Accepted Manuscript

Title: Adhesive interfacial characteristics and the related

bonding performance of four self-etching adhesives with
different functional monomers applied to dentin

Authors: Ruirui Wang, Yilin Shi, Tingting Li, Yueping Pan,

Yu Cui, Wenwei Xia

PII: S0300-5712(17)30120-3
DOI: http://dx.doi.org/doi:10.1016/j.jdent.2017.05.010
Reference: JJOD 2773

To appear in: Journal of Dentistry

Received date: 17-1-2017

Revised date: 6-5-2017
Accepted date: 15-5-2017

Please cite this article as: Wang Ruirui, Shi Yilin, Li Tingting, Pan Yueping,
Cui Yu, Xia Wenwei.Adhesive interfacial characteristics and the related bonding
performance of four self-etching adhesives with different functional monomers applied
to dentin.Journal of Dentistry http://dx.doi.org/10.1016/j.jdent.2017.05.010

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Adhesive interfacial characteristics and the related bonding

performance of four self-etching adhesives with different functional

monomers applied to dentin

Short title: interfacial characteristics, bonding performance, functional

monomers

Ruirui Wanga, Yilin Shia, Tingting Lia, Yueping Pana, Yu Cuia, Wenwei

Xiaa*

a
Department of Endodontics, Ninth People's Hospital, Shanghai Jiao

Tong University School of Medicine, Shanghai Key Laboratory of

Stomatology, Shanghai, China

*Corresponding author: Wenwei Xia, Address: 500, Quxi Road,

Shanghai 200011, China, Phone: +86 18621586202, E-mail address:

xiawenwei16@shsmu.edu.cn

Key words: functional monomer, hydrophilicity, chemical bonding, bond

strength, nanoleakage, durability.

Abstract
Objectives: To examine the interfacial chemical and morphological characteristics of
four self-etching adhesives bonded to dentin with different functional monomers.
Further, to evaluate the effects of this interaction between functional monomers and
dentin on short-term in vitro bonding performance of the four adhesives.
Methods: Clearfil SE Bond (CSE) and Scotchbond Universal (SU) containing
10-methacryloxydecyl dihydrogen phosphate (10-MDP), Optibond XTR (OX)
containing glycero-phosphate dimethacrylate (GPDM), and Adper Easy One (AEO)
containing 6-methacryloyloxyhexyl dihydrogen phosphate (6-MHP) were applied to
the dentin surface according to the instructions supplied with each. Interaction
between the functional monomers and dentin was characterized using thin-film X-ray
diffraction (TF-XRD) and scanning electron microscopy (SEM). The hydrophilicity
of each acidic monomer was also assessed by chemical structure drawing software.
Micro-tensile bond strength (μTBS) and nanoleakage were used to evaluate the
bonding effectiveness of the adhesives, either immediately or after thermo-cycling
(5°C–55°C) for 5,000 cycles.
Results: TF-XRD showed that both CSE and SU exhibited 10-MDP–Ca
nano-layering at the adhesive interface, but with different intensity when reacted with
dentin. OX, that contains GPDM, demineralized the dentin surface more severely,
forming long resin tags into the dentinal tubules, and gained the highest μTBS at the
immediate time-point. Thermo-cycling adversely affected the μTBS and nanoleakage
of AEO and OX, but had no significant influence on CSE and SU which contain
10-MDP.
Conclusions: Self-etching adhesives containing different structures/concentrations of
functional monomers produced adhesive interfaces with obviously different chemical
and morphological characteristics, which may have a direct impact on bonding
effectiveness.
Clinical relevance: Our findings support the concept that the stable chemical bonding
produced by 10-MDP to the Ca of hydroxyapatite is advantageous for durability of
adhesive–dentin bonds. In contrast a higher immediate bond strength was achieved
with the functional monomer GPDM that etched and wetted the dentin surface better.
Key words: functional monomer, chemical bonding, hydrophilicity, bond strength,
nanoleakage, durability.
1. Introduction
The bonding performance of the various commercially-available self-etching
adhesives evaluated either by laboratory or clinical studies varies greatly, relying on
the actual composition [1, 2] or rather the specific functional monomer included in it
[3]. With functional monomers, self-etching adhesives can simultaneously
demineralize and infiltrate the tooth surface. The chemical and morphological
characteristics of the adhesive–tooth interface and the closely-related quality of the
hybrid layer depend to a large extent on the interaction between functional monomers
and the tooth substrate [4, 5].
With different chemical structures, the functional monomers containing acidic
groups may interact quite differently with hydroxyapatite (HAp) and thus tooth tissue,
and even small changes may influence their polarity and consequently, influence their
interaction behavior and bonding efficacy [6, 7]. According to the concept of
‘Adhesion–Decalcification‘ (A–D concept) [8] that describes the way acidic molecules

