a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 1 1 5 5 – 1 1 6 0

Effect of calcium pre-rinse and fluoride dentifrice onremineralisation of artificially demineralised enamel andon the composition of the dental biofilm formed in situ

Ana Carolina Magalhaes a, Tatiana de Almeida Furlani b, Flavia de Moraes Italiani b,Flavia Godoy Iano b, Alberto Carlos Botazzo Delbem a, Marılia Afonso Rabelo Buzalaf b,*aDepartment of Pediatric Dentistry, Faculty of Dentistry of Aracatuba, UNESP – Sao Paulo State University, Aracatuba, SP,

Rua Jose Bonifacio, 1193 Aracatuba-SP 16015-050, BrazilbDepartment of Biological Sciences, Bauru School of Dentistry, University of Sao Paulo, Bauru, SP, Al. Octavio Pinheiro Brisolla,

9-75 Bauru-SP 17012-901, Brazil

a r t i c l e i n f o

Article history:

Accepted 4 June 2007

Keywords:

Enamel

Dental biofilm

Demineralisation

Calcium

Fluoride dentifrice

a b s t r a c t

Objective: This in situ blind crossover study investigated the effect of calcium (Ca) rinse prior

to the use fluoride (F) dentifrice on remineralisation of artificially demineralised enamel and

on the composition of biofilm. Design: During four phases of 14 days, 10 volunteers wore

appliances containing two artificially demineralised bovine enamel blocks. Three times a

day, they rinsed with 10 mL of Ca (150 mM) or placebo rinse (1 min). A slurry (1:3, w/v) of F

(1030 ppm) or placebo dentifrice was dripped onto the blocks. During 1 min, the volunteers

brushed their teeth with the respective dentifrice. The appliance was replaced into the

mouth and the volunteers rinsed with water. The biofilm formed on the blocks was analysed

for F and Ca. Enamel alterations were evaluated by the percentage of surface microhardness

change (%SMHC), cross-sectional microhardness (% mineral volume) and alkali-soluble F

analysis. Data were analysed by ANOVA (p < 0.05). Results: The use of the Ca pre-rinse before

the F dentifrice produced a six- and four-fold increase in biofilm F and Ca concentrations,

respectively. For enamel, the remineralisation was significantly improved by the Ca pre-

rinse when compared to the other treatments. There was a significantly higher concentra-

tion of alkali-soluble F in enamel when the F dentifrice was used, but the Ca pre-rinse did not

have any significant additive effect. Conclusions: According to our protocol, the Ca pre-rinse

significantly increased biofilm F concentration and, regardless the use of F dentifrice,

significantly enhanced the remineralisation of artificially demineralised enamel.

# 2007 Elsevier Ltd. All rights reserved.

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journal homepage: www. int l .e lsev ierhea l th .com/ journa ls /arob

* Corresponding author. Tel.: +55 14 32358246; fax: +55 14 32343164.E-mail address: mbuzalaf@fob.usp.br (M.A.R. Buzalaf).

0003–9969/$ – see front matter # 2007 Elsevier Ltd. All rights reservedoi:10.1016/j.archoralbio.2007.06.006

young adults in most industrialised countries during the last

decades.1

F may be deposited on the enamel by formation of a CaF2-

like reservoir. During a cariogenic challenge, F released from

this reservoir may diffuse into the enamel promoting

reformation of apatite.2 In addition, demineralised enamel

1. Introduction

The cariostatic effect of fluoride (F) dentifrices has been

recognised for a long time, and, in conjunction with improved

daily oral hygiene, F dentifrice is regarded as the major factor

responsible for the dramatic caries reduction in children and

d.

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 1 1 5 5 – 1 1 6 01156

acquires larger amounts of fluoride than does sound enamel.3,4

The dental biofilm is an important reservoir of this compound.

Clinical studies have demonstrated an inverse relationship

between the F concentrations in the dental biofilm and the

prevalence of dental caries.5–7 It now appears certain that the

uptake and retention of F by dental biofilm is mainly dependent

on biofilm calcium (Ca) concentrations.1,8–13

Following this rationale, attempts to increase the cario-

static effectiveness of F have focused on methods to increase

Ca concentrations in dental biofilm. It was reported that a

150 mmol/L Ca-lactate pre-rinse significantly increased sali-

vary fluoride concentrations after the use of a 228-ppm F

rinse.14 Significant increases in F concentrations in biofilm

fluid15 and in whole biofilm16 were also reported when the Ca-

lactate pre-rinse was used.

