An in vivo evaluation of bonding ability of comprehensive ...

dental materials xxx (2006) xxx–xxx
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An in vivo evaluation of bonding ability of comprehensive
antibacterial adhesive system incorporating MDPB
Satoshi Imazato a,∗ , Franklin R. Tay b , Andrea V. Kaneshiro a ,
Yusuke Takahashi a , Shigeyuki Ebisu a
a Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry,
1-8 Yamadaoka, Suita, Osaka 565-0871, Japan
b Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA
article info
Article history:
Received 6 September 2005
Accepted 5 January 2006
Keywords:
Antibacterial adhesive system
MDPB
Bonding interface
In vivo
1. Introduction
abstract
The challenge in the clinical treatment of dentinal caries is
the absence of a universally acceptable regime for the diagnosis
of carious lesions. Active bacteria may inadvertently be
left behind by incomplete caries removal. Thus, restorative
Objectives. This study examined the in vivo bonding ability to sound dentin of antibacterial
adhesive systems incorporating an antibacterial monomer MDPB based on morphological
evaluation of the resin–dentin interface.
Methods. Class V cavities were prepared on the buccal surfaces of the teeth of a beagle dog
and a composite filling performed using (1) commercial self-etching system Liner Bond
2 (LB primer + LB bond), (2) experimental primer containing 5% MDPB and LB bond, (3)
LB primer and experimental bonding-resin containing 2.5% MDPB, or (4) combination of
experimental primer and bonding-resin. After 7 days, the tooth crown was cut and fixed
in half-Karnovsky’s solution, and the sectioned surface observed under scanning electron
microscopy (SEM) after treatment with phosphoric acid and NaOCl. The ultrastructure of the
bonding interface was also examined by transmission electron microscopy (TEM). Microtensile
bond strengths (�TBS) of each group were measured using extracted teeth.
Results. SEM demonstrated that all groups produced a 1–2 �m thick hybrid layer with funnel
shaped resin tags, although the length of tags was shorter for the group in which MDPBcontaining
bonding-resin was used. TEM examination supported good adhesion of the
comprehensive adhesive system employing MDPB-containing primer/bonding-resin, showing
integrity between resin and dentin. There were no significant differences in �TBS among
the four groups tested (p > 0.05, ANOVA).
Significance. This study confirmed that the experimental antibacterial adhesive systems
employing MDPB-containing primer or/and bonding-resin could produce an effective bond
under in vivo conditions.
© 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
materials that exhibit antibacterial activity are useful for eliminating
the harmful effects caused either by residual bacteria
or bacterial microleakage.
The authors have previously reported that the incorporation
of an antibacterial monomer 12-methacryloyloxydodecylpyridinium
bromide (MDPB) was effective in providing
∗ Corresponding author. Tel.: +81 6 6879 2928; fax: +81 6 6879 2929.
E-mail address: imazato@dent.osaka-u.ac.jp (S. Imazato).
0109-5641/$ – see front matter © 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.dental.2006.01.005
DENTAL-906; No. of Pages 7

2 dental materials xxx (2006) xxx–xxx
a dentin primer and bonding-resin with antibacterial activity
[1–4]. The uncured dentin primer incorporating MDPB demonstrates
a bactericidal effect based on the strong antibacterial
activity of unpolymerized MDPB [1,3,5]. The ability of this
monomer to kill residual bacteria in the prepared cavity has
been also reported [6]. Bonding-resin containing MDPB could
inhibit the growth of bacteria on its surface by means of the
action of an immobilized bactericide after the resin is polymerized,
without adversely affecting its bonding characteristics
[4]. Therefore, it is considered that the use of a comprehensive
antibacterial adhesive system that employs MDPB-containing
primer and bonding-resin should be highly effective in achieving
the successful restorative treatment of caries. These favorable
in vitro results await in vivo confirmation. Thus, the
purpose of this study was to investigate the bonding ability
of an antibacterial adhesive system incorporating MDPB in
vivo by morphological evaluation of bonding interface using
a beagle dog model. The microtensile bond strength of an
experimental antibacterial adhesive system was also measured
under in vitro conditions. The null hypothesis tested
was that there is no difference in the bonding characteristics
of MDPB-containing primer/bonding-resin in vitro or
in vivo.
