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Effect of fluoride gels on occlusal fissures in primary molars: an in vitro study.

Introduction

The anti-caries effect of topical fluoride application depends on reaction products formed on the enamel during clinical treatment and the retention of these products over time. This process is dependent on the concentration of fluoride applied and the pH of the commercial product used [Villena et al., 2009]. A dose-dependent effect has been demonstrated in the analysis of enamel resistance to a cariogenic challenge following the application of an acidulated phosphate fluoride (APF) or sodium fluoride (NaF) gel, with greater fluoride absorption and a more efficient reduction in demineralisation in enamel treated with APF than with NaF [Delbem and Cury, 2002]. Concerning application time, studies have found a similar effect on enamel resistance to the formation of carious lesions with the application of acidulated fluoride gels for both one or four minutes [Garcia-Godoy et al., 1995; Delbem and Cury, 2002; Villena et al., 2009].

The efficacy of topically applied fluoride products in the prevention of caries in children and adolescents is clearly recognised from an evidence-based perspective [Marinho et al., 2002; Marinho, 2009; Poulsen, 2009]. While the use of high-frequency, low-concentration fluoride regimens for caries prevention and control is recommended daily regardless of the risk or activity of caries of the patient, the high-concentration, low-frequency fluoride application with APF or NaF gel is required in situations of high risk or carious activity.

As studies evaluating these fluoride compounds on the occlusal surfaces are scarce, the aim of the present study was to evaluate the effect of fluoride gels on the acid resistance of occlusal enamel in primary molars under dynamic pH-cycling conditions.

Material and methods

Ethical aspects

This study received approval from the Human Research Ethics Committee of the Universidade Federal de Santa Catarina (Brazil). The teeth were donated by the tooth bank of the School of Dentistry of the Universidade de Sao Paulo (Brazil).

Tooth selection and preparation of specimens

Forty-five extracted primary second molars were used (20 maxillary and 25 mandibular). The teeth displayed complete rhizolysis of the roots. The teeth were submitted to prophylaxis with a jet of bicarbonate and rinsed with de-ionised water. The occlusal surfaces were examined under a magnifying glass. A bitewing radiographic exam (0.8-s exposure time) was then performed to determine the presence of any hidden caries. The inclusion criteria were as follows: free of caries (visual and radiographic diagnosis), without fissure sealant, free of coloured or white spots, free of cracks and hypoplastic enamel. The teeth were stored in a 0.1% thymol solution (pH 7.0) until needed.

The surfaces were polished with pumice and water, rinsed with de-ionised water and blot dried. An orthodontic wire 0.6 mm in diameter was fixed to the pulp chamber with autopolymerising acrylic resin. All surfaces, except the occlusal surface, were coated twice with an acid-resistant varnish. The delimited occlusal area was 30 [mm.sup.2].

Experimental study design

The specimens were randomly assigned to three treatment groups (n = 15 each): a) control (submitted to pH-cycling); b) NaF gel (2% F, pH 7.0, Top Gel, Vigodent, Ind. e Com., Rio de Janeiro, RJ, Brazil)/pH-cycling; and c) APF gel (1.23%, pH 3.6 to 3.9, Top Gel, Vigodent, Ind. e Com., Rio de Janeiro, RJ, Brazil)/ pH-cycling. The experimental groups were treated with fluoride compounds for 4 min and then submitted to pH-cycling. The response variables were vol.% mineral and mineral loss (AZ).

Fluoride treatment

The occlusal surfaces were treated with 0.03ml fluoride gels applied with a disposable plastic syringe. The gels were applied for four minutes. The surfaces were then rinsed with de-ionised water for 30 s and blot dried.

