Fluoride Retention in Interdental Plaque and Saliva using Two NaF
Dentifrices 5000 and 1450 ppm F with and without Water Rinsing
A Nordström, D Birkhed
Department of Cariology, Institute of Odontology, Sahlgrenska Academy, University of
Gothenburg, Sweden
___________________________________________________________________________
Key words: Dental plaque, Fluoride dentifrice, Interdental sites, Tooth brushing, Water
rinsing
Short title: Two NaF Dentifrices 5000 and 1450 ppm F
Address: Dowen Birkhed, Department of Cariology, Institute of Odontology, Box 450,
SE-405 30 Göteborg, Sweden. E-Mail: birkhed@odontologi.gu.se
1
Abstract
A total of 26 healthy volunteers participated in this randomised 4-leg cross-over designed
study. The aim was to measure the fluoride (F) retention in interdental plaque and saliva. Two
NaF dentifrices (5000 and 1450 ppm F) were used, with and without post-brushing water
rinsing. The four tooth brushing procedures were carried out twice a day during two weeks.
Interdental plaque was collected from all proximal sites, after each test period, using a dental
floss. Immediately after the plaque sampling, the subjects were asked to brush their teeth with
the same toothpaste and post-brushing water rinsing procedure, as previously. Proximal saliva
was collected from four interdental sites, using small paper points, before and up to 60 min
after the brushing. The present study showed that the 5000 ppm F toothpaste without postbrushing water rinsing resulted in the highest F concentration in both plaque and saliva and
the 1450 ppm F toothpaste with water rinsing in the lowest (p < 0.05). The increase of F in
plaque between 1450 ppm with post-brushing water rinsing and 5000 without water rinsing
was 82%; the corresponding increase in saliva (AUC) was 112% (p < 0.05). Water rinsing
immediately after tooth brushing with 5000 ppm is reducing the F concentration in saliva with
32% (p < 0.05). We believe that 5000 ppm F toothpaste, especially without post-brushing
water rinsing, should be considered as an interesting product for daily use in high caries risk
patients.
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Introduction
The prevalence of caries has declined in many industrialised countries since the late 1960s.
This is mainly a result of the daily use of fluoride (F) dentifrices [Glass, 1982; Bratthall et al.,
1996; Marinho et al., 2003]. The Swedish Council on Technology Assessment in Health Care
(SBU, 2002) reported strong scientific evidence that daily use of F toothpaste is an effective
method for preventing dental caries in permanent teeth. Dentifrices with 1500 ppm F showed
a slightly better preventive effect (≈10%) compared with those with 1000 ppm F [Twetman et
al., 2003]. Several studies indicate that there is a linear relationship between F concentration
in toothpastes (0 to 1500 ppm) and caries reduction [Birkeland, 1972; White and Nancollas,
1990]. Various factors influence the efficacy of F toothpaste including the concentration of F
in the paste, the amount of toothpaste applied on the brush, the frequency of tooth brushing
and the post-brushing water rinsing [Davies et al., 2003].
In Sweden, the F concentration in dentifrices sold in ordinary stores varies between 400
and 1500 ppm. Toothpaste with a higher F concentration can only be obtained at a pharmacy
as an over-the-counter product. 5000 ppm F toothpaste has recently been introduced on the
Swedish market for high caries risk patients. There are few studies evaluating the F
concentration in saliva and plaque after using a dentifrice with 5000 ppm F [Tavss et al.,
2003]. In two clinical studies, a 5000 ppm fluoride gel was used daily in a similar way as
toothpaste with positive results [Englander et al., 1967, Dreizen et al., 1977]. The reversal of
root caries after using a dentifrice containing 5000 ppm F has been studied by Baysan et al.
[2001].
The intra-oral levels of F may differ in subjects with different post-brushing rinsing habits
[Collins et al., 1991; Duckworth et al., 1991; Richards et al., 1992] and the rinsing behaviour
may be related to the caries experience of the patient [Sjögren and Birkhed, 1993]. However,
Machiulskiene et al. [2002] showed limited support refraining from water rinsing after tooth
3
brushing. The difference between the mean caries increments in children with and without
water rinsing was only 0.6 DS and had no statistically significance.
The aim of this study was to investigate the F retention in interdental plaque and saliva
using two NaF dentifrices (5000 and 1450 ppm F), with and without post-brushing water
rinsing, in order to evaluate the potential benefit of a high F dentifrice without water rinsing.
