Journal of Dentistry 62 (2017) 54–63

Contents lists available at ScienceDirect

Journal of Dentistry
journal homepage: www.elsevier.com/locate/jdent

No-waiting dentine self-etch concept—Merit or hype MARK

a b c d e f,⁎⁎
Xue-qing Huang , César R. Pucci , Tao Luo , Lorenzo Breschi , David H. Pashley , Li-na Niu ,
⁎
Franklin R. Taye,
a
Department of Prosthodontics, Guanghua School and Hospital of Stomatology & Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University,
Guangzhou, Guangdong, PR China
b
Department of Restorative Dentistry, Institute of Science and Technology, São Paulo State University UNESP São Jose dos Campos, São Paulo, Brazil
c
Key Laboratory of Oral Medicine, Guangzhou Institute of Oral Disease, Stomatology Hospital of Guangzhou Medical University, Guangzhou, Guangdong, PR China
d
Department of Biomedical and Neuromotor Sciences, DIBINEM, University of Bologna – Alma Mater Studiorum, Bologna, Italy
e
College of Graduate Studies, Augusta University, Augusta, GA, USA
f
State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Oral Diseases, School of Stomatology,
The Fourth Military Medical University, Xi’an, Shaanxi, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Objective: A recently-launched universal adhesive, G-Premio Bond, provides clinicians with the alternative to use
Dentine the self-etch technique for bonding to dentine without waiting for the adhesive to interact with the bonding
Self-etch substrate (no-waiting self-etch; Japanese brochure), or after leaving the adhesive undisturbed for 10 s (10-s self-
Time factors etch; international brochure). The present study was performed to examine in vitro performance of this new
Universal adhesives
universal adhesive bonded to human coronal dentine using the two alternative self-etch modes.
Methods: One hundred and ten specimens were bonded using two self-etch application modes and examined
with or without thermomechanical cycling (10,000 thermal cycles and 240,000 mechanical cycles) to simulate
one year of intraoral functioning. The bonded specimens were sectioned for microtensile bond testing,
ultrastructural and nanoleakage examination using transmission electron microscopy. Changes in the composi-
tion of mineralised dentine after adhesive application were examined using Fourier transform infrared
spectroscopy.
Results: Both reduced application time and thermomechanical cycling resulted in significantly lower bond
strengths, thinner hybrid layers, and significantly more extensive nanoleakage after thermomechanical cycling.
Using the conventional 10-s application time improved bonding performance when compared with the no-
waiting self-etch technique. Nevertheless, nanoleakage was generally extensive under all testing parameters
employed for examining the adhesive.
Conclusion: Although sufficient bond strength to dentine may be achieved using the present universal adhesive
in the no-waiting self-etch mode that does not require clinicians to wait prior to polymerisation of the adhesive,
this self-etch concept requires further technological refinement before it can be recommended as a clinical
technique.
Clinical significance: Although the surge for cutting application time to increase user friendliness remains the
most frequently sought conduit for advancement of dentine bonding technology, the use of the present universal
adhesive in the no-waiting self-etch mode may not represent the best use of the adhesive.

1. Introduction switching and wide-area networking in the 1960s incited the emer-
gence of the internet and metamorphosed nearly all facets of human
Science and technology constitute a primary productive force that activity − society, politics, culture and the economy. On a lighter note,
stimulates society reform [1]. As early as the 1700s, technological the technology triggered by Doctor Michael Buonocore’s adaptation of
developments that led to the introduction of steam power catalysed the phosphoric acid treatment of metal surfaces to obtain better adhesion of
Industrial Revolution and provided the backdrop for modernisation on paint primers spurred the rise of the resin-bonding epoch in dentistry
a global scale. Technologies triggered by the concepts of packet [2].

⁎
Corresponding author at: The Dental College of Georgia, Augusta University, Augusta, Georgia, USA.
⁎⁎
Corresponding author.
E-mail addresses: [email protected] (L.-n. Niu), [email protected] (F.R. Tay).

http://dx.doi.org/10.1016/j.jdent.2017.05.007
Received 17 April 2017; Received in revised form 10 May 2017; Accepted 11 May 2017
0300-5712/ Published by Elsevier Ltd.
X.-q. Huang et al. Journal of Dentistry 62 (2017) 54–63

Table 1
Adhesive composition and application procedures.

Adhesive pH Composition Manufacturer’s instructions

G-Premio Bond 1.5 10-MDP, 4-META, 10-methacryoyloxydecyl dihydrogen thiophosphate, methacrylate acid ester, No-waiting self-etching mode
distilled water, acetone, phot initiators, silica
1. Apply using a microbrush
2. No waiting after applicatio
3. Dry thoroughly for 5 s with oil-free air under
maximum air pressur
4. Light-cure for 10 s
10-s self-etching mode

1. Apply using a microbrush

2. Leave undisturbed for 10 s after application
3. Dry thoroughly for 5 s with oil-free air under
maximum air pressure
4. Light-cure for 10 s

Abbreviations. 10-MDP: 10-methacryloxydecyl dihydrogen phosphate; 4-META: 4-methacryloyloxyethyl trimellitic anhydride.

