Dental Materials 39 (2023) 1113–1121

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Dental Materials
journal homepage: www.elsevier.com/locate/dental

Concentration effect of DMSO-dry bonding on the stability of

etch-and-rinse bonds
Omar Abdelaziz Ismail a, b, c, *, 1, Thiago Henrique Scarabello Stape a, d, Arzu Tezvergil-Mutluay a, d
a
Department of Restorative Dentistry and Cariology, Adhesive Dentistry Research Group, Institute of Dentistry, University of Turku, Turku, Finland
b
Department of Conservative Dentistry, Horus University in Egypt, Egypt
c
Department of Conservative Dentistry, Faculty of Dentistry, Cairo University, Egypt
d
Turku University Hospital, TYKS, University of Turku, Turku, Finland

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

Keywords: Objectives: To examine whether lower dimethyl sulfoxide (DMSO) concentrations would affect long-term bond
DMSO stability of simplified or multistep water-based adhesives to dry-etched dentin.
Dry bonding Methods: H3PO4-etched mid-coronal dentin surfaces from human molars were randomly blot- or air-dried for 30 s
Dental adhesives
and pretreated or not with 5 or 50 % (v/v) ethanolic DMSO solutions. Untreated samples served as control.
Dimethyl sulfoxide
Hybrid layer
Samples were bonded with a two-step or a three-step etch-and-rinse adhesive. Restored crown segments (n = 5/
group) were stored in distilled water for 24 h and sectioned for microtensile bond strength testing. Resin-dentin
beams (0.8 mm2) were tested under tension until fracture (0.5 mm/min) after 24 h and one year of storage in
artificial saliva at 37 ◦ C. Nanoleakage evaluation and hybrid layer characterization were performed by SEM.
Bond strength data was examined by three-way ANOVA followed by the Tukey test (α = 0.05). Results: Dry
bonding produced significantly lower bond strengths than conventional wet bonding for both water-based ad­
hesive systems (p < 0.05). DMSO-dry bonding restored bond strengths and reduced nanoleakage levels,
regardless of adhesive type or DMSO concentration (p < 0.05). Bond strengths of DMSO-dry bonded samples
were not significantly affected by long-term ageing regardless of adhesive type or DMSO concentration (p <
0.05).
Significance: Although bonding methacrylate-based resins to etched dentin is normally performed under wet
conditions, hybridization of air-dried collagen can outperform conventional wet bonding by employing water-
free DMSO solutions with concentrations as low as 5 %. Reduced moisture-related technique sensitivity,
higher bonding performance and improved hybrid layer stability may contribute to extend the service life of
resin-dentin bonding.

1. Introduction Resin-dentin bonding is a form of in situ dental tissue engineering, in

which methacrylate-based monomers penetrate the dentin matrix,
In the past decades, adhesive dentistry has revolutionized restorative become entangled and upon polymerization (e.g. hybrid layer forma­
procedures and extended the service life of tooth-bonded treatments [1]. tion) provide micromechanical retention [6]. Hybrid layer stability is
For such, adhesive procedures should maintain durable tooth-bonded mainly dictated by the quality of both the formed dimethacrylate
interfaces for a number of years to attain clinical durability. While polymer networks and their interaction with dentin [1–5]. Most of the
enamel bonding is predictable and enduring, considerable research “hydrophilicity” of etched dentin is due to the presence of bound (i.e. 21
effort has been devoted to extend the longevity of resin-dentin interfaces – 15 % w/w) and unbound (i.e. 75 – 79 % w/w) water molecules [7]. In
[1–5]. Despite improvements in resin formulations and better under­ H3PO4-etched dentin, hydroxyapatite minerals are initially dissolved
standing of degradation mechanisms, achieving stable resin-dentin and then replaced by water molecules (i.e. roughly 50 % v/v) [8]. Resin
bonding under clinical conditions remains a challenge. monomers must replace unbound water molecules for effective

* Corresponding author at: Department of Restorative Dentistry and Cariology, Adhesive Dentistry Research Group, Institute of Dentistry, University of Turku,
Turku, Finland.
E-mail address: oaisma@utu.fi (O.A. Ismail).
1
Institute of Dentistry, University of Turku. Lemminkaisenkatu 2, Turku, 20520, Finland.

