polymers
ArticleSodium p-Toluenesulfinate Enhances the Bonding Durability of
Universal Adhesives on Deproteinized Eroded Dentin
Yorichika Shioya 1 , Antonin Tichy 2, * , Kazuhide Yonekura 3 , Mayu Hasegawa 1 , Takashi Hatayama 1 ,Masaomi Ikeda 4 , Junji Tagami 1 , Masatoshi Nakajima 1 and Keiichi Hosaka 3, *
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Citation: Shioya, Y.; Tichy, A.;
Yonekura, K.; Hasegawa, M.;
Hatayama, T.; Ikeda, M.; Tagami, J.;
Nakajima, M.; Hosaka, K. Sodium
p-Toluenesulfinate Enhances theBonding Durability of Universal
Adhesives on Deproteinized Eroded
Dentin. Polymers 2021, 13, 3901.
https://doi.org/10.3390/polym13223901
Academic Editor:
Evangelia Vouvoudi
Received: 7 October 2021
Accepted: 8 November 2021
Published: 11 November 2021

Department of Cariology and Operative Dentistry, Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, Tokyo 113-8510, Japan;
shioya.ope@tmd.ac.jp (Y.S.); has.ope@tmd.ac.jp (M.H.); hatayama.ope@tmd.ac.jp (T.H.);tagami.ope@tmd.ac.jp (J.T.); nakajima.ope@tmd.ac.jp (M.N.)
Institute of Dental Medicine, First Faculty of Medicine of the Charles University and General University
Hospital in Prague, Karlovo namesti 32, 121-11 Prague, Czech Republic
Department of Regenerative Dental Medicine, Tokushima University Graduate School of Biomedical Sciences,
3-18-15, Kuramotocho, Tokushima 770-8504, Japan; k.yonekura@tokushima-u.ac.jp
Department of Oral Prosthetic Engineering, Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan;
ikedsdt@tmd.ac.jp
Correspondence: antonin.tichy@lf1.cuni.cz (A.T.); hosaka@tokushima-u.ac.jp (K.H.);
Tel.: +420-224-966-805 (A.T.); +81-088-633-7339 (K.H.)

Abstract: The effects of deproteinization using sodium hypochlorite (NaOCl) and the subsequent application of an antioxidant (sodium p-toluenesulfinate, STS) onto the bonding durability of universal
adhesives on eroded dentin were investigated. Untreated sound dentin served as the control, whereas
eroded dentin, which had been prepared by pH-cycling in 1% citric acid and a remineralization
solution, was either untreated, deproteinized with a 10% NaOCl gel or deproteinized with the 10%
NaOCl gel and subsequently treated with an STS-containing agent. The dentin surfaces were bonded
using a universal adhesive (Clearfil Universal Bond Quick, Scotchbond Universal or G-Premio Bond),
and the micro-tensile bond strength (µTBS) test was performed after 24 h or 10,000 thermal cycles.
The µTBS data were statistically analyzed using a three-way ANOVA and Tukey’s HSD post hoc
tests. The lowest µTBS was measured on untreated eroded dentin (p < 0.001). Deproteinization oferoded dentin resulted in µTBS similar to untreated sound dentin (p > 0.05), but the highest µTBS was
obtained if deproteinization was followed by the application of STS. Thermocycling significantly decreased µTBS in all groups (p < 0.001), except for STS-treated, deproteinized, eroded dentin (p > 0.05).
This indicated that deproteinization, followed by the application of STS, could enhance the bonding
durability of universal adhesives on eroded dentin.
Keywords: antioxidant; dental adhesives; sodium hypochlorite; tooth erosion

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1. Introduction
While the incidence of caries in developed countries tends to decrease, dental erosion
becomes increasingly frequent [1]. The prevalence of dental erosion in permanent dentitionis currently estimated to be 20–45% [1], and this is even more prevalent in risk groups,
such as patients with gastroesophageal reflux disease, eating disorders, special diets andthose who frequently consume acidic beverages, drugs or alcohol [1–4]. Dental erosion
manifests as erosive tooth wear, i.e., non-carious loss of hard dental tissues caused by theirdemineralization, which leads to a decrease in their mechanical properties and increased
susceptibility to abrasion or attrition [5]. Initially, only the enamel is affected, but longterm exposure to acids results in the involvement of dentin, which is less resistant todemineralization [6]. Consequently, the loss of dental tissues is accelerated, and restorative4.0/).

