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Effect of Rinsing with Toothpaste Slurries and Water Solutions with a
High Fluoride Concentration (5000 ppm) on de novo Plaque
Formation
Anna Nordström1, Chrysostomos Mystikos2, Per Ramberg2, Dowen Birkhed1
Departments of Cariology1 and Periodontology2, Institute of Odontology, Sahlgrenska
Academy, University of Gothenburg, Sweden
______________________________________________________________
Short title: Toothpaste with 5000 ppm F and Plaque Formation
Address: Dowen Birkhed, Department of Cariology, Institute of Odontology, Box 450,
SE-405 30 Göteborg, Sweden. E-mail: [email protected]
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Nordström A, Mystikos C, Ramberg P, Birkhed D. Effect of Rinsing with Toothpaste Slurries
and Water Solutions with a High Fluoride Concentration (5000 ppm) on de novo Plaque
Formation. Eur J Oral Sci
The aim of this study was to evaluate the effect of rinsing with toothpaste slurries and water
solutions with a high fluoride concentration on de novo plaque formation. 16 subjects rinsed 3
times/day for 4 d with the following dentifrice slurries: 5000, 1500 and 500 ppm F, while 12
subjects rinsed with water solutions: 5000, 1500, 500, 0 ppm F and 1.5% SLS (sodium lauryl
sulphate). Plaque was scored (QHI index) after each 4-d period. Plaque samples for F analysis
were collected. Significantly (P < 0.5) less plaque was scored for the dentifrice slurry with
5000 ppm F (buccal and all surfaces) and for 1.5% SLS (buccal surfaces). The differences in
plaque scores between dentifrice with 5000 and 1500 ppm F were 19% for all surfaces and
33% for buccal surfaces. The difference between the water solutions with 1.5% SLS and 1500
ppm F for buccal surfaces was 23%; the corresponding difference for 5000 ppm F was 17%
(P < 0.05). The dentifrice slurry with 5000 ppm F accumulated 56% more F in plaque (P <
0.05). The combination of high levels of F and SLS in dentifrice reduces de novo plaque
formation and increases the accumulation of F in plaque after 4 d.
Key words: Dental plaque, Dentifrice slurry, Fluoride, Plaque formation
Dowen Birkhed, Department of Cariology, Institute of Odontology, Box 450, SE-405 30
Göteborg , Sweden. Telefax: +45 31 82 57 33. E-mail: [email protected]
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The plaque-inhibiting effect of several antimicrobial substances, such as triclosan, essential
oils and chlorhexidine, has been reported (1-3). Fluoride (F), especially in toothpaste, is
widely used as a highly effective anti-caries agent. The daily supply of a low concentration of
F in saliva and plaque favours the remineralisation of enamel surface lesions (4). Fluoride in
the oral cavity may interact with the bacterial metabolism and its acid production and further
slow down the caries process (5). Moreover, F reduces the acid tolerance of the bacteria and
thereby decreases the cariogenicity of dental plaque (6). BAYSAN et al. (7) found significantly
lower plaque scores after 3 and 6 months in a group using a dentifrice with 5000 ppm F,
compared with a dentifrice with 1100 ppm F. According to LYNCH et al. (8), the level of F in
a 1500 ppm toothpaste is insufficient to interfere with bacterial growth and metabolism.
However, the anti-plaque effect of dentifrice slurries with a higher F concentration has so far
not been thoroughly investigated. The aim of the present investigation was therefore to study
the effect of daily rinsing with dentifrice slurries and water solutions with a high
concentration of F (5000 ppm) on de novo plaque formation during a 4-d period of no
mechanical cleaning. The accumulation of F in plaque was also studied.
Material and Methods
Two test series, here called I and II, were carried out with a randomised, cross-over design.
Series I was single blind with 3 cells and Series II was double blind with 5 cells. Sixteen
healthy dental students, aged 21-43 (mean age 27 yr), were recruited to Series I. Twelve of
these 16 subjects also participated in Series II (mean age 24 yr). Each volunteer had to fulfil
the following inclusion criteria: (i) good general health, (ii) no sign of destructive periodontal
disease, (iii) a minimum of 24 teeth with no extensive restorations and no teeth with exposed
root surfaces of > 1 mm, (iv) no antibiotic treatment during 3 months prior to the start of the
trial, (v) no regular medication with anti-inflammatory compounds, (vi) no use of tobacco
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products and (vii) no use of antiseptics. All subjects received a verbal and written description
of the study design and signed informed consent forms. This design, studying 4-d de novo
plaque formation, has previously been approved by the ethical review board at the University
of Gothenburg.
