Bio-Medical Materials and Engineering 19 (2009) 163–171 163

DOI 10.3233/BME-2009-0576
IOS Press

Assessment of the nanostructure of acid–base

resistant zone by the application of all-in-one
adhesive systems: Super dentin formation
Toru Nikaido a,∗ , Dinesh D.S. Weerasinghe a , Kanchana Waidyasekera a , Go Inoue a ,
Richard M. Foxton b and Junji Tagami a,c
a
Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School, Tokyo
Medical and Dental University, Tokyo, Japan
b
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, UK
c
Global Center of Excellence Program, International Research Center for Molecular Science in Tooth
and Bone Diseases, Tokyo, Japan

Received 4 August 2008

Accepted 3 October 2008

Abstract. An acid–base resistant zone (ABRZ) has been shown to be created under a hybrid layer in a self-etching adhesive
system at the adhesive/dentin interface. The purpose of this study was to assess the nanostructure of the ABRZ by applying
all-in-one adhesive systems. Human premolar dentin was treated with one of two all-in-one adhesive systems; Clearfil Tri-S
Bond and G-Bond according to the manufacturers’ instructions. After placement of a resin composite, the bonded interface was
vertically sectioned and subjected to an acid-base challenge. Following this, the nanostructure of the ABRZ was examined by
SEM and TEM. The SEM observations of the adhesive-dentin interface after the acid-base challenge indicated that a hybrid
layer less than 1 µm thick was created, and a ABRZ was formed beneath the hybrid layer for each adhesive system. The TEM
observations indicated that the ABRZ contained mineral components in both adhesive systems, however, the thickness of the
ABRZ was material dependent. The application of the all-in-one adhesive systems created an ABRZ at the underlying dentin,
which reinforced normal dentin against dental caries. Therefore, this zone was named ‘Super Dentin’. Formation of ‘Super
Dentin’ is a new approach in caries prevention.
Keywords: Interface, acid–base resistant zone, secondary caries, hybrid layer, all-in-one adhesive system

1. Introduction

Secondary caries is still one of the main reasons for the failure of restorations [1]. Key factors that
are associated with the prevention of secondary caries around restorations are the marginal integrity of
the restoration [2], durable adhesion [3], the physical properties of the restorative materials [4] and oral
hygiene [5].
*
Address for correspondence: 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.
Tel.: +81 3 5803 5483; Fax: +81 3 5803 0195; E-mail: nikaido.ope@tmd.ac.jp.

0959-2989/09/$17.00 © 2009 – IOS Press and the authors. All rights reserved
164 T. Nikaido et al. / Super dentin formation

Several in vitro secondary caries tests and evaluation methods have been developed that can assess
demineralized lesions and inhibition zones of dentin due to acid challenge. These methods include po-
larized light microscopy [6], the nanoindentation test [7], microradiography [8], confocal laser-scanning
microscopy and the X-ray analytical microscope [9]. However, the magnification of such methods is
insufficient if more detailed information at the interface between cavity and restoration is required.
Minimal intervention can be achieved using direct composite restorations in conjunction with an ad-
hesive system [10]. Dentin bonding systems have been dramatically simplified and improved during the
past decades. Scanning electron microscopy (SEM) has been shown to be a powerful device for ana-
lyzing the ultrastructure at the dentin/adhesive interface. Tsuchiya and others [11] observed artificial
secondary caries inhibition around adhesive restorations using SEM. They described the formation of an
acid–base resistant zone (ABRZ) adjacent to the hybrid layer after acid–base challenge. The results of
the study clearly showed the influence of adhesive material composition in the formation of ABRZ.
The mechanism of the acid–base resistant zone formation is still unclear. However, it was reported
that the ABRZ was formed only in a self-etching primer adhesive system and not in an acid etching
system [11]. In addition, the ABRZ was clearly observed beneath the hybrid layer in both intact and
caries-affected dentin using a self-etching primer system [12].
All-in-one adhesives contain acidic monomers, water and solvents in order to create a bond between
tooth substrate and resin composite by a single step. These systems are advantageous for clinicians
in saving time. However, the adhesive resin layer of all-in-one adhesives is permeable and allows the
formation of a water channel or water tree [13,14].
With the development of mild and simplified dentin bonding procedures, the method of observation
of the interfaces between tooth substrates and adhesive resins has shifted from SEM to transmission
electron microscopy (TEM). However, the bonding interfaces have been observed under a TEM without
demineralization in the previous studies. Therefore, the objectives of this study were to examine, the
ultramorphology of the interface of all-in-one adhesive systems after acid–base challenge using SEM
and TEM. The experimental hypothesis was that the morphology of the ABRZ formed by an all-in-one
adhesive was influenced by the adhesive material composition.

