Does the Conditioning Mode and Duration of Universal

Adhesives Affect the Bonding Effectiveness to Fluorotic

Enamel?
Fabiana Suelen Figueredo de Siqueiraa / Ana Armas-Vegab / Alexie Izquierdo-Buchelic /
Thaysa Fernandes Pintod / Taíse Alessandra Hanzene / José Bauerf / Andres Felipe Millan Cardenasg /
Alessandro D. Loguercioh

Purpose: To compare the adhesive-enamel microshear bond strength (μSBS), in situ degree of conversion (DC),
and the enamel-etching pattern of universal adhesives when applied for a prolonged period in the self-etch vs the
etch-and-rinse mode in fluorotic enamel.
Materials and Methods: Ninety-six human molars (48 with a Thylstrup and Fejerskov index [TFI] score of 0 and 48
with TFI score of 4) were sectioned into four parts and divided into 24 experimental groups based on enamel sur-
face (sound enamel or fluorotic enamel), adhesive (Clearfil Universal Bond, Futurabond U, iBond Universal, or
Scotchbond Universal), and enamel treatment/application time (etch-and-rinse mode [ER] or self-etch mode with
application times of 20 s [20SE] and 40 s [40SE]). The specimens were stored for 24 h and tested in shear at
1.0 mm/min (μSBS). Adhesive-enamel interfaces were evaluated for DC using micro-Raman spectroscopy. The
enamel-etching pattern was evaluated using SEM. For each adhesive, data from μSBS and DC were analyzed sepa-
rately using two-way ANOVA and Tukey’s post-hoc test at α = 0.05.
Results: On sound enamel, 40SE usually resulted in statistically similar mean μSBS (p > 0.52) and statistically
significantly improved mean DC values (p < 0.001) compared with the ER mode. Moreover, significantly improved
mean μSBS and DC values were obtained compared with 20SE (p < 0.01). On fluorotic enamel, there was no sta-
tistically significant difference in μSBS between the experimental groups (p > 0.76). However, for each applied
adhesive, 40SE resulted in improved mean DC values compared with 20SE or ER (p < 0.001). The deepest
enamel-etching pattern was obtained in ER mode, followed by 40SE in sound and fluorotic enamel.
Conclusion: Compared with ER mode, the prolonged application time of universal adhesives in SE mode in fluorotic
enamel increased the DC, enhanced enamel-etching pattern and promoted similar results in terms of adhesive-
enamel bond strength.
Keywords: adhesive bond strength, dental fluorosis, micro-Raman spectroscopy, scanning electron microscopy,
universal adhesive.

J Adhes Dent 2019; 21: 525–536. Submitted for publication: 15.09.18; accepted for publication: 04.08.19
doi: 10.3290/j.jad.a43695

a Professor, Department of Postgraduate Program in Dentistry, CEUMA Univer-
sity, São Luis, Maranhão, Brazil. Idea, designed testing assembly, performed
bond strength experiments, co-wrote and proofread the manuscript.
b Professor, Department of Dentistry, Equinoctial Technological University,
School of Dental Sciences Eugenio Espejo, Quito, Ecuador. Supervised bond
strength evaluation, proofread the manuscript.
c Professor, Department of Dentistry, Equinoctial Technological University,
School of Dental Sciences Salud Eugenio Espejo, Quito, Ecuador. Performed
degree of conversion test.
d MSc Student, Department of Postgraduate Program in Dentistry, CEUMA Uni-
versity, São Luis, Maranhão, Brazil. Performed etching pattern test, co-wrote
the manuscript.
e PhD Student, Department of Restorative Dentistry, State University of Ponta
Grossa, Ponta Grossa, Paraná, Brazil. Performed bond strength experiments
and co-wrote the manuscript.
f Professor, Department of Dentistry, Federal University of Maranhão, São Luis,
MA, Brazil. Provided consulting for statistical analysis, co-wrote the manu-
script.
g Professor, Department of Postgraduate Program in Dentistry, CEUMA Univer-
sity, São Luis, Maranhão, Brazil. Idea, designed testing assembly, co-wrote
and proofread the manuscript.
h Professor, Department of Dentistry, Equinoctial Technological University,
School of Dental Sciences Eugenio Espejo, Quito, Ecuador; Professor, Depart-
ment of Restorative Dentistry, State University of Ponta Grossa, Ponta Grossa,
Paraná, Brazil. Provided consulting for statistical analysis, contributed sub-
stantially to discussion, proofread the manuscript.
Correspondence: Andres Felipe Millan Cardenas, Universidade CEUMA. Departa-
mento de Pós-Graduação em Odontologia, Rua Josué Montello, 1 – Renascença
II, São Luís, Maranhão, Brazil, CEP: 65075-120. Tel: +55-98-3214-4164;
e-mail: andresfelipemillancardenas@hotmail.com