https://doi.org/10.1016/j.dental.2023.09.013
Received 13 May 2023; Accepted 28 September 2023
Available online 12 October 2023
0109-5641/© 2023 The Authors. Published by Elsevier Inc. on behalf of The Academy of Dental Materials. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
O.A. Ismail et al. Dental Materials 39 (2023) 1113–1121

resin-dentin bonding. Considering that the molar concentration of resin extractions.

monomers (0.3–5 mol/L) is only a fraction of the molar concertation of
water (55.6 mol/L) [9], complete water replacement by resin monomers 2.1. Experimental design and bonding procedures
is not easily achieved. Failure to do so results in porous water-rich
bonded interfaces highly susceptible to hydrolysis over time. The experimental design was composed of three study factors
Currently, hybridization of etched dentin is normally performed defined as: (i) “bonding protocol” at three levels (no treatment, 5 % or
under wet conditions. Water reduces collagen shrinkage by breaking 50 % DMSO-dry bonding), (ii) “bonding resin” at two levels (two-step or
interpeptide hydrogen bonding [10] maintaining interfibrillar spacing. three-step etch-and-rinse adhesive) and (iii) “storage time” at two levels
This allows better diffusion of methacrylate-based monomers and higher (24 h or one year). Conventional wet bonding following manufacturer’s
collagen fibril encapsulation [11], albeit percolation through collagen instructions served as isolated control groups at 24 h and one year
fibrils is mostly by hydrophilic monomers [12]. There is a consensus that without DMSO pretreatments. Molars were mounted in acrylic resin 2
etch-and-rinse resins are highly sensitive to dentin moisture [1,6,13]. mm below the cement-enamel junction and then sectioned perpendic­
Both overdry or overwet conditions strongly affect dentin-bonding ularly to their long axis under cooling to expose mid-coronal flat dentin
performance [14,15]. Water entrapment within the collagen matrix surfaces with a diamond saw (Isomet 1000 Precision Saw, Lake Bluff, IL,
limits the diffusion of cross-linking hydrophobic monomers into hybrid USA) [25]. Absence of remaining enamel on the dentin surfaces was
layers [12], contributes to phase separation of monomers [2], reduces verified with a stereomicroscope (Leica M60, Leica Microsystems,
monomer conversion thus resulting in mechanically weaker polymers Wetzlar, Germany) at 40 × magnification. Smear layer standardization
[16]. In fact, the critical amount of water necessary to produce optimal was done using 320-grit silicon carbide grinding paper (Buehler-Met) for
resin-dentin bonding is inferior than originally thought [17]. In attempt 60 s under water cooling. Crown segments (n = 5/group) were randomly
to reduce/eliminate the drawbacks of residual water on hybrid layer allocated to 8 groups according to dry-bonding protocols and bonding
quality, dry bonding has regained attention [18–24]. The main problem resin. Dentin surfaces were etched for 15 s with 32 % phosphoric acid
resides on preventing or effectively re-expanding collapsed collagen fi­ (Scotchbond Universal Etchant, 3 M ESPE, St. Paul, MN, USA), rinsed for
brils after air-drying for adequate hybridization. Methacrylate-based 15 s and either blot-dried, leaving the surface partially wet (Control
bonding agents do not promote adequate re-expansion of dried Wet), or air-dried for 30 s (dry bonding). DMSO 5 % and 50 % solutions
collagen [10], so additional measures are necessary to allow proper (v/v) were prepared by mixing DMSO (Dimethyl sulfoxide,
monomer-collagen interactions. However, bonding commonly available Sigma-Aldrich, St. Louis, MO, USA) with ethanol (Ethanol 99.8 %,
resins to etched-dry dentin is possible following newly proposed pro­ Sigma-Aldrich). 50 µL [21] of DMSO solutions (5 or 50 %) were actively
tocols [18–24]. Dry bonding not only facilitates application procedures, applied on the etched-dentin surfaces for 60 s according to bonding
but it can establish more reliable resin-dentin bonding given the protocols. Moisture control was performed by either blot-drying, until