interact with HAp, all acids bond ionically to the calcium of HAp in the first phase.
The molecule will then remain bonded if the ionic bond is hydrolytically stable, or it
will de-bond and calcium will be removed from deep within the tooth surface, leading
to a relatively deep demineralization of the dentin if the ionic bond is unstable in the
second phase.
A functional monomer such as 10-methacryloxydecyl dihydrogen phosphate
(10-MDP) can form stable calcium–phosphate complexes, and self-assemble into the
form of a regular layered structure at the apatite surface. The good hydrolytic stability
of 10-MDP-Ca salts may be attributed to the presence of long and relatively
hydrophobic spacer chains of 10-MDP. The unique chemical structure of 10-MDP and
the resultant intense and stable adhesion to the calcium in HAp has been shown to
contribute particularly to the durability of the bond as well as enhance the initial
bonding performance of self-etching adhesives [3, 9, 10]. Several recently marketed
‗universal‘ adhesives such as Scotchbond Universal (3M ESPE, Seefeld, Germany),
All-Bond Universal (Bisco, Schaumburg, IL, USA), and Clearfil 3S Bond Plus
(Kuraray Noritake Dental, Tokyo, Japan) have been formulated with 10-MDP. The
bonding effectiveness of universal adhesives has therefore been a hot topic since they
came into use [11–14].
Certainly, apart from 10-MDP, functional monomers such as glycero-phosphate
dimethacrylate (GPDM) also appear to be used in ‗universal‘ adhesives (OptiBond
XTR [Kerr, Orange, CA, USA]). However, to our knowledge there are no publications
concerning the characterization of the interaction of GPDM with HAp. In fact, current
work with regard to the chemical interaction of acid monomer with HAp-based
substrates mainly focused on 10-MDP, 4-methacryloxyethyl trimellitic acid (4-MET)
and 2-methacryloxyethyl phenyl hydrogen phosphate (Phenyl-P) [9, 15, 16].
Furthermore, self-etching adhesives are generally mixtures of components including
an acid functional monomer, hydrophobic monomers, water, and an organic solvent,
and thus functional monomers account for only a small proportion. Very few
investigations have specifically adopted commercial adhesives as the object of studies
to clarify the actual interaction between functional monomer and dentin and analyzed
their practical effects on bonding performance, considering that both the concentration
of functional monomer and other components in adhesives may influence the
interaction.
To address this issue, in the present study, we chose four commercial self-etching
adhesives: the two-step self-etching adhesives Clearfil SE Bond containing 10-MDP,
universal adhesives Scotchbond Universal also containing 10-MDP but at a lower
concentration and Optibond XTR containing GPDM, and the one-step self-etching
adhesive Adper Easy One containing 6-methacryloyloxyhexyl dihydrogen phosphate
(6-MHP). The aim of this study was first to characterize the interaction between the
functional monomers in the adhesives and the dentin chemically, using X-ray
diffraction (XRD), and also ultrastructurally, using scanning electron microscopy
(SEM). The effects of this interaction on short-term in vitro performance of the
adhesives bonded to human dentin were then analyzed, by assessing micro-tensile
bond strengths (μTBS) and nanoleakage. The null hypotheses were that：(1) adhesive
interfacial characteristics of the four self-etching adhesives with different functional
monomers applied to dentin are almost the same; (2) there is no difference in
short-term in vitro bonding performance of the four adhesives tested.

2. Materials and methods

One hundred and twenty-six caries-free extracted human third molars were
collected after obtaining informed consent from donors under a protocol approved by
the Ethics Committee for Human Studies of the Shanghai Ninth People‘s Hospital.
The teeth were cleaned, stored in 1% chloramine at 4°C and used within 1 month
following extraction. The compositions, manufacturers and application instructions of
the four adhesives examined in the present study are listed in Table 1.

