Bonding Performance of Simplified Dental Adhesives with

Three Application Protocols: An 18-month In Vitro Study

Ana Filipa Chasqueiraa / Sofia Arantes-Oliveirab / Jaime Portugalc

Purpose: To evaluate the influence of application protocol on the shear bond strength (SBS) and nanoleakage of
simplified adhesives over 18 months.
Materials and Methods: 810 dentin slices were obtained from 405 caries-free human molars. They were randomly
assigned to 54 experimental groups resulting from the combination of “adhesive” (Adper Scotchbond 1XT [S1XT],
Solobond M [SM], Scotchbond Universal Adhesive in etch-and-rinse mode [SUER] and self-etch mode [SUSE], Adper
Easy Bond [EB], Clearfil S3 Bond Plus [CS3]), “application protocol” (manufacturer’s instructions [MI], two extra
layers of adhesive [EL], hydrophobic resin layer [HL]), and “aging time” (24 h [24H], 6 months [6M], and
18 months [18M] in water). SBS tests were carried out using a Watanabe device followed by failure mode analysis.
For the nanoleakage study, specimens from 54 additional molars were prepared as previously described, im-
mersed in ammoniacal silver nitrate, and evaluated with SEM. SBS data were analyzed with ANOVA followed by
Tukey’s post-hoc tests; failure mode data were analyzed using chi-squared tests (α = 0.05). Nanoleakage data
were analyzed with the Kruskal-Wallis test followed by LSD tests (α = 0.05).
Results: After 18M, S1XT and SUSE achieved the highest mean SBS (p < 0.05). Regarding the application proto-
cols, EL and HL provided higher mean SBS than those obtained with MI (p < 0.05). HL resulted in the highest
mean SBS and the lowest mean nanoleakage after 18M.
Conclusion: Simplified adhesives may need an extra hydrophobic resin layer to achieve a stable and durable adhe-
sive interface. The self-etch approach should be recommended for the universal adhesive.
Keywords: dental bonding, dentin bonding agents, material testing, self-etch, etch-and-rinse, hydrophobic, water
storage, bond strength, nanoleakage.

J Adhes Dent 2020; 22: 255–264. Submitted for publication: 27.02.19; accepted for publication: 15.10.19
doi: 10.3290/j.jad.a44549

T he success of an adhesive restoration depends on the

integrity and longevity of the interface created between
the adhesive and the substrate.5,39 This adhesive joint is
Adhesives are classified according to the way they inter-
act with the smear layer: etch-and-rinse (ER) or self-etch
(SE) adhesives.28,55 For either approach, the adhesive can
r

held by an adhesive, which has been described as the most
vulnerable step of the clinical procedure.3,38
a Invited Assistant Professor, Department of Biomaterials, Faculty of Dental
Medicine, University of Lisbon, Lisbon, Portugal. Study design, performed the
experiments, wrote the manuscript.
b Associate Professor, Department of Geriatric Dentistry, Faculty of Dental Medi-i
cine, University of Lisbon, Lisbon, Portugal. Study design, wrote the manuscript.
c Full Professor, Department of Biomaterials, Faculty of Dental Medicine, Uni-i
versity of Lisbon, Lisbon, Portugal. Study design, performed the statistical
analysis, wrote the manuscript.
Correspondence: Ana Filipa Chasqueira, Faculdade de Medicina Dentária, Univer-
r
sidade de Lisboa, Rua Professora Teresa Ambrósio, 1649-003 Lisbon, Portugal.
Tel: +35-192-743-8727; e-mail: filipach@gmail.com
either be simplified (2-step etch-and-rinse and 1-step self-
etch) or traditional (3-step etch-and-rinse and 2-step self-
etch).55 Universal adhesives can be applied as 2-step etch-
and-rinse, 1-step self-etch, or 1-step self-etch adhesive on
dentin with selective enamel etching.13,28
Adhesives were simplified by incorporating hydrophilic
monomers and solvents into the adhesive solution and by
removing the hydrophobic resin layer, a characteristic final
step of traditional adhesives.55,60 These changes lead to
water seepage through the hybrid layer, which acts as a
semipermeable membrane, resulting in nanoleakage.41,48,54
This fluid movement also triggers the rupture of the covalent
bonds within the polymer, contributing to the deterioration of
the adhesive interface and decrease in bond strength over
time.3,10,13,17,48 In fact, despite an acceptable immediate
performance, studies have revealed low bond strengths for
simplified adhesives after artificial aging.3,10