Polymers 2021, 13, 3901. https://doi.org/10.3390/polym13223901

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Polymers 2021, 13, 3901

2 of 11treatment is required to reduce hypersensitivity and prevent further progression, leading
to the inflammation of the pulp [5,7].
According to the European consensus statement on managing severe tooth wear,
minimally invasive treatment options are recommended, such as direct composite restorations or partial crowns [8]. However, these restorations rely on adhesion, which may becompromised on eroded dentin [9,10] because a thick layer of exposed collagen fibrils
is present on the demineralized surface, interfering with the penetration of monomers
contained in dental adhesives [11]. As a result, defects are present in the hybrid layer, andthe bond strength may be lower than on sound dentin [9,10]. Moreover, the insufficient
penetration of the collagen mesh results in nanoleakage, and the exposed collagen becomesprone to degradation by enzymes such as matrix metalloproteinases (MMPs) [12]. The
decomposition of the collagen may contribute to the decreased durability of bonding toeroded dentin, as reported by numerous studies [9,10].
The available evidence was mainly obtained through in vitro studies, where erosion
was simulated using various pH-cycling protocols. However, the lack of standardization
complicates the interpretation of the results [9,10]. In previous studies, demineralization
was most commonly performed using citric acid of various concentrations or soft drinks
such as Coca-Cola and Sprite (The Coca-Cola Company, Atlanta, GA, USA). Based on theconsensus of the Workshop on Methodology in Erosion Research held in 2010, 1% citricacid should be preferred, as it is more reproducible and controllable than soft drinks, while
having a pH similar to that of orange juice [13]. The consensus also stated that the erosive
challenge should not exceed a few minutes, to be comparable with real-life situations.
For remineralization solutions, artificial saliva was recommended [13] and used in mostprevious studies, even though the composition was not standardized.
Various mechanical and chemical surface treatments were tested to improve theadhesion to eroded dentin. Mechanical treatments removed the superficial demineralized
layer and exposed the underlying sound dentin. As a result, increased bond strength
was reported after superficial preparation using a fine diamond bur [11,14] or laser [15].
Chemical treatments were non-invasive, i.e., the superficial dentin was modified rather thanremoved. Inhibitors of MMPs such as 2% chlorhexidine or benzalkonium chloride were
investigated in several studies, as they could prevent the enzymatic degradation of collagenand thus improve the bonding durability, however the results were conflicting [9,14,16–18].
On the other hand, deproteinization using sodium hypochlorite (NaOCl) for 40–60 s wasfound to improve durability [14,19,20], as this can remove the superficial collagen and,
therefore, improve the monomer penetration. However, previous reports have shown thatthe treatment of dentin with NaOCl harms the polymerization of adhesives, as the oxidizing
effect leads to premature chain termination [21] and hampers the bonding performance [22].
The effect is dependent on the application time of NaOCl [23] and to counteract this, theapplication of an antioxidant, e.g., sodium ascorbate, sodium thiosulfate, rosmarinic acidor sodium p-toluenesulfinate (STS), was shown to be effective in several studies [21,24–27].
Moreover, a recent study showed that the application of agents containing sulfinates
improved the adhesives’ degree of conversion even on untreated sound dentin [28].
Given the benefits of antioxidants on deproteinized sound dentin, the purpose ofthis study was to investigate whether the application of STS can improve the bonding
to deproteinized eroded dentin. As the combined pretreatment with NaOCl and STS isrelatively time-consuming, easy-to-use universal adhesives were employed in this study
so that the procedure was not excessively technique-sensitive. In addition, universal
adhesives are increasingly popular among clinicians, and recent studies suggest that their
durability may have improved [29–32]. The null hypothesis tested in this study was thatthe deproteinization using NaOCl and application of STS would not affect the bonding
performance of universal adhesives to eroded dentin.

