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Original Paper

Caries Res 2009;43:278285

DOI: 10.1159/000217860

Mechanism of Fluoride Dentifrice Effecton 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

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 impor-

tant role in dental caries decline observed in the last de-cades in either developed or developing countries [Brat-thall et al., 1996; Cury et al., 2004]. In addition to themechanical disruption of plaque by brushing, remnantsof plaque not removed by brushing are enriched with Ffrom these dentifrices, which could significantly inter-fere with subsequent de- and remineralization events atthe 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 dentifricesmaintains increased F levels in the whole plaque [Duck-worth and Morgan, 1991; Paes Leme et al., 2004; Cenci et

al., 2008] and in the fluid [Cenci et al., 2008] even 10 h ormore after brushing.

In addition to the anticaries effect of F dentifrices inresidual plaque, calcium fluoride (CaF2)-like materialcould be formed on enamel completely cleaned duringtoothbrushing and could also act as a F reservoir, slowlyreleasing F to newly formed plaque during the intervalswhen dentifrice is not being used [Rlla et al., 1991; ten

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 subse-quent inhibition of demineralization is not known. Both ef-

fects were evaluated using conventional (1,100 g F/g) and

low-F concentration (500 g F/g) dentifrices in a random-

ized, crossover, double-blind in situ study. Enamel blocks

not treated or pretreated with the dentifrices to form CaF 2-

like deposits were mounted in palatal appliances in contact

with a Streptococcus mutanstest plaque. Volunteers brushed

with non-F (negative control), low-F or conventional denti-

frices and inserted the appliance in the mouth. F concentra-

tion in the fluid and solid phases of the test plaque was de-

termined after 30 min, and a rinse with 20% sucrose solution

was performed. After additional 45 min, plaque was collect-ed 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 concen-

tration. Also, significant differences between the low-F and

Received: July 17, 2008

Accepted after revision: March 10, 2009

Published online: May 8, 2009

Livia M.A. TenutaFaculdade de Odontologia de PiracicabaPO Box 53

13414-903 Piracicaba, SP (Brazil)Tel. +55 19 2106 5393, Fax +55 19 2106 5302, E-Mail [email protected]

2009 S. Karger AG, Basel00086568/09/04340278$26.00/0

Accessible online at:www.karger.com/cre


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Anticaries Effect of F Dentifrice Caries Res 20 09;43:278285 279

Cate, 1997; Cury and Tenuta, 2008]. However, the forma-tion of CaF2-like material on enamel during clinically rel-evant exposure times to F products has been questioned[Harding et al., 1994; Petzold, 2001], and its role in theanticaries potential of F dentifrices has not been investi-gated under controlled conditions. Also, the relative im-

portance of CaF2-like material formed on enamel and ofF taken up by dental plaque during brushing on a subse-quent event of enamel demineralization is not known.

The study of these anticaries mechanisms should takeinto account differences in the cariogenic potential ofdental plaque. In a short-term intraoral enamel deminer-alization test [Zero et al., 1992, after Brudevold et al.,1984], an extracellular polysaccharide-rich test plaque isprepared from Streptococcus mutansand used to causeenamel demineralization at simulated increasing thick-nesses of plaque. Indeed, using this model it was possibleto 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 andthe consequent reduction of enamel demineralization[Tenuta et al., 2008]. Also, this model was successfullyused to evaluate the effect of a calcium abrasive-contain-ing F dentifrice on enamel de- and remineralization[Cury et al., 2003, 2005], and could be used to evaluateunder controlled conditions the mechanisms of inhibi-tion of enamel demineralization by F dentifrices.

Thus, in the present study a short-term in situ modelwas used to evaluate the anticaries effect of F dentifriceon remaining plaque and on plaque-free enamel. Denti-

frices with two F concentrations (500 and 1,100 g F/g)were used to evaluate the dose-response effect of bothmechanisms.