Vol 22, No 3, 2020 255

Chasqueira et al
Due to the limitations of simplified adhesives, alternative
protocols have been proposed, such as increasing the ap-
plication time of the adhesive,50 increasing evaporation time
of the solvent,20 application of an additional hydrophobic
resin layer over the adhesive,1,6,22,30,32,35 and application of
additional layers of the simplified adhesive.1,6,11,14,23,40
However, there is no consensus as to which is the most ef- f signed into 54 experimental groups according to the possi-
fective method.
The purpose of this study was to examine the effect of
two additional layers of the adhesive or an extra hydropho-
bic resin layer on the 24-h, 6-month, and 18-month dentin
shear bond strength (SBS) and nanoleakage of simplified
adhesives, according to the following hypotheses: SBS and
nanoleakage are not affected by adhesive, application pro-
tocol, or aging time.
MATERIALS AND METHODS
Specimen Preparation
After approval by the Institutional Ethics Committee, 459
caries-free human molars were used (405 for the SBS study
and 54 for the nanoleakage study) after storage in 0.5%
chloramine at 4ºC for a maximum of 6 months after extrac-
tion (ISO/TS 11405/2003). For the SBS test, two 2-mm-
thick dentin slices were obtained per tooth, removing the
proximal enamel with a diamond saw under water cooling
(Isomet 1000, Buehler; Lake Bluff, IL, USA). Regarding the
nanoleakage study, each crown was cut perpendicular to
the long axis of the tooth and, after the restorative proce-
dure, three specimens were obtained from the same dentin
substrate, one for each of the three aging times. A standard-
ized smear layer was created by polishing the dentin surface
under running water with 320-grit SiC paper (Struers; Copen-
hagen, Denmark) and rinsing with distilled water for 10 s.24
256
Fig 1 Experimental design. S1XT: Adper
Scotchbond 1 XT; SM: Solobond M: SUER:
Scotchbond Universal Adhesive applied
with etch-and-rinse strategy; SUSE: Scotch-
bond Universal Adhesive applied with self-
etch strategy; EB: Adper Easy Bond; CS3:
Clearfil S3 Bond Plus. MI: applied accord-
ing to manufacturer’s instructions; EL: two
extra adhesive applications; HL: hydrophobic
resin layer application; 24H: 24 hours;
6M: 6 months; 18M: 18 months; SBS,
shear bond strength.
Data from a pilot study were used for a power analysis to
estimate the sample size (SBS study, n = 15; nanoleakage
study, n = 3), providing statistical significance (_ = 0.05) at
a power of 80%.
The 810 dentin slices for SBS testing and the 54 dentin
surfaces for nanoleakage observation were randomly as-
ble combination of adhesive, application protocol, and aging
time (Fig 1).
The adhesives tested were: two 2-step etch-and-rinse
adhesives (Adper Scotchbond 1XT [S1XT, 3M Oral Care; St
Paul, MN, USA] and Solobond M [SM, Voco; Cuxhaven, Ger- r
many]); two 1-step self-etch adhesives (Adper Easy Bond
[EB, 3M Oral Care] and Clearfil S3 Bond Plus [CS3, Kuraray
Noritake; Okayama, Japan]); and a universal adhesive
(Scotchbond Universal Adhesive [SU, 3M Oral Care]) used
in etch-and-rinse (SUER) or in self-etch (SUSE) mode.
Adhesives were handled according to three different ap-
plication protocols: following the respective manufactur-
er’s instructions (MI) (Table 1); two extra layers of adhe-
sive (EL); or an extra hydrophobic resin layer20 (HL, Adper
Scotchbond Multi-Purpose Adhesive; 3M Oral Care). In the
two experimental protocols, photopolymerization was only
performed at the end of the adhesive procedure. No light
curing was performed between application of the different
layers.
Three aging times were defined: 24 h (24H), 6 months
(6M), and 18 months (18M).
Each adhesive was applied to the dentin and photopoly- y
merized with a light-curing unit (Bluephase 20i, Ivoclar Viva-
dent; Schaan, Liechtenstein) with an output of 1250 mW/
cm2. For the SBS specimens, a piece of adhesive tape
(Mylar, Dupont; Wilmington, NC, USA) bearing a perforation
with an internal diameter of 3 mm served as delimitation of
the bonding area. Two 2-mm increments of Filtek Z250 Uni-
The Journal of Adhesive Dentistry

Table 1 Materials used, chemical composition (batch numbers), and application procedure
Materials Composition (batch
(Manufacturer) number)
Adper Scotchbond 1XT* Bis-GMA, HEMA (5-15%),
(3M Oral Care; St Paul, dimethacrylate resins,
MN, USA) ethanol, water, functional
*also known as Adper methacrylate, Vitrebond
Single Bond Plus or copolymer, photoinitiation
Adper Single Bond 2 system, silica (N286534)
Solobond M Bis-GMA, HEMA (5-10%),
(VOCO; Cuxhaven, BHT, methacrylates,
Germany) acetone, organic acid
derivates, organic fluoride
component (1202181)
Adper Easy Bond** Bis-GMA, HEMA (15-25%),
(3M Oral Care) methacrylated phosphoric
** also known as esters, 1.6 hexanediol
Adper Easy One dimethacrylate, Vitrebond
copolymer, silica (7 nm),
ethanol, water,
camphorquinone,
stabilizers (480148)
Clearfil S3Bond Plus 10-MDP phosphate
(Kuraray Noritake; monomer, bis-GMA, HEMA
Tokyo, Japan) (10-35%) hydrophobic
dimethacrylate,
camphorquinone, ethyl
alcohol, water, silanated
colloidal silica (00009A)
Scotchbond Universal *** 10-MDP phosphate
(3M Oral Care) monomer, dimethacrylate
***also known as resins, HEMA (15-25%),
Single Bond Universal Vitrebond, copolymer, filler,
ethanol, water, initiators,
silane (468356)
Filtek Z250 XT Bis-GMA, UDMA, bis-EMA,
(3M Oral Care) PEG-DMA and TEG-DMA,
surface-modified zirconia/
silica (< 3 μm) and non-
agglomerated 20-nm
surface-modified silica
particles (68% by volume)
(N240749)
versal Restorative (3M Oral Care) were then applied and
light cured for 20 s each.
All specimens were initially stored in water at 37ºC for
24 h. One-third of the specimens were tested after this pe-
riod. The remainder were immersed in a sodium azide solu-
tion (pH = 7, 37ºC) for a period of 6 months or 18 months
before being subjected to shear bond strength testing. The
pH was controlled every week and the solution changed
monthly, or sooner given a pH < 7, indicating bacterial growth.
SBS Assay
SBS tests were performed with a single-plane Watanabe
device (Technical Section, Department of Dental Materials,
Univesity of Lisbon) in a universal testing machine (Instron
Vol 22, No 3, 2020
Chasqueira et al
Application procedure
– Apply Scothcbond etchant to enamel and dentin and leave it undisturbed for 15 s
– Rinse for 10 s
– Dry excess water with a cotton pellet, until the surface becomes bright without pooled
water
– Apply 2-3 consecutive adhesive coats, rubbing for 15 s
– Gently air dry the adhesive for 5 s to evaporate the solvents
– Light cure for 10 s
– Apply Vococid etchant, starting from the enamel margins, leave it undisturbed for 15 s
– Rinse the etchant for at least 15 s
– Dry the surface carefully; do not overdry dentin
– Apply the adhesive and leave undisturbed for 30 s
– Gently air dry to evaporate the solvent
– Light cure for 20 s
– Apply the adhesive with a microbrush and rub it for 20 s
– Gently air dry for approximately 5 s
– Light cure for 10 s
– Apply the adhesive, rubbing it for 10 s
– Dry with mild air stream > 5 s
– Light cure for 10 s
SE mode ER mode
– Apply the adhesive with a microbrush – Apply etchant agent for 15 s
and rub it for 20 s – Rinse for 10 s
– Gently air dry the adhesive for – Apply adhesive as for the SE mode
approximately 5 s, until no movement is
detected, indicating the complete
evaporation of the solvents
– Light cure for 10 s
– –
model 4502, Instron; High Wycombe, UK) with a 1 KN load
cell at a crosshead speed of 1 mm/min. Failure modes
were determined with a stereomicroscope (EMZ-8TRU, Meiji
Techno; Saitama, Japan) at 20X magnification, and classi-
fied as adhesive if the failure occurred totally at the adhe-
sive interface, cohesive if the failure was found exclusively
in dentin or restorative resin, and mixed when a combina-
tion of adhesive and cohesive failures was observed.19
Nanoleakage Study
After each aging time, nanoleakage specimens were coated
with two layers of nail polish up to 1 mm from the adhesive
interface, immersed in a 50% (w/v) ammoniacal silver ni-
trate (pH = 9.5) solution, and stored in the dark at 37ºC for
257
Chasqueira et al

