Abstract
Objective
This in situ study evaluated the effect of the association of low-F (4500 μg F/g) gel containing TMP and FT (1100 μg F/g) on enamel demineralization.
Methods
This crossover and double-blind study consisted of five phases of seven days each. Volunteers (n = 12) wore palatal appliances containing four enamel blocks. The cariogenic challenge was performed with 30% sucrose solution (six times/day). Treatments were: placebo toothpaste (PT, no fluoride/TMP); 1100 μg F/g toothpaste (FT); FT + 4500 μg F/g + 5%TMP gel (FT + TMP gel); FT + 9000 μg F/g gel (FT + 9000 gel) and FT + 12,300 μg F/g (FT + Acid gel). After topical application of treatments for one min, two blocks were removed for analysis of loosely bound fluoride (CaF 2 ), calcium (Ca), phosphorus (P) and firmly bound fluoride (FA) formed in enamel. After the seven-day experimental periods, the percentage of surface hardness loss (%SH), integrated subsurface hardness loss (ΔKHN), CaF 2 , Ca, P and FA retained were determined. Moreover, the biofilms formed on the blocks were analyzed for F, Ca, P and insoluble extracellular polysaccharide (EPS) concentrations.
Results
FT + TMP gel promoted the lowest%SH and ΔKHN (p < 0.001). The highest concentration of CaF 2 formed was observed for the FT + Acid gel (p < 0.001), followed by FT + 9000 gel > FT + TMP gel > FT > PT. CaF 2 retained on the blocks was reduced across all groups (p < 0.001). Similar values were observed for the Ca/P/F and EPS in enamel and biofilm for all fluoride groups.
Conclusion
The association of FT + TMP gel significantly reduced enamel demineralization in situ . Clinical Significance: The association of treatments may be an alternative for patients with high caries risk.
1
Introduction
Dental caries is considered a multifactorial disease that requires the complex interaction of several factors to develop clinical manifestation . Fluoride (F) has been the main agent used for dental caries prevention worldwide. The use of F toothpaste (FT) is considered the primary reason for the reduction in caries prevalence observed over the last decades .
Topical application of F (TAF) is often used in preventive programs as well as in patients at high risk of developing dental caries as an adjunct measure for the reduction of lesions . When a product with high F concentration is applied on the tooth surface, there is deposition of calcium fluoride (CaF 2 ), which is covered by calcium and phosphate ions, and saliva proteins that delay the solubility of the compound . Thus, it functions as a source of F, thereby interfering with the dynamics of the de-remineralization processes . However, the cost of products with high F concentration can be high, especially fluoridated varnishes that are the first choice for infants with high caries risk . This implies restricted access in terms of public health, especially in developing countries where fluoride gels are most commonly employed. Notwithstanding, the risk of ingestion and adverse events (mainly nausea and vomiting) can be present when fluoride gel is applied in children younger than six years, overcoming the potential benefits of its application .
One of the possible strategies that could be utilized to reduce the risk of acute toxicity is the reduction of F composition along with supplementation by calcium and/or phosphate salts. Among the phosphate salts with anticariogenic activity, sodium trimetaphosphate (TMP) appears to be the most effective . The addition of TMP to experimental gels with lower F content (4500 μg F/g) was shown to reduce demineralization and promote remineralization of tooth enamel similarly to conventional gels (9000 μg F/g and 12,300 μg F/g − Acid gel), as demonstrated in in vitro and in situ studies.
In situations of high caries risk, the combination of two topical methods, TAF and fluoride toothpaste (FT), has been suggested . A randomized controlled clinical trial demonstrated that TAF with Acid gel associated with supervised brushing with FT showed there to be the similar capacity of remineralizing initial carious lesions when compared with FT alone . On the other hand, an in situ study that evaluated the remineralization of enamel lesions after daily applications of high concentration gel (12,300 μg F/g, Acid gel) associated with FT (1450 μg F/g) indicated there was an increase in mineral volume for FT associated with acid gel compared to FT, concluding that there was an association of increased F incorporation into lesions for the FT + Acid gel group when compared to just FT. With this, there is no consensus on the additional effect of the association of F methods for dental caries control .
