DOI: https://doi.org/10.21203/rs.3.rs-2041868/v1
Power toothbrushes gain increasing acceptance for oral hygiene procedures. However, the brush head is generally smaller than that of a manual brush thus accommodating less toothpaste. This might influence salivary fluoride concentrations.
In a group of 20 adult volunteers in a 2-legged crossover study. They were instructed to use their habitual quantity of toothpaste (containing 1450ppm as NaF) ad libitum on a manual or power toothbrush. Salivary samples were taken at baseline, after 2 minutes brushing, after 5 minutes and then stepwise up to 60 minutes after brushing. Salivary samples were electrochemically analyzed for fluoride (ISE) for TF (total fluoride, whole sample after acid digestion) and IF (ionizable fluoride after centrifugation but without digestion), Area under the curve (AUC) and curve parameters after regression were calculated and compared using nonparametric statistical methods.
Toothpaste quantity was significantly (p < 0.05) higher with manual toothbrushes (manual ranging from 0.3-2.1g, electric from 0.2-1.2g). Volunteers placing low amounts of toothpaste on a manual brush also did so on the power brush (Spearman-rank correlation coefficient 0.503, p = 0.024). However, this difference in quantity was not reflected in AUC in saliva (p > 0.05). A small but significant difference was found between TF and IF for AUC, reflecting some interaction between saliva compounds and fluoride, independent of toothbrush type.
Interindividual variability is more important in fluoride availability in saliva than differences in toothpaste quantity between power and manual toothbrushing.
Trial registration: Belgian health authorities n° B670201836852
Trial registration: Belgian health authorities n° B670201836852
Toothbrushing with manual or electric toothbrushes has been shown to contribute to the maintenance of oral health by biofilm and debris removal [1]. If supplemented with a fluoride-containing toothpaste, the efficacy of brushing in prevention of dental caries is demonstrated [2].
Toothpastes with fluoride contain several possible compounds such as sodium fluoride (NaF), monofluorophosphate (MFP), amine fluoride or stannous fluoride (SnF2) [3]. NaF shows a high immediate bioavailability of ionized fluoride but is not compatible with some abrasives and tube materials
In 2015 a workshop was held about analytical procedures for fluoride-containing toothpastes [4]. NaF can be readily determined in toothpastes in vitro by direct ion-selective electrode potentiometry. Acid digestion was proposed for MFP-containing toothpastes in order to mobilize fluoride bound to calcium by interaction with abrasives [5]. Since saliva contains calcium, some interaction may be possible during in vivo toothpaste use.
There is some evidence attributing small but significant benefits to power toothbrushes regarding biofilm removal efficacy [1]. The influence of power toothbrushing on fluoride bioavailability was not explored in that paper, merely it was indicated that adjunctly using fluoride toothpaste had a demonstrated anti-caries effect. Studies regarding differences in fluoride bioavailability are lacking, however it may be that the generally smaller bristlehead of a rotating-oscillating power toothbrush reduces applied toothpaste quantities. This could be shown by a preliminary experiment performed prior to the main part of this study (Fig. 1). On the other hand, the mechanical action of higher movement frequency [6] may mobilize the applied toothpaste slurry more readily.
In contrast to former studies using standardized amounts of toothpaste [7] we attempted to investigate habitual toothpaste quantities applied in real-life condition by non-dental volunteers [8]. Our aim was to investigate the effect of brush-type on toothpaste quantity and salivary bioavailability of fluoride as main outcome in a group of volunteers.
The study protocol was approved by the University of Gent ethical committee (n° B670201836852) and 20 healthy human volunteers participated after written informed consent. Inclusion criteria were: being of an age > 18 years, ability to perform all 2 legs of the study, having no orthodontic or removable prosthetic appliances. Exclusion criteria were pregnancy, self-reported suffering from reduced salivary flow and known hypersensibility to the toothpaste ingredients. Participants had to be recruited outside university staff and students according to ethical committee specifications. A sample size of 20 was calculated based on a pilot study involving the applied quantity of toothpaste (Fig. 1), resulting in a power of 0.9 with α = 0.05.
The study protocol consisted of a two-legged crossover design. The volunteers brushed with one commercially available NaF toothpastes (Sensodyne, GSK Europe, Genval, Belgium). Brushing was performed at libitum using a power toothbrush (Oral-B® Sensitive Clean, Oral-B, Procter & Gamble, Marktheidenfeld, Germany) and a manual toothbrush (Tepe soft, TePe Munhygienprodukter AB, Malmö, Sweden) with a 5-day washout between legs. Foodstuffs with a known high fluoride content was to be avoided the day before and the day of the experiment. A fluoride-free toothpaste was given to the volunteers to be used the day before and the morning of the trial.