Fig 2 Influence of the adhesive on the

Influence of adhesive mean SBS. S1XT: Adper Scotchbond 1 XT
Adhesive; SM: Solobond M; SUER: Scotch-
60
bond Universal Adhesive applied with
Mean SBS, MPa (Error bar = SD)

etch-and-rinse strategy; SUSE: Scotchbond

50 Universal Adhesive applied with self-etch
strategy; EB: Adper Easy Bond Self-Etch
S1XT Adhesive; CS3: Clearfil S3 Bond Plus. For
40
each aging time, means with the same
SM
30 letter are not statistically different
SUER (p > 0.05).
20
SUSE

10 EB

CS3
0
24 h 6m 18 m
Aging time

Fig 3 Influence of the application protocol

Influence of application protocol on SBS. MI: applied as per manufacturer’s
instructions; EL: two extra adhesive appli-
60
cations; HL: extra hydrophobic adhesive
Mean SBS, MPa (Error bar = SD)

resin layer application. For each aging time,

50 means with the same letter are not statisti-
cally different (p > 0.05).
40

30 MI

EL
20
HL
10

0
24 h 6m 18 m
Aging time

Fig 4 Boxplot depicting the influence of

Influence of adhesive adhesive on nanoleakage. S1XT: Adper
Scotchbond 1 XT Adhesive; SM: Solobond
100
M; SUER: Scotchbond Universal Adhesive
applied with etch-and-rinse strategy; SUSE:
S1XT Scotchbond Universal Adhesive applied
80
with self-etch strategy; EB: Adper Easy
SM Bond Self-Etch Adhesive; CS3: Clearfil S3
Nanoleakage (%)