[FIGURE 1 OMITTED]

pH-cycling

After the fluoride treatment, the specimens were submitted to a pH-cycling model essentially based on work by Featherstone et al. [1986], but simulating a low cariogenic challenge. The specimens in each group were initially immersed in a demineralising solution (2.0 mM calcium, 2.0 mM phosphate in 75 mM acetate buffer, pH 4.3) for three hours at 37[degrees]c, then washed for one minute in de-ionised water, blot dried and immersed in a remineralising solution (1.5 mM calcium, 0.9 mM phosphate, 150 mM of KCl in 0.1M TRIS buffer, pH 7.0) for 21 hours at 37[degrees]c. These procedures were repeated until completing a total of 10 cycles. The specimens remained in the remineralising solution for 48 hrs and both solutions were changed before starting the next 5-day cycle. Residual fluoride (0.021mg[L.sup.-1]) was found in both the standard demineralising and remineralising solutions.

Acquisition of sections

At the end of the pH-cycling, the specimens were longitudinally sectioned with a wafering blade (#11-4253, Buehler[R], Lake Bluff, IL, USA). One bucco-lingual section of each specimen was obtained from the region of the central groove, side of the central fossa (primary mandibular second molar) and from the region of the mesial interlobal groove (primary maxillary second molar). The section was used for the cross-sectional microhardness (CSMH) test.

Preparation of sections and microhardness test

The sections for the CSMH test were embedded in epoxy resin and serially ground flat with 600 and 1,200 grit water-proof silicon carbide paper (Norton, Vinhedo, SP, Brazil), followed by polishing with 1/4 and 1[micro] diamond suspensions (Arotec, Cotia, SP, Brazil) and SUPRA felt (Arotec, Cotia, SP, Brazil) (Fig. 1a). Microhardness was measured using a Shimadzu HMV 2 version 1.23 (Shimadzu Corporation, Kyoto, Japan) coupled to the CAMS[TM]_WIN software program (Newage Testing Instruments, Inc., Southampton, PA, USA) with a Knoop diamond indenter under a 25g load for 10s.

Three rows of indentations were made in each fissure wall at 10, 30, 50, 70, 90, 110, 220 and 330[micro]m from the outer surface of the enamel. The distance between rows was 100[micro]m. As the base of the fissures was variable, a central point was established, from which a distance was run to the lateral wall, which was parallel to the ground surface, for initiating the indentations (Fig 1b-d). Following the determination of the Knoop hardness number (KHN), the mean values at each distance from the surface were averaged and converted into vol.% mineral, based on the empirical formula proposed by Featherstone et al. [1983]: vol.% mineral = 4.3 [[square root] KHN] + 11.3; r = 0.919. The set of data representing each artificial carious lesion in each enamel section was fitted to the curve and the area under the lesion tracing was calculated using the trapezoidal rule (in units of vol.% mineral x [micro]m). This area was then subtracted from the value of sound enamel in each group (sum of the values at 110, 220 and 330um) to determine the mineral loss ([DELTA]Z), which is the relative mineral loss for each lesion. The mean [DELTA]Z values from 15 lesions were averaged to obtain the [DELTA]Z value per group.

Statistical analysis

The data on vol.% mineral and [DELTA]Z displayed variance homogeneity and normal distribution and were submitted to one-way ANOVA followed by a post-hoc pairwise comparison using Tukey's test. The significance level was set at 5%. The analyses were performed with the Statistical Package for Social Sciences (SPSS for Windows, version 17.0, SPSS Inc, Chicago, USA).

Results

Vol.% mineral and [DELTA]Z

Mean ([+ or -] SD) and confidence interval (CI) values of vol.% mineral and [DELTA]Z are displayed in Tables 1 and 2, respectively. No statistically significant differences were found between groups for either vol.% mineral at the different depths or [DELTA]Z (p > 0.05).

At 50[micro]m from the surface the vol.% mineral was greater than 80 in all groups and remained above this value at greater depths (Table 1). Greater mineral loss ([DELTA]Z) was observed in the group treated with NaF, although a large CI is observed for the lesions in this group (Table 2).