2. Materials and methods
2.1. Adhesive systems
An experimental adhesive system was prepared by the addition
of the antibacterial monomer MDPB to a commercial
product (Clearfil Liner Bond 2, Kuraray Medical Inc., Tokyo,
Japan) consisting of two-liquid type self-etching primer (LB
primer) and a one-bottle bonding-resin (LB bond). For the
experimental primer, 10% (w/w) of MDPB was added to the Bliquid
of LB primer to give 5% (w/w) after mixing with A-liquid.
The experimental bonding-resin was prepared by incorporating
2.5% (w/w) MDPB into LB bond and used in combination
with the MDPB-containing primer (Table 1).
2.2. In vivo bonding procedures
A beagle dog (female, 13 months old, weight 10 kg) was housed
in the Osaka University Animal Facility, and used according to
the protocol approved by the ethical guidelines for animal care
of Osaka University Graduate School of Dentistry.
Table1–Materials tested in this study
The dog was subjected to general anesthesia by intramuscular
injection of 20 mg/kg ketamine and intravenous injection
of 10 mg/kg sodium pentobarbital. The teeth were cleaned
with 3% hydrogen peroxide and 5% tincture of iodine. Class V
cavities (3 mm × 4 mm, 1 mm depth) were prepared on the buccal
surfaces of molars, canines or incisors using a high-speed
diamond bur (D1, Shofu, Kyoto, Japan) under water spray.
The cavosurface margin was but-jointed and surrounded
by enamel. The cavities were then treated in four different
ways:
Group 1 (control): The cavity was treated with Clearfil Liner
Bond 2 according to the manufacturer’s instruction. A- and
B-liquids of LB primer were mixed and applied to the cavity
for 30 s. After drying with a gentle stream of air, LB bond
was applied and cured with a light-activation unit (Quick
Light, Morita, Kyoto, Japan) for 20 s. Then, a flowable composite
(Clearfil Protect Liner F, Kuraray Medical Inc.) was placed
in the cavity and light-cured for 40 s. The excess material
beyond the cavosurface margin was removed with a finishing
point (#60, Shofu).
Group 2: The cavity was restored in the same manner as
group 1 using the experimental primer instead of LB primer.
Group 3: The cavity was restored using the control primer
and the experimental bonding-resin.
Group 4: The cavity was restored in the same manner as the
above three groups using the experimental primer and the
experimental bonding-resin.
The animal was sacrificed after 7 days. Crowns of the
restored teeth were cut and immersed in half-Karnovsky’s
solution (pH 7.4) for 5 h at 4 ◦ C. The bonding interfaces were
examined by scanning electron microscopy (SEM) or transmission
electron microscopy (TEM).
2.3. SEM examination
The specimens were sectioned longitudinally through the
center of the restoration using a slow-speed saw equipped
with a diamond-impregnated disk (Isomet, Buehler, Lake
Bluff, IL, USA) under water cooling. The sectioned surface
was polished with silicon carbide papers of increasing fineness,
and finally on linen with 0.3 �m aluminum oxide.
The polished surfaces were treated with 50% phosphoric
acid for 30 s, then with 10% NaOCl for 2 min, followed by
rinsing with a copious amount of water after each treatment.
The fixed specimens were dehydrated in ascending
Materials Composition Batch number
Liner Bond 2 (Kuraray Medical Inc.)
LB primer HEMA, water, Phenyl-P A-liquid: 007; B-liquid: 009
LB bond Bis-GMA, HEMA, MDP, microfiller 0078
Experimental primer LB primer + 5% MDPB (10% MDPB in B-liquid)
Experimental bonding-resin LB bond + 2.5% MDPB
HEMA: 2-hydroxyethyl methacrylate; Phenyl-P: 2-methacryloyloxyethyl phenyl hydrogen phosphate; Bis-GMA: bisphenyl glycidyl methacrylate;
MDP: 10-methacryloyloxydecyl dihydrogen phosphate; MDPB: 12-metahcryloyloxydodecylpyridinium bromide.