Vol 21, No 6, 2019 525

Siqueira et al
D ental fluorosis is a developmental disturbance of
enamel caused by excessive fluoride exposure during
tooth formation.20 Although fluoride plays a key role in the
prevention and control of caries9 and has promoted a de-
crease in the incidence of caries over the last three de-
cades,9,34 the use of fluoride increases the risk of dental
fluorosis.56
Fluorosis of the enamel is characterized by a reduced
mineral content,4,21 resulting in surface and subsurface po-
rosities68 and subsequent visual, physical changes. Clini-
cally, dental fluorosis compromises the tooth’s aesthetics,
ranging from narrow white lines following the perikymata to
discrete white opaque areas, or to an entirely chalky-white
tooth surface, depending on the severity.2 In some patients,
the enamel may become so porous that the outer layers
break down and the exposed porous subsurface becomes
discolored22 from light to dark brown.28 The appearance of
this discoloration, as well as pitting of the enamel surface,
may be aesthetically objectionable to the extent that the
patient may experience social repercussions.13,80
Thus, several minimal-intervention treatments have been
proposed to improve the appearance of tooth stains such
as micro-abrasion, enamel resin infiltration, and bleach-
ing.2,44,53,62 However, in the most severe cases of fluor-
osis, restorative procedures are required to improve the
overall aesthetics and thus increase the psychosocial con-
fidence of affected individuals.66
However, due to the physical and morphological changes
induced by dental fluorosis,28 bonding to this substrate is a
clinical challenge,19,21,71 because fluorapatite is more re-
sistant to acid dissolution than hydroxyapatite.14 The well-
known and most commonly used technique for improving
bonding to enamel is to apply phosphoric acid before adhe-
sive application, in a technique known as etch-and-rinse
(ER).16,67 Although good results have been obtained on
sound enamel,17,49 there is no consensus regarding the ap-
plication of phosphoric acid to fluorotic enamel.6,47 In addi-
tion, it is worth mentioning that the application of phosphoric
acid adds an extra step to the bonding protocol, which does
not comply with the clinician’s preference for simplification.
This is the main reason for the recent advent of “univer-
sal” or “multimode” adhesives. Universal adhesives are
essentially 1-step self-etch (SE) adhesives with a functional
resin monomer that bonds chemically to apatite crystal-
lites.25,45,55 To date, 10-methacryloyloxydecyl dihydrogen
phosphate (MDP) is the most frequently reported functional
resin monomer that bonds chemically to teeth,81 and it is
present in the majority of universal adhesives available in
the market.35,65,69
A recent study35 found that the active application of uni-
versal adhesives containing MDP in the SE mode exhibited
similar bond strength results for enamel compared with the
same universal adhesives applied in the ER approach. In
this regard, some authors have suggested prolonging of
the application time or the application of multiple layers of
SE adhesives to improve bonding to the enamel.11,30,
33,52,78 The latter approach increases the contact between
acidic resin monomers and the enamel surface, creating a
526
more retentive pattern and increasing the bond strength to
enamel.11,30,33,52,78
In addition, the conversion of a monomer to a polymer
also plays an important role in successful dental restor-
ations10,57 and the quantitative measurement of the degree
of conversion inside the hybrid layer can provide relevant
information about adhesive performance.27,39 Cardenas et
al11 showed that an active and prolonged application period
may be an alternative means of increasing the in situ de-
gree of conversion of universal adhesives. However, this
was investigated in sound enamel. To the best of our knowl-
edge, no study has thus far evaluated the influence of the
application time on the bond strength of commercially avail-
able universal adhesives for fluorotic enamel.
Therefore, the aim of this study was to compare the ad-
hesive-enamel microshear bond strength (μSBS), enamel-
etching pattern, and in situ degree of conversion (DC) of
four universal adhesives when applied for a prolonged pe-
riod in the SE mode compared with the ER mode in fluorotic
enamel. The following null hypotheses were tested: pro-
longed application will not influence the μSBS or the DC of
the universal adhesives when applied in SE mode com-
pared with ER mode.
MATERIALS AND METHODS
Tooth Selection and Preparation
Dental fluorosis was diagnosed according to the severity of
the condition using the Thylstrup and Fejerskov index61 (TFI,
Table 1). Prior to selection of the teeth, two examiners were
trained and a calibration procedure was performed accord-
ing to Ermis et al.18 Ninety-six third maxillary and mandibu-
lar caries-free human molars were used. Forty-eight teeth
with a TFI score of 0 (without fluorosis) and 48 extracted
human teeth with a TFI score of 4 were used from subjects
living in fluorosis-endemic areas in Ecuador (Chimborazo,
2 ppm fluoride in the drinking water)5,7 (Fig 1). The teeth
were collected after the patients’ informed consent was ob-
tained under a protocol approved by the Ethics Committee
Review Board (#3.183.007). The teeth were disinfected in
0.5% chloramine, stored in distilled water, and used within
6 months of extraction.
The roots of all teeth were removed by sectioning at the
cementoenamel junction. Seventy-two teeth were used for
evaluation of μSBS and in situ DC at the adhesive-enamel
interface, and 24 teeth were used for the evaluation of etch-
ing pattern. Each dental crown was then sectioned diagonally
across the long axis of the tooth to produce four enamel
specimens (buccal, lingual, and proximals),35 for a total of
384 enamel surfaces: 288 enamel surfaces were used to
evaluate μSBS and in situ DC at the adhesive-enamel inter-
face, and 96 were used for the evaluation of the etching
pattern produced on the fluorotic enamel surfaces (Fig 2).
Experimental Design
The enamel specimens were assigned to 24 experimental
groups according to the combination of the independent
The Journal of Adhesive Dentistry
 Siqueira et al

Table 1 Clinical criteria and scoring for the Thylstrup and Fejerskov dental fluorosis index (TFI)68*

Score Criteria
0 Normal translucency of enamel remains after prolonged air drying.
1 Narrow white lines corresponding to the perikymata.
2 Smooth surfaces: More pronounced lines of opacity that follow the perikymata. Occasionally confluence of adjacent lines.
Occlusal surfaces: Scattered areas of opacity <2 mm in diameter and pronounced opacity of cuspal ridges.
3 Smooth surfaces: Merging and irregular cloudy areas of opacity. Accentuated drawing of perikymata often visible between
opacities.
Occlusal surfaces: Confluent areas of marked opacity. Worn areas appear almost normal but usually circumscribed by a rim of
opaque enamel.
4 Smooth surfaces: The entire surface exhibits marked opacity or appears chalky white. Parts of surface exposed to attrition
appear less affected.
Occlusal surfaces: Entire surface exhibits marked opacity. Attrition is often pronounced shortly after eruption.
5 Smooth surfaces and occlusal surfaces: Entire surface displays marked opacity with focal loss of outermost enamel (pits) <2 mm
in diameter.
6 Smooth surfaces: Pits are regularly arranged in horizontal bands <2 mm in vertical extension.
Occlusal surfaces: Confluent areas <3 mm in diameter exhibit loss of enamel. Marked attrition.
7 Smooth surfaces: Loss of outermost enamel in irregular areas involving <1/2 of entire surface.
Occlusal surfaces: Changes in the morphology caused by merging pits and marked attrition.
8 Smooth and occlusal surfaces: Loss of outermost enamel involving >1/2 of surface.
9 Smooth and occlusal surfaces: Loss of main part of enamel with change in anatomic appearance of surface. Cervical rim of
almost unafffected enamel is often noted.
*Adapted from Rozier.61

Fig 1 Images of a fluorotic tooth with Thylstrup and Fejerskov index score 4,68 exhibiting chalky white surfaces (buccal, lingual, and proximal).

variables: enamel surface (sound enamel and fluorotic
enamel), adhesive – Clearfil Universal Bond (CUB, Kuraray
Noritake Dental; Tokyo, Japan), Futurabond U (FBU, Voco;
Cuxhaven, Germany), iBond Universal (IBU, Heraeus Kulzer;
Hanau, Germany), and Scotchbond Universal (SBU, 3M Oral
Care; St Paul, MN, USA) – and enamel treatment/applica-
tion time (ER and SE mode with application times of 20 s
and 40 s). Simple randomization of the enamel specimens
for all testing was performed for each substrate (sound
enamel and fluorotic enamel). An individual who was not
involved in the research protocol performed this procedure
using computer-generated numbers.
Microshear Bond Strength Test (μSBS)
Enamel specimens (n = 8 per group) were embedded in a
polyvinyl chloride tube (10 mm high x 13 mm diameter)
using a chemically cured acrylic resin (Jet Clássico, São
Paulo, SP, Brazil) so that the enamel surface was left ex-
posed at the top of the cylinder. The bonding area was iso-
lated according to the protocol proposed by Shimaoka et
al.64 Six to eight perforations with an internal diameter of
0.8 mm were made in a piece of acid-resistant, double-
faced adhesive tape (Adelbras Ind e Com Adesivos; São
Paulo, SP, Brazil) that was adapted to the enamel surface.
This procedure was performed using the Hygienic Ainsworth

Vol 21, No 6, 2019 527

Siqueira et al

Microshear bond strength

a d e f g

Ligh Cure Ligh Cure

Enamel specimen Adhesive Resin composite Microshear bond

was mounted in application packed inside strength test
PVC tube each Tygon tube
b
In situ degree of conversion

h i j k l
DC evaluation by
Polishing with
Ligh Cure Ligh Cure micro-Raman
SiC paper
spectroscopy

c
Enamel-etching pattern

m n o p
Dehydrated 12 h SEM

Acetone 24 h

Fig 2 Schematic diagram showing the preparation of enamel specimens for the different tests. (a) Ninety-six human molars (48 with TFI of 0
and 48 with TFI of 4) were sectioned diagonally across the occlusal surface (b) to obtain four enamel specimens (buccal, lingual, and proximal)
(c). For the microshear bond strength test (d), an enamel specimen was embedded in a PVC tube with acrylic resin so that the enamel surface
was left exposed at the top of the cylinder. Six to eight perforations with an internal diameter of 0.8 mm were made in an acid-resistant,
double-faced adhesive tape (yellow in [e]) and adapted to the enamel’s surface. After adhesive application (e) and light curing, the Tygon
tubes were placed on the enamel surface, filled with resin composite (f) and light cured for 20 s. After 24 h of water storage, the Tygon tubes
and the double-faced adhesive tape were carefully removed to expose the composite cylinders. Each enamel specimen was assembled in a uni-
versal testing machine for microshear bond strength testing with an orthodontic loop around the composite resin specimens (g). In the enamel
specimens for in situ degree of conversion testing (h), the adhesives were applied, and composite resin buildups were constructed on the
bonded enamel (i and j). After storage of the restored teeth for 24 h, the adhesive-enamel specimens were longitudinally sectioned across the
interface using a diamond saw to obtain two adhesive-enamel slices (k) that were wet polished with SiC paper and analyzed via micro-Raman
spectroscopy (l). For the enamel-etching pattern, after sectioning of the enamel (m), the adhesives were applied according to the experimental
groups but not light cured (n) and immediately stored in acetone for 24 h to remove all the resin monomers from the surface (o). Then, the
enamel specimens were dehydrated for 12 h, sputter coated with gold-palladium and the entire surface of treated enamel was examined using
SEM (p).

