Efficacy of 1 . 23 % APF gel applications on incipient carious lesions : a double-blind randomized clinical trial

The aim of this double-blind randomized clinical trial was to evaluate the efficacy of 1.23% APF gel application on the arrest of active incipient carious lesions in children. Sixty 7to 12-year-old children, with active incipient lesions were included in the study. Children were divided randomly into 2 groups: 1.23% APF gel and placebo gel applications. Each group received 8 weekly applications of treatment. The lesions were re-evaluated at the 4th and 8th appointments. Poisson regression analysis was used to estimate relative risks of the presence of active white spot lesions. Groups showed similar results (PR = 1.67; CI 95% 0.69–3.98). The persistence of at least 1 active lesion was associated with a higher number of lesions in the baseline (PR = 2.67; CI 95% 1.19–6.03), but not with sugar intake (PR = 1.06; CI 95% 0.56–2.86) and previous exposure to fluoride dentifrice (PR = 1.26; CI 95% 0.49–2.29). The trial demonstrates the equivalence of the treatments. The use of the APF gel showed no additional benefits in this sample of children exposed to fluoridated water and dentifrice. The professional dental plaque removal in both groups may also account for the resulting equivalence of the treatments. Descriptors: Fluorides, Topical; Dental Caries; Toothbrushing. Introduction Despite the decline of the prevalence of dental caries observed worldwide, there is still a considerable number of children presenting with the disease. The clinical features of dental caries may vary from a white spot to a cavity, if the disease is not controlled. Studies have indicated that initial carious lesions are significantly more prevalent than cavitated lesions1,2 and that the presence of initial lesions is a predictor of caries increment in the permanent dentition.3 Preventive strategies must be employed to revert the changes provoked by the disease in enamel through a noninvasive treatment. Topical fluoride has been used as an adjunct in the treatment of initial caries lesions. Among the fluoride-containing applications, the use of professional acidulated phosphate fluoride (APF) is used widely. In an in situ study, the presence of fluoride played an important role in rehardening enamel.4 Without plaque removal and under cariogenic challenge, the APF gel reduced demineralization in enamel when used alone. However, the use of fluoride dentifrice alone or in combination with APF gel showed no differences between groups regarding the reduction of enamel mineral loss. Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript. Submitted: Oct 02, 2012 Accepted for publication: Feb 06, 2013 Last revision: Mar 08, 2013 Efficacy of 1.23% APF gel applications on incipient carious lesions: a double-blind randomized clinical trial 280 Braz Oral Res., (São Paulo) 2013 May-Jun;27(3):279-85 Similar results were found in another study that assessed the reduction of lesion size.5 A randomized controlled clinical trial (RCT) showed no additional benefits after treating incipient carious lesions with APF, suggesting that the weekly supervised toothbrushing played a more important role.6 Another study has shown the relevance of plaque disturbance in the arrest of incipient carious lesions, regardless of the use of fluoride.7 Most results, however, were obtained from in situ studies4,5,8,9 and only a few of the studies were RCT,6,7 which is recognized to be the most appropriate tool for comparing therapies. Further research on non-surgical treatment of incipient carious lesions is still needed.10,11 A research on the best fluoride regimen to assist in the remineralization of early carious lesions is important in order to provide a stronger evidence-base for the use of professionally applied topical fluoride.11 Furthermore, head-tohead comparisons of regimens and agents may provide more useful information.12 The aim of this double-blind RCT was to evaluate the efficacy of the application of 1.23% APF gel on the arrest of active incipient carious lesions in children receiving professional toothbrushing with fluoride dentifrice. Methodology Subjects and study design A double-blind RCT trial was carried out in a public school in the city of Pelotas, Brazil. This research was approved by the Ethics Committee of the School of Dentistry of the University of São Paulo and the parents of the children signed a written consent form. All children from 7 to 12 years old were recruited and examined. To be included in the study children had to have at least 1 active incipient non cavitated carious lesions on the buccal surfaces of upper permanent incisors and/or of lower permanent first molars. Incipient carious lesions were considered to be active if they showed a whitish, opaque (chalky) coloration, if they felt rough when the probe was gently moved across the surface,13 and if they were close to the gingival margin. Children participating in any