All 16 subjects were considered to be “heavy plaque formers” and were selected from 30
subjects during a 4-wk screening period, preceding the start of the trial. The definition of
heavy plaque formers was based on the criteria specified by MAGNUSSON et al. (9), who
evaluated the rate of plaque formation after 16-20 h of no oral hygiene. During a 2-wk
preparatory period, each subject received a series of thorough dental prophylaxis, including
scaling and professional mechanical tooth cleaning, (PMTC) (10), in order to establish
healthy gingival conditions. The teeth were professionally cleaned with a hand-piece and a
rubber cup, using RDA 170 polish paste (CCS AB, Borlänge, Sweden) and flossed. In
addition, the subjects were given instruction in proper oral hygiene measures and were told to
use an F-free dentifrice, prior to and between each experimental period. The PMTC was
repeated during a 2-wk preparatory period when indicated.
Series I (toothpaste slurries)
On a given day (Day 0), dental plaque was disclosed with erythrosine (Diaplac®, LIC AB,
Enköping, Sweden) and all the participants received PMTC. They were asked to refrain from
tooth brushing and proximal cleaning but to rinse with 1 ml of the assigned toothpaste slurry,
3 times/day, for 2 min and for 4 d (in the morning after breakfast, after lunch and in the
evening, just before bedtime). 1 ml of the dentifrice slurry was corresponding to the amount
of 1 g toothpaste used for ordinary tooth brushing. The rinsing procedure was carried out with
active movements of the lips and cheeks in order to enforce penetration of the slurry into
proximal sites. The subjects were instructed not to rinse with water after expectorating the
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slurry and not to eat or drink for 2 h. The following three Duraphat (Colgate-Palmolive AB,
Danderyd, Sweden) dentifrice slurries were used in random order: (1) dentifrice, 5000 ppm F,
(2) dentifrice plus water (dilution 1:2), 1500 ppm F and (3) dentifrice plus water (dilution
1:9), 500 ppm F. Each subject was provided with the slurries in 12 disposable syringes of 1
ml and was instructed to mix them for 10 s before application in the mouth. Plaque sampling
and plaque scoring were performed at the end of each experimental 4-d period. The test
periods were separated by a 3-d wash-out period using F-free toothpaste.
Series II (water solutions)
Series II was carried out 6 months later and comprised 12 of the 16 subjects who participated
in Series I. The dentifrice slurries were changed to water solutions with F, in order to show
the effect of sodium lauryl sulphate (SLS) in the dentifrice on plaque formation. The
experiment was similar to Series I, except that no plaque sampling was carried out. The
following five water solutions were used in random order: 1) 5000 ppm F, 2) 1500 ppm F, 3)
500 ppm F, 4) 0 ppm F and 5) 1.5% SLS and 0 ppm F.
Plaque registration (Series I & II)
Plaque was registered at the end of the 4-d period in two quadrants, by one of the authors
(C.M.). After disclosing with erythrosine (Diaplac®, LIC AB, Enköping, Sweden), plaque
was scored at six sites (mesiobuccal, buccal, distobuccal, mesiolingual, lingual and
distolingual) on all the teeth, according to the criteria specified in the Turesky modification of
the Quigley & Hein Index (QHI) (11, 12). Plaque sampling in Series I was performed prior to
the disclosure procedure in order to avoid any possible interaction with and influence of
erythrosine.
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Plaque sampling (Series I)
Plaque was collected 2 h after the last rinsing procedure, from all tooth surfaces in the 1st and
3rd quadrants in 8 subjects and in the 2nd and 4th quadrants in the remaining 8 subjects.
Proximal plaque was collected from all proximal sites, except the lower anterior region, using
extra–wide, waxed fluoride-free dental floss. Plaque on smooth surfaces (buccal and lingual)
was collected with an amalgam carver. The plaque material was transferred to a pre-weighed
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 and weighed. The total amount of plaque,
collected from proximal and smooth surfaces, was weighed and expressed as mg wet weight
and determined to the nearest 0.1 mg. The samples were frozen at –20˚C pending further
analysis. The samples were processed and analysed with respect to F concentration according
to FURUICHI & BIRKHED (2). A volume of 200 µl of liquid, consisting of TISAB III (Thermo
Electron Corp., Waltham, USA) and distilled water (1:10), was added to the plaque samples.
The plaque suspension was homogenised by sonification for 20 s (Branson W185D,
Dansbury, Connecticut, USA) and kept in a refrigerator (+4˚C) over night, in order to disperse
the plaque. The tubes were 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 and the F concentration was
measured 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 ion-specific electrode (model 96-09, Orion Research), three standard solutions
(0.1, 1.0, and 10 ppm F) were analysed. The F concentration was expressed as both ppm F
and ng F/mg plaque wet weight.
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Statistical analysis
The mean QHI scores for all surfaces (Series I & II) and the mean F concentration in plaque
(Series I) were the outcome variables. Power analyses were based on a standard deviation of
0.8 QHI score units. There was a 80% power to detect a 20% difference in mean plaque
reduction including 12 subjects. Analysis of variance (two-way ANOVA), followed by
Student-Neumann-Keul’s (SNK) test, were applied to evaluate if there were significant
differences between the 4-day periods. P < 0.05 were considered statistically significant.