rubber-dam punch (Coltene; Altstätten, Switzerland). The
variation in the number of perforations for each enamel sur-
face was dependent on the dimensions of the enamel spec-
imens. The universal adhesives were applied to the enamel
surface in accordance with the manufacturer’s instructions,
as described in Table 2. A single operator performed all
bonding procedures, as follows:
y Etch-and-rinse (ER mode): The phosphoric acid gel of each
adhesive was applied and left undisturbed for the time
period recommended by each manufacturer. Then, the
surface was rinsed with water using an air-water syringe.
y SE application for 20 s (20SE): Each adhesive was ac-
tively applied on the enamel surface for 20 s. The man-
ual pressure exerted on the microbrush (Microbrush In-
ternational; Grafton, WI, USA) during the application
process was equivalent to 35 g (Table 2).36,59
y SE application for 40 s (40SE): Each adhesive was ac-
tively applied on the enamel surface for 40 s as de-
scribed for the previous group (Table 2).
After application of the adhesive, transparent polyethylene
Tygon tubes (Tygon Medical Tubing Formulations 54-HL,
Saint Gobain Performance Plastics; Akron, OH, USA) with
the same internal diameter as the perforations and a height
of 0.5 mm were positioned on the perforations over the
double-faced tape. This ensured that their lumen were con-
gruent with the circular areas exposed by the perforations.
The resin composite (Opallis, FGM; Joinville, SC, Brazil) was

528 The Journal of Adhesive Dentistry


Table 2 Adhesive (batch number), composition, and application mode of the adhesives according to manufacturers’
instructions
Adhesive
(batch number) Composition
Clearfil Universal 1. Etchant: 35% phosphoric acid,
Bond (CUB) colloidal silica, polyethyleneglycol,
(700018) pigment, and water (K-ETCHANT)
2. Adhesive: HEMA, MDP, bis-GMA,
ethanol, camphorquinone, hydrophilic
aliphatic dimethacrylate, silane
coupling agent, colloidal silica, water,
and accelerators
Futurabond U 1. Etchant: 34% phosphoric acid, water,
(FBU) synthetic amorphous silica,
(1705288) polyethylene glycol, aluminum oxide
(Scotchbond Universal Etchant)
2. Adhesive:
Liquid 1: Acidic adhesive monomer
HEMA, bis-GMA, HEDMA, UDMA catalyst
Liquid 2: Ethanol initiator, catalyst
iBond Universal 1. Etchant: 35% phosphoric acid,
(IBU) colloidal silica,
(010024) polyethyleneglycol, pigment, and
water (iBOND Etch 35 Gel)
2. Adhesive: Acetone, UDMA, TEGDMA,
4-methacryloxyethyltrimellitic anhydride,
photoinitiator.
Scotchbond 1. Etchant: 34% phosphoric acid, water,
Universal (SBU) synthetic amorphous silica,
(638367) polyethylene glycol, aluminum oxide
(Scotchbond Universal Etchant)
2. Adhesive: MDP phosphate monomer,
dimethacrylate resins, bis-GMA, HEMA,
methacrylatemodified polyalkenoic acid
copolymer, camphorquinone, filler,
ethanol, water, initiators, silane
Abbreviations: HEMA, 2-hydroxyethyl methacrylate; MDP, methacryloyloxydecyldihydrogen phosphate; bis-GMA, bisphenolglycidyl methacrylate;
UDMA, urethanedimethacrylate; HEDMA, hexamethylenedimethacrylate. a As per manufacturer’s instructions.
carefully packed inside each tube and a clear Mylar matrix
strip was placed over the filled Tygon tube and gently
pressed into position. The resin composite was cured for
20 s using an LED light-curing unit set at 1200 mW/cm2
(Radii-cal, SDI; Bayswater, Victoria, Australia). A radiometer
(Demetron L.E.D. Radiometer, Kerr Sybron Dental Special-
ties; Middleton, WI, USA) was used to examine the light in-
tensity after every five specimens. These procedures were
performed with the aid of 10X magnifying loupes.
After storage of the specimens in distilled water for 24 h
at 37ºC, the Tygon tubes and the double-faced adhesive
tape were carefully removed using a blade to expose the
composite cylinders. Each specimen was examined using a
stereomicroscope at 10X magnification. The bonded cylin-
Vol 21, No 6, 2019
Siqueira et al
Application mode
Self-etch (SE) Etch-and-rinse (ER)
1. Apply bond and rub for 20 sa or for 1. Apply etchant for 10 s
40 s 2. Rinse thoroughly
2. Dry by mild air blowing for 5 s 3. Dry
3. Light cure for 10 s at 1200 mW/cm2 4. Apply adhesive as for the
self-etch mode
1. Apply the adhesive to the entire 1. Apply etchant for 15 s
preparation with a microbrush and rub 2. Rinse for 10 s
for 20 sa or for 40 s. If necessary, 3. Air dry 2 s
rewet the disposable applicator during 4. Apply adhesive as for the
treatment self-etch mode
2. Direct a gentle stream of air over the
liquid for about 5 s until it no longer
moves and the solvent is evaporated
completely
3. Light cure for 10 s at 1200 mW/cm2
1. Apply the adhesive to the entire 1. Apply etchant for 15 s
preparation with a microbrush and rub 2. Rinse for 10 s
it for 20 sa or for 40 s. If necessary, 3. Dry
rewet the disposable applicator during 4. Apply adhesive as for the
treatment self-etch mode
2. Direct a gentle stream of air over the
liquid for about 5 s until it no longer
moves and the solvent is evaporated
completely
3. Light cure for 10 s at 1200 mW/cm2
1. Apply the adhesive to the entire 1. Apply etchant for 15 s
preparation with a microbrush and rub 2. Rinse for 10 s
for 20 sa or for 40 s. If necessary, 3. Air dry 2 s
rewet the disposable applicator during 4. Apply adhesive as for the
treatment self-etch mode
2. Direct a gentle stream of air over the
liquid for about 5 s until it no longer
moves and the solvent is evaporated
completely
3. Light cure for 10 s at 1200 mW/cm2
der was discarded if there was evidence of porosities or
gaps at the interface.46
The specimens were attached to a shear-testing jig
(Odeme Biotechnology; Joaçaba, SC, Brazil) and tested in a
universal testing machine (Kratos IKCL 3-USB, Kratos Equi-
pamentos Industriais; Cotia, São Paulo, Brazil). Each speci-
men was positioned in the universal testing machine and a
thin wire (0.2 mm diameter) was looped around the base of
each composite cylinder. The wire contacted the composite
resin cylinder along half of its circumference. The setup
(adhesive-enamel interface, the wire loop, and the center of
the load cell) was maintained in alignment to ensure the
correct orientation of the shear forces.63 The crosshead
speed was set at 1 mm/min until failure (Fig 2).
529
Siqueira et al