Beginning with the initial technology trigger, the platform asso- etch; Japanese version of manufacturer’s instructions), or after leaving
ciated with the development and coming-of-age of new technologies the adhesive undisturbed for 10 s (10-s self-etch; international version
appears to follow a similar cycle, irrespective of the field of endeavour of manufacturer’s instructions) [24]. Introduction of the no-waiting
[3–5]. In the context of dentine bonding, the enthusiasms associated concept, while appealing to clinicians, should not compromise adhesive
with the advent of simultaneous etching of enamel and dentine [6], bonding effectiveness and the durability of resin-dentine bonds. Hence,
multi-step etch-and-rinse adhesives [7] and the water-wet bonding the present study was performed to examine in vitro performance of the
etch-and-rinse technique [8] produced a plethora of successful dental single-component, universal adhesive bonded to human coronal den-
adhesion stories, reaching a peak of inflated expectations during the tine using the two aforementioned alternative self-etch modes. Accord-
early 1990s [9]. No sooner had clinicians been besotted with these ingly, two null hypotheses were tested: 1) there is no difference
excitements than reports of post-operative sensitivity and adversarial between the use of no-waiting self-etch and 10-s self-etch on the
pulpal responses associated with the use of those adhesives began to microtensile bond strength of the universal adhesive to dentine; and
surface, particularly when the adhesives were applied to deep, vital 2) thermomechanical cycling has no effect on the microtensile bond
dentine [10,11]. By etching and priming tooth substrates simulta- strength to dentine when the universal adhesive is used in either the no-
neously, two-step self-etch adhesives were subsequently introduced to waiting or the 10-s self-etch mode.
solve those problems by reducing dentine permeability through the
incorporation of smear layer components into the hybrid layer [12]. 2. Materials and methods
Paradoxically, the problem-solving strategy contributed to unforeseen
problems. Increases in water sorption associated with incorporating 2.1. Dentine disc preparation
increasingly hydrophilic adhesive resin monomers [13], adhesive coat-
ings with semi-permeable membrane-like characteristics [14], as well One hundred and ten freshly extracted intact human third molars
as bond degradation over time [15,16] became the “Achilles heel” of were collected according to a protocol approved by the Human
both etch-and-rinse and self-etch adhesives. Waxing and waning of the Assurance Committee of the Augusta University, with informed consent
promises to deliver speedier, easier and more user-friendly adhesive obtained from the donating subjects with respect to the use of human
versions precipitated a trough of disillusionment in adhesive dentistry. tissues. The extracted teeth were stored in 0.9% (w/v) NaCl containing
As more is known about the control of resin-dentine bond degradation 0.02% sodium azide at 4 °C for no longer than one month. The roots of
via the use of the water-compatible catalytic systems [17], less the teeth were removed using a water-cooled low-speed saw (Isomet,
hydrophilic adhesive resin monomers [16] and inhibition of adhesive- Buehler Ltd, Evanston, IL, USA). For each tooth, a flat bonding surface
activated endogenous proteases [16], clinicians begin to appreciate the was prepared by removing the occlusal third of each tooth crown to
benefits associated with step reduction for both the etch-and-rinse [7] expose the mid-coronal dentine. The dentine surfaces were polished
and self-etch adhesives [18]. The simplified clinical manipulation and with 320-grit silicon carbide paper under water irrigation for 1 min.
shortened application time associated with these new adhesive genres
moved the industry to a slope of enlightenment. Universal adhesives
emerged as the latest player in this arena [19]. They provided dentists 2.2. Bonding procedures
with the option in choosing adhesive strategies (i.e. etch-and-rinse, self-
etch or selective etch), the versatility in bonding to zirconia [20] and The teeth were randomly assigned to two groups (50 teeth per
the purported advantage of bonding to silica-based ceramics without group). The composition of G-Premio Bond (GP) is listed in Table 1.
the use of additional primers [21]. Nevertheless, universal adhesives Self-etch bonding procedures were performed according to the Japa-
have been perceived as old wines in new bottles [22] because they nese version of the manufacturer’s instructions (no-waiting self-etch
inherit the limitations of previous adhesive categories [23]. In the mode) or the international version of the manufacturer’s instructions
absence of further enlightenment, it appears that dentine adhesive (10-s self-etch mode). For the no-waiting self-etch mode, the adhesive
development has entered the plateau stage of a classical technology was applied on the dentine surface and immediately air-dried without
development cycle. New insights are thus required to transcend waiting (see below). For 10-s self-etch mode, the adhesive was applied
technological development of dentine adhesives to new heights. on the dentine surface and left undisturbed for 10 s. For both modes,
A recently-launched universal adhesive (G-Premio Bond, GC the adhesive was dried for 5 s using oil-free air from the triple syringe of
Corporation, Tokyo, Japan) provides the clinician with the alternative a clinical operatory unit under maximum air pressure. After light-curing
to use the self-etch technique for bonding to dentine without waiting for of the adhesive, resin composite build-ups were constructed in four 1-
the adhesive to interact with the bonding substrate (no-waiting self- mm thick increments using G-ænial Sculpt (GC Corporation). Each
increment was light-cured for 20 s using a light-emitting diode