other preventive program were not included, as well as children with neurological or systemic diseases. A pre-test was conducted on 30 children. After 8 weeks, the incidence of active lesions was 33.3% in the group of professional toothbrushing and placebo gel and 60% in the group of professional toothbrushing and fluoride gel application. Assuming that the difference in incidence between groups would be 27%, α = 0.05 and β = 0.20, the sample size for each group was 30 children with at least 1 lesion. Examination methods and evaluation One previously trained dentist performed clinical examinations of the children at school under artificial illumination in regular chairs. The Plaque Index System,14 with scores ranging from 0 to 3, was used to assess the buccal surfaces of teeth 16, 11, 26 and 31 and the lingual surfaces of teeth 36 and 46.15 The children were classified as presenting a score  <  1, 1–1.9 or  ≥  2. Dental caries were recorded using WHO criteria, as follows: decayed, missing or filled surface (DMFS),16 and the corresponding severity of dental caries in children were categorized as 0–3 or > 3 surfaces. The clinical diagnosis of white spot lesions was made after adequately cleaning and drying the surface with compressed air. A visual-tactile examination was performed. A probe was used to gently check  for surface texture and loss of tooth structure.13 The reliability of the calibration was assessed by the Kappa test (kappa 0.80). Regarding the number of lesions, the children were classified as presenting 1–2 or > 2 non cavitated lesions. The children were assigned randomly to either the placebo group (Group I) or the 1.23% APF (DFL, Sultan Topex, Rio de Janeiro, Brazil) treatment group (Group II). The allocation was concealed by opening an opaque randomization envelope containing papers with only the number of the group, 30 for Group I and 30 for Group II. Both the placebo and the fluoride gels were placed in similar bottles with different labels (I and II). The placebo gel was manufactured by a compounding pharmacy and both gels displayed the same characteristics (color, smell, consistency). The following procedures were performed at each weekly appointment for 8 weeks6 by the operator: • toothbrushing instructions, Bonow MLM, Azevedo MS, Goettems ML, Rodrigues CRMD 281 Braz Oral Res., (São Paulo) 2013 May-Jun;27(3):279-85 • never = 0, • rarely = 1, • weekly = 2, and • daily = 5. When the pattern of consumption scored 30 points or over, it was considered to be high and, when it scored lower than 30 points, it was considered to be moderate.18 Statistical analysis Data were analyzed using Stata 10.0 (Stata Corporation, College Station, USA). Differences between groups were assessed using the chi-square test, Fisher’s exact test, and t-test with Welch’s correction. Poisson regression analysis was used to estimate relative risks of the children presenting at least 1 active white spot lesion after 8 weeks. First, the relative risk and 95% CI of each variable were estimated separately. Then, multivariate modeling was performed. The level of significance was set at 5%. Results Four hundred and thirty children were examined until the sample of 60 children was attained. Figure 1 shows the diagram of the RCT. One of the participants of the placebo group dropped out. A total of 112 active white spot lesions were present in 59 children. No adverse effects occurred. The groups showed similarities in all the characteristics that were assessed (Table 1). The groups were also similar in relation to the DMFS caries index (p = 0.74, t-test with Welch’s correction) which was around 7.5. Table 2 presents the crude and multivariate model for the presence of white spot lesions after 8 weeks. The treatments showed similar results. The persistence of at least 1 active lesion was associated with a higher number of white spot lesions in baseline in crude analysis and also when adjusted for the group and sugar intake. The majority of lesions (62%) showed arrest in the final evaluation. There was an increase in the percentage of inactive lesions when the results from the 4th and the 8th sessions were compared in both groups (p < 0.01) (Figure 2). • professional toothbrushing of the lesion areas for 15 s, • drying of the buccal surface, and • topical application of a 1.23% APF, according to the manufacturer’s instructions, or placebo gel on the lesion, according to each group, using cotton swabs to minimize the ingestion of the gel. Children were given toothbrushes (Colgate/Palmolive, São Bernardo do Campo, Brazil) and fluoridated dentifrice (1100 ppm NaF; Tandy, Colgate/ Palmolive, São Bernardo do Campo, Brazil) to be used throughout this trial. It was emphasized that the children had to brush their teeth at home with the dentifrice. Furthermore, mothers were asked to control their children’s dental hygiene procedure. Children did not participate in any additional type of preventive program. The examiner performed the eva