2. Materials and methods

2.1. Materials used in this study

Two all-in-one adhesive systems, Clearfil Tri-S Bond (Kuraray Medical, Tokyo, Japan) and G-Bond
(GC, Tokyo, Japan) were used in this study (Table 1). A total of twelve caries-free human premolars
stored frozen after extraction, were used for this study. Verbal agreement was obtained for their use in
research.

2.2. SEM observation

The method of sample preparation for SEM examination is illustrated in Fig. 1. Human premolars
were sectioned at the mid coronal portion, vertical to the tooth axis, with a low-speed diamond saw
(Isomet, Buehler, Lake Bluff, IL, USA) to obtain 1-mm-thick dentin disks. The dentin surfaces were
ground with #600-grit silicon carbide paper under running water. One surface of each disk was applied
with one of two adhesive systems, Clearfil Tri-S Bond and G-Bond according to the manufacturers’
instructions (Table 1). A flowable resin composite (Metafil Flo, shade A2, Sun Medical, Moriyama,
 T. Nikaido et al. / Super dentin formation 165

Table 1
All-in-one adhesive systems used in this study
Adhesive Manufacturer Composition Directions
Clearfil Tri-S Bond Kuraray Medical, MDP, HEMA, Bis-GMA, hydrophobic Apply for 10 s, gently air spread, and
Tokyo, Japan dimethacrylate, silanated colloidal sil- remove excess of agent with strong
ica, camphorquinone, ethanol, water air. Light cure for 10 s
G-Bond GC, Tokyo, Japan 4-MET, phosphate acid monomer, Apply for 20 s, air dry for 5 s to evap-
UDMA, silica, photo-initiator, acetone, orate the solvent. Light cure for 10 s
water
MDP: 10-methacryloyloxydecyl dihydrogen phosphate; HEMA: 2-hydroxyethyl methacrylates;
Bis-GMA: bisphenol A-glycidyl methacrylate; 4-MET: 4-methacryloxyethyltrimellitic acid; UDMA: urethane dimethacrylate.

Fig. 1. Specimen preparation for SEM observation.

Japan) was then placed between pairs of prepared dentin disks and light-cured for 40 s from the top and
bottom surfaces to make a dentin disk sandwich [12].
After storage for 24 h in distilled water, each prepared specimen was sectioned perpendicular to the
dentin–adhesive interface with a diamond saw and embedded in epoxy resin (Epoxicure Resin, Buehler).
Each specimen was first stored in 100 ml of a buffered demineralizing solution, containing 2.2 mmol/l
CaCl2 , 2.2 mmol/l NaH2 PO4 and 50 mmol/l acetic acid adjusted to pH 4.5 for 90 min to create artificial
secondary caries [15]. The specimens were then immersed in 5% NaClO for 20 min in an attempt to
remove any demineralized dentin collagen fibrils, and rinsed with running water for 30 s. Following
this, 4-META/MMA-TBB resin (Super Bond C&B, Sun Medical) was applied without acid-etching of
the treated surface in order to prevent wear of the adhesive during polishing because the edge of the
adhesive was torn away during specimen polishing [12]. 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, then polished with diamond pastes (Struers A/S, Copenhagen, Denmark) down
166 T. Nikaido et al. / Super dentin formation

Fig. 2. Specimen preparation for TEM observation.

to 0.25 µm. The polished surfaces were etched with an argon-ion beam (EIS-IE, Elionix, Tokyo, Japan)
for 7 min to bring the hybrid layer into sharp relief. The 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 surface [16]. 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).

2.3. TEM observation

Sample preparation for TEM examination is illustrated in Fig. 2. Six non-carious human premolars,
extracted for orthodontic reasons were used in this study. The occlusal enamel was removed by means
of a slow-speed saw (Isomet, Buehler) under water cooling. A smear layer was created by grinding
the surface with wet 600-grit silicon carbide paper. The dentin surfaces were treated with each dentin
adhesive according to the manufacturers’ recommendations (Table 1). After the applying the dentin
adhesives, a 2-mm thick layer of resin composite (Clearfil AP-X, Kuraray Medical) was applied. The
bonded specimens were stored in water at 37◦ C for one week. The roots were sectioned and removed
and then the crowns were sectioned perpendicular to the long axis of the tooth using a slow speed saw
to expose the adhesive–dentin interface.
Each specimen was then stored in 50 ml of a buffered demineralizing solution for 90 min, 5% NaClO
for 20 min, and rinsed with running water for 30 s in the same manner as described above. Following
this, bonded specimens (approximately 1 mm × 1.5 mm rectangular, 2 mm long) were prepared using
a slow speed diamond saw. They were fixed for 2 hours in 2.5% glutaraldehyde. Following the osmium
treatment, the samples were dehydrated in an ascending ethanol series (50%, 70%, 80%, 90% and 95%)
for 25 min in each and in 100% with two changes. The dehydrated specimens were then embedded in
epoxy resin (TAAB 812, TAAB Laboratories, Aldermaston, UK). Further TEM specimen preparation
was performed in accordance with established procedures for ultra structural TEM examination of bio-
logical tissues. Twenty, 100-nm-thick specimens treated with each bonding agent, were observed using a
 T. Nikaido et al. / Super dentin formation 167

transmission electron microscope (Hitachi H-600, Hitachi, Tokyo, Japan) with an accelerating potential
of 75 kV; objective aperture 100 µm in diameter.