Polymers 2021, 13, 3901

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2. Materials and Methods
One hundred and twenty-four extracted sound human molars were used in this study,
following protocol number D2013-22, approved by the Human Research Ethics Committeeof Tokyo Medical and Dental University. They were stored in a periodically exchanged
0.1% thymol solution at 4 ◦ C and used within 6 months of extraction. Their occlusal enamel
was removed using a model trimmer, and the exposed mid-coronal dentin surfaces wereground with a wet 320-grit SiC paper (DCCS, Sankyo Fuji Star, Saitama, Japan) for 30 s
to create a standardized smear layer. A quarter of the teeth served as a control group,
while the rest were subjected to pH-cycling, according to Zimmerli et al. [11]. In brief, theteeth with exposed dentin surfaces were immersed in 1% citric acid (pH = 3.5; Wako Pure
Chemical Industries, Osaka, Japan) for 5 min, rinsed with distilled water, and immersed ina remineralization solution with pH adjusted to 6.4 (Table 1) for 3.5 h. The pH-cycling was
conducted for 8 consecutive days with 6 cycles per day. The pH of the demineralization
and remineralization solution was periodically monitored with a pH meter.
Table 1. Composition of the demineralization and remineralization solution (pH-cycling).
Solution

Composition

Demineralization

1% citric acid (pH = 3.5)

Remineralization

0.002 g ascorbic acid, 0.58 g NaCl, 0.17 g CaCl2 , 0.16 g
NH4 Cl, 1.27 g KCl, 0.16 g NaSCN, 0.33 g KH2 PO4 , 0.34 g
Na2 HPO4 dissolved in 1 L of demineralized water (pHadjusted to 6.4 with HCl)

For the micro-tensile bond strength (µTBS) test, 120 teeth were divided into fourgroups as follows: (1) untreated sound dentin (control group), (2) untreated eroded dentin,(3) eroded dentin deproteinized using a 10% NaOCl gel (AD gel; Kuraray Noritake Dental,
Tokyo, Japan) and (4) eroded dentin deproteinized using the 10% NaOCl gel followed by
the application of an STS-containing agent (Accel; Sun Medical, Kyoto, Japan). AD gel wasapplied with rubbing motion for 60 s, rinsed off with water for 15 s, and the surfaces weremildly air-blown for 10 s. Accel was applied using a disposable microbrush for 10 s andgently air-dried for 10 s. The dentin surfaces were further divided into three subgroups
and bonded using the universal adhesives Clearfil Universal Bond Quick (UBQ; Kuraray
Noritake Dental, Tokyo, Japan), Scotchbond Universal (SBU; 3M, St. Paul, MN, USA) or
G-Premio Bond (GPB, GC, Tokyo, Japan). These universal adhesives were then used in
the self-etching mode according to the manufacturers’ instructions, which are listed inTable 2 along with the composition of the materials used. Light-curing was performed
using an LED light-curing unit (Valo; Ultradent Products, South Jordan, UT, USA) for
10 s at 1000 mWcm−2 . The bonded surfaces were built up with a hybrid resin composite(Clearfil AP-X, shade A2; Kuraray Noritake Dental, Tokyo, Japan) to a height of 6 mm inthree increments. Each increment was light-cured for 40 s with the Valo light-curing unit.
Table 2. Composition and application procedure of materials used in this study.
Material(Manufacturer)

Composition

Clearfil Universal
Bond Quick (UBQ;
Kuraray Noritake
Dental, Tokyo, Japan)

10-MDP, Bis-GMA,
HEMA, hydrophilic
amide monomers,
colloidal silica, coupling
agent, sodium fluoride,
CQ, ethanol, waterpH

Application Procedure
1.

2.3

2.
3.

Apply to dentin withrubbing motion
Dry with mild airpressure for 5 s
Light cure for 10 s

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Table 2. Cont.
Material(Manufacturer)

Composition

Scotchbond Universal
Adhesive (SBU; 3M,
St.Paul, MN, USA)

10-MDP, dimethacrylate
resins, Bis-GMA, HEMA,
Vitrebond copolymer,
silane, ethanol, water,
filler, initiator

G-Premio Bond (GPB;
GC, Tokyo, Japan)

Accel (Sun Medical,
Kyoto, Japan)

10-MDP, 4-MET, MEPS,
methacrylate monomer,
acetone, water,
initiator, silica
sodiump-toluenesulfinate,ethanol, water

AD gel (Kuraray
Noritake Dental,
Tokyo, Japan)

10% sodium
hypochlorite, thickener

Clearfil AP-X (shade
A2; Kuraray Noritake
Dental, Tokyo, Japan)

Bis-GMA, TEGDMA,
silanated barium glassfiller, silanated silica filler,silanated colloidal silica,
CQ, initiators,
accelerators, pigments

pH

Application Procedure
1.

