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Hospitals, King’s College London, Floor 25, London Bridge, London SE1-9RT, UK
c Instrumental Analysis Research Center, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo
113-8549, Japan
a r t i c l e i n f o a b s t r a c t
Article history: Objectives. Smear layer deproteinizing was proved to reduce the organic phase of smear layer
Received 28 May 2017 covered on dentin surface. It was shown to eliminate hybridized smear layer and nanoleak-
Received in revised form age expression in resin–dentin bonding interface of two-step self-etch adhesive. This study
8 October 2017 aimed to investigate those effects on various one-step self-etch adhesives.
Accepted 24 November 2017 Methods. Four different one-step self-etch adhesives were used in this study; SE One (SE),
ScotchbondTM Universal (SU), BeautiBond Multi (BB), and Bond Force (BF). Flat human dentin
surfaces with standardized smear layer were prepared. Smear layer deproteinizing was car-
Keywords: ried out by the application of 50 ppm hypochlorous acid (HOCl) on dentin surface for 15 s
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Smear layer deproteinizing followed by Accel (p-toluenesulfinic acid salt) for 5 s prior to adhesive application. No sur-
Hypochlorous acid face pretreatment was used as control. Microtensile bond strength (TBS) and nanoleakage
One-step self-etch adhesive under TEM observation were investigated. The data were analyzed by two-way ANOVA and
Microtensile bond strength Tukey’s post-hoc test and t-test at the significant level of 0.05.
Nanoleakage Results. Smear layer deproteinizing significantly improved TBS of SE, SU, and BB (p < 0.001).
Smear layer Hybridized smear layer observed in control groups of SE, BB, and BF, and reticular nanoleak-
Hybridized smear layer age presented throughout the hybridized complex in control groups of BB and BF were
Monomer infiltration eliminated upon the smear layer deproteinizing.
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Significance. Smear layer deproteinizing by HOCl and Accel application could enhance the
quality of dentin for bonding to one-step self-etch adhesives, resulting in the improving
∗
Corresponding author at: Faculty of Dentistry, Bangkokthonburi University, 16/10 Leabklongtaweewatana Rd. Khet Taweewatana,
Bangkok 10170, Thailand.
E-mail address: ornnicha.tha@bkkthon.ac.th (O. Thanatvarakorn).
1
Faculty of Dentistry, Bangkokthonburi University, 16/10 Leabklongtaweewatana Rd., Khet Taweewatana, Bangkok 10170, Thailand.
2
Department of Restorative Dentistry and Periodontology, Faculty of Dentistry, Chiang Mai University, T. Suthep, A. Muang, Chiang Mai
50200, Thailand.
3
Department of Operative Dentistry and Endodontics, Faculty of Dentistry, Mahidol University, Phayathai, Rajthewee, Bangkok 10400,
Thailand.
https://doi.org/10.1016/j.dental.2017.11.023
0109-5641/© 2018 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) 434–441 435
TBS, eliminating hybridized smear layer and preventing reticular nanoleakage formation
in resin–dentin bonding interface.
© 2018 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
2.2. Microtensile bond strength (TBS) test staining technique (n = 2 per subgroup). The specimens were
cut into two 0.9-mm thick slabs perpendicular to the bonded
Each bonded specimen was sectioned parallel to the long interface from the center of the tooth. After nail varnish
axis of the tooth into beams with a bonded surface area of coating, leaving a 1-mm window around the bonded inter-
1.0 ± 0.1 mm2 using a slow-speed water-cooled diamond saw face, the slabs were immersed in ammoniacal silver nitrate
(Isomet, Buehler Ltd., Lake Bluff, IL, USA). Four beams at the solution for 24 h, rinsed thoroughly with distilled water, and
center of each bonded specimen were subjected to TBS test- then immersed in photo-developing solution for 8 h under
ing in a universal testing machine (EZ test; Shimadzu, Kyoto, a fluorescent light. Specimen fixation was achieved by pro-
Japan) at a crosshead speed of 1 mm/min. The data were ana- cessing through Karnovsky’s solution, osmium tetroxide, and
lyzed using two-way ANOVA, post-hoc Tukey HSD test, and dehydrated in ascending ethanol series (50–100%) before
t-test with the significant level of 0.05. Statistical analysis was embedding in epoxy resin [25]. Using an ultramicrotome and
carried out using SPSS version 22.0 (SPSS, Chicago, IL, USA). a diamond knife (Diatome Ltd., Bienne, Switzerland), 70-nm
thick ultrathin sections were prepared and collected on 150-
mesh copper grids. The nanoleakage expression of the bonded
2.3. Failure mode analysis
interface was examined using a TEM operating at 75 kV.
