Response of carious enamel to TiF 4varnish treatment under diverse cariogenic activities in situ

Abstract

Objective

To compare the effect of TiF 4 and NaF varnishes on demineralized bovine enamel under different cariogenic activities in situ .

Methods

Twenty subjects participated of this in situ study with 3 crossover phases (14 days each), in which they wore palatal appliances containing demineralized bovine enamel samples (8 samples/appliance for phase) treated with TiF 4 , NaF (all with 2.45% F) or placebo varnish. The samples were subjected to different cariogenic conditions (1. absence of biofilm accumulation and sucrose exposure; 2. presence of biofilm and absence of sucrose exposure; 3. presence of biofilm and 20% sucrose exposure 4×/day; 4. presence of biofilm and 20% sucrose exposure 8×/day). All were exposed to fluoride dentifrice (2×/day). The mineral content and lesion depth were evaluated using transverse microradiography (TMR) and the data were subjected to RM two-way ANOVA/Bonferroni tests (p < 0.05).

Results

TiF 4 varnish significantly increased the remineralization of artificial carious lesions compared to placebo, regardless of the cariogenic activity. On the other hand, the remineralizing effect of NaF varnish was dependent on the cariogenic activity. For NaF, remineralization happened only in conditions 1 and 3 compared to placebo varnish (p < 0.0001). NaF was unable to prevent further demineralization under biofilm accumulation and sucrose exposure 8×/day (condition 4). In the absence of fluoride treatment, demineralization happened in all conditions, except in the condition 1.

Conclusion

Therefore, 4% TiF 4 varnish was the only treatment able to improve enamel remineralization regardless of the cariogenic activity, while NaF varnish failed in preventing further demineralization under high cariogenic activity in situ .

Clinical significance

4% TiF 4 varnish showed better remineralizing effect compared to NaF varnish, which was seen regardless of the cariogenic activity. This is a promising finding to support the indication of TiF 4 in the clinic.

Introduction

In the last years, polyvalent metals fluorides, such as stannous fluoride (SnF 2 ) and titanium tetrafluoride (TiF 4 ), have became of great interest again due to new formulations that have shown to be more effective against tooth demineralization than NaF formulations . Previous works from our group have shown superiority of 4% TiF 4 varnish compared to NaF varnish against dental caries and erosion , with similar cytotoxicity .

The inclusion of TiF 4 into a varnish allows longer time of contact with enamel, improving the reaction of titanium with the apatite phosphate and allowing the formation of a “glaze”-like layer on the tooth surface rich in titanium dioxide, hydrated titanium phosphate and calcium fluoride . Due to its low pH, TiF 4 varnish is able to enhance the enamel fluoride uptake compared to NaF varnish , showing a greater fluoride release into artificial saliva and a higher fluoride availability to react with tooth . The varnish may also reduce the contact of TiF 4 with soft tissues compared to a rinse solution, reducing the possibility of cytotoxicity.

Despite we have shown that TiF 4 is able to improve remineralization under controlled conditions in vitro and in situ , we have no information if its positive effect could be modulated by the cariogenic activity of the patient. Therefore, this in situ study aimed to compare the effect of TiF 4 and NaF varnishes in respect to bovine enamel remineralization under different cariogenic activities (4 different conditions ranging from no to high cariogenic activity). The hypothesis is that the positive effect of TiF 4 varnish on enamel remineralization is not influenced by the cariogenic activity.

Materials and methods

Ethical aspects and subjects

The study followed a double-blind, randomized and crossover protocol, comprising of 3 phases (14 days each), with an interval of 7 days between them. Twenty healthy adults (19–42 years old) were included according to the study inclusion and exclusion criteria as described elsewhere . The sample size calculation was based on previous work from our group considering a power of 80% and a α error of 5%.

This study was conducted according to the guidelines of good clinical practice and conformed to the Declaration of Helsinki. Ethical approval for this study involving human subjects was granted by the local Ethics Committee (Process n. 114/2011. Bauru School of Dentistry, University of São Paulo, SP, Brazil). The subjects received written instructions, including schedules, and were trained for all procedures required during the study. Informed consent was obtained from all subjects before starting the study.

Samples preparation

Five hundred and forty-four enamel samples (4 mm × 4 mm × 3 mm) were prepared from labial surface of the bovine crowns as described in elsewhere . All enamel samples were submitted to the baseline surface microhardness analysis and protected on 1/3 of the surface with cosmetic nail varnish (Risque, São Paulo, Brazil) to obtain a sound control area (S). They were then demineralized using a lactic buffer solution containing calcium, phosphate and 6 μM MDHP (Sigma-Aldrich, St. Louis, USA), during 6 days at 37 °C . Four hundred and eighty samples were selected and distributed to the groups according to the percentage of surface hardness change (% SHC mean 87.2 ± 7.6%). The samples had a further 1/3 of the surface protected with cosmetic nail varnish to preserve an untreated pre-demineralized area (DE).

