d e n t a l m a t e r i a l s 3 7 ( 2 0 2 1 ) e109–e117

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Comparison of different etch-and-rinse adhesive

systems based on shear fatigue dentin bond
strength and morphological features the interface

Yuta Kasahara a , Toshiki Takamizawa a,∗ , Eizo Hirokane a ,

Akimasa Tsujimoto a , Ryo Ishii a , Wayne W. Barkmeier b , Mark A. Latta b ,
Masashi Miyazaki a
a Department of Operative Dentistry, Nihon University School of Dentistry, Tokyo, Japan
b Department of General Dentistry, Creighton University School of Dentistry, Omaha, Nebraska, USA

a r t i c l e i n f o a b s t r a c t

Keywords: Objectives. The aim of this study was to investigate dentin bonding durability of different
Dentin bond durability etch-and-rinse (ER) adhesive systems under fatigue stress and to compare morphological
Etch-&-rise systems features of resin/dentin interfaces using SEM.
Biomechanical stress Methods. Two three-step ER adhesives, a two-step ER adhesive, and a universal adhesive
Reaction layer in ER mode were evaluated. Before application of either primer or adhesive, phosphoric
acid etching of human dentin was completed. Fifteen bonded specimens for each adhesive
system were stored in distilled water at 37 ◦ C for 24 h, then subjected to a shear bond strength
(SBS) test. Bonding durability was assessed from the perspective of biomechanical stress. 25
bonded specimens for each adhesive system were subjected to shear fatigue strength (SFS)
testing with a repeated subcritical load at a frequency of 20 Hz for 50,000 cycles or until
failure.
Results. Mean SBS and SFS values ranged from 33.3 to 41.2 MPa, and from 18.3 to 20.3 MPa,
respectively. Three-step adhesives showed higher SBS and SFS values than the other adhe-
sive systems. Under SEM, resin tags in different adhesive systems showed similar features,
but morphology below the hybrid layer was material dependent. The universal adhesive in
ER mode showed an obvious thin, high-density reaction layer below the hybrid layer.
Significance. Three-step adhesives showed higher dentin bond durability than the other ER
adhesives; no significant differences in SFS were found between the universal adhesive in
ER mode and the three-step ER adhesives. The results of this in vitro study indicate that
some ER adhesives might establish chemical bonding with intact dentin below the hybrid
layer in addition to micromechanical retention.
© 2020 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.

∗
Corresponding author at: Department of Operative Dentistry, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku,
Tokyo 101-8310, Japan.
E-mail address: takamizawa.toshiki@nihon-u.ac.jp (T. Takamizawa).
https://doi.org/10.1016/j.dental.2020.11.006
0109-5641/© 2020 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.
e110 d e n t a l m a t e r i a l s 3 7 ( 2 0 2 1 ) e109–e117

ical features to conventional single-step self-etch adhesives.

