Microtensile Bond Strength and Micromorphology of

Bur-cut Enamel Using Five Adhesive Systems

Alexandra Vinagrea / João Ramosb / Ana Messiasc / Fernando Marquesd /
Francisco Carameloe / António Mataf

Purpose: This study compared the microtensile bond strengths (μTBS) of two etch-and-rinse (ER) (OptiBond FL
[OBFL]; Prime & Bond NT [PBNT]) and three self-etching (SE) (Clearfil SE Bond [CSEB]; Xeno III [XIII]; Xeno V+
[XV+]) adhesives systems to bur-prepared human enamel considering active (AA) and passive (PA) application of
the self-etching systems.
Materials and Methods: Ninety-six enamel surfaces were prepared with a medium-grit diamond bur and ran-
domly allocated into 8 groups to receive adhesive restorations: G1: OBFL; G2: PBNT; G3: CSEB/PA; G4: CSEB/
AA; G5: XIII/PA; G6: XIII/AA; G7: XV+/PA; G8: XV+/AA. After composite buildup, samples were sectioned to
obtain a total of 279 bonded sticks (1 mm2) that were submitted to microtensile testing (μTBS; 0.5 mm/min)
after 24-h water storage (37ºC). Etching patterns and adhesive interfacial ultramorphology were also evaluated
with confocal laser scanning (CLSM) and scanning electron microscopy (SEM). Data was analyzed with one-way
ANOVA (α = 0.05). Weibull probabilistic distribution was also determined.
Results: Regarding μTBS, both adhesive system and application mode yielded statistically significant differ-
ences (p < 0.05) among groups. ER adhesive systems together with CSEB/AA and XIII/PA recorded the highest
and statistically similar bond strength results. XV+ presented very low bond strength values, regardless of the
application mode. Among self-etching adhesives, CSEB produced significantly higher μTBS values when applied
actively. Qualitative evaluation by SEM and CLSM revealed substantial differences between groups both in adhe-
sive interfaces and enamel conditioning patterns.
Conclusions: ER and SE adhesive systems presented distinctive bond strengths to bur-cut enamel. The applica-
tion mode effect was adhesive dependent. Active application improved etching patterns and resin interfaces
micromorphology.
Keywords: adhesive systems, microtensile bond strength, application mode, enamel, morphology.

J Adhes Dent 2015; 17: 107–116. Submitted for publication: 05.02.15; accepted for publication: 31.03.15
doi: 10.3290/j.jad.a34060

adhesion of composite resins to dental substrates.26,46

C urrently, etch-and-rinse and self-etching adhesive sys-
tems represent the two main strategies to promote
a Guest Professor, Faculty of Medicine, University of Coimbra, Coimbra, Portu-
gal. Concept, hypothesis, experimental design, performed the experiments in
partial fulfillment of requirements for a degree, wrote the manuscript.
b Auxiliary Professor, Faculty of Medicine, University of Coimbra, Coimbra, Por-
tugal. Concept, hypothesis, experimental design, contributed to discussion,
proofread the manuscript.
c
PhD Student, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
Performed statistical evaluation, proofread the manuscript.
d Lecturer, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. Per-
formed experiment, proofread the manuscript.
e Auxiliary Professor, Faculty of Medicine, University of Coimbra, Coimbra, Por-
tugal. Performed statistical evaluation, proofread the manuscript.
f Full Professor, School of Dental Medicine, Lisbon University, Lisbon, Portu-
gal. Proofread the manuscript.
Correspondence: Dr. Alexandra Vinagre, Faculty of Medicine, University of
Coimbra, Av. Bissaya Barreto, Blocos de Celas, 3000-075 Coimbra, Portugal.
Tel: +35-191-263-8914, Fax: +35-123-985-7777. e-mail: avinagre@fmed.uc.pt
Phosphoric acid continues to be the approach of choice
for optimization of enamel surface conditioning, 26
whereas the application of self-etching adhesive systems
in either prepared or unprepared enamel is still a contro-
versial issue. Several studies report the unpredictability
of the performance of self-etching adhesives in intact
enamel, presenting evidence that previous mechanical
preparation could potentiate the adhesion capacity of
these systems.10,15,24,27,33 Besides, some researchers
report that the type of instrumentation used to prepare
dental substrates could interfere with the performance
of self-etching adhesives.5,21,37,48 Diamond bur prep-
aration of cavities produces specific effects on enamel
surfaces, resulting in the formation of a thick and ir-
regular smear layer.21 Since enamel contains a high
mineral fraction and larger hydroxyapatite crystals than
dentin, and because the demineralization potential of
self-etching systems is more limited than that provided
by phosphoric acid, it is possible that more hydrogen ions