Introduction
Despite the decline of the prevalence of dental caries observed worldwide, there is still a considerable number of children presenting with the disease.The clinical features of dental caries may vary from a white spot to a cavity, if the disease is not controlled.Studies have indicated that initial carious lesions are significantly more prevalent than cavitated lesions 1,2 and that the presence of initial lesions is a predictor of caries increment in the permanent dentition. 3reventive strategies must be employed to revert the changes provoked by the disease in enamel through a noninvasive treatment.Topical fluoride has been used as an adjunct in the treatment of initial caries lesions.Among the fluoride-containing applications, the use of professional acidulated phosphate fluoride (APF) is used widely.In an in situ study, the presence of fluoride played an important role in rehardening enamel. 4ithout plaque removal and under cariogenic challenge, the APF gel reduced demineralization in enamel when used alone.However, the use of fluoride dentifrice alone or in combination with APF gel showed no differences between groups regarding the reduction of enamel mineral loss.

Declaration of Interests:
The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.
Similar results were found in another study that assessed the reduction of lesion size. 5 randomized controlled clinical trial (RCT) showed no additional benefits after treating incipient carious lesions with APF, suggesting that the weekly supervised toothbrushing played a more important role.6 Another study has shown the relevance of plaque disturbance in the arrest of incipient carious lesions, regardless of the use of fluoride.7 Most results, however, were obtained from in situ studies 4,5,8,9 and only a few of the studies were RCT, 6,7 which is recognized to be the most appropriate tool for comparing therapies.Further research on non-surgical treatment of incipient carious lesions is still needed.10,11 A research on the best fluoride regimen to assist in the remineralization of early carious lesions is important in order to provide a stronger evidence-base for the use of professionally applied topical fluoride. The aim of this double-blind RCT was to evaluate the efficacy of the application of 1.23% APF gel on the arrest of active incipient carious lesions in children receiving professional toothbrushing with fluoride dentifrice.

Methodology Subjects and study design
A double-blind RCT trial was carried out in a public school in the city of Pelotas, Brazil.This research was approved by the Ethics Committee of the School of Dentistry of the University of São Paulo and the parents of the children signed a written consent form.
All children from 7 to 12 years old were recruited and examined.To be included in the study children had to have at least 1 active incipient non cavitated carious lesions on the buccal surfaces of upper permanent incisors and/or of lower permanent first molars.Incipient carious lesions were considered to be active if they showed a whitish, opaque (chalky) coloration, if they felt rough when the probe was gently moved across the surface, 13 and if they were close to the gingival margin.Children participating in any other preventive program were not included, as well as children with neurological or systemic diseases.
A pre-test was conducted on 30 children.After 8 weeks, the incidence of active lesions was 33.3% in the group of professional toothbrushing and placebo gel and 60% in the group of professional toothbrushing and fluoride gel application.Assuming that the difference in incidence between groups would be 27%, α = 0.05 and β = 0.20, the sample size for each group was 30 children with at least 1 lesion.

Examination methods and evaluation
One previously trained dentist performed clinical examinations of the children at school under artificial illumination in regular chairs.The Plaque Index System, 14 with scores ranging from 0 to 3, was used to assess the buccal surfaces of teeth 16, 11, 26 and 31 and the lingual surfaces of teeth 36 and 46. 15The children were classified as presenting a score < 1, 1-1.9 or ≥ 2. Dental caries were recorded using WHO criteria, as follows: decayed, missing or filled surface (DMFS), 16 and the corresponding severity of dental caries in children were categorized as 0-3 or > 3 surfaces.The clinical diagnosis of white spot lesions was made after adequately cleaning and drying the surface with compressed air.A visual-tactile examination was performed.A probe was used to gently check for surface texture and loss of tooth structure. 13The reliability of the calibration was assessed by the Kappa test (kappa 0.80).Regarding the number of lesions, the children were classified as presenting 1-2 or > 2 non cavitated lesions.
The children were assigned randomly to either the placebo group (Group I) or the 1.23% APF (DFL, Sultan Topex, Rio de Janeiro, Brazil) treatment group (Group II).The allocation was concealed by opening an opaque randomization envelope containing papers with only the number of the group, 30 for Group I and 30 for Group II.Both the placebo and the fluoride gels were placed in similar bottles with different labels (I and II).The placebo gel was manufactured by a compounding pharmacy and both gels displayed the same characteristics (color, smell, consistency).The following procedures were performed at each weekly appointment for 8 weeks 6 by the operator: • toothbrushing instructions, • never = 0, • rarely = 1, • weekly = 2, and • daily = 5.
When the pattern of consumption scored 30 points or over, it was considered to be high and, when it scored lower than 30 points, it was considered to be moderate. 18

