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OFInfluence of human dentine on the antibacterial activity of

self-etching adhesive systems against cariogenic bacteria

Juliana O. Gondim a, Cristiane Duque b, Josimeri Hebling a, Elisa M.A. Giro a,*aDepartment of Paediatric Dentistry, School of Dentistry of Araraquara, Sao Paulo State University, UNESP, Rua Humaita,

1680 Araraquara, SP, BrazilbDepartment of Oral Diagnosis, School of Dentistry of Piracicaba, Sao Paulo State University, UNICAMP, Av. Limeira,

907 Piracicaba, SP, Brazil

j o u r n a l o f d e n t i s t r y x x x ( 2 0 0 8 ) x x x – x x x

a r t i c l e i n f o

Article history:

Received 27 September 2007

Received in revised form

7 December 2007

Accepted 22 December 2007

Keywords:

Dentine bonding agents

Bacteria

Antibacterial agents

Dentine

Dentine permeability

a b s t r a c t

Objectives: The incorporation of antibacterial agents into adhesive systems has been pro-

posed to eliminate residual bacteria from dentine. This study used the agar diffusion

method to evaluate the antibacterial activity of clearfil protect bond (CPB), clearfil SE bond

(CSEB), clearfil Tri-S bond (C3SB) and Xeno-III (XIII) self-etching adhesive systems, with or

without light-activation, against cariogenic bacteria, and to assess the influence of human

dentine on the antibacterial activity of these materials.

Methods: An aliquot of 10 ml per material (and individual components) were pipetted onto

paper and dentine discs distributed in Petri dishes containing bacterial culture in BHI agar.

Positive control was 0.2% chlorhexidine digluconate (CHX).

Results: After incubation, the adhesive components of CPB and CSEB, liquid A of XIII and

C3SB did not present antibacterial activity when applied to paper discs. The non-light-

activated CPB primer + adhesive promoted the greatest inhibition of Streptococcus mutans

( p < 0.05), whereas with light-activation, there was no significant difference between

primer + adhesive and primer alone. For Lactobacillus acidophilus, CPB primer presented

the greatest antibacterial activity in both light-activation conditions (p < 0.05). Regarding

the dentine discs, only CHX promoted an inhibitory effect, though less intense than on paper

discs ( p < 0.05). CHX presented greater antibacterial activity against S.mutans than against L.

acidophilus ( p < 0.05).

Conclusions: Light-activation significantly reduced the antibacterial activity of the self-

etching adhesive systems; MDPB incorporation contributed to the effect of adhesive systems

against cariogenic bacteria; the components eluted from the adhesive systems were not

capable to diffuse through 400 mm-thick dentine disc to exert their antibacterial activity

against cariogenic bacteria.

# 2008 Elsevier Ltd. All rights reserved.

avai lab le at www.sc iencedi rec t .com

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

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U1. Introduction

Although the etiological factors of caries disease and the

methods for its prevention have been widely investigated,

* Corresponding author at: Faculdade de Odontologia de AraraquarAraraquara, SP, CEP 14801-903, Brazil. Tel.: +55 16 3301 6336.

E-mail address: egiro@foar.unesp.br (Elisa M.A. Giro).

0300-5712/$ – see front matter # 2008 Elsevier Ltd. All rights reserveddoi:10.1016/j.jdent.2007.12.007

Please cite this article in press as: Gondim JO, et al., Influence of hu

systems against cariogenic bacteria, Journal of Dentistry (2008), do

secondary caries is still the main cause for replacement of

restorations.1,2 During removal of carious tissue, bacteria

invariably remain entrapped in the dentinal substrate because

neither the clinical parameters of dentine hardness and colour

a, Departamento de Clınica Infantil Rua Humaita, 1680 Centro

.

