Effect of toothbrushing duration and dentifrice quantity on enamel remineralisation: An in siturandomized clinical trial

Abstract

Objectives

The influence of toothbrushing duration and dentifrice quantity on fluoride efficacy against dental caries is poorly understood. This study investigated effects of these two oral hygiene factors on enamel remineralisation (measured as surface microhardness recovery [SMHR]), enamel fluoride uptake (EFU), and net acid resistance (NAR) post-remineralisation in a randomized clinical study using an in situ caries model.

Methods

Subjects (n = 63) wore their partial dentures holding partially demineralised human enamel specimens and brushed twice-daily for two weeks, following each of five regimens: brushing for 120 or 45 s with 1.5 g of 1150 ppm F (as NaF) dentifrice; for 120 or 45 s with 0.5 g of this dentifrice; and for 120 s with 1.5 g of 250 ppm F (NaF) dentifrice.

Results

Comparing brushing for 120 s against brushing for 45 s, SMHR and EFU increased by 20.0% and 26.9% respectively when 1.5 g dentifrice was used; and by 22.8% and 19.9% respectively when 0.5 g dentifrice was used. Comparing brushing with 1.5 g against brushing with 0.5 g dentifrice, SMHR and EFU increased by 35.3% and 51.3% respectively when brushing for 120 s, and by 38.4% and 43.0% respectively when brushing for 45 s. Increasing brushing duration and dentifrice quantity also increased the NAR value. The effects of these two oral hygiene factors on SMHR, EFU, and NAR were statistically significant (p < 0.05 in all cases).

Conclusion

Brushing duration and dentifrice quantity have the potential to influence the anti-caries effectiveness of fluoride dentifrices. Study NCT01563172 on ClinicalTrials.gov .

Clinical significance

The effect of two key oral hygiene regimen factors – toothbrushing duration and dentifrice quantity – on fluoride’s anticaries effectiveness is unclear. This 2-week home-use in situ remineralisation clinical study showed both these factors can influence fluoride bioactivity, and so can potentially affect fluoride’s ability to protect against caries.

Introduction

Regular brushing with a fluoride-containing dentifrice has been convincingly demonstrated to reduce the development of dental caries . Its mode of action is at least two-fold: firstly, fluoride can reduce demineralisation when bound to the enamel surface, as fluoridated enamel is less acid-soluble than native enamel; secondly, it can promote remineralisation of partially demineralised enamel in the presence of saliva-derived calcium and phosphate ions, both when bound to the enamel surface and when present in solution in fluid overlying the lesion . Fluoride is delivered from a dentifrice into the oral cavity only during the brief periods of toothbrushing. Evidence indicates fluoride is incorporated into enamel during the time of brushing driven by high concentration in the dentifrice slurry, but is also taken up by oral soft tissues and plaque . These reservoirs slowly liberate fluoride into the oral fluids over several hours post-brushing, boosting its protective effect on enamel .

The protection fluoride provides against caries will therefore be influenced by the intra-oral fluoride concentration achieved during brushing and by the frequency and duration of fluoride exposure. Simply increasing fluoride concentration in the dentifrice is an effective way to decrease caries incidence ; however, fluoride concentration is limited in mass-market dentifrices to 1500 ppm or less. Other ways to potentially increase fluoride protection include increasing frequency of use, decreasing post-brushing rinsing, increasing the amount of dentifrice used, and increasing brushing duration . Consequently, how an individual uses a fluoride dentifrice – i.e. their oral hygiene routine – is very important to how much protection toothbrushing can afford against caries .

Surprisingly, to date only frequency of brushing and degree of rinsing have been extensively studied in relation to fluoride effectiveness. Several correlation studies have between them indicated that these two factors are important in determining caries risk , though it should be noted a later, prospective study did not show an effect of post-brushing rinsing regimen on caries incidence .