grades of ethanol, and freeze-dried. After being sputter-coated
with gold, the resin–dentin interfaces were examined under
a scanning electron microscope (JSM-5310LV, JEOL, Tokyo,
Japan) at 20–25 kV. Three restorations were examined for each
group.
2.4. TEM examination
The specimens of groups 1 and 4 were prepared according to
the TEM protocol reported by Tay et al. [7]. Briefly, the specimens
were completely demineralized in ethylenediamine
tetraacetic acid (pH 7.0), post-fixed in 1% osmium tetroxide,
and dehydrated in ascending grades of ethanol (30–100%).
After embedding in epoxy resin, 90–100 nm thick sections
were prepared with an ultramicrotome. Sections were doublestained
with uranyl acetate and Reynold’s lead citrate to
examine the ultrastructural characteristics of the resin–dentin
interfaces, using a transmission electron microscope (Philips
EM208S, Eindhoven, Netherlands) operating at 80 kV.
2.5. Microtensile bond strength tests
Forty extracted human third molars were employed for
microtensile bond testing. The teeth were collected after the
patients’ informed consents were obtained under a protocol
reviewed and approved by the institutional review board from
the Medical College of Georgia, USA. These teeth were stored
in 0.5% chloramines T until use. Occlusal enamel was removed
from each tooth using the Isomet saw under water cooling,
creating flat surfaces for bonding in mid-coronal dentin.
Each surface was further abraded with 180-grit silicon carbide
papers to create clinically relevant smear layers for dentin
bonding. The four groups of commercial/experimental twostep
self-etch adhesives were used in the manner previously
described, with 10 teeth employed for each group. Incremental
composite build up was performed on each bonded tooth
surface using a light-cured micro-hybrid composite (Clearfil
AP-X, Kuraray Medical Inc.) to a height of 4 mm.
After 24 h of water storage, two 0.9-mm thick slabs were
obtained from each bonded tooth using the Isomet saw under
water cooling. These slabs were further sectioned to produce
0.9 mm × 0.9 mm beams containing the resin–dentin interface.
The four longest beams were selected from those acquired
from each tooth. Each beam was fixed to a modified Bencor
Multi-T testing assembly (Danville Engineering, San Ramon,
CA, USA) using cyanoacrylate adhesive (Zapit; DVA, Corona,
CA, USA). The beams were pulled to failure under tension
using a universal testing machine (Model 4440; Instron Inc.,
Canton, MA, USA) at a crosshead speed of 1 mm per minute.
The exact dimension of each fractured beam was then individually
measured using a digital caliper (Model CD-6BS; Mitsutoyo,
Tokyo, Japan), from which the tensile bond strength was
calculated. Means were taken from the four beams derived
from the same tooth and the bond strength data for each
group was expressed with the tooth instead of an individual
beam as the testing unit (N = 10). Data from the four
groups were statistically analyzed using one way analysis
of variance and Tukey’s post hoc multiple comparison test
with ˛ = 0.05.
dental materials xxx (2006) xxx–xxx 3
Fig. 1 – Scanning electron micrographs of the resin–dentin
interface produced by the control adhesive system Liner
Bond 2.
3. Results
3.1. SEM observation
Representative bonded interfaces of each group are shown in
Figs. 1–4. Groups 1 and 2 demonstrated interfaces with approximately
a 1–2 �m thick hybrid layer and resin tags extending
5–8 �m into the dentin (Figs. 1 and 2). A filigree pattern of
the resin-infiltrated layer and cone shape of resin tags, were
clearly observed.
The thickness of the hybrid layer produced for groups 3 and
4 was similar to those of groups 1 and 2. However, the resin
tags extended up to only 2–3 �m for these groups and were
shorter compared with those with LB bond (groups 1 and 2),
although a funnel-shaped configuration of the tag necks was
apparent (Figs. 3 and 4).
3.2. TEM observation
TEMs of resin–dentin interfaces of groups 1 and 4 are shown in
Figs. 5 and 6, respectively. Hybrid layers within the underlying
intact dentin in both groups were about 1 �m thick. Formation
of a hybridized smear layer incorporating smear layer remnants
were observed at the top part of the hybridized complex.