Volunteers were instructed to donate a salivary sample at baseline, after 2 minutes of toothbrushing, and, following a 10 second rinse with 10 ml of deionized water, after 3, 5, 10, 20 30, 40 and 60 minutes. Saliva was collected by drooling in a disposable vessel until a volume of 2.5 ml was reached. Fluoride concentration of saliva samples was measured as quickly as possible, if analysis on the same day could not be done, samples were kept frozen at -18°C overnight.
Saliva analysis was performed on duplicate samples according to the protocol developed by Cury and al [5] for in-vitro determination of fluoride in toothpaste. 2 aliquots of whole saliva + toothpaste slurry were pipetted first and subjected to acid digestion with 2N HCl at 45°C for 1 hour, then neutralized with 2.5 ml of 2N NaOH. This is the total amount of fluoride potentially available (total fluoride, TF). 2 more aliquots were analyzed after centrifugation without acid digestion as ionizable fluoride (IF) by only adding 0.5 ml of an equinormal solution of NaOH and HCl. All aliquots were buffered by 0.5 ml of TISAB 2 (Fischer Scientific, Chelmsford, USA) and assayed for fluoride using direct potentiometry with an ion-specific electrode (ISE, Mettler-Toledo, Greifensee, CH). The electrode was calibrated before measurements using appropriate standards prepared with a NaF solution. The mV-concentration graph was plotted by semi-logarithmic regression using GraphPad Prism software (GraphPad, La Jolla, USA) and concentration of the unknown solution was obtained by interpolation.
The protocol was validated internally by always analyzing duplicate samples and calculating correlation coefficients.
For each volunteer (mean of duplicates), a fluoride-time curve was established. The maximum concentration (Cmax) was established from the curves and the Area under the Curve without baseline was established by triangulation (Prism V6.0, GraphPad, La Jolla, CA, USA). Logarithmic decay curves were established from the moment brushing was terminated until the end of the experiment on the shifted fluoride measurements based on a power function. A curve fit through a non-linear least squares regression (R version 3.6.1 package nls.multstart 1.0.0) was performed on the fluoride-time data according to the equation: F = e^(a tµ) – 1,
ln(y + 1) = a tb, with y being fluoride concentration (mg/L), t time (min) and a as the overall scaling factor and b the exponent, indicating the rate of the concentration decay. Parameters a and b were compared using non-parametric statistics.
For the statistical analysis, AUC and Cmax values were normalized by logarithmic transformation and compared using parametric one-way ANOVA. Applied toothpaste quantities were compared Wilcoxon tests and correlated between the manual and power toothbrush using Spearman-Rank correlation.
All volunteers completed both legs of the study. No adverse effects were recorded. In three samples (taken after brushing) the fluoride concentration exceeded the measuring range of the standard series. Correlation between duplicate measurements ranged between r2 = 0.949 to 0.998 (Pearson’s correlation, p < 0.001).
The applied quantities ranged from 0.6 gram for MFP/power to 1 gram for NaF/manual (Table 1).
|
Fluoride compound |
Other ingredients |
Abrasive system |
Quantities applied g, median (IQR), n = 20 |
||
|---|---|---|---|---|---|
|
Manual toothbrush Bristle surface: 2.4 cm2 |
Power toothbrush Bristle surface: 1.3 cm2 |
||||
|
NaF (Sensodyne), Batch nr 2088BKWB |
Sodium fluoride 1450mg/kg |
Potassium nitrate |
SiO2 |
1.02 (0.75;1.47) |
0.65 (0.48;0.85) |
In Fig. 2, fluoride-time curves for the different toothbrush type and TF/IF are shown. In all cases, a peak value was observed after toothbrushing and fluoride concentrations dropped towards the end of the experiment.
Fluoride concentrations remained significantly elevated with respect to baseline until 10 minutes after the end of brushing for all experiments (Wilcoxon, p < 0.05).
Cmax values (Table 2) were somewhat higher after manual toothbrushing compared to power, however this effect was not significant (p > 0.05). When comparing the different fluoride analysis modes, it was shown that in the NaF toothpaste, a difference in Cmax was found between TF and IF, although not significantly so.
Area under curve (AUC) values (Table 2) were higher, although not significantly so for manual versus power toothbrushing. However, a significant difference (p < 0.05) was found for AUC between IF and TF per toothbrush type.