Statistical analysis
Data were analyzed using Stata 10.0 (Stata Corporation, College Station, USA).Differences between groups were assessed using the chi-square test, Fisher's exact test, and t-test with Welch's correction.Poisson regression analysis was used to estimate relative risks of the children presenting at least 1 active white spot lesion after 8 weeks.First, the relative risk and 95% CI of each variable were estimated separately.Then, multivariate modeling was performed.The level of significance was set at 5%.

Results
Four hundred and thirty children were examined until the sample of 60 children was attained.Figure 1 shows the diagram of the RCT.One of the participants of the placebo group dropped out.A total of 112 active white spot lesions were present in 59 children.No adverse effects occurred.
The groups showed similarities in all the characteristics that were assessed (Table 1).The groups were also similar in relation to the DMFS caries index (p = 0.74, t-test with Welch's correction) which was around 7.5.
Table 2 presents the crude and multivariate model for the presence of white spot lesions after 8 weeks.The treatments showed similar results.The persistence of at least 1 active lesion was associated with a higher number of white spot lesions in baseline in crude analysis and also when adjusted for the group and sugar intake.
The majority of lesions (62%) showed arrest in the final evaluation.There was an increase in the percentage of inactive lesions when the results from the 4 th and the 8 th sessions were compared in both groups (p < 0.01) (Figure 2).
• professional toothbrushing of the lesion areas for 15 s, • drying of the buccal surface, and • topical application of a 1.23% APF, according to the manufacturer's instructions, or placebo gel on the lesion, according to each group, using cotton swabs to minimize the ingestion of the gel.
Children were given toothbrushes (Colgate/Palmolive, São Bernardo do Campo, Brazil) and fluoridated dentifrice (1100 ppm NaF; Tandy, Colgate/ Palmolive, São Bernardo do Campo, Brazil) to be used throughout this trial.It was emphasized that the children had to brush their teeth at home with the dentifrice.Furthermore, mothers were asked to control their children's dental hygiene procedure.Children did not participate in any additional type of preventive program.
The examiner performed the evaluation of the white spots after the 4 th and 8 th treatment sessions.Lesions were categorized as inactive if: • they showed whitish, brownish or black coloration, • enamel was shiny and felt hard or smooth when the tip of the probe was moved gently across the surface, • there was no clinically detectable loss of substance, and • lesion was located at some distance from the gingival margin, 13 The participants, the care providers and the examiner who assessed the outcome, did not know which gel was used in each group, characterizing a double-blind study.

Questionnaire
A questionnaire was answered by the parents.To determine the socioeconomic status of the child's family, the scale of the ABIPEME (Associação Brasileira de Institutos de Pesquisa de Mercado) was used. 17The children's diets were classified at baseline according to the frequency of sugar intake.Twenty most consumed foods containing sugar were selected, and each was given a value according to the frequency of consumption, as follows:

Discussion
The results did not show any additional effect of the APF gel on the arrest of enamel lesions compared to placebo gel after 8 weeks.Both groups received professional toothbrushing before the application of the gel at all sessions, and were advised to use fluoridated dentifrice at home.In both groups, the arrest of white spot lesions was observed.The biofilm control itself is an effective way to obtain arrest of lesions, 6 since it promotes mechanical wear of the lesion, what contributes to the improvement of its final aspect. 19urface abrasion with some redeposition of minerals is believed to be responsible for the regression of lesions. 19Holmen et al. 20 observed that functional wear and usual toothbrushing, with no additional fluoride, were responsible for the arrest by the disturbance and removal of bacterial deposits.Experimentally induced enamel lesions were subsequently remineralized in vivo without any fluoride treatment. 8The enamel demineralization or remineralization effect can only be quantified in in situ and in vitro studies using invasive and analysis methods, 21 and not in in vivo studies, like the present one.
The caries-inhibiting effect of the fluoride gel was not clinically relevant in a study with children with low incidence of caries who were regular attendees of pediatric dental clinics and were receiving semiannual check-ups with oral hygiene instruction and supervised toothbrushing with fluoride toothpaste. 22thers studies 4,23,24 showed that toothbrushing with fluoridated dentifrices alone is able to lead to the arrest of white spot lesions.
It is noteworthy that children at high risk for dental caries might benefit from additional exposure to fluoride. 11In a RCT of the efficacy of a high-fluoride gel self-applied by toothbrushing in children at high caries risk, it was found that self-     application of fluoride gel at school benefits children from more disadvantaged communities who may brush less frequently at home, and therefore have the greatest potential to benefit from this program. 25Children recruited for this study had active dental caries, an individual factor that increases risk.Besides this, children were recruited from a public school and had low socioeconomic status, characteristics recognized as risk factors for dental caries.
The professional toothbrushing throughout the clinical study and oral hygiene supervision of children by parents can mask the real benefit of fluoride gel application; thus, our results should be interpreted with caution.There was evidence from nine trials showing that the simultaneous use of a topical fluoride treatment with fluoride toothpaste results in an enhanced caries-inhibiting effect, compared with the use of toothpaste alone. 26However, these results refer to preventive measures, whereas the present study assessed the arrest of incipient caries lesions.It is currently recognized that the effect of fluoride on the dynamics of the caries process and its success in preventing caries should not be confused with its arrest or reversal of carious lesions.
The evaluation of the use of fluoridated gel was short-term, enabling intense supervision and effective control of plaque removal.This period of time would be enough to allow the observation of clinical alterations showing the arrest of further demineralization of the lesions. 19It is worth mentioning that among the lesions classified as active in the final examination, some presented clinical features of arrested caries, which reflects the dynamic nature of the caries process. 27However, as improvement occurred between 4 and 8 weeks it is possible that better results could have been achieved with a longer period of follow-up.This seems to be particularly true in cases of children with a higher number of incipient carious lesions.
High numbers of active lesions may represent a "risk" to maintain white spot activity. 6In this study, the numbers of lesions at baseline were also verified to interfere with the presence of white spot lesions after 8 weeks.Incipient carious lesions in children with many lesions would take longer to become inactive when compared to those of children with fewer lesions.Arrow 28 concluded that enamel caries progression was associated with baseline caries experience, showing that individuals with a high number of lesions are at higher caries risk.Further studies should address this question.
The sugar intake showed no influence on persistence of active white spot lesions.The relationship between sugar intake and incipient carious lesions occurs only when oral hygiene is poor. 29A study with 6 year-old children showed that sweet consumption at least once a day was associated with a higher caries index.However, a low toothbrushing frequency was the main behavior risk factor for high levels of caries. 30ample size calculation was conducted to detect a true difference based on the results of a pilot test.However results showed great variation from the pilot study.A limitation is that smaller differences, although important, would not be detected.However, this sample size calculation method was adopted because no other study was found with similar intervention tested.Thus, the results should be interpreted with caution.

Conclusion
The trial failed to demonstrate clear evidence of a difference in efficacy between groups and, thus, the equivalence of the treatments can be concluded.The use of fluoride gel showed no additional benefits in this sample of children exposed to fluoridated water and dentifrice.The professional dental plaque removal in both groups may also account for the equivalence of treatments obtained in this study.

Figure 2 -
Figure 2 -Distribution of lesions after 4 and 8 weeks by the number of white spot lesions that the patient presented at baseline.

Table 1 -
Characteristics of children by treatment group.