ppm F, NaF, Colgate ® , São Bernardo, Brazil) per sample for 15 s [ ]. The appliances were then washed with tap water for 5 s and replaced in the mouth before performing the rinsing. The subjects immediately rinsed the tested solution (v = 10 ml) for 60 s. After rinsing, the subjects were instructed to keep the appliance inside the mouth for at least 30 min and to refrain from eating or drinking during this period. At the end of each in situ phase, the appliances were collected from all subjects and the samples prepared for the contact profile analysis. Fig. 2 summarizes the experimental protocol.
The subjects were instructed to keep their usual eating habits and to perform oral hygiene using a toothbrush (Curaprox ® , Kriens, Switzerland), dental floss (Jadefrog ® , Londrina, Brazil) and fluoride toothpaste (1100 ppm F, NaF, Colgate, São Bernardo, Brazil) provided by the researchers during all phases of the study. The hygiene of the appliance was restricted to the palatal area. The subjects were also instructed to refrain from using any other type of fluoride or antimicrobial product during the in situ phases and the washout periods.
During the in situ phases, two meals with a duration of 30 min each were allowed (12:00 p.m. and 8:00 p.m.). During the meal periods, the appliance had to be kept in gauze moistened with water, and oral hygiene with fluoride toothpaste was mandatory 5 min before the appliance was placed back into the mouth (in the case of the 1st meal).
After each phase, a visual scale (Wong-Baker Pain Scale [WBPS]) was applied to assess the subject’s degree of satisfaction with the treatment ( Fig. 3 ). The scale is known to be one of the most effective tools for self-rated patient pain [ ]. A questionnaire about the taste of the mouth rinse, possible side effects and difficulties with the in situ protocol was also applied.
2.4
Tooth wear measurement
Tooth wear was determined using a contact profilometer (Mahr Perthometer, Göttingen, Germany). Five equidistant surface scans of each sample were performed (3.5 mm of reading, 250 μm apart from each other), under 100% humidity in case of dentine, at the baseline and after the in situ phases. After the in situ phases, the nail polish was removed as previously described [ ]. To achieve the repeatability of the measurement, the samples presented an identification mark (small drilling made with ¼ drill) and were inserted into a metal device (x and y axis determination) to allow the stylus to be accurately repositioned at each measurement.
The baseline profile was compared to the final profile for the calculation of tooth wear using the software Mahr Surf XCR20. The scans were superposed, and the average depth of the area under the curve was calculated (μm) considering 0.1 μm as the limit of detection [ ].
2.5
Statistical analysis
The data were statistically analysed using the software GraphPad Prism for Windows (GraphPad Softwares, San Diego, USA). The assumptions of equality of variances and normal distribution of data were checked using the Bartlett and Kolmogorov-Smirnov tests respectively. Two-way RM ANOVA was applied, considering treatment (3 levels) and challenge (2 levels) as criteria at a 5% level of significance. The individual comparisons were done using the post-hoc Bonferroni test.
2
Material and methods
2.1
Ethical aspects and subject selection
The study followed a double blind, randomized, crossover in situ protocol, comprising 3 phases of 5 days each, with an interval of 10 days among them. Firstly, the study protocol was submitted and approved by the local Ethics Committee (no. 67610417.3.0000.5417; Ethics Committee of the Bauru School of Dentistry, University of São Paulo, SP, Brazil).
Fifteen healthy adults (11 women and four men, 18–27 years old) were then selected according to the study inclusion and exclusion criteria. The inclusion criteria were good general and oral health (no caries or significant gingivitis/periodontitis), stimulated salivary flow rate > 1 ml/min and unstimulated salivary flow rate > 0.25 ml/min. The exclusion criteria were the presence of systemic illness, pregnancy or breastfeeding, use of orthodontic appliances, professional fluoride application within 2 months prior to the study and smoking habit. The subjects received written instructions and the schedules, and they were trained for all procedures required during the study (e.g., how to brush the samples). Informed consent was obtained from all subjects before the beginning of the study.
2.2
Preparation of samples
One hundred eighty enamel and 180 dentine samples (4 mm × 4 mm × 3 mm) were prepared from the labial surfaces of bovine crowns and labial/lingual surfaces of the cervical portion of bovine roots respectively. The teeth were stored in 0.1% buffered thymol solution (pH 7.0). The samples were cut using an ISOMET Low Speed Saw cutting machine (Buehler Ltd., Lake Bluff, USA) separated by a 4-mm-thick spacer. The surfaces were ground flat with water-cooled silicon-carbide discs (320, 600 and 1200 grades of Al 2 O 3 papers; Buehler, Lake Bluff, USA). Thereafter, the baseline profile was measured using a contact profilometer (Mahr Perthometer, Göttingen, Germany), and two thirds of the outer samples’ surfaces were protected with red nail polish (Risque, São Paulo, Brazil) to obtain two control areas for the determination of tooth wear. Before the in situ protocol, the samples were sterilized using ethylene oxide [Gas exposure time (30% ETO/70% CO 2 ) for 4 h under a pressure of 0.5 ± 0.1 kgF/cm 2 ].
2.3
In situ protocol and experimental groups
Acrylic palatal appliances were prepared individually for each subject and for each in situ phase. They were made with eight cavities (5 mm × 5 mm × 4 mm), which were distributed on the left and right sides. Enamel and dentine samples were randomly accommodated into the cavities and fixed in place with wax, resulting in a total of four samples subjected to erosion only (2 enamel and 2 dentine samples) and four samples subjected to erosion plus abrasion (2 enamel and 2 dentine samples). All samples were placed at the same level of the acrylic resin and were replaced for each in situ phase.
The study comprised 3 crossover phases of 5 days each (12 h of use a day, from 8:00 a.m. to 8:00 p.m. During the night, the appliance was stored in gauze moistened with tap water), with an interval period of 10 days between each phase. In each phase, five subjects were assigned to one of the three treatments, as follows: the experimental solution containing 0.042% NaF and 0.049% TiF 4 (NaF: 200 ppm F − , TiF 4 : 189 ppm Ti +4 and 300 ppm F − , pH 4.4); commercial solution containing SnCl 2 /NaF/AmF (800 ppm Sn +2 , 500 ppm F − , pH 4.5, Elmex ® − GABA International AG, Therwil, Switzerland, positive control); and distilled water (negative control). The experimental fluoride solution was prepared a day before the beginning of each phase. Fig. 1 summarizes the study design.