man dentine on the antibacterial activity of self-etching adhesive

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JJOD 1183 1–8

nor the caries-detector dyes are able to ensure complete

elimination of microorganisms.3,4 As the contemporary

dentine adhesive systems have not proved completely

effective in eliminating microleakage at tooth/restoration

interface, the incorporation of antibacterial agents into these

materials has been proposed in an attempt to prevent caries

recurrence.5–15

Among the antibacterial agents incorporated into adhesive

materials, the resin monomer 12-methacryloyloxydodecyl-

pyridinium bromide, known as MDPB, stands out.8,9,13 The

main advantage of MDPB is its capacity to copolymerize with

other resin monomers being immobilised within the polymer

matrix, which confers safety and prolonged antibacterial

action to this agent, as it does not leach to the medium. This

characteristic also ensures a good survival rate for the

restoration, as MDPB, unlike soluble antibacterial agents, is

not deleterious to the physical and mechanical proprieties of

the materials to which it is incorporated.8,9,16

The incorporation of MDPB into self-etching adhesive

systems has been advocated as extremely advantageous

because, in the clinical practice, these materials are directly

applied to the dentine with some level of contamination and,

unlike the two-step or three-step total-etching adhesive

systems, they do not have a separate acid etching step in

their protocol, which could reduce significantly the number of

residual microorganisms.14,17,18 In spite of the acidic nature of

self-etching adhesive systems, this characteristic does not

seem sufficient to completely eliminate residual bacteria from

dentine because this tissue acts as a solid buffering medium to

acidic monomers.15,19 Therefore, for the self-etching adhesive

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Table 1 – Specifications, main components and manufacturer

Material (manufacturer) Type

Clearfill protect bond (CPB)

(Kuraray Medical Inc.

Okayama, Japan)

Two-step self-etching

adhesive system

Prim

dimet

Adhes

hydro

N,N-d

CQ, si

BisGM

Clearfil SE bond (CSEB),

(Kuraray Medical Inc.,

Okayama, Japan)

Two-step self-etching

adhesive system

Prim

dimet

p-tolu

HEMA

dimet

N,N-d

CQ, si

silica,

Clearfil Tri-S bond (C3SB)

(Kuraray Medical Inc.

Okayama, Japan)

One-step self-etching

adhesive system

HEM

dimet

silica,

Xeno-III (XIII) (Dentsply,

Konstanz, Germany)

One-step self-etching

adhesive system

Liqui

THB, a

B: Piro

Ethyl

0.2% chlorhexidine digluconate

(CHX) (University Pharmacy,

UNESP, Araraquara, SP, Brazil)

Antibacterial solution 0.2%

aqueo

BisGMA: bisphenol A glycidyl dimethacrylate (BisGMA); THB: toluene

methacrylate; MDP: 10-methacryloxydecyl dihydrogen phosphate; MD

fluoro phosphazene modified methacrylate; Piro-EMA: phosphoric acid m

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systems against cariogenic bacteria, Journal of Dentistry (2008), do

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systems to inhibit the microorganisms that remain lodged

inside the dentinal tubules, they should present antibacterial

activity and capacity to diffuse to some extent through the

dentin.19

However, there is little published information on the

influence of dentine on the antibacterial properties of self-

etching adhesive systems.13,19,20 Therefore, the aim of this

study was to evaluate the antibacterial activity of self-etching

adhesive systems, with or without light-activation, against

cariogenic bacteria, and to assess the influence of the

interposition of 400 mm-thick human dentine discs on the

antibacterial activity of these materials using the agar

diffusion method.

2. Materials and methods

The specifications, main components and manufacturers of

the materials used in this study are presented in Table 1. The

antibacterial activity of the materials was evaluated against

the following bacterial strains: Streptococcus mutans (UA-159)

and Lactobacillus acidophilus (ATCC #IAL-523).

2.1. Agar diffusion method: application on paper discs

CPB, CSEB, C3SB and XIII self-etching adhesive systems, as

well as their individual components, were submitted to the

agar diffusion method. The positive and negative controls

were, respectively, 0.2% chlorhexidine digluconate (CHX) and

paper discs not impregnated with any material (PD).