Materials and Methods

Experimental DesignThis was a crossover, double-blind, split-mouth, short-term in

situ study, approved by the Research and Ethics Committee ofPiracicaba Dental School (Protocol No. 007/2006). Twelve adult

volunteers, aged 2145 years, with past caries experience rangingfrom 0 to 38 lesions (mean DMFS = 11.3) and normal salivary f low

rate (mean unstimulated = 0.58 and stimulated = 1.62 ml/min)signed a written, informed consent to participate. Three denti-frices with distinct F concentrations were studied: non-F placebodentifrice (negative control); 500 g F/g dentifrice; 1,100 g F/gdentifrice. They were evaluated with respect to inhibition ofenamel demineralization (hardness loss) by a S. mutans testplaque after one cariogenic challenge due to a sucrose rinse. Twodistinct anticaries mechanisms and their interaction were evalu-ated:

Experiment 1: Effect of CaF2-Like Material Preformed onEnamel by Dentifrices.Enamel blocks were treated with a slurryof dentifrice for 5 min before mounting in the in situ appliance,and at the beginning of the intraoral test volunteers brushed theirown teeth with non-F dentifrice (fig. 1).

Experiment 2: Effect of F in Plaque after Brushing with the Den-tifrices and Its Association with Preformed CaF2-Like Material.

Volunteers brushed their own teeth with one of the test denti-frices and immediately after spitting out the dentifrice foam, in-serted into the oral cavity the palatal appliance containing fourblocks previously treated with the slurry of designated dentifriceand four nontreated blocks (fig. 1).

In both experiments, eight bovine enamel blocks with knownsurface hardness (SH) were mounted in two holders (4 blocks ineach), in contact with a test plaque prepared from S. mutansIB1600, which were fixed in a palatal appliance [Zero et al., 1992;Cury et al., 2003] used by the volunteers in four experimentalphases. The F dentifrices used were similar in composition, ex-cept for the F content (added as NaF) and were all silica-based.After 30 min of intraoral appliance use, immediately before a1-min rinse with 20% sucrose solution, half of the enamel blockswere removed and test plaque was collected for analysis of F in thefluid and solid phases. Forty-five minutes after the cariogenicchallenge, the remaining enamel blocks were collected for mea-surement of percentage change in SH and the test plaque was col-lected and analyzed for F in the fluid and solids. The sequence oftreatments tested in each phase was randomized among volun-teers using a computer-allocated randomization list. All volun-teers lived in an optimally fluoridated area (0.60.8 g F/ml) andfor a 7-day lead-in period prior to each experimental phase, theyused the dentifrice that would be used for brushing in the nextphase.

Specimen Preparation and Treatment with DentifricesEnamel blocks (5!5!2 mm) obtained from bovine incisors

were polished f lat and their SH was determined using a Shimadzu

HMV-2000 microhardness tester with a Knoop indenter using a50-gram load for 5 s. In each enamel block, 11 indentations weremade at 50, 75, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000 and2,500 m from one block edge, which was marked for future ref-erence. The mean hardness of these 11 indentations was calcu-lated and a total of 384 blocks presenting an average hardness of340.5 kg/mm2(SD 30.3) were randomly assigned to the treatmentgroups.

Blocks used to evaluate the effect of F products on enamel wereimmersed in a slurr y (1: 3 w/w in distilled water) of the assigneddentifrice for 5 min under agitation (80 rpm), then gently washedfor 10 s with distilled water and dried with soft paper. This treat-ment was performed immediately before mounting the enamelblocks in the in situ appliance.

The surface concentration of CaF2-like material formed onenamel blocks by this procedure was determined in an extra setof 30 enamel blocks (10 treated with each dentifrice), which wereisolated with wax leaving only the enamel surface exposed. Blockswere individually immersed in 1.0 M KOH (0.04 ml/mm2) for24 h [Caslavska et al., 1975]. After buffering with TISAB II con-taining 1.0 MHCl, F was measured using an ion-selective elec-trode (Orion 96-09) and an ion analyzer (Orion EA-940) and thesurface concentration of CaF2-like material was ca lculated fromthe amount of solubilized fluoride and the exposed surface area.


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Tenuta/Zamataro/Del Bel Cury/Tabchoury/Cury

Caries Res 2009;43:278285280

Palatal Appliance Mounting

Test plaque was prepared from S. mutansIngbritt-1600 (kind-ly donated by the Eastman Department of Dentistry, Rochester,N.Y., USA) [Cury et al., 2003]. Briefly, bacteria were grown inTodd-Hewitt broth containing 1% sucrose (to allow the produc-tion of extracellular polysaccharides) for 18 h, at 37 C and under10% CO2. Bacteria were harvested by centrifugation and seriallywashed in cold buffer (100 mMKCl, 20 mMKHCO3, pH 7.0) toremove remnants of culture medium. Palatal appliances carryingtwo plastic holders were constructed for each volunteer. Fourenamel blocks were mounted in each holder, with enamel sur-faces in contact with the test plaque. The plastic holders weremounted with the marked edge of the enamel blocks, where thebaseline hardness measurements were made, facing the center ofthe palatal appliance. For further details see Cury et a l. [2003].