2.1. Thin-film X-ray diffraction (TF-XRD) analysis

Fifteen dentin disks (10 × 8 × 1 mm) were cut using a low-speed diamond saw
(ISO Met 4000, Buehler Ltd, Lake Bluff, IL, USA) under water cooling. The prepared
disks were polished with wet 600-grit SiC paper for 1 min. All dentin surfaces were
carefully verified, by stereomicroscopy, for the absence of enamel/pulp tissue. Each of
the four self-etching adhesives (Table 1) were applied to dentin disks by lightly
rubbing each dentin surface using a microbrush according to the instructions supplied
with each (n = 3, referred to as ‗CSE_dentin‘, ‗SU_dentin‘, ‗OX_dentin‘ and
‗AEO_dentin‘). The samples were then vigorously air-dried before being analyzed by
thin-film XRD (Bruker AXS D8, Germany) operated at 40 kV acceleration and 200
mA current, with the angle of the incident X-ray beam fixed at 2.0° and a scanning
time of 0.02°/sec for 2θscan). Untreated dentin (‗dentin‘) specimens were examined
by XRD as reference.

2.2. Morphological assessment under SEM

 The morphological assessment consisted of two parts: the first part consisted of
observation of the surfaces of adhesive-treated dentin, with and without rinsing with
ethanol or distilled water following a protocol similar to that described by Yoshihara
et al. [6]. In the second part, vertical sections of the adhesive interface were further
prepared to assess the extent of adhesive infiltration into dentin.
Part 1: Further 600-grit SiC-paper-ground thirty-nine dentin specimens were
prepared. Thirty-six of the disks were treated with each of the four self-etching
adhesives whilst the other three dentin disks were used as untreated controls (n=9).
The ―self-etching adhesives + dentin specimens‖ and ―self-etching adhesives + dentin
ethanol/water rinsed specimens‖ (three specimens per subgroup) were mounted on
aluminum stubs, dehydrated in silica gel, gold sputter-coated, and imaged by SEM
(S3400, Hitachi, Tokyo, Japan) at 15 kV.
Part 2: Twelve teeth were selected for this part. The enamel and superficial
dentin of these teeth were removed to expose the mid-coronal dentin. The exposed
dentin surfaces were then polished using 600-grit SiC paper for 60 s under running
water to create standardized surfaces. The adhesives were applied to the dentin by
lightly rubbing each dentin surface using a microbrush following the respective
manufacturers‘ instructions, then air-dried and light-cured for 10 s under an LED
light-curing unit (Elipar Trilight, 3M ESPE) with an output intensity of 750 mW/cm2 .
Composite resin crowns were built with a nanofilled composite resin (Filtek Z350 XT,
3M ESPE, St. Paul, MN, USA) in two increments of 2 mm each. Each increment was
light-cured for 40 s under the LED light-curing unit mentioned above. After storing in
distilled water for 24 h at 37°C, all resin–dentin specimens were longitudinally
sectioned perpendicular to the bonded interface with a low-speed diamond saw (ISO
Met 4000) under sustained water-cooling to obtain resin–dentin slabs (the central
slabs, three specimens per group) with a thickness of 1 mm.
The surfaces of the slabs were treated with 37% phosphoric acid for 30 s to
completely eliminate any smear layer created by cutting. Surfaces were then treated
with 5% sodium hypochlorite for 5 min to provide evidence of infiltration of the
adhesive into dentin. Finally, prepared slabs were sputter-coated with gold and
examined under SEM at an accelerating voltage of 15 kV.

2.3. Hydrophilicity assessment of acidic functional monomer

The number of carbon atoms and/or ester/polyether groups in spacer chains may
influence the hydrophilicity of the functional monomers and also their interaction
with calcium and dentin [7]. As displayed in Fig. 1, the chemical structures of the
three phosphate functional monomers evaluated in the present investigation were
different from each other considering both of number of carbon atoms and presence of
hydrophilicity group in spacer chains. The hydrophilicity of each acidic functional
monomer was assessed using the chemical structure drawing software ChemBioDraw
Ultra 14.0 (Perkin Elmer, Waltham, MA, USA) which calculated the estimated
hydrophobicity of each structure by means of Viswanadhan‘s fragmentation method
[17]. Higher logarithms of partition coefficient indicate more hydrophobic structures.

