Original Paper

Caries Res 2009;43:278285

DOI: 10.1159/000217860

Received: July 17, 2008

Accepted after revision: March 10, 2009
Published online: May 8, 2009

Mechanism of Fluoride Dentifrice Effect

on Enamel Demineralization
L.M.A. Tenuta C.B. Zamataro A.A. Del Bel Cury C.P.M. Tabchoury J.A. Cury
Piracicaba Dental School, UNICAMP, Piracicaba, Brazil

Key Words
Demineralization Dentifrice Fluoride Low fluoride
concentration Plaque fluid

Abstract
Although the anticaries effect of fluoride (F) dentifrices is
clearly established, the relative importance of F taken up by
dental plaque not removed by brushing and of F products
(CaF2-like) formed on totally cleaned enamel for the subsequent inhibition of demineralization is not known. Both effects were evaluated using conventional (1,100 g F/g) and
low-F concentration (500 g F/g) dentifrices in a randomized, crossover, double-blind in situ study. Enamel blocks
not treated or pretreated with the dentifrices to form CaF2like deposits were mounted in palatal appliances in contact
with a Streptococcus mutans test plaque. Volunteers brushed
with non-F (negative control), low-F or conventional dentifrices and inserted the appliance in the mouth. F concentration in the fluid and solid phases of the test plaque was determined after 30 min, and a rinse with 20% sucrose solution
was performed. After additional 45 min, plaque was collected and the loss of surface hardness at different test-plaque
depths was measured. CaF2-like deposition on enamel and
F taken up by plaque due to the use of F dentifrices were able
to significantly increase F concentration in the fluid phase of
the test plaque, but only the latter significantly reduced the
loss of hardness because of the 2030 times higher F concentration. Also, significant differences between the low-F and

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conventional dentifrices were observed for F on enamel, in

plaque and on the subsequent loss of hardness. The results
suggest that uptake of F by dental plaque not removed by
brushing may be the main cause of the anticaries effect of F
dentifrices.
Copyright 2009 S. Karger AG, Basel

Fluoride (F) dentifrice is considered to have an important role in dental caries decline observed in the last decades in either developed or developing countries [Bratthall et al., 1996; Cury et al., 2004]. In addition to the
mechanical disruption of plaque by brushing, remnants
of plaque not removed by brushing are enriched with F
from these dentifrices, which could significantly interfere with subsequent de- and remineralization events at
the tooth-plaque interface [Duckworth and Morgan,
1991; Vogel et al., 1992; Ekstrand, 1997; ten Cate, 1997;
Cury and Tenuta, 2008]. In fact, the use of F dentifrices
maintains increased F levels in the whole plaque [Duckworth and Morgan, 1991; Paes Leme et al., 2004; Cenci et
al., 2008] and in the fluid [Cenci et al., 2008] even 10 h or
more after brushing.
In addition to the anticaries effect of F dentifrices in
residual plaque, calcium fluoride (CaF2)-like material
could be formed on enamel completely cleaned during
toothbrushing and could also act as a F reservoir, slowly
releasing F to newly formed plaque during the intervals
when dentifrice is not being used [Rlla et al., 1991; ten
Livia M.A. Tenuta
Faculdade de Odontologia de Piracicaba
PO Box 53
13414-903 Piracicaba, SP (Brazil)
Tel. +55 19 2106 5393, Fax +55 19 2106 5302, E-Mail litenuta@fop.unicamp.br

Cate, 1997; Cury and Tenuta, 2008]. However, the formation of CaF2-like material on enamel during clinically relevant exposure times to F products has been questioned
[Harding et al., 1994; Petzold, 2001], and its role in the
anticaries potential of F dentifrices has not been investigated under controlled conditions. Also, the relative importance of CaF2-like material formed on enamel and of
F taken up by dental plaque during brushing on a subsequent event of enamel demineralization is not known.
The study of these anticaries mechanisms should take
into account differences in the cariogenic potential of
dental plaque. In a short-term intraoral enamel demineralization test [Zero et al., 1992, after Brudevold et al.,
1984], an extracellular polysaccharide-rich test plaque is
prepared from Streptococcus mutans and used to cause
enamel demineralization at simulated increasing thicknesses of plaque. Indeed, using this model it was possible
to show the relationship between CaF2-like material
formed on enamel by professional fluoride application,
the release of F to the fluid phase of the test plaque and
the consequent reduction of enamel demineralization
[Tenuta et al., 2008]. Also, this model was successfully
used to evaluate the effect of a calcium abrasive-containing F dentifrice on enamel de- and remineralization
[Cury et al., 2003, 2005], and could be used to evaluate
under controlled conditions the mechanisms of inhibition of enamel demineralization by F dentifrices.
Thus, in the present study a short-term in situ model
was used to evaluate the anticaries effect of F dentifrice
on remaining plaque and on plaque-free enamel. Dentifrices with two F concentrations (500 and 1,100 g F/g)
were used to evaluate the dose-response effect of both
mechanisms.