Intraoral TestVolunteers brushed their teeth with 1.0 g of the assigned den-

tifrice for 1 min, and immediately after spitting out the dentifricefoam (without rinsing), the appliance was inserted into the mouth.After 30 min, two enamel blocks in each holder were removed andtest plaque collected for analysis of F concentration in the fluidand solid phases. A cariogenic challenge was then conducted withthe appliance in situ, by gently rinsing with 15 ml 20% sucrosesolution for 1 min. Forty-five minutes later, the appliance was re-

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

was collected for analysis of F in the fluid and solids. During theintraoral test, subjects refrained from talking, drinking or eat-ing.

Collection and Fluoride Analysis of PlaqueTest plaque samples were collected using a plast ic spatula, and

immediately placed inside an oil-filled centrifuge tube [Vogel etal., 1997]. The tube was centrifuged for 5 min (21,000g) at 4 C toseparate the fluid from the plaque solids.

The f luid phase was recovered with oil-filled capillary micro-pipettes, and the F concentration immediately determined usingan inverted F electrode [Vogel et al., 1997]. Samples were appliedon the surface of the oil-covered F electrode and diluted withTISAB III (1: 10) under a microscope. A micro-reference electrode

was used to close the circuit, and the signal was read using a high-impedance electrometer (WPI, FD223, Sarasota, Fla., USA) cou-pled to the computer software Plot 1 (Paffenbarger Research Cen-ter, ADA Foundation, Gaithersburg, Md., USA).

After f luid extraction, plaque solids were kept frozen until theextraction of total F with acid. For extraction, the tip of the cen-trifuge tube was cut, and the remaining plaque was centrifugedinto another microcentrifuge tube containing 0.5 M HCl (0.1ml/10 mg of biofilm wet weight) [Tenuta et al., 2006]. Sampleswere agitated for 3 h at room temperature, centrifuged, and the

Volunteers brushed their own teeth for 1 min and immediately after spitting out the foam, started using the appliance

384 enamel blocks

Randomization

Treatment

with a slurryof the 500 g

F/g dentifrice

Treatment with

a slurry of the1,100 g F/g

dentifrice

Treatment with

a slurry of the1,100 g F/g

dentifrice

Treatment with

a slurry of theplacebo

dentifrice

No

treatment

No

treatment

No

treatment

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

I

n

s

i

t

u

t

e

s

t

Placebo dentifrice

Treatment

with a slurryof the 500 g

F/g dentifrice

Time =

00:00

Time =

00:75 Analysis of hardness loss in enamelblocks and F in the test plaque

30 min

Test plaque collected for F analysis in the fluid and solid phases

1 min rinse with 20%sucrose solution

45 min

Placebo dentifrice

Effect of CaF2formed on enamelEffect of increased F concentration in plaquedue to brushing with the distinct

dentifrices, associated or not with CaF2formed on enamel

P

r

e

tr

e

a

t

m

e

n

t

of

e

n

a

m

e

l

Time =

00:30

Fig. 1.Experimental design of the study.

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supernatant neutralized with fresh TISAB III containing 2.5 MNaOH (0.02 ml/10 mg biofilm). F in the acid extract was measured

as described above, but the standards used contained HCl andNaOH in the same proportion as the samples.

Analysis of SH LossEnamel blocks removed from the holders were washed with

deionized water and their SH was again measured, at 100 mfrom the initial indentations, at 11 points at corresponding dis-tances from the block edge. From this block edge, F from denti-frice used for brushing and the sucrose solution diffused throughthe test plaque in contact with the enamel surface, reaching deep-er plaque areas. This al lowed the diffusion of substrates throughdental plaque at thicknesses of up to 2.5 mm to be simulated, sincethe positions of hardness measurements on enamel in contactwith the test plaque can be used as a reference for test plaquethickness [Zero, 1995]. The percentage change in surface hard-

ness [%SHC = 100 (SH after in situ test baseline SH)/baselineSH] was calculated at each distance.

