MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION References

 
Experimental design. The Research and Ethics Committee of the Bauru School of Dentistry, University of São Paulo, Brazil (process number 104/2006), approved this study. The study was a crossover, double-blinded design conducted in three phases consisting of seven days each, with a washout period of seven days between each phase. We based the sample size calculation on a previous study¹⁴ and designed the study to have a statistical power of 0.75 with an of .05. Ten postgraduate students with a mean age of 24 years (range, 19–30 years) who were in good oral health and residing in an area with fluoridated water (0.70 mg F/liter) volunteered to participate in this study.

The subjects wore acrylic palatal appliances, which contained four bovine enamel slabs divided into two rows (one on each side of the mouth): erosion and erosion plus abrasion. The use of two conditions in the same intraoral palatal appliance was supported by the absence of a crossover effect in previous studies.^14,17 The tested dentifrices were Duraphat 5000 ppm Fluoride Toothpaste (Colgate-Palmolive, New York City, 5,000 ppm F, sodium fluoride [NaF], silica, mean ± SD, relative dentin abrasion [RDA]: 77 ± 11, pH 8.0, not marketed in the United States and available only by prescription); Crest Cavity Protection (Procter & Gamble, Cincinnati) (1,100 ppm F, NaF, silica, RDA 100, pH 7.0); and a placebo dentifrice (formulated exactly like the Duraphat dentifrice except that it contained no F, silica, pH 8.0).

Researchers at the University of Zürich, Switzerland, determined the RDA of the Duraphat 5,000 ppm Fluoride Toothpaste by using the same methodology as that used by Barbakow and colleagues,¹⁸ and we expect the RDA of the placebo dentifrice to be similar to that of the 5,000 ppm dentifrice, because they have the same formulation. Rice and colleagues¹⁹ reported the RDA of the 1,100 ppm dentifrice (Crest Cavity Protection). The subjects consumed cola to achieve the erosion (pH 2.6, 60 seconds), and they brushed with the randomly assigned dentifrice slurry for 30 seconds four times a day to achieve abrasion. After each phase, we determined enamel loss via profilometry.

Enamel slabs and palatal appliance preparation. We prepared 120 enamel slabs (4 x 4 millimeters) from extracted bovine incisors, which we sterilized by storing in 2 percent formaldehyde solution (pH 7.0) for 30 days at room temperature. We ground flat the enamel surfaces of the slabs with water-cooled carborundum discs (320, 600 and 1,200 grades of aluminium oxide papers, Buehler, Lake Bluff, Ill.) and polished them with 1-micrometer diamond spray (Buehler).

To distribute the samples to the three dentifrice groups, we determined the surface microhardness by creating five indentations in different regions of the slabs using a diamond indenter (Knoop hardness test; test force, 25 grams; five-second testing [HMV-2 microhardness tester, Shimadzu, Kyoto, Japan]). To maintain reference surfaces for determination of lesion depth via profilometry, we applied two layers of nail varnish on one-half of the surface of each slab. We used wax to bind two of the slabs into cavities (5 x 5 x 3 mm) on either side of the intra-oral palatal appliances.

Seven days before the beginning of the study and throughout the three experimental phases (seven days each), subjects brushed their teeth with one of the dentifrices. In this crossover protocol, we randomly assigned them to the three treatments. During the first 12 hours of each intraoral phase, the slabs were not subjected to erosive or abrasive challenges to allow the formation of a salivary pellicle.^7,14 According to Amaechi and colleagues,²⁰ after only one hour, pellicle exhibits a protective effect against erosion. For the next seven days, erosive and abrasive challenges were performed extraorally four times a day at predetermined times (8:00 a.m., 12:00 p.m., 4:00 p.m. and 8:00 p.m.) after meals.^7,14

Erosion. To achieve erosion of the enamel slabs, we instructed subjects to remove the appliances and immerse them for one minute in a cup containing 150 milliliters of cola poured from a freshly opened bottle (regular Coke, Coca-Cola, Atlanta, pH 2.6, distributed by Spal, Porto Real, RJ, Brazil) at room temperature. During this ex vivo erosion, subjects brushed their teeth with one of the dentifrices by using a soft toothbrush with rounded filament ends and a small amount of dentifrice (approximately 0.3 g).