Materials and Methods

Experimental Design
This was a crossover, double-blind, split-mouth, short-term in
situ study, approved by the Research and Ethics Committee of
Piracicaba Dental School (Protocol No. 007/2006). Twelve adult
volunteers, aged 2145 years, with past caries experience ranging
from 0 to 38 lesions (mean DMFS = 11.3) and normal salivary flow
rate (mean unstimulated = 0.58 and stimulated = 1.62 ml/min)
signed a written, informed consent to participate. Three dentifrices with distinct F concentrations were studied: non-F placebo
dentifrice (negative control); 500 g F/g dentifrice; 1,100 g F/g
dentifrice. They were evaluated with respect to inhibition of
enamel demineralization (hardness loss) by a S. mutans test
plaque after one cariogenic challenge due to a sucrose rinse. Two
distinct anticaries mechanisms and their interaction were evaluated:

Anticaries Effect of F Dentifrice

Experiment 1: Effect of CaF2-Like Material Preformed on

Enamel by Dentifrices. Enamel blocks were treated with a slurry
of dentifrice for 5 min before mounting in the in situ appliance,
and at the beginning of the intraoral test volunteers brushed their
own teeth with non-F dentifrice (fig. 1).
Experiment 2: Effect of F in Plaque after Brushing with the Dentifrices and Its Association with Preformed CaF2-Like Material.
Volunteers brushed their own teeth with one of the test dentifrices and immediately after spitting out the dentifrice foam, inserted into the oral cavity the palatal appliance containing four
blocks previously treated with the slurry of designated dentifrice
and four nontreated blocks (fig. 1).
In both experiments, eight bovine enamel blocks with known
surface hardness (SH) were mounted in two holders (4 blocks in
each), in contact with a test plaque prepared from S. mutans IB
1600, which were fixed in a palatal appliance [Zero et al., 1992;
Cury et al., 2003] used by the volunteers in four experimental
phases. The F dentifrices used were similar in composition, except for the F content (added as NaF) and were all silica-based.
After 30 min of intraoral appliance use, immediately before a
1-min rinse with 20% sucrose solution, half of the enamel blocks
were removed and test plaque was collected for analysis of F in the
fluid and solid phases. Forty-five minutes after the cariogenic
challenge, the remaining enamel blocks were collected for measurement of percentage change in SH and the test plaque was collected and analyzed for F in the fluid and solids. The sequence of
treatments tested in each phase was randomized among volunteers using a computer-allocated randomization list. All volunteers lived in an optimally fluoridated area (0.60.8 g F/ml) and
for a 7-day lead-in period prior to each experimental phase, they
used the dentifrice that would be used for brushing in the next
phase.
Specimen Preparation and Treatment with Dentifrices
Enamel blocks (5 ! 5 ! 2 mm) obtained from bovine incisors
were polished flat and their SH was determined using a Shimadzu
HMV-2000 microhardness tester with a Knoop indenter using a
50-gram load for 5 s. In each enamel block, 11 indentations were
made at 50, 75, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000 and
2,500 m from one block edge, which was marked for future reference. The mean hardness of these 11 indentations was calculated and a total of 384 blocks presenting an average hardness of
340.5 kg/mm2 (SD 30.3) were randomly assigned to the treatment
groups.
Blocks used to evaluate the effect of F products on enamel were
immersed in a slurry (1:3 w/w in distilled water) of the assigned
dentifrice for 5 min under agitation (80 rpm), then gently washed
for 10 s with distilled water and dried with soft paper. This treatment was performed immediately before mounting the enamel
blocks in the in situ appliance.
The surface concentration of CaF2-like material formed on
enamel blocks by this procedure was determined in an extra set
of 30 enamel blocks (10 treated with each dentifrice), which were
isolated with wax leaving only the enamel surface exposed. Blocks
were individually immersed in 1.0 M KOH (0.04 ml/mm2) for
24 h [Caslavska et al., 1975]. After buffering with TISAB II containing 1.0 M HCl, F was measured using an ion-selective electrode (Orion 96-09) and an ion analyzer (Orion EA-940) and the
surface concentration of CaF2-like material was calculated from
the amount of solubilized fluoride and the exposed surface area.