appropriate conditions [18–21]. paper filters presented no visible moisture creating a partially wet sur­
Among the recently proposed bonding techniques, dimethyl sulf­ face (Control Wet) or by air-drying for 30 s (Control Dry and DMSO-dry
oxide (DMSO)-dry bonding emerges as an alternative to bond relatively bonding) [18–21]. Two water-based bonding resins were used for hy­
hydrophilic resins to extensively air-dried dentin [18–21]. Unlike pre­ bridization either a simplified two-step etch-and-rinse adhesive (Adper
viously proposed bonding protocols, which indeed produced improved Single Bond Plus, 3 M-ESPE; SB) or a three-step etch-and-rinse adhesive
outcomes [22–24], DMSO-dry bonding acts in several fronts to facilitate (Scotchbond Multi-Purpose, 3 M-ESPE; SBMP). Composition of adhesive
bonding to H3PO4-etched dentin. Improved hybrid layer formation [20], systems and bonding procedures are shown in Table 1. All bonding
better collagen wettability [19], more uniform conversion [18] and resins were actively applied with light pressure of approximately 4 g
lower endogenous collagenolytic activity [21] contribute to the
extended durability of resin-dentin interfaces [18]. One main advantage
Table 1
is that DMSO-dry bonding can effectively displace residual water within Adhesive Systems, main components, and application mode of bonding agents.
collagen. This results in bonded interfaces with reduced water entrap­
Adhesive system Components Bonding Mode of
ment and lower porosity when water-free DMSO pretreatments are used
protocol application*
[18,19]. To date, reports assessing DMSO-dry bonding have mainly
Scotchbond Multi- Primer: water (>40 %), Control a, b, c, f, g, h, j
focused on relatively high DMSO concentrations (i.e., 50 % v/v)
Purpose (3 M/ HEMA, polyalkenoic acid Wet and k
[18–21]. While substantially higher DMSO concentrations can be dele­ ESPE; SBMP) methacrylate copolymer Control a, b, d, f, g, h, j
terious to resin-dentin interfaces [18], lower thresholds for dry bonding Batch # Dry and k
have not been investigated. Reducing DMSO content could potentially Bond: bis-GMA, HEMA, 5% a, b, d, e; f, g,
facilitate overall solvent removal and avoid any potential DMSO-related dimethacrylates DMSO-dry h, j and k
photoinitiators 50 %
cytotoxicity issues, which may be empirically attributed to higher DMSO
DMSO-dry
concentrations. Therefore, the primary aim of this study was to inves­ Single Bond plus Ethyl alcohol; bis-GMA; Control a, b, c, i, g and
tigate whether DMSO-dry bonding using water-free low-DMSO contents (3 M/ESPE; SB) silane-treated silica; HEMA; Wet k
would affect etch-and-rinse bonding of methacrylate-based resins. The Batch # water (<10 %); copolymer of Control a, b, c, d, i, g
acrylic and itaconic acid; Dry and k
objectives were to evaluate whether dentin pretreatments with reduced
UDMA 5% a, b, d, e, i, g
DMSO content would affect immediate or long-term bonding of different DMSO-dry and k
water-containing resins to extensively air-dried dentin. The tested null 50 %
hypotheses were that DMSO-dry bonding with low-DMSO contents DMSO-dry
would have no effect on (i) resin-dentin bonding or (ii) hybrid layer Scotchbond 37 % phosphoric acid, a and b
Universal fumared silica (pH 0.6)
quality and stability of etched dentin.
Etchant (3 M-
ESPE)
2. Materials and methods
Abbreviations: HEMA = 2-hydroxyethyl methacrylate; bis-GMA = bis-phenol
diglycidylmethacrylate; UDMA = diurethane dimethacrylate
Forty-eight sound third molars were selected for this study under a * a: dentin etching for 15 s; b: water rinsing for 15 s; c: blot-drying leaving dentin
protocol approved by the Ethical Committee, University of Oulu, slight moist; d: air-drying for 30 s; e: DMSO-pretreatment application for 60 s
Finland (#23–2003). Teeth were extracted for clinical reasons that were according to bonding protocol; f: active Primer active application for 10 s; g:
not related to this study. Molars were stored in 0.5 % Chloramine-T gentle blow-drying for 10 s; h: active Bond application for 10 s; i: active adhesive
immediately after extraction at 4 ◦ C and used within 3 months of application for 10 s; j: air-drying for 5 s and k: light curing for 10 s

