Dental Materials Journal 2009; 28(6): 717–721

The acid-base resistant zone in three dentin bonding systems

Go INOUE1, Toru NIKAIDO1, Richard M. FOXTON2 and Junji TAGAMI1,3
1
Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and
Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
2
Division of Conservative Dentistry, King’s College London Dental Institute at Guy’s, King’s College and St. Thomas’ Hospitals, King’s College
London, London Bridge, London SE1-9RT, United Kingdom
3
Global Center of Excellence Program; International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental
University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
Corresponding author,  Go INOUE;  E-mail:  inoue.ope@tmd.ac.jp


An acid-base resistant zone has been found to exist after acid-base challenge adjacent to the hybrid layer using SEM. The aim of this
study was to examine the acid-base resistant zone using three different bonding systems. Dentin disks were applied with three
different bonding systems, and then a resin composite was light-cured to make dentin disk sandwiches. After acid-base challenge,
the polished surfaces were observed using SEM. For both one- and two-step self-etching primer systems, an acid-base resistant zone
was clearly observed adjacent to the hybrid layer — but with differing appearances. For the wet bonding system, the presence of an
acid-base resistant zone was unclear. This was because the self-etching primer systems etched the dentin surface mildly, such that
the remaining mineral phase of dentin and the bonding agent yielded clear acid-base resistant zones. In conclusion, the acid-base
resistant zone was clearly observed when self-etching primer systems were used, but not so for the wet bonding system.