Caries Res 2009;43:278285

279

Randomization
P
r
e
t
r
e
a
t
m
e
n
t
I
n
s
i
t
u
t
e
s
t

o
f
e
n
a
m
e
l
Time =
00:00

Treatment
with a slurry
of the 500 g
F/g dentifrice

Treatment with
a slurry of the
1,100 g F/g
dentifrice

Treatment with
a slurry of the
placebo
dentifrice

Effect of CaF2 formed on enamel

Placebo dentifrice

No
treatment

Treatment
with a slurry
of the 500 g
F/g dentifrice

No
treatment

Treatment with
a slurry of the
1,100 g F/g
dentifrice

No
treatment

Effect of increased F concentration in plaque due to brushing with the distinct

dentifrices, associated or not with CaF2 formed on enamel
500 g F/g dentifrice

Placebo dentifrice

1,100 g F/g dentifrice

Volunteers brushed their own teeth for 1 min and immediately after spitting out the foam, started using the appliance
30 min
Time =
00:30

Test plaque collected for F analysis in the fluid and solid phases
1 min rinse with 20% sucrose solution
45 min

Time =
00:75

Analysis of hardness loss in enamel blocks and F in the test plaque

Fig. 1. Experimental design of the study.

Palatal Appliance Mounting

Test plaque was prepared from S. mutans Ingbritt-1600 (kindly donated by the Eastman Department of Dentistry, Rochester,
N.Y., USA) [Cury et al., 2003]. Briefly, bacteria were grown in
Todd-Hewitt broth containing 1% sucrose (to allow the production of extracellular polysaccharides) for 18 h, at 37 C and under
10% CO2. Bacteria were harvested by centrifugation and serially
washed in cold buffer (100 mM KCl, 20 mM KHCO3, pH 7.0) to
remove remnants of culture medium. Palatal appliances carrying
two plastic holders were constructed for each volunteer. Four
enamel blocks were mounted in each holder, with enamel surfaces in contact with the test plaque. The plastic holders were
mounted with the marked edge of the enamel blocks, where the
baseline hardness measurements were made, facing the center of
the palatal appliance. For further details see Cury et al. [2003].
Intraoral Test
Volunteers brushed their teeth with 1.0 g of the assigned dentifrice for 1 min, and immediately after spitting out the dentifrice
foam (without rinsing), the appliance was inserted into the mouth.
After 30 min, two enamel blocks in each holder were removed and
test plaque collected for analysis of F concentration in the fluid
and solid phases. A cariogenic challenge was then conducted with
the appliance in situ, by gently rinsing with 15 ml 20% sucrose
solution for 1 min. Forty-five minutes later, the appliance was re-

280

Caries Res 2009;43:278285

moved and SH of the enamel blocks again determined. Test plaque

was collected for analysis of F in the fluid and solids. During the
intraoral test, subjects refrained from talking, drinking or eating.
Collection and Fluoride Analysis of Plaque
Test plaque samples were collected using a plastic spatula, and
immediately placed inside an oil-filled centrifuge tube [Vogel et
al., 1997]. The tube was centrifuged for 5 min (21,000 g) at 4 C to
separate the fluid from the plaque solids.
The fluid phase was recovered with oil-filled capillary micropipettes, and the F concentration immediately determined using
an inverted F electrode [Vogel et al., 1997]. Samples were applied
on the surface of the oil-covered F electrode and diluted with
TISAB III (1:10) under a microscope. A micro-reference electrode
was used to close the circuit, and the signal was read using a highimpedance electrometer (WPI, FD223, Sarasota, Fla., USA) coupled to the computer software Plot 1 (Paffenbarger Research Center, ADA Foundation, Gaithersburg, Md., USA).
After fluid extraction, plaque solids were kept frozen until the
extraction of total F with acid. For extraction, the tip of the centrifuge tube was cut, and the remaining plaque was centrifuged
into another microcentrifuge tube containing 0.5 M HCl (0.1
ml/10 mg of biofilm wet weight) [Tenuta et al., 2006]. Samples
were agitated for 3 h at room temperature, centrifuged, and the