Statistical AnalysisThe surface concentrations of CaF2-like material formed on

enamel after treatment with the dentifrices were compared usingANOVA. For the in situ test variables, as all volunteers used allcombinations of treatments, they were considered as statisticalblocks to decrease unknown variability in the experimental error.The isolated effect of CaF2-like material formed on enamel by thedentifrices (experiment 1) was tested by ANOVA. A split-plotANOVA was used to analyze the concomitant effect (experiment2) of F in plaque due to brushing (dentifrice used as plot) andCaF2-like material formed on enamel by the dentifrices (pretreat-

ment of blocks as subplot). Data for %SHC were also analyzed bysplit-plot ANOVA, considering the depths of the hardness mea-surements as subplots. The normality of error distribution andthe homogeneity of variance were checked for each response vari-able using the SAS/LAB package (SAS software, version 8.01, SASInstitute Inc., Cary, N.C., USA) and data were transformed assuggested by the software, according to Box et al. [1978]. Post-ANOVA comparisons were performed using Tukey test. SAS soft-ware was used for all analyses and the significance limit was setat 5%.

Results

Experiment 1: Effect of Preformed CaF2-Like MaterialPretreatment of enamel blocks with the dentifrices re-

sulted in significantly different surface concentrations ofCaF2-like material on enamel (p !0.05), with mean val-ues (8SD) of 0.2480.09, 0.5080.25 and 0.8780.42g F/cm2for enamel blocks treated with the placebo, 500and 1,100g F/g dentifrices, respectively. Also, F concen-tration in the f luid phase of plaque collected after 30 minin situ was significantly different for enamel blocks pre-treated with the different dentifrices (p!0.05, table 1). Inplaque solids, F concentrations were significantly higherfor enamel blocks pretreated with F dentifrices (p!0.05),

but no significant difference with respect to dentifrice Fconcentration was observed (table 1).

Forty-five minutes after the cariogenic challenge, Fconcentration in plaque fluid was still higher over blockspretreated with both F dentifrices when compared to theplacebo (p !0.05), but no significant difference was ob-served between them (table 1). In the solids, the differ-ences between the groups were not significant.

Although pretreatment significantly increased F con-centration in the f luid and solid phases of the plaque, nosignificant differences in loss of enamel hardness wereobserved among the three tested dentifrices (fig. 2). Less

loss of hardness was observed at 2,000 and 2,500 mfrom the block edge (p !0.05).

Experiment 2: Effect of F in Plaque after BrushingBrushing with the different dentifrices at the begin-

ning of the in situ test produced distinct levels of F con-centration in the f luid and solid phases of test plaque col-lected 30 min later, the highest for the 1,100 g F/g den-

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

Pretreatmentof enamelblocks

30 min after dentifrice use, at the momentof the cariogenic challenge

75 min after dentifrice use(45 min after cariogenic challenge)

F in the fluidphase, M

F in the solidphase, nmol/g



Placebo 2.180.6a 41.988.2a 1.180.4a 39.2811.6a

500 g F/g 9.683.1b 54.188.6 b, 1 3.782.6b 52.9822.1a

1,100 g F/g 17.284.4c 57.3810.3b 4.681.8b 48.3810.1a

Among dentifrices (within columns), means with distinct letters are significantly different (p < 0.05). Valueswere transformed to log10for statistical analysis.

1One outlier removed (F in solids = 110.0 nmol/g).


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Caries Res 2009;43:278285282

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 andsolid phases of the plaque were still signif icantly differentamong the three dentifrices (p !0.05, table 2). At thistime, the effect of pretreatment of enamel blocks with thedentifrices was signif icant, since a higher F concentrationwas observed in the plaque fluid and solids in contact

with enamel blocks that had been pretreated with thedentifrices (p !0.05, table 2).

No significant effect of pretreatment of the enamelblocks on %SHC was observed. Thus, data for nontreatedand treated enamel blocks are combined in figure 3 forclarity. The pattern of variation of %SHC with distancefrom the block edge was different according to the denti-frice used. For both F dentifrices, the distance from theblock edge did not affect %SHC, but for the placebo den-tifrice hardness loss was smaller at 2,000 and 2,500 m

ABAB

AB

AB A AA

AB

0

10

20

30

40

50

60

0 500 1,000 1,500

Distance from the block edge (m)

2,000 2,500

Placebo

500 g F/g

1,100 g F/g

%SHC

B

C

C

Fig. 2.Experiment 1: Mean and SD (n = 12)of %SHC, according to the dentifrice usedfor pretreating enamel blocks used in thein situ demineralization test. Placebo den-tifrice was used for brushing at the begin-ning of the test. No significant differencewas found among dentifrices (p 1 0.05).Uppercase letters represent the differencesbetween depths: values sharing the sameletter are not significantly different.