Thus, the aim of this in situ study was to assess the effect of the association of low-F (4500 μg F/g) gel containing TMP and FT (1100 μg F/g) on enamel demineralization. The null hypothesis was that the association of treatments with low-F/TMP gel and FT would promote a similar reduction in demineralization when compared to the association of 9000 μg F/g gel and FT treatments.
2
Material and methods
2.1
Experimental design
This study was approved by the Institutional Review Board of São Paulo State University (Unesp), School of Dentistry, Araçatuba, Brazil (Protocol: 50723315.1.0000.5420), and all participants read and signed an informed consent form prior to study onset. This crossover double-blind study was conducted in five phases of seven days each. The sample size of volunteers was based on a previous study , considering the primary outcome from surface and cross-sectional hardness analysis, in terms of the mean difference between groups (30 and 1300, respectively), standard deviation (20 and 900, respectively), an α-error of 5% and a β-error of 20%. Volunteers (n = 12) aged 20 to 30 years who were in good general and oral health wore palatal appliances initially containing four bovine enamel blocks ( Fig. 1 A) selected by initial surface hardness (SHi). The cariogenic challenge was performed with 30% sucrose (six times/day). Treatments were: no fluoride/TMP toothpaste − Placebo (PT); 1100 μg F/g toothpaste (FT); FT and 4500 μg F/g gel containing 5%TMP (FT + TMP gel); FT and 9000 μg F/g gel (FT + 9000 gel) and FT and 12,300 μg F/g (FT + Acid gel). After topical application of treatments for one min, two blocks were removed for analysis of loosely bound (CaF 2 ) and firmly bound (FA) fluoride formed in enamel; and calcium (Ca) and phosphorus (P) on the same blocks. After the seven-day experimental period, the percentage of surface hardness loss (%SH), integrated subsurface hardness loss (ΔKHN), CaF 2 and FA retained as well as Ca and P in enamel were determined. Moreover, biofilms were analyzed for F, Ca, P and insoluble extracellular polysaccharide (EPS) concentrations. Data were assess using one-way and two-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test (p < 0.001).
2.2
Gel/toothpaste formulation and fluoride/pH assessment
An experimental gel (i.e., 100 g) with neutral pH was prepared in the laboratory with the following ingredients: 8.0 g of carboxymethyl cellulose (Synth, Diadema, São Paulo, Brazil), 0.1 g sodium saccharin (Vetec, Duque de Caxias, Rio de Janeiro, Brazil), 28.0 g glycerol (Sigma-Aldrich Co., St. Louis, MO, USA) and 0.5 g of peppermint oil (Synth, Diadema, São Paulo, Brazil) adjusted with deionized water to 100 g. F (NaF, Merck, Darmstadt, Germany) was added to the gel at concentrations of 4500 (1.0 g of NaF) or 9000 μg F/g (2.0 g of NaF). Subsequently, TMP (Sigma-Aldrich Co., St. Louis, MO, USA) was added at a 5% concentration (5.0 g) to the gel with F concentrations of 4500 μg F/g. A commercial acid gel was used as positive control (12,300 μg F/g, Acid gel, pH = 4.5, DFL Indústria e Comércio S.A., Rio de Janeiro, RJ, Brazil). In addition, toothpastes were produced with the following constituents: 0.5 g of titanium dioxide (Sigma-Aldrich Co., St. Louis, MO, USA), 1.7 g of carboxymethyl cellulose (Sigma-Aldrich Co., St. Louis, MO, USA), 0.08 g of methyl p -hydroxybenzoate sodium (Sigma-Aldrich Co., St. Louis, MO, USA), 0.1 g of saccharin (Vetec, Duque de Caxias, Rio de Janeiro, Brazil), 0.5 g of peppermint oil (Synth, Diadema, São Paulo, Brazil), 26.6 g of glycerol (Sigma-Aldrich Co., St. Louis, MO, USA), 10 g of abrasive silica (Tixosil 73 ® , Rhodia, São Paulo, Brazil) and 1.7 g of sodium lauryl sulfate (Sigma-Aldrich Co., St. Louis, MO, USA) adjusted with deionized water to 100 g. NaF (Merck, Darmstadt, Germany) was added to the F toothpaste to reach a concentration of 1100 μg F/g. A toothpaste without F (Placebo) was also prepared.
