Preventive effect of iron gel with or without fluoride on bovine enamel erosion in vitro
Dr SHC Sales-Peres
Department of Pediatric Dentistry
Orthodontics and Public Health
Bauru School of Dentistry
University of São Paulo
Al. Octávio Pinheiro Brisolla
9-75. Bauru-SP 17012-901
Background: The aim of this study was to evaluate the preventive effect in vitro of experimental gel containing iron and/or fluoride on the erosion of bovine enamel.
Methods: To standardize the blocks (n = 80), specimens (4 × 4 mm) were previously selected to measure the initial microhardness. The blocks were randomly allocated into four groups of 20 samples each: C (control, placebo gel); F (fluoride gel, 1.23% NaF); Fe (iron gel, 10 mmol/L FeSO4) and F + Fe (fluoride + iron gel). The gels were applied and removed after 1 minute. The blocks were then submitted to six alternating remineralization and demineralization cycles. The beverage Coca-Cola® (10 minutes, 30 mL) was used for demineralization, and artificial saliva (1 hour) for remineralization. The effect of erosion was measured by wear analysis (profilometry). Data were analysed by ANOVA and the Tukey test for individual comparisons (p <0.05).
Results: The mean wear (± SD, μm) was C: 0.94 ± 0.22; F: 0.55 ± 0.12; Fe: 0.49 ± 0.11 and F + Fe: 0.55 ± 0.13. When the experimental gels were used, there was statistically significant reduction in enamel wear in comparison with the control (p <0.001). However, the experimental gels did not differ significantly among them.
Conclusions: The gels containing iron with or without fluoride are capable of interfering with the dissolution dental enamel in the presence of erosive challenge.
analysis of variance
The loss of substance by erosion is a dynamic process with periods of demineralization and remineralization. Thus, preventive measures against erosion are required. Studies on the influence of fluoridation measures on tooth wear of tooth substance damaged by erosion are scarce, but in an in vitro demineralization and remineralization model, showed the protective effect of an acidified fluoride gel on enamel abrasion.1 When a cyclic demineralization and remineralization model was used in vitro, it was concluded that an intensive fluoridation measure is most effective in reducing the progression of erosion.2 Attin et al.1 showed a protective effect in vitro of an acidified fluoride gel against abrasion in eroded enamel. Jones et al.3 exposed enamel coated with fluoride gels to stimulated gastric acid in situ. On the other hand, the study of fluoride action in reducing dental erosion demonstrated that the erosive potential was not minimized with the use of fluoride. The study showed that fluoride was unable to provide a preventive effect against erosion.4 The in situ study by Lussi et al.5 showed that a single fluoride rinse had no significant effect on the prevention of toothbrush abrasion of softened enamel.
It has been suggested that iron has the effect of reducing enamel demineralization.6 Some in situ studies have shown that iron reduces the demineralization of enamel in a situation of high cariogenic challenge.7,8 The possibility that iron could be used to reduce an erosive challenge was based on studies using abiotic models, which showed that iron was effective in inhibiting enamel dissolution.9–11 In a pioneer study, Sales-Peres et al.12 showed the effectiveness in situ of iron rinsing for inhibiting enamel dissolution. However, possible adverse effects of iron, such as tooth staining, were identified when the preventive vehicle was supplemented with this ion.
Thus, the aim of the present in vitro study was to evaluate the efficacy of an experimental iron gel with or without fluoride, for the prevention of erosive wear on bovine enamel blocks exposed to a soft drink.
Materials and methods
Bovine enamel preparation
Fragments of permanent bovine enamel were obtained from bovine incisors. The teeth were sterilized by storage in 10% formalin buffered solution, pH 7, for 30 days. The crowns were sectioned from the roots with a diamond disk (Isomet 1000; Buehler, Lake Bluff, IL, USA). Two parallel diamond discs, separated by a 4 mm spacer, were then used to cut 1–2 fragments from the crown of each bovine incisor. The enamel surface of the blocks was ground flat with water-cooled carborundum discs (320, 600 and 1200 grit Al2O3 papers; Buehler, Lake Bluff, IL, USA), and polished with felt pads with diamond spray (1 μm; Buehler, Lake Bluff, IL, USA).