Vol 17, No 2, 2015 107

Vinagre et al
released from the adhesive are neutralized by the enamel
smear, limiting their interaction with the subsurface
enamel.6,21,36 These conditions could compromise the
effectiveness of adhesion to ground enamel conditioned
by self-etching adhesives, particularly the least acidic
ones. Active application of self-etching adhesives seems
to favor the interaction with substrates by improving bond
strength.2,7,8,19,22,30,39,47 This is considered a simple
and rapid technique to increase mixture entropy and
improve solvent evaporation before the light-curing step
by increasing the kinetics of the moieties.31 Moreover,
active rubbing application is thought to improve depth of
demineralization, infiltration, and chemical interaction as
it promotes a more effective contact of monomers at the
surface.7,8,19 Thus, an active application of self-etching
adhesive by rubbing it into ground enamel is expected to
produce significant improvements in bonding efficacy.
To date, no studies have evaluated the effect of differ-
ent application modes of self-etching adhesive systems to
bur-treated enamel surfaces on the bond strength and sur-
face micromorphology. The present study aimed to evalu-
ate the resin-enamel bond strength of two etch-and-rinse
systems and three self-etching systems applied with dif-
ferent modes, either active or inactive, to bur-cut enamel,
and to examine the enamel surface micromorphology and
resin/enamel interfaces produced under the same condi-
tions. The null hypotheses tested in this study are that (1)
no differences in bur-cut enamel bond strength are found
between etch-and-rinse and self-etching bonding systems;
(2) surface application modes do not affect bur-cut enamel
bond strength of self-etching adhesive systems.
MATERIALS AND METHODS
Specimen Preparation
Forty-eight noncarious human molars were collected
after patients’ informed consent, as approved by the
Ethics Committee of the Faculty of Medicine, University
of Coimbra, Portugal (CE-001/2013). The teeth were
cleaned of all surrounding soft tissues or debris and
stored in a 10% buffered formalin solution (pH 7.0) at
room temperature for up to six months after extraction.
All molars were sectioned mesiodistally parallel to the
long axis with a water-cooled diamond saw (Accutom 50,
Struers; Ballerup, Denmark) in order to obtain two speci-
mens per tooth. The buccal and lingual surfaces were
carefully identified by labelling the corresponding tooth
and surface. Roots were then partially removed and pulp
debris detached from the remnant root canal pathway,
ensuring sufficient retention for subsequent tooth em-
bedding. Each sample was individually embedded in an
acrylic resin (Vertex, Vertex-Dental; Zeist, Netherlands)
for better handling, leaving the coronal portion above
the acrylic resin.
All buccal and lingual enamel surfaces were flattened
using a medium-grit (105 to 125 μm) diamond bur (G837-
314-018-8-ML, Diatech, Coltène Whaledent; Altstätten,
Switzerland) for 5 s with a water-cooled high-speed tur-
bine. For each sample, the maximum surface area of flat
108
enamel was exposed. Prepared specimens were carefully
observed under a stereomicroscope at 20X magnification
to ensure absence of dentin, cracks, or defects. Enamel
surfaces were then demarcated to outline the flattest pre-
pared area for bonding using a flowable blue light-curing
resin (LC Block-Out Resin, Ultradent; South Jordan, UT,
USA), which also served to facilitate, a posteriori, the
identification of useful and eligible samples for testing.
An even number of buccal- and lingual-faced speci-
mens was randomly divided into groups according to the
adhesive system tested and protocol of application (AA
– active application, or PA – passive application mode).
Twelve halves were used for each experimental group.
Two etch-and-rinse (OptiBond FL [OBFL] and Prime &
Bond NT [PBNT]) and three self-etching adhesive systems
(Clearfil SE Bond [CSEB], Xeno III [XIII], and Xeno V+ [XV+])
were used in this study. Their composition and application
methods are described in Table 1.
A single operator applied the adhesives according
to the specific group protocol. An LED light-curing unit
(Bluephase, Ivoclar Vivadent; Schaan, Liechtenstein) was
set to the low power mode with a light intensity of 650
mW/cm2 and used throughout the adhesive application
and restorative procedure. Following adhesive application,
a microhybrid resin composite (Esthet·X HD, A2, Dentsply
DeTrey; Konstanz, Germany) was used to create resin
composite buildups in three layers of 1.5 mm each. Each
layer was light cured for 20 s, followed by a final polymer-
ization of 60 s. The specimens were then stored at 100%
humidity at 37ºC for 24 h.
Microtensile Bond Strength Test (μTBS)
After storage, specimens were sectioned horizontally with
a low-speed cutting saw (Accutom 50) perpendicular to
the adhesive/tooth interface under water cooling in order
to obtain three or four 1-mm parallel slices. The spaces
between slices were dried and filled with a light-body poly-
vinylsiloxane (Aquasil Ultra XLV; Dentsply DeTrey; Kon-
stanz, Germany) for specimen stabilization prior to the
next set of cuts. Then, specimens were sectioned verti-
cally, dividing the horizontal parallel slices into sticks with
a cross-sectional area of approximately 1.00 ± 0.2 mm2.
Each specimen yielded a set of 3 to 6 useful sticks. The
interface perimeter of all sticks was inspected under
an optical microscope (Leica CLS 150 MR; Heerbrugg,
Switzerland) set at 40X magnification to exclude those
with any kind of defect or failure. Additionally, each stick
was measured with a digital caliper (Mitutoyo; Kawasaki,
Japan) to determine the mean bonding area within each
group. All sticks were constantly stored in tap water.
For microtensile measurement, each stick was fixed
on a testing jig with a cyanoacrylate adhesive (Permabond
735, Permabond International; Englewood, NJ, USA) and
stressed at a crosshead speed of 0.5 mm/min until fail-
ure in a specific device (Od04-Plus, Odeme Prod. Med.
Odont.; Luzerna, SC, Brasil) linked to a universal testing
machine (Model AG-I, Shimadzu; Kyoto, Japan). The μTBS
values were expressed in MPa and were calculated by
dividing the imposed force (N) at the time of fracture by
the bonded area (mm2). The occurrence of failure prior to
The Journal of Adhesive Dentistry
 Vinagre et al