The F concentrations in the gels and toothpastes were determined with a F ion-specific electrode (9609 BN; Orion Research Inc., Beverly, MA, USA) attached to an ion analyzer (Orion 720 A+; Orion Research Inc.) and calibrated with standards containing 0.125–2.000 μg F/g. The pH levels of the gels and toothpaste were determined with a pH electrode (2A09E, Analyser, São Paulo, Brazil) that was calibrated with standard pH levels of 7.0 and 4.0 .
2.3
Clinical phase of experimental groups
The palatal appliance was prepared in acrylic resin (Jet, Articles Classic Odontológico, São Paulo, Brazil), and initially, four enamel blocks were fixed with a different device used during each phase of the experiment. Next, prophylaxis was performed using a non-F paste and rubber cup. At the beginning of each experimental phase, an amount of gel sufficient to fill stock trays to approximately one-third of their capacity was placed. Teeth were air-dried, the trays were placed over them, and the subject was instructed to close their jaws with the trays in contact for one min for treatments with gels. In toothpaste-only groups, the volunteers were instructed to brush their natural teeth for one minute with PF or FT. The same treatments (gels and/or toothpastes) were simultaneously performed on enamel blocks extraorally ( Fig. 1 C). To simulate the initial treatment with the toothpastes, the blocks were treated with slurries of toothpastes (toothpaste/deionized suspension, 1:3 w/w) over the course of one minute. Immediately after the treatments, two blocks from each device were removed for determination of CaF 2 and FA formed in enamel ( Fig. 1 H) as described subsequently. Ca and P in enamel were measured on the same blocks ( Fig. 1 I).
Next, to allow biofilm accumulation on the enamel blocks, a piece of plastic mesh was fixed to the acrylic appliance with the specific leaving of a one-mm space from the block surface . To provide a cariogenic challenge able to promote the formation of subsurface lesions ( Fig. 2 ), the volunteers were instructed to remove the device and drip 30% sucrose solution (Sucrose, Synth, Diadema, Brazil) ( Fig. 1 D) onto each enamel block six times/day at predetermined times (8:00 am, 11:00 am, 2:00 pm, 5:00 pm, 7:00 pm and 9:00 pm) . Subjects were instructed to use the appliances during the entire day (including at night) except when drinking or eating. At these moments, the devices were to remain covered by gauze moistened in deionized water. The volunteers brushed their natural teeth three times/day (at 07:30, 12:30 and 21:30 h) with PT or FT during a habitual oral hygiene routine for two min as previously described for the treatments. Brushing was performed with the acrylic appliance in the oral cavity, allowing the natural saliva/toothpaste slurry to come into contact with the enamel blocks by gently swishing the slurry in the mouth . During the seven-day pre-experimental period and washout periods, the volunteers brushed their teeth with toothpaste without F (Placebo).