Materials and Methods
Subjects
A total of 26 healthy volunteers participated in this randomised 4-leg cross-over designed
study. 16 students at the Dental Hygienist School in Göteborg aged 19-35 years (mean 24
years) and 10 subjects from the staff at the Public Dental Service Clinic in the city of Varberg
aged 34-55 years (mean 47 years) Sweden were recruited. The subjects were carefully
selected in order to be capable of following the instructions given. They were at general good
health and had at least 24 teeth. The subjects were instructed not to use any other F products
apart from the test toothpaste, not to eat or drink within two hours after last tooth brushing,
not to use any proximal cleaning and not to use chewing gum or snuff, during test periods.
The study has been approved by the Ethics Committee of Sahlgrenska Academy at Göteborg
University (Dnr 188-05).
Study Design
One week prior to each test period, the subjects used F-free toothpaste (Aloe Vera
Tandkräm, Forever Living Products, Göteborg, Sweden) twice daily and a waxed, F-free
dental floss (Johnson & Johnson Products, New Brunswick, N.J., USA, or Sunstar Butler
Laholm, Sweden) once daily. Just before starting the experimental period, the teeth were
cleaned professionally with a hand-piece and a rubber cup, using RDA 170 polish paste (CCS
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AB, Borlänge, Sweden) and flossed. The subjects were then carefully instructed to use one
gram of toothpaste on their toothbrushes and to brush for two minutes, twice a day [SganCohen, 2004]. Each test period lasted for two weeks, followed by a wash-out period of one
week, using F-free toothpaste. There were some minor differences in procedures between the
subjects. The 10 volunteers from Varberg were given stricter instructions regarding the
amount of toothpaste applied on their toothbrushes and the amount of water for the rinsing
procedure. This was considered as an improved standardization.
Two dentifrices were used, Duraphat 5000 ppm F as NaF (Colgate-Palmolive AB,
Danderyd, Sweden) and Pepsodent Superfluor 1450 ppm F as NaF (Lever Fabergé,
Stockholm, Sweden). The subjects participated in four test periods. The toothpaste and the
rinsing procedure were carried out using a randomised cross-over table. The study was not
blinded. 1) Duraphat toothpaste without post-brushing water rinsing. 2) Duraphat toothpaste
followed by 10 ml water rinsing three times, each time for 10 s. 3) Pepsodent toothpaste
without post-brushing water rinsing. 4) Pepsodent toothpaste followed by 10 ml water rinsing
as in No 2. The water rinsing was carried out with a 10-ml plastic cup. This rinsing procedure
has also been used by Issa and Toumba, [2004]. During test period No 1 and 3, the subjects
simply spat out the dentifrice-saliva slurry after brushing and no post-brushing water rinsing
was allowed. The outcome measure was accumulation of F in the proximal plaque (expressed
as ppm F or F per mg plaque) and retention of F in proximal saliva, based on the area under
the curve (AUC).
Fluoride Accumulation in Interdental Plaque
Interdental plaque was collected 2 h after the last brushing occasion (in order to measure
the F accumulated in plaque during the two weeks), using an F-free, extra-wide, waxed dental
floss (Johnson & Johnson or Sunstar Butler), according to Sjögren and Birkhed [1993].
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Plaque was collected from all proximal sites, except in the lower anterior region. The
collected plaque was transferred from the floss to a 2-ml plastic tube (Eppendorf PRC,
Sarstedt, Nümbrecht, Germany). This was done by drawing the floss through a slit, which had
been cut in the lid of the tube. Plaque adhering to the floss was thereby left on the inside of
the tube. The samples were frozen at -20˚C until analysed. In 16 of the 26 subjects, the
Eppendorf tubes were weighted before and after the plaque sampling and the plaque wet
weight determined to the nearest 0.1 mg. No plaque weight data was available on the
remaining 10 subjects. Thus, the accumulated F in the extracted plaque suspension was
measured and expressed as ppm F in these 10 subjects and as both ppm F and F per mg plaque
in the remaining 16.