1. Introduction However, universal adhesives in ER mode exhibit different
morphological features from three- and two-step ER adhesive
Etch-and-rinse adhesive systems (ER) use phosphoric acid
systems. Most universal adhesives show a thin high-density
etching to treat enamel and dentin before application of adhe-
layer (reaction layer) underneath HL that is not visible in
sive [1]. ER adhesive systems are grouped into three-step and
three and two-step ER adhesive systems [22]. Most universal
two-step systems based on the use of a priming step [1]. ER
adhesives use an effective functional monomer that creates
adhesive systems are thought to enhance enamel bond effec-
a stable self-assembled nano-layering at the bonding inter-
tiveness [2,3], and remove bonding inhibitors such as saliva,
face, 10-methacryloyloxydecyl dihydrogen phosphate (MDP)
blood, gingival crevicular fluid, and dental plaque owing to the
[14]. Some conventional ER adhesive systems do not form this
use of strong acid and a water rinse after etching [4]. Based
layering. Therefore, functional monomers of universal adhe-
on a systematic review and meta-analysis of dentin bonding
sives in ER mode interact in some way with HAp underneath
performance with caries-affected dentin, ER adhesive systems
the demineralized dentin. In contrast, conventional three- and
show better dentin bonding than self-etch (SE) adhesive sys-
two-step ER adhesive systems contain different functional
tems [5]. Furthermore, a systematic review of post-operative
monomers or a polyalkenoic acid copolymer that might inter-
sensitivity for posterior resin composite restorations found
act with HAp after acid etching. However, little information is
that adhesive strategy (ER or SE) for posterior resin compos-
available to date concerning dentin bonding mechanisms of
ite restorations does not influence the risk and intensity of
universal adhesives in ER mode and conventional ER adhesive
post-operative sensitivity [6]. Despite these adhesive systems
systems.
being used worldwide in dentistry, bonding strategies using
The purpose of this study was to investigate the differences
phosphoric acid pre-etching before application of adhesive are
in dentin bonding mechanisms among different ER adhesive
not the most recent technology and increase the number of
systems based on shear fatigue strength tests and SEM obser-
clinical steps.
vation of the resin/dentin interface. The null hypothesis to
Fundamentally, the bonding mechanism of ER adhe-
be tested was: the dentin bond durability and morphological
sive systems is thought to be micromechanical interlocking
features of resin/dentin interfaces of different ER adhesives,
between cured adhesive and hard tissue [1,7]. The strong
including universal adhesives in ER mode, would not differ.
acid etching not only increases surface energy and bond-
ing area but also enhances porosity in mineralized tissue
[1,7]. Different enamel etching patterns exist [8], and very 2. Materials and methods
fine undulations created on enamel surfaces are effective for
establishing strong and durable enamel bonds [9,10]. The for- 2.1. Study materials
mation of a hybrid layer (HL) and of resin tags in dentinal
tubules plays some role in maintaining mechanical inter- Materials used in this study are shown in Table 1. Two three-
locking for dentin bonding. However, in contrast to enamel step ER adhesives, OptiBond FL (OL, Kerr, Orange, CA, USA) and
bonding, concerns remain for dentin bonding due to the pos- Adper Scotchbond Multi-Purpose Plus (SM, 3M Oral Care, St.
sibility of naked collagen fibrils that are not protected by resin Paul, MN, USA), and a two-step ER adhesive, Single Bond Plus
monomers [11,12]. From biomechanical and biological degra- (SB, 3M Oral Care), were used. A universal adhesive, Scotch-
dation perspectives, insufficiently resin-impregnated areas bond Universal (SU, 3M Oral Care), was used as a comparison
may be vulnerable and thus more susceptible to structural adhesive. Phosphoric acid pre-etching was performed using
deterioration. Ultra-Etch (Ultradent Products, South Jordan, UT, USA). The
In contrast, bonding of self-etch (SE) adhesive systems microhybrid resin composite Clearfil AP-X (Kuraray Noritake
involves a chemical interaction between hydroxyapatite (HAp) Dental, Tokyo, Japan) was used for bonding to dentin. A tung-
and functional resin monomers, followed by micromechanical sten halogen visible-light curing unit, Spectrum 800 Curing
interlocking with etched mineralized tissue [7,13]. In particu- Unit (Dentsply Sirona, York, PA, USA), was used, and light
lar, the Ca salt formed by reaction between HAp and functional irradiance (average 600 mW/cm2 ) was checked during the
resin monomer is both water-insoluble, and acid-resistant experiment.
[14,15]. Further, the Ca salt can be created within a clinically
realistic timeframe and shows excellent stability even after
2.2. Specimen preparation
ultrasonication [16].
Universal adhesive systems are widely accepted because
Extracted and de-identified human molar teeth were used.
of their simplified bonding procedures, similar to single-step
After extraction, soft tissue attached to the root was imme-
SE adhesive systems [17,18]. Furthermore, they have broad
diately removed with hand instruments, and teeth were then
applicability because they can be used in either ER or SE
stored frozen (−20 ◦ C) until use. To assure homogeneity, teeth
mode [3,19]. Although the dentin bonding mechanisms of uni-
with almost the same size and shape were carefully selected
versal adhesives in different etching modes are dissimilar,
from stored extracted teeth, and teeth with any sign of caries
previous studies have shown that dentin bond strength in ER
or cracking of the enamel were discarded. Approximately two-
mode is almost unchanged in SE mode [19–21]. A scanning
thirds of apical root structures were then removed using a
electron microscopy (SEM) investigation observed universal
low-speed saw (IsoMet 1000 Precision Sectioning Saw, Buehler,
adhesive/dentin interfaces in different etching modes, and
Lake Bluff, IL, USA). Selected teeth were prepared by sec-
universal adhesives in SE mode exhibited similar morpholog-
tioning the teeth mesiodistally. Each tooth segment was then
 d e n t a l m a t e r i a l s 3 7 ( 2 0 2 1 ) e109–e117 e111