To standardize the blocks, specimens were previously selected to measure the initial microhardness (five indentations in different regions of the blocks, 25 g, 10 s, HMV-2000; Shimadzu Corporation, Tokyo, Japan). Blocks that presented 10% below and 10% above the mean value were excluded from the sample.
In order to maintain reference surfaces for determining lesion depth, two layers of nail varnish (Risque, Niasi, Taboão da Serra, São Paulo, Brazil) were applied on half of the specimen surfaces.
Eighty specimens with a mean surface microhardness of between 291 and 382 KHN (mean = 344.19) were randomly divided into four groups: C (control, placebo gel, neutral pH, manipulation); F (fluoride gel, 1.23% NaF, neutral pH, manipulation); Fe (iron gel, 10 mmol/L FeSO4, neutral pH, manipulation) and F + Fe (fluoride + iron gel, 1.23% and 10 mmol/L FeSO4, neutral pH, manipulation). Before exposure to acid, the samples were pre-treated with one of the respective gels. A thin layer of gel was applied to the enamel surface with a microbrush. After 1 minute, the gel was removed from the surface with a cotton roll and the samples were stored in artificial saliva for 12 hours at 37°C to allow superficial hydration. The composition of the artificial saliva was 1.5 mmol/L Ca(NO3)2. H2O; 0.9 mmol/L Na2HPO4.2H2O; 150 mmol/L KCl; 0.1 mol/L H2NC(CH2OH)3 (TRIS); 0.05 NaF pH 7.0.13
The samples of all groups (n = 20) were submitted to demineralization–remineralization regimes. Specimens were consecutively cycled through this regimen six times. One complete cycle consisted of the following steps: (1) demineralization in 30 mL of the beverage (Coca-Cola®, Cia de Bebidas Ipiranga, Ribeirão Preto, Brazil, pH = 2.6) for 10 minutes under gentle agitation; (2) remineralization in 30 mL of artificial saliva for 60 minutes at 37°C. After each treatment, specimens were rinsed with deionized water.
At the end of experimental phase, the nail varnish over the reference surfaces was carefully removed with acetone-soaked cotton wool.14 Surface profiles of the enamel specimens were obtained with a contact profilometer (Hommel Tester T 1000, Hommelwerke, VS, Schwenningen, Germany). For the determination of enamel loss, the tape was removed and five profiles were recorded at exactly the same sites as for baseline measurement. The profile scans were performed in the centre of each specimen at intervals of 250 μm. Treatment scans were superimposed and the average depth of the area under curve in the eroded area was calculated with specially designed software. The results of the five scans were averaged for each specimen.
The assumptions of equality of variances and normal distribution of errors were checked. Since the assumptions were satisfied, data were analysed by one-way analysis of variance (ANOVA). For individual comparisons among the groups, Tukey’s test was used. The level of significance was set at 5%.
The experimental groups presented a significantly lower mean wear than that observed for the control group (Tables 1 and 2). In this study, the tissue loss (mean ± SD) was highest in the control group (0.94 ± 0.22). All gels tested reduced tissue loss significantly compared with the control group (p <0.001). Values obtained for fluoride gel, iron gel and fluoride + iron gel were 0.55 ± 0.12, 0.49 ± 0.11, 0.55 ± 0.13, respectively.
Table 1. Wear (μm) and standard deviation (± SD) of enamel, with different treatments
|Control gel||20||0.94 ± 0.22|
|Fluoride gel||20||0.55 ± 0.12|
|Iron gel||20||0.49 ± 0.11|
|Fluoride + iron gel||20||0.55 ± 0.13|
Table 2. Tukey multiple comparisons test among the groups
|C × F||0.39||p < 0.001|
|C × Fe||0.45||p < 0.001|
|C × F + Fe||0.39||p < 0.001 |
|F × Fe||0.06||p > 0.05|
|F × F + Fe||−0.003||p > 0.05|
|Fe × F + Fe||−0.06||p > 0.05|
Although the iron gel showed the best result, it did not differ significantly when compared with the other groups (p >0.05) (Table 2).