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

Dentifrice usedfor brushing andpretreatingenamel blocks

Pretreatmentof enamelblocks

30 min after dentifrice use, at the momentof the cariogenic challenge

75 min after dentifrice use(45 min after cariogenic challenge)





Placebo no 2.080.4 40.487.6 0.980.5 35.5810.1yes 2.180.6 41.988.2 1.180.4 39.2811.6

500 g F/g no 2458290 1938150 22.9836.1 67.3835.6yes 2038222 1788122 28.4839.1 83.1839.6

1,100 g F/g no 5088

437 3258

202 60.28

74.0 107.28

68.4yes 5258408 3768225 75.4867.1 121.3861.5

For the variables F in the f luid and solids analyzed 30 min after dentifrice use, a ll 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 denti-frices differed from each other (p < 0.05) and the effect of pretreatment of blocks was statistically significant (p < 0.05). Values weretransformed to the log10for the statistical analysis, except for F in solids at 30 min, which were transformed to the inverse of the squareroot of original data.

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from the block edge (p !0.05), without significant differ-ences among the other distances. At up to 500 m fromthe block edge, no significant difference was observed be-tween the F dentifrices, and %SHC was less than afterbrushing 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 1,100g F/g dentifrice wasused for brushing and the highest when the placebo den-tifrice was used. Also, at 2,500 m, the 1,100g F/g den-tifrice presented less loss of hardness than either of theother dentifrices, which did not differ significantly.

Discussion

The experimental model used in the present study al-lowed the distinction of different mechanisms of inhibi-tion of enamel hardness loss by F dentifrices, i.e. F in re-sidual plaque, F products formed on cleaned enamel bydentifrice application, or both. This would simulate clin-ical conditions where: (1) some plaque is not removed bybrushing, but is enriched with F available from the den-tifrice; (2) plaque is completely removed and F reacts with

enamel, but a new biofilm is formed over it; or (3) F fromdentifrice reacts with a clean enamel surface, a new bio-film is formed over it and it is exposed to F from denti-frice. However, limitations of the model mean that theresults cannot be extrapolated directly to in vivo condi-tions. One limitation is the monospecific artificial testplaque used, and its limited exposure to the dentifrices bythe contact of the dentifrice foam remaining in the mouth

after brushing and the small area of test plaque exposedto the oral cavity. Also, the cariogenic challenge is per-formed only 30 min after dentifrice use or application tothe enamel blocks, which could cause the effect of thedentifrices to be overestimated. However, the model al-lows the factors under investigation to be studied sepa-rately and, given the short-term effect observed for Fproducts formed on cleaned enamel on subsequent lossof hardness, which could not have been evaluated usinga long-term model, it was a useful method for comparingthe mechanisms involved in the anticaries effect of F den-

tifrices. Furthermore, the results give new insights for fu-ture research on the anticaries effect of F dentifrices,mouthrinses and product development.

Pretreatment of enamel blocks for 5 min with a slurryof the F dentifrices resulted in deposition of CaF2-likematerial, which was proportional to the F concentrationin the dentifrice. Although the exposure time of 5 mincould be considered too long compared to conventionalbrushing habits, in vivo the first brushing strokes wouldpromote the reaction of the undiluted dentifrice withenamel, which could increase the amount of CaF2-likematerial formed. However, the CaF2-like material formed

by the treatment with the dentifrice slurry in the presentstudy could be expected to have a different solubility be-havior of F deposits formed in vivo on enamel, in thepresence of saliva, due to phosphate contamination[Christoffersen et al., 1988].

It has been questioned whether CaF2-like materialforms after treatment with F products within clinicallyrelevant exposure times [Harding et al., 1994; ten Cate,

Placebo

500 g F/g

1,100 g F/g

0

10

10

20

30

40

50

60

%SHC

0 500 1,000 1,500

Distance from the block edge (m)

2,000 2,500

a

a

b

a

a

a

a

a

a

a

a

a

a

a

a

a

b

b bb b b b c

a

b

c

a

a

b

a

b

b

Fig. 3.Experiment 2: Mean and SD (n = 12)of %SHC in enamel blocks used in the insitu demineralization test, according tothe dentifrice used for brushing at the be-

ginning of the test. Since no significant ef-fect of pretreatment of enamel blocks wasobserved, data of pretreated and non-pre-treated blocks were combined. At each dis-tance, dentifrice means with the same low-ercase letters are not significantly differ-ent. No significant effect of distance fromthe block edge was observed for the 500-and 1,100-g F/g dentifrices, but for theplacebo dentifrice, hardness loss at 2,000and 2,500m was significantly lower thanat the other distances (p !0.05).