After the TBS test, both the dentin and composite surfaces of
the fractured specimens underwent a serial dehydration pro- 3. Results
cess and were observed using a scanning electron microscope
(SEM; JSM-5310LV, JEOL, Tokyo, Japan) for failure mode deter- 3.1. Microtensile bond strength (TBS) test
mination. The predominant failure over 80% of entire surface
area was considered and was classified as one of the following; The results of the TBS test are summarized in Table 2. Two-
cohesive within dentin, adhesive at dentin/adhesive inter- way ANOVA revealed that TBS was influenced by smear layer
face, adhesive at adhesive/resin composite interface, cohesive deproteinizing (p < 0.001) and by type of adhesive (p < 0.001).
within resin composite, or mixed (adhesive and cohesive fail- There was a significant interaction between two independent
ure occurred). Failure modes were analyzed for statistically variables (p = 0.028). Smear layer deproteinizing with HOCl
significant differences by the non-parametric Pearson chi- solution could significantly improve TBS of SE, SU, and BB
square test at a significant level of 0.05. (p < 0.05), but not for BF (p > 0.05).
Additional specimens prepared as described above, using The percentage of failure modes in each group is summarized
low-viscosity composite (Protect Liner F; Kuraray Noritake in Fig. 1. There were significant differences in failure mode
Dental Inc., Tokyo, Japan), were subjected to a silver nitrate distribution among the groups (p < 0.05). For SE, SU, and BB,
d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) 434–441 437
Fig 1 – Bar graph shows percentage of failure modes in each group. Chi-square test revealed significant differences in failure
mode distribution among the groups (p < 0.05). Comparing the control and smear layer deproteinizing groups, only BB
showed significant differences (decrease in adhesive failure at dentin/adhesive interface) (p = 0.011), whereas it was not
significantly different in other adhesives (p > 0.05). The failure mode of BF was predominantly adhesive failure at
adhesive/resin composite interface, irrespective of surface pretreatment. CR: cohesive failure within resin composite; Ad
(ad/com): adhesive failure at adhesive/resin composite interface; Ad (dent/ad): adhesive failure at dentin/adhesive interface;
CD: cohesive failure within dentin; Mixed: mixed failure.
failure at dentin/adhesive interface or mixed-failure mainly hybrid layer, whereas in SU, nanoleakage was hardly exhib-
occurred. On the other hand, the BF specimens predomi- ited. (Fig. 2E–H)
nantly failed at the adhesive/resin composite interface. For
SE, SU and BF, there were no significant differences in failure
mode distribution between the control and smear layer depro- 4. Discussion
teinizing groups (p > 0.05). For BB, failure at dentin/adhesive
interface significantly decreased in the smear layer depro- The results of this study showed that smear layer deproteiniz-
teinizing group (p = 0.011). ing could significantly improve bond strength in SE, SU and BB,
eliminate the hybridized smear layer in SE, BB and BF, as well
3.3. Nanoleakage evaluation as eliminate reticular nanoleakage in BB and BF. Thus, the null
hypothesis can be rejected.