Table 1 – Materials used in this study.

Code Three-step ER adhesives Main components Manufacturer
Lot No.
OL OptiBond FL Primer: HEMA, GPDM, BHT, Kerr
ethanol, water, CQ
6902900 (Primer) Adhesive: bis-GMA, UDMA, Orange, CA, USA
TEGDMA, GDMA, HEMA, filler, CQ,
ODMAB, filler (fumed SiO2 , barium
aluminoborosilicate, Na2 SiF6 ),
ytterbium trifluoride, coupling
factor A174
6911571 (Adhesive)
SM Scotchbond Multi-purpose plus Primer: HEMA, polyalkenoic acid, 3 M Oral Care, St. Paul, MN, USA
water
N852287 (Primer) Adhesive: bis-GMA, HEMA,
triphenylantimony, amines
N86909 (Adhesive)
Code Two-step ER adhesive
SB Adper Single Bond Plus bis-GMA, HEMA, UDMA, Vitrebond 3 M Oral Care
copolymer, GDMA, ethanol, water,
silane treated silica,
diphenyliodonium
hexafluorophosphate, EDMAB, CQ
N898889
Code Universal adhesive in ER mode
SU Scotchbond Universal bis-GMA, MDP, HEMA, Vitrebond 3 M Oral Care
copolymer, silane treated silica,
ethanol, water, CQ, silane
666964
Etching agent
Ultra-Etch G017 35% phosphoric acid Ultradent Products, South
Jordan, UT, USA
ER: etch-and-rinse, HEMA: 2-hydroxyethyl methacrylate, GPDM: glycerol dimethacrylate dihydrogen phosphate, BHT: butylated hydroxy-
toluene, CQ: dl-camphorquinone, bis-GMA: 2,2-bis[4-(2-hydroxy-3-methacryloyloxypropoxy)phenyl] propane, UDMA: urethane dimethacrylate,
TEGDMA: triethyleneglycol dimethacrylate, GDMA: glycerol dimethacrylate, ODMAB: 2-ethylhexyl-4-(dimethylamino)benzoate, EDMAB: ethyl
4-dimethyl aminobenzoate, MDP: 10-methacryloyloxydecyl dihydrogen phosphate.