CT

s of the tested materials

Composition pH Batch#

er: HEMA, MDP, hydrophilic

hacrylate, MDPB, water.

ive: HEMA, MDP,

phobic dimethacrylate,

iethanol-p-toluidine,

lanized colloidal silica,

A, sodium fluoride

P: 1.9. A: 2.8 41,137

er: HEMA, MDP, hydrophilic

hacrylate, N,N-diethanol-

idine, CQ, water. Adhesive:

, MDP, hydrophobic

hacrylate,

iethanol-p-toluidine,

lanized colloidal

BisGMA

P: 1.9. A: 2.8 51,308

A, BisGMA, MDP, hydrophobic

hacrylate, silanized colloidal

CQ, ethanol, water

2.4 61,113

d A: HEMA, water, ethanol,

morphous silica. Liquid

-EMA, PEM-F, UDMA, THB, CQ,

4-dimethylaminobenzoate

1.0 (mixed) 0,504,002,710

chlorhexidine digluconate

us solution

5.9 –

hydroxybutyrate; CQ: D,1-camphorquinone; HEMA: 2-hydroxyethyl

PB: 12-methacryloyloxydodecyl pyridinium bromide; PEM-F: mono

odified methacrylacte; UDMA: urethane dimethacrylate.

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Fig. 1 – (A) 400 mm-thick dentine disc with a diameter of

8 mm. (B) Dentine disc with a protective barrier delimiting

a 6 mm diameter area. (C) Distribution of the specimens in

agar plate.

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For the agar diffusion method, the standard bacterial strains

were first reactivated in 5 ml of BHI broth (Brain Heart Infusion,

BHITM; Difco Laboratories, Detroit, MI, USA) for 24 h at 37 8C

under conditions of microaerophilia. From this culture, another

replication was performed in 5 ml of BHI broth and maintained

for 24 h at 37 8C in microaerophilia, which allowed obtaining an

inoculum containing 109 CFU/ml. To verify and standardize the

number of microorganisms present in the inoculum, serial

dilutions were performed up to 10�6.

An aliquot of 250 ml of one of the bacterial inocula was

spread onto the surface of Petri dishes containing 15 ml of BHI

agar. Because of the number of groups (n = 12), two Petri dishes

were necessary to distribute the specimens in such way that

on each dish seven paper discs (6 mm diameter; 1.5 mm thick)

were used. Five discs represented five different adhesive

groups, and two discs the control groups, which were repeated

for each Petri dish. A total of 8 specimens were used per group,

except for the positive and negative controls (n = 16). In the

group of specimens that were light activated, all materials

(including the positive and negative controls) were irradiated

for 10 s with a halogen light curing unit (Optilux 500; Kerr

Company, Orange, CA, USA) with a light intensity not lower

than 400 mW/cm2.

The dishes were maintained at room temperature for 2 h

for pre-diffusion of the materials and were incubated at 37 8C

in microaerophilia during 24 h for S. mutans and 48 h for L.

acidophilus. The diameter of the bacterial growth inhibition

zones produced around the paper discs impregnated with the

tested materials was measured at two regions in millimetres

using a digital calipter (Mitutoyo, Kawasaki, Japan). The largest

diameters measured horizontally and vertically were recorded

and an average was calculated for each inhibition zone. The

diameter of the paper disc (6 mm) was subtracted from this

value and the result was divided by two to obtain the width of

the inhibition zone of each specimen.

For positive and negative control groups eight paper discs

were randomly selected for analysis.

2.2. Agar diffusion method: application on dentine discs

In this assay, only the materials that presented antibacterial

activity by the agar diffusion method on paper discs were used

with the same bacterial strains. CHX was used as a positive

control applied onto dentine discs (CHXd) and paper discs

(CHXp).

Eight-four sound human third molars obtained from the

tooth bank of the School of Dentistry of Araraquara (UNESP,

Araraquara, SP, Brazil) were used after approval of the

research project by the institutional Ethics in Human Research

Committee (Process #54/05). The teeth were used within 6

months after extraction. For each tooth, a dentine disc of

approximately 500 mm thickness was obtained by transversal

section of the tooth at the cervical third using a water-cooled

diamond saw (Series 15 LC Diamond; Buehler, Lake Buff, IL,

USA) in a low-speed cutting machine (Isomet 1000; Buehler,

Lake Buff, IL, USA). The dentine discs were manually ground

with 320-grit silicon carbide paper until reaching a thickness

of 400 mm and were stored in phosphate buffer saline (PBS), pH

7.2, at �48C until the moment of dentine permeability

determination.