55
X.-q. Huang et al. Journal of Dentistry 62 (2017) 54–63

(440–480 nm range) curing unit with an output intensity of 1200 mW/ which the specimens were bonded and tested, and the category of
cm2. To facilitate sectioning of transmission electron microscopy (TEM) failure (adhesive vs non-adhesive).
specimens, the bonded specimens were coupled with a 2 mm thick layer
of Protect Liner F flowable resin composite (Kuraray Noritake Dental
2.4. Transmission electron microscopy
Inc., Tokyo, Japan) and light-cured for 20 s.
Because the manufacturer emphasised the mandatory use of max-
Five of the 10 beams designated for TEM were used for nanoleakage
imum air pressure for drying the adhesive in the bonding instructions, a
examination. Each beam was coated with two layers of nail varnish
pilot study was also performed to understand the consequence of not
applied to within 1 mm of the bonded interface. After drying, the
adhering to this instruction. In the pilot study, additional dentine discs
varnish-coated beams were immersed in 50% ammoniacal silver nitrate
were bonded using the two aforementioned self-etch protocols (N = 2),
solution for 48 hours. The silver-impregnated specimens were thor-
but with the adhesive dried with 5 s of gentle air flow. After coupling
oughly rinsed with deionised water and placed in photo-developing
with the flowable resin composite, the bonded specimens were aged for
solution for 8 h, under a fluorescent light, to facilitate reduction of the
24 h and prepared for nanoleakage examination of the resin-dentine
diaminesilver ions into metallic silver grains [29]. The silver-impreg-
interfaces (methods described in the TEM section).
nated specimens were polished, cleaned ultrasonically, dehydrated in
After bonding, the specimens from each experimental group (i.e.
an ascending ethanol series (50–100%), immersed in propylene oxide
those not related to the pilot study) were divided into two batches. One
as transition medium, and embedded in epoxy resin. Ninety nanometre-
batch was stored in deionised water at 37 °C for 24 h; the other batch
thick sections were prepared using an ultramicrotome and examined
was subjected to thermomechanical challenge, using 10,000 thermal
without staining, using a JEM-1230 TEM (JEOL, Tokyo, Japan) at
cycles (10 °C for one min, 25 °C for one min and 55 °C for one min) and
110 kV.
240,000 mechanical cycles, corresponding to one year of intraoral
For each beam, 5 images taken at 10,000× magnification were
functioning [25,26]. The resin-dentine specimens were sectioned in
analysed with the Scion Image software (Scion Corp., Frederick, MD)
both x and y directions across the adhesive interface to obtain beams
for the percentage area within the resin-dentine interface (i.e. the
with cross-sectional areas of approximately 0.9 mm × 0.9 mm using
adhesive layer and hybrid layer) occupied by the silver tracer. The
the ‘non-trimming’ version of the microtensile bond testing procedures
mean of the percentage of interface containing silver deposits from the
[27]. Simulated ageing was performed in a thermomechanical wear
5 images was used to represent the overall nanoleakage of a subgroup.
system (Model ER-37000; Erios, São Paulo, SP, Brazil). The four longest
Because the homogeneity of variance assumption of the data sets
beams were obtained from the two central slabs of each bonded tooth
derived from the 4 subgroups was violated, the interval data (in
for bond strength testing and one beam each was obtained from the two
percentages) were logarithmically transformed to satisfy both normal-
central slab of each bonded tooth for TEM. Hence, for each of the four
ity and equal variance assumptions. The transformed data were
subgroups, 80 beams from 20 teeth were used for bond strength testing
analysed with two-factor analysis of variance to examine the effects
and 10 beams from 5 teeth were used for TEM examination of the resin-
of adhesive application time and simulated ageing, and the interaction
dentine interface.
of those two factors on the nanoleakage results. Post-hoc pairwise
comparisons were performed using the Tukey statistic. Statistical
2.3. Microtensile bond strength
significance was set at α = 0.05.
The other five beams from each subgroup were completely demi-
Each beam from a subgroup (N = 20) was secured with cyanoacry-
neralised in 0.1 M formic acid/sodium formate (pH 2.5). The end point
late glue (Zapit; Dental Ventures of America, Corona, CA, USA) to a
of demineralisation was determined by drop-wise addition of a 10%
testing jig and stressed to failure under tension in a universal testing
potassium oxalate solution to the demineralisation medium, which
machine (Vitrodyne V1000; Liveco Inc., Burlington, VT, USA) at a
formed a white calcium oxalate precipitate when calcium ions were
cross-head speed of 1 mm/min. The tensile load at failure was recorded
present. Completely demineralised beams containing the resin-dentine
and divided by the measured cross-sectioned area of each beam to yield
interface were fixed with Karnovsky’s fixative (2.5 wt% glutaraldehyde
the tensile bond strength in megaPascals (MPa). The mean bond
and 2% paraformaldehyde in 0.1 mol/L cacodylate buffer; pH, 7.3) for
strength of the 4 beams derived from one tooth was used to represent
8 h, and post-fixed in 1% osmium tetroxide for 1 h. The fixed specimens
the tensile bond strength of that tooth, yielding 20 values per subgroup.
were dehydrated in an ascending ethanol series (30–100%), immersed
Data were analysed with parametric statistical methods after validating
in propylene oxide as a transition medium and ultimately embedded in
the normality (Shapiro-Wilk test) and homogeneity of variance (mod-
pure epoxy resin. Ninety-nanometre thick sections were prepared,
ified Levene test) assumptions of the data sets. A two-factor analysis of
stained with 2% aqueous uranyl acetate and Reynold’s lead citrate,
variance was used to examine the effects of adhesive application time
and examined using the JEM-1230 TEM at 110 kV.
(i.e. no-waiting or 10-s self-etch) and simulated ageing (i.e. without or
with thermomechanical cycling), and the interaction of those two
factors on the bond strength results. Post-hoc pairwise comparisons 2.5. Attenuated total reflection-Fourier transform infrared spectroscopy
were performed using the Tukey statistic. Statistical significance was set
at α = 0.05. A Nicolet 6700 spectrophotometer (Thermo Scientific Inc.,
After bond strength testing, the two ends of a fractured stick were Waltham, MA, USA) with an attenuated total reflection setup was used
retrieved and examined with 10× magnification using a stereoscopical to collect infrared spectra from dentine discs before self-etching
microscope to determine the mode of failure. Failure modes were (control), and after etching the mineralised dentine without waiting
classified as adhesive failure (failure along the adhesive interface), or for 10 s (N = 2 dentine discs). The universal adhesive was applied
mixed failure (failure within the adhesive joint with failure within the respectively to each disc without light-curing. The etched dentine
resin composite or dentine), or cohesive failure (failure within the resin surface was rinsed with absolute ethanol to completely remove the
composite or dentine). For statistical analysis, the number of specimens uncured adhesive prior to scanning. The rationale of adhesive dissolu-
exhibiting mixed and cohesive failures were combined into the non- tion was to prevent interference of the adhesive vibrational peaks with
adhesive category. Adhesive and non-adhesive failure modes in the 4 the peaks characteristic of mineralised dentine. Spectra were collected
subgroups (zero-second or 10-s etch; with or without thermomechani- between 4000 and 500 cm−1 at 4 cm−1 resolution using 32 scans. The
cal cycling) were arranged into a 2 × 4 contingency table and analysed spectra were superimposed after correction of their baseline shifts, so
with the Fisher-Freeman-Halton statistic [28]. A 95% confidence level that the intensities of the carbonated apatite-associated peaks could be
was used to determine if an association existed between the method in compared.

56
X.-q. Huang et al. Journal of Dentistry 62 (2017) 54–63

Table 2
2 × 4 contingency table showing the distribution of adhesive and non-adhesive (i.e.
mixed and cohesive combined) failures in the 4 subgroups.

No-waiting No-waiting 10-s 10-s Totals

self-etch self-etch self-etch self-etch
No TMC After TMC No TMC TMC

Adhesive 15 19 12 18 64
failure
Non-adhesive 5 1 8 2 16
failure
Totals 20 20 20 20 80

Abbreviation: TMC: thermomechanical cycling

Fisher-Freeman-Halton statistic
PA = 0.0354, where
PA: the probability of the observed array of cell frequencies plus the sum of the
probabilities of all other cell-frequency arrays (such as would be consistent with the
observed marginal totals) that are ≤ the probability of the observed array.
PB = 0.0291, where
PB: the probability of the observed array of cell frequencies plus the sum of the
probabilities of all other cell-frequency arrays (such as would be consistent with the
observed marginal totals) that are < the probability of the observed array.
Number of tables evaluated = 969
The chi-square test was not performed because more than 20% of the cells have an
expected frequency of less than 5.