mounted in an aluminum ring with a diameter of 25 mm using
Triad DuaLine (Dentsply Sirona, Charlotte, NC, USA). Dentin
bonding surfaces were ground flat using a water coolant and a
sequence of silicone carbide (SiC) papers (Struers, Cleveland,
OH, USA) ending at 4000 grit. The study protocol was reviewed
and approved by the Ethics Committee for Human Studies at
our institution (#2015-06).
2.3. Bonding procedures and shear bond strength
(SBS)
Experimental protocols for dentin bonding procedures are
provided in Table 2. Forty specimens were used for each test
group: 15 to determine dentin SBS and 25 to determine the
SFS in ER mode (phosphoric acid application for 15 s before
adhesive application). The sample size for SBS was based
on ISO specification 29022 (Dentistry-Adhesion-Notched-edge
shear bond strength test) [23]. It indicates that the sample size
should be fifteen, and we adopted this number as our SBS
testing protocol is a modified version of ISO 29022. All bond-
ing procedures were performed following the manufacturer’s
instructions (Table 2), and were conducted by a single oper-
ator. After bonding, a stainless steel metal ring was set over
the bonding site and held in place with a custom fixture in
a modified Ultradent Bonding Jig (Ultradent Products). Metal
rings were used to place resin composite on dentin surfaces
for shear bond strength (SBS) and shear fatigue strength (SFS)
tests. The bonding procedure resulted in a resin composite
cylinder inside the ring 2.36 mm in diameter and approxi-
mately 2.5 mm in height. The ring was left in place for both
tests.
Resin composite was condensed in the ring and light-
irradiated for 30 s. Fifteen bonded specimens for each group
were stored in distilled water at 37 ◦ C for 24 h, then sub-
jected to the SBS test. A metal rod with a chisel-shaped end
was used to apply load to the metal ring immediately adja-
cent to the flat tooth surface. The specimens were loaded to
failure at 1.0 mm per min with a universal testing machine
(Type 5500R, Instron, Canton, MA, USA). The SBS values were
calculated from peak load at failure divided by the bonded sur-
face area. To determine bond failure mode, bonding sites on
tooth surfaces and resin composite cylinders were observed
after testing under an optical microscope (SZH-131, Olympus,
Tokyo, Japan) at a magnification of ×10. Based on the percent-
age of substrate area (adhesive, resin composite, and dentin)
observed in de-bonded resin composites and tooth bonding
sites, bond failure was classified into: (1) adhesive failure; (2)
cohesive failure in the composite; (3) cohesive failure in the
dentin; or (4) mixed failure, defined as partially adhesive and
partially cohesive.
e112 d e n t a l m a t e r i a l s 3 7 ( 2 0 2 1 ) e109–e117

Table 2 – Bonding procedures for the tested adhesives.

Adhesive application protocol
Three-step ER adhesive
OL Dentin surface was phosphoric acid etched for 15 s. Etched surface was rinsed with water for 15 s. Dried gently for 3 s (do
not desiccate). Primer was applied to dentin surface with light scrubbing motion for 15 s. Air dried for 5 s. Using same
applicator, adhesive was applied with light scrubbing motion for 5 s. Air thinned for 3 s. Light irradiated for 20 s.
SM Dentin surface was phosphoric acid etched for 15 s. Etched surface was rinsed with water for 15 s. Air dried gently for 2 s.
Left moist. Primer was applied to dentin. Air dried gently for 5 s. Adhesive was applied to dentin. Light cured for 10 s.
Two-step ER adhesive
SB Dentin surface was phosphoric acid etched for 15 s. Etched surface was rinsed with water for 10 s and blotted dry. Apply
2−3 consecutive coats of adhesive for 15 s with gentle agitation. Air dried gently for 5 s. Light cured for 10 s.
Universal adhesive
SU in ER mode Dentin surface was phosphoric acid etched for 15 s. Etched surface was rinsed with water for 10 s. Adhesive was applied to
air-dried dentin surface with rubbing motion for 20 s and then medium air pressure applied to surface for 5 s. Light
irradiated for 10 s.
ER: etch-and-rinse.

2.4. Shear fatigue strength (SFS) tests to a freeze-drying system (Model ID-3; Elionix, Tokyo, Japan)
for 30 min. Specimens were then subjected to argon-ion beam
Bonded specimens were stored in 37 ◦ C distilled water for etching (EIS-200ER, Elionix) for 40 s with an accelerating volt-
24 h before testing. The staircase method of fatigue testing age of 1.0 kV and ion current density of 0.4 mA/cm2 directed
reported by Draughn [24] was used for the SFS test proce- perpendicularly to polished surfaces. Finally, SEM specimens
dure. The initial maximum load applied was 50 %–60% of were coated with a thin film of gold in a vacuum evaporator
the baseline SBS value for each group tested and the lower (Quick Coater, Type SC-701, Sanyu Denchi, Tokyo, Japan) and
load limit was set near zero (0.4 N). Using a sine wave for observed with a FE-SEM (ERA-8800FE, Elionix) at an operating
50,000 cycles or until failure, load was applied at a frequency voltage of 10 kV.
of 20 Hz with an all-electric tabletop test instrument (Elec-
troPuls E1000, Instron). The load was incrementally increased
2.6. Statistical analysis
or decreased (depending on survival or failure) by approxi-
mately 10% of the initial load. The stress that produces 50%
For SBS data, homogeneity of variance (Bartlett’s test) and nor-
failure is termed fatigue strength, in adaptation of the calcu-
mal distribution (Kolmogorov-Smirnov test) were confirmed
lation described by Draughn [24]. Sample size for SFS testing is
before analysis of variance (ANOVA). One-way ANOVA, fol-
difficult to determine, as samples that survive are not reflected
lowed by Tukey’s honestly significant difference (HSD) test (˛
in the calculation, and the number of samples that survive
= 0.05), was used. Statistical analysis for SBS used statistical
cannot be precisely predicted in advance. An initial sample
analysis software (Sigma Plot, ver. 11.0, SPSS, Chicago, IL, USA).
size of 25 typically gives good results for SFS. To determine
A modified t-test with Bonferroni correction was used for SFS
bond failure mode, the bonding sites of failed specimens were
data with a custom program implemented in a spreadsheet
observed using the same procedures as described above for
(Excel, Microsoft, Redmond, WA, USA).
the SBS test.