2.7

2.
3.
1.

1.5

2.
3.

Apply to dentin withrubbing motion for 20 s
Dry with mild airpressure for 5 s
Light cure for 10 s
Apply to dentin withrubbing motion for 10 s
Dry with strong air
pressure for 5 s
Light cure for 10 s

1.
2.

Apply to dentin for 10 s
Dry with gentle airpressure for 10 s

1.

Apply to dentin withrubbing motion for 60 s
Rinse off with water for
15 s
Dry with mild airpressure for 10 s

2.
3.

1.

2.

Place AP-X onto thebonded dentin surface inincrements of
2 mm thickness
Light cure eachincrement for 40 s

Abbreviations: 10-MDP: 10-methacryloyloxydecyl dihydrogen phosphate; Bis-GMA: bisphenol A diglycidylmethacrylate; HEMA: 2-hydroxyethyl methacrylate; CQ: camphorquinone; 4-MET: 4-methacryloxyethyl trimellitic acid;
MEPS: methacryloyloxyalkyl thiophosphate methylmethacrylate; TEGDMA: triethyleneglycol dimethacrylate.

After storage in distilled water at 37 ◦ C for 24 h, the specimens were sectioned
in two directions perpendicular to the adhesive layer into beams (cross-sectional area:
1.0 × 1.0 mm) using a low-speed diamond saw (Isomet 1000; Buehler, Lake Bluff, IL, USA)
under water cooling. The beams’ dimensions were measured using a digital calliper(Mitutoyo CD15; Mitutoyo, Kawasaki, Japan) for precise bonding area calculation. Fourbeams from the central area of each specimen were used for the testing. Half of the beams
were tested immediately, while the other half underwent artificial aging simulated by
10,000 thermal cycles between 5 ◦ C and 55 ◦ C (dwell time 30 s, transfer time 5 s). After therespective storage condition, the beams were individually glued to a µTBS testing jig using
a cyanoacrylate adhesive (Model Repair II Blue; Dentsply Sirona, Charlotte, NC, USA),
mounted in a tabletop testing machine (EZ-SX; Shimadzu, Kyoto, Japan), and subjected tothe µTBS test at a crosshead speed of 1.0 mm/min.
The fractured specimens were mounted on brass stubs, desiccated for 24 h, sputtercoated with gold, and their failure mode was determined using a scanning electron microscope (SEM; JSM-5310; JEOL, Tokyo, Japan) at 75× magnification. The failure modes
were classified as: (1) a cohesive failure in dentin (70–100% of the failure occurred in
dentin), (2) a dentin/adhesive interfacial failure (70–100% of the failure occurred at theinterface between dentin and the adhesive), (3) an adhesive/composite interfacial failure(70–100% of the failure occurred at the interface between the resin composite and adhesive),(4) a cohesive failure in the resin composite (70–100% of the failure occurred in the resin