Failure mode analysis for all subgroups indicates that more than
60% of the specimens exhibited adhesive failure, while the remaining
were mixed failure or cohesive failure in resin or dentine. Because only
a few incidents of cohesive failure were identified in all subgroups,
cohesive failures were combined with mixed failures in each subgroup
(i.e. non-adhesive failures) for statistical analysis. A 2 × 4 contingency
table depicting the frequency of adhesive and non-adhesive failures in
the beams derived from the 4 subgroups is shown in Table 2. The sum of
the probabilities of the observed array of cell frequencies, together with
the probabilities of all other cell-frequency arrays that are less than or
equal to the probability of the observed array (i.e. PA) is 0.0354. There
was a statistically significant association between the method in which
Fig. 1. Comparison of A. microtensile bond strength (in MPa), and B. nanoleakage (in
the specimens were bonded and tested, and the category of failure
percentages) of the universal adhesive (G-Premio Bond, GC Corp., Tokyo, Japan) bonded
to dentine using the no-waiting or the 10-s self-etching mode, with or without (P < 0.05).
thermomechanical cycling. For each chart, data represent mean values and standard The ultrastructure of the resin-dentine interface and representative
deviations. for the factor “adhesive application time”, subgroups labelled with different images of nanoleakage that occurred within the interface of specimens
letter superscripts (upper case for without cycling and lower case for after cycling) are bonded using the no-waiting self-etch mode are shown in Fig. 2 (no
significantly different (P < 0.01). For the factor “simulated ageing”, subgroups con- thermomechanical cycling) and Fig. 3 (after thermomechanical cy-
nected by a bar with asterisk are significantly different (P < 0.01).
cling). The hybrid layers were generally less than 200 nm thick in most
of the sections, spanning the width of two layers of collagen fibrils
3. Results (Fig. 2, left column). The thickness of the hybrid layer was uneven in
some sections and there were regions where the hybrid layer was very
Microtensile bond strengths of the four subgroups bonded with the thin or missing (Fig. 3, left column), with occasional remnants of the
universal adhesive (no-waiting self-etch, 10-s self-etch, with or without smear layer. Despite the application of a single coat of adhesive
thermomechanical cycling) are summarised in Fig. 1A. Bond strength (3–5 μm thick), the basal portion of the polymerised, filled adhesive
was significantly affected by the adhesive application time that approximated the hybrid layer and dentinal tubules was more
(P < 0.001) and whether simulated ageing was performed electron-dense after staining with the water-based heavy metal stains,
(P < 0.001). There was a statistically significant interaction between when compared with the coronal portion of the stained adhesive. In
the two factors (P = 0.001). For pairwise comparisons within the factor unstained, non-demineralised sections (right column of both figures),
adhesive application time, bond strength of the no-waiting self-etch nanoleakage was manifested as extensive patches of electron-dense,
subgroup (34.4 ± 3.9 MPa) was significantly lower (P < 0.001) than reticular silver deposits that were the most extensive at the base of the
the 10-s self-etch subgroup (50.2 ± 4.0 MPa) when thermomechanical adhesive layer above the hybrid layer. Specimens after thermomecha-
cycling was not performed. Likewise, bond strength of the no-waiting nical cycling exhibited almost continuous silver tracer infusion (Fig. 3)
self-etch subgroup (28.3 ± 4.1 MPa) was significantly lower along the base of the adhesive layer, when compared with specimens
(P < 0.001) than the 10-s self-etch subgroup (38.3 ± 3.0 MPa), after that had not been subjected to thermomechanical cycling (Fig. 2).
the specimens were thermomechanically cycled. In both the no-waiting Although extensive nanoleakage was observed, all the intensely air-
self-etch subgroup and the 10-s self-etch subgroup, the bond strength dried specimens (i.e. maximum air pressure for 5 s) were devoid of
decreased significantly after thermomechanical cycling (P < 0.001). water bubble entrapment within the adhesive layer. In contrast,
For pairwise comparisons within the factor simulated ageing, signifi- abundant silver-impregnated water bubbles could be identified from
cant differences were identified between the no-waiting and 10-s self- the thicker adhesive layers (> 10 μm thick) present in the pilot study
etch subgroups irrespective of whether thermomechanical cycling was specimens which were dried with gentle air pressure only (Supplemen-
performed (P < 0.001 for both pairwise comparisons). tary information Fig. S-1).

57
X.-q. Huang et al. Journal of Dentistry 62 (2017) 54–63

Fig. 2. Representative TEM images of resin-dentine interfaces created by the application of a universal, single component, light-cured adhesive to human coronal dentine using the no-
waiting self-etch mode, without thermomechanical cycling. Note that nanoleakage (right column) is most the extensive at the base of the adhesive layer above the hybrid layer.
Abbreviations: Afilled adhesive ; Between open arrowheadshybrid layer ; Cresin composite ; Ddemineralised dentine ; Mmineralised dentine ; Tdentinal tubule.

The ultrastructure of the resin-dentine interface and representative there was no significant difference in the nanoleakage expression from
images of nanoleakage that occurred within the interface in specimens specimens bonded with the two self-etch modes when thermomecha-
bonded using the 10-s self-etch mode are shown in Fig. 4 (no nical cycling was not performed (P = 0.906); however, there was
thermomechanical cycling) and Fig. 5 (after thermomechanical cy- significantly more nanoleakage in specimens bonded with the no-
cling). In stained, demineralised sections (left columns), electron-dense waiting self-etch mode after the specimen were thermomechanically
hybrid layers that were generally 500 nm thick could be identified. cycled (P = 0.015). For the factor simulated ageing, thermomechanical
Similar to the no-waiting self-etch subgroups, the adhesive layer was cycling had a significant influence on the extent of nanoleakage
approximately 3–6 μm thick. Although only a single coat of adhesive expression (P = 0.005). Pairwise comparisons indicated that nanoleak-
was applied, a lightly-stained upper layer and a more heavily-stained age of specimens bonded with the no-waiting self-etch mode were
lower layer were apparent after the polymerised adhesive was stained significantly higher after thermomechanical cycling (P = 0.012); by
with uranyl acetate and lead citrate. In unstained, non-demineralised contrast, nanoleakage in specimens bonded with the 10-s self-etch
sections (right columns), nanoleakage in both subgroups was mani- mode was not significantly increased after thermomechanical cycling
fested as discontinuous islands of reticular silver deposits within the (P = 0.083).
hybrid layer and the basal part of the adhesive layer. Fig. 6 shows superimposed infrared spectra generated from dentine
The percentage distributions of nanoleakage with the adhesive and discs without adhesive application (control), and after the adhesive was
hybrid layers of the four subgroups are depicted in Fig. 1B. The extent applied in the no-waiting or 10-s self-etch mode, followed by dissolu-
of nanoleakage was significantly affected by the adhesive application tion of the adhesive with ethanol. When no adhesive was applied, the
time (P = 0.006) as well as whether simulated ageing was performed mineralised dentine exhibited intense apatite-associated phosphate
(P = 0.004). There was no statistically significant interaction between bands at ∼1000–1100 cm−1 (υ3 asymmetric P-O stretch),
the two factors (P = 0.501). For the factor adhesive application time, ∼560–600 cm−1 (υ4 asymmetric O-P-O bending) and ∼960 cm−1
significant difference was identified between the no-waiting and the 10- (υ1 symmetric P-O stretch) [30,31]. Carbonate bands characteristic of
s self-etch modes (P = 0.006). Pairwise comparisons indicated that carbonated apatite could be detected at ∼875 cm−1 (υ2 out-of-plane

58
X.-q. Huang et al. Journal of Dentistry 62 (2017) 54–63

Fig. 3. Representative TEM images of resin-dentine interfaces created by the application of the universal adhesive to human coronal dentine using the no-waiting self-etching mode, after
thermomechanical cycling. Note that the thickness of the hybrid layer is uneven in some sections and there are regions where the hybrid layer is very thin or missing. Abbreviations: A:
filled adhesive; C: resin composite; D: demineralised dentine; H: hybrid layer; M: mineralised dentine; P: smear plug; T: dentinal tubule.

