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

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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 post-

brushing 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

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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 [Sgan-

Cohen, 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

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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 caries-

prevention 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)

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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 1.1 ±0.9 1.3 ± 0.9 0.04 ± 0.02

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