2.5. SEM observations

3. Results
Specimens for SEM observation were fabricated following
3.1. SBS and SFS
the procedures described earlier to evaluate the resin/dentin
interface thickness of adhesive layers (AL), thickness of the
Mean SBS and SFS values ranged from 33.3 to 41.2 MPa
HL, lengths of internalized resin tags, and alterations near the
(Table 3). The three-step ER adhesives OL and MP showed sig-
interface between AL and dentin substrate.
nificantly higher SBS values than the universal adhesive SU
Bonded specimens embedded in epoxy resin were stored at
and the two-step adhesive SB. No significant difference in SBS
37 ◦ C in distilled water for 24 h, then sectioned longitudinally
was observed between SB and SU. Mean SFS values ranged
with a low-speed saw. Sectioned surfaces were polished with
from 18.3 to 20.3 MPa. MP showed the highest SFS value among
4000 grit SiC papers, then mirror polished with abrasive discs
the tested adhesives and showed a significantly higher SFS
(Fuji Star Type DDC, Sankyo Rikagaku, Saitama, Japan) and
value than SB.
diamond pastes (DP-Paste; Struers, Ballerup, Denmark) with a
final particle size of 0.25 ␮m. Polished specimens were cleaned
ultrasonically for 20 min. Half of the polished specimens were 3.2. Failure mode analysis of de-bonded specimens
etched with HCl solution (6 mol/L) for 25 s and deproteinized
by immersion in a 6% NaOCl solution for three minutes to Failure mode frequencies for each group in SBS and SFS
visualize internalized resin tags. All SEM specimens were showed adhesive failure as the predominant failure mode for
dehydrated in ascending grades of tert-butyl alcohol (50%, 75%, all adhesives (Table 3). All adhesives showed adhesive failure
95% for 20 min each and 100% for two hours), then transferred fin all SFS de-bonded specimens, while some de-bonded spec-
 d e n t a l m a t e r i a l s 3 7 ( 2 0 2 1 ) e109–e117
Table 3 – Comparison of SBS and SFS in different
etch-and-rinse adhesive systems.
SBS SFL Ratio
SFS/SBS
OL 41.2 (5.3)A [93/0/7/0] 19.1 (1.9)ab [100/0/0/0] .464
SM 39.8 (4.7)A [93/0/7/0] 20.3 (3.3)a [100/0/0/0] .510
SB 34.2 (4.6)B [93/0/0/7] 18.3 (1.8)b [100/0/0/0] .535
SU 33.3 (6.3)B [100/0/0/0] 19.0 (2.0)ab [100/0/0/0] .570
Same upper case letter in vertical columns (SBS) indicates no dif-
ference at 5% significance level.
Same lower case letter in vertical columns (SFS) indicates no differ-
ence at 5% significance level.
Values in parenthesis indicate standard deviation.
SBS: shear bond strength, SFS: shear fatigue strength, n = 15 for SBS,
n = 25 for SFS, mean (SD) in MPa.
Failure mode: [adhesive failure/cohesive failure in resin compos-
ite/cohesive failure in dentin/mixed failure] percentage of each
failure mode.
imens for OL, SM, and SB showed cohesive failure in dentin or
mixed failure in SBS.
3.3. SEM observations
Representative SEM images of demineralized and depro-
teinized interfaces showed no differences among adhesive
systems in morphological features near the interface (Fig. 1).
However, irregular filler particles were observed in the resin
tags of OL. For all adhesives, dense resin tags longer than 50
␮m and a 1–2 ␮m hybrid layer (HL) were observed. Additionally,
penetration into branches of dentinal tubules was observed for
all adhesives.
SEM images of resin/dentin interfaces after argon-ion etch-
ing are provided in Fig. 2. The adhesive layer (AL) thickness of
the three-step adhesive OL was approximately 20 ␮m (Fig. 2A).
However, the AL of the three-step adhesive SM (40–50 ␮m,
Fig. 2B) was four to five times thicker than the AL of SU and
SB (10–15 ␮m, Fig. 2C and D). SM showed a homogeneous AL
without inorganic fillers, while other adhesives showed a het-
erogeneous AL. In particular, inorganic fillers were obvious in
OL and SU, and various sizes of irregular fillers (Fig. 2A) were
observed in OL. Uniform spherical nanofillers were observed
in SU (Fig. 2D). All tested adhesives showed a 1- to 2-␮m-thick
HL between AL and dentin substrates. A high-density layer
below the HL was not observed clearly in SM and SB, but OL
and SU showed a thin, high-density layer as a reaction layer
(Fig. 2A and D, indicated by arrow heads). The reaction layer