In the control groups, TEM micrographs of the resin–dentin NaOCl solution can effectively dissolve organic substrates
interfaces of SE, BB and BF showed the presence of hybridized from biological materials. NaOCl dissociates to Na+ and OCl−
smear layers, whereas the SU group exhibited only a thin and establishes HOCl in water. The proportion of HOCl and the
authentic hybrid layer (approximately 0.15 m) with the OCl− in the solution depends on the pH [26,27]. That is, above
absence of a hybridized smear layer. For BB and BF, retic- pH 9, almost all HOCl ionizes to OCl− , and at an approximate
ular nanoleakage patterns were observed throughout the pH of 6, the concentration of HOCl is optimal and its dissocia-
hybridized complex, whereas for SE and SU, spotted pat- tion is minimal [26,28]. The OCl− ion is the determining factor
terns of nanoleakage were observed at the adhesive interface for proteolytic activity, because OCl− exerts the chlorination
(Fig. 2A–D) to protein, giving chloramines, which are then broken down
In the smear layer deproteinizing groups, there was no into nitrogen-centered radicals and induce the fragmentation
hybridized smear layer in all the adhesives. Nanoleakage in SE, of proteins [29]. Additionally, OCl− ion is capable of infiltrat-
BB, and BF was observed as a spotted pattern in the authentic ing mineralized collagen because of its small size [30]. On
No HOCl 15 s and
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pretreatment Accel 5 s
(control)
SE 54.2 (7.1)A,1 63.2 (9.3)a,2
SU 64.2 (8.0)B,1 72.8 (8.6)b,2
BB 22.2 (2.6)C,1 30.9 (5.0)c,2
BF 29.8 (4.7)D,1 32.1 (7.0)c,1
Significant differences in each column were represented by the different superscript letters. Significant differences in each row were represented
by the different superscript numbers.
438 d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) 434–441
Fig. 2 – TEM micrographs revealed the hybridized smear layer (arrows) in the control groups of SE (A), BB (C), and BF (D).
Reticular nanoleakage (hand pointers) was expressed within the hybridized complex in the control groups of BB and BF,
whereas it was spotted nanoleakage (arrow heads) in SE and SU (B). Upon smear layer deproteinization, the hybridized
smear layer and reticular nanoleakage were eliminated. Only spotted nanoleakage was seen in SE (E), BB (G), and BF (H).
d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) 434–441 439
the other hand, the “top-down” removal of the organic phase eral phase, leading to elimination of hybridized smear layer
from the mineralized dentin by NaOCl is diffusion-controlled [33]. For SE, smear layer deproteinizing could eliminate the
and is both time- and concentration-dependent [30]. When hybridized smear layer by the organic dissolubility of HOCl
exposed to 2% NaOCl for 4 h, dentinal erosion was not evi- solution. Interestingly, there were no obvious differences in
dent by TEM observation, as indicated by the presence of an nanoleakage expression within the hybrid layer between the
intact dentin surface, and dentinal tubules were of normal control and smear layer deproteinizing groups in both SE and
dimension with barely visible lateral branches of the tubules SU, but smear layer deproteinizing could significantly increase
[30]. In this study, a 50 ppm HOCl solution (pH = 7.0) by acidify- the TBS of both adhesives to dentin. These results indicate
ing NaOCl with HCl was used as a smear layer deproteinizing that smear layer deproteinizing could enhance the dentin
agent. In this pH condition, most of chlorine exists as HOCl, bonding performance of SE and SU.
where OCl− is minor species [29]. The Cl atom in HOCl behaves For BB and BF in the control groups, there were severe retic-
as Cl+ , which is a strong electrophile. Upon contacting to ular nanoleakage formations within the hybrid layers. This
collagen, amino groups within the collagen triple helix, which pattern of nanoleakage indicated the area of water retained
are susceptible to electrophilic attack of Cl+ , are chlorinated within the hybrid and adhesive layers, originating from poor
and fragmentation occurs [31]. The dissolution of organic monomer infiltration [34,35]. On the other hand, smear layer
phase in the mineralized dentin by HOCl solution would be deproteinizing could improve nanoleakage expression with
influenced by diffusion of HOCl molecule from the smear an absence of reticular pattern nanoleakage, eliminating the
layer-covered dentin surface, which is dependent upon the hybridized smear layer. These results indicate that disso-
concentration and the application time. The 15 s treatment lution/removal of the organic phase of smear layer could
of 50 ppm HOCl solution has a comparable organic dissolu- facilitate monomer infiltration into the underlying dentin.