Please cite this article in press as: Gondim JO, et al., Influence of hu

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2.3. Determination of dentine permeability

The dentine discs were bilaterally etched with 0.5 M EDTA

solution, pH 7.2, for 30 s and thoroughly washed with deionised

water to remove the smear layer created during grounding.

Thereafter, the discs were individually held in a device known

as in vitro pulp chamber, modified from the original apparatus

developed by Hanks et al.,21 which allowed standardisation of a

dentine area of 0.28 cm2 for assessment of permeability. The

artificial pulp chamber was connected to an appliance (Flodec;

De Marco Engineering, Geneva, Switzerland) by means of a

0.5 mm-diameter polyethylene tube for dentine permeability

reading. A small air bubble was introduced into the system and

15 cm H2O hydrostatic pressure was applied. The air bubble

movement was recorded by an appliance’s infrared sensor and

transformed in dentine permeability value using proprietary

software (De Marco Engineering, Geneva, Switzerland). Based

onthe results of these readings, the dentinediscswereallocated

in 14 experimental groups with six discs each in such a way that

the mean dentine permeability of the groups did not differ

statisticaly (ANOVA; p > 0.05).

2.4. Determination of antibacterial activity

The 400 mm-thick dentine discs were reduced using #1090

diamond burs (KG Sorensen, Barueri, SP, Brazil) and 320-grit

SiC paper to a standardized diameter of 8 mm (Fig. 1A). The

occlusal surface of the discs was wet ground with 320-grit SiC

paper for 5 s to produce a new smear layer.22 On that surface a

dam was built up surrounding the periphery of the disc with

polyether impression material (Impregum Penta Soft; 3M

ESPE, Seefeld, Germany) using a PentaTM Elastomer syringe

(3M ESPE, Seefeld, Germany), in such a way to delimit a 6 mm

diameter area for application of the tested materials and to

avoid extravasation of the adhesives (Fig. 1B). The discs were

stored for 24 h in saline for complete setting of the impression

material and were thereafter autoclaved at 120 8C for 20 min.

The bacterial suspensions of S. mutans and L. acidophilus

were seeded onto six Petri dishes each, containing 15 ml of BHI

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Table 2 – Width of the inhibition zones (mm) obtained for S. mutans (median, minimum and maximum) according to thematerial and light-activation condition by the agar diffusion test in paper discs

Material Without light-activation With light-activation

Clorexidina (CHX) (n = 8) 4.65 (3.88–5.00) Bb 5.09 (4.40–5.88) Aa

Paper disc (PD) (n = 8) 0 0

Clearfil protect bond-primer (CPB-P) (n = 8) 5.05 (3.65–6.56) Ab 4.75 (3.84–5.54) Aa

Clearfil protect bond-adhesive (CPB-A) (n = 8) 0 0

Clearfil protect bond-primer + adhesive (CPB-P + A) (n = 8) 6.92 (6.20–7.74) Aa 5.04 (4.08–6.13) Ba

Clearfil SE bond-primer (CSEB-P) (n = 8) 4.06 (3.40–4.56) Ac 1.96 (1.68–2.23) Bc

Clearfil SE bond-adhesive (CSEB-A) (n = 8) 0 0

Clearfil SE bond-primer + adhesive (CSEB-P + A) (n = 8) 3.39 (3.01–3.68) Ad 2.43 (1.73–3.19) Bb

Clearfil Tri S bond (C3SB) (n = 8) 0 0

Xeno-III liquid A (XIII-A) (n = 8) 0 0

Xeno-III liquid B (XIII-B) (n = 7) 3.04 (2.38–3,33) Ae 2.06 (1.79–3.21) Bbc

Xeno-III liquid A + liquid B (XIII-A + B) (n = 7) 2.19 (1.06–2.81) Af 1.46 (1.16–1.51) Bd

Different uppercase letters in lines and lowercase letters in rows indicate statistically significant difference at 5%.

j o u r n a l o f d e n t i s t r y x x x ( 2 0 0 8 ) x x x – x x x4

JJOD 1183 1–8

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agar. One paper and seven dentine discs were distributed in

each dish. After that 10 ml of the positive control (CHX) and the

other tested materials were pipetted on their surface. The

adhesive systems were not light-activated. After incubation of

the Petri dishes at 37 8C under conditions of microaerophilia

during 24 h (S. mutans) and 48 h (L. acidophilus), the width of the

inhibition zones produced around each specimen was

calculated in the same way as described for the agar diffusion

method with paper discs (Fig. 1C).