OeCeO bend) and between ∼1440–1550 cm−1 (υ3 asymmetric CeO self-etch adhesive and the thickness of the smear layer [32–34]. In the
stretch) [31]. The intensities of these bands decreased gradually with present work, 320-grit abrasive papers were used to create smear layers
increases in adhesive application time. with thickness approximating those created by tungsten carbide burs
[33]. Transmission electron microscopy of specimens in the no-waiting
self-etch, no thermomechanical cycling group (Fig. 2) showed that
4. Discussion there was inadequate etching of the smear layer-covered dentine by the
acidic monomers within the limited time frame. This probably
Significantly lower microtensile bond strengths were identified accounted for the lower microtensile bond strengths obtained with
when the adhesive was used in the no-waiting self-etch mode, when the no-waiting self-etch mode (Fig. 1A). The present result differs from
compared with using the universal adhesive in the 10-s self-etch mode. that supplied by the manufacturer http://www.gcamerica.com/
This warrants rejection of the first null hypothesis that “there is no products/operatory/G-Premio_BOND/GCA_G-Premio_BOND_SellSheet-
difference between the use of no-waiting self-etch and 10-s self-etch on 11-04-2016-iPad.pdf). The manufacturer claimed that there is no
the bond strength to dentine”. The use of simulated ageing resulted in difference in bonding performance between no-waiting (0-s) and 10-s
reduced bond strengths for the universal adhesive irrespective of self-etch mode. However, the manufacturer did not reveal details on the
application time. Thus, the second null hypothesis that “thermomecha- method of dentine preparation. For example, the thickness of the smear
nical cycling has no effect on the microtensile bond strength to dentine layer might have been different from that created in the present study.
when the universal adhesive is used in either the no-waiting or the 10-s Such an issue is important especially when adhesives are applied with
self-etch mode” also has to be rejected. reduced time [24]. G-Premio Bond is a moderately aggressive self-etch
Adhesives used in the self-etch mode are designed to bond to tooth adhesive because of its low pH value (1.5; Table 1) [24]. Even though
substrates by simultaneous etching and resin replacement of the the manufacturer advocates “no-waiting” in its Japanese version of
dissolved mineral component [12]. The ability of these adhesives to instructions, 2–3 s will have elapsed during the period of adhesive
etch through the smear layer and hybridise the underlying dentine is a application before the operator begins to evaporate the solvent. This
time-dependent process that depends on both the aggressiveness of the

59
X.-q. Huang et al. Journal of Dentistry 62 (2017) 54–63

Fig. 4. Representative TEM images of resin-dentine interfaces created by the application of the universal adhesive to human coronal dentine using the 10-s self-etch mode, without
thermomechanical cycling. Abbreviations: A: filled adhesive; C: resin composite; D: demineralised dentine; H: hybrid layer; M: mineralised dentine; P: smear plug; T: dentinal tubule.

provides a narrow window of opportunity for the adhesive to etch 1). Evaporation of acetone causes water derived from the adhesive or
through the smear layer and demineralise the underlying dentine to the underlying dentine to be trapped as water bubbles within the water-
produce a shallow and uneven hybrid layer (Fig. 3). When application immiscible adhesive components [38]. Evaporation of the adhesive
time was increased to 10 s, thicker and more uniform hybrid layers solvent with strong air-blasts removed the bulk of the interfacial water,
were observed (Fig. 4). Information derived from TEM examination was leaving adhesive layers free of water droplets (Figs. 2–5). Even when
also supported the infrared spectroscopy data (Fig. 6). The intensities of microscopical phase separation is eliminated with strong air-blow,
apatite-associated phosphate bands and carbonate bands decreased separation of the adhesive may occur at the nanoscale after solvent
with increases in adhesive application time. evaporation. Nanoscopical phase separation has been described as
Self-etch and universal adhesives contain hydrophilic and hydro- undesirable flaws in adhesive design that creates hydrophilic resinous
phobic resin monomer blends dissolved in miscible solvents [22]. When domains that are susceptible to expedited degradation by salivary
these water-containing adhesives are formulated without using 2- esterases [39,40]. In the present context, although only a single coat
hydroxyethyl methacrylate (HEMA) as a co-solvent, microscopical of adhesive was applied, a less electron-dense coronal adhesive zone
and even macroscopical phase separation of water-immiscible resin and a more electron-dense basal adhesive zone were apparent after the
monomers readily occurs after solvent evaporation [35]. For example, resin-dentine interface was stained with uranyl acetate and lead citrate
phase separation in G-Bond (GC Corporation) is manifested as large (Figs. 2–5). Application of polymer hydrogels for extraction of uranium
globules of water-immiscible resins that are randomly dispersed within from sea water as an environmental conservation measure sheds light
the adhesive layer; the phenomenon arises when the adhesive is used on the possible mechanism that produces this phenomenon. Uptake of
against the manufacturer’s recommendation in using strong air-blow uranyl cations is augmented as the hydrophilicity of the polymer
during the bonding procedures [36,37]. As the latest successor of G- increases, due to faster diffusion and adsorption of aqueous solutions
Bond, G-Premio Bond inherits the same feature and requires strong air- into a hydrophilic polymer, compared with a hydrophobic polymer
blow to reduce microscopical phase separation. Silver-filled water [41,42]. In the present context, a diffusion gradient for the uranyl
bubbles could be identified within the adhesive layer when only gentle acetate stain was probably created within the adhesive layer based on
air-blow was used in the pilot study (Supplementary information Fig. S- the differential quantity of hydrophilic resin components across the

60
X.-q. Huang et al. Journal of Dentistry 62 (2017) 54–63

Fig. 5. Representative TEM images of resin-dentine interfaces created by the application of the universal adhesive to human coronal dentine using a 10-s self-etching mode, after
thermomechanical cycling. Abbreviations: A: filled adhesive; C: resin composite; D: demineralised dentine; H: hybrid layer; M: mineralised dentine; P: smear plug; T: dentinal tubule.