tion ability to dentin surface to the same time treatment of 6% Additionally, TBS of BB to dentin significantly increased with
NaOCl solution, in which the mineral to matrix ratio increased the smear layer deproteinizing. On the other hand, for BF,
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at the smear layer-covered dentin surface [15] without the HOCl and Accel pretreatment could not improve the TBS
appreciable morphological alterations at smear layer-covered to dentin, in which the specimens predominantly failed at
dentin surface on SEM images [16,17]. Presumably, the organic the interface between adhesive/resin composite. In a pre-
dissolution on smear layer-covered dentin surface by 50 ppm vious study, it was reported that BF often exhibited a gap
HOCl solution in this study, would be limited in the smear between the composite and the adhesive layer at the cavity
layer and not extend to underlying mineralized collagen due floor in a Class I cavity [36]. BF contains HEMA/water and
to lower concentration of chlorine (50 ppm) and shorter appli- utilizes isopropyl alcohol as an organic solvent. HEMA is a
cation time (15 s). hydrophilic monomer and attracts water, which cannot be
Smear layer deproteinizing by 50 ppm HOCl for 15 or 30 s completely removed from the adhesive layer by air-drying.
could reduce reticular nanoleakage formation and eliminate Moreover, isopropyl alcohol makes evaporation more diffi-
hybridized smear layer at the adhesive interface with two-step cult by air-drying than acetone and ethanol [37]. Therefore,
self-etch adhesive, Clearfil SE Bond [15]. However, in our pilot in BF, the oxygen inhibition layer on the light-cured adhesive
study using the one-step self-etch adhesive, SE, the reticu- layer would include larger amounts of residual water/solvents,
lar pattern of nanoleakage within the hybrid layer severely which would impair co-polymerization with the hydrophobic
increased (not shown). Generally, it is more difficult for one- resin composite. This might be a reason why in the case of
step self-etch adhesives to polymerize than two-step self-etch BF, improvement of the quality of hybrid layer by smear layer
adhesives because water/solvent in one-step self-etch adhe- deproteinizing could not lead to significant increase of TBS
sives cannot be completely removed from the adhesive layer, value.
which hampers the polymerization process [32]. The results of Larger amount of nanoleakage can increase fluid pen-
the pilot study would be due to the interference of monomer etration within the hybrid layer and facilitate hydrolytic
polymerization of SE with the remaining oxidizing effect on degradation at the adhesive interface over time [38]. Therefore,
the HOCl-pretreated dentin surface. Therefore, in this study, the reduction of nanoleakage formation with the elimination
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application with the reducing agent, Accel , was undertaken of hybridized smear layer by the application of deproteiniz-
in order to neutralize the oxidizing effect on the HOCl-treated ing agent would lead to an improvement in the long-term
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dentin surface. The subsequent application of Accel could durability of dentin bond of one-step self-etch adhesives. In
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reduce nanoleakage formation at the adhesive interface with this study, Accel containing p-toluenesulfinic acid sodium
SE, compared with the single pretreatment of HOCl solution. salt, which was introduced as a pretreatment with adhe-
In the no-pretreatment control groups, SE and SU produced sive root canal sealer after endodontic irrigation with NaOCl,
hybrid layers of superior quality as the reticular pattern of was used to reverse the residual oxidizing effect on HOCl-
nanoleakage was absent, but only for SU, whose hybridized pretreated dentin. The salt of p-toluenesulfinic acid was
smear layer could not be observed. It should be noted that reported to accelerate the polymerization of methyl methacry-
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SU created a very thin authentic hybrid layer (approximately late [39], therefore pretreatment of Accel might promote
0.15 m) due to the higher pH of the SU adhesive (pH = 2.7). polymerization of the adhesive on dentin surface, leading to
Generally, the higher the adhesive pH, the lower the deminer- improvement of long-term bonding performance of one-step
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alization ability of the smear layer and the underlying dentin. self-etch adhesives to dentin [40]. However, Accel might lose
Thus, the scrubbing technique is required on the application its polymerization-accelerating ability in this study on HOCl-
procedure of SU in order to facilitate the removal of the organic pretreated dentin due to the neutralization with oxidants via
phase of the smear layer as well as dissolution of the min- free radical quenching mechanism. Further researches are
440 d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) 434–441
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