Table 1 Adhesive system: groups, composition and application mode

Group Material Composition and pH Application mode

(abbreviation),
manufacturer,
batch No.
1 Optibond FL Etchant: 37.5% phosphoric acid (Kerr Gel Apply etchant for 15 s; rinse for 15 s; air dry for 5 s; scrub
(OBFL) Etchant) surface for 15 s with primer; gently air blow for 5 s; apply
Kerr; Orange, FL Primer: HEMA, GPDM, PAMA, ethanol, bonding agent; gently air blow for 3 s; light cure for 20 s
CA, USA water, CQ, BHT (pH ca 1.9)
FL Adhesive: bis-GMA, HEMA, GDMA, CQ,
4677483 ODMAB, filler (fumed silica, barium aluminobo-
rosilicate glass, disodium hexafluorosilicate),
coupling factor A174

2 Prime&Bond NT Etchant: 36% phosphoric acid (Conditioner 36)
(PBNT) Dentsply Adhesive: PENTA, UDMA, resin R5-62-1, T-resin
DeTrey; Kon- (cross-linking agent), D-resin (small hydrophilic
stanz, Germany molecule), cetylamine hydrofluoride, acetone,
butylated hydroxytoluene, 4-ethyl dimethyl ami-
1206000730 nobenzoate, silica nanofiller (pH ca 2.7)
Apply etchant for 15 s; rinse for 15 s; air dry for 5 s; apply
adhesive on surface and wait 20 s; gently air blow for 5 s;
light cure for 10 s; apply second coat; gently air blow for
5 s; light cure for 10 s

3 Clearfill SE Bond Primer: 10- MDP, HEMA, hydrophilic aliphatic Passive Apply primer and leave undisturbed for 20 s;
(CSEB) dimethacrylate, CQ, DET, water (pH ca 2.1) (CSEB/PA) gently air blow for 5 s; apply bonding agent;
Kuraray Medical; gently air blow for 5 s; light cure for 10 s
Tokyo, Japan Bond: 10-MDP, bis-GMA, HEMA, hydrophobic (manufacturer’s directions)
aliphatic dimethacrylate, DET, silanated col-
4 041931 loidal silica Active Apply primer with a rubbing motion for 20 s;
(CSEB/AA) gently air blow for 5 s; apply bonding agent;
gently air blow for 5 s; light cure for 10 s

5 Xeno III Bottle A: HEMA, ethanol, purified water, BHT, Passive Mix liquids A and B for 5 s; apply adhesive
Dentsply DeTrey stabilizers, nanofiller (XIII /PA) on surface and leave undisturbed for 20 s;
gently air blow for 5 s; light cure for 10 s
1302000019 Bottle B: Pyro-EMA, PEM-F; UDMA, BHT, CQ, (manufacturer’s directions)
EPD (mixture pH < 1)
6 Active Mix liquids A and B for 5 s; apply adhesive
(XIII/AA) on surface and scrub it in a rubbing motion
for 20 s; gently air blow for 5 s; light cure
for 10 s

7 Xeno V + Bifunctional acrylic amides, acryloamido alcyl- Passive Apply adhesive on surface and leave undis-
Dentsply DeTrey sulfonic acid, “inverse” functionalized phos- (XV+/PA) turbed for 20 s; gently air blow for 5 s; light
phoric acid esters, camphorquinone, butylated cure for 10 s
1209000038 benzenediol, water, tert-butanol (pH ca 1.3)
8 Active Apply adhesive on surface and scrub it in a
(XV+/AA) rubbing motion for 20 s; gently air blow for 5 s;
light cure for 10 s (manufacturer’s directions)

Bis-GMA: bisphenol A diglycidyl methacrylate; BHT: butylhydroxytoluene; CQ: camphorquinone (photo-initiator); DET: N,N-diethanol p-toluidine; EPD: p-
dimethylamino ethyl benzoate; GDMA: glycerol dimethacrylate; GPDM: glycerol phosphate dimethacrylate; HEMA: 2-hydroxyethyl methacrylate; MDP:
10-methacryloyloxydecyl dihydrogenphosphate; ODMAB: 2-(ethylhexyl)-4-(dimethylamino)benzoate; PAMA: phthalic acid monoethyl methacrylated; PEM-F:
pentamethacryloyloxyethyl cyclohexaphosphazene monofluoride; PENTA: dipentaerythritol pentaacrylate phosphate; pyro-EMA: tetramethacryloyloxyethyl pyro-
phosphate; UDMA: urethane dimethacrylate.