4 dental materials xxx (2006) xxx–xxx
Fig. 2 – Scanning electron micrographs of the resin–dentin
interface produced by the experimental primer containing
MDPB and LB bond.
Dentinal tubules were invariably occupied by smear plugs in
both groups.
As the experimental bonding-resin was prepared by handmixing
of MDPB with LB bond, many voids were observed in
the bonding-resin layer for group 4 due to the entrapment of
air bubbles (Fig. 6a).
3.3. Microtensile bond strength tests
Fig. 7 indicates the results of the microtensile bond strength
test. There were no significant differences among the four
groups (p > 0.05). The mean tensile bond strength values
ranged from 30.8 to 34.9 MPa.
4. Discussion
The microtensile test performed on human teeth demonstrated
that usage of 2.5% MDPB-containing bonding-resin in
combination with the control or a 5% MDPB-containing primer
did not adversely affect the dentin bonding ability of the parent
commercial system without MDPB under in vitro conditions.
These results supported the previous findings obtained
by conventional tensile bond strength tests [4]. The addition of
Fig. 3 – Scanning electron micrographs of the resin–dentin
interface produced by the control primer (LB primer) and
the experimental bonding-resin containing MDPB.
extra components such as free, non-polymerizable antimicrobials
frequently interrupts the physical form of the polymers,
leading to a reduction in their physical properties [8]. However,
the synthetic monomer MDPB is a polymerizable bactericide
and does not adversely affect curing of the primer and
bonding-resin [4]. Curing ability of bonding-resin is one of the
important factors for obtaining a strong bond to dentinal substrate
[9,10]. Hence, incorporation of MDPB is advantageous
over the addition of free antimicrobials such as chlorhexidine.
The pH value of the primer in the control self-etching system
Liner Bond 2 is about 1.4. The thickness of the hybrid layer
produced in vivo for all groups in the beagle dog model was
1–2 �m when examined under SEM. This value was similar to
that reported for Liner Bond 2 by in vitro tests [11]. Since the
addition of MDPB does not affect the pH value of the primer
(data not shown), it is speculated that the same degree of demineralization
of the smear layer and intertubular dentin as the
control primer was obtained for the MDPB-containing primer.
The fact that there were no differences in the appearance and
thickness of the hybrid layers among all groups indicates that
the experimental bonding-resin containing MDPB could penetrate
well into the demineralized dentinal surface to produce
optimal hybridization with exposed collagen. However, resin

Fig. 4 – Scanning electron micrographs of the resin–dentin
interface produced by the combination of the experimental
primer/bonding-resin containing MDPB.
tags formed in the tubules showed an apparent difference
between the control (groups 1 and 2) and the MDPB-containing
experimental bonding-resin (groups 3 and 4), the latter being
shorter than the former. This discrepancy was not observed
dental materials xxx (2006) xxx–xxx 5
when the experimental bonding-resin was examined under in
vitro condition using extracted human teeth [4]. In this study,
cavities were prepared in sound dentin and it seems that the
dentinal fluid in the tubule caused a disturbance of the infiltration
of bonding-resin for groups 3 and 4. One of the possible
reasons for this is the greater viscosity of MDPB-containing
bonding-resin compared with the control. Incorporation of
MDPB increases the viscosity of Bis-GMA based resin probably
due to molecular interaction. The concentration of MDPB
added to bonding-resin was set at 2.5% as this was the maximum
amount of MDPB which would not to cause an adverse
influence on handling properties. However, a slight increase
in the viscosity was obtained even at 2.5% MDPB. In addition,
MDPB is more hydrophobic than HEMA, so that the hydrophobicity
of the experimental bonding-resin is greater than that
of the control. Although infiltration into the demineralized
smear layer and intertubular dentin was not affected, it is
likely that penetration of MDPB-containing bonding-resin into
the dentinal tubule was limited under in vivo conditions due
to outflow of dentinal fluid.