2.4. Micro-tensile bond strength (μTBS) testing

Another 48 teeth were divided into four groups of 12 each and randomly
assigned to one of the four test adhesives. Resin–dentin bonded specimens were
prepared as previously described (2.2 Part 2). After storing in distilled water for 24 h
at 37°C, specimens were then longitudinally sectioned in mesio-distal and
buccal-lingual directions across the bonded interface to obtain resin–dentin sticks with
a cross-sectional area of approximately 1 mm2. Only sticks (2–3 sticks) from the
central two slabs of each tooth were used, and 30 sticks of each group were obtained.
Half of the sticks were used immediately (24 h) for the μTBS test, whilst the
remaining half were thermo-cycled (TC-501F, Suzhou Weier laboratory equipment
Co., Ltd., Suzhou, China) for 5,000 cycles at 5°C –55°C with a dwell time of 30 s in
each water bath, and then subject to μTBS testing.
For μTBS testing, the sticks were fixed to a testing jig with cyanoacrylate glue
and tested to failure using a universal tester (Vitrodyne V1000, Liveco Inc.,
Burlington, VT, USA) at a crosshead speed of 1 mm/min. The μTBS values (MPa)
were calculated by dividing the load at failure by the cross-sectional bonding area.
Statistical analysis was performed using sticks as the statistical unit and carried out
with SPSS version 20.0 (SPSS, Chicago, IL, USA). Data were analyzed using
two-way analysis of variance (with the type of self-etching adhesives and testing
condition as the variables) and Tukey‘s test at a = 0.05.

2.5. Nano-leakage evaluation

Twelve teeth were used for nano-leakage evaluation. Resin-bonded sticks of each
self-etching adhesive were prepared as previously described (2.4) with half of them
having been thermo-cycled for 5,000 cycles (three sticks for each subgroup) for
nanoleakage evaluation. The sticks were immersed in a 50wt% ammoniacal silver
nitrate solution [18] in darkness for 24 h, rinsed thoroughly in distilled water, and then
immersed in photographic developing solution for 8 h under a fluorescent light to
reduce silver ions into metallic silver grains within voids along the bonded interface.
Thereafter, the specimens were rinsed with distilled water and polished with wet
2000-grit SiC paper before being ultrasonically cleaned and air dried. Resin–dentin
interfaces were analyzed using the aforementioned SEM in the backscatter mode. The
nanoleakage pattern was only qualitatively observed.

3. Results
3.1. TF-XRD analysis
The untreated dentin samples (Fig. 2, ‗dentin‘) showed XRD peaks at 2θ =
26.14°, 28.86°, 31.80°, 32.2°, and 33.00°, which must be ascribed to dentinal HAp.
The dentin specimens treated with CSE revealed three characteristic peaks at 2θ =
2.28° (d = 3.87 nm), 4.52° (d = 1.95 nm), and 6.70° (d = 1.32 nm) (Fig. 2,
‗CSE-dentin‘), representing the formation of 10-MDP–Ca salts [9] and thus the
so-called ‗nano-layering‘. Likewise, three characteristic peaks at 2θ = 2.48° (d =
3.56nm), 5.08° (d = 1.74 nm), and 7.64° (d = 1.16 nm) showing a slight shift and
decreased intensity were detected in the SU group (Fig. 2, ‗SU-dentin‘). These peaks
were not detected in the untreated dentin reference. No characteristic peaks that could
be assigned to the products of chemical interactions between the functional monomer
and dentin in the OX and AEO groups were observed.

3.2. Morphological assessment under SEM

Figure 3 shows the surfaces of adhesive-treated dentin following ethanol rinsing.
Some distinct structural differences originating from the interaction of the different
monomers with dentin were observed. In the CSE and SU groups, most of the
adhesives were retained on the surfaces of the dentin even after rinsing with ethanol,
which may be attributed to the stable 10-MDP–Ca formed. In contrast, in the AEO
group, adhesives were easily removed by ethanol, but some smear debris that may not
be fully demineralized remained in the dentinal tubules. In the OX group, the smear
layer was completely removed, and the dentinal tubules were patent after rinsing with
ethanol.
The SEM results in Figure 4 show vertical sections of the adhesive interface and
to some extent support the observations shown in Figure 3 according to the ‗A–D
concept‘. As all of the specimens were etched with 37% phosphoric acid to eliminate
any smear layer and superficial dentin, adhesives which penetrated into the dentin
were clearly exposed. As measured roughly in the 5000× image, the mean infiltration
depth of both CSE and SU was 1 μm, while for AEO it was less than 1 μm. In contrast,
deep penetration of OX was discernible by formation of resin tags of 15–30 μm in
length into the dentinal tubules.