Table 3 Number of specimens (%) according to fracture mode* for all experimental groups

Sound enamel Fluorotic enamel

Application
Adhesives mode A/M CE CR PF A/M CE CR PF
CUB ER mode 56 (100) 0 (0) 0 (0) 0 (0) 50 (92) 2(4) 1 (2) 1 (2)
20SE 59 (98) 0 (0) 0 (0) 1 (2) 60 (98) 1 (2) 0 (0) 0 (0)
40SE 60 (100) 0 (0) 0 (0) 0 (0) 55 (100) 0 (0) 0 (0) 0 (0)
FBU ER mode 54 (96) 0 (0) 2 (4) 0 (0) 53 (96) 0 (0) 0 (0) 2 (4)
20SE 50 (100) 0 (0) 0 (0) 0 (0) 55 (95) 2 (3) 0 (0) 1 (2)
40SE 52 (96) 1 (2) 0 (0) 1 (2) 60 (100) 0 (0) 0 (0) 0 (0)
IBU ER mode 60 (100) 0 (0) 0 (0) 0 (0) 58 (96) 0 () 0 (0) 2 (4)
20SE 50 (96) 0 (0) 0 (0) 2 (4) 54 (96) 2 (4) 0 (0) 0 (0)
40SE 60 (98) 0 (0) 1 (2) 0 (0) 54 (96) 1 (2) 0 (0) 1 (2)
SBU ER mode 54 (100) 0 (0) 0 (0) 0 (0) 64 (100) 0 (0) 0 (0) 0 (0)
20SE 54 (96) 1 (2) 0 (0) 1 (2) 51 (98) 1 (2) 0 (0) 0 (0)
40SE 61(98) 0 (0) 0 (0) 1 (2) 58 (98) 1 (2) 0 (0) 0 (0)
* A/M – adhesive/mixed; CE – cohesive-enamel; CR – cohesive-resin; PF – premature failure.

The μSBSs (MPa) were calculated by dividing the load at
failure by the surface area (mm2). After testing, the speci-
mens were examined using an optical microscope (SZH-
131, Olympus; Tokyo, Japan) at 100X magnification to de-
fine the location of bond failure. The type of failure was
determined based on the percentage of substrate-free ma-
terial as follows: cohesive ([CE] failure exclusively within
enamel or [CR] failure exclusively within resin composite),
adhesive/mixed ([A/M] failure at the adhesive-enamel inter-
face, or failure at the adhesive-enamel interface with partial
cohesive failure of the neighboring substrates), and prema-
ture failures (PF).
In Situ Degree of Conversion (DC)
Enamel specimens (n = 4 per group) were prepared accord-
ing to previous studies.11,35 The adhesives were applied
and composite resin buildups were constructed on the
bonded enamel using the same materials and protocols
used fo the μSBS test. After storage of the restored teeth in
distilled water at 37°C for 24 h, the adhesive-enamel spec-
imens were longitudinally sectioned across the bonded in-
terface using a slow-speed diamond saw (Isomet, Buehler;
Lake Bluff, IL, USA) to obtain two adhesive-enamel slices.
The adhesive-enamel slices were wet polished with
1500-, 2000-, and 2500-grit SiC paper for 15 s each. Then,
they were ultrasonically cleaned for 20 min in distilled water
and stored in water for 24 h at 37ºC (Fig 2). A micro-Raman
microscope (XploRA ONE Raman microscope, HORIBA Sci-
entific, New Jersey, NY, USA) was first calibrated for zero
and then for coefficient values using a silicon sample. The
samples were analyzed using a 638-nm diode laser and an
X100/0.9 NA air objective. The Raman signal was acquired
using a 600-line/mm grafting centered between 500 and
2000 cm-1 with the parameters of 100 mW, spatial resolu-
tion of 3 μm, spectral resolution of 5 cm-1, and an accumu-
lation time of 25 s, with three co-additions.
Spectra were acquired at the adhesive-enamel adhesive
interface at three distinct sites for each specimen. The spec-
tra of uncured adhesives were acquired as references. The
ratio of the double-bond content of monomer to polymer in the
adhesive was calculated according to the following formula:
DC (%) = (1 - [Rcured / Runcured]) x 100, where R is the ratio of
the aliphatic and aromatic peak intensities at 1639 cm-1 and
1609 cm-1 in cured and uncured adhesives, respectively.
Enamel-etching Pattern
The enamel-etching pattern (n = 4 enamel surfaces per
group) was evaluated on the enamel surface using a scan-
ning electron microscope (SEM; VEGA 3 TESCAN; Shi-
madzu, Tokyo, Japan). For this purpose, in ER mode, the
phosphoric acid gel was applied on the enamel for 30 s,
rinsed for 10 s, and air dried according to each manufactur-
er’s instructions. Thereafter, the adhesives were applied
using the ER and SE strategy under the different experimental
conditions (Table 2), but the adhesives were not light cured.
The enamel surfaces were immediately stored in ace-
tone for 24 h to dissolve the resinous material that was
present.54 Then, the specimens were rinsed in deionized
water (5 min), a 96% alcohol bath (5 min), and deionized
water (5 min) to dissolve and remove the SE primer and the
adhesive resins.54
All specimens were dried and dehydrated in a desiccator
for 12 h, and the conditioned enamel surfaces were sputter-
coated with gold-palladium in a vacuum evaporator (SCD
050, Balzers; Schaan, Liechtenstein). The entire surface of
the treated enamel was examined in an SEM (VEGA 3 TES-
CAN, Shimadzu) (Fig 2). Images of representative surface
areas were acquired at 5000X magnification.

530 The Journal of Adhesive Dentistry

 Siqueira et al

Table 4 Microshear bond strengths in MPa (means ± standard deviations) of the different experimental groups*

Sound enamel Fluorotic enamel

Adhesives ER mode 20SE 40SE ER mode 20SE 40SE

CUB 19.45 ±1.5B 19.35 ± 2.1A,B 22.66 ± 1.5A 15.94 ± 1.1C 14.38 ± 1.2C 15.16 ± 1.9C
FBU 17.21 ± 2.0a 14.78 ± 1.9b 18.10 ±1.6a 14.56 ± 1.9b 13.89 ± 2.1b 13.17 ± 1.7b
IBU 15.31 ± 2.0A,B 14.36 ± 1.9B 17.34 ± 1.4A 11.59 ± 1.2C 12.12 ± 2.5C 12.71 ± 1.3C
SBU 19.43 ± 2.1a 17.17 ± 2.2b 20.56 ± 1.1a 16.76 ± 1.8b 16.07 ± 2.1b 16.83 ± 2.2b
*For each adhesive, different superscript letters indicate statistically significant differences between groups (two-way ANOVA; Tukey’s test, p < 0.05).

Table 5 In situ degree of conversion (%) (means ± standard deviations) of the different experimental groups*

Sound enamel Fluorotic enamel

Adhesives ER mode 20SE 40SE ER mode 20SE 40SE

CUB 63.09 ± 2.8B,C 69.10 ± 1.7B 74.08 ± 1.4A 53.32 ± 1.9D 52.25 ± 2.7D 58.26 ± 2.7C
FBU 58.26 ± 2.6b 56.30 ± 1.9b 64.47 ± 2.4a 48.47 ± 1.8c 49.75 ± 2.4c 55.21 ± 2.1b
IBU 55.46 ± 2.2B 56.52 ± 2.1B 61.16 ± 2.6A 47.51 ± 1.8c 49.33 ± 2.0B,C 53.21 ± 1.6B
SBU 69.61 ± 2.2b 68.65 ± 2.3b 76.03 ± 2.5a 57.62 ± 2.6c 59.34 ± 1.4c 65.12 ± 1.9b
*For each adhesive, different letters indicate statistically significant differences between groups (two-way ANOVA; Tukey’s test, p < 0.05).