60 Bond Plus. For each aging time, means

SUER with the same letter are not statistically
different (p > 0.05).
SUSE
40
EB

20 CS3

0
24 h 6m 18 m
Aging time

258 The Journal of Adhesive Dentistry


24 h. The specimens were then washed with distilled water
and immersed in a photodeveloping solution under fluores-
cent light for 8 h.42 After this procedure, specimens were
processed for SEM. They were fixed with 2.5% glutaralde-
hyde in a 0.1 M sodium cacodylate solution (pH = 7.2) at
4°C for 24 h, and washed three times for 20 min each with
0.1 M sodium cacodylate buffer. Specimens were polished
under running water with SiC abrasive paper of decreasing
abrasiveness (800-, 1200-, and 2500-grit, HV 30-800, Stru-
ers; Ballerup, Denmark) followed by polishing cloths
(Buehler) with 3-μm, 1-μm and 0.25-μm diamond polishing
paste for 1 min each (Electron Microscopy Sciences; Hat-
field, PA, USA). Finally, specimens were dehydrated by im-
mersion in ascending concentrations of ethanol (25%, 50%,
and 75% for 20 min each, 96% for 30 min, and 100% for
60 min), ending with 10 min in HMDS (Sigma Aldrich; St
Louis, MO, USA).27 The specimens were left to dry over-
night and made conductive by the deposition of gold-palla-
dium (JEOL Fine Coat Ion Sputter JFC-1100E; Tokyo, Japan).
Nanoleakage in the adhesive interface was analyzed with
SEM (JEOL JSM-5200LV) at 20 kV in backscattered mode.
The nanoleakage in each specimen was measured by calcu-
lating the ratio between length of silver-ion-infiltrated seg-
g mixed (43%), and was statistically significantly (p < 0.001)
ments and the entire length of the adhesive interface.
Statistical Analysis
Data were analyzed with IBM SPSS Statistics, v 21.0 (IBM
SPSS; Armonk, NY, USA). After assessing normality and
homoscedasticity with Shapiro-Wilk tests, SBS data were
submitted to a 3-way ANOVA followed by a 1-way ANOVA to
detect the main effect of aging time, and 2-way ANOVAs to
detect the interaction effect between adhesive and applica-
tion protocol for each aging time. When appropriate,
Tukey’s post-hoc tests were performed (_ = 0.05). Chi-
squared tests were used to analyze failure mode data
(_ = 0.05). Nanoleakage data were statistically analyzed
with the Friedman non-parametric test for repeated mea-
sures to determine the influence of aging duration. Non-
parametric Kruskal-Wallis tests followed by multiple com-
parisons with the LSD test to rank the effects of the
adhesive and application protocol (_ = 0.05).
RESULTS
SBS Assay
Mean SBS ranged from 15.4 MPa for the EB-MI-18M group
to 51.5 MPa for the S1XT-HL-24H group.
Three-way ANOVA showed a statistically significant
(p < 0.001) influence of the three independent variables on
SBS and statistically significant (p ≤ 0.01) interactions be-
tween all factors. One-way ANOVA, subsequently performed,
demonstrated a significant decrease in the mean SBS be-
tween 24H and 6M (p < 0.001), and between 6M and at
18M (p = 0.016).
Additionally, 2-way ANOVAs showed a statistically signifi-
cant (p < 0.05) influence of both the adhesive and the ap-
plication protocol on mean SBS, but no interaction between
Vol 22, No 3, 2020
Chasqueira et al
factors (p > 0.05), for each of the three aging times. In
terms of the influence of adhesive on mean SBS (Fig 2), no
differences were found between the mean SBS of S1XT,
SM, and SUSE at 24H (p > 0.05), but S1XT yielded signifi-
cantly (p < 0.05) higher SBS than did the other adhesives.
The mean SBS obtained with SUSE was statistically signifi-
cantly higher (p < 0.001) than that of SUER. At 6M, CS3
showed a statistically significantly (p = 0.013) lower mean
SBS than S1XT. No other differences were observed be-
tween adhesives at 6M (p > 0.05). At 18M, S1XT and SUSE
reached higher mean SBS than all the other adhesives
(p < 0.001), but no other significant (p > 0.05) differences
were observed between adhesives. Regarding the effect of
the application protocol on mean SBS (Fig 3), no significant
(p > 0.05) differences were found between the EL and HL
protocols, but both resulted in statistically significantly
greater SBS than MI, both at 24H and 6M. However, at
18M, all application protocols resulted in mean SBS that
were statistically significantly different (p < 0.001) from
each other, the highest mean being obtained with the HL
and the lowest with the MI application protocol.
Failure mode was predominantly adhesive (39%) or
influenced by the three factors.
Nanoleakage Study
The S1XT-MI-18M experimental condition showed the high-
est median nanoleakage (51%). Only 7% of the specimens
revealed a complete absence of silver ions in the adhesive
interface (EB-EL-24H, one specimen; EB-HL-24H, three
specimens; CS3-MI-24H, one specimen; CS3-EL-24H, two
specimens; CS3-HL-24H, two specimens; CS3-HL-6M, one
specimen; CS3-HL-18M, two specimens).
Statistical analysis revealed that aging time had no sig-g
nificant (p = 0.464) influence on nanoleakage. On the other
hand, nanoleakage was statistically influenced by adhesive
at each aging time (24H, p < 0.001; 6M, p = 0.003; 18M,
p = 0.032) (Fig 4). S1XT and SM revealed significantly
greater (p < 0.05) nanoleakage than did EB and CS3 at 6M
and 18M (Figs 5–8). However, no differences (p > 0.05)
were found between the two self-etch adhesives (EB vs
CS3), between the two etch-and-rinse adhesives (S1XT vs
SM), or between the two application modes of SU (SUSE
vs. SUER) at any aging time (Figs 9 and 10).
For the factor application protocol, no significant differ-