3.3. Hydrophilicity assessment of acidic functional monomers

The hydrophobicity of each functional monomer shown by estimation of the
logarithm of the partition coefficient (noctanol/water: log (p)) was higher for 10-MDP
(4.09) and lower for GPDM (1.99), whereas 6-MHP (2.5) presented intermediate
hydrophobicity.

3.4. Micro-tensile bond strength testing

Micro-tensile bond strength data of the four self-etching adhesives are presented
in Table 2. The variables the type of self-etching adhesives (F = 43.443; P < 0.001)
and testing condition (F = 9.181; P < 0.001) influenced bond strength significantly.
The interactions between the type of self-etching adhesives and testing condition were
not significant (P = 0.296). The μTBS to dentin of both OX and SU was significantly
higher than that of CSE and AEO at 24 h, with OX being the highest. A significant
reduction (P = 0.009) in μTBS was recorded for AEO after thermo-cycling for 5,000
cycles. In addition an impressive drop was observed for OX after aging, although this
difference was not significant (P = 0.073). The μTBS of CSE and SU remained stable
before and after thermo-cycling (P > 0.05).

3.5. Nano-leakage evaluation

Representative backscattered SEM images of the resin–dentin interfaces for all
adhesives used in this study before and after thermo-cycling are depicted in Figure 5.
All of the adhesives exhibited spotted or linear discontinuous silver nitrate uptake
along the hybrid layer before thermo-cycling. There was only a slight increase of
silver impregnation after thermo-cycling in the CSE and SU groups. In contrast, the
OX and AEO groups showed obviously increased silver infiltration after
thermo-cycling, and in many cases the entire length of the hybrid layer was infiltrated
with silver deposits of the specimens prepared. Obviously, the area ratio of
silver-infused regions in the hybrid layer of the OX and AEO groups was
considerably higher than that observed in the CSE and SU groups after
thermo-cycling despite the lack of statistical analysis.