hydrophobic resin components. This nanoscopical phase separation

phenomenon implies that phase separation not only occurs between
water and resin monomers, but also between resin monomers with
different degrees of hydrophilicity. This type of phase separation may
be more prominent in adhesive systems without HEMA as co-solvent to
improve miscibility of hydrophobic and hydrophilic components after
evaporation of the acetone solvent [43].
Ideally, volatile adhesive solvents should be completely evaporated
by air-blowing before light-curing. However, a previous study showed
that complete solvent evaporation cannot be achieved with less than
10 min of air-blowing [44]. Remnant water within the adhesive layer
was manifested as “water tree” nanoleakage expression in both the no-
waiting and the 10-s self-etch modes (Figs. 2 and 4) [45]. The extensive
silver uptake along the adhesive layer, irrespective of adhesive applica-
tion time, is likely attributed to fluid movement caused by evaporative,
Fig. 6. Representative infrared spectra collected from the surface of mineralised dentine
osmotic and convective water fluxes across dentine [46]. Although it is
discs without adhesive application (control), and from dentine discs that had been etched
with the universal adhesive without waiting or for 10 s. The adhesive was removed prior
necessary to remove solvents from adhesives before light-curing, the
to spectrum acquisition. The spectra were superimposed after correction of the baseline same air-blowing process induces outward evaporative water flux from
shifts, so that the intensities of the carbonated apatite-associated peaks may be compared. the smear-layer-covered dentine [47]. Moreover, acidic and ionic resin
monomers present in an adhesive may also induce osmotic water flux
thickness of the adhesive layer. The more heavily-stained adhesive zone from the underlying hydrated dentine [46]. The osmolatity of G-Premio
on top of hybrid layer probably contained more hydrophilic compo- Bond is 4600 milliosmoles/kg (mOsm/kg; unpublished results) com-
nents, whilst the less heavily-stained coronal zone contained more pared to that of plasma (290 mOsm/kg). Not surprisingly, once this

61
X.-q. Huang et al. Journal of Dentistry 62 (2017) 54–63

hypertonic adhesive is dispensed, it would extract water/dentinal fluid gical wave, it is prudent to recollect that the benefits associated with
from the host dentine into the adhesive, producing water trees within low hanging fruit style innovations are likely to be over-estimated. For
the polymerised adhesive [46]. Even in the presence of smear plugs innovation to take root in any technological advancement, it must focus
(Fig. 4), slow convective water flux permits water/dentinal fluid to on the long-term opportunities where unique ideas and value proposi-
permeate the relatively viscous adhesive following evaporation of the tions prevail. Whereas low hanging fruits are tempting and easy to pick,
adhesive solvent [48]. In addition, a thick/porous smear layer acts as a true rewards ultimately come from the willingness to climb higher and
water reservoir on the dentine surface that enables water accumulation stretch for the real treasure. In the context of dentine bonding, realistic
within the adhesive layer [49]. Collectively, these different types of challenges remain atop in the arena of improving bond durability. Only
water fluxes are responsible for the extensive nanoleakage associated when these challenges are met can users of dentine adhesives expect to
with the use of either self-etch mode. find real value from innovation.
Bond durability of adhesives is critical for clinical success of direct
and indirect bonded restorations. Thermal, mechanical or thermome- 5. Conclusion
chanical cycling may be conducted to simulate changes in materials
caused by intraoral functioning and ageing [50]. A recent study Within the limits of the present study, it may be concluded that
examined the long-term tensile bond strength of resin composite fillings sufficient short-term bond strength to dentine may be achieved using
performed in real life and under simulated ageing conditions to identify the universal adhesive investigated in the no-waiting self-etch mode
parameters that may be used to forecast the durability of resin-dentine technique prior to polymerisation of the adhesive. Nevertheless,
bonds [51]. Based on the results of that study, a thermomechanical extended application time with the 10-s self-etch mode improves its
cycling protocol consisting of 10,000 thermal cycles and 240,000 short-term bonding performance. In addition, thermomechanical cy-
mechanical cycles was used in the present study, corresponding to cling decreases the bond strength of the adhesive system when dentine
one year of intraoral functioning [30,31]. This dynamic testing is bonded with either of the two self-etch modes.
procedure also enables extraction of poorly-polymerised resin oligo-
mers from the resin-dentine interface [39]. This accounts for the Acknowledgements
increase in the propensity of silver impregnation along the interface
after thermomechanical cycling (Fig. 1B) [26]; the phenomenon was This work was supported by National High Technology Research
more pronounced when the adhesive was applied with reduced and Development Program of China grant 2015AA020942, National
application time. Nature Science Foundation of China grant 81400555 and Natural
Admittedly, it is not usual to test only one adhesive without Science Basic Research Plan in Shaanxi Province of China grant
comparing the bonding performance with other commercial adhesives 2015JM8383 (PI: Li-na Niu, The Fourth Military Medical University),
to minimise product bias. This may be regarded as the limitation of the National Nature Science Foundation of China grant 81500883 and
present study. An attempt was made to compare G-Premio Bond with grant 2015A030401035 from Guangdong Public Research and
two other universal adhesives (Supplementary information, Table S-1). Competence Special Fund Expenditure (PI: Xue-qing Huang, Sun Yat-
The results indicate that prior to thermomechanical cycling, the bond sen University).
strength for the G-Premio Bond used in the 10-s self-etch is not
significantly different from the other two commercially available Appendix A. Supplementary data
universal adhesives. Because G-Premio Bond is currently the only
product available in the market in which an adhesive is recommended Supplementary data associated with this article can be found, in the
by the manufacturer to be used in the no-waiting self-etch mode, the online version, at 10.1016/j.jdent.2017.05.007.
most appropriate solution to test the hypotheses in the present study
was to use G-Premio Bond in the 10-s self-etch mode as its own control. References
Revisit of this issue is necessary when similar products are available in
the future. Although the thermomechanical cycling regime employed in [1] P. Deng, Science and technology constitute a primary productive force, The Selected
present study has been reported to approximate 1 year of physiological Works of Deng Xiao Peng, (2008) (Accessed 04 December 2017), https://
dengxiaopingworks.wordpress.com/2013/03/18/science-and-technology-
ageing in the oral cavity, there is no literature to support that constitute-a-primary-productive-force/.
thermomechanical cycling alone is sufficient to activate matrix-bound [2] N.V. Babu, R. Joseph, Dr. michael buonocore – ‘Adhesive dentistry – 1955', J.
matrix metalloproteinases and cysteine cathepsins present in human Conserv. Dent. 8 (2005) 43–44.
[3] C. Mason, E. Manzotti, Induced pluripotent stem cells: an emerging technology
dentine [52]. Hence, in-situ zymography as well as long-term water- platform and the Gartner hype cycle, Regen. Med. 4 (2009) 329–331.
ageing studies are required to evaluate the resistance of resin-dentine [4] A.T. Johnson, The technology hype cycle, IEEE. Pulse. 6 (2015) 50.
interfaces created by the two self-etch modes to degradation by [5] A.A. Ronfard, M.H. Vertès, A. May, M.E. Dupraz, Y. Bayon van Dyke, Evaluating the
past, present, and future of regenerative medicine: a global view, Tissue Eng. Part B
adhesive-activated endogenous proteases [53]. Rev. 23 (2017) 199–210.
Beginning with the acid-etching trigger that was initiated more than [6] T. Fusayama, M. Nakamura, N. Kurosaki, M. Iwaku, Non-pressure adhesion of a new
six decades ago, technological developments in dentine bonding have adhesive restorative resin, J. Dent. Res. 58 (1979) 1364–1370.
[7] D.H. Pashley, F.R. Tay, L. Breschi, L. Tjäderhane, R.M. Carvalho, M. Carrilho,
witnessed a “peak of inflated expectations” and subsequently a “trough
A. Tezvergil-Mutluay, State of the art etch-and-rinse adhesives, Dent. Mater. 27
of disillusionment”, before reaching the “slope of enlightenment” and (2011) 1–16.
eventually the “plateau of productivity”. These phenomena are just [8] J. Kanca, Improved bond strength through acid-etching of dentin and bonding to
about true of the progress of any major technology, as summarised by wet dentin surfaces, J. Am. Dent. Assoc. 123 (1992) 35–43.
[9] R. Bertolotti, Total etch total seal, total success, Dent. Dimens. 23 (1990) 3–4.
the Amara’s law, which states that “we tend to overestimate the effect of a [10] D.H. Pashley, The effects of acid-etching on the pulpodentin complex. Buonocore
technology in the short run and underestimate the effect in the long run” Memorial Lecture, 1992, Oper. Dent. 17 (1992) 229–242.
[54]. Dentine bonding has progressed to the stage where it normalises [11] E. Asmussen, E.C. Munksgaard, Adhesion of restorative resins to dentinal tissues, in:
G. Vanherle, D.C. Smith (Eds.), Posterior Composite Resin Dental Restorative
with best clinical practices and becomes an expectation. It is thought- Materials, Peter Szule Publishing Co., 1985, p. 228.
provoking that the surge for cutting application time to increase user [12] B. Van Meerbeek, J. De Munck, Y. Yoshida, S. Inoue, M. Vargas, P. Vijay, K. Van
friendliness remains the most frequently sought conduit for advance- Landuyt, P. Lambrechts, G. Vanherle, Buonocore memorial lecture. Adhesion to
enamel and dentin: current status and future challenges, Oper. Dent. 28 (2003)
ment of this technology. The pursuit of low hanging fruit represents a 215–235.
manufacturer’s business obligation to fulfil consumers’ demands, and is [13] F.R. Tay, D.H. Pashley, Have dentin adhesives become too hydrophilic? J. Can.
brought to dénouement with the advent of the so-called no-waiting Dent. Assoc. 69 (2003) 726–731.
[14] F.R. Tay, D.H. Pashley, B.I. Suh, R.M. Carvalho, A. Itthagarun, Single-step adhesives
dentine self-etch concept. Prior to diving head first into this technolo-