the actual testing was included in the calculation of the
mean μTBS as 0 MPa, with an explicit note of the number
of such pre-testing failures (PTF) in each group. Figure 1
shows a schematic diagram of tooth preparation, restor-
ation, specimen sectioning, and subsequent testing.
After μTBS measurement, the fractured sticks were
evaluated under an optical microscope (Leica CLS 150
MR) set at 40X magnification to determine the failure
mode, which was classified as follows: cohesive failure
in enamel (CE), cohesive failure in composite resin (CR),
adhesive (A), or mixed (M), when adhesive and cohesive
failures simultaneously occurred and cohesive failures
occupied more than 10% of the total area.
Surface Enamel Micromorphology
Qualitative assessment of conditioning effects created
on bur-cut enamel by the different adhesive systems
and methodologies described for bond strength test-
ing was conducted under scanning electron microscopy
(SEM). Enamel samples were obtained from two large
molars, previously bur-prepared on all their surfaces so
that substrate homogeneity could be attained across
groups. For purposes of comparison, one sample was
always reserved with no treatment. Self-etching systems
were applied to enamel surfaces but not light cured, so
that resin could be eliminated. For CSEB, only the primer
was applied. Surfaces were rinsed with acetone for

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Vinagre et al
Fig 1 Schematic diagram of specimen preparation. From top
left to bottom right: embedding 96 buccal and lingual surfaces;
grinding enamel with a diamond bur; outlining the flattest surface,
bonding and restorative procedures; specimen preparation; sec-
tioning; resin-enamel bonded sticks; microtensile bond strength.
30 s, immersed in an acetone bath and sonicated con-
tinuously for 15 min, followed by immersion for 10 min
in a 95% ethanol solution and then 10 min more in a
100% ethanol solution. A 12-h acetone bath completed
the dehydration process. For samples etched with 37%
phosphoric acid, after the 15-s conditioning period, the
acid was rinsed off with distilled water for 20 s and the
sample surface dehydrated similarly. Then all samples
were mounted on aluminum stubs, sputter coated with
gold-paladium (Polaron E-5000, Sputter-Coater, Polaron
Equipment; Watford, UK), observed and photographed in
a scanning electron microscope (Hitachi S-4100, Hitachi;
Tokyo, Japan) at an acceleration voltage of 25 kV. In
order to standardize and compare, the micrographs were
taken at magnifications of 1000X and 5000X.
Resin/Enamel Interfacial Micromorphology
Morphological observation of the resin/enamel inter-
faces was performed in 4 additional samples of each of
the 8 group combinations that were prepared as for the
microtensile specimens, except that two to three grains
of rhodamine B were added to the adhesive to provide a
fluorescent label. The teeth were sectioned longitudinally
through the restoration, polished with 1200-, 2500-, and
4000-grit silicone carbide paper under refrigeration and
sonicated for 15 min. The fluorescent specimens were
observed with a confocal laser scanning microscope
(LSM 710, emission 561 nm, pass filter 570 nm; Carl
Zeiss; Göttingen, Germany). Micrographs were taken at
magnifications of 400X and 800X. The same specimens
were then conditioned with a 37% phosphoric acid solu-
tion for 10 s to remove the inorganic component and
washed with distilled water. They were then immersed
in a sodium hypochlorite solution to dissolve the organic
110
components. The specimens were dehydrated in ascend-
ing concentrations of ethanol and then gold sputter-
coated prior to SEM observation (Hitachi S-4100, Hitachi)
at an acceleration voltage of 25 kV. Micrographs were
taken at magnifications of 800X and 2000X.
Statistical Analysis
Statistical analysis was performed with IBM SPSS Sta-
tistics 20.0 (SPSS; Chicago, IL, USA). One-way ANOVA
was used to compare means of microtensile bond
strength data between groups. Post-hoc pairwise com-
parisons were performed using the Games Howell
correction. The chi-square test was used to compare
between failure modes of the eight groups. The signifi-
cance level was set at α = 0.05. For each group, Weibull
distribution parameters were also determined using the
linear regression method at a 95% confidence level.
This analysis was performed using MATLAB R2012 (The
Mathworks; Natick, MA, USA).
RESULTS
A total of 276 specimens were available for microten-
sile testing. Descriptive statistics, the number of tested
specimens and pre-test failures (PTF) are depicted in
Table 2. One-way ANOVA revealed statistically significant
differences among groups (F[7, 105.213] = 333.636,
p < 0.01). Pairwise comparisons between groups indi-
cated no significant differences among the mean μTBS
values of OBFL, PBNT, CSEB/AA, and XIII/PA, which re-
corded the highest bond strength values. The interaction
between self-etching adhesive system and application
mode was only statistically significant for CSEB (CSEB/
AA vs CSEB/PA, p < 0.01). For this adhesive, the ac-
tive application mode demonstrated an increase in the
mean of μTBS of 9.51 MPa (4.79-14.22, 95% CI). Re-
garding XIII, no significant differences in bond strength
were observed between application modes, although a
higher result variability was recorded for XIII/PA. CSEB/
PA showed significantly lower bond strength (p < 0.01)
than OBFL, PBNT, CSEB/AA, XIII/PA, and XIII/AA, and
significantly higher bond strength (p < 0.01) than XV+.
XV+ also revealed significant differences from all other
groups (p < 0.01), regardless of the application mode.
In fact, this adhesive system generated almost null bond
strength values and numerous pre-test failures (PTF). As
statistically significant differences were detected for both
μTBS data between groups and application modes of self-
etching systems, both null hypotheses were rejected.
The results of the Weibull survival analysis are pre-
sented in Fig 2 and Table 3. Weibull analysis indicated
the highest characteristic strength and Weibull modulus
for OBFL, PBNT, and CSEB/AA among all other groups.
The failure pattern frequency and distribution can be
analysed in Table 4. Etch-and-rinse adhesives OBFL and
PBNT followed a similar trend, in which mixed, adhesive,
and enamel cohesive failure modes were more often de-
tected. For the self-etching adhesives XIII and CSEB in ei-
ther application mode, failures were predominantly adhe-
The Journal of Adhesive Dentistry
 Vinagre et al