Statistical Analysis ally, each universal adhesive applied in SE mode for 40 s

The μSBS of all the adhesive-enamel specimens with adhe-
sive/mixed failure mode from the same enamel specimens
were averaged for statistical purposes. Similarly, the same
procedure was performed for the DC measurements, so
that the experimental unit in this study was the enamel
specimens. Specimens with cohesive and premature fail-
ures were not included in the data analysis.
For each adhesive, data from μSBS and in situ DC were
analyzed separately using two-way ANOVA (enamel surface
vs enamel treatment/application time) and Tukey’s post-
hoc test at _ = 0.05. The enamel-etching pattern was only
evaluated qualitatively.
(40SE) exhibited statistically significantly higher mean bond
strengths compared with the SE mode for an application of
20 s (20SE) (p < 0.01; Table 4), with the exception of CUB.
For CUB, no significant difference was observed when 20SE
and 40SE were compared in the sound enamel (p = 0.35,
Table 4).
Conversely, no significant difference was observed be-
tween the experimental groups in the case of the fluorotic
enamel (p > 0.76; Table 4). When sound and fluorotic
enamel were compared, for all the adhesives and strat-
egies, statistically significantly higher mean bond strengths
were obtained to sound enamel than to fluorotic enamel
(p < 0.001; Table 4).

RESULTS In Situ Degree of Conversion (DC)

Microshear Bond Strength
Most of the specimens (98%) exhibited adhesive/mixed fail-
ures, 1% showed premature failure, and 1% displayed cohe-
sive failure exclusively within the enamel, or cohesive fail-
ure exclusively within the resin composite (Table 3). When
sound enamel was evaluated, the application of each uni-
versal adhesive under SE mode for 40 s (40SE) resulted in
mean bond strengths that were statistically similar to those
obtained for the respective ER application mode (p > 0.52;
Table 4). The exception was CUB, for which the 40SE ap-
plication technique statistically signifcantly increased the
mean bond strengths compared to those obtained with the
respective ER application mode (p < 0.01; Table 4). Usu-
For each universal adhesive applied in the SE mode for 40 s
(40SE) on sound enamel, the mean DCs were statistically
significantly higher than those obtained with the respective
ER application mode (p < 0.001; Table 5). For all evaluated
universal adhesives, the application in SE mode for 20 s
(20SE) resulted in mean DCs that were statistically similar
to those obtained for the ER groups (p > 0.62; Table 5).
For each universal adhesive applied in SE mode for 40 s
(40SE) on fluorotic enamel, the mean DCs exhibited statis-
tically significant improvements compared to those ob-
tained with the respective SE mode application for 20 s
(20SE) and the ER application mode (p < 0.001; Table 5).
Conversely, no significant difference was observed on fluo-
rotic enamel between the SE mode applied for 20 s (20SE)

Vol 21, No 6, 2019 531

Siqueira et al

Fig 3 Representative morphol-

ogy of sound and fluorotic Sound Enamel
enamel treated with four univer-
ER mode 20SE 40SE
sal adhesives for different
enamel treatment/application A B C
times. An increase of irregulari-
ties and micro-porosities on the
fluorotic enamel surface of was
visible compared to sound
enamel. A more pronounced CUB
prism dissolution was observed
for ER mode for sound enamel
(A, D, G, J) and fluorotic enamel
(a, d, g, j). For both sound (B, E,
H, K) and fluorotic enamel (b, e,
h, k), 20SE application resulted D E F
in a less pronounced etching
pattern with less exposure of
the underlying enamel com-
pared with 40SE.
FBU

G H I

IBU

J K L

SBU

and the ER mode (p > 0.86; Table 5). When sound and
fluorotic enamel were compared, for all adhesives and strat-
egies, significantly higher mean DCs were obtained in
sound enamel (p < 0.001; Table 5).
Enamel-etching pattern
Representative SEM images of the enamel-etching pattern
of the different experimental groups are shown in Fig 3. In-
dependent of the enamel type, ER promoted a more pro-
nounced and differential enamel dissolution with complete
elimination of the smear layer and increased exposure of
the prism periphery.
SE mode applied for 20 s (20SE) resulted in a mild mor-
phological alteration in which the superficial enamel layer
was removed without exposure of the subsurface enamel
(Fig 3). However, for both sound and fluorotic enamel, a
more pronounced etching pattern was observed with in-
creased exposure of the enamel prisms and more signs of
hydroxyapatite dissolution for all universal adhesives used
in the 40SE mode (Fig 3). Moreover, an increase of micro-
irregularities and porosities on the entire fluorotic enamel
surface was obvious compared to sound enamel (Fig 3).

532 The Journal of Adhesive Dentistry

 Siqueira et al

Fluorotic Enamel
ER mode 20SE 40SE

a b c

CUB

d e f

FBU

g h i

IBU

j k l

SBU

DISCUSSION lower mean bond strengths for each universal adhesive in

The results of the present study indicated that the applica-
tion of universal adhesives for 20 s (20SE) in SE mode pro-
duced lower bond strengths and less pronounced demineral-
ization patterns in sound enamel compared to the 40SE or
ER groups. It is known that universal adhesives applied in SE
mode do not contain enough acidic monomers to produce an
etching pattern similar to that produced by phosphoric
acid.26,32,51,75 Indeed, shallow intercrystallite infiltration of
the monomers and slight interprismatic resin tag formation
were reported in several studies.26,51 This could explain the
the 20SE group than in the 40SE and ER groups.
Conversely, the application of a universal adhesive in SE
mode for 40 s (40SE) resulted in bond strengths similar to
those applied in ER mode in sound enamel, except for CUB.
In that case, when applied in the 40SE mode, there was a
significant improvement of mean μSBS compared to the ER
mode. This led to a rejection of the first null hypothesis.
Thus, we speculate that the prolonged application time
could have compensated for the pH of the universal adhe-
sives, as previously reported by Cardenas et al11 and Logu-
ercio et al,35 thereby increasing the bond strength and DC