62
X.-q. Huang et al. Journal of Dentistry 62 (2017) 54–63

are permeable membranes, J. Dent. 30 (2002) 371–382. micro-and nanophase separation of dentin bonding agents by stereoscopy and
[15] Y.L. Liu, L. Tjäderhane, L. Breschi, A. Mazzoni, N. Li, J. Mao, D.H. Pashley, F.R. Tay, atomic force microscopy, Microsc. Microanal. 18 (2012) 279–288.
Limitations in bonding to dentin and experimental strategies to prevent bond [36] K.L. Van Landuyt, J. De Munck, J. Snauwaert, E. Coutinho, A. Poitevin, Y. Yoshida,
degradation, J. Dent. Res. 90 (2011) 953–968. S. Inoue, M. Peumans, K. Suzuki, P. Lambrechts, B. Van Meerbeek, Monomer-
[16] L. Tjäderhane, Dentin bonding: can we make it last? Oper. Dent. 40 (2015) 4–18. solvent phase separation in one-step self-etch adhesives, J. Dent. Res. 84 (2005)
[17] F. Abedin, B. Roughton, Q. Ye, P. Spencer, K. Camarda, Computer-aided molecular 183–188.
design of water compatible visible light photosensitizers for dental adhesive, Chem. [37] S. Sauro, F. Mannocci, M. Toledano, R. Osorio, I. Thompson, T.F. Watson, Influence
Eng. Sci. 159 (2017) 131–139. of the hydrostatic pulpal pressure on droplets formation in current etch-and-rinse
[18] B. Van Meerbeek, K. Yoshihara, Y. Yoshida, A. Mine, J. De Munck, K.L. Van and self-etch adhesives: a video rate/TSM microscopy and fluid filtration study,
Landuyt, State of the art of self-etch adhesives, Dent. Mater. 27 (2011) 17–28. Dent. Mater. 25 (2009) 1392–1402.
[19] G. Alex, Universal adhesives: the next evolution in adhesive dentistry? Compend. [38] K.L. Van Landuyt, J. Snauwaert, J. De Munck, E. Coutinho, A. Poitevin, Y. Yoshida,
Contin. Educ. Dent. 36 (2015) 15–26. K. Suzuki, P. Lambrechts, B. Van Meerbeek, Origin of interfacial droplets with one-
[20] H. Xie, Q. Li, F. Zhang, Y. Lu, F.R. Tay, M. Qian, C. Chen, Comparison of resin step adhesives, J. Dent. Res. 86 (2007) 739–744.
bonding improvements to zirconia between one-bottle universal adhesives and [39] Q. Ye, Y. Wang, P. Spencer, Nanophase separation of polymers exposed to simulated
tribochemical silica coating which is better? Dent. Mater. 32 (2016) 403–411. bonding conditions, J. Biomed. Mater. Res. B Appl. Biomater. 88 (2009) 339–348.
[21] K. Yoshihara, N. Nagaoka, A. Sonoda, Y. Maruo, Y. Makita, T. Okihara, M. Irie, [40] F. Abedin, Q. Ye, R. Parthasarathy, A. Misra, P. Spencer, Polymerization behavior of
Y. Yoshida, B. Van Meerbeek, Effectiveness and stability of silane coupling agent hydrophilic-rich phase of dentin adhesive, J. Dent. Res. 94 (2015) 500–507.
incorporated in ‘universal' adhesives, Dent. Mater. 32 (2016) 1218–1225. [41] S. Ören, T. Çaykara, Ö. Kantoglu, O. Güven, Effect of pH, ionic strength, and
[22] C. Chen, L.N. Niu, H. Xie, Z.Y. Zhang, L.Q. Zhou, K. Jiao, J.H. Chen, D.H. Pashley, temperature on uranyl ion adsorption by poly (N-vinyl 2-pyrrolidone-g-tartaric
F.R. Tay, Bonding of universal adhesives to dentine—old wine in new bottles? J. acid) hydrogels, J. Appl. Polym. Sci. 78 (2000) 2219–2226.
Dent. 43 (2015) 525–536. [42] G.S. Chauhan, A. Kumar, A study in the uranyl ions uptake on acrylic acid and
[23] Z.Y. Zhang, F.C. Tian, L.N. Niu, K. Ochala, C. Chen, B.P. Fu, X.Y. Wang, acrylamide copolymeric hydrogels, J. Appl. Polym. Sci. 110 (2008) 3795–3803.
D.H. Pashley, F.R. Tay, Defying ageing: an expectation for dentine bonding with [43] K.L. Van Landuyt, J. Snauwaert, M. Peumans, J. De Munck, P. Lambrechts, B. Van
universal adhesives? J. Dent. 45 (2016) 43–52. Meerbeek, The role of HEMA in one-step self-etch adhesives, Dent. Mater. 24 (2008)
[24] P. Saikaew, A.F. Chowdhury, M. Fukuyama, S. Kakuda, R.M. Carvalho, H. Sano, The 1412–1419.
effect of dentine surface preparation and reduced application time of adhesive on [44] A.F. Reis, M.T. Oliveira, M. Giannini, M.F. De Goes, F.A. Rueggeberg, The effect of
bonding strength, J. Dent. 47 (2016) 63–70. organic solvents on one-bottle adhesives’ bond strength to enamel and dentin, Oper.