r
ences in nanoleakage were found at either 24H (p = 0.97) or
6M (p = 0.335). At 18M, HL yielded statistically significantly
(p = 0.034) less nanoleakage than did MI (Fig 11). Nano-
leakage was detected essentially at the adhesive interface.
DISCUSSION
The degradation of the adhesive-dentin interface created
with simplified adhesives occurs due to the presence of hy- y
drophilic domains that allow water into the interface.41,52
This behavior is typical of adhesives with high concentrations
of HEMA, large amount of solvents, or solvents with lower
259
Chasqueira et al
Fig 5 Representative backscattered Fig 6 Representative backscattered
micrographs of S1XT-18M nanoleakage at micrographs of SM-18M nanoleakage at
the resin-dentin adhesive interface. the resin-dentin adhesive interface.
vapor pressure.26 Complete evaporation of these solvents
is difficult when the manufacturer’s recommended adhesive
evaporation time is used, as this has been shown to be
clearly insufficient.16,20 The presence of residual solvents at
the adhesive interface leads to incomplete polymerization of
the resin, decreased degree of conversion, formation of
pores, and loss of mechanical properties over time.45,53
Thus, in the present study, dentin SBS and nanoleakage
were recorded for five simplified adhesives (two 1-step self-
etch, two 2-step etch-and-rinse, and one universal) over
18M of aging, using three application protocols (MI, EL, and
HL). All the adhesives studied contain HEMA, since we in-
tended to study the influence of the handling protocol – not
the composition of the adhesive – on the quality of the ad-
hesion interface over time. HEMA is a very hydrophilic
monomer, which leads to water sorption and can lead to
the formation of poly-HEMA hydrogels in the hybrid layer and
overlying adhesive layer.37,44 These unpolymerized domains
are highly degradation prone over time.20
Regarding the EL experimental application protocol, in-
creasing the number of adhesive layers with the conse-
quent extended application time increases the concentra-
tion of monomers on the substrate and facilitates solvent
evaporation by drying the adhesive between each layer ap-
plied.14,17,34 However, this phenomenon only occurs if the
additional layers are copolymerized with the preceding
one,40 as in the present study. Increasing the number of
layers 2-step etch-and-rinse adhesives also contributes to a
better monomer impregnation into the collagen network,17
minimizing the possibility of unsupported collagen under the
hybrid layer.3,18 One-step self-etch adhesives form thin lay-y
ers at the adhesive interface; therefore, they are very sus-
ceptible to oxygen inhibition of polymerization.12,23 Contain-
ing water or residual fluid from the dentin substrate may
also interfere with the polymerization of acidic mono-
mers.58,64 Therefore, increasing the number of adhesive
layers may contribute to a higher degree of conversion of
the adhesive inside the interface. The performance of sim-
plified adhesives seemed to improve when two additional
layers were applied.17
260
Fig 7 Representative backscattered
micrographs of EB-18M nanoleakage at the
resin-dentin adhesive interface.
The application of HL waterproofs the permeable inter-
face that is created with simplified adhesives.22,30,35 The
HL also reduces the retention of solvents and unreacted
monomers within the adhesive interface.2,3 In the present
study, HL was copolymerized with the simplified adhesive,
based on the results of a pilot study which obtained higher
bond strengths by copolymerization than independent po-
lymerization of the two materials. Adper Scotchbond Multi-
Purpose, used as HL, contains HEMA (30-40 wt%), which
seemed to allow successful copolymerization with the sim-
plified or universal adhesive previously applied.
The mean SBS gradually decreased with aging. This deg-
radation may be due to the elution of free monomers at the
interface during aging in water, since the adhesives studied
create permeable interfaces.48,54 The decrease in mean
SBS over time may also be explained by hydrolytic degrada-
tion at the adhesive interface or immediately below it in an
unsupported collagen zone. This zone is often found in the
simplified etch-and-rinse adhesives, but may also occur in
self-etch adhesives, as described in the literature.13,34
Aging was conducted by immersion in sodium azide solu-
tion, since ISO/TS 11405 (2003) recommends it, and it
has been used in several long-term studies. This protocol
seems to exert the greatest influence on the adhesive inter-
r
face in terms of chemical degradation, simulating the aging
of the restoration in vivo.15
While S1XT and SUSE revealed the highest mean SBS at
18M, the differences between adhesives became more pro-
nounced at 18 months. Relative to SU, higher mean SBSs
were obtained when SU was applied in self-etch mode com-
pared to the etch-and-rinse mode. Based on its composi-
tion, SU may offer the advantage of forming chemical bond-
ing between 10-MDP and Ca2+ in hydroxyapatite, with the
consequent formation of insoluble salts of 10-MDP-Ca in a
structure of nanolayers.61,62 However, nanolayering has
been reported to be sparse in adhesives containing HEMA.
In fact, this molecule interferes with the formation of
10-MDP-Ca, even when present in amounts as low as 8%.61
Since all the adhesives in our study contain HEMA, the as-
sociation between the relatively high mean SBS of SUSE
The Journal of Adhesive Dentistry