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[26] in circular rubbing movements. For SB groups, SB was applied in a USA) for 8 h under fluorescent light. Silver impregnated resin-dentin
single coat for 10 s and gently air-blown for 10 s. For SBMP groups, the beams were embedded in epoxy resin, wet polished with 600-, 1000-,
Primer was applied for 10 s and gently air-blown for 10 s. The Bond was and 2000-grit SiC paper (Carbimet, Buehler Ltd.,) and 1, 0.25 (MetaDi,
subsequently applied for 10 s and gently air-blown for 5 s. Adhesive Buehler Ltd) and 0.05 µm (MasterPrep, Buehler Ltd) polishing pastes,
procedures were carried out in a controlled environment with a tem­ ultrasonically cleaned in distilled water after each polishing step for 2
perature of 23 ℃ and a relative humidity between 45 % and 55 %. min, air-dried for 2 h, mounted on aluminum stubs, dried in silica
Adhesives were light cured for 10 s using a LED light-curing unit (Elipar overnight and carbon sputtered. Nanoleakage was qualitatively
Deepcure, 3 M ESPE) at 1200 mW/cm2. Composite blocks were built analyzed using a scanning electron microscope (Phenom ProX,
with a nanofilled composite resin (Filtek Supreme XTE, 3 M ESPE) in two Phenom-World) on backscattering mode at 10 kV. Silver uptake patterns
increments of approximately 2 mm. Each increment was light cured for and extensions were evaluated by a blinded operator at magnifications
20 s. All bonding procedures were carried out by a single operator. The ranging from 1000 – 10,000×.
restored crown segments were stored in distilled water for 24 h at 37 ◦ C
and longitudinally sectioned with a slow-speed diamond saw (Isomet, 2.5. Hybrid layer characterization (SEM)
Buehler Ltd) into resin-dentin beams with cross sectional area of
approximately 0.8 mm2. A minimum of 18 resin-dentin beams were Two central resin-dentin beams from each tooth were randomly
sectioned per tooth. selected for hybrid layer evaluation under SEM. Beams were embedded
in epoxy resin and wet-polished with 600-, 1200-, 2000- and 4000-grit
2.2. Resin-dentin beam storage SiC paper (Buehler Ltd., Lake Bluff, IL, USA). Specimens were ultra­
sonically cleaned in distilled water after each polishing step for 5 min.
Resin-dentin beams were randomly selected for the microtensile test Bonded interfaces were then treated with 50 % H3PO4 for 5 s and 3 %
and nanoleakage analyses under two conditions: immediate, after 24 h NaOCl for 10 min followed by dehydration in ascending ethanol series
of storage in distilled water, or long-term ageing, after one year storage (50, 70, 80, 90 and 3 × 100 %), fixed in hexamethyldisilazane, sputtered
in artificial saliva at 37 ◦ C. Half of the beams were used for immediate with gold/palladium and analyzed on backscattering mode at 10 kV
testing and the other half for long-term ageing. The artificial saliva (pH (Phenom ProX, Phenom-World). A series of sequential micrographs of
7.4) was composed of 5 mM HEPES, 2.5 mM CaCl2⋅H2O, 0.05 mM ZnCl2 the entire bonded interfaces (5000 × magnification) were obtained from
and 0.3 mM NaN3 at 37 ◦ C [27]. It was changed biweekly to avoid pH each resin-dentin beam. Three randomly selected areas on each micro­
changes [18,25]. graph, located between adjacent resin tags, were analyzed and measured
by a blinded examiner. Hybrid layer thickness was measured using an
2.3. Microtensile bond strength test (µTBS) open-source image software (ImageJ, National Institute of Health,
Bethesda, MD, USA). Measurements obtained from beams were aver­
Microtensile bond strength testing followed the Academy of Dental aged and corresponded for the hybrid layer thickness of each specific
Materials guidelines for non-trimmed µTBS testing [25]. A minimum of tooth (n = 5).
7 beams per tooth (n = 5 teeth/group) were tested at each storage
