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Effect of topical application of fluoride gel on artificial secondary caries
inhibition
Shigemitsu Toba, DDS,a Patricia N. R. Pereira, DDS, PhD,b Toru Nikaido, DDS,
PhD,a and Junji Tagami, DDS, PhDaa

Cariology and Operative Dentistry, Department of Restorative Sciences,
Graduate School, Tokyo Medical and Dental University, Tokyo, Japan, andb
Department of Operative Dentistry, School of Dentistry, University of North
Carolina at Chapel Hill, Chapel Hill, NC, USA
Purpose: To evaluate the effect of topical application of fluoride gel on artificial secondary caries aroundadhesive restorations.
Materials and Methods: Two box-shaped cavities were prepared on both buccal and lingual dentin
surfaces of bovine roots. Each cavity was restored with either a non fluoride-releasing adhesive, Clearfil
SE Bond/a resin composite, Clearfil AP-X (AP-X), or an experimental fluoride-releasing adhesive,
ABF/AP-X. Acidulated phosphate fluoride gel (pH 4.2) was topically applied to the restorations and cavity
margins for 0 (not applying fluoride at all), 1, 2 and 4 minutes, immediately before immersion in adecalcifying solution (pH 4.5). Longitudinal sections were then cut in half through each restoration.
Depths of outer lesion and thickness of inhibition zone around restorations were determined by a confocal
laser scanning microscope. The data were analyzed by one- and two-way ANOVA and Fisher’s PLSD test
(p<0.05).
Results: Inhibition zone was observed only in ABF/AP-X with and without topical application of fluoride
gel. Topical applications of fluoride for 2 and 4 minutes significantly decreased the depth of outer lesion inboth restoration systems (p<0.05).
Conclusion: Inhibition zone formation was observed in ABF, while it was not observed in Clearfil SE
Bond. Topical application of acidulated phosphate fluoride gel for over 2 minutes was effective to decrease
the depth of the outer lesion in both adhesive systems. (Int Chin J Dent 2003; 3: 53-61.)
Clinical Significance: Application of a fluoride-releasing dentin bonding system and a fluoride gel may beclinically beneficial for root caries prevention.
Key Words: dentin bonding system, fluoride gel, fluoride-release, secondary caries inhibition.

Introduction
Recent advances in adhesive technology for use with resin composite restorative systems have resulted inroutine usage of resin-based restorations in clinical dentistry. It has been extensively reported that dentin
adhesive systems bond very well to dentin.1 However, secondary caries is still one of the main reasons for53

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the replacement of composite restorations.2
The caries process is a dynamic balance between demineralization and remineralization of the harddental tissues that may eventually result in cavitation.3 Establishment of caries that is clinically detectabledepends on both the acid and the enzymes produced by bacteria within the plaque.4-6 The products ofplaque bacteria, including lactic, acetic, and citric acid, reduce the pH on the tooth surface and demineralize
the dental tissues. Fluoride has significant effect in inhibiting caries.5,7,8 As a result, fluoride-releasing
restorative materials have been developed, with the intent to help prevent recurrent caries.9-12 Many
researchers have studied the effect of fluoride-releasing restorative materials. It has been then reported thatfluoride-releasing restorative materials were effective to inhibit secondary caries in vitro.13,14 Therefore,
fluoride-releasing restorative materials can be expected to inhibit the secondary caries.
As fluoride-releasing adhesives directly contact the cavity wall, fluoride ions released from them easily
penetrate and diffuse into the dentin at the cavity wall. Ferracane et al.15 detected fluoride ion release froma fluoride-releasing adhesive into the hybrid layer created at the resin-dentin interface. The fluoride ionspenetrating into the dentin enhance mineralization of the dentin and reduce the demineralization of thedentin.16 Therefore, the dentin penetrated by fluoride ions offers greater resistance against secondary caries
as compared to dentin that has not contacted fluoride. Topical application of a fluoride gel has also been
focused upon as a means to prevent primary caries.17 Therefore, the combination of fluoride-releasingmaterials and fluoride gel is expected to be more effective in secondary caries control.
A new evaluation method using a confocal laser scanning microscopy (CLSM) was developed toevaluate in vitro secondary caries formation and inhibition.18 Specimen preparation for CLSM is simple,
and the specimen can be observed under environmental conditions. The aim of this study was to evaluate
the effect of topical application of fluoride gel on artificial secondary caries inhibition around resin
composite restorations with non fluoride- or fluoride-releasing dentin bonding systems using CLSM.