Tenuta/Zamataro/Del Bel Cury/

Tabchoury/Cury

Color version available online

384 enamel blocks

Table 1. Experiment 1: F concentrations in the fluid and solid phases of the test plaque (mean 8 SD; n = 12)
according to the dentifrice used for pretreatment of the enamel blocks

Pretreatment
of enamel
blocks

30 min after dentifrice use, at the moment

of the cariogenic challenge

75 min after dentifrice use

(45 min after cariogenic challenge)

F in the fluid
phase, M

F in the solid
phase, nmol/g

F in the fluid
phase, M

F in the solid
phase, nmol/g

Placebo
500 g F/g
1,100 g F/g

2.180.6a
9.683.1b
17.284.4c

41.988.2a
54.188.6 b, 1
57.3810.3b

1.180.4a
3.782.6b
4.681.8b

39.2811.6a
52.9822.1a
48.3810.1a

Among dentifrices (within columns), means with distinct letters are significantly different (p < 0.05). Values
were transformed to log10 for statistical analysis. 1 One outlier removed (F in solids = 110.0 nmol/g).

supernatant neutralized with fresh TISAB III containing 2.5 M

NaOH (0.02 ml/10 mg biofilm). F in the acid extract was measured
as described above, but the standards used contained HCl and
NaOH in the same proportion as the samples.
Analysis of SH Loss
Enamel blocks removed from the holders were washed with
deionized water and their SH was again measured, at 100 m
from the initial indentations, at 11 points at corresponding distances from the block edge. From this block edge, F from dentifrice used for brushing and the sucrose solution diffused through
the test plaque in contact with the enamel surface, reaching deeper plaque areas. This allowed the diffusion of substrates through
dental plaque at thicknesses of up to 2.5 mm to be simulated, since
the positions of hardness measurements on enamel in contact
with the test plaque can be used as a reference for test plaque
thickness [Zero, 1995]. The percentage change in surface hardness [%SHC = 100 (SH after in situ test baseline SH)/baseline
SH] was calculated at each distance.
Statistical Analysis
The surface concentrations of CaF2-like material formed on
enamel after treatment with the dentifrices were compared using
ANOVA. For the in situ test variables, as all volunteers used all
combinations of treatments, they were considered as statistical
blocks to decrease unknown variability in the experimental error.
The isolated effect of CaF2-like material formed on enamel by the
dentifrices (experiment 1) was tested by ANOVA. A split-plot
ANOVA was used to analyze the concomitant effect (experiment
2) of F in plaque due to brushing (dentifrice used as plot) and
CaF2-like material formed on enamel by the dentifrices (pretreatment of blocks as subplot). Data for %SHC were also analyzed by
split-plot ANOVA, considering the depths of the hardness measurements as subplots. The normality of error distribution and
the homogeneity of variance were checked for each response variable using the SAS/LAB package (SAS software, version 8.01, SAS
Institute Inc., Cary, N.C., USA) and data were transformed as
suggested by the software, according to Box et al. [1978]. PostANOVA comparisons were performed using Tukey test. SAS software was used for all analyses and the significance limit was set
at 5%.