Fluoride Retention in Interdental Saliva
This experiment was carried out after each 2-week test period and was directly following
the collection of plaque. Interdental saliva was sampled from the proximal sites 16/15, 25/26,
35/36, 45/46, using a standardised, triangular-shaped paper point. The paper point was
punched from filter paper (Laboratorieläsk, Munktell Filter, Grycksbo, Sweden) to a size of
5x2 mm, using a special punch instrument. Each paper point absorbs approximately 4 μl
(±10%) of fluid, when kept in place for 20 s, using a pair of forceps. Immediately after
removal from the interdental area, the paper point was transferred to a 0.5-ml Eppendorf tube
(Sjögren and Birkhed, 1994; Särner et al., 2003). The samples were frozen at -20˚C until
analysed. After collection of the baseline samples, the subjects brushed their teeth with the
same toothpaste and the same post-brushing water rinsing procedure, as during the 2-week
test period. Sampling was then repeated after 1, 3, 5, 10, 30 and 60 min.
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Fluoride Analysis
A volume of 200 μl of liquid, consisting of distilled water and TISAB III (10:1) (Thermo
Electron Corp., Waltham, USA) was added to the plaque and saliva samples. The plaque
suspension was homogenised by sonification for 20 s (Branson W185D, Dansbury,
Connecticut, USA) in order to disperse the plaque. The plaque and saliva samples were kept
in a refrigerator at +4˚C over night. The tubes were then vibrated in a Minishaker MS1 (IKA,
Wilmington, USA) for 20 s. 100 μl of the solution was placed as a drop on a Petri dish. The F
concentration was measured by an ion-specific electrode (model 96-09, Orion Research Inc.)
by carefully lowering the electrode into the fluid. The surface tension of the drop ensured that
the liquid enclosed the entire membrane surface of the electrode. In order to calibrate the
electrode, several standard solutions were used. The F concentration was expressed as ppm.
Statistical Methods
Two-way ANOVA followed by Scheffe´s test were used to compare the four test periods,
regarding accumulation of F in the proximal plaque (expressed as ppm F or F per mg plaque)
and the retention of F in proximal saliva, based on the area under the curve (AUC). AUC was
calculated by a computer program (KaleidaGraph 3.01, Synergy Software, Reading, PA,
USA). P-values < 0.05 were considered statistically significant.
Results
Fluoride Accumulation in Interdental Plaque
The mean values (± SD) of F concentration in the extracted plaque suspension, expressed
as ppm F and F per mg plaque wet weight are presented in table 1. The highest accumulation
of F in proximal plaque was found using toothpaste with 5000 ppm F, without post-brushing
water rinsing (No 1) and the lowest using 1450 ppm F with 3x10 ml water rinsing (No 4).
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This trend was the same even if the F accumulation was expressed as ppm F in the suspension
or F per mg plaque wet weight. The increase of F in plaque between 1450 ppm with rinsing
and 5000 without rinsing was 82% (2.0-1.1/1.1x100; p < 0.05; n=26). No other statistical
significant differences were found.
Fluoride Retention in Interdental Saliva
The mean values of the F concentration at the various time points and the AUC values are
shown in Fig. 1. The difference between the four methods was most pronounced within the
first 30 min. The increase of F in saliva (AUC) between 1450 ppm with rinsing and 5000
without rinsing was 112% (40.0-18.9/18.9x100). The toothpaste with 5000 ppm F without
post-brushing water rinsing gave the highest F concentration in proximal saliva and differed
significantly from the other three procedures (p < 0.001). The toothpaste with 1450 ppm
followed by 3x10 ml water rinsing resulted in the lowest F concentrations during the entire
60-min period. Comparing 5000 ppm F toothpaste without post-brushing water rinsing (No 1)
with 1450 ppm F also without water rinsing (No 3), a mean increase (AUC) of 55% was
found (40.0-25.7/25.7x100). The corresponding increase, rinsing with 3x10 ml water was
44% (27.2-18.9/18.9x100); also this difference was statistically significant (p < 0.001).
Discussion
The main result, considering the retention of F in saliva was that the content of F in the
dentifrice had a great influence. The 5000 ppm F toothpaste compared to 1450 ppm F
increased the F level in the interdental area with more than 50%, independent of water rinsing
method. Regarding the accumulated F in plaque, the increase between the 1450 and the 5000
ppm F toothpaste was up to 0.8 times. This value should be compared with the level of F in
the two toothpastes (5000 vs. 1450 ppm = 3.5 times). An explanation could be that F does not
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accumulate in plaque over time. Heijnsbroek et al. [2006] found no F accumulation in plaque
after 6 hr using an AmF/SnF2 dentifrice. These differences, both with respect to F retention
immediately after brushing and the accumulation of F in interdental plaque over time, could
be of clinical importance for the prevention of proximal caries.