Vol 21, No 6, 2019 533

Siqueira et al
in the sound enamel interface when universal adhesives
were applied for 40SE.
It is known that prolonged application times can improve
the diffusion and interactions of acidic monomers, thereby
increasing the potential of etching and impregnation of the
resin into the underlying enamel.52,73 Although the etching
pattern achieved with 40SE was still not as retentive as
that produced in ER mode, SEM analysis supports the as-
sertion that deep demineralization occurred with the univer-
sal adhesives when applied in 40SE mode (Fig 3).
In addition, the active application of a universal adhesive
in SE mode may increase outward solvent diffusion, mainly
for adhesives composed of solvent with low vapor pres-
sures, such as water/ethanol.75 Solvent evaporation may
facilitate changes in polymer topology by reducing the intrin-
sic fraction of the nanopores, thereby promoting increased
cross linking and improved mechanical properties of the
polymer inside the adhesive layer.10,57 This may explain the
observation that the highest DC was obtained for all adhe-
sives when applied in 40SE active mode compared with the
20SE and ER groups for both enamel substrates. This led
to a rejection of the second null hypothesis.
However, active, prolonged application time (40SE) was
still more effective for CUB, mainly because the mean bond
strength in this case improved statistically significantly com-
pared to that obtained with the respective ER application
mode. Clearfil Universal Bond (pH 2.3), Futurabond (pH
2.3), and iBond Universal (pH 1.6) are more acidic than
Scotchbond Universal (pH 2.7),11,35 and this lower pH can
potentiate the demineralization mechanism and improve
the superficial interaction of the adhesive.8,32,43,48,60
However, the manufacturer’s material safety data sheet
is not clear on whether FBU contains 10-MDP. The pres-
ence of 10-MDP improves mechanical strength82,83 and
protects the adhesive interface against hydrolysis,23 since
it forms a nanolayer with calcium by chemically interacting
with hydroxyapatite.29,81
Moreover, although iBond Universal has a lower pH than
that of the universal adhesives used in the present study, it
is a HEMA-free adhesive. Although promising bond strength
results have been reported for HEMA-free adhesives,74,76
HEMA-free universal adhesives are usually more prone to
phase separation at the interface.35 Moreover, no signifi-
cant clinical improvement was observed in a recently pub-
lished systematic review that compared HEMA-free adhe-
sives with HEMA-containing adhesives with respect to
restoring noncarious cervical lesions.15
In addition, IBU is a unique acetone-based universal ad-
hesive. Although acetone has a higher vapor pressure than
ethanol and water,50 which theoretically results in a better
water evaporation rate due to the “azeotrope effect”42 or
“water-chasing effect”,31 acetone-based adhesives require
a longer evaporation time than that recommended by the
manufacturer.38 Therefore, rapid acetone evaporation in-
creases the concentration of monomers in the adhesives,40
which lowers the vapor pressure of the remaining residual
solvents, especially water. Thus, it is more difficult to evap-
orate residual water.40,74
534
When sound and fluorotic enamel are compared, lower
bond strengths are obtained to fluorotic enamel, indepen-
dent of the application technique. These results should be
examined further, because fluorotic enamel is more resis-
tant to acidic dissolution due to the presence of fluorapa-
tite,14,20 which adversely affects the performance of the
adhesive.1,6,24,71,79
Although fluorapatite may be more acid resistant, other
structural changes, such as fluorotic enamel’s outer hy-
permineralized layer with a porous hypomineralized subsur-
face and the low surface energy, impair surface wetting.
This results in a decrease in the bond strength.72 This mor-
phological alteration, as shown in Fig 3, can also be caused
by the lower performance of all universal adhesives on fluor-
otic vs sound enamel, independent of the treatment/appli-
cation time.
However, a more detailed examination of the fluorotic
enamel results revealed that although fluorotic enamel was
more acid resistant, the prolonged application mode (40SE)
improved demineralization, observable as an increase of
micro-irregularities and porosities of the prisms in the mi-
croscopic analysis. Further, 40SE mode also improved the
interaction of acidic monomers with prismatic and interpris-
matic areas by carrying fresh monomers to the underlying
enamel during active application compared to 20SE, as pre-
viously observed for sound enamel.3,12,35,41,70,78
Although the bond strength to fluorotic enamel did not
increase significantly, active and prolonged application
(40SE) revealed a significant increase of DC inside the hy-
brid layer with fluorotic enamel compared to the 20SE or ER
groups. This could be explained by the lower amount of
solvent inside the adhesive layer after light curing, during
which the adhesive was applied actively for a prolonged
time, as previously indicated. The presence of a solvent
after light curing increases the number of voids and porosi-
ties. Consequently, the DC of the adhesive layer decreases,
as previously shown in the sound enamel.11,35
It is worth mentioning that in the present study, the num-
ber of premature failures was under 1%. There is no con-
sensus in the literature on the correct treatment of prema-
ture failure. A critical review by Van Meerbeek et al77
reported that researchers may either exclude all pre-test
failures from the analysis, or assign a pre-determined value
to pre-test failures, for example, the mean between the low-
est bond strength measured and zero.37,58 As the number
of premature failures was low (±1%) in this study, the as-
signment of a bond strength to this small percentage could
skew the data distribution, which could affect the statistical
analysis.77
In summary, the results of the present study confirmed
the previous findings that active and prolonged application of
universal adhesives in SE mode may be a viable alternative
to increase the bonding properties to sound enamel.11,35 In
the case of fluorotic enamel, this can be an alternative to
achieve results similar to that of the etch-and-rinse ap-
proach. However, further studies are needed to evaluate
other clinical alternatives or etching/conditioning agents to
increase the bonding properties to fluorotic enamel.
The Journal of Adhesive Dentistry