2.4
Hardness analysis
Enamel surface hardness was measured before (SHi) ( Fig. 1 B) and after pH cycling (SHf) ( Fig. 1 F) using a Micromet 5114 hardness tester (Buehler, Lake Bluff, IL, USA) and Buehler OmniMet software (Buehler) followed by calculation of the percentage of surface hardness loss (%SH = [(SHf-SHi)/SHi] *100). For the cross-sectional hardness measurements, the enamel blocks were longitudinally sectioned through their center and embedded in acrylic resin with the cut face exposed and gradually polished. A sequence of 14 indentations was created 100 μm apart at different distances (5, 10, 15, 20, 25, 30, 40, 50, 70, 90, 110, 130, 220 and 330 μm) from the outer enamel surface using a Micromet 5114 hardness tester (Buehler, Lake Bluff, USA) and Buehler OmniMet software (Buehler) with a Knoop diamond indenter under a 5 g load for 10 s . Integrated hardness (KHN x μm) of the lesion into sound enamel was calculated according to the trapezoidal rule (GraphPad Prism, version 3.02) and subtracted from the integrated hardness for sound enamel to yield the integrated area of the subsurface regions in enamel, which was dubbed the integrated loss of subsurface hardness (ΔKHN; KHN x μm) ( Fig. 1 G) .
2.5
Analysis of loosely bound fluoride (CaF 2 ) formed and retained on enamel
The concentration of CaF 2 deposited on enamel was analyzed after treatments (CaF 2 formed) and after in situ challenge (CaF 2 retained). A digital caliper (Mitutoyo CD-15B, Mitutoyo Corporation, Japan) was employed to measure the surface area of enamel blocks (n = 240) . The surface of each specimen, except for enamel, was coated with wax. They were then immersed in 0.5 mL of 1.0 mol/L KOH solution under constant agitation ( Fig. 1 H) . After 24 h, the solution was neutralized and buffered with 0.5 mL of TISAB II (total ionic strength adjustment buffer) modified with HCl. F content was determined with an ion-specific electrode (9409BN, Thermo Scientific, Beverly, MA, USA) and a microelectrode reference (Analyser, São Paulo, Brazil) coupled to an ion analyzer (Orion 720 A plus , Thermo Scientific, Beverly, MA, USA) calibrated by standards containing 4.00 to 64.00 μg F/mL (100 μg F/g, Orion 940907). Data obtained in mV were converted to μg F/cm 2 using Microsoft Excel.
2.6
Analysis of firmly bound fluoride (FA) formed and retained; Ca and P in enamel
After extraction of the CaF 2 , enamel blocks were fixed to a modified microscope with a micrometer (Micrometer 733 MEXFLZ-50, Starret, Athol, MA, USA) to measure enamel wear. One layer of enamel (50.4 ± 0.4 μm) was removed from each block using self-adhesive polishing discs (13 mm in diameter, 400-grit silicon carbide, Buehler) fixed to the bottom of polystyrene crystal tubes (J-10; Injeplast, Sao Paulo, SP, Brazil) . The tubes containing enamel powder received an addition of 0.5 mL of 1.0 mol/L HCl, and these were kept under constant stirring for one hour. The analysis of FA formed/retained was performed in 0.25 mL of that solution after addition of the same volume of TISAB II, modified with NaOH, as previously described. Ca analysis in enamel after treatments and subsequent to in situ challenge, considered as formed and retained, respectively, was performed according to the Arsenazo III colorimetric method . The absorbance readings were recorded at 650 nm using a plate reader (Microplate Spectrophotometer EONC, Biotek, Winooski, VT, USA). P was measured according to Fiske and Subbarow , and the absorbance readings were recorded at 660 nm. All results were expressed as μg/mm 3 ( Fig. 1 I).
2.7
Analysis of dental biofilm composition
The biofilm formed on enamel was collected and stored in microcentrifuge tubes. The biofilm samples were dried in vacuum over P 2 O 5 (Vetec Quimica Fina Ltda., Duque de Caxias, Rio de Janeiro, Brazil) for 12 h at room temperature. After extraction for 3 h at room temperature with 0.5 mol/L hydrochloric acid (250 μL/mg, biofilm wet weight) under constant agitation, the same volume of NaOH (0.5 mol/L) was added . The samples were then centrifuged (11,000 × g ) for 1 min and the supernatant retained for determination of F, Ca and P. F was analyzed using an ion-specific electrode (Orion 9409 BN) and a potentiometer (Orion 720 A plus ) . Ca concentration was evaluated by the Arsenazo III colorimetric test . P concentration was measured via the molybdate colorimetric method . EPS was extracted by adding 1.0 mol/L NaOH (10 μL/mg dry weight) to the biofilm . The amount of EPS was determined according to the phenol-sulfuric acid method . The results were expressed as mol/kg of F, Ca and P; and mg/g of EPS (dry weight) ( Fig. 1 E).