Anticaries Effect of F Dentifrice

Results

Experiment 1: Effect of Preformed CaF2-Like Material

Pretreatment of enamel blocks with the dentifrices resulted in significantly different surface concentrations of
CaF2-like material on enamel (p ! 0.05), with mean values (8SD) of 0.24 8 0.09, 0.50 8 0.25 and 0.87 8 0.42
g F/cm2 for enamel blocks treated with the placebo, 500
and 1,100 g F/g dentifrices, respectively. Also, F concentration in the fluid phase of plaque collected after 30 min
in situ was significantly different for enamel blocks pretreated with the different dentifrices (p ! 0.05, table 1). In
plaque solids, F concentrations were significantly higher
for enamel blocks pretreated with F dentifrices (p ! 0.05),
but no significant difference with respect to dentifrice F
concentration was observed (table 1).
Forty-five minutes after the cariogenic challenge, F
concentration in plaque fluid was still higher over blocks
pretreated with both F dentifrices when compared to the
placebo (p ! 0.05), but no significant difference was observed between them (table 1). In the solids, the differences between the groups were not significant.
Although pretreatment significantly increased F concentration in the fluid and solid phases of the plaque, no
significant differences in loss of enamel hardness were
observed among the three tested dentifrices (fig. 2). Less
loss of hardness was observed at 2,000 and 2,500 m
from the block edge (p ! 0.05).
Experiment 2: Effect of F in Plaque after Brushing
Brushing with the different dentifrices at the beginning of the in situ test produced distinct levels of F concentration in the fluid and solid phases of test plaque collected 30 min later, the highest for the 1,100 g F/g denCaries Res 2009;43:278285

281

%SHC

AB
10 AB
AB
AB A A
20
A

Fig. 2. Experiment 1: Mean and SD (n = 12)

of %SHC, according to the dentifrice used

for pretreating enamel blocks used in the
in situ demineralization test. Placebo dentifrice was used for brushing at the beginning of the test. No significant difference
was found among dentifrices (p 1 0.05).
Uppercase letters represent the differences
between depths: values sharing the same
letter are not significantly different.

C
B
AB

30
40
Placebo
50

500 g F/g
1,100 g F/g

60
0

500

1,000
1,500
Distance from the block edge (m)

2,000

2,500

Table 2. Experiment 2: F concentration in the fluid and solid phase of the test plaque (mean 8 SD; n = 12) according to the dentifrice
used for brushing at the beginning of the in situ test and the pretreatment of the enamel blocks

Dentifrice used
for brushing and
pretreating
enamel blocks

Pretreatment
of enamel
blocks

30 min after dentifrice use, at the moment

of the cariogenic challenge

75 min after dentifrice use

(45 min after cariogenic challenge)

F in the fluid
phase, M

F in the solid
phase, nmol/g

F in the fluid
phase, M

F in the solid
phase, nmol/g

Placebo

no
yes
no
yes
no
yes

2.080.4
2.180.6
2458290
2038222
5088437
5258408

40.487.6
41.988.2
1938150
1788122
3258202
3768225

0.980.5
1.180.4
22.9836.1
28.4839.1
60.2874.0
75.4867.1

35.5810.1
39.2811.6
67.3835.6
83.1839.6
107.2868.4
121.3861.5

500 g F/g
1,100 g F/g

For the variables F in the fluid and solids analyzed 30 min after dentifrice use, all dentifrices differed from each other (p < 0.05),
but no effect of pretreatment of enamel blocks was found. For F in the fluid and solids analyzed 75 min after dentifrice use, all dentifrices differed from each other (p < 0.05) and the effect of pretreatment of blocks was statistically significant (p < 0.05). Values were
transformed to the log10 for the statistical analysis, except for F in solids at 30 min, which were transformed to the inverse of the square
root of original data.

tifrice and the lowest for the placebo dentifrice (p ! 0.05,

table 2), irrespective of the pretreatment of enamel blocks
with dentifrices.
Forty-five minutes after the cariogenic challenge (75
min after brushing), F concentrations in the fluid and
solid phases of the plaque were still significantly different
among the three dentifrices (p ! 0.05, table 2). At this
time, the effect of pretreatment of enamel blocks with the
dentifrices was significant, since a higher F concentration
was observed in the plaque fluid and solids in contact
282

Caries Res 2009;43:278285

with enamel blocks that had been pretreated with the

dentifrices (p ! 0.05, table 2).
No significant effect of pretreatment of the enamel
blocks on %SHC was observed. Thus, data for nontreated
and treated enamel blocks are combined in figure 3 for
clarity. The pattern of variation of %SHC with distance
from the block edge was different according to the dentifrice used. For both F dentifrices, the distance from the
block edge did not affect %SHC, but for the placebo dentifrice hardness loss was smaller at 2,000 and 2,500 m
Tenuta/Zamataro/Del Bel Cury/
Tabchoury/Cury