4. Discussion
Results from both chemical and morphologic evaluations suggest that interfacial
characteristics of the four self-etching adhesives tested applied to dentin were
different, leading us to reject the first null hypothesis. The bonding mechanism of
self-etching adhesive systems has been intensely investigated and two types of
bonding mechanisms–micro-mechanical and chemical bonding–have been established.
It has been reported that chemical bonding between functional monomers and the
calcium of HAp can significantly improve both bonding strength and bonding
durability. Among the functional monomers so far tested, 10-MDP was found to
adhere to calcium and dentin most readily and strongly [6, 16]. In the present study,
three characteristic peaks representing the formation of nanolayered 10-MDP–Ca salts
could be detected by XRD when the 10-MDP-containing adhesives CSE and SU were
applied to dentin according to the instructions supplied with each. Though both of the
adhesives contain 2-hydroxyethyl methacrylate, and SU also contains polyalkenoic
acid copolymer [14], both of which have been reported to interfere with nano-layering
of 10-MDP–Ca salts at the interface by reducing the HAp-demineralization rate or
competing with 10-MDP for the same Ca-bonding sites in HAp [19].
It is worth noting that the three characteristic XRD peaks detected in the CSE
group are almost the same as the peaks observed in a synthesized MDP-0.5 Ca salt
(with a molar ratio of MDP to calcium of 2) which showed three characteristic XRD
peaks at 2.24o, 4.51o, and 6.76° in a more recent study [20]. Meanwhile the
XRD-peaks in the SU group were closer to that of synthesized MDP-1 Ca (with a
molar ratio of MDP to calcium of 1) at 2.42°, 4.88°, and 7.34°. These results were
consistent with the fact that the concentration of 10-MDP in CSE, a ‗two-bottle‘
self-etching adhesive that has 10-MDP incorporated into both the primer and the bond,
is higher than that in SU, a ‗one-bottle‘ universal adhesive, and thus contains
proportionally less 10-MDP. The relatively decreased intensity revealed in the SU
group by XRD also confirmed this, since the XRD peak intensity is a measure of the
quantity of a specific compound present within a certain substrate [21].
No characteristic peaks that may be assigned to formation of GPDM-Ca salts in
the OX group appeared. GPDM is one type of phosphoric functional monomer and it
is one of the first chemical compounds proposed to improve bonding with human
dentin [22]. Recent research has indicated that formation of monomer–calcium salts is
influenced by the length (number of carbons) and hydrophilicity of the spacer chain of
functional monomers [7]. Functional monomers with a long and rather hydrophobic
spacer chain such as 10-MDP and 12-methacryloyloxy-dodecyl-dihydrogen
Phosphate (12-MDDP) [7], or that contain an additional hydrophobic trimethylphenyl
group such as MAEPA [6], can form stable ionic bonds with the Ca of HAp. In
contrast, the shorter spacer chain and the higher hydrophilicity of GPDM compared to
10-MDP, calculated according to Viswanadhan‘s fragmentation method, may not
favor such stable monomer–calcium formation. Taking these data together with the
findings of previous studies [6, 7], it could reasonably be inferred that the chemical
bonding efficacy of GPDM may be very limited compared with that of 10-MDP.
The SEM results shown in Figures 3 and 4 show the surfaces of adhesive-treated
dentin with ethanol rinsing and a vertical section of the adhesive interface,
respectively, and support the results obtained in TF-XRD analyses according to the
‗A–D concept‘. In the CSE and SU groups, most of the adhesives were retained on the
surfaces of dentin even after rinsing with ethanol, which may be attributed to the
stable 10-MDP–Ca formed. In contrast, hardly any adhesive remained on the dentin
surface and the smear layer was completely removed, clearly exposing the dentinal
tubules, in the GPDM-based group. Indeed, the peritubular dentin and the intertubular
dentin were demineralized severely and the smear layer on the dentin surface was
dissolved thoroughly after treatment with a ‗primer‘ of OX compared with that of
CSE (our unpublished data). Deep penetration of OX formed resin tags of 15–30 μm
in length extending into dentinal tubules as revealed in Fig. 4, and could, at least in
part, be attributed to the relatively intense ‗etching‘ effect of GPDM, though also to
the strong hydrophilicity itself that induced better dentin wettability [23]. While for
10-MDP-based CSE and SU, the mean infiltration depth was approximately 1 μm.
Taken together with the results from TF-XRD, we can conclude that the chemical
bonding potential of GPDM is relatively weak and it tends to demineralize the dentin
in the second phase according to the ‗A–D concept‘.
The structure of 6-MHP is similar to that of 10-MDP apart from a shorter
carbon-chain spacer which consists of only six methylene groups. Consequently the
hydrophobicity of 6-MHP is lower than that of 10-MDP. A previous investigation
reported that XRD of an experimental 6-MHP primer revealed three characteristic
peaks, indicating deposition of 6-MHP–Ca salts but less intensively in comparison
with 10-MDP [24]. The absence of similar characteristic peaks in the present study
could probably be attributed to the low concentration of 6-MHP in ―one-bottle‖ AEO
formulations, and thus the little 6-MHP–Ca generated may not meet the requirements
for the amounts (3–5wt.% of a material) detectable by XRD [6]. A previous study also