2.5. Statistical analysis

The Mann–Whitney non-parametric test was used to evaluate

the influence of light-activation on the antibacterial activity of

each adhesive system when using the agar diffusion method

with paper discs, and Kruskal–Wallis and Mann–Whitney tests

were used to compare the materials under each light-

activation condition. The evaluation of the influence of human

dentine on the materials’ antibacterial activity was performed

by comparing the widths of the bacterial growth inhibition

zones of the groups, using the Mann–Whitney test. Spear-

man’s correlation was also used to verify the existence of a

correlation between dentine permeability and inhibition zone

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Table 3 – Width of the inhibition zones (mm) obtained for L. athe material and light-activation condition by the agar diffusi

Material

Chlorhexidine (CHX) (n = 8)

Paper disc (PD) (n = 8)

Clearfil protect bond-primer (CPB-P) (n = 8)

Clearfil protect bond-adhesive (CPB-A) (n = 8)

Clearfil protect bond-primer + adhesive (CPB-P + A) (n = 8)

Clearfil SE bond-primer (CSEB-P) (n = 8)

Clearfil SE bond-adhesive (CSEB-A) (n = 8)

Clearfil SE bond-primer + adhesive (CSEB-P + A) (n = 8)

Clearfil Tri S bond (C3SB) (n = 8)

Xeno-III liquid A (XIII-A) (n = 8)

Xeno-III liquid B (XIII-B) (n = 6)

Xeno-III liquid A + liquid B (XIII-A + B) (n = 6)

Different uppercase letters in lines and lowercase letters in rows indicat

Please cite this article in press as: Gondim JO, et al., Influence of hu

systems against cariogenic bacteria, Journal of Dentistry (2008), do

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Rwidths. A significance level of 5% (a = 0.05) was set for all

statistical tests.

3. Results

3.1. Agar diffusion method: application to paper discs

The medians of inhibition zone widths and the minimum and

maximum values according to the bacterial strain, material

and light-activation condition are presented in Tables 2 and 3.

Regardless of the type of microorganism and presence/

absence of light-activation, no inhibition zone was produced

with the adhesive components of clearfil protect bond (CPB-A)

and Clearfil SE bond (CSEB-A), liquid A of Xeno-III (XIII-A) and

clearfil Tri-S bond adhesive system (C3SB).

3.2. S. mutans

Clearfil protect bond (primer + adhesive) without light-activa-

tion produced the largest inhibition zone (p < 0.05), followed

by CPB primer (CPB-P) and CHX, which did not differ to each

other (p > 0.05). With light-activation, there was no statisti-

cidophilus (median, minimum and maximum) according toon test in paper discs

Without light-activation With light-activation

5.06 (4.03–5.44) Ac 4.47 (4.03–5.93) Ad

0 0

7.15 (6.99–8.37) Aa 6.93 (6.34–7.64) Aa

0 0

6.52 (6.21–7.00) Ab 6.12 (5.62–6.59) Bb

6.37 (5.84–7.46) Ab 5.59 (3.65–7.48) Abcd

0 0

6.52 (6.19–7.05) Ab 5.62 (5.19–6.13) Bc

0 0

0 0

0.68 (0.40–0.78) Ad 0.80 (0.42–0.96) Ae

0.66 (0.58–1.10) Ad 0.58 (0.49–0.73) Ae

e statistically significant difference at 5%.

man dentine on the antibacterial activity of self-etching adhesive

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cally significant difference between CPB components together

(CPB-P + A) and the primer alone (CPB-P), which presented

similar inhibition to that of CHX (p > 0.05). Regarding the

effect of light-activation on the inhibitory potential, the

materials, in general, exhibited lower antibacterial activity

after light-activation (p < 0.05), except for CPB-P, which

results with and without halogen light curing did not differ

significantly to each other (Table 2).