period. Beams were individually attached to a custom-made testing jig 2.6. Statistical analysis
using a cyanoacrylate adhesive (Loctite 416, Henkel Corp., Dublin,
Ireland) and tested under tension on a mechanical testing machine Bond strength data was normally distributed (Shapiro-Wilk; p =
(Shimadzu, AGS-X, Maryland, USA) at a crosshead speed of 0.5 mm/min 0.091) and homoscedastic (Levene Test; p = 0.064). Three-way ANOVA
until failure. The cross-sectional area (CA) in mm2 of each beam was followed by the Tukey test. Hybrid layer thickness data (Levene Test; p
measured with a digital caliper to nearest 0.01 mm. The formula uTBS = = 0.137) was analyzed by two-way ANOVA followed by the Tukey test.
P/CA was used to calculate µTBS values in MPa. For the statistical Statistical significance was set at α = 0.05. Calculations were performed
analysis, pre-test failures were considered as 0 MPa. Since tooth was by IBM SPSS Statistics for Windows, version 26 (IBM Corp., Armonk, NY,
considered as the statistical unit, bond strengths of resin-dentin beams USA).
from each tooth were averaged to represent the mean bond strength
value at each specific storage period [25]. All measurements were per­ 3. Results
formed by a blinded operator. Fractured resin-dentin beams were
analyzed with a stereomicroscope (Leica MD60, Leica Microsystems) at 3.1. Microtensile bond strength
40 × magnification to determine fracture patterns. Unidentifiable sur­
faces were examined by scanning electron microscopy (SEM) (Phenom The cross-sectional area of resin-dentin beams (0.84 mm2 ± 0.1)
ProX, Phenom-World, Eindhoven, Netherlands). Fracture modes were ranged from 0.70 to 0.99 mm2 without significant differences regarding
classified as (A) adhesive failure, (C) cohesive failure in composite or specimen size between groups (p = 0.092). Bond strengths are reported
dentin, (M) mixed failure (failure at composite/dentin interface with in Fig. 1. Three-way ANOVA revealed that “bonding protocol” (p <
cohesive failure of any substrates) and (P) pretest failure samples [18]. 0.001; η2 = 0.908), “bonding resin” (p < 0.001; η2 = 0.258), “storage
time” (p = 0.005; η2 = 0.115) and the interactions between “bonding
2.4. Interfacial nanoleakage evaluation (SEM) protocol” * “storage time” (p = 0.046; η2 = 0.116) had significant effects
on resin-dentin bond strengths. No significant differences were observed
Two resin-dentin beams per tooth (n = 5 teeth/group) were between the two-step (SB) and three-step (SBMP) etch-and-rinse adhe­
randomly selected to evaluate silver nitrate uptake at bonded interfaces sives under wet bonding at 24 h. Dry bonding (Control Dry) produced
after 24 h or one-year storage in artificial saliva at 37 ◦ C. Nanoleakage the lowest bond strengths. Significant reductions in the order of roughly
evaluation was performed according to a protocol previously described 85 % (p < 0.05) were observed for both bonding resins at 24 h compared
by Tay et al. [28]. Resin-dentin beams were initially wet polished with to the conventional wet-bonding technique (Control Wet). DMSO-dry
600-grit and 2000-grit SiC paper and coated with two layers of nail bonding protocols produced significantly higher bond strengths than
varnish applied up to 1 mm of the bonded interfaces. Coated samples dry bonding (Control Dry) for both adhesives (p < 0.05). For the two-
were rehydrated in distilled water for 1 h and immersed in 50 % (w/v) step etch-and-rinse adhesive (SB), both 5 % and 50 % DMSO-dry
ammoniacal silver nitrate (pH 9.5) for 24 h. Samples were thoroughly bonding produced comparable immediate bond strengths to wet
rinsed in distilled water for 120 s and immersed in photo-developing bonding (Control Wet). While ageing for one year significantly reduced
solution (Kodak Professional D-76 developer, Kodak Rochester, NY, bond strengths of the two-step etch-and-rinse adhesive by roughly 46 %