In situ protocol

Acrylic palatal appliances were prepared with four cavities (11 mm × 6 mm × 4 mm) distributed on both left and right sides, able to receive 2 samples each, for each phase and subject. The samples were disinfected as previously described . After the disinfection, the samples were randomly distributed in one of 3 randomized, double-blind and crossover phases: Phase A- 5.45% NaF varnish (pH 5, 2.45% F); Phase B- 4.00% TiF 4 experimental varnish (pH 1, 2.45% F) and Phase C- placebo varnish (pH 5, without fluoride). All varnishes were prepared by FGM-Denstcare (Joinvile, Brazil).

The treatments with the varnishes were carried out in vitro at the day before the beginning of each in situ phase. The fluoride varnishes were applied on the enamel surface (1/3 central enamel area) for 6 h under artificial saliva exposure and then removed as described previously .

The treated samples were allocated into the appliance according to 4 cariogenic conditions (n = 2 for each condition): 1. absence of biofilm accumulation and absence of sucrose exposure; 2. presence of biofilm and absence of sucrose exposure; 3. presence of biofilm and 20% sucrose exposure 4×/day; 4. presence of biofilm and 20% sucrose exposure 8×/day. For the biofilm formation, a 4 mm-deep space was created in the acrylic appliance, leaving a 1.0 mm space. Additionally, the samples were protected from mechanical disturbance using a plastic mesh .

The subjects were instructed to wear the appliance for 20 h a day, removing it only for the 4 daily meals (1 h each), during 14 days. The subjects were instructed to maintain the usual eating habits and to perform oral hygiene using only the products provided by the researchers (tooth floss, toothbrushing and F toothpaste, Colgate-Palmovile, São Bernardo dos Campos, Brazil) and not to use any other type of fluoride or antibacterial product during the in situ phases. Furthermore, the samples should be not brushed during oral hygiene.

The sucrose exposure was done for 5 min ex vivo , 4 times per day (condition 3) or 8 times per day (condition 4), with an interval of at least 1 h between each challenge. All samples were exposed to fluoride toothpaste slurry (1100 ppm F, NaF, Sorriso Fresh plus menthol impact, Colgate-Palmolive, São Bernardo dos Campos, Brazil, diluted 1:3 water), 2 times per day, for 1 min, ex vivo .

TMR analysis

After the in situ phases, the samples were transversally sectioned to the orientation of the protective nail varnish, allowing all areas were presented in the TMR specimen: sound (S), demineralized in vitro (DE), demineralized in vitro , treated and further de-remineralized in situ (DE-RE). Fragments were polished to obtain specimens with thickness around 80–100 μm. Microradiographs were made of each tooth fragment in combination with “stepwedge” (14 slices, ± 30 μm thick, 99.9% Al) for calibration, using glass plates that were exposed to x-ray Cu Ka (20 KV and 20 mA) for 13 min. The glass plate was developed (4 min, 20 °C), fixed (6 min, 20 °C) and washed using running water (10 min). The developed plate was analysed using a transmitted light microscope fitted with a 20 x objective (Axioplan, Zeiss, Oberkochen, Germany) and a CCD camera (XC-77 CE, Sony, Tokyo, Japan) coupled to a computer with software for calculations (TMR 2012 and TMR 2006 Inspector Research BV, Amsterdam, Netherlands).

The integrated mineral loss (ΔZ, %vol·μm) and the lesion depth (LD, μm) were measured in all enamel areas to allow the calculation of ΔΔZ [ΔZ lesion (DE area) – ΔZ effect (DE-RE area)] and ΔLD [LD lesion (DE area) – LD effect (DE-RE area)] .

Statistical analysis

Data were statistically analysed using the software Graph Pad Prism for Windows (GraphPad Software, San Diego, USA). Repeated measures two-way ANOVA followed by Bonferroni’s test were applied to compare the criteria (the treatments and the cariogenic activities). The significance level was set at 5%.

Materials and methods

Ethical aspects and subjects

The study followed a double-blind, randomized and crossover protocol, comprising of 3 phases (14 days each), with an interval of 7 days between them. Twenty healthy adults (19–42 years old) were included according to the study inclusion and exclusion criteria as described elsewhere . The sample size calculation was based on previous work from our group considering a power of 80% and a α error of 5%.

This study was conducted according to the guidelines of good clinical practice and conformed to the Declaration of Helsinki. Ethical approval for this study involving human subjects was granted by the local Ethics Committee (Process n. 114/2011. Bauru School of Dentistry, University of São Paulo, SP, Brazil). The subjects received written instructions, including schedules, and were trained for all procedures required during the study. Informed consent was obtained from all subjects before starting the study.