Fig 8 Representative backscattered Fig 9 Representative backscattered
micrographs of CS3-18M nanoleakage at micrographs of SUSE-18M nanoleakage at
the resin-dentin adhesive interface. the resin-dentin adhesive interface.
and the chemical stability promoted by the 10-MDP-Ca may
need to be viewed with caution, until further studies shed
light on this controversial topic.49
Etching dentin with phosphoric acid, as with SUER, re-
sults in surface dentin decalcification due to the dissolution
of hydroxyapatite. When applied to this decalcified sub-
strate, SU is basically a traditional 2-step ER adhesive that
contains water and HEMA, making the interface very perme-
able.37 For this adhesive, the manufacturer recommends
gently air drying the adhesive for 5 s. According to the litera-
ture, this evaporation protocol is clearly insufficient for ad-
hesives that contain water, including universal adhe-
sives.16,20 The increase in hydrophilicity promoted by HEMA
has, in fact, been a long-term problem due to water sorp-
tion, solubility, lower degree of conversion, and consequent
hydrolytic degradation of the adhesive interface.9,47,59 The
results obtained with S1XT in the present study may be
partially attributed to the low technique sensitivity of this
adhesive. The presence of water and ethanol as solvents
allows expansion of the collagen network, even if it has col-
lapsed upon drying the water used to rinse off acid.54 S1XT
contains < 5 wt% water and 25-35 wt% ethanol, while SU
contains 10-15 wt% water and 10-15 wt% ethanol.36 The
higher concentration of ethanol in S1XT may be responsible
for the higher mean SBS of S1XT at 18M relative to that of
SUER, as bond strengths are related to the ability of the
primer to support the re-expansion of collapsed dentinal
collagen upon etching. In addition, the lower concentration
of water in S1XT makes this adhesive less susceptible to
hydrolytic degradation caused by residual water over time.
The other etch-and-rinse adhesive, SM, contains acetone as
a solvent, but no water. Acetone is very volatile; its vapor
pressure is higher than that of water and ethanol, which are
present in S1XT adhesive.31 In fact, acetone-based adhe-
sives need a large amount of residual surface water to
achieve high bond strengths.26 Therefore, SM does not
have the ability to re-expand the collapsed collagen net-
work, which would explain the results obtained in the pres-
ent study. In addition, acetone-based adhesives do not per- r able, allowing the movement of water into the interstices
form as well clinically as ethanol-based adhesives, 33
Vol 22, No 3, 2020
Chasqueira et al
Fig 10 Representative backscattered
micrographs of SUER-18M nanoleakage at
the resin-dentin adhesive interface.
probably as a result of forming thin adhesive layers on the
etched dentin surface.7
At 18M, the HL protocol resulted in the highest mean
SBS. The increased SBS may be a consequence of the in-
creased thickness of the adhesive layer, which is known to
reduces the detrimental effects of polymerization stress of
composites resins.8 In addition, the HL may have trans-
formed the permeable adhesive interface into a better-
sealed interface with an increase in adhesive thickness,
higher conversion rate, better mechanical properties, and
less potential for hydrolytic degradation of resin and colla-
gen.2,4,21 An increase in the quality and durability of the
entire restoration may be achieved when using an extra hy- y
drophobic resin layer.22,23,43
Considering the failure mode, and regardless of the ad-
hesive or application protocol, the percentage of adhesive
failures increased over time, while the mixed failure rate
decreased proportionally. This change may have been a re-
sult of the adhesive interface degradation which could have
occurred during 18-month water storage.
As for the nanoleakage results, no differences were
found with different aging durations, and similar results
were obtained for adhesives with the same adhesive strat-
egy. Differences between the simplified 2-step ER adhe-
sives (S1XT and SM) and the 1-step SE adhesives (EB and
CS3) became evident at 6M, with the ER adhesives pre-
senting the highest nanoleakage. For the universal adhe-
sive SU, no differences in nanoleakage were found between
SUER and SUSE at 18M.
The differences found between simplified ER and SE ad-
hesives point to a possible discrepancy between the demin-
eralization of the substrate and the impregnation of the
resin, since etching dentin with phosphoric acid leaves col-
lagen unprotected. The resin may not be able to infiltrate
through the entire depth of demineralized substrate. This
results in a lower concentration of monomers at depth and
the possible creation of an unsupported collagen zone
under the hybrid layer.25,37 This region is extremely perme-
and consequent hydrolysis of collagen and resin over
261
Chasqueira et al
Influence of application protocol
100
80
Nanoleakage (%)
60
40
20
0
24 h 6m
Aging time
time.25,34 SE adhesives contain a solution of acidic mono-
mers that allows simultaneous demineralization and impreg- g
nation of the dentin, creating interfaces with fewer defects
and lower permeability, compared to those created by ER
adhesives.55,56 The existence of an unsupported collagen
zone under the adhesive interface is less likely to occur for
SE adhesives, which may lead to less nanoleakage.3,10,41
Comparing the results for the two one-step SE adhesives
EB and CS3 at each evaluation time for each application
protocol, the respective mean SBSs and nanoleakage were
very similar (data not shown), in spite of having different
functional monomers. CS3 is an MDP-based adhesive,
while EB contains 6-MHP. CS3 results in 10-MDP-Ca salt
formation or nanolayering within the hybrid layer, whereas
EB does not form 10-MDP-Ca salts.57,63
Both adhesives are considered “ultra-mild” SE adhe-
sives, as their pH is ≥ 2.5. This mild pH is responsible for
identical morphological ultrastructures of the hybrid layer
and possibly similar bond strengths. In fact, the pH of SE
adhesives plays a significant role in dentin bond strengths,
as the mean bond strength of CS3 was higher than that of
the predecessor of EB, Adper Prompt L-Pop, a strong SE
adhesive (pH = 1).51 SUSE and CS3 are both ultra-mild
10-MDP-based 1-step SE adhesives with pH = 2.7. Never- r The self-etch approach should be recommended for the uni-
theless, SUSE resulted in significantly higher SBS than
CS3. The higher concentration of HEMA in CS3 may have
resulted in a more hydrophilic solution, and therefore less
stable bonding.
Regarding the two adhesion strategies used with SU, a
greater percentage of nanoleakage would be expected with
the ER strategy at 18M, due to the prior application of
phosphoric acid which removes some of the dentin miner-
als, hampering the possibility of nanolayering formation.
262
Fig 11 Boxplot of the influence of applica-
tion protocol on nanoleakage. MI: applied
as per manufacturer’s instructions; EL: two
extra adhesive applications; HL: extra hy-
drophobic adhesive resin layer application.
For each aging time, means with the same
letter are not statistically different
(p > 0.05).
MI
EL
HL
18 m
However, similar results were achieved with both ER and SE
strategies, probably because SU also contains a polyalke-
noic acid copolymer (also known as Vitrebond copolymer)
and HEMA, all of which compete for binding to calcium.
In the present study, the application of a hydrophobic
resin layer led to the lowest values of nanoleakage, while
the manufacturer’s protocol resulted in the highest nano-
leakage. The HL may have protected the adhesive interface
from contact with water from the outside, as well as the
from the elution of free monomers promoted by hydrophilic
monomers.29,46 The HL protocol improved nanoleakage
only after 18M, which validates the aging period used in
this work. In fact, the results of in vitro studies performed
immediately after the adhesive procedure or with relatively
short aging times are not entirely valid for predicting the
performance of adhesive resin composite restorations over
time.
CONCLUSION
Simplified adhesives may need an extra hydrophobic resin
layer to achieve a stable and durable adhesive interface.
versal adhesive.
ACKNOWLEDGMENTS
The authors thank Prof. Jorge Perdigão for his critical review of the
manuscript and his suggestions. The authors would also like to
thank VOCO, 3M Oral Care, and Kuraray Noritake for providing the
materials.
The Journal of Adhesive Dentistry