Abrasion. After eroding the slabs with the cola, subjects placed one drop (approximately 35 micro-liters) of dentifrice slurry on the enamel surface of each slab. The subjects did not brush the first row, but they brushed the second row using an electric toothbrush with soft rounded filament ends for 30 seconds (166 oscillations per second). We trained and instructed subjects to perform this procedure carefully to avoid a crossover effect of the treatments. Subjects then returned the appliances to their mouths and rinsed for five seconds with 10 mL of water.

To achieve erosion of the enamel slabs, subjects removed the appliances and immersed them for one minute in a cup containing 150 milliliters of cola poured from a freshly opened bottle.

We prepared the dentifrice slurry consisting of the dentifrice and deionized water in a 1:3 (g/mL) proportion. We placed all solutions and dentifrices into separate unlabeled vials to conform to the blinded protocol of the study. In addition, we instructed participants to wear the appliances continuously day and night, but to remove them during meals (four times a day for one hour each time). During this period, they stored the appliance in wet gauze. The subjects received oral and written instructions to refrain from using any fluoridated product other than the assigned dentifrice.

Enamel loss assessment. After seven days, we removed the enamel slabs from the appliances and carefully removed the nail varnish on the reference surfaces by using acetone-soaked cotton wool.⁶ We obtained surface profiles of the enamel samples by using a stylus profilometer (Perthometer M2, Mahr, Göttingen, Germany). To determine the enamel loss, we recorded four profiles across the protected and eroded surfaces. We recorded one profile on the protected surface only, which served as a baseline control for determination of enamel loss. We performed the profile scans in the center of each specimen at intervals of about 250 µm. We superimposed scans of the control and eroded areas over one another and calculated the mean depth of the area under the curve in the eroded area by using specially designed software (Mahr). We averaged the results of the four scans for each specimen.

Statistical analysis. We used software (GraphPad Prism 4, version 4.0 for Windows, GraphPad Software, La Jolla, Calif.). We checked our assumptions of equality of variances and normal distribution of errors for all of the variables tested. Because the assumptions were satisfied, we used two-way repeated-measures analysis of variance (ANOVA) and Bonferroni post hoc test. We set the significance level at .05.

	RESULTS

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Two-way repeated-measures ANOVA revealed a significant difference between the conditions (that is, erosion plus abrasion versus erosion only) (F₁ = 17.48, P = .0003) but not between the dentifrices (F₂ = 1.099, P = .3476). The interaction between the criteria (that is, the condition [erosion plus abrasion versus erosion only] and the type of dentifrice) was not significant (F₂ = 0.0446, P = .957). The table shows that for the erosion-plus-abrasion condition, the enamel wear was significantly higher than that for the erosion-only condition (P < .05). We found no significant differences in enamel wear between the two fluoridated dentifrices, and the results did not differ from those for the placebo dentifrice (P > .05).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION References

However, the results of this in situ investigation need to be interpreted within the framework of the study design. We might assume that the tongue has an abrasive effect on the samples located in the palate. However, Gregg and colleagues²² showed that licking of enamel samples had only a minor abrasive effect on eroded enamel. Still, we advised subjects to avoid licking or touching the enamel blocks to minimize abrasion. Participants performed the erosive and abrasive protocols extraorally, and these were not counterbalanced by salivary properties such as buffering capacity and salivary flow rate,^23,24 which might have reduced the demineralization and enhanced the remineralization of the eroded surfaces.^7,24 Moreover, we must keep in mind that the toothpaste slurry was prepared via dilution with water instead of saliva. We used this design to eliminate the influence of minerals present in saliva and to focus only on the effect of fluoride concentrations in the toothpastes.