Keywords: Acid-base resistant zone, Secondary caries, Self-etching primer


Received Apr 28, 2009: Accepted Jul 27, 2009

challenge. Moreover, although the acid-base resistant

INTRODUCTION
Dentin bonding systems are routinely used for the
clinical placement of resin composite restorations.
Good adhesion has been ascribed as the primary factor
for enhancing the long-term sealing of the cavity
margin, thereby protecting the restoration against
secondary caries1-3). However, replacement of
restorations due to secondary caries is still a prevalent
problem in restorative dentistry.
To date, many studies have been devoted to the
prevention of secondary caries formation around
composite restorations4). However, limited work has
been done in the detailed observation of the dentin-
adhesive interface. In terms of in vitro secondary
caries assessment, it has been carried out using
different methods such as polarized light microscopy5),
microhardness analysis6), microradiography7), confocal
laser scanning microscopy, and X-ray analytical
microscopy8).
In addition to the abovementioned methods,
scanning electron microscopy (SEM) has been used to
observe the artificial secondary caries around adhesive
restorations placed in bovine root dentin9). It was
found that the change in ultrastructure of the cavity
margins after acid-base challenge was adhesive
material-dependent. When self-etching primer
adhesive systems were used, a thin layer — so-called
the “acid-base resistant zone (ABRZ) — was observed
beneath the hybrid layer after acid-base challenge10).
The adhesive resin had impregnated the exposed
collagen bundles and became entangled with them to
create a hybrid layer, which was distinguished by
argon-ion etching and was resistant to acid-base
zone was similarly observed after acid-base challenge
with different self-etching primer systems, differences
in terms of morphology and physical properties were
detected among the different bonding systems.
In another study11), it was found that an acid-base
resistant zone was clearly observed beneath the hybrid
layer when a two-step self-etching primer system was
used and that its nanohardness was higher than the
hybrid layer. However, with a fluoride-releasing
bonding system, the acid-base resistant zone was
thicker at the base7). As for total-etch bonding system,
two-step and one-step self-etching primer bonding
systems, no studies have been conducted to characterize
and compare the acid-base resistant zones formed by
the three bonding systems.
Therefore, the purpose of this study was to
examine, using SEM, the ultrastructure of ABRZ at the
interface of human dentin specimens when three
different bonding systems were used.
MATERIALS AND METHODS
Materials
Clearfil SE Bond (Kuraray Medical, Tokyo, Japan) was
used as a two-step self-etching primer bonding system
in this study. It comprised an acid monomer, 10-
methacryloxydecyl dihydrogen phosphate (MDP), in the
primer which dissolved the smear layer and
demineralized the underlying dentin, resulting in mild
surface etching. AQ Bond Plus (Sun Medical,
Moriyama, Japan) was used as a one-step self-etching
primer bonding system. It contained 4-META, which
was acidic and simultaneously dissolved the smear
718 Dent Mater J 2009; 28(6): 717–721
Fig. 1 Schematic illustration of specimen preparation method.
layer and etched the dentin surface mildly. This was
followed by copolymerization with other monomer
components for photoactivation and hybrid layer
formation. Single Bond (3M ESPE, Minneapolis, MN,
USA) was used as a total-etch bonding system with
phosphoric acid etching. Since the collagen fibrils were
exposed after etching, the wet bonding technique was
used to prevent collapse of the collagen fibrils. All the
materials were fluoride-free. A conventional tungsten-
halogen light curing unit, XL3000 (3M ESPE,
Minneapolis, MN, USA), was used to cure the adhesives
and the resin composite.
Specimen preparation
Specimen preparation was performed as described in a
previous study11), which is illustrated in Fig. 1. Twelve
caries-free human third molars, stored frozen after
extraction, were used for this study. Verbal consent
was obtained for their use in laboratory research under
a protocol approved by the Ethics Committee of the
Tokyo Medical and Dental University.
Molars were sectioned in the mid coronal region,
perpendicular to the long axis, with a low-speed
diamond saw (Isomet, Buehler, Lake Bluff, IL, USA) to
obtain two 1-mm-thick dentin disks. The dentin
surfaces were ground with wet 600-grit silicon carbide
paper under running water. One surface of each disk
was applied with one of the three bonding systems
according to manufacturers’ instructions. A flowable
resin composite (Metafil Flo, shade A2, Sun Medical,
Moriyama, Japan) was then placed between each pair
of prepared dentin disks and light-cured for 40 seconds
from the top and bottom surfaces to make a dentin disk
sandwich12).
After 24 hours of storage in distilled water, each
prepared specimen was sectioned perpendicular to the
dentin-adhesive interface with the diamond saw and
embedded in an epoxy resin (EpoxiCure Resin, Buehler,
Lake Bluff, IL, USA).
Each specimen was first stored in 100 ml of a
buffered demineralizing solution, containing 2.2 mmol/
L CaCl2, 2.2 mmol/L NaH2PO4, and 50 mmol/L acetic
acid adjusted to a pH 4.5 for 90 minutes to create
artificial secondary caries13). The specimens were then
immersed in 5% sodium hypochloride for 20 minutes to
remove any demineralized dentin collagen fibrils,
followed by rinsing under running water for 30
seconds.
To protect the outer edge of the adhesive from
wear which might occur during specimen polishing, 4-
META/MMA-TBB resin (Super-Bond C&B, Sun
Medical, Moriyama, Japan) was applied onto the
treated dentin surface without acid-etching9). After
curing the 4-META/MMA-TBB resin, the specimens
were sectioned perpendicular to the dentin-adhesive
interface and reduced to a thickness of approximately 1
mm. Polishing was done with diamond pastes (Struers
A/S, Ballerup, Denmark) down to 0.25 µm, and then
the polished surfaces were etched with an argon ion
beam (EIS-IE, Elionix, Tokyo, Japan) for 7 minutes to
bring the hybrid layer into sharp relief. Operating
conditions for the argon ion beam etching were an
accelerating voltage of 1 kV and an ion current density
of 0.2 mA/cm2 with the ion beam directed perpendicular
to the polished surface12). The specimens were then
gold-sputter coated, and morphological changes to the
dentin-adhesive interface due to acid-base challenge
were observed using a SEM (JSM-5310LV, JEOL,
Tokyo, Japan).
RESULTS
Figures 2–4 show the ultrastructures around the
dentin-adhesive interfaces after acid-base challenge. A
total of 16 specimens were observed for each bonding
system, and these selected figures were typical
 Dent Mater J 2009; 28(6): 717–721 719

Fig. 2 Ultrastructures around the adhesive-dentin interface for Clearfil SE Bond. (A) Depth of outer lesion was
approximately 15 µm under ×2000 magnification. An acid-base resistant zone (white arrowheads) was
clearly observed beneath the hybrid layer. (B) Close-up of the top surface of hybrid layer under ×7500
magnification, which was approximately 1 µm thick. An acid-base resistant zone was observed.