An interesting observation in this study was that the post-brushing water rinsing had a
great influence on the retention of F in the proximal area. Previous studies have produced
similar results. Duckworth et al. [1991] and Sjögren and Birkhed [1993] observed an increase
in F concentrations in saliva when the frequency and the amount of rinsing water were
reduced. Sjögren and Melin [2001] found a tendency towards an increased concentration of F
in saliva and plaque when the amount of post-brushing water was reduced.
The method of collecting proximal saliva with paper points has been used in several
studies [Sjögren and Birkhed, 1994; Kashani et al., 1998; Särner et al., 2003]. In our
experience, this sampling method was found reproducible. The oral motor activity influences
the production of saliva and thereby the clearance. In order to eliminate this factor, it was
considered important to avoid frequent talking during the collection of the samples. The
method for sampling and analysing F in plaque was originally described by Wilson and
Ashley [1988]. Using TISAB III and distilled water for extracting the F as shown by
Duckworth et al [1994] seems to release as much F from young plaque (24 h old) as
perchlorid acid. This method was also used by Sjögren et al., [1996] in a similar experiment.
Some toxicological concern has been reported using high-F toothpaste (5000 ppm)
[Ekstrand, 2006]. Approximately 5-10% of the dentifrice is swallowed during tooth brushing
with a toothpaste technique using a minimum amount of water [Sjögren et al., 1994]. The
amount of swallowed fluoride is thereby 0.25-0.50 mg F (thus 0.05-0.10x5=0.25-0.5 mg F),
when using 1 g of a toothpaste containing 5 mg F/g toothpaste. This quantity corresponds to
the amount of F in 1-2 fluoride tablets containing 0.25 mg F and is considered to have no
9
toxic effect in teenagers and adults. Children under 16 years should not use toothpaste with
5000 ppm F according to instructions given by the manufacturer. Perhaps the age of 12 years
would be a more appropriate age of limit, to be able to influence the newly erupted premolars
and second molars.
Adults and teenagers with a high caries risk are a suitable target group for using a
dentifrice with 5000 ppm F. Adolescents in particular run a certain caries risk when their teeth
have just erupted. A high-F dentifrice has also been recommended for optimal cariesprevention strategies during orthodontic treatment [Derks et al., 2004]. Elderly people with
hypo salivation due to medication or radiation are another interesting group to keep in mind.
The reversal of root caries has already been documented by Baysan et al. [2001], using a
toothpaste with 5000 ppm F.
To conclude, 5000 ppm F toothpaste, without post-brushing water rinsing resulted in the
highest F concentration in both plaque and saliva and 1450 ppm F toothpaste with 3x10 ml
post brushing water rinsing in the lowest. 5000 ppm F toothpaste, especially without rinsing
seems thereby to be a potentially important vehicle for caries prevention and treatment.
Further long-term clinical studies are needed to evaluate the effect of 5000 ppm F toothpaste
on dental caries. A long-term clinical study on caries active teenagers comparing 5000 and
1450 ppm F dentifrices is in progress in our department.
Acknowledgements
We gratefully acknowledge the technical and statistical assistance of Ann-Britt Lundberg
and Tommy Johnsson. This study was supported by grants from the Region of Västra
Götaland, the Swedish Patent Revenue Research Fund and the Institute of Odontology,
University of Gothenburg.
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Fig. 1. Mean values (n = 26) of F concentration in proximal saliva (expressed as log ppm) at
various time points up to 60 min after using two NaF dentifrices with 1450 and 5000 ppm F,
with and without post-brushing water rinsing. Inserted is also the AUC values (0-60 min),
expressed as mean ± SD. The bars indicate the statistical differences (p < 0.001)
14
Table 1. Mean values and standard deviations of F accumulation in approximal plaque,
expressed as ppm F in the extracted plaque suspension (n = 26) or as F per mg plaque wet
weight (n = 16). The samples were collected after two weeks´s experimental period (1-4) and
2 h after last brushing occasion.
ppm F (n = 26)
ppm F (n = 16)
F/mg plaque (n =16)
5000 No rinsing 2.0 ± 2.1
2.7 ± 2.4
0.11 ± 0.16
5000 Rinsing
1.6 ± 1.5
1.9 ± 1.5
0.06 ± 0.05
1450 No rinsing 1.4 ± 1.3
1.7 ± 1.5
0.07 ± 0.12
1450 Rinsing
1.3 ± 0.9
0.04 ± 0.02
1.1 ±0.9
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