showed that 6-MHP had low decalcification ability [24], which agreed well with our
SEM results showing that the adhesive AEO was easily removed and dentinal tubules
exposed with some smear debris left after rinsing with ethanol, and that virtually no
resin tags formed in a vertical section of the adhesive interface. The μTBS of AEO
was the lowest whether before or after aging, and thermo-cycling had a negative
impact on the bonding performance of AEO, which might result from the weak ionic
bonding of 6-MHP and calcium in dentin as well as the comparatively inapparent
mechanical retention at the interface.
It has been demonstrated that functional monomers showing more intense
monomer–calcium formation are more likely to produce high initial bond strength and
durability, compared with those attaining lower monomer–calcium formation [3, 7,
10]. However, in the current study, GPDM containing adhesive OX gained the highest
bonding strength before thermo-cycling, although GPDM exhibited lower formation
of ionic bonds with calcium compared to 10-MDP. This might be attributable to the
strong micromechanical interlocking resulting from resin tags at the interface,
seemingly like the reported ‗gold standard‘ of etch-and-rinse adhesives. The
micro-mechanical interlocking is considered as a prerequisite to achieve good
bonding [5]. Another special advantage of GPDM is that it has two polymerizable
groups, and thus tends to react with the other monomers in the adhesives and the
restorative material more strongly than other functional monomers used in the study
with only one polymerizable group. A higher degree of polymerization is correlated
with increased quality of the polymer network and improved mechanical properties of
resin-based materials [25–27].
However, an impressive drop in bond strength after aging was recorded for OX
although this was not statistically significant, and OX showed obviously increased
silver infiltration after thermo-cycling, both of which were signals of degradation at
the adhesive interface. GPDM exhibited greater demineralization of dentin rather than
bonding to calcium of HAp, in which process the unstable calcium phosphate
dicalcium phosphate dehydrate is produced and embedded in the thin hybrid layer [9].
In an aqueous environment, these products dissolve out gradually, thereby seriously
weakening the interfacial integrity. In addition, adhesives containing more hydrophilic
monomers produce resin–dentin interfaces more prone to water contamination and
hydrolytic degradation, and this may thus explain the measured increase in
nanoleakage of OX after aging, as GPDM shows the highest hydrophilicity of the
adhesives tested in the present investigation.
By contrast, the presence of 10-MDP in the composition of CSE and SU may
well explain the stable bond strength and nanoleakage before and after thermo-cycling.
The hydrophobic long alkyl chain in 10-MDP that may keep the water molecules at a
distance, coupled with the stable nano-layering of 10-MDP-Ca, can effectively protect
the created adhesive interface from biological degradation. Besides 10-MDP, SU also
contains a polyalkenoic acid copolymer. As the main components of glass-ionomers,
polyalkenoic acid has been reported to bond chemically and spontaneously to HAp.
The basic difference between polyalkenoic acid and 10-MDP is that the latter consists
of individual monomers that upon polymerization become a polymer linked to HAp,
while polyalkenoic acid is a polymer with a multitude of carboxyl functional groups
attached to the polymer backbone and can ―grab‖ Ca at different and remote sites [4].
Glass-ionomers have indeed been observed to have the lowest annual failure rate with
regard to Class-V adhesive restorations [28]. In this study, SU showed a significantly
higher μTBS than CSE (which only contains 10-MDP) with or without
thermo-cycling, a property which may be attributed to the existence of polyalkenoic
acid. On the whole, difference in short-term micro-tensile bond strengths and
nanoleakage of the four adhesives tested were observed, and we may also reject the
second null hypothesis.
In general, the findings of this study support the concept that the stable chemical
bonding produced by 10-MDP to the Ca of HAp is advantageous for the durability of
adhesive–dentin bonds. In contrast, a higher immediate bond strength results from the
functional monomer GPDM, that etches and wets the dentin surface better. In addition,
the two recently marketed universal adhesives SU and OX gained higher bonding
strength even after aging than the traditional CSE and AEO, although OX showed
increased nanoleakage after thermo-cycling, which represented the progress of
bonding technology.
In addition, although the ‗Adhesion–Decalcification‘ model regarding interaction
of acidic molecules with apatite has been verified by many researchers and their
studies [6–8, 21, 24, 29–31] since it was put forward, the actual chemical reaction
process between acidic molecules and apatite changes dynamically and new
discoveries into the reaction mechanism have emerged recently. Tian et al. [32]
reported that 10-MDP and its analog MDA are eluted completely from mineralized
dentin powder in column chromatography experiments using an ethanol–water mobile
phase, which indicated that the interactions between tested monomers and apatite are
most likely to be reversible and challenges the previously proposed
adhesion–decalcification concept. The chemical mechanism of the interaction of
acidic monomers with HAp/tooth hard substance would be of great interest to
investigate further.