Table 2 Descriptive statistics of bur-cut enamel μTBS as mean ± standard deviation for all groups

Group n Mean ± SD (MPa) Min (MPa) Max (MPa) PTF (n)

1: OBFL 33 26.86 ± 7.71a 12.02 39.84 0

2: PBNT 31 27.10 ± 7.17a 14.74 41.19 0

3: CSEB/PA 40 15.63 ± 6.51b 5.60 29.97 0

4: CSEB/AA 41 25.14 ± 7.11a,c 11.35 41.14 0

5: XIII/PA 35 22.65 ± 8.58a,c 9.03 43.62 0

6: XIII/AA 32 21.56 ± 4.28c 14.27 29.71 0

7: XV+/PA 36 2.38 ± 3.03d 0.00 12.26 14

8: XV+/AA 28 0.24 ± 0.62d 0.00 2.37 22

Means with different superscript letters indicate significant difference (p < 0.05). n: number of specimens; Min: lowest bond strength value; Max: highest
bond strength value; PTF: pretest failures. PA: passive application mode; AA: active application mode. OptiBond FL: OBFL; Prime & Bond NT: PBNT; Clearfil
SE Bond: CSEB; Xeno III: XIII; Xeno V+: XV+.

0.9

0.8

0.7
Probability of failure

0.6

0.5 OBFL
PBNT
0.4 XV+/PS
XV+/AT
0.3 XIII/PS
XIII/AT
0.2 CSEB/PA
CSEB/AA
0.1

0
0 5 10 15 20 25 30 35 40 45 50
Fig 2 Weibull distribution: probabil-
Applied stress
ity of failure vs applied stress (MPa).

Table 3 Weibull modulus (m) and 95% confidence interval (CI) of m. characteristic strength (σ0) and 95% confi-
dence interval (CI) of σ0, and Weibull coefficient of correlation (r) of experimental groups

Group m m 95% CI σ0 σ0 95% CI r

1: OBFL 3.75 3.11; 4.88 29.60 26.17; 32.58 0.97

2: PBNT 3.14 2.83; 3.63 30.98 26.58; 36.24 0.52

3: CS:EB/PA 2.23 2.02; 2.55 18.50 15.14; 22.32 0.57

4: CSEB/AA 3.40 2.95; 4.08 28.20 24.93; 31.57 0.76

5: XIII/PA 2.59 2.29; 3.09 25.96 21.76; 30.59 0.69

6: XIII/AA 4.99 4.37; 6.03 23.62 21.39; 25.68 0.72

7: XV+/PA 1.18 0.95; 1.62 4.20 2.57; 6.08 0.94

8: XV+/AA 0.89 0.67; 1.43 1.30 0.38; 2.71 0.96

For group abbreviations, see Table 2.