2.8
Statistical analysis
SigmaPlot 12.0 software was used for statistical analysis and the significance level was set at 5%. All variables exhibited a normal (Shapiro–Wilk) and homogeneous (Bartlet) distribution. Data from the dental biofilm analysis (F, Ca, P and EPS) and enamel analysis (%SH and ΔKHN) were subjected to one-way repeated measures analysis of variance (ANOVA). The results of CaF 2 , FA, Ca and P formed/retained (enamel analysis) were submitted to two-way repeated measures ANOVA. The Student–Newman–Keuls post hoc test was performed for multiple comparisons.
2
Material and methods
2.1
Experimental design
This study was approved by the Institutional Review Board of São Paulo State University (Unesp), School of Dentistry, Araçatuba, Brazil (Protocol: 50723315.1.0000.5420), and all participants read and signed an informed consent form prior to study onset. This crossover double-blind study was conducted in five phases of seven days each. The sample size of volunteers was based on a previous study , considering the primary outcome from surface and cross-sectional hardness analysis, in terms of the mean difference between groups (30 and 1300, respectively), standard deviation (20 and 900, respectively), an α-error of 5% and a β-error of 20%. Volunteers (n = 12) aged 20 to 30 years who were in good general and oral health wore palatal appliances initially containing four bovine enamel blocks ( Fig. 1 A) selected by initial surface hardness (SHi). The cariogenic challenge was performed with 30% sucrose (six times/day). Treatments were: no fluoride/TMP toothpaste − Placebo (PT); 1100 μg F/g toothpaste (FT); FT and 4500 μg F/g gel containing 5%TMP (FT + TMP gel); FT and 9000 μg F/g gel (FT + 9000 gel) and FT and 12,300 μg F/g (FT + Acid gel). After topical application of treatments for one min, two blocks were removed for analysis of loosely bound (CaF 2 ) and firmly bound (FA) fluoride formed in enamel; and calcium (Ca) and phosphorus (P) on the same blocks. After the seven-day experimental period, the percentage of surface hardness loss (%SH), integrated subsurface hardness loss (ΔKHN), CaF 2 and FA retained as well as Ca and P in enamel were determined. Moreover, biofilms were analyzed for F, Ca, P and insoluble extracellular polysaccharide (EPS) concentrations. Data were assess using one-way and two-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test (p < 0.001).