Bovine enamel has been used widely in dental research as a model for human enamel. Even so, Rios and colleagues⁷ reported that bovine enamel exhibited a higher susceptibility to erosion and abrasion compared with human enamel. Moreover, polishing the slabs might have affected our results, because polished surfaces have been shown to be more susceptible to acids than are natural surfaces.²⁵ However, for exact profilometric analysis, a polished surface is necessary.

The data showed that the erosion-plus-abrasion condition resulted in greater wear than did the erosion-only condition. This finding is consistent with findings in the literature,^4,6,7 because the erosive attack causes softening of the enamel, thus leading to an increased susceptibility to abrasive wear.^3,4,6,7

The 5,000 and 1,100 ppm fluoridated tooth-pastes (Duraphat 5000 ppm Fluoride Toothpaste and Crest Cavity Protection, respectively) groups showed a tendency for less enamel wear compared with the placebo group in both the erosion-only and erosion-plus-abrasion conditions. However, the results showed no significant differences between the groups. However, we must interpret the lack of significant differences with caution, because significant differences between groups might have existed, but were not possible to achieve in this study owing to its low power (approximately 40 percent). Thus, it is possible to infer that the presence of fluoride in the dentifrice is important, regardless of the concentration.

We might have expected to observe better protection from the fluoride dentifrices, as previous studies have shown.^9,12–14 The greater fluoride concentration (5,000 ppm F) was not better able to protect enamel against erosion or even erosion plus abrasion compared with the 1,100 ppm F dentifrice. We first hypothesized that the differences in pH between the two fluoride dentifrices (5,000 and 1,100 ppm) might have masked the effects of the dentifrice with the higher fluoride concentration. However, none of the dentifrices used in our study had a low pH that could have enhanced the fluoride effect, as was the case in the study by Ganss and colleagues.⁹

In addition, we might have speculated that the dilution of the dentifrice and the short time of fluoride application did not allow for the deposit of a calcium-fluoridelike layer that was able to prevent erosive or abrasive attack. In both the erosion and erosion-plus-abrasion conditions, fluoride contacted the enamel for 30 seconds only, and subjects rinsed their mouths afterward with water. This hypothesis also could explain the absence of a significant difference between the highly concentrated (5,000 ppm F) and regular (1,100 ppm F) fluoridated dentifrices. Magalhães and colleagues¹⁴ and Ganss and colleagues⁹ conducted studies in which they used pure dentifrices, not the slurries used in our study, which might have contributed to their better results with regard to fluoride dentifrices.

The setup of our study probably resulted in a lower fluoride concentration, which did not enable the adequate deposit of the calcium-fluoridelike layer. However, in the clinical situation, the dentifrice always is diluted by saliva. In fact, our study results showed a tendency for a better effect with the 5,000 ppm F dentifrice than with the 1,100 ppm F dentifrice in both conditions; moreover, when we consider that the power of the study was low (about 40 percent), the lack of significant differences between the dentifrices must be analyzed carefully.

Another factor that might have contributed to our results is the abrasivity of the toothpastes. However, Hooper and colleagues²⁶ reported that the abrasivity of the toothpastes is of minor relevance with regard to the abrasion of eroded enamel. In addition, we used a short erosive period (four episodes of one minute each) to simulate a clinical condition of frequent consumption of soft drinks. One can speculate that the fluoride effects might be enhanced if longer erosive periods are used.^9,14 Future research should evaluate the fluoride response during different periods of acidic challenge.

	CONCLUSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION References

 
Within the in situ, ex vivo conditions of this study, the highly concentrated F dentifrice (5,000 ppm F) did not have a significant protective effect on enamel against erosion and erosion plus toothbrush abrasion. Consequently, patients at risk of experiencing enamel erosion should benefit from other preventive measures. However, future studies are needed to confirm these findings.