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Vinagre et al
Table 4 Distribution of the failure patterns of the experimental groups in absolute number of specimens (percentage)
Group 1 2 3 4
OBFL PBNT CSEB/PA
Adhesive 9 (27.3) 7 (22.6) 18 (45)
Mixed 13 (39.4) 9 (29) 16 (40)
Cohesive in resin 3 (9.1) 1 (3.2) 0 (0)
Cohesive in enamel 8 (24.2) 14 (45.2) 6 (15)
PTF 0 (0) 0 (0) 0 (0)
For group abbreviations, see Table 2.
sive or mixed. PTF were recorded only for XV+, all of which
were adhesive, which indicated a significant association
between groups and failure pattern (χ2[28] = 223.76;
p < 0.01).
The surface micromorphology of enamel rendered by bur
and conditioning with different adhesives is shown in Fig 3.
The SEM micrograph of enamel prepared with a 125-μm
diamond bur shows an irregular, rough surface covered with
a dense smear layer that prohibits identification of exposed
enamel prisms (Fig 3a). The etching pattern obtained with
phosphoric acid induced a clearer dissolution of enamel
prisms vs that found with self-etching systems. The enamel
surface appeared very porous, and numerous enamel crys-
tallites could be observed (Fig 3b). Morphological changes
in enamel surfaces were more distinct when self-etching
adhesives were applied actively than passively. Neverthe-
less, demineralization depth seems to be adhesive related.
In specimens etched with passively applied CSEB or XIII,
smear layer debris and bur scratches can still be identified
on enamel surfaces (Figs 3c and 3e). A few areas revealed
a discrete degree of interprismatic demineralization, which
can be better recognized at higher magnifications. No dis-
tinct morphological prismatic features were observed on
the enamel surface where XV+ was applied passively (Fig
3 g). The enamel surface etched with actively applied CSEB
shows a mild interprismatic dissolution of surface crystal-
lites that create microporosities within enamel prisms (Fig
3d). For actively applied XV+, a less distinct pattern was
observed (Fig 3 h). In contrast, actively applied XIII seemed
to show a greater demineralization depth, mainly at inter-
prismatic areas (Fig 3f).
The resin/enamel interfaces obtained with SEM and
fluorescence microscopy are shown in Fig 4. For both of the
etch-and-rinse adhesives OBFL and PBNT a higher level of
inter- and intraprismatic resin penetration into demineral-
ized enamel was observed, compared to the self-etching
adhesives, as confirmed by fluorescence images (Figs 4
and 5). CSEB/AA, CSEB/PA, or XIII/PA resin/enamel in-
terfaces showed a very thin hybridized layer with poorly
defined resin tags (Figs 6 to 8). Nevertheless, actively ap-
plied self-etching systems seemed to enhance monomer
penetration (Figs 7 and 9). For XV+ in both application
modes, interfacial gaps were noticeable, probably related
to cohesive failures in the hybrid layer, as some enamel
interprismatic penetration was evident (Figs 10 and 11).
112
5 6 7 8
CSEB/AA XIII/PA XIII/AA XV+/PA XV+/AA
20 (48.8) 14 (40) 15 (46.9) 22 (61.1) 6 (21.4)
17 (41.5) 11 (31.4) 13 (40.6) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
4 (9.8) 10 (28.6) 4 (12.5) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0) 14 (38.9) 22 (78.6)
DISCUSSION
Although clinical trials remain the gold standard in evalu-
ating the performance of dental materials, useful clinical
data for each new individual product is difficult to obtain.
Therefore, laboratory tests are still beneficial tools to
evaluate and explore methodologies concerning the use
of dental adhesives. Within the bond testing literature,
every research group produces individualized datasets,
because of the many variables associated with this field;
thus, caution is advisable when interpreting bonding data
across separate studies.37,38 Because of the brittleness
of the tissue, enamel microtensile specimens are intrinsi-
cally more prone to failure.34 Nevertheless, this meth-
odology is frequently used, even for testing adhesion
to enamel. Van Meerbeek et al45 proposed the use of
alginate or gypsum to fill up the space between the slabs
during microspecimen processing to better support the
slabs during the second, 90-degree-turned cut. Due to
better handling, extra-low viscosity polyvinylsiloxane was
used for space filling in the present study. Considering
that premature debonding only occurred in the group of
the self-etching adhesive XV+ at such a high frequency, it
is probably due to an intrinsic problem of adhesive.
For self-etching adhesives, some original protocol varia-
tions have been proposed to improve their performance in
both enamel and dentin in terms of conditioning time,28,47
previous phosphoric acid etching,11,41 passive vs active
application mode,2,7,8,19,22,30,39,47 number of adhesive
layers applied, or placement of a final, separate hydropho-
bic coating.1,32,42 In the present study, as expected, both
etch-and-rinse systems yielded the highest bond strengths
to bur-cut enamel. Nevertheless, they did not differ sig-
nificantly from those obtained for XIII regardless of the
application mode, or from CSEB when actively applied. In
fact, in the present study, the active application mode only
increased enamel bond strength for the mild self-etching
adhesive CSEB. In contrast, noticeable changes in enamel
surface micromorphology could be perceived when the ac-
tive application mode was employed regardless of the self-
etching system studied. Generally, greater dissolution was
achieved, although to different degrees depending on the
adhesive system, confirming that active application allows
fresh acidic monomers to progressively reach the bottom
of the smear layer and interact with subsurface enamel,
The Journal of Adhesive Dentistry
 Vinagre et al