3.3. L. acidophilus

CPB-P presented the greatest antibacterial activity under both

conditions of curing (p < 0.05). Regarding the effect of light-

activation, there was no statistically significant difference

(p > 0.05) among the tested materials, except for CPB-P + A

and CSEB-P + A, which exhibited greater inhibitory potential

without light-activation (p < 0.05) (Table 3).

3.4. Agar diffusion method: application to dentine discs

When the dentine disc was interposed, none of the tested self-

etching adhesive systems presented antibacterial activity.

Although CHX presented an inhibitory effect against S. mutans

and L. acidophilus, it was less intense than that observed on the

paper disc (p < 0.05). In addition, CHX showed greater

antibacterial activity against S. mutans than against L.

acidophilus (p < 0.05) (Table 4). There was no positive correla-

tion between the degree of permeability of the dentine discs

and the produced antibacterial effect (r = �0.119; p > 0.05).

4. Discussion

The antibacterial activity of the tested self-etching adhesive

systems and their individual components with and without

light-activation was evaluated against S. mutans and L.

acidophilus, the main pathogens directly related to the

development of dental caries.23 The agar diffusion method

was used for being a widely employed methodology for

determination of the inhibitory activity of liquid substances, in

addition to being a simple and easy-to-handle technique and

facilitate the comparison to other studies.9,13,15,19,20,24

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Table 4 – Widths of the inhibition zones (mm) obtained for S. maccording to the group by the agar diffusion test in dentin dis

Group

Chlorhexidine in paper disc (CLXp)

Chlorhexidine in dentin disc (CLXd)

Clearfil protect bond-primer (CPB-P)a

Clearfil protect bond-primer + adhesive (CPB-P + A)a

Clearfil SE bond-primer (CSEB-P)a

Clearfil SE bond-primer + adhesive (CSEB-P + A)a

Xeno-III liquid B (XIII-B)a

Xeno-III liquid A + liquid B (XIII-A + B)a

Different uppercase letters in lines and lowercase letters in rows indicata Uncured dentine bonding agents.

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systems against cariogenic bacteria, Journal of Dentistry (2008), do

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Corroborating the findings of Imazato et al.,24 it was

observed that the non-light-activated materials presented

significantly greater antibacterial activity in the groups CPB-

P + A, CSEB-P, CSEB-P + A, XIII-B and XIII-A + B when the

bacterial strain was S. mutans, and for the groups CPB-P + A

and CSEB-P + A, when the bacterial strain was L. acidophilus

(p < 0.05). Nevertheless, even with light-activation, there was

some antibacterial activity for these materials when applied to

paper discs. The polymerization of adhesive materials causes

entrapment in the polymeric matrix and decreases the release

of polymerizable antibacterial components, as well as adhe-

sion-promoting acidic monomers.24 Therefore, the formation

of inhibition zones of microbial growth when the adhesive

systems were light-activated may be explained by the fact that

a complete conversion of monomers into polymers does not

occur and hence residual monomers can be released to the

medium.25 Furthermore, the halogen light is not capable of

passing through the paper disc impregnated with the adhesive

components adequately, what added to the deficient blown off

water present in the primer composition when it is applied

onto paper discs, prevents great part of the monomers from

forming polymeric chains.24 The antibacterial effect might

also be due to the release of residual monomers present in the

oxygen-inhibited layer.26

The antibacterial action of adhesive systems may be

influenced by factors inherent to the material itself (pH,

viscosity, diffusion capacity and presence of antibacterial

agents) and factors related to dentine substrate (thickness and

permeability).14,15,19,24,27–31 The acidic nature of the primer of

self-etching adhesive systems has been considered as one of

the key factors related to bacterial inhbition.24,25,27,29 Phenyl-P,

4-META and MDP are the main acidic monomers contained in

the formulation of self-etching adhesive systems and are

capable of demineralizing and infiltrating simultaneously the

dentine substrate. According to Ohmori et al.,32 MDP has a

higher inhibitory action against microorganisms than Phenyl-

P. MDP is present both in the primer and in the adhesive

components of CPB and CSEB, and in C3SB (single-bottle).