of SU was clearer and thicker than the layer of OL.
4. Discussion
We should consider dental adhesives from the perspectives
of biological, biochemical, and biomechanical factors for
resin/tooth interfaces when bond durability is evaluated. In
this study, we evaluated bonding durability using an SFS test,
which simulates the oral environment in terms of biomechan-
ical stress from repeated subcritical loading on resin/dentin
interfaces [10,25,26]. The bond strength tests in this study
showed that the three-step adhesives OL and SM had higher
e113
SBS and SFS values than the other adhesive systems, and
significant differences were observed in SBS. Therefore, the
null hypothesis that the dentin bond durability of differ-
ent ER adhesives, including universal adhesives in ER mode,
would not differ, was rejected. s. In this study, the two three-
step ER adhesives showed higher SBS and SFS values than
the other adhesive systems. Although the method of bond
strength measurement differed from the present study, pre-
vious investigations using a ␮-TBS test have compared three-
and two-step ER adhesives, and they showed similar results
to this study. [27–29]. On the other hand, a previous clin-
ical trial compared four adhesives for non-carious cervical
lesions (NCCL), representing all adhesion strategies, and only
the three-step ER adhesive displayed a retention rate above
90% at 18 months. However, it is important to bear in mind that
the dentin substrate in NCCL tends to be sclerotic, unlike this
study [30]. The gold standard three-step ER adhesive OL shows
the highest immediate (after 24 h storage) SBS value among
the tested adhesives in the present study. This result may be
attributed to high filler loading of adhesive resin and high
mechanical strength of cured adhesive layers. SEM images
of resin/dentin interfaces of OL showed irregular filler parti-
cles in resin tags (Fig. 2A). Infiltrating filler particles in resin
tags might thus help establish micromechanical interlock-
ing within dentinal tubules. Also, the functional monomer
glycerol dimethacrylate dihydrogen phosphate (GPDM) may
establish chemical bonds with intact dentin beneath the
hybrid layer [31].
On the other hand, Kawazu et al. [32] have reported a com-
parison of the dentin bond durability after thermal stress (TC)
or long-term water storage (WS) in three different types of ER
adhesive systems from the same manufacture. At the end of
these tests, the three-step ER adhesive SM showed decreased
dentin SBS after prolonged degradation in both TC and WS,
and significantly lower SBS values than the two-step ER adhe-
sive system SB and universal adhesive SU in ER mode. The
thicker adhesive layer of SM might induce greater dimen-
sional alteration from contraction and expansion in response
to temperature variation, resulting in degradation of bonded
interfaces. Further, among the tested adhesives, SM has by
far the highest level of HEMA (30−40 wt%). High HEMA may
increase susceptibility to hydrolytic degradation over time in
WS. However, SM shows a higher SFS than the other adhe-
sives in the present study. A possible explanation is that the
thicker and homogenous adhesive layer of SM may distribute
stress at bond interfaces during repeated subcritical fatigue
stress [33,34]. The AL thickness of SM was approximately twice
that of OL (Fig. 2A and B) and four to five times that of SU
and SB (Fig. 2C and D). Load stress from SFS testing generates
cracks in the vicinity of the adhesive/dentin bonding inter-
face, and plastic deformation zones are formed near the ends
of the cracks. A plastic deformation zone is a zone formed
in the crack tip region that shows permanent deformation or
alterations of solid material without fracture under sustained
stress [35]. Further, the size of plastic deformation zones in
bond areas depends on the elasticity (E) of these areas as
well as E (elasticity)/HD (hardness) ratios. Thus, resistance to
crack propagation may increase when the thickness of cured
adhesive layers is much greater than plastic deformation zone
size. This hypothesis is supported by a previous study that
e114 d e n t a l m a t e r i a l s 3 7 ( 2 0 2 1 ) e109–e117