2.2
Gel/toothpaste formulation and fluoride/pH assessment
An experimental gel (i.e., 100 g) with neutral pH was prepared in the laboratory with the following ingredients: 8.0 g of carboxymethyl cellulose (Synth, Diadema, São Paulo, Brazil), 0.1 g sodium saccharin (Vetec, Duque de Caxias, Rio de Janeiro, Brazil), 28.0 g glycerol (Sigma-Aldrich Co., St. Louis, MO, USA) and 0.5 g of peppermint oil (Synth, Diadema, São Paulo, Brazil) adjusted with deionized water to 100 g. F (NaF, Merck, Darmstadt, Germany) was added to the gel at concentrations of 4500 (1.0 g of NaF) or 9000 μg F/g (2.0 g of NaF). Subsequently, TMP (Sigma-Aldrich Co., St. Louis, MO, USA) was added at a 5% concentration (5.0 g) to the gel with F concentrations of 4500 μg F/g. A commercial acid gel was used as positive control (12,300 μg F/g, Acid gel, pH = 4.5, DFL Indústria e Comércio S.A., Rio de Janeiro, RJ, Brazil). In addition, toothpastes were produced with the following constituents: 0.5 g of titanium dioxide (Sigma-Aldrich Co., St. Louis, MO, USA), 1.7 g of carboxymethyl cellulose (Sigma-Aldrich Co., St. Louis, MO, USA), 0.08 g of methyl p -hydroxybenzoate sodium (Sigma-Aldrich Co., St. Louis, MO, USA), 0.1 g of saccharin (Vetec, Duque de Caxias, Rio de Janeiro, Brazil), 0.5 g of peppermint oil (Synth, Diadema, São Paulo, Brazil), 26.6 g of glycerol (Sigma-Aldrich Co., St. Louis, MO, USA), 10 g of abrasive silica (Tixosil 73 ® , Rhodia, São Paulo, Brazil) and 1.7 g of sodium lauryl sulfate (Sigma-Aldrich Co., St. Louis, MO, USA) adjusted with deionized water to 100 g. NaF (Merck, Darmstadt, Germany) was added to the F toothpaste to reach a concentration of 1100 μg F/g. A toothpaste without F (Placebo) was also prepared.
The F concentrations in the gels and toothpastes were determined with a F ion-specific electrode (9609 BN; Orion Research Inc., Beverly, MA, USA) attached to an ion analyzer (Orion 720 A+; Orion Research Inc.) and calibrated with standards containing 0.125–2.000 μg F/g. The pH levels of the gels and toothpaste were determined with a pH electrode (2A09E, Analyser, São Paulo, Brazil) that was calibrated with standard pH levels of 7.0 and 4.0 .
2.3
Clinical phase of experimental groups
The palatal appliance was prepared in acrylic resin (Jet, Articles Classic Odontológico, São Paulo, Brazil), and initially, four enamel blocks were fixed with a different device used during each phase of the experiment. Next, prophylaxis was performed using a non-F paste and rubber cup. At the beginning of each experimental phase, an amount of gel sufficient to fill stock trays to approximately one-third of their capacity was placed. Teeth were air-dried, the trays were placed over them, and the subject was instructed to close their jaws with the trays in contact for one min for treatments with gels. In toothpaste-only groups, the volunteers were instructed to brush their natural teeth for one minute with PF or FT. The same treatments (gels and/or toothpastes) were simultaneously performed on enamel blocks extraorally ( Fig. 1 C). To simulate the initial treatment with the toothpastes, the blocks were treated with slurries of toothpastes (toothpaste/deionized suspension, 1:3 w/w) over the course of one minute. Immediately after the treatments, two blocks from each device were removed for determination of CaF 2 and FA formed in enamel ( Fig. 1 H) as described subsequently. Ca and P in enamel were measured on the same blocks ( Fig. 1 I).
Next, to allow biofilm accumulation on the enamel blocks, a piece of plastic mesh was fixed to the acrylic appliance with the specific leaving of a one-mm space from the block surface . To provide a cariogenic challenge able to promote the formation of subsurface lesions ( Fig. 2 ), the volunteers were instructed to remove the device and drip 30% sucrose solution (Sucrose, Synth, Diadema, Brazil) ( Fig. 1 D) onto each enamel block six times/day at predetermined times (8:00 am, 11:00 am, 2:00 pm, 5:00 pm, 7:00 pm and 9:00 pm) . Subjects were instructed to use the appliances during the entire day (including at night) except when drinking or eating. At these moments, the devices were to remain covered by gauze moistened in deionized water. The volunteers brushed their natural teeth three times/day (at 07:30, 12:30 and 21:30 h) with PT or FT during a habitual oral hygiene routine for two min as previously described for the treatments. Brushing was performed with the acrylic appliance in the oral cavity, allowing the natural saliva/toothpaste slurry to come into contact with the enamel blocks by gently swishing the slurry in the mouth . During the seven-day pre-experimental period and washout periods, the volunteers brushed their teeth with toothpaste without F (Placebo).