Results
Plaque index (Series I and II)
The mean QHI scores for buccal and all surfaces in Series I and II are presented in Fig. 1. In
Series I, the difference in mean QHI scores between the dentifrice slurry with 5000 ppm F
and the 1500, 500 ppm F slurries was statistically significant for buccal and all surfaces (P <
0.05). The differences in plaque scores between the dentifrice slurry with 5000 and 1500 ppm
F were 19% for all surfaces and 33% for buccal surfaces. In Series II, the difference in mean
QHI scores between the solution with 1.5% SLS, and the 5000, 1500, 500 0 ppm F solutions
was statistically significant for buccal surfaces (P < 0.05). The difference between the 1.5%
SLS solution and 1500 ppm F was 23% and the corresponding difference for 5000 ppm F was
17%.
Fluoride accumulation in plaque (Series I)
The amount of plaque and the accumulation of F in plaque for the three dentifrice slurries are
presented in Table 1. There was no significant difference regarding the amount of plaque (wet
weight). The difference in the accumulation of F in plaque (expressed as ppm F) between
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dentifrice slurries with 5000 ppm F and 1500 ppm F was 56%, which was statistically
significant (P < 0.05). The corresponding difference for 500 ppm F was 63%, which was also
statistically significant (P < 0.01).
Discussion
In the present 4-d experimental study, significantly less de novo plaque was scored for the
dentifrice slurry with 5000 ppm F and for the water solution with 1.5% SLS. This is in
agreement with BAYSAN et al. (7), who observed that the plaque index after 3 and 6 months
was significantly lower in a 5000 ppm F compared with an 1100 ppm F group. However, the
study was originally designed to compare the ability of two sodium fluoride dentifrices to
reverse primary root caries lesions and the reduction of plaque scores was not the primary
outcome. No other studies have investigated the anti-plaque effect of dentifrice slurries with a
high F concentration (5000 ppm F), hence a number of studies have described the anti-caries
effect of F.
Bacteria grow preferentially attached to surfaces embedded in an exopolysaccharide matrix
to form biofilms. In this mode of growth, bacteria often show reduced susceptibility to
antimicrobials and a higher concentration of such detergents is needed. The anti-caries actions
of F appear to be complex, involving both on bacteria and on mineral phases. Fluoride acts to
reduce the acid tolerance of the bacteria and thereby decreases the cariogenicity of dental
plaque and the effect is most persistent at low pH values (5). Fluoride in the oral cavity may
also interact with the bacterial metabolism and its acid production and further slow down the
caries process (6). Both F and SLS in dentifrices are usually regarded as weak plaque
inhibitors and LYNCH et al. (8) concluded that the level of F in 1500 ppm toothpaste is
insufficient to interfere with bacterial growth and metabolism. In addition, VAN LOVEREN et
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al. (13) observed that F dentifrice does not affect plaque composition or F tolerance or the
acidogenicity of mutans streptococci.
The present study also showed a reduction of plaque scores for water solution with 1.5%
SLS. The anionic detergent SLS is a common compound in dentifrices. It is included mainly
because of its foaming activity, but effects on dental plaque have also been reported. The
antimicrobial action of SLS is related to its adsorption and penetration through the porous cell
wall following by interaction with components of the cell membrane, lipids and proteins (14,
15). The penetration of SLS into the membrane causes an increase in cell permeability, which
may result in leakage of intracellular components and cell lysis (14). This result is in
agreement with data presented by WAALER et al. (16), who concluded that a mouthwash
containing 1.5% SLS inhibit plaque formation after 4 d. FERNANDA et al. (17) has reported
that SLS alone and in combination with NaF reduce the bacterial viability, lactate formation
and extracellular polysaccharide formation both in vitro and in vivo. It was also concluded
that SLS and NaF in combination had additive effects (17). The viability of bacteria in newly
formed plaque may therefore be of some interest. Dental plaque bacteria forms complex and
robust cell aggregates which cannot be counted accurately using epifluorescence microscopy
and this causes a problem for quantifying their viability. FILOCHE et al. (18) developed a
fluorescence assay to quantify the viable biomass of dental plaque biofilms. The conflicting
result from this study showed that F at 1000, 3000 and 5000 ppm promote plaque viability.
Further studies evaluating the viability of bacteria in newly formed plaque after using a
dentifrice with 5000 ppm F are therefore needed.
We noticed that the reduction of plaque scores in the present study was more extended for
buccal surfaces. An explanation could be that the dentifrice slurry and water solutions more
easily had access to and penetrated this surface by the active movements of the lips and
cheeks.