Polymers 2021, 13, 3901

5 of 11composite build-up) or (5) a mixed failure (at least two of the failure patterns mentioned
above were observed, but none of them covered over 70%). In addition, SEM was used
to observe morphological changes on the dentin surface in the experimental groups. The
remaining four teeth (one sound and three eroded) were used and pretreated as describedabove. Initially, the specimens were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate (Na(CH3 )2 AsO2 ) buffer at pH 7.4 for 12 h at 48 ◦ C and rinsed with 20 mL of
0.2 M sodium cacodylate buffer at pH 7.4 for 1 h. The specimens were then dehydrated
in ascending grades of ethanol: 25% (20 min), 50% (20 min), 75% (20 min), 95% (30 min)and 100% (60 min), followed by immersion in hexamethyldisilazane for 10 min [33]. After
sputter-coating with gold, the dentin surfaces were observed at 5000x magnification.
Statistical analyses were performed using the SPSS Statistics software (version 27.0
for Windows; IBM, Armonk, NY, USA) at the significance level of 0.05. For the µTBS
data, the tooth was considered a statistical unit (n = 5), i.e., µTBS values of the four beams
originating from each tooth were averaged, and the mean was processed statistically. Since
the Shapiro–Wilk test indicated that µTBS data were normally distributed and Levene’s
test confirmed the homogeneity of variances, the results were analyzed with a three-way
ANOVA (variables: dentin pretreatment, adhesive, aging). Pairwise comparisons were
performed using Tukey’s HSD post hoc tests. Failure mode distributions were analyzed
using Fisher’s exact test.
3. Results
3.1. µTBS
The µTBS results are presented in Table 3. The three-way ANOVA revealed that theeffects of dentin pretreatment (p < 0.001), aging (p < 0.001) and adhesive (p = 0.006) weresignificant. The interaction between dentin pretreatment and aging was significant as well(p = 0.003), whereas interactions between dentin pretreatment and material (p = 0.908), andbetween material and aging (p = 0.448) were not significant. The three-way interaction was
not significant either (p = 0.869). Regardless of the adhesive and aging, µTBS to eroded
dentin was significantly lower compared to other groups (p < 0.001). µTBS to sound dentin(control group) was similar to deproteinized eroded dentin, yet decreased significantly afterthermocycling (p < 0.001). The application of the STS-containing agent increased µTBS withall adhesives, but the difference was not significant with GPB (p > 0.05). Thermocycling
had no significant effect on µTBS to deproteinized eroded dentin treated with the STScontaining agent (p > 0.05). Among the adhesives, the µTBS of UBQ was significantly
higher compared to SBU (p = 0.049) and GPB (p = 0.006), which did not significantly differ
from each other (p = 0.736).
Table 3. µTBS results: Mean (SD) in MPa.
Adhesives

UBQ

SBU

GPB

Dentin Pretreatment

24 h

10,000 Thermal Cycles

Control

63.1 (10.7) A,b

50.1 (7.6) B,b

Eroded49.4 (9.7) A,c

22.1 (5.7) B,c

NaOCl

61.0 (8.5) A,b

44.3 (8.3) B,b

NaOCl + STS

71.0 (9.3) A,a

64.5 (9.8) A,a

Control

57.6 (6.7) A,ab

47.0 (5.8) B,ab

Eroded39.2 (8.7) A,c

23.5 (5.8) B,c

NaOCl

55.9 (11.9) A,b

43.8 (7.1) B,b

NaOCl + STS

63.9 (7.6) A,a

58.2 (11.7) A,a

Control

53.1 (9.3) A,b

42.9 (7.2) B,b

Eroded40.4 (8.5) A,c

20.4 (4.1) B,c

NaOCl

60.9 (8.7) A,ab

40.1 (10.2) B,abNaOCl + STS

61.3 (8.8) A,a

58.7 (8.2) A,a

Different uppercase letters indicate a statistically significant difference between aging conditions (in rows).
Different lowercase letters indicate a statistically significant difference between dentin pretreatments (in columns)
for each adhesive.

Polymers 2021, 13, 3901
Polymers 2021, 13, x FOR PEER REVIEW

6 of 11
6 of 113.2. Failure
Failure Mode
Mode Analysis
Analysis
3.2.
The failure
failure mode
differThemode distributions
distributionsare
areshown
shownininFigure
Figure1.1.There
Therewerewerenonosignificant
significant
difences
betweentheexperimentalgroups(p>0.05).
However,slightlymoredentin/adhesive
ferences between the experimental groups (p > 0.05). However, slightly more dentin/adinterfacial failures were observed on untreated eroded dentin, whereas cohesive failures inhesive interfacial failures were observed on untreated eroded dentin, whereas cohesive
dentin and the resin composite tended to be more frequent on deproteinized eroded dentin
failures in dentin and the resin composite tended to be more frequent on deproteinizedtreated with the STS-containing agent. No pre-testing failures occurred.
eroded dentin treated with the STS-containing agent. No pre-testing failures occurred.

FigureFigure 1.
1. Distribution
Distribution ofof failure
failure modes
modes inin percentage.
percentage.