TEM images of the bonding interface demonstrated continuity
of resin and dentin with the production of hybridized
dentin and hybridized smear layers for both the control and
the experimental antibacterial adhesive systems employing
MDPB-containing primer/bonding-resin (Figs. 5 and 6). As
depicted by SEM, formation of 1–2 �m thick hybridized layers
with irregular surfaces was clearly observed. Occlusion of
dentinal tubules was manifested by the SEM observation of
funnel-shaped resin tags even for the experimental system.
This morphologic feature is indicative of the creation of tight
seals at the tubule orifices. Thus, it may be concluded from
the SEM and TEM evaluation that ultrastructure of the interface
of the experimental adhesive system containing MDPB
was not different from that of the control, with the exception
that shorter resin tags were identified for experimental systems
with the MDPB-containing bonding-resin.
It is well known that in vitro evaluation of the bonding
ability of adhesive systems using extracted teeth sometimes
does not reflect reality. For example, smear layers produced
by 600-grit abrasive papers in vitro does not simulate what
Fig. 5 – Transmission electron micrograph of the resin–dentin interface produced by Liner Bond 2 system (control). (a) Low
magnification view of the resin–dentin interface. (b) High magnification view of the hybrid layer. A: filled adhesive; P:
experimental MDPB-primer; Hs: loose, discontinuous hybridized smear layer; H: 1 �m thick hybrid layer formed in the
intertubular dentin (D); T: dentinal tubule.

6 dental materials xxx (2006) xxx–xxx
Fig. 6 – Transmission electron micrograph of the resin–dentin interface produced by the experimental primer and the
experimental bonding-resin containing MDPB. (a) Low magnification view. As the experimental bonding-resin was
prepared by hand-mixing of MPDB with LB Bond, numerous air voids (pointer) could be seen within the adhesive layer (A).
C: flowable composite. (b) A high magnification view of the previous figure, showing the hybridized complex that comprised
the surface hybridized smear layer (Hs) and the underlying hybrid layer (H) in intact intertubular dentin (D).
dentists produce in a clinical setting, and bond strengths of
mild self-etching primers measured under such in vitro conditions
are suggested to be overestimated [12]. Most in vitro
bond strength tests, including the one in the present study,
were performed on flat tooth surfaces that are highly compliant
and with minimal polymerization shrinkage stresses
[13]. Recent studies showed that bond strengths of the adhesives
were substantially reduced when testing was performed
on cavities with low compliance such as Class I and Class II
cavities [14,15]. Therefore, observation of resin–dentin interfaces
under in vivo conditions provides important information
on the clinical efficacy of bonding. Although we have to
reject the null hypothesis, since in vivo resin infiltration into
dentinal tubules was slightly affected for MDPB-containing
Fig. 7 – Microtensile bond strengths of each group
measured using extracted teeth. There were no significant
differences among all specimens (p > 0.05, ANOVA).
bonding-resin, the present results using a beagle dog model
demonstrated that experimental adhesive systems employing
MDPB-containing primer/bonding-resin were able to exhibit
satisfactory performance in vivo. Self-etching systems are
claimed to have advantages over etch-and-rinse systems as
there is little discrepancy between the depth of decalcification
and bonding-resin penetration [16]. This is also expected
to be valid for the experimental adhesive systems.
It has been reported that polymerized resin matrices
are vulnerable to hydrolytic degradation after water sorption
[17,18], and bonding agents that contain hydrophilic
monomers exhibit lower bond durability compared with
those containing hydrophobic monomers [19]. Incorporation
of MDPB, which increases the hydrophobicity of the adhesives,
may result in greater hydrolytic stability of the bonding interface.
It would be of interest in the future to examine the durability
of resin–dentin bonds made by the MDPB-containing
primer/bonding-resin. Further investigations should be also
aimed at assessing the antibacterial effects of this adhesive
system against microorganisms that are capable of penetrating
these interfaces by microleakage, as well as its clinical
performance.
Acknowledgments
This work was supported by a Grant-in-aid for Scientific
Research (15209066, 16390545) from the Japan Society for the
Promotion of Science and the 21st Century COE at Osaka University
Graduate School of Dentistry supported by the Ministry
of Education, Culture, Sports, Science and Technology.
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