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The subjects in the present study were all dental students and had relatively low levels of
cariogenic bacteria, even though they were regarded as “heavy plaque formers”. Thirteen of
16 volunteers had none or less than 100,000 mutans streptococci/ml. Only 3 subjects had
more than 1,000,000 CFU/ml. The lactobacillus scores were extremely low; 15 subjects of 16
had less than 10,000 CFU/ml. According to SEKINO et al. (1), the number of salivary
bacteria may influence the amount of plaque formed during the early phase of no oral
hygiene. In the present study, the mean QHI plaque scores after 4 d (around 2.5-3; Fig.1) are
in agreement with findings in other studies using the 4-d de novo plaque formation model
(19).
An experimental period of 4 d may at first be regarded as short for detecting major
differences in de novo plaque formation for weak plaque inhibitors. However, according to
FURUICHI & BIRKHED (20) and RAMBERG et al. (19), no major difference was observed in
plaque formation after 4 or 14 d of no mechanical cleaning. The plaque-inhibiting effect
observed by BYASAN et al. (7) for a dentifrice with 5000 ppm F was actually detected after a
period of 3 and 6 months. It has been suggested that intra-oral fluoride reservoirs may take
weeks to “fill”, possible due to the time taken for establishment of steady-state F
concentration in plaque and saliva (21). On the other hand, CLAYDON & ADDY (22) concluded
that even a 24-h plaque re-growth study design, using conventional measurements of plaque
accumulation, could be useful as a rapid method of screening potential inhibitory agents and
formulations. From an ethical point of view, it is preferable to use a short experimental
period, since the subjects had to refrain from all oral hygiene. As previously mentioned, both
F and SLS in dentifrices are usually regarded as weak plaque inhibitors. However, in the
present study, the concentration of F in the dentifrice (5000 ppm F) was 3.5 times higher than
that in a standard dentifrice (1500 ppm F) and the rinsing procedure was extended from 2 to 3
times a day. According to WATSON et al (5), the penetration of F into natural plaque biofilms
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increased with duration of NaF exposure. Furthermore, the Duraphat dentifrice is
recommended to be used 3 times a day for patients with a high caries risk, according to the
manufacturer.
The difference in the accumulation of F in plaque (Series I) between dentifrice slurries
with 5000 and 1500 ppm F was 56%. The results of earlier studies of F accumulation diverge.
FURUICHI & BIRKHED (2) observed that the retention of F in plaque was less, when F was
delivered in a dentifrice rather than in a mouth rinse. According to PESSAN et al. (23), the
difference between dentifrices and solutions, in terms of their ability to increase plaque F
concentration, may be due to the presence of SLS. The use of F in dentifrice is likely to
increase the plaque F concentration significantly for up to 12 h (23). In contrast, HEIJNSBROEK
et al. (24) reported that F does not accumulate in plaque over time. The concentration of F in
a dentifrice may influence the retention of F in both plaque and saliva. In a recent study, we
found that the accumulation of F in plaque was 2 times higher for a dentifrice with 5000 than
for the dentifrice with 1450 ppm F, after 14 d (25).
In summary, significantly less de novo plaque was observed for the dentifrice slurry with
5000 ppm F (on buccal and all surfaces), for the water solution with 1.5% SLS (on buccal
surfaces) and the reduction ranged from 17 to 33%. We therefore conclude that the
combination of high levels of F (5000 ppm) and 1.5% SLS in a dentifrice reduces de novo
plaque formation and increases the accumulation of F in plaque, after 4 d with daily rinsing, 3
times a day. We believe that this reduction of de novo plaque formation have a clinical
importance.
Acknowledgements
We gratefully acknowledge the technical assistance of Ann-Britt Lundberg and the statistical
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assistance of Tommy Johnsson. This study was supported by the Institute of Odontology,
University of Gothenburg.
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Legend to figure
Fig. 1. The mean plaque index (QHI) scores ± SD for buccal and all surfaces, representing
each experimental 4-d period in Series I (Toothpaste slurries; n=16) and in Series II (Water
solutions; n=12).
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Table 1. The amount of collected plaque (expressed as mg wet weight) and the accumulation
of F in plaque, expressed as ppm F in the extracted plaque suspension or as ng F per mg
plaque wet weight (mean ± SD of 16 subjects), for the three dentifrice slurries in Series I. The
samples were collected after a 4-d experimental period. The difference between 5000 and
1500 ppm F was statistically significant (P < 0.05) and the corresponding difference for 500
ppm F was also statistically significant (P < 0.01).
F concentration mg wet weight ppm F ng F/mg plaque
5000 ppm F 9.56 ± 3.3 3.86 ± 2.6 0.44 ± 0.34
1500 ppm F 7.31 ± 3.7 1.68 ± 1.6 0.34 ± 0.57
500 ppm F 9.36 ± 4.3 1.41 ± 1.2 0.16 ± 0.11