3.3.
3.3. MorphologicalMorphological Analysis
Analysis ofof thethe Dentin
Dentin Surfaces
Surfaces
Representative
Representative SEM
SEM images
images ofof thethe dentin
dentin surfaces
surfaces areare presented
presented in Figure 2. In thecontrol
control group
group (Figure 2a), the dentin surface was covered with a compact smear layer anddentinal
dentinal tubules
tubules were
were obliterated.
obliterated. pH-cycling
pH-cycling removed
removed thethe smear
smear layer
layer and exposed theorifices ofof dentinal
dentinal tubules
tubules (Figure(Figure 2b–d).
2b–d). On
On thethe untreateduntreated eroded
eroded dentin,
dentin, collagencollagen fibrils
fibrilsorificeswere observed
observed (Figure(Figure 2b); however, deproteinization
deproteinization with
with 10% NaOCl removed them
were
(Figure 2c,d). The
The deproteinizeddeproteinized surfaces
surfaces were
were very porous,
porous, and dentinal tubules’ orifices(Figurewere enlarged to a funnel
funnel shape (Figure 2c,d). The
The application
application ofof thethe STS-containing
STS-containing agent
were
did
not
significantly
alter
the
surfacemorphology
ofthedeproteinizederoded dentin.
did not significantlyPolymers 2021, 13, 3901

1, 13, x FOR PEER REVIEW

7 of 11

7 of 11

2. Scanningelectron micrographs
the dentin
surfaces.
The dentin
surfacegroupin thewas
conFigure 2.Figure
Scanning
electron micrographsof the dentinofsurfaces.
(a) Thedentin(a)surfacein the control
covered
trol
group
was
coveredbyasmear
layer.
(b)
Smear
layer
was
not
present
onerodeddentin,andby a smear layer. (b) Smear layer was not present on eroded dentin, and collagen fibrils were observed on the untreatedfibrils were observed
on dentin
the untreateddeproteinizationof eroded
dentin to asurface. collagen(c) After deproteinizationof erodedwith 10%surface.
NaOCl,(c)theAfterorificesof dentinal tubules
were enlarged
with
10%
NaOCl,
theorificesofdentinal
tubuleswereenlargedtoafunnelshape(indicatedby thefunnel shape (indicated by the white arrow), and the surface was very porous. (d) The application of the STS-containing
white
arrow),
andthesurfacewasveryporous.
(d):Theapplicationofthe
STS-containing
agent
agent did not alter the surface morphology.
did not alter the surface morphology.
4. Discussion

4. Discussion

While the achievement of durable adhesion on sound dentin is challenging, bonding
toerodeddentinis even more
complexas the cyclic
demineralization
While the achievementof durable
adhesionon sounddentinis challenging, exposesbondinga layer ofdisorganized collagen network on the eroded dentin surface [6] (Figure 2b). As a result,to eroded dentin is even more complex as the cyclic demineralization exposes a layer ofthe penetration of adhesives into the underlying dentin is hindered, and a defective hybrid
disorganized collagen network on the eroded dentin surface [6] (Figure 2b). As a result,layer is formed, leading to lower bond strengths and reduced durability [9,10]. This was
the penetration confirmedof adhesives
intopresent
the underlying
dentin isand a defective
hy- in theby thestudy, as bonding
to hindered,untreated eroded
dentin resulted
brid layer is formed,
lower
bond
and reduced
durability
ThisHowever,lowestleading
24 h andtoagedµTBS,
andstrengths
dentin/adhesiveinterfacial
failures [9,10].
prevailed.
was confirmed bythepresent
study,
asbonding
tountreated
erodeddentinresultedin using
the objective of this study was to examine the effect of a deproteinizing pretreatment
10%agedNaOCl
gel andthedentin/adhesivesubsequent applicationof an STS-containing
agent onthe bonding
the lowest 24 h aand
µTBS,
and
interfacial
failures prevailed.
Howperformanceof universal
adhesives
erodedAs deproteinization
significantly
ever, the objectiveof this study
was to examine
thetoeffectof dentin.
a deproteinizing
pretreatment
increasedµTBSµTBS afterapplication
the applicationof STS-containing
STS surpassed thatof untreatedusing a 10% NaOCl
gel andtheandsubsequent
of an
agent
on the sounddentin,thenullhypothesiswasrejected.
bonding performance of universal adhesives to eroded dentin. As deproteinization sigNaOCl is a strongly alkaline, non-specific proteolytic compound that can decompose
nificantly increased µTBS and µTBS after the application of STS surpassed that of unthe exposed collagen layer on eroded dentin. The deproteinizing ability of NaOCl was contreated sound dentin,
null
hypothesiswasrejected.
firmed the
using
SEM
(Figure 2c,d),which
revealed that collagen was removed, funnel-shaped
NaOCl is a orificesstrongly
alkaline,
non-specificproteolytic
thatmorecan decomposeof dentinal tubules were exposed,and compoundthe surface wasporous compared tothe exposed collagen
layer
on or
erodedThe deproteinizing
abilitypresumably
of NaOClfacilitatedwasuntreatedsounderodeddentin.
dentin (Figure2a,b). These changes
theconfirmed usingpenetration
SEM (Figure2c,d),
whichandrevealed
that collagenwas removed,
funnelof theadhesives
hybridization,
thus leading
to a significant
increase inµTBS
after deproteinization.
There wassignificant
difference
betweenshaped orifices ofdentinal
tubules were exposed,
andalsotheno
surfacewas moreporouscom- µTBS to
deproteinizederodeddentinandthecontrolgroup.
These
findings
agree
witha study bypared to untreated sound or eroded dentin (Figure 2a,b). These changes presumably faDeari et al.,which
a similarand
deproteinizing
pretreatment
was used
However, Dearicilitated the penetration
ofintheadhesives
hybridization,
thus leading
to a[14].
significant