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Caries Res 2009;43:278285284

1997; Petzold, 2001] and our results agree with those ofBruun and Givskov [1993], who found a small increase inKOH-soluble F in enamel treated with a slurry of NaFdentifrice for 2 min. This result suggests that only smallamounts of CaF2-like material can be deposited oncleaned enamel by brushing with a fluoride dentifrice.

However, the increase of F in the fluid phase of the testplaque in contact with enamel blocks pretreated with theF dentifrices (table 1) was probably due to F release fromthe CaF2-like products formed, as previously observedfor professional F application [Tenuta et al., 2008]. On theother hand, the maintenance of these enamel reservoirsin the mouth is not expected to be long, considering thelow concentration found and the decrease in F concentra-tion in the f luid phase of plaque from the measurementsat 30 and 75 min. Additionally, the role of the CaF2-likereservoirs formed by one dentifrice application on a sub-sequent event of enamel demineralization was negligible

(fig. 2). This agrees with previous observations by others[Bruun and Givskov, 1993; ten Cate, 1997] that the lowsurface concentration of CaF2-like material formed oncleaned enamel would have an unimportant effect onsubsequent loss of enamel hardness once new plaque isformed. However, in the present study only one exposureto dentifrice was simulated, and CaF2-like material depo-sition was shown to increase by daily rinses with sodiumfluoride [Saxegaard and Rlla, 1989], and also to be high-er in caries-like lesions than on sound enamel [Bruun andGivskov, 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). Thegreater demineralization after the use of the placebo den-tifrice is in agreement with the high cariogenicity of themodel used, which allows the diffusion of sucrose throughthe extracellular polysaccharide-rich test plaque and achange in SH of 40% at the first depths of sugar penetra-tion and around 20% deeper [Zero, 1995; Cury et al.,2003]. However, after brushing with both F dentifrices,demineralization was significantly less than after brush-ing with placebo, and no significant effect of test plaquethickness (sugar diffusion distance) was observed. Also,

the results demonstrate a higher anticaries effect for theconventional F concentration dentifrice at greater thick-nesses of the test plaque, and agree with recent findingsshowing that the effectiveness of low-F dentifrices maydepend on the caries activity of the study population[Lima et al., 2008].

In fact, although the test plaque was not collected as afunction of plaque thickness, it is assumed that F from

the dentifrice foam remaining in the volunteers mouthdiffused through the plaque, since inhibition of demin-eralization was seen at greater plaque thicknesses. Also,a clear dose-response effect was observed in plaque F con-centration after brushing with both F dentifrices, whichcould cause the observed effect on enamel hardness.

The increase in F concentration in the fluid and solidphase of the test plaque after brushing suggests that everytime that toothbrushing is made with F dentifrices, rem-nants of plaque are enriched with F, which could be sus-tained by F being released from oral soft tissues [Duck-worth and Morgan, 1991]. F in the fluid phase of the bac-terial test plaque (10 times lower after 45 min, table 2)decreased more sharply than F in the solid phase (3 timeslower after 45 min), suggesting that the slow release of Ffrom bacterial reservoirs in the plaque could be respon-sible for the high F levels found in plaque fluid even 10 hafter F dentifrice use [Cenci et al., 2008]. Indeed, the

model used seems promising to study bacterial F reser-voirs and their biological role, since it excludes othertypes of mineral reservoirs potentially present in naturalplaque [Tenuta et al., 2006].

In conclusion, the results suggest that F productsformed on enamel by dentifrice application can increaseF concentration in the f luid of plaque formed on it, but toa much lower extent and with minimal effect on loss ofenamel hardness when compared to the burst of F inplaque fluid after brushing with F dentifrices. Sinceplaque-free teeth would not demineralize, the resultssuggest that F uptake by dental plaque not removed by

brushing may be the main cause of the anticaries effectof F dentifrices. Also, the higher F availability in the flu-id phase of plaque after use of conventional F concentra-tion dentifrice, and the smaller demineralization at agreater depth when compared to low-F dentifrice, suggestthat the latter should be prescribed with caution.

Acknowledgments

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


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