Color version available online

Color version available online

10
0
10
%SHC

Fig. 3. Experiment 2: Mean and SD (n = 12)

of %SHC in enamel blocks used in the in
situ demineralization test, according to
the dentifrice used for brushing at the beginning of the test. Since no significant effect of pretreatment of enamel blocks was
observed, data of pretreated and non-pretreated blocks were combined. At each distance, dentifrice means with the same lowercase letters are not significantly different. No significant effect of distance from
the block edge was observed for the 500and 1,100-g F/g dentifrices, but for the
placebo dentifrice, hardness loss at 2,000
and 2,500 m was significantly lower than
at the other distances (p ! 0.05).

aa a

20 a a a

a a

30
b
40 b b

c
b b b

Placebo

500 g F/g

50

1,100 g F/g
60
0

from the block edge (p ! 0.05), without significant differences among the other distances. At up to 500 m from
the block edge, no significant difference was observed between the F dentifrices, and %SHC was less than after
brushing with the placebo dentifrice (p ! 0.05). However,
at 1,000 and 1,500 m from the block edge, the lowest
%SHC was observed when the 1,100 g F/g dentifrice was
used for brushing and the highest when the placebo dentifrice was used. Also, at 2,500 m, the 1,100 g F/g dentifrice presented less loss of hardness than either of the
other dentifrices, which did not differ significantly.

500

1,000
1,500
Distance from the block edge (m)

2,000

2,500

The experimental model used in the present study allowed the distinction of different mechanisms of inhibition of enamel hardness loss by F dentifrices, i.e. F in residual plaque, F products formed on cleaned enamel by
dentifrice application, or both. This would simulate clinical conditions where: (1) some plaque is not removed by
brushing, but is enriched with F available from the dentifrice; (2) plaque is completely removed and F reacts with
enamel, but a new biofilm is formed over it; or (3) F from
dentifrice reacts with a clean enamel surface, a new biofilm is formed over it and it is exposed to F from dentifrice. However, limitations of the model mean that the
results cannot be extrapolated directly to in vivo conditions. One limitation is the monospecific artificial test
plaque used, and its limited exposure to the dentifrices by
the contact of the dentifrice foam remaining in the mouth

after brushing and the small area of test plaque exposed

to the oral cavity. Also, the cariogenic challenge is performed only 30 min after dentifrice use or application to
the enamel blocks, which could cause the effect of the
dentifrices to be overestimated. However, the model allows the factors under investigation to be studied separately and, given the short-term effect observed for F
products formed on cleaned enamel on subsequent loss
of hardness, which could not have been evaluated using
a long-term model, it was a useful method for comparing
the mechanisms involved in the anticaries effect of F dentifrices. Furthermore, the results give new insights for future research on the anticaries effect of F dentifrices,
mouthrinses and product development.
Pretreatment of enamel blocks for 5 min with a slurry
of the F dentifrices resulted in deposition of CaF2-like
material, which was proportional to the F concentration
in the dentifrice. Although the exposure time of 5 min
could be considered too long compared to conventional
brushing habits, in vivo the first brushing strokes would
promote the reaction of the undiluted dentifrice with
enamel, which could increase the amount of CaF2-like
material formed. However, the CaF2-like material formed
by the treatment with the dentifrice slurry in the present
study could be expected to have a different solubility behavior of F deposits formed in vivo on enamel, in the
presence of saliva, due to phosphate contamination
[Christoffersen et al., 1988].
It has been questioned whether CaF2-like material
forms after treatment with F products within clinically
relevant exposure times [Harding et al., 1994; ten Cate,