REFERENCES
1. Albuquerque M, Pegoraro M, Mattei G, Reis A, Loguercio AD. Effect of
double-application or the application of a hydrophobic layer for improved
efficacy of one-step self-etch systems in enamel and dentin. Oper Dent
2008;33:564–570.
2. Andrade e Silva SM, Carrilho MR, Marquezini L Jr, Garcia FC, Manso AP,
Alves MC, Carvalho RM. Effect of additional hydrophilic versus hydropho-
bic coat on the quality of dentinal sealing provided by two-step etch-and-
rinse adhesives. J Appl Oral Sci 2008;17:184–189.
3. Breschi L, Mazzonni A, Ruggeri A, Cadenaro M, Di Lenarda R, Dorigo E.
Dental adhesion review: aging and stability of the bonded interface. Dent
Mater 2008;24:90–101.
4. Cadenaro M, Antoniolli F, Sauro S, Tay FR, De Lenarda R, Prati C, Bia-
sotto M, Contardo L, Breschi L. Degree of conversion and permeability of
dental adhesives. Eur J Oral Sci 2005;113:525–530.
5. Cardoso MV, de Almeida Neves VA, Mine A, Coutinho E, Van Lanuyt K,
De Munck J, Van Meerbeek. Current aspects on bonding effectiveness
and stability in adhesive dentistry. Aust Dent J 2011;56(suppl1):31–44.
6. Chasqueira AF, Arantes-Oliveira S, Portugal J. Effect of changes to the
manufacturer application techniques on the shear bond strength of simpli-
fied dental adhesives. J Appl Biomater Funct Mater 2013;11:e117–121.
7. Cho B, Dickens SH. Effects of the acetone content of single solution den-
tin bonding agent on the adhesive layer thickness and microtensile bond
strength. Dent Mater 2004;20:107–115.
8. Choi KK, Condon JR, Ferracane JL. The effects of adhesive thickness on
the polymerization contraction stress of composite. J Dent Res
2000;79:812–817.
9. Collares FM, Ogliari FA, Zanchi CH, Petzhold CL, Piva E, Samuel SM. In-
fluence of 2-hydroxyethyl methacrylate concentration on polymer network
of adhesive resin. J Adhes Dent 2011;13:125–129.
10. De Munck J, Vargas M, Iracki J, Van Landuyt K, Poitevin A, Lambrecht P,
Van Meerbeek B. One-day bonding effectiveness of new self-etch adhe-
sives to bur-cut enamel and dentin. Oper Dent 2005;30:39–49.
11. Fujiwara S, Takamizawa T, Barkmeier WW, Tsujimoto A, Imai A, Watanabe
H, Erickson RL, Latta MA, Nakatsuka T, Miyazaki M. Effect of double-layer
application on bond quality of adhesive systems. J Mech Behav Biomed
Mater 2018;77:501–509.
12. Gauthier MA, Stangel I, Ellis TH, Zhu XX. Oxygen inhibition in dental res-
ins. J Dent Res 2005;84:725–729.
13. Hashimoto M, Nagano F, Endo K, Ohno H. A review: Biodegradation of
resin-dentin bonds. Jpn Dent Sci Rev 2011;47:5–12.
14. Hashimoto M, Sano H, Yoshida E, Hori M, Kaga M, Oguchi H, Pashley
DH. Effects of multiple adhesive coatings on dentin bonding. Oper Dent
2004;29:416–423.
15. Hashimoto M. A review – micromorphological evidence of degradation in
resin-dentin bonds and potential preventional solutions. J Biomed Mater
Res 2010;92B:268–280.
16. Hiraishi N, Breschi L, Prati C, Ferrari M, Tagami J, King NM. Technique
sensitivity associated with air-drying of HEMA-free, single-bottle, one-step
self-etch adhesives. Dent Mater 2007;23:498–505.
17. Ito S, Tay FR, Hashimoto M, Yoshiyama M, Saito T, Brackett WW, Waller
JL, Pashey DM. Effects of multiple coatings of two all-in-one adhesives on
dentin bonding. J Adhes Dent 2005;7:133–141.
18. King NM, Tay FR, Pashley DH, Hashimoto M, Ito S, Brackett WW, García-
Godoy F, Sunico M. Conversion of one-step to two-step self-etch adhe-
sives for improved efficacy and extended application. Am J Dent 2005;
18:126–134.
19. Luque-Martinez IV, Perdigão J, Muñoz MA, Sezinando A, Reis A, Loguercio
AD. Effects of solvent evaporation time on immediate adhesive proper-
ties of universal adhesives to dentin. Dent Mater 2014;30:1126–1135.
20. Malacarne J, Carvalho RM, de Goes MF, Svizero N, Pashley DH, Tay FR,
Yiu CK, Carrilho MR. Water sorption/solubility of dental adhesive resins.
Dent Mater 2006;22:973–980.
21. Manso AP, Marquezini L Jr, Silva SM, Pashley DH, Tay FR, Carvalho RM.
Stability of wet versus dry bonding with different solvent-based adhe-
sives. Dent Mater 2008;24:476–482.
22. Muñoz MA, Sezinando A, Luque-Martinez I, Szesz AL, Reis A, Loguercio
AD, Bombarda NH, Perdigão J. Influence of a hydrophobic resin coating
on the bonding efficacy of three universal adhesives. J Dent 2014;42:
595–602.
23. Nunes TG, Ceballos L, Osorio R, Toledano M. Spatially resolved photopo-
lymerization kinetics and oxygen inhibition in dental adhesives. Biomateri-
als 2005;26:1809–1817.
Vol 22, No 3, 2020
Chasqueira et al
24. Oliveira SSA, Pugach MK, Hilton JF, Watanabe L, Marshall SJ, Marshall
GW. The influence of the dentin smear-layer on adhesion: a self-etching
primer vs a total-etch system. Dent Mater 2003;19:758–767.
25. Pashley DH, Tay FR, Breschi L, Tjäderhane L, Carvalho RM, Carrilho M,
Tezvergil-Mutluay A. State of the art etch-and-rinse adhesives. Dent Mater
2011;27:1–16.
26. Pashley EL, Zhang Y, Lockwood PE, Rueggeberg FA, Pashley DH. Effects
of HEMA on water evaporation from water-HEMA mixtures. Dent Mater
1998;14:6–10.
27. Perdigão J, Lambrechts P, Van Meerbeek B, Vanherle G, Lopes AL. Field
emission SEM comparison of four postfixation drying techniques for
human dentin. J Biomed Mater Res 1995; 29:1111–1120.
28. Perdigão J, Muñoz MA, Sezinando A, Luque-Martinez IV, Staichak R, Reis
A, Loguercio A. Immediate adhesive properties to dentin and enamel of a
universal adhesive associated with a hydrophobic resin coat. Oper Dent
2014;39:489–499.
29. Pushpa R, Suresh BS. Marginal permeability of one step self-etch adhe-
sives: Effects of double application or the application of hydrophobic
layer. J Conserv Dent 2010;13:141–144.
30. Reis A, Albuquerque M, Pegoraro M, Mattei G, Bauer JR, Grande RH,
Klein-Junior CA, Baumhardt-Neto R, Loguercio AD. Can the durability of
one-step self-etch adhesives be improved by double application or by an
extra layer of hydrophobic resin? J Dent 2008;36:309–315.
31. Reis A, Cardoso PC, Vieira LCC, Baratieri LN, Grande RH, Loguercio AD.
Effect of prolonged application times on the durability of resin-dentin
bonds. Dent Mater 2008;24:639–644.