Nevertheless, in the present study, as previously pub-

lished,19,20,27 only the primer alone and the primer + adhesive

association of CPB and CSEB exhibited antibacterial effect

against the tested bacterial strains when applied to paper

utans and L. acidophilus (median, minimum and maximum)cs

Microorganism

S. mutans (n = 6) L. acidophilus (n = 6)

6.34 (5.68–7.17) Aa 5.45 (4.50–6.31) Ba

2.80 (1.12–3.70) Ab 1.02 (0.78–1.61) Bb

0 0

0 0

0 0

0 0

0 0

0 0

e statistically significant difference at 5%.

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discs. In spite of containing MDP, the lack of antibacterial

activity of C3SB and the adhesive component alone of CPB and

CSEB might be attributed to the presence of hydrophobic

molecules in their formulation, which may impair their

diffusion in the agar medium.24

When applied to 400 mm-thick dentine discs, neither of the

tested materials presented antibacterial activity. This lack of

antibacterial activity may be attributed to the protective effect

produced by the buffer capacity of dentine against acidic

agents, that is, the dissolution of dentine apatite by the action

of acids may be neutralised by the bonds between dentine

phosphate and carboxylic ions with H+ protons. In addition,

the thicker the remaining dentine, the greater the buffering

effect.33 Therefore, the deeper the microorganisms are lodged

into the dentinal tubules, the lower the effectiveness of these

materials to successfully eliminate them.

Fluoride has been incorporated into adhesive systems and

restorative materials, but its activity is more related to the

inhibition of the demineralisation process and optimisation of

remineralisation than to its antibacterial activity.12 In accor-

dance with the findings of previous studies,19,20 in the present

investigation Xeno-III showed low inhibitory activity when

applied to paper discs, mainly against L. acidophilus. This

antibacterial effect has been attributed to the liquid B of this

adhesive system that contains fluoride among other compo-

nents. Schmalz et al.19 have stated that the removal of fluoride

annulled the antibacterial effect of Prime&Bond NT. Never-

theless, in spite of containing fluoride and having the lowest

pH (pH 1) among the materials evaluated in the present study,

Xeno-III has a greater viscosity due to the presence of UDMA in

its formulation, which is a hydrophobic resin monomer with

high molecular weight. Therefore, it is speculated that the

viscosity may modulate the antibacterial activity of this

material20 and hinder its diffusion through the dentinal

tubules, which could explain the lack of inhibition of bacterial

growth observed when applied to dentine discs.

Among the self-etching adhesive systems evaluated in this

study, CPB presented the greatest antibacterial activity when

applied onto paper discs. Similar effect has been reported in

previous studies.9,11,14,15,17,19,20 In addition to MDP, the primer

of this adhesive system contains another resin monomer,

known as MDPB, which is an antibacterial agent composed of a

quaternary ammonium with a methacryloyl group.8 The

incorporation of MDPB into adhesive systems does not

interfere with their acidic potential and viscosity.30 An

advantage of MDPB in relation to other antibacterial agents

is the fact that it is copolymerized together with other

monomers and does not leach from the material after light-

activation. In addition, this monomer maintains its antibac-

terial activity, though to a less extent, when the microorgan-

ism gets in contact with the material surface.8,11,13,15,28

In an attempt to elucidate the real contribution of MDPB,

Imazato et al.13 compared the antibacterial potential of the

primer of clearfil protect bond before and after removal of this

resin monomer from the primer’s composition. The authors

observed that, in the absence of MDPB, the primer did not exert

antibacterial effect against S. mutans and Lactobacillus. This

findings demonstrates that the antibacterial effect of CPB

depends not only on the acidity of the material, but also (and

mainly) on the presence of MDPB.31,34

Please cite this article in press as: Gondim JO, et al., Influence of hu

systems against cariogenic bacteria, Journal of Dentistry (2008), do

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Schmalz et al.19 evaluated the effect of dentine on the