|
Manual toothbrush |
|||||
|---|---|---|---|---|---|
|
mode |
Cmax (mgL-1) |
AUC (mgL-1*min) |
Scaling parameter a |
Exponent b |
|
|
TF |
84 (36;141) |
310 (149;407) |
9.64 (7.32;12,09) |
0.99 (0.74;1.38) |
|
|
IF |
70 (32;108) |
119 (35; 232) |
8.74 (7.52;10.98) |
1.14 (0.83;1.26) |
|
|
Power toothbrush |
|||||
|
mode |
Cmax (mgL-1) |
AUC (mgL-1*min) |
Scaling parameter a |
Exponent b |
|
|
TF |
61 (30;155) |
196 (108;443) |
8.44 (7.53;11.79) |
1.07 (0.93;1.27) |
|
|
IF |
65 (29;125) |
104 (50;209) |
8.50 (7.61;11.37 |
1.03 (0.93;1.28) |
|
This study tested the hypothesis that the size of the toothbrush head influences applied toothpaste quantity and possibly bioavailability. It was also intended to test the different forms of fluoride as determined by the acid digestion method in an in-vivo test with human saliva. From these hypotheses one could be verified, the larger brush head of the manual toothbrush yielded consistent and significant higher quantities of toothpaste applied. However, a dose-response relationship for salivary fluoride could not be established, the interindividual variations and sample size of 20 persons reduced the power of the study so that differences in results were not significant when subgroups were concerned. However, the study more closely approaches realistic circumstances of brushing habits. Ad libitum toothpaste use was described by [8]. They could not show an increase of plaque fluoride, admittedly a very indirect measure of fluoride in saliva with higher quantities. A higher brushing frequency outweighed the effect of quantity of toothpaste, furthermore omitting outliers lead to a higher standardization of the results. In our study, also large interindividual differences were found. In contrast to the findings of [8], quantities of toothpaste employed in our study were about half. Nevertheless, the results here may lead to possible further research hypotheses which may be confirmed using a higher standardization of independent variables [7]. If quantities were standardized among participants a certain dose-response effect with different toothpaste quantities and fluoride concentrations could be demonstrated [9]. Using a larger panel of volunteers and standardized quantities of toothpaste with a 1:3 quantity difference there was a dose-response relationship between quantity and AUC [10]. Later, these authors found also this dose-response relationship in enamel fluoride uptake and protection against demineralization [11]. As in many studies involving fluoride in saliva, their data showed high variability, as in the present study.
The acid digestion protocol is generally applied to MFP toothpastes due to their low ionizability [12]. However, applying it to NaF toothpastes showed that there may be an interaction between fluoride and salivary calcium [13]. From AUC differences between uncentrifuged and centrifuged samples (Table 2) it may be concluded that especially in the time shortly after toothpaste application some fluoride remains bound in the formulation (binders, abrasive) or bound to salivary constituents before being released into saliva [14]. This phenomenon, generally reported for MFP toothpastes is also, be it to a lesser extent, true for the more readily soluble NaF. It can therefore be said that the technique may be of value for the determination of clinical efficacy of toothpastes as discussed in the workshop in 2015 [4]. The fact that these differences AUC values between TF and IF were smaller in power than manual toothbrushing may indicate that mechanical mobilization by toothbrush movement [6] could have dispersed toothpaste ingredients more effectively. Dispersion of toothpaste is also influenced by their rheology (flow properties) although not much attention has been given to the subject in the literature.
In this study it could be shown that using a power toothbrush with a smaller (Table 1) rotating head decreased the amount of applied fluoride significantly compared to a manual toothbrush. This may be advantageous for young children in whom a reduction of administered amounts of fluoride is desirable [15]. If the calculation of fluoride uptake by toothbrushing [16] is applied on our study results, using a power toothbrush would result in a swallowed amount of 0.05mg of fluoride versus 0.08mg with a manual toothbrush. It must be noted that this study was done in adults who generally apply larger quantities than they might apply to their children. Power toothbrushes can safely be used by children with a high rate of acceptance [17].
In contrast to studies using standardized quantities of toothpaste, a clear dose-response relationship could not be established. This may also be explained by the variable amount of paste being lost with rinsing and retained on the brush head (30–40%) [18, 19]. Furthermore, large differences existed between the volunteers due to brushing technique [20], salivary secretion, swallowing and potentially the quantity of biofilm in the mouth. These variables were however not measured and may be of interest for further research. Fluoride bioavailability seems to be a complex matter depending on applied quantity, brushing and rinsing habits, the dynamics of the brush movements and compositional aspects of the toothpastes such as rheology.
To the best of our knowledge, a comparison between manual and power toothbrushing on salivary fluoride has not yet been performed. Power toothbrushes with a smaller head generally accommodate smaller toothpaste quantities. However, due to interindividual variability there was no straight-forward effect on bioavailability as expressed by several pharmacodynamic parameters tested in the present study.
Ethics: the project was submitted to the UZ Ghent ethics committee and received approval (registration n° B670201836852) . Consent to participation was obtained in written form from all participants prior to the start of the study. All clinical and laboratory procedures were performed according to the applicable legislation and guidelines.
Consent for publication: not applicable
Availability: the dataset is available from the authors on reasonable request from the corresponding author (PB) via [email protected]
Competing interests: the authors have no competing iterests to declare
Funding: the project received no specific funding. Materials were acquired by department’s general funds
Author’s contributions: PB formulated the research question and drafted the final manuscript, HV and ADB performed the clinical and laboratory work, WJ performed data analysis and participated in writing the manuscript. All authors read and approved the final manuscript.
Acknowledgements: the authors are grateful to C. Vercruysse for support during laboratory work.