Figure 2 displays the mean ([+ or -] SD) values of vol.% mineral in the enamel at each depth after the pH-cycling in each treatment group. The similar performance among groups is plotted on the graph (p > 0.05). Above 50[micro]m from the surface, the vol.% mineral was homogeneous in all groups. A carious lesion of approximately 30[micro]m in depth was produced.

[FIGURE 2 OMITTED]

Discussion

The administration of high-concentration fluoride, together with other preventive and control measures, is necessary in individuals with high carious activity [Altenburger et al., 2008]. The effectiveness of such compounds regarding enamel resistance to demineralisation has been proven in vitro on enamel with a smooth surface [Garcia-Godoy et al., 1995; Delbem and Cury, 2002; Wiegand et al., 2005]. However, data on the occlusal surface are scarce in the literature.

In the present study, the demineralisation and remineralisation process provided mineral volume percentages suitable for artificial carious lesions of up to 30[micro]m from the surface in all groups. Although mineral loss was greater in the group treated with NaF, the high standard deviation value led to a non-significant difference comparison with the other groups. A likely hypothesis for this is the variability in the mineral content in the teeth, which constitutes one of the limitations of the present study, as it was not possible to determine the initial mineral content in the surfaces investigated in order to obtain a homogeneous sample. In the analysis of mineral loss, the control group did not differ significantly from the APF and NaF groups, suggesting that the residual fluoride (0.02 mg[L.sup.-1]) in the standard solutions could have influenced the demineralisation pattern in the specimens in this group. The literature reports an increase in fluoride content in enamel untreated with fluoridated products and submitted to pH-cycling [Delbem and Cury, 2002].

In a study carried out by Tanaka et al. [1993], the high degree of fluoride adsorption by the surface enamel came from solutions with low concentrations of fluoride ion and low pH. The reduction in the demineralisation of the enamel was proportional to the concentration of fluoride in the solution. Besides low pH, fluoride in solution and over a prolonged period of time provides more satisfactory results than the application of highly concentrated agents, even those with acid pH, such as APF. In a study involving highly concentrated fluoridated products, the authors demonstrated that the pH and vehicle of these products are more important than the fluoride content [Hayacibara et al., 2004]. The low pH of the product has the function of providing calcium ions for the interaction with the fluoride ions applied and reducing the amount of HP[O.sub.4.sup.-2], which inhibits the deposition of calcium fluoride [Lagerlof et al., 1988; Alves et al., 2007]. Maia et al. [2003] found that the frequent use of a fluoride dentifrice enables a greater degree of enamel hardness than the combination of the dentifrice with a fluoride varnish. Paes Leme et al. [2003] also found that a single application of fluoride gel was less efficient than the regular use of a fluoride dentifrice. These studies also demonstrate that greater fluoride absorption by carious enamel does not enhance the remineralisation capacity of saliva. These findings may partially explain the results of the present study. Although the APF group exhibited less mineral loss than the other groups, this difference did not achieve statistical significance.

A likely hypothesis for the results of the present study is the deposition of fluoride ions in the outermost layers of the enamel [Wiegand et al., 2005], impeding the action of fluoride in the mineral deposition in the inner portion of the lesion. The outer layers of human enamel are especially sensitive to fluoride and might be stabilised and condensed due to incorporation of this element [Karlinsey et al., 2011].

A study carried out with human molars (sound and with visible carious lesion in fissures) mapped the distribution of fluoride and calcium in the surface enamel of the occlusal fissures and determined the association between variations in fluoride concentration and the occurrence of demineralised areas in the enamel and subjacent dentine. In initial lesions, the fluoride concentration ranged from 1,900-7,200 ppmF, with an increase in fluoride occasionally found in the body of the lesion. In advanced carious lesions, fluoride concentrations were considerably high, ranging from 2,700-10,000 ppmF. The present authors suggest that the high concentration of fluoride detected in the outermost enamel is acquired during pre-eruptive maturation, when the enamel is more porous. Increased concentrations of fluoride were not found in subsurface lesions of the fissure enamel, which presupposes that fluoride does not diffuse to the interior of the deepest part of the lesion and therefore has little effect on the progression of such lesions [Pearce et al., 1999].