The quantity of dentifrice used must at some point affect fluoride efficacy. However, in the conventional usage range of approximately 0.5–1.5 g ( i.e. from a typical pea-sized amount to an amount sufficient to cover the length of a typical toothbrush head ), the effect on fluoride efficacy in vivo is controversial. Clinical studies utilizing the proxy caries measures of fluoride delivery and remineralisation in situ , in which quantity was prospectively controlled or carefully monitored, have consistently shown positive correlations with dentifrice quantity . In contrast, two caries clinical studies that investigated the correlation of dentifrice quantity with caries prevention , and a study by Sjögren and Birkhed correlating oral hygiene practice with caries incidence , found no evidence for a link, though in none of the clinical studies was dentifrice quantity controlled, nor measured on more than one occasion.

The effect of toothbrushing duration on fluoride effectiveness has received little attention. As noted for dentifrice quantity, at some point toothbrushing duration must influence fluoride effectiveness, but it is not clear whether there is any benefit from increasing brushing time from the typical value of about 45 s to the recommended 2 min or more ( e.g. British Dental Association recommendation ). Fluoride penetration through plaque in situ , uptake to demineralised enamel in vitro , and fluoride delivery to the oral soft tissues in vivo are all time-dependent in this range. Consistent with these results, a pilot single-use study by this group demonstrated that brushing duration can influence in situ enamel fluoride uptake and remineralisation in the absence of plaque .

Building on this limited evidence base, the aim of this study was to determine whether two key aspects of dentifrice usage regimen, brushing duration and dentifrice quantity, can – in ranges relevant to typical oral hygiene practice – affect fluoride performance using an in situ clinical design as representative as practicable of the in vivo oral situation. ‘Fluoride performance’ encompassed measuring effects on enamel remineralisation (by surface microhardness recovery, SMHR ); enamel fluoride uptake (by EFU ); and enamel acid resistance post-remineralisation (by net acid resistance, NAR ). The primary objective was to determine whether brushing for 120 s versus 45 s with 1.5 g of 1150 ppm F dentifrice can increase SMHR. The effect of dentifrice dose was also examined by brushing with 0.5 g of this dentifrice for the same time periods. The null hypotheses were that neither brushing duration nor dentifrice quantity would have an effect on the study end-points.

Materials and methods

This was an examiner- and analyst-blind, five-treatment regimen, cross-over, in situ model study [30], performed at the Oral Health Research Institute, Indiana, USA. The study was approved by the Indiana University Institutional Review Board (approval number 0812-55) and carried out according to guidelines set out in the Declaration of Helsinki. This study is registered on ClinicalTrials.gov , study number NCT01563172 . There was one amendment to the study to more explicitly explain brushing instructions to subjects.

Study population

Individuals 18–80 years old were recruited to the study from the Indianapolis area (where community water contains about 1 μg/mL fluoride). Inclusion criteria included wearing a removable mandibular partial denture, being otherwise in good general and dental health and having unstimulated saliva flow ≥0.2 mL/min, stimulated saliva flow ≥0.8 mL/min. Subjects were recruited who granted written informed consent, demonstrated understanding of the protocol and were considered willing, able and likely to comply with all study procedures. Potential subjects were excluded if they reported they were pregnant, were intending to become pregnant during the study period, or were lactating. Subjects were enrolled and allocated to treatment by clinical personnel under supervision of the Principal Investigator (DTZ).

The recruitment sample size target of 65 was based on 45 subjects completing all treatments assuming a drop-out rate of 30%. This sample size gave 90% power at p < 0.05, using two-sided testing, to detect a mean difference in % SMHR of 8.36, based on within-subject standard deviation of 11.96 (determined from previous, unpublished data). Randomisation was performed via a Williams’ square approach, generated by GlaxoSmithKline Consumer Healthcare Biostatistics department.

Experimental design

The study flow is summarized in Fig. 1 . Each subject undertook treatments in a cross-over design in five successive 3-week cycles. At the start of each 3-week cycle, subjects followed their normal oral hygiene regimen, then, following a prophylaxis, they used a fluoride-free ‘washout’ dentifrice twice daily during the final 2–3 days of the first week. The treatment phase started at the beginning of Week 2 and involved subjects using their assigned dentifrice (1150 ppm F or 250 ppm F) twice daily, brushing for the specified brushing duration and using the specified dentifrice quantity. Treatment continued until the end of Week 3, after which subjects started the next cycle. Adverse events (AEs) were recorded at each visit to the clinic.