Fig. 1 – Representative SEM images of resin/dentin interface after HCL and NaOCl treatment. (A) Resin dentin interface with
OL (1000× and 10,000×). (B) Resin dentin interface with SM (1000× and 10,000×). (C) Resin dentin interface with SB (1000×
and 10,000×). (D) Resin dentin interface with SU (1000× and 10,000×).
OL: OptiBond FL, SM: Scotchbond Multi-purpose plus, SB: Single Bond Plus, SU: Scotchbond Universal.
The visible material is indicated by abbreviations: AL: adhesive layer, HL: hybrid layer (white arrowheads), RT: resin tag.

reported the effects of double-layer application on the bonding
effectiveness of universal adhesive systems [36]. Therefore,
understanding the main degradation factor in the experimen-
tal protocol is important when evaluating bond durability in a
simulated oral environment.
This study employed two SEM sample preparation tech-
niques for resin dentin interfaces in order to identify dentin
bond mechanisms in different ER adhesive systems. Firstly,
dentin bond specimens were treated with HCl and NaOCl
solutions to observe patterns of resin monomer infiltration
into dentin. This technique allows imaging of resin tags and
hybrid layers after demineralization and deproteinization. The
other technique uses argon-ion-beam etching, a standard
method in dental SEM research since 1962 [37]. Integrating
these two techniques reveals the ultrastructure of resin/dentin
interfaces and promotes deep insight into dentin bonding
mechanisms.
SEM observations using demineralization and depro-
teinization showed resin tags in different adhesive systems
with similar features. All adhesives exhibited obvious adhe-
sive penetration into branches of dentinal tubules. Further,
lengths of resin tags were more than 50 ␮m (Fig. 1). SEM images
of argon-ion-beam etched resin/dentin interfaces indicated
that the appearances of surfaces below the HL are material
dependent. These images permit the categorization of adhe-
sives into three types based on morphological features of the
reaction layer [22] (Fig. 2). SU showed an obvious thin, high-
density reaction layer that was not visible in SM or SB. For OL,
this layer was less clear than for SU. We speculate that this
layer might be evidence of Ca salt formation during chemi-
cal bonding, and the different appearances of reaction layers
among adhesives might be attributed to the strength of chem-
ical interactions.
All tested adhesives likely possess chemical bonding abil-
ity. OL contains GPDM and SU contains MDP as functional
monomers that are thought to establish chemical bonds with
hydroxyapatite [14–16,31]. In contrast, SM and SB do not con-
tain functional monomers, but do include a polyalkenoic acid
copolymer, namely Vitrebond, that may bond chemically with
dentin HAp [36,37]. A possible reason for differences among
adhesives in morphological appearance near the interface
between HL and intact dentin is different levels of Ca2+ salt
stability during argon-ion etching. Argon-ion-beam etching
is an effective technique for visualizing complex structures
in sharp contrast by selectively removing softer substances
[38]. Therefore, if Ca salts formed in the reaction layer resist
argon-ion-beam etching, the reaction layer will be empha-
sized. A study of the stabilities of Ca salts formed with
GPDM and MDP, using nuclear magnetic resonance (NMR)
spectroscopy, confirmed the adsorption of GPDM onto HAp.
However, this salt was more easily removed than MDP [39].
Yoshida et al. [16] reported that the Ca salt formed between
MDP and synthetic hydroxyapatite showed higher resistance
to ultrasonic vibration and was more stable in an aque-
 d e n t a l m a t e r i a l s 3 7 ( 2 0 2 1 ) e109–e117 e115