increase in µTBS after deproteinization. There was also no significant difference betweenµTBS to deproteinized eroded dentin and the control group. These findings agree with astudy by Deari et al., in which a similar deproteinizing pretreatment was used [14]. However, Deari et al. did not measure aged bond strength, which highlights a limitation of

Polymers 2021, 13, 3901

8 of 11et al. did not measure aged bond strength, which highlights a limitation of their study, as
the correlation of immediate bond strength with clinical outcomes is limited [34]. Augusto
et al. used 10% NaOCl for 60 s and revealed that deproteinization significantly improvedµTBS to eroded dentin [19]. Furthermore, µTBS to deproteinized eroded dentin was not
significantly affected by 5000 thermal cycles, and it was significantly higher than the control
group after aging, regardless of etching mode [19]. Siqueira et al. used a 5.2% solutionof NaOCl for 40 s and found that the µTBS of a self-etching adhesive to deproteinizederoded dentin exceeded µTBS to sound dentin both immediately and after 3 years of water
storage [20]. It was also found that the deproteinization of eroded dentin significantly
decreased nanoleakage [20], and therefore contributed to excellent bonding durability. In
this study, µTBS to deproteinized dentin significantly decreased after 10,000 thermal cycles.
We speculate that the differences might influence this in pH-cycling protocols and theadhesive systems used.
As a part of the deproteinizing action of NaOCl, free radicals were formed, and theyinterfered with the propagation of vinyl radicals, which in turn resulted in premature
chain termination and the incomplete polymerization of adhesives [21,24–27]. This was therationale for using STS, as previous studies showed that applying antioxidants or reducing
agents could improve the bond strength to NaOCl-deproteinized dentin [21,24–27]. Furthermore, sulfinates could substitute tertiary amines as co-initiators of camphorquinone [35],which is used as a photoinitiator in most adhesive systems, and sulfinate-containing
agents were recently reported to increase the degree of conversion of light-cured adhesives on dentin [28]. In this study, the treatment of deproteinized eroded dentin with the
STS-containing agent resulted in the highest µTBS. Moreover, the bond strength was not
significantly affected by thermocycling, as opposed to other tested groups, including sounddentin. Our assumption is that STS improved the degree of conversion of the tested adhesives [28], thus contributing to the improved µTBS, particularly after thermocycling [31].
Notably, while the bonding durability was improved, this was at the expense of two additional steps, which made the bonding procedure more elaborate and time-consuming.As
such, a slight preparation of the eroded dentin using a diamond bur [11,14], followed by
the standard application of any adhesive may be preferred by some clinicians, even thoughsuch an approach is slightly more invasive.
In the control group, µTBS was similar to deproteinized eroded dentin, but the characteristics of the bonding surface were dissimilar. On eroded dentin, the smear layer was
removed by pH-cycling (Figure 2c), and deproteinization created a mineral-rich porous
surface suitable for the formation of micromechanical and chemical bonding. In contrast,
the dentin surface in the control group was entirely covered with a compact smear layer(Figure 2a), the presence of which could hinder ideal bonding as the smear layer interferes with monomer infiltration and the chemical interaction of the adhesive monomers
with the underlying intact dentin [36,37]. Additionally, the formation of the hybridized
smear layer above the authentic hybrid layer [38] was thought to have an adverse effecton bonding durability [37]. While these factors could have harmed immediate and aged
µTBS, they were probably balanced by sound dentin acting as a better substrate for bonding, and the absence of radicals, which could impede the polymerization of adhesives on
deproteinized dentin.
While the effect of dentin pretreatment was similar for all the tested adhesives,
their µTBS differed, as UBQ exhibited significantly higher values than SBU and GPB.
Even though all the adhesives contained 10-methacryloyloxydecyl dihydrogen phosphate(10-MDP), impurities were found to adversely affect hybridization, the formation of calcium salts, and nanolayering [39]. Since Kuraray Noritake Dental reportedly produced
the purest 10-MDP [39], this could have contributed to the high µTBS of UBQ. UBQ also
contains novel hydrophilic amide monomers [30], allowing for the reduced content of
2-hydroxyethyl methacrylate (HEMA). This may support the durability of UBQ, as HEMA
was associated with increased water sorption and a decreased degree of conversion [40,41].
However, the HEMA-free adhesive GPB did not exhibit superior durability. This may bePolymers 2021, 13, 3901