Another hypothesis for the results is that a single application of the fluoridated agent on the occlusal surface does not enhance enamel acid resistance. One study in smooth surface reports that the administration of a 1.23% APF gel one or two times and associated with the daily use of a fluoridated dentifrice was not capable of significantly enhancing surface hardness and fluoride content in bovine enamel in comparison to blocks submitted to demineralisation alone [Jardim et al., 2008].

The findings of the present study are in disagreement with those described by Delbem and Cury [2002], who found that APF was more effective at reducing enamel demineralisation under a cariogenic challenge than the NaF gel. In this study, fluoride deposition was greater in the APF group prior to the pH-cycling. A greater fluoride concentration in the APF gel, its lower pH, application regimen and porosity of the lesion favours greater fluoride absorption [Mellberg et al., 1988]. However, a greater fluoride concentration does not contribute to greater efficacy, as demonstrated by Maia et al. [2003]. Fluoride from highly concentrated products is deposited in the outer layers of the enamel and does not hinder the exit of calcium and phosphate ions from the body of the lesion. In carious enamel, a high fluoride concentration is found at the surface of the enamel and occasionally within the body of the lesion [Pearce et al., 1999]. Unlike the studies of Mellberg et al. [1988], Delbem and Cury [2002] and Maia et al. [2003], fluoride treatments in the present study were performed on pit and fissures, which are morphologically different from a smooth surface.

Even in sound enamel, APF treatments produce enamel dissolution, with the precipitation of fluoride-rich reaction products on the surface of the enamel [Margolis and Moreno, 1990]. This initial fluoride absorption is followed by a lower and more controlled diffusion stemming from the coverage of calcium fluoride on the surface of the enamel [Hicks, 1986]. Based on the literature, which reports an inhibitory action of the demineralisation process by APF [Mellberg et al., 1988; Delbem and Cury, 2002], and founded on the dissolution mechanism of enamel through treatment with APF, a significantly greater effect of resistance to demineralisation was expected from the APF gel in relation to the NaF gel.

Fluoride applied to sound enamel surfaces produces lower amounts of calcium fluoride [ten Cate, 1997; Jardim et al., 2008]. Moreover, mature enamel has less permeability [Kotsanos and Darling, 1991], which can influence the formation of calcium fluoride on the enamel. These variables could have affected the inhibition of demineralisation in the present study, as exfoliated primary teeth were used and the application of the fluoride compounds was performed prior to the cariogenic challenge.

The use of completely mature teeth with previous contact with fluoride is a limitation of the present study that should be considered in future investigations. An alternative would be the use of permanent third molars that were completely embedded prior to surgical removal. Embedded teeth would eliminate this type of bias, as they would not have undergone post-eruption maturation or cycles of demineralisation and remineralisation and would not have had any contact with fluoridated agents.

The results of the present in vitro study should be interpreted with caution, as they do not portray a real clinical situation. The oral environment houses cariogenic bacteria and salivary components that affect the outcome when fluoridated agents are administered. Moreover, the actual process of demineralisation and remineralisation occurs in the long term, whereas the in vitro simulation of this process was limited to 10 cycles. Thus, further studies should be conducted in order to confirm or refute the findings of the present study. The results suggest that a single application of a high-concentration fluoride compound does not promote a greater resistance to demineralisation in the enamel of pits and fissures, regardless the product used.

Conclusion

A single application of a high concentration fluoride compound does not promote greater resistance to demineralisation in enamel pits and fissures, regardless of the product used.

Acknowledgments

The authors are grateful to the tooth bank of the School of Dentistry of the Universidade de Sao Paulo (Brazil) for the donation of the teeth used in this study. The authors also would like to thank professors Dr. Maria Letfcia Ramos-Jorge and Dr. Fernanda de Oliveira Ferreira (Universidade Federal dos Vales do Jequitinhonha e Mucuri, Brazil) for assistance in the statistical analysis of the data. This research was supported by Brazilian Coordination of Higher Education (CAPES), Ministry of Education, Brazil.