Anticaries Effect of F Dentifrice

Caries Res 2009;43:278285

Discussion

283

1997; Petzold, 2001] and our results agree with those of

Bruun and Givskov [1993], who found a small increase in
KOH-soluble F in enamel treated with a slurry of NaF
dentifrice for 2 min. This result suggests that only small
amounts of CaF2-like material can be deposited on
cleaned enamel by brushing with a fluoride dentifrice.
However, the increase of F in the fluid phase of the test
plaque in contact with enamel blocks pretreated with the
F dentifrices (table 1) was probably due to F release from
the CaF2-like products formed, as previously observed
for professional F application [Tenuta et al., 2008]. On the
other hand, the maintenance of these enamel reservoirs
in the mouth is not expected to be long, considering the
low concentration found and the decrease in F concentration in the fluid phase of plaque from the measurements
at 30 and 75 min. Additionally, the role of the CaF2-like
reservoirs formed by one dentifrice application on a subsequent event of enamel demineralization was negligible
(fig. 2). This agrees with previous observations by others
[Bruun and Givskov, 1993; ten Cate, 1997] that the low
surface concentration of CaF2-like material formed on
cleaned enamel would have an unimportant effect on
subsequent loss of enamel hardness once new plaque is
formed. However, in the present study only one exposure
to dentifrice was simulated, and CaF2-like material deposition was shown to increase by daily rinses with sodium
fluoride [Saxegaard and Rlla, 1989], and also to be higher in caries-like lesions than on sound enamel [Bruun and
Givskov, 1993], which could be further studied.
The effect of F uptake in plaque by brushing with F
dentifrices on the inhibition of enamel demineralization,
measured as loss of hardness, is clearly shown (fig. 3). The
greater demineralization after the use of the placebo dentifrice is in agreement with the high cariogenicity of the
model used, which allows the diffusion of sucrose through
the extracellular polysaccharide-rich test plaque and a
change in SH of 40% at the first depths of sugar penetration and around 20% deeper [Zero, 1995; Cury et al.,
2003]. However, after brushing with both F dentifrices,
demineralization was significantly less than after brushing with placebo, and no significant effect of test plaque
thickness (sugar diffusion distance) was observed. Also,
the results demonstrate a higher anticaries effect for the
conventional F concentration dentifrice at greater thicknesses of the test plaque, and agree with recent findings
showing that the effectiveness of low-F dentifrices may
depend on the caries activity of the study population
[Lima et al., 2008].
In fact, although the test plaque was not collected as a
function of plaque thickness, it is assumed that F from
284

Caries Res 2009;43:278285

the dentifrice foam remaining in the volunteers mouth

diffused through the plaque, since inhibition of demineralization was seen at greater plaque thicknesses. Also,
a clear dose-response effect was observed in plaque F concentration after brushing with both F dentifrices, which
could cause the observed effect on enamel hardness.
The increase in F concentration in the fluid and solid
phase of the test plaque after brushing suggests that every
time that toothbrushing is made with F dentifrices, remnants of plaque are enriched with F, which could be sustained by F being released from oral soft tissues [Duckworth and Morgan, 1991]. F in the fluid phase of the bacterial test plaque (10 times lower after 45 min, table 2)
decreased more sharply than F in the solid phase (3 times
lower after 45 min), suggesting that the slow release of F
from bacterial reservoirs in the plaque could be responsible for the high F levels found in plaque fluid even 10 h
after F dentifrice use [Cenci et al., 2008]. Indeed, the
model used seems promising to study bacterial F reservoirs and their biological role, since it excludes other
types of mineral reservoirs potentially present in natural
plaque [Tenuta et al., 2006].
In conclusion, the results suggest that F products
formed on enamel by dentifrice application can increase
F concentration in the fluid of plaque formed on it, but to
a much lower extent and with minimal effect on loss of
enamel hardness when compared to the burst of F in
plaque fluid after brushing with F dentifrices. Since
plaque-free teeth would not demineralize, the results
suggest that F uptake by dental plaque not removed by
brushing may be the main cause of the anticaries effect
of F dentifrices. Also, the higher F availability in the fluid phase of plaque after use of conventional F concentration dentifrice, and the smaller demineralization at a
greater depth when compared to low-F dentifrice, suggest
that the latter should be prescribed with caution.

Acknowledgments
We thank the volunteers for their valuable participation, Colgate (So Paulo, SP, Brazil) for the dentifrice formulations, and
FAPESP (05/04703-0 and 06/01193-3) for financial support.

Tenuta/Zamataro/Del Bel Cury/

Tabchoury/Cury

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