Fig. 1
Subject study flow chart showing recruitment and treatment details.

Treatment regimens

Three dentifrices were used in this study: (i) an 1150 ppm fluoride (as NaF) dentifrice (Aquafresh ® Advanced 2 x Enamel Strengthening Action, silica-based; GSK Consumer Healthcare, Weybridge, Surrey, UK); (ii) a 250 ppm fluoride (as NaF) dentifrice, prepared in the same base; (iii) a variant of this base with no added fluoride, used as a washout product. For (ii) and (iii), sodium fluoride was replaced with water.

The single experimental factor analysed was treatment regimen, as follows:

  • i.

    45 s brushing, 0.5 g of 1150 ppm F dentifrice;

  • ii.

    120 s brushing, 0.5 g of 1150 ppm F dentifrice;

  • iii.

    45 s brushing, 1.5 g of 1150 ppm F dentifrice;

  • iv.

    120 s brushing, 1.5 g of 1150 ppm F dentifrice;

  • v.

    120 s brushing, 1.5 g of 250 ppm F dentifrice.

A new Aquafresh ® Flex soft toothbrush was supplied to each subject for each treatment cycle.

Preparation and use of in situ devices

Measurements on enamel blocks were made in vitro , separated from the clinical environment. Human enamel specimens were cut and polished, and their SMH measured as previously described . Five baseline indentations were placed on each specimen; only those with mean indentation lengths of 43 ±3 μm were accepted. Specimens were then subjected to pre-treatment demineralisation by immersion in 0.05 M/L lactic acid, 50% saturated with hydroxyapatite, containing 0.1% w/v Carbopol ® 907 (BF Goodrich Co., Cleveland, OH, USA), pH 5.0, at 37 °C for 24 h, following which SMH was again measured by placing five indentations to the left of the baseline indentations. This process created very shallow lesions (25–30 μm depth) in which a surface layer had not completely formed . Specimens with mean indentation lengths of 120 ±20 μm were accepted for the study.

The specimens were randomly divided into five equally sized groups and graded by post-demineralisation SMH, then randomly assigned to the 65 subjects, who were considered as statistical blocks. Prior to insertion, specimens were sterilized by ethylene oxide. Two specimens were mounted in the subjects’ appliances, as shown in Fig. 2 , and covered with gauze (Polyester Knit Fabric; Item# 401628, Impra, Tempe, USA). Subjects wore their appliance continuously throughout each 2-week treatment period , but could remove it to clean it with water and to rinse their mouth after meals and snacks. Prior to brushing, subjects weighed the dentifrice to within 0.1 g of their assigned target weight. They spent one quarter of their assigned brushing time brushing each of the four quadrants of their dentition, taking care not to brush the enamel specimens. Subjects expectorated immediately following the end of their timed brushing period and without delay rinsed their mouths by swilling with 15 mL tap water continuously for approximately 10 s.

Fig. 2
An example partial denture with gauze-covered enamel specimens as used in this study.

After the 2-week period, subjects removed their appliance and the enamel specimens were excised. The specimens were re-analysed for SMH and then tested for EFU using micro-drill enamel biopsy as previously described , taking four 100 μm cores per specimen. Core diameters were determined using a calibrated microscope. The specimens were then subjected to post-treatment demineralisation using the same acid challenge as for the pre-treatment demineralisation and SMH was again recorded.

Subject adherence

Subjects undertook an extensive training and tracking program to promote protocol adherence. Five pre-study training sessions covered the use of study weighing scales and timers, which were subsequently used by each subject at home to weigh dentifrice and time each brushing during treatment periods. Subjects recorded the time of day, dentifrice weight and brushing duration in a diary. Each subject also performed a single supervised brushing at the study site at the beginning and on the eighth day of each test period (±1 day). Compliance was assessed by weighing of dentifrice tubes before and after each treatment period and comparison to product usage diaries.