9 of 11attributed to the content of 4-methacryloxyethyl trimellitic acid, whose bonding potential
was lower compared to 10-MDP [42]. In addition, the use of acetone as a solvent in GPBmight be suboptimal on eroded dentin since its ability to re-expand collapsed collagenfibers is limited due to its low hydrogen-bonding capacity [43]. On the other hand, GPB
was the only tested adhesive not affected by the application of STS, which might indicate that its polymerization was not impaired by the presence of radicals produced by
NaOCl. This could be due to the content of diphenyl(2,4,6-trimethylbenzoyl)phosphineoxide known as TPO, a very efficient photoinitiator, which does not require tertiary amines
as co-initiators [31].
The main limitation of this study is that it was performed in vitro on artificiallyeroded dentin surfaces. While the pH-cycling models attempted to mimic the process of
tooth erosion by cyclic demineralization and remineralization, the intraoral conditions
were much more complex. Firstly, the collagen network on eroded surfaces may havebeen decomposed by various enzymes, or as a result of chewing and the brushing ofteeth. Secondly, the in vivo process of erosion was much longer compared to pH-cycling,
and provided the eroded teeth were vital, the structure of dentin may have changed inreaction to the cyclic acid attacks. Thirdly, the bonding surfaces involved both dentin andenamel, but in vitro studies usually investigated these substrates separately. Therefore,
their outcomes should be interpreted with caution, and they should be confirmed byin vivo studies.
5. Conclusions
Within the limitations of this in vitro study, we concluded that deproteinization with
NaOCl significantly increased the bond strength of universal adhesives to eroded dentin.
When combined with the subsequent application of a sodium p-toluenesulfinate-containing
agent, the bond strength and durability were even superior to sound dentin.
Author Contributions: Conceptualization, Y.S. and K.H.; methodology, Y.S., K.Y., M.N. and K.H.;
validation, Y.S. and A.T.; formal analysis, M.I. and A.T.; investigation, Y.S., K.Y. and M.H.; resources, J.T., M.N., and K.H.; data curation, K.Y. and M.H.; writing—original draft preparation, Y.S.;
writing—review and editing, A.T., K.Y., M.H., T.H., M.I., J.T., M.N. and K.H.; visualization, Y.S. andT.H.; supervision, J.T. and K.H.; project administration, Y.S. and K.H.; funding acquisition, A.T. and
K.H. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Ministry of Education, Culture, Sports, Science, andTechnology of Japan (Grants-in-Aid for Scientific Research 18K09571 and 21K16988) and Charles
University (grant Progres Q29/1LF).
Institutional Review Board Statement: The use of extracted teeth was approved by the Human
Research Ethics Committee of Tokyo Medical and Dental University (protocol number D2013-22).
Data Availability Statement: Data available on request from the corresponding author.
Acknowledgments: The authors acknowledge the manufacturers for donating materials for thisresearch. Monica Yamauti is gratefully acknowledged for her assistance with the statistical analysis.
Conflicts of Interest: The authors declare no conflict of interest.

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