3. Results

3.1. SEM observations

Representative SEM pictures of the dentin–adhesive interfaces after acid–base challenge are shown in
Fig. 3. The outer lesion (OL), which is the dentin surface demineralized after acid–base challenge, was
observed in both adhesive systems. The depth of the outer lesion ranged from 10 to 15 µm in which there
was no difference between each specimen.
For Clearfil Tri-S Bond (Fig. 3(a)), the top surfaces of the resin composite, Metafil Flo (CR) and the
adhesive (B) were not damaged by the acid–base challenge. The thickness of the adhesive was less than
10 µm. A hybrid layer distinguished by argon ion beam etching (H) was hardly observed at the interface.
An ABRZ was observed beneath the hybrid layer, which was approximately 1 µm thick (white triangles).
For G Bond (Fig. 3(b)), the top surfaces of the resin composite, Metafil Flo (CR) and the adhesive (B)
were not altered by the acid–base challenge. The adhesive was approximately 10 µm thick. However,
it was very hard to detect the hybrid layer (H) under ×3500 magnification. An ABRZ was recognized
beneath the hybrid layer, which was less than 1 µm thick (white triangles).

3.2. TEM observations

Representative TEM pictures of the dentin–adhesive interfaces after acid–base challenge at the middle
portion of the ABRZ are shown in Fig. 4. The outer lesions (OL) were clearly identified in both adhesive
systems.

Fig. 3. SEM observations of adhesive–dentin interface after acid–base challenge (×3,500). OL: outer lesion, CR: resin com-
posite, B: adhesive, H: hybrid layer, ABRZ: acid–base resistant zone. (a) Clearfil Tri-S Bond. A hybrid layer distinguished by
argon ion beam etching (H) was hardly observed at the interface. An ABRZ was recognized beneath the hybrid layer, which
was approximately 1 µm thick (white triangles). (b) G-Bond. It was very hard to detect the hybrid layer (H). An ABRZ was
recognized beneath the hybrid layer, which was less than 1 µm thick (white triangles).
168 T. Nikaido et al. / Super dentin formation

Fig. 4. TEM observations of adhesive–dentin interface after acid–base challenge (×50,000). OL: outer lesion, B: adhesive, H:
hybrid layer distinguished by argon ion etching in SEM, ABRZ: acid–base resistant zone. (a) Clearfil Tri-S Bond. Two layers
with totally 400 nm thick were recognized at the interface. An unstained layer (H) with maximum thickness of 100 nm was
partially identified adjacent to the adhesive, which is possibly coincident to the hybrid layer visualized by argon ion etching in
SEM. An electron dense area (ABRZ) with approximately 300 nm thick, was observed adjacent to the hybrid layer, which is
probably coincident with the ABRZ. (b) G-Bond. Two stained/unstained layers were recognized at the interface. The unstained
layer (H) was thicker than that of Clearfil Tri-S Bond, ranging from 500 to 700 nm. G-Bond created a thin electron dense area
with approximately 100 nm.

Clearfil Tri-S Bond (Fig. 4(a)) contains nanofiller particles, which are distributed in the adhesive (B).
Two layers, 400 nm thick, were identified at the interface. An unstained layer (H) with a maximum thick-
ness of 100 nm was partially identified adjacent to the adhesive, which is possibly coincident with the
hybrid layer visualized by argon ion etching and SEM. An electron dense area (ABRZ), approximately
300 nm thick, was observed adjacent to the hybrid layer, which is probably coincident with the ABRZ
observed using SEM.
In the case of G-Bond (Fig. 4(b)) nano-particles were observed in the adhesive (B). Also, two
stained/unstained layers were identified at the interface. However, the unstained layer (H) was thicker
than that of Clearfil Tri-S Bond, ranging from 500 to 700 nm. G-Bond created a thin electron dense area,
approximately 100 nm thick.