In the studies mentioned above, the vehicle for use of F was

a rinse. However, F dentifrice is by far the most frequently

used topical fluoride agent.17 Despite some authors have

reported a significant increase in salivary F levels after the use

of F dentifrice preceded by a calcium pre-rinse,18 others have

not13 or have identified an increase only in the short time.19

This study was designed in an attempt to conciliate these

different findings. For this purpose, an in situ model using

artificially demineralised enamel was employed, in order that

besides the evaluation of biofilm F and Ca concentrations, the

associated enamel alterations could also be assessed.

2. Materials and methods

2.1. Experimental design

This study was approved by the Research and Ethics

Committee of the Bauru School of Dentistry, University of

Sao Paulo (Proc. no. 318/2003). Ten volunteers20,21 with good

oral and general health (five male and five female, mean age 24

years, mean DMFT 7.4, PHP index between 0 and 1) took part in

a blind crossover protocol conducted in four phases of 14 days,

after signing an informed consent. The following treatments

were used: Ca solution rinse and F dentifrice (Ca–F), Ca

solution rinse and placebo dentifrice (Ca), placebo solution

rinse and F dentifrice (F) and placebo solution rinse and

placebo dentifrice (P). For this purpose, the volunteers wore

appliances containing two artificially demineralised enamel

blocks. Three times/day, they rinsed with 10 mL of Ca

(150 mM) or placebo rinse (1 min). A slurry (1:3, w/v) of F

(1030 ppm) or placebo dentifrice was dripped onto the blocks.

During 1 min, the volunteers brushed their teeth with the

respective dentifrice. The volunteers replaced the appliance in

the mouth and rinsed with water. The response variables

evaluated were the percentage of surface microhardness

change (%SMHC), cross-sectional microhardness (% mineral

volume) and alkali-soluble F analysis for enamel alterations,

as well as the Ca and F concentrations present in the biofilm

formed on the blocks.

2.2. Enamel blocks and palatal appliance preparation

Enamel blocks (4 mm � 4 mm � 2.5 mm) were prepared from

incisor bovine teeth, freshly extracted, sterilised by storage in

2% formaldehyde solution (pH 7.0) for 30 days at room

temperature. The enamel surface of the blocks was ground

flat with water-cooled carborundum discs (320, 600 and 1200

grades of Al2O3 papers; Buehler, Lake Bluff, IL, USA) and

polished with felt paper wet by diamond spray (1 mm, Buehler),

resulting in removal of about 100 mm depth of the enamel. This

was controlled with a micrometer. The surface microhardness

determination was performed by five indentations (Knoop

diamond, 25 g, 10 s, HMV-2000; Shimadzu Corporation, Tokyo,

Japan).

Eighty blocks were obtained and subjected to artificial

demineralisation by immersion in 32 mL of 50 mM buffer

acetate solution [1.28 mM Ca(NO3)2�4H2O, 0.74 mM NaH2PO4�2H2O, 0.03 ppm F, pH 5.0, 37 8C], during 16 h.22 After that, the

microhardness was again evaluated and the percentage of

surface microhardness change was calculated [%SMHC

demin = 100(SMH demin � SMH sound)/SMH sound]. Blocks

with mean %SMHC around 65–90 were selected and randomly

allocated for the intra-oral phases. In order to evaluate the

profile of the lesion formed, 10 blocks were analysed for cross-

sectional microhardness (Fig. 1).

One cavity of 5 mm � 5 mm � 3 mm was made on the left

and right sides of the acrylic palatal appliances and in each

of them one block of enamel was fixed with wax. For this, a

4-mm-deep space was created in the acrylic appliance,

leaving a 1.5-mm space for plaque accumulation.23 For the

formation of dental biofilm on the enamel block, it was

protected from mechanical disturbance by a plastic mesh

fixed in the acrylic surface. The blocks were replaced after

each phase.

2.3. Treatment

Three times a day, after the meals, the volunteers rinsed with

10 mL of a 150 mM calcium lactate (Sigma–Aldrich, Atlanta,

Georgia, USA) or placebo (deionised water) solution, during

1 min. In sequence, the solution was expectorated and a slurry

(1:3, w/v) of fluoride (Crest1, NaF, 1030 ppm) or placebo

dentifrice (Crest1, Proctor & Gamble, Cincinnati, Ohio, United

States) was dripped onto the enamel blocks (3 drops/block).

During 1 min, the volunteers brushed their teeth with a pea-

size amount (�0.3 g) of the respective dentifrice. The Crest1

placebo was identical to Crest1 except that it contained no

detectable fluoride. The abrasive system in the products is

hydrated silica and they contain no detectable calcium. After

the time elapsed, the volunteers replaced the appliance in the

mouth and rinsed their mouth with 5 mL of drinking water

(0.7 ppm F). All solutions and dentifrices used were placed in

separated vials which did not allow their identification by the

volunteers, in order to conform with the blind protocol of the

study.