Data analysis

The response factor for the primary objective was SMHR, calculated as:

% SMHR = [(D1 − R)/(D1 − B)] × 100

Where B = indentation length (μm) of sound enamel specimen at baseline; D1 = indentation length (μm) after pre-treatment in vitro demineralisation; R = indentation length (μm) after in situ remineralisation . For each indentation measurement, the five indentations within each of the two demineralised enamel blocks were averaged and used in the calculation of % SMHR on an enamel block level. These results were averaged over the two blocks to give the subject-level value. If a subject was missing an enamel block, the data from the remaining block was used to perform calculations.

Secondary objective response factors included EFU and NAR of the experimental specimens. EFU was calculated based on the amount of fluoride divided by the area of the enamel cores, expressed as μg F/cm 2 . NAR was calculated as:

% NAR = [(D1 − D2)/(D1 − B)] × 100

Where D1 and B are defined as above and D2 = indentation length (μm) after post-treatment in vitro demineralisation .

Statistical analysis was performed on the per-protocol (PP) population, which included all randomized subjects who had no major protocol deviations. For each efficacy parameter, the study treatment regimens were compared using a mixed-model analysis of variance (ANOVA). The model included fixed effects for study period and treatment regimen, and a random effect for subject. The analysis of the data included the estimation of the least-square means for the treatments and the comparison of the least-square means for the study treatment regimes. Post-ANOVA, selected pair-wise treatment comparisons were performed. All statistical tests of hypotheses were two-sided and employed a level of significance of α = 0.05. No adjustment was made for multiplicity as the primary endpoint and comparisons were pre-defined.

Materials and methods

This was an examiner- and analyst-blind, five-treatment regimen, cross-over, in situ model study [30], performed at the Oral Health Research Institute, Indiana, USA. The study was approved by the Indiana University Institutional Review Board (approval number 0812-55) and carried out according to guidelines set out in the Declaration of Helsinki. This study is registered on ClinicalTrials.gov , study number NCT01563172 . There was one amendment to the study to more explicitly explain brushing instructions to subjects.

Study population

Individuals 18–80 years old were recruited to the study from the Indianapolis area (where community water contains about 1 μg/mL fluoride). Inclusion criteria included wearing a removable mandibular partial denture, being otherwise in good general and dental health and having unstimulated saliva flow ≥0.2 mL/min, stimulated saliva flow ≥0.8 mL/min. Subjects were recruited who granted written informed consent, demonstrated understanding of the protocol and were considered willing, able and likely to comply with all study procedures. Potential subjects were excluded if they reported they were pregnant, were intending to become pregnant during the study period, or were lactating. Subjects were enrolled and allocated to treatment by clinical personnel under supervision of the Principal Investigator (DTZ).

The recruitment sample size target of 65 was based on 45 subjects completing all treatments assuming a drop-out rate of 30%. This sample size gave 90% power at p < 0.05, using two-sided testing, to detect a mean difference in % SMHR of 8.36, based on within-subject standard deviation of 11.96 (determined from previous, unpublished data). Randomisation was performed via a Williams’ square approach, generated by GlaxoSmithKline Consumer Healthcare Biostatistics department.

Experimental design

The study flow is summarized in Fig. 1 . Each subject undertook treatments in a cross-over design in five successive 3-week cycles. At the start of each 3-week cycle, subjects followed their normal oral hygiene regimen, then, following a prophylaxis, they used a fluoride-free ‘washout’ dentifrice twice daily during the final 2–3 days of the first week. The treatment phase started at the beginning of Week 2 and involved subjects using their assigned dentifrice (1150 ppm F or 250 ppm F) twice daily, brushing for the specified brushing duration and using the specified dentifrice quantity. Treatment continued until the end of Week 3, after which subjects started the next cycle. Adverse events (AEs) were recorded at each visit to the clinic.

Jun 19, 2018 | Posted by in General Dentistry | Comments Off on Effect of toothbrushing duration and dentifrice quantity on enamel remineralisation: An in siturandomized clinical trial
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