Fig. 3 Ultrastructures around the adhesive-dentin interface for AQ Bond Plus. (A) Depth of outer lesion was
almost the same as Clearfil SE Bond under ×3500 magnification. A thin acid-base resistant zone (white
arrowheads) was also observed beneath the hybrid layer, as well as erosion around the bottom of ABRZ
(circle). (B) Close-up of the top surface of hybrid layer under ×7500 magnification, which was less than 1
µm thick. An acid-base resistant zone was observed.

examples which exhibited a trend of more than 50%.
An outer lesion, which was the demineralized dentin
surface after acid-base challenge, was observed for all
the bonding systems. The depth of the outer lesion
ranged from 10 µm to 15 µm with no significant
differences among the three bonding systems.
For Clearfil SE Bond, the top surface of the
adhesive layer (Ad) was not damaged by acid-base
challenge (Figs. 2a and 2b). Thickness of the adhesive
layer was approximately 25 µm. A hybrid layer (HA)
distinguished by argon ion beam etching was observed
at the interface between the adhesive and dentin.
Using ×7500 magnification (Fig. 2b), the thickness of
the hybrid layer (HA) was observed to be approximately
1 µm. Similarly, the top surface of the hybrid layer
(HA) was not damaged by acid-base challenge. Beneath
the hybrid layer (HA), an acid-base resistant zone
(ABRZ) of approximately 1 µm thickness was observed.
For AQ Bond Plus, the top surfaces of the resin
composite, Metafil Flo (C), and the adhesive layer (Ad)
were not damaged by acid-base challenge (Fig. 3a).
Thickness of the adhesive layer was approximately 7
µm. However, it was very hard to detect the hybrid
layer (HA) under ×3500 magnification. Using ×7500
magnification (Fig. 3b), the thickness of the hybrid
layer (HA) was observed to be less than 1 µm, and that
the top surface was not damaged by acid-base
challenge. Beneath the hybrid layer (HA), an acid-base
resistant zone (ABRZ) was identified along the
interface, which was less than 1 µm thick. The dentin
surface adjacent to the acid-base resistant zone (arrow)
was observed to be more eroded than other areas of the
dentin surface.
For Single Bond, the top surfaces of the resin
composite, Metafil Flo (C), and the adhesive layer (Ad)
were not damaged by acid-base challenge (Fig. 4a).
720 Dent Mater J 2009; 28(6): 717–721
Fig. 4 Ultrastructures around the adhesive-dentin interface for Single Bond. (A) Depth of outer lesion was almost
the same as Clearfil SE Bond under ×3500 magnification. However, no acid-base resistant zone was
observed. (B) Close-up of the top surface of hybrid layer under ×5000 magnification.
Thickness of the adhesive layer was approximately 10
µm. A hybrid layer (HA) of approximately 5 µm
thickness, as distinguished by argon ion beam etching,
was observed at the interface. The top surface of the
hybrid layer (HA) seemed to be damaged by acid-base
challenge, which also seemed to slope. An acid-base
resistant zone (ABRZ), which was observed for Clearfil
SE Bond and AQ Bond Plus, was not detected beneath
the hybrid layer (HA) (Fig. 4b).
DISCUSSION
The hybrid layer was defined as a layer where the
functional monomer penetrated the dentin and cured in
situ14). To distinguish the hybrid layer from dentin and
adhesive resin, acid treatments such as hydrochloric
acid14) and phosphoric acid15,16) have been routinely used
in previous studies. Therefore, the hybrid layer was
defined as an acid-base resistant zone with respect to
its characteristics against acid treatments. Argon ion
beam etching has been used as another method to
clearly reveal the hybrid layer at the adhesive-dentin
interface12). Since the etching outcome with argon ion
beam etching is material-dependent, the hybrid layer
can be clearly distinguished in sharp relief. However,
the hybrid layer is not a uniform layer, especially with
respect to the existence of nanospaces within the
remaining demineralized dentin after phosphoric acid
etching16). Previous studies using Raman spectroscopy
have suggested that the resin monomers of some self-
etching primer bonding systems penetrate deeper than
the hybrid layer distinguished by argon ion etching17,18).
Clearfil SE Bond was a fluoride-free, two-step self-