References

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Alves KMRP, Pessan JP, Brighenti FL, et al. In vitro evaluation of the effectiveness of acidic fluoride dentifrices. Caries Res 2007; 41:263-267.

Delbem ACB, Cury JA. Effect of application time of APF and NaF gels on microhardness and fluoride uptake of in vitro enamel caries. Am J Dent 2002; 15:169-172.

Featherstone JDB, ten Cate JM, Shariati M, et al. Comparison of artificial caries: like lesions by quantitative microradiography and microhardness profiles. Caries Res 1983; 17:385-391.

Featherstone JDB, O'Reilly MMO, Shariati M, et al. Enhancement of remineralisation in vitro and in vivo. In: Leach SA (ed): Factors relating to demineralisation and remineralisation of the teeth. Washington: IRL Press; 1986, pp 23-34.

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M.C. Ferreira *, M.C.M. Calvo **, R.S. Vieira ***

* Department of Health Sciences, Universidade Federal dos Vales do Jequitinhonha e Mucuri, ** Public Health Department, *** Stomatology Department, Universidade Federal de Santa Catarina; Brazil.

Postal address: Dr M.C. Ferreira, Rua 12, 648, Governador Valadares, MG, 35020-690, Brazil.

Email: meirecofe@ig.com.br
Table 1. Vol.% mineral of cross-sectional enamel
according to group and depth; mean ([+ or -] SD)
and 95% confidence intervals.

 Control NaF 2%

Depth Mean CI Mean CI
 ([+ or -] SD) ([+ or -] SD)

10 [micro]m 64.9 (13.5) 57.4-72.4 61.5 (15.2) 53.0-69.9
30 [micro]m 81.2 (10) 75.7-86.7 76.1 (15.9) 67.3-84.8
50 [micro]m 85.6 (7.1) 81.6-89.5 88.5 (7) 84.7-92.4
70 [micro]m 87.3 (7.4) 83.1-91.4 92.2 (5.3) 89.3-95.2
90 [micro]m 88.2 (7) 84.4-92.1 92.9 (5.9) 90.0-96.1
110 [micro]m 88.1 (6.9) 84.3-92.0 92.9 (6.1) 89.5-96.2
220 [micro]m 87 (6.8) 83.2-91.0 91.4 (5.9) 88.2-94.7
330 [micro]m 88.7 (6.7) 85.0-92.4 90.6 (3.6) 88.6-92.6

 APF 1.23%

Depth Mean CI
 ([+ or -] SD)

10 [micro]m 68.4 (10.8) 62.4-74.3
30 [micro]m 84.1 (6.7) 80.4-87.8
50 [micro]m 87.1 (5.2) 84.2-90.0
70 [micro]m 87.5 (4.6) 85.0-90.1
90 [micro]m 88.8 (4.6) 86.3-91.3
110 [micro]m 88.5 (5.4) 85.5-91.5
220 [micro]m 86.5 (6.1) 83.2-90.0
330 [micro]m 87.8 (4.2) 85.5-90.1

Standard deviation (SD)
Confidence intervals (CI)

Table 2. Mean mineral loss ([DELTA]Z,
vol.% [micro]m) according to treatment;
mean ([+ or -] SD) and 95% confidence
intervals

 [DELTA]Z

Group Mean [+ or -] SD CI

Control 446.83 [+ or -] 418.54 (a) 215.1-678.6
NaF 2% 759.35 [+ or -] 608.72 (a) 422.3-1096.5
APF 1.23% 346.7 [+ or -] 353.94 (a) 150.7-542.7

Values with same lowercase letter in
same column do not differ significantly
statistical analysis
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Author:Ferreira, M.C.; Calvo, M.C.M.; Vieira, R.S.
Publication:European Archives of Paediatric Dentistry
Article Type:Report
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Date:Dec 1, 2011
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