A 7-day washout period was allowed before the beginning

of the study and between the phases to eliminate possible

residual effect from the previous treatment. During the

experimental period, the volunteers received instructions to

wear the appliance all the time, including at night, but to

remove it during meals (1 h � 3 meals/day), when it was

involved in a gauze wet with deionised water. The volunteers

received oral and written information to refrain from using

any antibacterial or fluoridated product.

Fig. 1 – Mean % mineral volume vs. distance results. P,

placebo rinse and placebo dentifrice; Ca, Ca rinse and

placebo dentifrice; F, placebo rinse and F dentifrice; Ca–F,

Ca rinse and F dentifrice. Lesion refers to blocks submitted

to in vitro demineralisation which remained untreated.

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 1 1 5 5 – 1 1 6 0 1157

2.4. Analysis of dental biofilm

After each phase, the plastic meshes were removed, and

the dental biofilm formed on all the enamel blocks was

collected with plastic curettes, 12 h after the last exposure

to the solutions. The material was placed in preweighed

microcentrifuge tubes. The wet weight of each sample

was determined to �10 mg. For inorganic composition

analysis, 0.5 mmol/L HCl was added to the tube in the

proportion of 0.5 mL/10.0 mg plaque wet weight. After

extraction for 3 h at room temperature under constant

agitation, the same volume of TISAB II, pH 5.0, was added to

the tube as a buffer.23,24 The samples were centrifuged

(11,000 � g) for 1 min and the supernatant retained for

determination of F and Ca. In the acid extract of biofilm,

fluoride was determined with an ion-selective electrode

Orion 96-09 and an ion analyser EA-940, using the Halls-

worth et al.25 method. Ca was determined by atomic

absorption spectrometry (AAS vario 6, Analytik Jena AG,

Germany), using lantanium to suppress the phosphate

interference.

2.5. Microhardness analysis

Enamel surface microhardness was measured as described in

the Section 2.2. Five indentations, at distances of 100 mm from

each other, were made in the centre of enamel blocks before

the treatments (SMH). The SMH lesion was evaluated as

described before the in situ phase. After the in situ phase, final

microhardness test (SMH effect) was made. The percentage of

surface microhardness change was calculated as follows:

%SMHC effect = (SMH effect � SMH demin)/(SMH sound �SMH demin) � 100.

To perform cross-sectional microhardness (CSMH) tests,

the blocks of each volunteer in each treatment were long-

itudinally sectioned through the centre, embedded and

polished. Three rows of eight indentations each were made,

one in the central region of the dental enamel exposed and the

other two 100 mm below and above this, under a 25-g load for

10 s. The indentations were made at 10, 30, 50, 70, 90, 110, 220

and 330 mm from the outer enamel surface. The mean values

at all three measuring points at each distance from the surface

were then averaged. The area under the KHN � mm curve was

calculated using the trapezoidal rule, according to Feather-

stone et al.26

2.6. Determination of alkali-soluble F in enamel

The concentration of F was evaluated in the other half of the

blocks that was not used for CSMH analysis.27 A circular

hole (2.0 mm diameter) was punched in adhesive tape,

which was applied firmly to the centre of the enamel block.

The remaining surfaces of the block were painted with wax

so that only a 3.14 mm2 surface area was exposed. The block

was then placed in a plastic test tube containing 0.30 mL of

1 M KOH for 24 h under constant agitation. An equal volume

of TISAB II (containing HCl) was added. This solution

was analysed with the electrode, along with standards

containing from 0.025 to 3.2 mg F/mL. Data were expressed

as mgF/mm2.

2.7. Statistical analysis

Data passed normality and homogeneity tests and were then

analysed by repeated measures two-way ANOVA and Bonfer-

roni test (for CSMH) and repeated measures ANOVA and

Tukey–Kramer test (for the other variables), using the Soft-

wares GraphPad Prism 4 and GraphPad Instat, respectively. A

significance level of 0.05 was selected a priori.

3. Results

According to Fig. 1, the enamel softening after artificial

demineralisation (baseline) was more superficial, characteris-

ing a surface-softened lesion. The mean %SMHC for all the

groups was similar (around 73%), due to the randomisation

process. The % mineral volume at 10 mm depth was around

40% and the mineral loss occurred approximately up to 90 mm

depth (78% mineral volume).

Table 1 shows the F and Ca concentrations in biofilm,

according to the treatments. The mean F concentration was

higher for the groups where F dentifrice was used (F and Ca–F).