32. Reis A, Leite TM, Matte K, Michels R, Amaral RC, Geraldeli S, Loguercio
AD. Improving clinical retention of one-step self-etching adhesive systems
with an additional hydrophobic adhesive layer. J Am Dent Assoc
2009;140:877–885.
33. Reis A,  Loguercio AD. A 36-month clinical evaluation of ethanol/water
and acetone-based etch-and-rinse adhesives in non-carious cervical le-
sions. Oper Dent 2009;34:384–391.
34. Sano H, Takatsu T, Ciucchi B, Horner JA, Matthews WG, Pashley DH.
Nanoleakage: leakage within the hybrid layer. Oper Dent 1995;20:18–25.
35. Sezinando A, Luque-Martinez I, Muñoz MA, Reis A, Loguercio AD, Per- r
digão J. Influence of a hydrophobic resin coating on the immediate and
6-month dentin bonding of three universal adhesives. Dent Mater 2015;
31:e236–246.
36. Sezinando A, Perdigão J, Ceballos L. Long-term in vitro adhesion of poly-y
alkenoate-based adhesives to dentin. J Adhes Dent 2017;19:305–316.
37. Spencer P, Wang Y. Adhesive phase separation at the dentin interface
under wet bonding conditions. J Biomed Mater Res 2002;62:447–456.
38. Strassler HE. Applications of total-etch adhesive bonding. Comp Contin
Educ Dent 2003;24:427–436.
39. Swift EJ. Dentin/enamel adhesives: review of the literature. Pediatr Dent
2002;24:456–461.
40. Taschner M, Kummerling M, Lohbauer U, Breschi L, Petschelt A, Franken-
berger R. Effect of double-layer application on dentin bond durability of
one-step self-etch adhesives. Oper Dent 2014;39:416–426.
41. Tay F, Pashley D, Suh B, Carvalho R, Itthagarun A. Single-step adhesives
are permeable membranes. J Dent 2002;30:371–382.
42. Tay FR, Hashimoto M, Pashley DH, Peters MC, Lai SC, Yiu CK, Cheong C.
Aging affects two modes of nanoleakage expression in bonded dentin. J
Dent Res 2003;82:537–541.
43. Tay FR, Frakenberger R, Kreijci I, Bouillaguet S, Pashley DH, Carvalho
RM, Lay CN. Single-bottle adhesives behave as permeable membranes
after polymerization. I. In vivo evidence. J Dent 2004;32:611–621.
44. Tay FR, Pashley DH, Suh B, Carvalho RM, Miller M. Single step, self-etch
adhesives behave as permeable membranes after polymerization – Part I
– Bond strength and morphologic evidence. Am J Dent 2004;17:271–278.
45. Tay FR, Pashley DH, Suh BI, Hiraishi N, Yiu CK. Buonocore memorial lec-
ture. Water treeing in simplified dentin adhesives – déjà vu? Oper Dent
2005;30:561–579.
46. Tay FR, Pashley DH, Yoshiyama M. Two modes of nanoleakage expres-
sion in single-step adhesives. J Dent Res 2002;81:472–476.
47. Tay FR, Pashley DH. Have dentin adhesives become too hydrophilic? J
Can Dent Assoc 2003;69:726–731.
48. Tay FR, Pashley DH. Water treeing – a potential mechanism for degrada-
tion of dentin adhesives. Am J Dent 2003;16:6–12.
49. Tian F, Zhou L, Zhang Z, Niu L, Zhang L, Chen C, Zhou J, Yang H, Wang X,
Fu B, Huang C, Pashley DH, Tay FR. Paucity of nanolayering in resin-den-
tin interfaces of MDP-based adhesives. J Dent Res 2016;95:380–387.
263
Chasqueira et al
50. Toledano M, Proenca JP, Erhardt MCG, Osorio E, Aguilera FS, Osorio R,
Tay FR. Increases in dentin-bond strength if doubling application time of
an acetone-containing one-step adhesive. Oper Dent 2007;32:133–137.
51. Van Landuyt KL,  Mine A,  De Munck J,  Jaecques S,  Peumans M,  Lam-
brechts P, Van Meerbeek. Are one-step adhesives easier to use and bet-
ter performing? Multifactorial assessment of contemporary one-step
self-etching adhesives. J Adhes Dent 2009;11:175–190.
52. Van Landuyt KL, Snauwaert J, De Munck J, Coutinho E, Poitevin A, Yo-
shida Y Suzuki K, Lambrechts P, Van Meerbeek B. Origin of interfacial
droplets with one-step adhesives. J Dent Res 2007;86:739–744.
53. Van Landuyt KL, Snauwaert J, De Munck L, Peumans M, Yoshida Y,
Poitevin A, Coutinho E, Suzuki K, Lambrechts P, Van Meerbeek B. Sys-
tematic review of the chemical composition of contemporary dental adhe-
sives. Biomaterials 2007;28:3757–3785.
54. Van Landuyt KL, Snauwaert J, Peumans M, De Munck J, Lambrechts P,
Van Meerbeek B. The role of HEMA in one-step self-etch adhesives. Dent
Mater 2008;24:1412–1419.
55. Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P. Bu-
onocore memorial lecture. Adhesion to enamel and dentin: current status
and future challenges. Oper Dent 2003;28:215–235.
56. Van Meerbeek B, Yoshihara K, Yoshida Y, Mine A, De Munck J, Van Lan-
duyt K. State of the art of self-etch adhesives. Dent Mater 2011;27:17–28.
57. Wang R, Shi Y, Li T, Pan Y, Cui Y, Xia W. Adhesive interfacial characteris-
tics and the related bonding performance of four self-etching adhesives
with different functional monomers applied to dentin. J Dent
2017;62:72–80.
58. Wang Y,  Spencer P. Continuing etching of an all-in-one adhesive in wet
dentin tubules. J Dent Res 2005;84:350–354.
59. Ye Q, Park JG, Topp E, Wang Y, Misra A, Spencer P. In vitro performance
of nano-heterogeneous dentin adhesive. J Dent Res 2008;87:829–833.
264
60. Yiu CKY, Pashley EL, Hiraishi N, King NM, Goracci C, Ferrari M, Carvalho
RM, Pashley DH, Tay FR. Solvent and water retention in dental adhesive
blendsafter evaporation. Biomaterials 2005;26:6863–6872.
61. Yoshida Y, Yoshihara K, Hayakawa S, Nagaoka N, Okihara T, Matsumoto
T, Minagi S, Osaka A, Van Landuyt K, Van Meerbeek B. HEMA inhibits in-
terfacial nano-layering of the functional monomer MDP. J Dent
Res 2012;91:1060–1065.
62. Yoshihara K, Yoshida Y, Hayakawa S, Nagaoka N, Torii Y, Osaka A, Suzuki
K, Minagi S, Van Meerbeek B, Van Landuyt KL. Self-etch monomer-cal-
cium salt deposition on dentin. J Dent Res 2011;90:602–606.
63. Yoshihara K, Yoshida Y, Nagaoka N, Hayakawa S, Okihara T, De Munck
J, Maruo Y, Nishigawa G, Minagi S, Osaka A, Van Meerbeek B. Adhesive
interfacial interaction affected by different carbon-chain monomers. Dent
Mater 2013;29:888–897.
64. Zhang Y, Wang Y. Photopolymerization of phosphoric acid ester-based
self-etch dental adhesives. Dent Mater J 2013;32:10–18.
Clinical relevance: Simplified adhesives may need an
extra hydrophobic resin layer to achieve a stable and
durable adhesive interface. The self-etch approach
should be recommended for use with the universal
adhesive.
The Journal of Adhesive Dentistry