etching primer system. Good dentin bonding with self-
etching primer systems has already been demonstrated
in numerous laboratory studies19,20). In the present
study, SEM observation revealed that the SE Bond
adhesive demonstrated good resistance against the
acid-base challenge. This could be due to good
mechanical properties of the adhesive rendered by
ingredients such as multi-functional methacrylates and
microfillers9,11).
AQ Bond Plus was a one-step self-etching primer
system. SEM observation revealed a very thin ABRZ
along the interface. However, an eroded dentin area
adjacent to ABRZ was observed, indicating weaker acid
resistance than intact dentin. It was thus suggested
that nanospaces might be present beneath ABRZ
because of incomplete monomer penetration into the
demineralized dentin.
Single Bond was a one-bottle adhesive system
which required phosphoric acid etching. Degree of
exposure of the collagen fibrils depends on the
demineralization of the dentin substrate via acid-
etching with mineral acids. Following rinsing, the
mineral phase of the superficial dentin is completely
removed, leaving the collagen fibrils literally suspended
in water21,22). With Single Bond, the phosphoric acid
etched the smear layer and demineralized the dentin
deeper than Clearfil SE Bond and AQ Bond Plus,
thereby resulting in a thick hybrid layer. However, the
phosphoric acid etched too strongly, which then
resulted in the disappearance of ABRZ.
Previous studies have reported that when self-
etching primer adhesive systems were used for bonding,
an acid-base resistant zone (ABRZ) was observed
beneath the hybrid layer after acid-base challenge9,11).
From a clinical standpoint, creation of an ABRZ lends
aid to the prevention of secondary caries. Although the
mechanism of acid-base resistant zone formation
remains unclear; it is decidedly different from the
inhibition zone which is formed due to fluoride release.
This was because the ABRZ was formed in this study
despite the adhesives being fluoride-free. It was thus
speculated that ABRZ formation could be related not
only to the amount of fluoride released from a fluoride-
containing adhesive, but also to the ability of an
adhesives to infiltrate dentin.
On the characterization of ABRZ, it was speculated
that ABRZ was a mixture of partially demineralized
 Dent Mater J 2009; 28(6): 717–721
dentin with self-etching primer and resin monomers.
An ABRZ was observed beneath the hybrid layer for
both one-step and two-step self-etching primer systems.
The ABRZ created by Clearfil SE Bond and AQ Bond
Plus would be related not only to adhesive penetration,
but also to the quality of the hybrid layer (HA). In a
study by Watanabe et al.13), a self-etching primer
bonding system was used after the dentin surface was
subjected to mild acid treatment. By means of TEM to
observe the adhesive-dentin interface, hydroxyapatite
particles wrapped with resin monomer were found to
remain below the hybrid layer. Therefore, with regard
to self-etching primer bonding systems, the mechanism
of ABRZ formation depended on the kind of adhesive
system used. In a previous study, ABRZ was identified
as part of the hybrid layer, whereby the higher
microhardness of caries-affected dentin 2 µm beneath
the hybrid layer supported the theory that the acid-
base resistant zone was composed of penetrated
monomer and dentin11).
Secondary caries begins at the margin between
dentin and the restorative material. Pertaining to this
problem, bonding systems have been used — and
perennially improved upon in a pursuit for better
marginal integrity — to prevent the occurrence of
marginal gaps. In the present study, the adhesive-
dentin interfaces demonstrated resistance to acid-base
challenge. Therefore, formation of an ABRZ is
important in the prevention of secondary caries around
a restoration. As for the formation of an outer lesion
around a restoration, which is due to primary caries
but not secondary caries, it was not inhibited by
bonding systems. Leveraging on this study’s results,
further studies should be performed to characterize the
acid-base resistant zones formed using various adhesive
materials.
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