[25] F. Lutz Krejci, In-vitro test results of the evaluation of dental restoration systems Dent. 28 (2003) 700–706.
Correlation with in-vivo results, Schweiz. Monatsschr. Zahnmed. 100 (1990) [45] F.R. Tay, D.H. Pashley, Water treeing—a potential mechanism for degradation of
1445–1449. dentin adhesives, Am. J. Dent. 16 (2003) 6–12.
[26] G.C. Martins, A. Sánchez-Ayala, P.H. D'Alpino, A.L. Calixto, J.C. Gomes, [46] F.R. Tay, D.H. Pashley, B.I. Su, N. Hiraishi, C.K.Y. Yiu, Water treeing in simplified
O.M. Gomes, Interfacial integrity of bonded restorations with self-etching adhe- dentin adhesives-deja vu? Oper. Dent. 30 (2005) 561–579.
sives: water storage and thermo-mechanical cycling, Eur. J. Dent. 6 (2012) [47] M. Hashimoto, S. Ito, F.R. Tay, N.R. Svizero, H. Sano, M. Kaga, D.H. Pashley, Fluid
169–177. movement across resin-dentin interface during and after bonding, J. Dent. Res. 83
[27] D.H. Pashley, R.M. Carvalho, H. Sano, M. Nakajima, M. Yoshiyama, Y. Shono, (2004) 843–848.
C.A. Fernandes, F.R. Tay, The microtensile bond test: a review, J. Adhes. Dent. 1 [48] F.R. Tay, D.H. Pashley, M.C. Peters, Adhesive permeability affects composite
(1999) 299–309. coupling to dentin treated with a self-etch adhesive, Oper. Dent. 28 (2003)
[28] S. Lydersen, V. Pradhan, P. Senchaudhuri, P. Laake, Choice of test for association in 610–621.
small sample unordered r x c tables, Stat. Med. 26 (2007) 4328–4343. [49] M.H. Mahdan, M. Nakajima, R.M. Foxton, J. Tagami, Combined effect of smear
[29] F.R. Tay, D.H. Pashley, M. Yoshiyama, Two modes of nanoleakage expression in layer characteristics and hydrostatic pulpal pressure on dentine bond strength of
single-step adhesives, J. Dent. Res. 81 (2002) 472–476. HEMA-free and HEMA-containing adhesives, J. Dent. 41 (2013) 861–871.
[30] A. Destainville, E. Champion, D. Bernache-Assollant, E. Laborde, Synthesis, [50] L. Mair, P. Padipatvuthikul, Variables related to materials and preparing for bond
characterization and thermal behaviour of apatitic tricalcium phosphate, Mater. strength testing irrespective of the test protocol, Dent. Mater. 26 (2010) e17–23.
Chem. Phys. 80 (2003) 269–277. [51] H.M. Rêgo, T.S. Alves, E. Bresciani, L.N. Niu, F.R. Tay, C.R. Pucci, Can long-term
[31] M. Markovic, B.O. Fowler, M.S. Tung, Preparation and comprehensive character- dentine bonding created in real life be forecasted by parameters established in the
ization of a calcium hydroxyapatite reference material, J. Res. Natl. Inst. Stand. laboratory? Sci. Rep. 6 (2016) 37799.
Technol. 109 (2004) 553–568. [52] P.M. Scaffa, L. Breschi, A. Mazzoni, C.M. Vidal, R. Curci, F. Apolonio, P. Gobbi,
[32] F.R. Tay, D.H. Pashley, Aggressiveness of contemporary self-etching systems. I: D.H. Pashley, L. Tjäderhane, I.L. Tersariol, F.D. Nascimento, M.R. Carrilho, Co-
depth of penetration beyond dentin smear layers, Dent. Mater. 17 (2001) 296–308. distribution of cysteine cathepsins and matrix metalloproteases in human dentin,
[33] S.S. Oliveira, M.K. Pugach, J.F. Hilton, L.G. Watanabe, S.J. Marshall, G.W. Marshall Arch. Oral Biol. 74 (2017) 101–107.
Jr., The influence of the dentin smear layer on adhesion: a self-etching primer vs. a [53] R. Seseogullari-Dirihan, F. Apollonio, A. Mazzoni, L. Tjaderhane, D.H. Pashley,
total-etch system, Dent. Mater. 19 (2003) 758–767. L. Breschi, A. Tezvergil-Mutluay, Use of crosslinkers to inactivate dentin MMPs,
[34] J. De Munck, K. Van Landuyt, M. Peumans, A. Poitevin, P. Lambrechts, M. Braem, Dent. Mater. 32 (2016) 423–432.
B. Van Meerbeek, A critical review of the durability of adhesion to tooth tissue: [54] Encyclopedia. Definition Of: Amara's Law, PC Magazine, 2017 (Accessed 04
methods and results, J. Dent. Res. 84 (2005) 118–132. November 2017).
[35] M. Toledano, M. Yamauti, E. Osorio, F. Monticelli, R. Osorio, Characterization of

63