antibacterial activity of adhesive systems, TEGDMA and HEMA

against S. mutans, S. sobrinus and L. acidophilus using different

agar diffusion methods. Unlike the results of the present

study, those authors found that the tested adhesive systems

produced bacterial growth inhibition zones around bovine

dentine discs. However, their results also demonstrated that

the dentine influences the antibacterial effect of the adhesive

systems reducing or annulling it. These findings are consistent

with those of the present study and prior investigations,18

which have also shown the modulation effect exerted by the

dentine substrate. It should be mentioned, however, that

some methodological differences existing between the pre-

sent study and the study conducted by Schmalz et al.19 may

possibly explain the distinct results obtained in both studies,

among which, dentine disc type and thickness and the

amount of adhesive system applied to disc surface. In the

present study, 400 mm-thick human dentine discs were used

and 10 ml of material (adhesive system or individual compo-

nents) were pipetted on the surface of each specimen, while

Schmalz et al.19 used 200–500 mm-thick bovine dentine speci-

mens that were impregnated with 20 ml of the tested

materials. In addition, in the present study, a protective

barrier was built to avoid overflow of the material applied to

the surface of the dentine discs.

Dentine has a recognized protective action, however this

effect is limited.33 The dentinal substrate may impede or

difficult the diffusion of large-sized molecules, while allows

the diffusion of toxins resulting from the bacterial metabolism

and small-sized hydrophilic molecules, such as TEGDMA and

HEMA, which, in spite of not presenting bactericidal effect are

cytotoxic.35,36 The lack of production of inhibition zones

observed in this study indicates that the components eluted

from the adhesives were not able to diffuse through 400 mm-

thick dentine or did not exert inhibitory effect against

cariogenic bacteria on the agar.

Dentine permeability varies from tooth to tooth and from

one region to another in the same tooth.37 Some factors, such

as the anatomic arrangement, diameter, length and density of

the dentinal tubules, may be related to this variation.37 Thus,

the permeability of the dentine discs used in this study was

measured in order to obtain groups with statistically similar

dentine permeability means and hence avoid the influence of

this confounder in group comparisons.

Material’s viscosity may also interfere with its diffusion

through the dentinal tubules.37,38 Therefore, the difference of

viscosity of the materials evaluated in this study might have

influenced the obtained results, as only CHX (pH 5.9), which is

a low-viscosity material, was able to permeate the smear layer

and penetrate the dentinal tubules, exerting an antibacterial

effect. Although this effect was lower than that produced

when the material was applied to paper discs (p < 0.05),

demonstrating the influence of dentine substrate on the

material’s antibacterial activity.

The methodology employed in the present experiment has

some limitations in reproducing the physiological conditions

of the pulpodentinal complex, such as the presence of

intratubular fluid and intra-pulpal pressure. Due to these

limitations, the results obtained in in vitro studies cannot be

directly extrapolated to the in vivo situation because they may

man dentine on the antibacterial activity of self-etching adhesive

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not reflect the actual effect of the material when applied under

clinical conditions. However, laboratorial tests are indispen-

sable because they are at the base of the scientific evidence

pyramid providing research-based support and serving as a

reference for clinical investigations. Therefore, although the

present study demonstrated in vitro the antibacterial action of

some self-etching adhesive systems with and without light-

activation and the modulation effect of dentine substrate,

further clinical trials with human patients should be con-

ducted to determine the ultimate inhibitory effect of adhesive

systems against cariogenic microorganisms.

5. Conclusions

(1) Light-activation significantly reduced the antibacterial

activity of the tested adhesive systems;

(2) The presence of the antibacterial monomer MDPB in the

clearfil protect bond adhesive system contributed to the

inhibitory activity of the material against cariogenic

bacteria;

(3) The components eluted from the adhesive systems were

not capable of diffusing through 400 mm-thick dentine disc

to exert their antibacterial activity against cariogenic

bacteria.

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man dentine on the antibacterial activity of self-etching adhesive

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