The results of various in vitro studies suggest that the presence of increasing iron concentrations can inhibit the dissolution of enamel under acidic conditions.8–10,15,16 For the first time, a study evaluated the action the iron by dental erosion.12 Based on the action of iron in reducing the in vitro demineralization of enamel, Sales-Peres et al.12 proposed an in situ study to evaluate the effect of an iron mouthrinse after acid challenge. The in situ research showed that a 10 mmol/L iron rinse after an acidic challenge with Coca-Cola® was capable of reducing the change in surface microhardness and wear of human enamel and dentine blocks, respectively.
As far as we are aware, this was the first time that iron gel with or without fluoride was tested for the prevention of enamel demineralization. The mechanism involved in this protection of iron against mineral dissolution is not completely understood. Table 1 showed that the different treatments were capable of reducing tooth wear and found similar effects among the experimental groups.
Data showed that the effect on experimental groups was better when compared with the control (Table 2). Sales-Peres et al.12 observed that the iron rinse caused tooth staining, which could alter application of the rinse. This is another point in favour of adding the iron to gel, because the brown colour is caused by iron oxidation. All available gels were also applied only once on the specimens. For a better comparison between the rinse and the gel with iron, we need to correlate the application method. The rinse was used several times and the gel for only one application. This fact reduces the risk of the occurrence of brown spots on the teeth. It seems worth investigating whether carriers with a better capability to adhere to dental surfaces, such as gels, might increase their protective effect. Moreover, the efficacy of iron gel agents has to be confirmed in vivo.
Gels containing iron, with or without fluoride, significantly reduced erosion (around 40%) when compared to the control, confirming previous studies on the effect of iron on erosion.12
In this study, the combined effect of fluoride and iron in the same product found no cumulative effects of iron and fluoride when added into gel on the reduction of erosive wear of enamel blocks. Despite the mean wear found for the fluoride, the fluoride + iron and the iron gels was similar (0.55 ± 0.12 μm; 0.55 ± 0.13 μm; 0.49 ± 0.11 μm, respectively), the mechanism of action between these products was the difference. It is possible that the formation of a thin acid-resistant coating of hydrous iron oxide on the enamel mineral surface may be a possible factor.6 Previous studies indicate the effect of fluoride in the remineralization of enamel and in the nucleation of apatite, substitution of calcium in apatite17 increased concentration in the remineralization of dentine and enamel and inhibition of demineralization.18
Hughes et al.19 conducted research with the aim of determining the protective effect of fluoride treatments against enamel erosion in vitro. The findings indicated that fluoride applied to enamel erosion either in acidic solutions or as a pre-treatment, reduced enamel erosion. A recent study evaluated the relevance of cations in different fluoride compounds (amine, sodium, and stannous) with regard to their effectiveness as anti-erosive agents. Considerable differences were found between the fluoride compounds tested. Treatment with solutions containing stannous fluoride was the most effective.20
On the other hand, the use of fluorides, such as sodium fluoride, was tested for reducing dentine sensitivity.21 Fluorides decrease the permeability of dentine in vitro,22 possibly by precipitation of insoluble calcium fluoride within the tubules. In this study, the substrate evaluated was enamel. However, further studies are required, including investigations concerning reduction of tooth wear in dentine and products able to reduce dentine permeability, such as iron with or without fluoride compounds.
The fluoride containing gels could have added effect and hence the reason for the insignificant results among the groups with this compound. However, the group with only iron presented the superior action in enamel erosion.
The experimental gels tested in the present study were used with formulations containing iron and/or fluoride to reduce dental erosion. This factor is particularly important if one regards the possible clinical applicability of such iron gel.
Within the limitations of an in vitro study, one can conclude from the results of this study that iron is capable of interfering in the dissolution of dental enamel when used in the form of iron gel with or without fluoride, by increasing the enamel resistance to acidic drinks in order to prevent dental erosion. Further in situ and in vivo studies are required to substantiate the effects of iron in the clinical environment.
This study was supported by a grant from PIBIC/CNPq (National Council of Scientific and Technological Development) and FAPESP (Foundation for Research Support of the State of São Paulo). The authors wish to thank Professor Marília Afonso Rabelo Buzalaf for permission to use the laboratory.