Samples preparation

Five hundred and forty-four enamel samples (4 mm × 4 mm × 3 mm) were prepared from labial surface of the bovine crowns as described in elsewhere . All enamel samples were submitted to the baseline surface microhardness analysis and protected on 1/3 of the surface with cosmetic nail varnish (Risque, São Paulo, Brazil) to obtain a sound control area (S). They were then demineralized using a lactic buffer solution containing calcium, phosphate and 6 μM MDHP (Sigma-Aldrich, St. Louis, USA), during 6 days at 37 °C . Four hundred and eighty samples were selected and distributed to the groups according to the percentage of surface hardness change (% SHC mean 87.2 ± 7.6%). The samples had a further 1/3 of the surface protected with cosmetic nail varnish to preserve an untreated pre-demineralized area (DE).

In situ protocol

Acrylic palatal appliances were prepared with four cavities (11 mm × 6 mm × 4 mm) distributed on both left and right sides, able to receive 2 samples each, for each phase and subject. The samples were disinfected as previously described . After the disinfection, the samples were randomly distributed in one of 3 randomized, double-blind and crossover phases: Phase A- 5.45% NaF varnish (pH 5, 2.45% F); Phase B- 4.00% TiF 4 experimental varnish (pH 1, 2.45% F) and Phase C- placebo varnish (pH 5, without fluoride). All varnishes were prepared by FGM-Denstcare (Joinvile, Brazil).

The treatments with the varnishes were carried out in vitro at the day before the beginning of each in situ phase. The fluoride varnishes were applied on the enamel surface (1/3 central enamel area) for 6 h under artificial saliva exposure and then removed as described previously .

The treated samples were allocated into the appliance according to 4 cariogenic conditions (n = 2 for each condition): 1. absence of biofilm accumulation and absence of sucrose exposure; 2. presence of biofilm and absence of sucrose exposure; 3. presence of biofilm and 20% sucrose exposure 4×/day; 4. presence of biofilm and 20% sucrose exposure 8×/day. For the biofilm formation, a 4 mm-deep space was created in the acrylic appliance, leaving a 1.0 mm space. Additionally, the samples were protected from mechanical disturbance using a plastic mesh .

The subjects were instructed to wear the appliance for 20 h a day, removing it only for the 4 daily meals (1 h each), during 14 days. The subjects were instructed to maintain the usual eating habits and to perform oral hygiene using only the products provided by the researchers (tooth floss, toothbrushing and F toothpaste, Colgate-Palmovile, São Bernardo dos Campos, Brazil) and not to use any other type of fluoride or antibacterial product during the in situ phases. Furthermore, the samples should be not brushed during oral hygiene.

The sucrose exposure was done for 5 min ex vivo , 4 times per day (condition 3) or 8 times per day (condition 4), with an interval of at least 1 h between each challenge. All samples were exposed to fluoride toothpaste slurry (1100 ppm F, NaF, Sorriso Fresh plus menthol impact, Colgate-Palmolive, São Bernardo dos Campos, Brazil, diluted 1:3 water), 2 times per day, for 1 min, ex vivo .

TMR analysis

After the in situ phases, the samples were transversally sectioned to the orientation of the protective nail varnish, allowing all areas were presented in the TMR specimen: sound (S), demineralized in vitro (DE), demineralized in vitro , treated and further de-remineralized in situ (DE-RE). Fragments were polished to obtain specimens with thickness around 80–100 μm. Microradiographs were made of each tooth fragment in combination with “stepwedge” (14 slices, ± 30 μm thick, 99.9% Al) for calibration, using glass plates that were exposed to x-ray Cu Ka (20 KV and 20 mA) for 13 min. The glass plate was developed (4 min, 20 °C), fixed (6 min, 20 °C) and washed using running water (10 min). The developed plate was analysed using a transmitted light microscope fitted with a 20 x objective (Axioplan, Zeiss, Oberkochen, Germany) and a CCD camera (XC-77 CE, Sony, Tokyo, Japan) coupled to a computer with software for calculations (TMR 2012 and TMR 2006 Inspector Research BV, Amsterdam, Netherlands).

The integrated mineral loss (ΔZ, %vol·μm) and the lesion depth (LD, μm) were measured in all enamel areas to allow the calculation of ΔΔZ [ΔZ lesion (DE area) – ΔZ effect (DE-RE area)] and ΔLD [LD lesion (DE area) – LD effect (DE-RE area)] .

Statistical analysis

Data were statistically analysed using the software Graph Pad Prism for Windows (GraphPad Software, San Diego, USA). Repeated measures two-way ANOVA followed by Bonferroni’s test were applied to compare the criteria (the treatments and the cariogenic activities). The significance level was set at 5%.

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Jun 17, 2018 | Posted by in General Dentistry | Comments Off on Response of carious enamel to TiF 4varnish treatment under diverse cariogenic activities in situ
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