Materials and Methods
The materials used in this study are summarized in Table 1. The dentin bonding systems used were nonfluoride-releasing dentin adhesive system, Clearfil SE Bond (SE, Kuraray Medical, Tokyo, Japan) and an
experimental fluoride-releasing adhesive system, ABF (Kuraray Medical). Both systems are two-step
self-etching primer systems. The resin composite was Clearfil AP-X (Kuraray Medical). An acidulated
phosphate fluoride gel, 2.2% APF (pH 4.2) was prepared at Department of Pharmacy in the dental hospital,
Tokyo Medical and Dental University.
Fig. 1 shows the method of specimen preparations. Sixteen bovine incisors from 20-24 month old cattle
were used in this study. The cementum was removed using a carborundum point (HP #20) to expose root
dentin. The roots were obtained by separating them from the crown segments at the cementum-enamel
junction with a low-speed diamond saw under water spray coolant. The pulp tissue was left in situ toprevent dehydration of the root dentin during the restorative procedure, and the cut surface and root apex
were sealed with utility wax, and coated with two layers of nail varnish. Two box-shaped cavities54

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Int Chin J Dent 2003; 3: 53-61.

Table 1. Materials used.
Material

Components

Batch No.

Primer: MDP, HEMA, Water, PI, Accelerators, CA

011151

Adhesive system
Clearfil SE Bond*

Bond: MDP, HEMA, MFM, PI, Accelerators, CA,
Microfiller
ABF (Experimental)*

Primer: MDPB, MDP, HEMA, MFM, PI, Water

991130

Bond: MDP, HEMA, MFM, PI, Microfiller, NaF
Resin composite
Clearfil AP-X*

Bis-GMA,TEGDMA,

Bariumglass

filler,PI,

00686A

Accelerators
Fluoride gel

2.2% NaF, Phosphoric acid, Distilled water
Sodium carboxy methyl cellulose, Glycerin (pH 4.2)

*Manufacturer; Kuraray Medical, Tokyo, Japan.
MDP; 10-methacryloyloxydecyl dihydrogen phosphate, HEMA; 2-hydroxyethyl methacrylate, PI; photoinitiator, CA;
catalyst; MFM; multi-functional methacrylate, MDPB; methacryloyloxydodecylpyridinium bromide, Bis-GMA; bisphenol
A-diglycidyl methacrylate, TEGDMA; triethyleneglycol dimethacrylate.

(approximately 3 mm long, 2 mm wide, 1.5 mm deep) were prepared on buccal and lingual dentin surfacesof the roots of bovine incisors, using a diamond bur (ISO #106) mounted in high-speed turbine with
air-water coolant. The distance between the two cavities prepared on the each buccal or lingual surface wasabout 4 mm. The cavity margins were finished with a straight fissure steel bur (ISO #010) in a slow speed
handpiece under copious water spray, to produce a cavosurface angle as close as possible to 90˚. Eightcavities were prepared for each group. Each cavity was restored with ABF/AP-X or SE/AP-X according to
the manufacturer’s instructions and stored in water for 1 week at 37˚C,12 then polished flat with polishingdisks (Rainbow Polishing Kit, Shofu, Kyoto, Japan) under running water to expose the cavity margins. The
margins were checked with a light stereomicroscope at 10 x magnification to discard any specimens withcracks evident at the margins. The teeth were coated with nail varnish except for the restorations and a 1
mm-window around cavity margins. Fluoride gel was applied to the restorations and cavity margins for 0,
1, 2, and 4 minutes, immediately before immersion in a decalcifying solution (100 mL) that was adopted
from Ten Cate et al.19 for 3.5 days at 37˚C. The acid buffer solution contained 2.2 mmol/L CaCl2, 2.2mmol/L NaH2PO4 and 50 mmol/L acetic acid adjusted to pH value of 4.5 with NaOH.
The specimens were removed from the decalcifying solution and thoroughly rinsed in running water.
Longitudinal sections were then cut through each restoration by means of a water-cooled diamond saw
microtome (Leitz 1600 Microtome, Ernst Leitz, Wetzlar, Germany) to form 2 halves. The cut surfaces ofthe specimens were polished to high gloss with abrasive discs and diamond pastes successively from 6 µm
to 0.25 µm grit size (DP-Paste, Struers, Denmark). The polished surfaces were cleaned in ultrasonic bath55

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Int Chin J Dent 2003; 3: 53-61.