Fig 3 Representative SEM photomicro-

graphs (magnification 5000X) of (a) a
bur-prepared enamel surface where sur-
face appears irregular and rough with
a thick smear layer that inhibits iden-
tification of exposed enamel rods; (b) a b
bur-prepared enamel surface conditioned
with 37% phosphoric acid showing a geo-
metrical, uniform and regular etching
pattern with a widespread removal of
apatite crystals, generating a classical
honeycomb configuration. A multitude
of deep intercrystallite pits could be ob-
served; (c) bur-prepared enamel surface
conditioned with CSEB/PA where globular
debris on the surface resembling dis-
crete signs of smear layer are present.
Cutting scratches are mostly seen and
an unclear and inhomogeneous etching c d
effect can be observed; (d) bur-prepared
enamel surface conditioned with CSEB/
AA exhibiting a superficial but regular
etching effect in which selective demin-
eralization of the interprismatic enamel
was preferential. Cutting scratches could
be identified; (e) bur-prepared enamel
surface conditioned with XIII/PA showing
a shallow, non-defined etching effect,
where cutting scratches could be easily
identified; (f) bur-prepared enamel sur-
face conditioned with XIII/AA with a well-
defined etching pattern, where substrate e f
was etched to a certain depth, resulting
in a “keyhole” configuration, in which
the etching of prism peripheries was
more clearly observed; (g) bur-prepared
enamel surface conditioned with XV+/
PA showing clear signs of smear layer
and cutting scratches. An indistinguish-
able etching effect could be observed; (h)
bur-prepared enamel surface conditioned
with XV+/AA revealing a slight, poorly de-
fined etching effect, in which occasional
prism periphery demineralization could be
observed. g h

leading to more aggressive demineralization. Thus, in the
present investigation, a positive correlation could be found
between the active mode of application and etching capac-
ity of the self-etching systems, but this was not always
accompanied by improved enamel bond strengths. Similar
results were reported by other authors.2,22,39 However,
there was a great qualitative difference in the morphology
of enamel surfaces conditioned by phosphoric acid and
self-etching adhesive systems, as shown in the SEM im-
ages. Phosphoric acid created the most regular and deep-
est etching pattern involving both inter- and intraprismatic
enamel, as previously reported.12,29
The CSEB adhesive system is a two-step self-etching
system that has become one of the most promising mater-
ials regarding adhesion to dentin.9,33,44 In line with three-
step etch-and-rinse systems such as the OBFL system,
this system is considered a benchmark in the adhesion
field.45 However, the use of CSEB according to the man-
ufacturer’s recommendation (passive mode) on bur-cut
enamel led to low μTBS values in this study. The surface
preparation method can significantly affect the nature of
the enamel smear layer; using a bur in clinical situations,
a thicker, rougher surface is produced than that obtained
in the laboratory using SiC papers.21 It is hypothesized

Vol 17, No 2, 2015 113

Vinagre et al
Fig 4 SEM (left) and Confocal Laser Scanning Microscope
(CLSM) fluorescent (right) photographs of the enamel/resin in-
terfaces in a cross-sectioned specimen treated with OBFL where
both inter- and intraprismatic adhesive penetration in demineral-
ized enamel is evident.
Fig 6 SEM (left) and CLSM fluorescent (right) photographs
of the enamel/resin interfaces in a cross-sectioned specimen
treated with CSEB/PA showing a very shallow and superficial
adhesive penetration in demineralized enamel.
Fig 8 SEM (left) and CLSM fluorescent (right) photographs
of the enamel/resin interfaces in a cross-sectioned specimen
treated with XIII/PA showing a shallow and inhomogeneous
adhesive intra- and interprismatic penetration in demineralized
enamel.
Fig 10 SEM (left) and CLSM fluorescent (right) photographs
of the enamel/resin interfaces in a cross-sectioned specimen
treated with XV+/PA showing an inconsistent adhesive penetra-
tion with an evident interfacial gap.
114
Fig 5 SEM (left) and CLSM fluorescent (right) photographs
of the enamel/resin interfaces in a cross-sectioned specimen
treated with PBNT where both inter- and intraprismatic adhesive
penetration in demineralized enamel is evident.
Fig 7 SEM (left) and CLSM fluorescent (right) photographs
of the enamel/resin interfaces in a cross-sectioned specimen
treated with CSEB/AA showing a shallow adhesive intaprismatic
penetration in demineralized enamel.
Fig 9 SEM (left) and CLSM fluorescent (right) photographs
of the enamel/resin interfaces in a cross-sectioned specimen
treated with XIII/AA showing a shallow and consistent adhesive
intra- and interprismatic penetration in demineralized enamel.
Fig 11 SEM (left) and CLSM fluorescent (right) photographs
of the enamel/resin interfaces in a cross-sectioned specimen
treated with XV+/AA showing intra- and interprismatic adhesive
penetration in demineralized enamel along with interfacial gap
formation indicating debonding beyond the adhesive layer.
The Journal of Adhesive Dentistry