Fig. 2 – Representative SEM images of resin/dentin interface after argon-ion etching. (A) Resin/dentin interface with OL
(2500× and 20,000×). (B) Resin/dentin interface with SM (2500× and 20,000×). (C) Resin/dentin interface with SB (2500× and
20,000×). (D) Resin/dentin interface with SU (2500× and 20,000×).
OL: OptiBond FL, SM: Scotchbond Multi-purpose plus, SB: Single Bond Plus, SU: Scotchbond Universal.
The visible material is indicated by abbreviations: AL: adhesive layer, HL: hybrid layer (white arrowheads), RC: resin
composite, RL: reaction layer (yellow arrowheads).

ous environment than salts formed with other functional micromechanical interlocking. Further research is needed to
monomers—4-methacryloxyethyl trimellitic acid (4-MET) and investigate the dentin bond mechanisms of ER adhesive sys-
2-methacryloxyethyl phenyl hydrogen phosphate (phenyl-P). tems.
Further, an investigation of chemical interactions between
synthetic HAp and Vitrebond-copolymer-containing (VCP)
5. Conclusions
adhesives, including SM, SB, and SU, found that a chemi-
cal interaction occurred between VCP-containing adhesives
In the results of this in vitro study, three-step ER adhesives
and synthetic HAp. However, the polyalkenoate reaction may
showed significantly higher SBS values than both a two-step
have been retarded or hindered by the addition of specific
ER adhesive and a universal adhesive in ER mode. Dentin bond
resin components such as MDP in SU [37]. Therefore, MDP
durability measured by the SFS test showed a higher SFS for
has a stronger affinity for dentin HAp than other functional
three-step ER adhesives than for other ER adhesives. Still,
monomers or VCP, which may explain the strong expression
no significant differences were found between the universal
of the reaction layer in SU. However, morphological features
adhesive in ER mode and the three-step ER adhesives. Further-
around the interface between HL and intact dentin may not be
more, although no apparent differences were found between
directly connected to dentin bond strength. Mechanical test-
adhesive systems in internalized resin tags, as shown by SEM,
ing of adhesion, such as SBS or SFS, may be influenced by both
their morphological appearance below the HL was material
chemical bond strength and the mechanical properties of the
dependent. The thin high-density reaction layer in SU was
adhesive layer and its thickness.
more evident than in other adhesives.
Because of the presence of vulnerable regions in the
vicinity of adhesive/dentin interfaces, ER adhesive systems
raise concern for dentin bond durability [40]. In general, the Funding
main dentin bonding mechanism of ER adhesive systems
is micromechanical interlocking between porous dentin tis- This work was supported in part by Grants-in-Aid for Scien-
sue after decalcification and cured resin monomers. However, tific Research, No. 19K10158, from the Japan Society for the
given the results of SEM observations, some ER adhe- Promotion of Science. This project was also supported in part
sives might establish chemical bonding between functional by the Sato and Uemura Fund by a grant from Nihon University
monomers and dentin HAp below the HL in addition to School of Dentistry, Japan.
e116 d e n t a l m a t e r i a l s 3 7 ( 2 0 2 1 ) e109–e117
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific
Research, No. 19K10158, from the Japan Society for the Promo-
tion of Science. This project was also supported in part by the
Sato Fund, Uemura Yasuo•Haruko fund, and by a grant from
the Dental Research Center of the Nihon University School of
Dentistry, Japan.
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