and then observed using the confocal laser scanning microscope (CLSM, 1LM15W, Lasertec, Yokohama,Japan) at 500 x magnification (20 x objective lens). The CLSM is a video rate instrument, achieving aframe time of 33 ms by the use of an accousto-optic deflector for high speed scanning in one axis. This
allows rapid three-dimensional assessment of samples without damage from prolonged drying or exposure
to laser illumination. The instrument was initially developed for non-contact, high-resolution inspection of
surfaces such as integrated circuits. The images appear as surface reflection images of the sectioned
sample achieved without the use of an immersion medium.
Composite restoration
Bovine root

Nail varnish

Exposed area
1 mm-window around cavity margins

Cutting

Resin composite

Adhesive

DentinFig. 1. Sample preparation.
Adhesive

NailvarnishResin composite

Inhibitionzone (IZ)
Outer
lesion
(OL)

Denti
n

Fig. 2. Measurement of inhibition zone and outer lesion using a CLSM.

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Fig. 2 shows the measurement method of the inhibition zone and outer lesion. The features evaluatedwere depth and shape of the lesion, and presence or absence of an inhibition zone adjacent to the cavity
wall for each restoration. Since the demineralized tissue may collapse and shrink, a straight line was
extended from the top surface of the restorative material, and the depth of the outer lesion was measured
from the height to the deepest demineralized front. The height of the inhibition zone was measured fromthe top of the inhibition zone (the width of approximately 6 µm ) to the deepest demineralized front. Eightcavities from two bovine incisors were used for each group. The data were analyzed by two-way analysis
of variance (ANOVA). The two factors analyzed were material, topical application of fluoride gel or no
topical application. After that, one-way ANOVA and Fisher’s PLSD test at the confidence level of 95%
were performed (p<0.05).

Results
Fig. 3 shows a typical CLSM image of ABF/AP-X restoration. Table 2 shows the depths of outer lesion
and the heights of inhibition zone for SE and ABF.

For SE, the depths of the outer lesion wereapproximately 160 µm, when the APF gel was not applied. When acidulated phosphate fluoride gel was
topically applied to the restoration for 2 minutes, the depths of the outer lesion significantly decreased toapproximately 120 µm. There were no significant differences of the application period between 2 minutes
and 4 minutes. An inhibition area was not observed in any group.

OL
AP-X
ABF

IZ

DentinFig. 3. A typical CLSM image of ABF/AP-X restoration with topical application of the APF gel for 2
minutes. The depth of the outer lesion was approximately 120 µm. An inhibition area createdadjacent to the bonding agent was observed.

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Table 2.

Effect of topical application of fluoride gel on the depths of outer lesion and the heights
of inhibition zone for Clearfil SE Bond and ABF.

Topicalapplication

time (minutes)

Depth of outer lesion (mm)SE

Heigths of inhibition zone (mm)ABF
a169.6 (4.3)

a

SE

ABF

n.d.

62.4 (8.3) c

0159.1 (18.8)

1

146.1 (10.8)

158.3 (7.2)

n.d.

63.5 (2.4) c2

123.8 (20.0) b

115.7 (6.9) bn.d.

62.9 (3.2) c

4

113.8 (13.6) b

115.2 (19.6) bn.d.

63.7 (10.3) c

Values are mean (SD), n=8, n.d.; not determined.
Groups identified by the same superscript letters are not statistically significantly different (p>0.05).

For ABF, the depths of the outer lesion were also approximately 170 µm when the APF gel was notapplied. However, the depths of the outer lesion significantly decreased when the fluoride gel was topicallyapplied to the restoration. The depths of the outer lesion for 2 minutes and 4 minutes were approximately
120 µm. There were no significant differences of the depth of outer lesion between 2 minutes and 4
minutes. An inhibition area created adjacent to the bonding agent was observed. The heights of theinhibition zone were approximately 60 µm in all the cases.