There was a six-fold increase in F concentration when the Ca

pre-rinse was used along with the F dentifrice (F compared to

Ca–F, p < 0.05). A similar result was found for Ca concentra-

tions. When the Ca pre-rinse was used along with F dentifrice,

a four-fold increase in biofilm Ca concentration was seen,

when compared to the Ca rinse alone (Ca compared to Ca–F,

p < 0.05).

Table 2 shows the results of enamel blocks analysis with

respect to %SMHC, % mineral volume, as well as alkali-soluble

F concentrations, according to the treatments. In respect to

%SMHC, the use of Ca rinse alone significantly enhanced the

rehardening of enamel when compared to all the other groups

(p < 0.05). As for CSMH, all the treatments increased the %

mineral volume, but only the groups where the Ca rinse was

used (Ca and Ca–F) presented a significant increase when

compared to placebo (p < 0.05) at a distance of 10 mm from the

surface. For the other distances, no significant differences

among the groups were seen (Fig. 1). The area under the

KHN � mm curve was similar for all groups (data not shown).

Table 1 – Mean (WS.E.) Ca and F concentrations in thebiofilm, according to the different treatments

Treatmentsa Calcium (mg/mg) Fluoride (ng/mg)b

P 1.76 � 0.45a 11.89 � 1.50a

Ca 7.17 � 2.09b 11.59 � 2.48a

F 2.99 � 0.36a 176.09 � 32.72b

Ca–F 26.20 � 3.63c 1079.10 � 184.69c

Values in the same column followed by distinct superscript letters

indicate statistical significance ( p < 0.05).a P, placebo rinse and placebo dentifrice; Ca, Ca rinse and placebo

dentifrice; F, placebo rinse and F dentifrice; Ca–F, Ca rinse and F

dentifrice.b Data were submitted to log transformation before analysis.

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 1 1 5 5 – 1 1 6 01158

There was a significantly higher concentration of alkali-

soluble F in enamel when the F dentifrice was used, but the Ca

pre-rinse did not have any significant additive effect.

4. Discussion

Tooth brushing with F dentifrice is an important public health

measure for the control of dental caries since it combines the

removal or disruption of dental biofilm with the cariostatic

effect of F. Because it is not possible to completely remove

biofilm by brushing, the amount of F incorporated can play an

important role in caries control.

There is considerable evidence showing that the uptake

and retention of F are directly related to the amount of Ca in

biofilm.8–13,28–31 In this study, the Ca lactate rinse prior to the

use of F caused a six-fold increase in biofilm F concentrations,

which are lower than the findings reported in a study in which

the effect of a 150 mM calcium lactate pre-rinse on F uptake

from a 228-ppm F rinse by whole biofilm was tested.14 This

difference may be attributed to the different F vehicles used (F

dentifrice � F rinse), since the surfactant sodium lauryl

sulphate present in dentifrice has been reported to have a

strong affinity for Ca,32,33 which could affect the availability of

ionic Ca and, hence the uptake of F fluoride by biofilm during

the use of the dentifrice. In addition, the results of the study

mentioned above14 refer to the biofilm collected 1 h after the

Table 2 – Mean %SMHC (WS.D.), % mineral volume (WS.D.)and F concentrations (WS.E.) in the enamel

Treatmentsa %SMHC % Mineralvolume (10 mm)

F (mg/mm2)

P 48.23 � 19.08a 58.06 � 14.3a 1.47 � 0.32a

Ca 67.69 � 16.01b 73.71 � 9.3b 1.17 � 0.49a

F 53.82 � 16.30a 63.80 � 13.9a 8.48 � 1.72b

Ca–F 52.98 � 8.87a 74.09 � 11.3b 9.14 � 2.53b

Values in the same column followed by distinct superscript letters

indicate statistical significance ( p < 0.05). The mean %SMHC and

mineral volume (10 mm) of the surface-softened specimens with-

out treatment was 73% and 37%, respectively.a P, placebo rinse and placebo dentifrice; Ca, Ca rinse and placebo

dentifrice; F, placebo rinse and F dentifrice; Ca–F, Ca rinse and F

dentifrice.