4. Discussion

The presence of an acid-base resistant zone below the hybrid layer has been documented with self-
etching adhesive systems in a secondary caries attack [11,12,17]. The morphology of this zone was
highly material dependant [11,17]. However, the ultrastructural features of this zone are not still clear.
 T. Nikaido et al. / Super dentin formation 169

We used two all-in-one adhesive systems with different compositions to elucidate the in vitro artificial
caries inhibition properties using observations from SEM and TEM examination.
The hybrid layer was originally defined as a layer that monomer penetrated into dentin and cured in
situ [18]. Argon-ion beam etching has been used for specimen preparation for SEM to clearly reveal
the hybrid layer at the resin–dentin interface [16]. However, a micro-laser Raman spectroscopic study
of the interface between a self-etching primer system and dentin demonstrated that the resin monomer
penetrated deeper than the hybrid layer identified by an argon-ion beam etching [19]. Watanabe et al.
[20] observed a self-etching primer adhesive system-dentin interface after mild acidic treatment by TEM.
They reported hydroxyapatite particles wrapped with resin monomer remaining below the hybrid layer.
Clearfil Tri-S Bond and G-Bond contain the acidic monomers MDP and 4-MET, respectively. Acidic
monomers simultaneously condition and prime the dentin. However, the acidity of these monomers is not
as high as that of inorganic acids, such as phosphoric and citric acids [21]. Therefore, all-in-one adhesive
systems partially demineralize dentin, leaving mineral crystals in the hybrid layer. Yoshida et al. [22]
claimed that interactions between hydroxyapatite crystals and acidic monomers of self-etching primers
may create calcium salts with different rates. The acidic monomer, MDP, adheres to hydroxyapatite
readily and intensively, forming a less soluble salt [23]. This fact may contribute to the stability of the
interface against acid–base challenge in all-in-one adhesive systems.
Koshiro et al. [24] reported that the interface formed by the all-in-one adhesives, GB and SSB200
(code name of Clearfil Tri-S Bond before marketing) were extremely thin (300 nm or less). They pro-
posed that these adhesive systems should be categorized according to “Nanointeraction Zone” type.
Yuan et al. [25] reported that the thickness of the hybrid layer of Clearfil Tri-S Bond was less than
1.0 µm, through which a homogeneous and complete resin infiltration could be expected to occur. These
results were determined using a TEM without demineralization of the specimens. TEM observation of
the interface after silver staining revealed that nanoleakage was mostly occurred at the top of the hybrid
layer [25]. This fact suggests that the possible area for nanoleakage may be different from the area of
the ABRZ.
In the present study, ABRZ formation was confirmed in both Clearfil Tri-S Bond and G-Bond by
SEM. However, the thickness of the hybrid layer and the ABRZ observed using SEM and TEM are
not comparable because of the different methods of specimen preparation. There were differences in
the ultra-morphology of the interface after acid–base challenge between Clearfil Tri-S Bond and G-
Bond in the TEM observations, which express a different potential for demineralization inhibition. The
TEM pictures demonstrated that there were two different layers identified at the interface. One layer
adjacent to the adhesive was an unstained layer, which is thought to be coincident with the hybrid layer
distinguished by argon ion etching. Another layer showed densely packed mineral crystallites, which
is thought to be coincident with the ABRZ in SEM. This suggests that the acid–base resistant zone
is not purely dentin, but is a combination of dentin and the adjacent hybrid layer. Those regions with
apatite crystallites were expected to be continuous with the dentin. Therefore, the hypothesis of this
study was accepted. If the acid–base resistant zone is assumed to be made of resin-infiltrated dentin,
the same chemical reaction of hydroxyapatite and an acidic monomer in the adhesive may take place
in this zone, giving rise to the ability to resist demineralization from an acid attack. The thickness and
density of the mineral crystals in the ABRZ was different in Clearfil Tri-S Bond and G-Bond. This may
be due to different chemical reactions of the acidic monomers present in the adhesives. Shinohara et al.
[17] reported that a thicker ABRZ adjacent to the hybrid layer could be observed only when a fluoride-
releasing adhesive was used. This fact suggested that fluoride-ions released from an adhesive system
170 T. Nikaido et al. / Super dentin formation

could also contribute to increasing the rate of calcium phosphate crystallization and reducing the rate of
apatite dissolution in the ABRZ.
The exact mechanism of the formation of the acid–base resistant zone is still unclear. However, these
findings strongly suggest that the dentin adjacent to the adhesive–dentin interface is different from the
intact dentin and contains a limited potential to resist an acid attack from secondary caries. Therefore,
the reinforced dentin may be termed ‘Super Dentin’ which has an ability to prevent primary caries.

5. Conclusions

1. The SEM and TEM observations indicated that an acid–base resistant zone was found adjacent to
the hybrid layer in both Clearfil Tri-S Bond and G-Bond.
2. The morphologies of the ABRZ were material dependent.
3. It is suggested that this layer be named ‘Super Dentin’, which is created by a self-etching primer
adhesive system.

Acknowledgements

This work was supported by the Global Center of Excellence (GCOE) Program, International Research
Center for Molecular Science in Tooth and Bone Diseases at Tokyo Medical and Dental University and
a Grant-in Aid from the Japan Society for the Promotion of Science (JSPS No. 19209059).

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