CONCLUSION
The active and prolonged application of universal adhesives
on fluorotic enamel for 40 s in self-etch mode resulted in
increased DC and a more pronounced enamel-etching pat-
tern. In addition, it yielded similar results in terms of adhe-
sive-enamel bond strength when compared to the use of
phosphoric acid.
ACKNOWLEDGMENTS
This study was partially supported by The National Council for Scien-
tific and Technological Development (CNPq) under grants 305588/
2014-1 and 304105/2013-9 from Brazil.
REFERENCES
1. Adanir N, Turkkahraman H, Yalcin Gungor A. Effects of adhesion promoters
on the shear bond strengths of orthodontic brackets to fluorosed enamel.
Eur J Orthod 2009;31:276–280.
2. Akpata ES. Occurrence and management of dental fluorosis. Int Dent J
2001;51:325–333.
3. Ando S, Watanabe T, Tsubota K, Yoshida T, Irokawa A, Takamizawa T,
Kurokawa H, Miyazaki M. Effect of adhesive application methods on bond
strength to bovine enamel. J Oral Sci 2008;50:181–186.
4. Aoba T, Fejerskov O. Dental fluorosis: chemistry and biology. Crit Rev
Oral Biol Med 2002;13:155–170.
5. Armas-Vega ADC, Gonzalez-Martinez FD, Rivera-Martinez MS, Mayorga-
Solorzano MF, Banderas-Benitez VE, Guevara-Cabrera OF. Factors associ-
ated with dental fluorosis in three zones of Ecuador. J Clin Exp Dent
2019;11:e42–e48.
6. Ateyah N, Akpata E. Factors affecting shear bond strength of composite
resin to fluorosed human enamel. Oper Dent 2000;25:216–222.
7. Basantes EFB, Falconí PRP, Yungan RGA. Estudio toxicológico de las
fuentes de agua de las comunidades Atapo Culebrillas y Santa Teresa
por presencia de fluorosis dental. Revista Caribeña de Ciencias Sociales
2018;abril 2018.
8. Breschi L, Gobbi P, Falconi M, Mazzotti G, Prati C, Perdigao J. Ultra-mor-
phology of self-etching adhesives on ground enamel: a high resolution SEM
study. Am J Dent 2003;16(Spec No):57A–62A.
9. Buzalaf MA, Levy SM. Fluoride intake of children: considerations for den-
tal caries and dental fluorosis. Monogr Oral Sci 2011;22:1–19.
10. Cadenaro M, Breschi L, Rueggeberg FA, Suchko M, Grodin E, Agee K, Di
Lenarda R, Tay FR, Pashley DH. Effects of residual ethanol on the rate
and degree of conversion of five experimental resins. Dent Mater
2009;25:621–628.
11. Cardenas AM, Siqueira F, Rocha J, Szesz AL, Anwar M, El-Askary F, Reis
A, Loguercio A. Influence of conditioning time of universal adhesives on
adhesive properties and enamel-etching pattern. Oper Dent 2016;41:
481–490.
12. Cehreli SB, Eminkahyagil N. Effect of active pretreatment of self-etching
primers on the ultramorphology of intact primary and permanent tooth
enamel. J Dent Child (Chic) 2006;73:86–90.
13. Chankanka O, Levy SM, Warren JJ, Chalmers JM. A literature review of
aesthetic perceptions of dental fluorosis and relationships with psycho-
social aspects/oral health-related quality of life. Community Dent Oral Ep-
idemiol 2010;38:97–109.
14. Christensen GJ. Clinical factors affecting adhesion. Oper Dent 1992;
(suppl 5):24–31.
15. da Silva TSP, de Castro RF, Magno MB, Maia LC, Silva ESMHDJ. Do
HEMA-free adhesive systems have better clinical performance than
HEMA-containing systems in noncarious cervical lesions? A systematic
review and meta-analysis. J Dent 2018;74:1–14.
16. De Munck J, Van Landuyt K, Peumans M, Poitevin A, Lambrechts P,
Braem M, Van Meerbeek B. A critical review of the durability of adhesion
to tooth tissue: methods and results. J Dent Res 2005;84:118–132.
17. De Munck J, Van Meerbeek B, Yoshida Y, Inoue S, Vargas M, Suzuki K,
Lambrechts P, Vanherle G. Four-year water degradation of total-etch ad-
hesives bonded to dentin. J Dent Res 2003;82:136–140.
Vol 21, No 6, 2019
Siqueira et al
18. Ermis RB, De Munck J, Cardoso MV, Coutinho E, Van Landuyt KL,
Poitevin A, Lambrechts P, Van Meerbeek B. Bonding to ground versus un-
ground enamel in fluorosed teeth. Dent Mater 2007;23:1250–1255.
19. Ertugrul F, Turkun M, Turkun LS, Toman M, Cal E. Bond strength of differ-
ent dentin bonding systems to fluorotic enamel. J Adhes Dent 2009;11:
299–303.
20. Fejerskov O, Larsen MJ, Richards A, Baelum V. Dental tissue effects of
fluoride. Adv Dent Res 1994;8:15–31.
21. Fejerskov O, Manji F, Baelum V. The nature and mechanisms of dental
fluorosis in man. J Dent Res 1990;69(Spec No):692–700.
22. Fomon SJ, Ekstrand J, Ziegler EE. Fluoride intake and prevalence of den-
tal fluorosis: trends in fluoride intake with special attention to infants. J
Public Health Dent 2000;60:131–139.
23. Fukegawa D, Hayakawa S, Yoshida Y, Suzuki K, Osaka A, Van Meerbeek
B. Chemical interaction of phosphoric acid ester with hydroxyapatite. J
Dent Res 2006;85:941–944.
24. Gungor AY, Turkkahraman H, Adanir N, Alkis H. Effects of fluorosis and
self etching primers on shear bond strengths of orthodontic brackets. Eur
J Dent 2009;3:173–177.
25. Hanabusa M, Mine A, Kuboki T, Momoi Y, Van Ende A, Van Meerbeek B,
De Munck J. Bonding effectiveness of a new ‘multi-mode’ adhesive to
enamel and dentine. J Dent 2012;40:475–484.
26. Hannig M, Bock H, Bott B, Hoth-Hannig W. Inter-crystallite nanoretention
of self-etching adhesives at enamel imaged by transmission electron mi-
croscopy. Eur J Oral Sci 2002;110:464–470.
27. Hass V, Dobrovolski M, Zander-Grande C, Martins GC, Gordillo LA,
Rodrigues Accorinte Mde L, Gomes OM, Loguercio AD, Reis A. Correlation
between degree of conversion, resin-dentin bond strength and nanoleak-
age of simplified etch-and-rinse adhesives. Dent Mater 2013;29:921–928.
28. Horowitz HS, Driscoll WS, Meyers RJ, Heifetz SB, Kingman A. A new
method for assessing the prevalence of dental fluorosis – the Tooth Sur-
face Index of Fluorosis. J Am Dent Assoc 1984;109:37–41.
29. Inoue S, Koshiro K, Yoshida Y, De Munck J, Nagakane K, Suzuki K, Sano
H, Van Meerbeek B. Hydrolytic stability of self-etch adhesives bonded to
dentin. J Dent Res 2005;84:1160–1164.
30. Itou K, Torii Y, Takimura T, Chikami K, Ishikawa K, Suzuki K. Effect of
priming time on tensile bond strength to bovine teeth and morphologic
structure of interfaces created by self-etching primers. Int J Prosthodont
2001;14:225–230.
31. Jacobsen T SK. Some effects of water on dentin bonding. Dent Mater
1995;11:132–136.
32. Kenshima S, Reis A, Uceda-Gomez N, Tancredo Lde L, Filho LE, Nogueira
FN, Loguercio AD. Effect of smear layer thickness and pH of self-etching
adhesive systems on the bond strength and gap formation to dentin. J
Adhes Dent 2005;7:117–126.
33. Kimmes NS, Barkmeier WW, Erickson RL, Latta MA. Adhesive bond
strengths to enamel and dentin using recommended and extended treat-
ment times. Oper Dent 2010;35:112–119.
34. Kukleva MP, Kondeva VK, Isheva AV, Rimalovska SI. Comparative study
of dental caries and dental fluorosis in populations of different dental
fluorosis prevalence. Folia Med (Plovdiv) 2009;51:45–52.
35. Loguercio AD, Munoz MA, Luque-Martinez I, Hass V, Reis A, Perdigao J.
Does active application of universal adhesives to enamel in self-etch
mode improve their performance? J Dent 2015;43:1060–1070.
36. Loguercio AD, Stanislawczuk R, Mena-Serrano A, Reis A. Effect of 3-year
water storage on the performance of one-step self-etch adhesives ap-
plied actively on dentine. J Dent 2011;39:578–587.
37. Loguercio AD, Uceda-Gomez N, Carrilho MR, Reis A. Influence of speci-
men size and regional variation on long-term resin-dentin bond strength.
Dent Mater 2005;21:224–231.
38. Luque-Martinez IV, Perdigao J, Munoz 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.
39. 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.
40. Miettinen VM, Vallittu PK, Docent DT. Water sorption and solubility of
glass fiber-reinforced denture polymethyl methacrylate resin. J Prosthet
Dent 1997;77:531–534.
41. Miyazaki M, Hinoura K, Honjo G, Onose H. Effect of self-etching primer ap-