Discussion
This study investigated the inhibitory effect of topical application of fluoride gel on artificial secondary
caries inhibition around resin composite restorations with non fluoride- or fluoride-releasing dentin bonding
systems using a CLSM. Pereira et al.12 and Okuda et al.18 focused on the caries-inhibiting effect of
restorative materials performed in a chemical-caries inducing test system. Previously, polarized lightmicroscopy (PLM) was used to evaluate outer lesions and inhibition zones. This has been a common
method for evaluation of demineralization of dental tissues and inhibition of secondary caries.11,12,20,21 Thismethod requires extensive specimen preparation procedures, such as thin slicing of the teeth, dehydration
with ascending grades of ethanol and immersion and observation in an imbibing medium of quinoline. On
the other hand, a CLSM evaluation can be performed under near normal environmental conditions resulting
in fewer artifacts. The sample preparation for CLSM is simpler than that for PLM because specimen
preparation procedures that produce artifacts are not required. In this study, outer lesions and inhibitionzone were positively detected with the CLSM just after simple sectioning and polishing procedures.
The fluoride-releasing dentin adhesive system, ABF, demonstrated the potential for artificial secondary
caries inhibition around the restoration. However, the thickness of the inhibition zone is relatively thinnerthan those created with a conventional glass-ionomer cement (GIC).18 This result agreed with those ofprevious studies.22,23 The source of the fluoride in ABF is by a proprietary treatment of NaF, which is able
to penetrate into the dentin adjacent to the restoration through the matrix. The factor that is most likely to
explain the differences is not so much the presence of fluoride, but the way in which it can be released.

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Torii et al.22 mentioned that the amount of fluoride released from the material is related not only to theconcentration of fluoride in the material, but also to whether it can spread and diffuse out from within thematerial. The latter is determined by the permeability of the materials since the presence of water in amaterial facilitates both the inward and outward diffusion of ions. The fluoride source surrounded by resin
matrix may have difficulty being in contact with water because the free movement of water is probably
limited in the cured resin matrix. This is probably related to the composition and content of the fluoride
source and hydrophilic monomers, such as HEMA.
An experimental self-etching primer system of this fluoride-releasing adhesive system is ABF, which
includes the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) against
bacteria in human dentinal carious lesions. For the non-fluoride-releasing dentin adhesive system, Clearfil
SE Bond, inhibition zones were not observed in all groups. However, Torii et al.22 noted that a thin andweak radio-opaque zone was micrographically observed using a non-fluoride-releasing dentin adhesive
system, Clearfil Linerbond II (Kuraray Medical). They suggested that this fact should be associated withthe adhesion of the restoration-to-cavity wall promoted by the adhesive system. The formation of a hybrid
layer by the infiltration of adhesive resin into the dentin is an essential mechanism for dentin adhesion.24,25
Therefore, the hybrid layer may present acid-resistance against the secondary caries formation.
Topical application of acidulated phosphate fluoride gel for over 2 minutes was effective in decreasing
the depths of the outer lesion in both ABF and SE. It was reported that topical application of fluoride gel
was effective in protection against root-surface caries.26 Garcia-Godoy et al.27 demonstrated the ability of ashorter APF application time to affect caries-like lesion depth in enamel. These findings agree with theresults of this study. The role of fluoride in preventing root-surface lesions has been shown by in vitro
studies.26,27 The mechanisms of protection against root-surface caries depend on the deposition of fluoride
within and on the root surfaces. Calcium fluoride and various fluoridated mineral phases are created assurface deposits and are incorporated into the superficial tooth surface. These fluoride-rich deposits
enhance root surface caries resistance.28 Fluoride provides an inhibitory effect against bacteria in plaqueand demineralization of root surfaces, while promoting root caries remineralization. This emphasizes theneed for fluoride in the prevention of clinically undetected root-surface lesion and benefit of various
fluoride modalities in preventing primary root-surface and enamel caries, as well as secondary caries.
Within the limitations of this laboratory study, the combination of a fluoride-releasing adhesive system andtopical application of fluoride gel may protect secondary caries formation around restorations on root
surfaces.

Conclusion
1.

Inhibition zone formation was observed in ABF using a CLSM, while it was not observed in Clearfil
SE Bond.

2.

Topical application of acidulated phosphate fluoride gel for over 2 minutes was effective to decrease
the depth of the outer lesion in both ABF and Clearfil SE Bond.

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3.

Int Chin J Dent 2003; 3: 53-61.

Application of a fluoride-releasing dentin bonding system and a fluoride gel may be clinicallybeneficial for root caries prevention.

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Reprint request to:
Dr. Toru Nikaido
Cariology and Operative Dentistry, Department of Restorative Sciences
Graduate School, Tokyo Medical and Dental University
1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan
FAX: +81-3-5803-0195 E-mail: nikaido.ope@tmd.ac.jp
Received on May 20, 2003. Revised on June 10, 2003. Accepted on June 25, 2003.
Copyright ©2003 by the Editorial Council of the International Chinese Journal of Dentistry.

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