that the compromised enamel bonding could to a cer-
tain extent be attributed to interference of bur debris
smeared across enamel during cavity preparation, par-
ticularly with mild self-etching adhesives, such as CSEB,
as their demineralization capacity is limited.12,20,36 This
was corroborated in this study, since its application in
the passive mode showed an enamel surface with persis-
tent signs of smear layer, bur-cutting scratches, and an
unclear and inhomogeneous etching effect. On the other
hand, 24-h enamel bond strength of actively applied CSEB
was found to be significantly higher than that obtained
with the same adhesive system applied passively; the
increase of bond strength was about 10 MPa. In addition,
a more regular etching effect and more pronounced in-
traprismatic resin penetration into demineralized enamel
was observed. These improved results support the active
application mode on bur-cut enamel for this particular ad-
hesive system, as it can probably help increase the quan-
tity of hydrogen ions available at the surface, enhancing
the demineralization process and the penetration of the
primer into subsurface demineralized enamel.22 Further,
this procedure may potentiate chemical interaction with
underlying enamel by forming calcium phosphate salts as
a result of the chemical bond of hydroxyapatite calcium
with the functional monomer 10-MDP.18,33
Active application of XIII did not reveal an increase in
enamel bond strengths values, as also reported by other
authors.47 Also, when applied passively according to the
manufacturer’s instructions and compared with CSEB, no
significant differences in ground enamel bond strengths
were reported between those two systems, despite conflict-
ing reports of the superiority of XIII17,43 vs reported higher
bond strengths for CSEB.1,3 XIII is a strong two-component,
one-step self-etching adhesive system, having a pH of the
mixture < 1 and the ability to moderately demineralize
enamel.12,43 Although agitation of XIII produced a more
defined etching pattern, the inherent demineralization ef-
fect of this system must be sufficient to promote adequate
micromechanical retention of monomers. Nevertheless,
less variability of μTBS values, a high Weibull modulus,
and more defined interfacial morphological features were
reported for XIII in the active application mode, which can
indicate a more reliable and consistent adhesion ability.
Moreover, when applied to dentin, significantly better im-
mediate and long-term bond strengths were reported when
this system was applied actively.7,19,30
The self-etching system XV+ yielded results that may
be cause for concern. Objectively, almost no enamel μTBS
was measurable, regardless of application mode, since
adhesive mechanically-induced or pre-testing failures read-
ily occurred. XV+ is a HEMA-free, water-based one-bottle
self-etching adhesive that incorporates tert-butanol as a co-
solvent and is considered an intermediately strong system
with a pH around 1.3.4 Although limited research has been
conducted with this specific adhesive system, it is consid-
ered an optimized version of its predecessor Xeno V, where
the main differences lie in the absence of acrylic acid and
a wettability agent, but better curing efficiency. Neverthe-
less, both formulations contain the most important, similar
components with potential innovative functions, namely,
Vol 17, No 2, 2015
Vinagre et al
“inverse” functionalized phosphoric acid esters, bifunc-
tional acrylic amides, and tertiary butanol. These patented
monomers are claimed to be more stable in aqueous acidic
solution than are acrylic ester functions of methacrylates. In
addition, the methyl groups surrounding the alcohol group
of tert-butanol would prevent an addition chemical reaction
with the polymerizable acrylic groups, keeping this function
intact.23,35 Despite these potential advantages, several fac-
tors can be pointed out for the failure observed in this study
for the self-etching system XV+. The first is related to the
composition that, if equivalent to the predecessor Xeno
V, includes a high solvent (73  wt%) and low hydrophobic
(3 wt%) proportion, accounting for the highly hydrophilic be-
havior.13 The second is related to the absence of HEMA,
which can predispose to phase separation, requiring strong
air drying of the adhesive before curing.40 Nonetheless,
this procedure can lead to a critical reduction of adhesive
layer thickness which, combined with the low hydrophobic
fraction and the presence of oxygen, reduces polymerization
efficiency and, consequently, bonding effectiveness. More
studies should be performed to better understand the bond-
ing mechanism of this specific system and others like it,
especially because simplified adhesives are being increas-
ingly used in patients without any proof of clinical efficacy.
Previous studies have shown that ultramorphological
features on enamel depend mainly on the pH of the so-
lution.12,14,25 Instead, the composition and mechanical
properties of the adhesive layer achieved after curing may
contribute in a major way to the bond strength accom-
plished for each specific material.16,43
CONCLUSIONS
The results of the present study lead to the rejection of
the null hypotheses. Both the adhesive system and the
application method had a significant effect on the bond
strengths to bur-prepared enamel. The etch-and-rinse
adhesive systems OBFL and PBNT together with CSEB
used actively and XIII used passively exhibited the high-
est and statistically similar bond strengths. XV+ showed
significantly lower bond strengths compared to all other
materials, regardless of the application mode. In respect
to application mode, active application demonstrated a
significant and positive influence on CSEB bond strength.
ACKNOWLEDGMENTS
The authors would like to express their gratitude to Kerr, Kuraray
Medical Inc, and Dentsply DeTrey for providing the materials used
in this study.
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Clinical relevance: The trend in bonding has been to-
wards simplification; nevertheless this may compromise
the bonding performance of some self-etching sys-
tems to bur-prepared enamel. Active application on the
enamel surface can be a valuable tool to improve the
performance of some self-etching adhesive systems.
The Journal of Adhesive Dentistry