5. Conclusions
Within the limits of the present study, it may be concluded that self-etch
adhesives containing different structures/concentrations of functional monomers
produced adhesive interfaces with obviously different chemical and morphological
characteristics, which may have a direct impact on bonding effectiveness.

Conflict of interest
The authors declare no potential conflict of interest in this study.

Acknowledgements
Materials in this study were kindly donated by Kuraray, 3M ESPE and Kerr. This
study was supported by STCSM (No. 15140902402).
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Tables
Table 1 - Adhesives investigated in the present study
Adhesive pH Lot No. Composition Application
procedure
Clearfil SE primer: primer: 1.Primer:10-MDP,water,HEMA,camphorquinone, 1. Apply primer to
Bond 1.9 01218A hydrophilic dimethacrylate tooth surface and
(Kuraray bond: 2.Bonding: 10-MDP, Bis-GMA, HEMA, leave in place for 20s.
Medical 01841A camphorquinone, hydrophobic dimethacrylate, 2. Dry with air stream
Inc., N,N-diethanol p-toluidine bond, colloidal silica to evaporate
Tokyo, the volatile
Japan) ingredients.
3. Apply bond to the
tooth surface
and then create a
uniform film using
a gentle air stream.
4. Light-cure for 10 s.
Scotchbond 2.7 527687 10-MDP, HEMA, silane, dimethacrylate resins, Self-etch (SE)
Universal VitrebondTM copolymer, filler, ethanol, water, 1. Apply the adhesive
(3M ESPS, initiators or adhesive
Seefeld, mixture to the
Germany) prepared tooth and
rub
it in for 20 s.
2. Gently air-dry the
adhesive for
approximately 5s for
the solvent to
evaporate.
3.Light cure for 10 s.
Optibond primer: primer: 1.primer: GPDM Dimethacrylate, HEMA, 1.Apply primer to the
XTR (Kerr, 2.4 5033284 Acetone, Ethanol, Water, CQ Initiator enamel/ dentin
Orange, adhesive: adhesive 2. adhesive: Bis-GMA, Tri-functional monomer, surface using the
CA, USA ) 3.4 5033285 HEMA, Ethanol, CQ Initiator Barium glass filler, disposable applicator
nano-filler. brush; scrub the
surface with a
brushing
motion for 20 s.
2. Air thin for 5
seconds with medium
air pressure;
3.Apply adhesive to
 the enamel/dentin
surface with
light-brushing motion
for
15s; air thin for 5 s.
4. Light cure for 10 s.

Adper Easy 2.4 558422 HEMA, Bis-GMA, water, phosphoric acid- 1. Dispense one drop
Bond (3M -methacryloxy-hexylwsters, ethanol, of the adhesive
ESPS, silane-treated silica, HDDMA, copolymer of into a dappen dish
Seefeld, acrylic and itaconic acid, DMAEMA, phosphine and apply liberally
Germany) oxide, CQ with an applicator for
15 s using rubbing
motion.
2. Gently air-blow
until the liquid does
not move anymore.
3. Light-cure for 10s.

Table 2 - Micro-tensile bond strengths of the four self-etching adhesives bonded to dentin

before and after thermo-cycling (MPa, Means ± Standard Deviations).

Adhesive Immediate After thermo-cycling P value

CSE 37.20±5.79a 36.48±7.24a 0.807

SU 54.25±8.61b 51.10±8.25b 0.314

OX 57.72±9.75b 50.71±9.33b 0.073

AEO 37.08±6.51a 27.66±8.48c 0.009

F value 21.396 23.749

P value 0.000 0.000

a,b,c——the same superscript letters above the columns indicate no significant difference (P >

0.05).
Figure Legends
Fig. 1- Chemical structures of the three different phosphate functional monomers

Fig. 2- TF-XRD patterns of untreated dentin and adhesive-treated dentin. The 10-MDP-based CSE
group and SU group exhibited three characteristic peaks representing 10-MDP-Ca salts formation
but with different intensity. In the 6-MHP-based AEO group and the GPDM-based OX group, no
peaks that could be attributed to monomer-Ca salts could be identified.

Fig. 3-SEM micrographs of dentin treated with each self-etching adhesive and rinsed with ethanol.
In the CSE and SU groups, most of the adhesives were retained on the surfaces of dentin even
after rinsing with ethanol. In the OX group, the smear layer was completely removed, and the
dentinal tubules were patent after rinsing with ethanol. In the AEO group, adhesive was easily
removed by ethanol, but some smear debris that may not be fully demineralized remained in the
dentinal tubules.

Fig. 4-SEM micrographs of the adhesive–dentin interface indicating that the self-etching
adhesives applied infiltrated into dentin, forming resin tags. As measured roughly in the images,
the mean infiltration depths of CSE (a, b) and SU (c, d) were 1 μm, while that of AEO was less
than 1 μm (g, h). Deep penetration of OX was discernible by formation of resin tags of 15–30 μm
length into dentinal tubules (e, f). (Co: composite, Al: adhesive layer, De: dentin).

Fig. 5-Backscattered SEM micrographs illustrating the most common nano-leakage features
traced by silver uptake. Arrows indicate silver deposits at the adhesive–dentin interfaces. The
interface of all the adhesives with dentin showed spotted or linear discontinuous silver nitrate
uptake along the hybrid layer at the immediate time-point (a, c, e, g). For CSE and SU, this
nano-leakage remained stable after thermo-cycling (b, d), compared to the interface of OX and
AEO which showed obviously increased silver infiltration after aging (f, h).