plication method on enamel bond strength. Am J Dent 2002;15:412–416.
42. Moszner N, Salz U, Zimmermann J. Chemical aspects of self-etching
enamel-dentin adhesives: a systematic review. Dent Mater 2005;21:
895–910.
535
Siqueira et al
43. Moura SK, Pelizzaro A, Dal Bianco K, de Goes MF, Loguercio AD, Reis A,
Grande RH. Does the acidity of self-etching primers affect bond strength
and surface morphology of enamel? J Adhes Dent 2006;8:75–83.
44. Munoz MA, Arana-Gordillo LA, Gomes GM, Gomes OM, Bombarda NH,
Reis A, Loguercio AD. Alternative esthetic management of fluorosis and
hypoplasia stains: blending effect obtained with resin infiltration tech-
niques. J Esthet Restor Dent 2013;25:32–39.
45. Munoz MA, Luque I, Hass V, Reis A, Loguercio AD, Bombarda NH. Imme-
diate bonding properties of universal adhesives to dentine. J Dent
2013;41:404–411.
46. Muñoz MA, Baggio R, Emilio Mendes YB, Gomes GM, Luque-Martinez I,
Loguercio AD, Reis A. The effect of the loading method and cross-head
speed on resin-dentin microshear bond strength. Int J Adhes Adhes
2014;50:136–141.
47. Ng’ang’a PM, Ogaard B, Cruz R, Chindia ML, Aasrum E. Tensile strength
of orthodontic brackets bonded directly to fluorotic and nonfluorotic
teeth: an in vitro comparative study. Am J Orthod Dentofacial Orthop
1992;102:244–250.
48. Pashley DH, Tay FR. Aggressiveness of contemporary self-etching adhe-
sives. Part II: etching effects on unground enamel. Dent Mater 2001;17:
430–444.
49. 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.
50. 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.
51. Perdigao J, Geraldeli S. Bonding characteristics of self-etching adhesives
to intact versus prepared enamel. J Esthet Restor Dent 2003;15:32–41.
52. Perdigao J, Gomes G, Lopes MM. Influence of conditioning time on
enamel adhesion. Quintessence Int 2006;37:35–41.
53. Perdigao J, Lam VQ, Burseth BG, Real C. Masking of enamel fluorosis
discolorations and tooth misalignment with a combination of at-home
whitening, resin infiltration, and direct composite restorations. Oper Dent
2017;42:347–356.
54. Perdigao J, Lopes MM, Gomes G. In vitro bonding performance of self-
etch adhesives: II--ultramorphological evaluation. Oper Dent 2008;33:
534–549.
55. Perdigao J, Sezinando A, Monteiro PC. Effect of substrate age and adhe-
sive composition on dentin bonding. Oper Dent 2013;38:267–274.
56. Petersen PE, Lennon MA. Effective use of fluorides for the prevention of
dental caries in the 21st century: the WHO approach. Community Dent
Oral Epidemiol 2004;32:319–321.
57. Reis A, Ferreira SQ, Costa TR, Klein-Junior CA, Meier MM, Loguercio AD.
Effects of increased exposure times of simplified etch-and-rinse adhe-
sives on the degradation of resin-dentin bonds and quality of the polymer
network. Eur J Oral Sci 2010;118:502–509.
58. Reis A, Loguercio AD, Azevedo CL, de Carvalho RM, da Julio Singer M,
Grande RH. Moisture spectrum of demineralized dentin for adhesive sys-
tems with different solvent bases. J Adhes Dent 2003;5:183–192.
59. Reis A, Pellizzaro A, Dal-Bianco K, Gones OM, Patzlaff R, Loguercio AD.
Impact of adhesive application to wet and dry dentin on long-term resin-
dentin bond strengths. Oper Dent 2007;32:380–387.
60. Rotta M, Bresciani P, Moura SK, Grande RH, Hilgert LA, Baratieri LN,
Loguercio AD, Reis A. Effects of phosphoric acid pretreatment and substi-
tution of bonding resin on bonding effectiveness of self-etching systems
to enamel. J Adhes Dent 2007;9:537–545.
61. Rozier RG. Epidemiologic indices for measuring the clinical manifestations
of dental fluorosis: overview and critique. Adv Dent Res 1994;8:39–55.
62. Sherwood IA. Fluorosis varied treatment options. J Conserv Dent 2010;13:
47–53.
63. Shimada Y, Yamaguchi S, Tagami J. Micro-shear bond strength of dual-
cured resin cement to glass ceramics. Dent Mater 2002;18:380–388.
64. Shimaoka AM, de Andrade AP, Cardoso MV, de Carvalho RC. The impor-
tance of adhesive area delimitation in a microshear bond strength experi-
mental design. J Adhes Dent 2011;13:307–314.
65. Siqueira FSF, Cardenas AM, Ocampo JB, Hass V, Bandeca MC, Gomes
JC, Reis A, Loguercio AD. Bonding performance of universal adhesives to
eroded dentin. J Adhes Dent 2018;20:121–132.
536
66. Slaska B, Liebman AI, Kukleris D. Restoration of fluorosis stained teeth:
a case study. Dent Clin North Am 2015;59:583–591.
67. Swift EJ, Jr., Perdigao J, Heymann HO. Bonding to enamel and dentin: a
brief history and state of the art, 1995. Quintessence Int 1995;26:95–110.
68. Thylstrup A, Fejerskov O. Clinical appearance of dental fluorosis in per-
manent teeth in relation to histologic changes. Community Dent Oral Epi-
demiol 1978;6:315–328.
69. 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.
70. Torres CR, Barcellos DC, Pucci CR, Lima Gde M, Rodrigues CM, Siviero
M. Influence of methods of application of self-etching adhesive systems
on adhesive bond strength to enamel. J Adhes Dent 2009;11:279–286.
71. Torres-Gallegos I, G AM-C, Loyola-Rodriguez JP, Patino-Marin N, Encinas
A, Ruiz F, Anusavice K. Effectiveness of bonding resin-based composite
to healthy and fluorotic enamel using total-etch and two self-etch adhe-
sive systems. Dent Mater J 2012;31:1021–1027.
72. Torres-Mendez F, Martinez-Castanon GA, Torres-Gallegos I, Zavala-Alonso
NV, Patino-Marin N, Nino-Martinez N, Ruiz F. Effects of silver nanoparti-
cles on the bonding of three adhesive systems to fluorotic enamel. Dent
Mater J 2017;36:266–274.
73. Tsuchiya H, Tsubota K, Iwasa M, Ando S, Miyazaki M, Platt JA. Influence
of adhesive application time on enamel bond strength of single-step self-
etch adhesive systems. Oper Dent 2010;35:77–83.
74. Van Landuyt KL, De Munck J, Snauwaert J, Coutinho E, Poitevin A, Yo-
shida Y, Inoue S, Peumans M, Suzuki K, Lambrechts P, Van Meerbeek B.
Monomer-solvent phase separation in one-step self-etch adhesives. J
Dent Res 2005;84:183–188.
75. Van Landuyt KL, Snauwaert J, De Munck J, 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.
76. 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.
77. Van Meerbeek B, Peumans M, Poitevin A, Mine A, Van Ende A, Neves A,
De Munck J. Relationship between bond-strength tests and clinical out-
comes. Dent Mater 2010;26:e100–121.
78. Velasquez LM, Sergent RS, Burgess JO, Mercante DE. Effect of place-
ment agitation and placement time on the shear bond strength of 3 self-
etching adhesives. Oper Dent 2006;31:426–430.
79. Weerasinghe DS, Nikaido T, Wettasinghe KA, Abayakoon JB, Tagami J.
Micro-shear bond strength and morphological analysis of a self-etching
primer adhesive system to fluorosed enamel. J Dent 2005;33:419–426.
80. Yildiz G, Celik EU. A minimally invasive technique for the management of
severely fluorosed teeth: A two-year follow-up. Eur J Dent 2013;7:504–508.
81. Yoshida Y, Nagakane K, Fukuda R, Nakayama Y, Okazaki M, Shintani H,
Inoue S, Tagawa Y, Suzuki K, De Munck J, Van Meerbeek B. Comparative
study on adhesive performance of functional monomers. J Dent Res
2004;83:454–458.
82. Yoshihara K, Yoshida Y, Hayakawa S, Nagaoka N, Torii Y, Osaka A, Su-
zuki K, Minagi S, Van Meerbeek B, Van Landuyt KL. Self-etch monomer-
calcium salt deposition on dentin. J Dent Res 2011;90:602–606.
83. Yoshihara K, Yoshida Y, Nagaoka N, Fukegawa D, Hayakawa S, Mine A,
Nakamura M, Minagi S, Osaka A, Suzuki K, Van Meerbeek B. Nano-con-
trolled molecular interaction at adhesive interfaces for hard tissue recon-
struction. Acta Biomater 2010;6:3573–3582.
Clinical relevance: The active and prolonged application
time of universal adhesives applied in SE mode to
fluorotic enamel may be an alternative to increase the
DC and enhance the enamel-etching pattern. This a
pproach yielded similar results in terms of adhesive-
enamel bond strength compared to the use of
phosphoric acid.
The Journal of Adhesive Dentistry