exposure to F, whilst in our study the biofilm was collected

12 h after the exposure. On the other hand, the increase seen

in biofilm F concentrations when the Ca pre-rinse was used

may seem contradictory to the results reported previously by

our research group.19 However, it must be pointed out that in

the study by Pessan et al.19 the biofilm was allowed to

accumulate only for 12 h before collection, because the teeth

surfaces were regularly brushed. In the present study, the

biofilm remained undisturbed and protected by the plastic

mesh during the whole experimental period (14 days), which

allowed a cumulative effect. This, in addition to the reduction

in the bioavailability of F present in the dentifrice (due to the

existence of sodium lauryl sulphate), may have led to a smaller

F uptake in the study by Pessan et al.19

A similar result was found for Ca concentrations. When the

Ca pre-rinse was used along with F dentifrice, a four-fold

increase in biofilm Ca concentration was seen, when

compared to the Ca rinse alone (p < 0.05). This was expected,

due to the close association between biofilm F and Ca

concentrations, which have been extensively reported.1,8–13

In fact, in the present study, the increase in biofilm Ca by the

Ca–F treatment was around 610 nmol/mg, whilst the increase

in biofilm F was around 56 nmol/mg (Table 1). If the only

increase in plaque Ca were due to the formation of CaF2, then

this would account for around 28 nmol Ca/mg, which is only

5% of the calcium incorporated. This is consistent with the

observation that the fraction of biofilm calcium involved in the

retention and uptake of F does not exceed 1–4%. 12,13,19 Thus,

most of the Ca incorporated after the Ca–F treatment was not

bonded to F (CaF2). Additional studies are required to clarify

the mechanism of Ca uptake in dental biofilm when a Ca rinse

is used prior to an F dentifrice. Some authors have suggested

that the high F concentration in the F dentifrice causes the

formation of large amounts of CaF2, which accumulates on the

surface of the biofilm and could act as a diffusion barrier.13,19

This could restrict the loss of Ca previously incorporated in the

biofilm due to the Ca rinse, thus considerably increasing the

measured Ca concentration.

The alkali-soluble F concentration in enamel, similarly to

what was reported for the biofilm, significantly increased when

the F dentifrice was used, but the Ca pre-rinse did not

significantly improve the performance of the F dentifrice. The

in situ design employed may have contributed to this result,

since a space was created between the enamel block surface

and the plastic mesh in order to allow biofilm accumulation.

Thus, it is possible that the layer of biofilm deposited on the

enamel block may have impaired the deposition of CaF2

globules on enamel surface. In addition, it is well known that

F present in phosphate- and protein-covered CaF2 deposits

formed after a topical F application is released by decreasing the

pH, when the phosphate groups become protonated.34 In the

present study, the absence of cariogenic challenges may have

diminished the dissolution of the CaF2 deposits present in the

biofilm, thus reducing the release of the respective ions to

enamel, which is consistent with the higher Ca and F

concentrations found in the biofilm formed in the Ca–F group

(Table 1). Further clinical trials should evaluate if higher degrees

of fluoride uptake in enamel are obtained when the biofilm is

constantly beingremovedbytoothbrushingand/ormastication

and when cariogenic challenges are normally performed.

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 1 1 5 5 – 1 1 6 0 1159

Despite the increase in the biofilm F and Ca concentrations

caused by the use of the Ca pre-rinse before the F dentifrice,

the degree of surface remineralisation was not significantly

increased. It was also noteworthy that the Ca rinse alone had a

better performance than when in association with the F

dentifrice. It is important to mention that there was a

rehardening around 50% of the enamel surface for the placebo

group. In addition, the lesion, even for this group, was

restricted to the outer 10 mm of enamel. This indicates that

the exposure of the blocks to saliva alone during 14 days was

able to remineralise the enamel blocks to a high extent. If this

study had been conducted for a shorter period of time, the

detection of the differences in the degree of remineralisation

of the deeper layers of enamel could have been possible. It is

noteworthy that the Ca pre-rinse before the use of F dentifrice

was also effective in order to remineralise the subsurface, but

was not significantly different from the Ca rinse alone. Thus, it

seems that this effect was more due to the use of Ca than to the

use of F. In this regard, it must be noted that the use of an intra-

oral Ca rinse and an extra-oral F exposure could have favoured

the Ca rinse alone since the enamel samples have had a better

exposure to the Ca than to the F.

In conclusion, according to our protocol, the use of the Ca

pre-rinse before the F dentifrice significantly increased biofilm

F and Ca concentrations – and regardless the use of F dentifrice

– it significantly enhanced the remineralisation of artificially

demineralised enamel.

Acknowledgements

We thank FAPESP for the concession of a grant to the last

author (Proc. 01/13588-9) and also a scholarship to the second

author (Proc. 04/11653-6), which permitted the conclusion of

the study. This publication was based on a thesis submitted by

the second author to Bauru Dental School, University of Sao

Paulo, in partial fulfilment of the requirements for a MS degree

in Oral Biology. We are grateful to Dr. Vanessa Eid da Silva

Cardoso for the calcium analysis.

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