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Fig. 1. Microtensile bond strength (MPa) means and standard deviations of resin-dentin interfaces bonded to dry dentin using 5 % or 50 % DMSO/EtOH solutions as
pretreatments. A two-step (SB) or a three-step (SBMP) etch-and-rinse adhesive were used for bonding. Resin-dentin beams were tested at 24 h or after 1 year of ageing
in artificial saliva at 37 ◦ C. Tooth was considered the statistical unit (n = 5/group). Different upper-case letters indicate significant differences between groups within
the 24 h testing period. Different lower-case letters indicate significant differences between groups after ageing for 1 year. * indicates significant differences between
ageing periods within similar treatments. Statistical comparisons were performed by the Tukey test (α = 0.05).

(p < 0.05) under wet bonding (Control Wet), no significant reductions under wet bonding (p > 0.05); however, 50 % DMSO-dry bonding
occurred for DMSO-dry bonded samples regardless of DMSO concen­ produced significantly higher bond strengths than wet bonding in order
tration (p < 0.05). For the three-step etch-and-rinse adhesive, 5 % of 50 % (p < 0.05). Fracture pattern distributions (%) for all groups are
DMSO-dry bonding produced comparable immediate bond strengths to shown in Fig. 2 and Table 2 shows the number of fractured resin-dentin
wet bonding (p > 0.05) and 50 % DMSO-dry bonding produced signif­ beams. The predominant failure mode at 24 h was mixed failure for both
icantly higher values (p < 0.05). Ageing for one year had no significant adhesive systems for all bonding protocols except for dry bonding
effects on the bond strengths of the three-step etch-and-rinse adhesive (Control Dry), which presented a high number of pre-test failures. After

Fig. 2. Fracture patterns in percentages (%) of tested specimens after the bond strength test at 24 h or 1 year of aging in artificial saliva at 37 ◦ C. Fracture patterns
were classified as: cohesive failure = failure exclusive within dentin or resin composite; adhesive failure = failure at resin/dentin interface and mixed failure
= failure at resin/dentin interface with cohesive failure of the neighboring substrates.

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Table 2
Microtensile bond strength means (MPa), standard deviations ( ± SD) and number of specimens according to fracture mode.
Simplified E&R adhesive (SB) Multistep E&R adhesive (SBMP)

Control Wet Control Dry 5 %DMSO-dry 50 %DMSO-dry Control Wet Control Dry 5 %DMSO -dry 50 %DMSO-dry

24 h 39.52 3.37 47.3 46.81 42.94 6.32 52.90 61.68

± 7.05 ± 0.65 (0/11/5/ 16/ ± 8.77 ± 5.6 ± 7.55 ± 0.9 (1/8/10/ 16/ ± 6.9 ± 10.3
(3/12/20/ 1/ 32) (1/12/23/ 0/ (2/8/27/ 1/38) (2/12/20/ 2/ 35) (1/7/28/ 1/ (3/10/22/ 1/
36) 36) 36) 37) 36)
1 year 21.22 2.38 46.76 47.86 37.07 5.51 51.39 55.46
± 5.3 ± 0.49 (0/11/3/ 22/ ± 9.38 ± 8.49 ± 6.83 ± 1.01 (0/7/8/ 20/ ± 8.96 ± 7.64
(4/19/11/ 1/ 36) (2/7/29/ 1/39) (1/11/25/ 0/ (2/22/8/ 4/36) 35) (1/7/30/ 1/ (3/10/24/ 1/
35) 37) 39) 38)

Tooth was considered the statistical unit (n = 5). Numbers between quotation marks represent the number of specimens (resin-dentin beams) following the fracture
mode classification (1/2/3/4/5): (1) cohesive failure; (2) adhesive failure; (3) mixed failure; (4) pre-test failure and (5) total number of tested specimens.

3.3. Hybrid layer characterization (SEM)

Table 3
Hybrid layer thickness (µm) and standard deviations.
Representative SEM micrographs showing hybrid layers of resin-
Adhesive Control Wet Control 5 % DMSO- 50 % DMSO- dentin interfaces are shown in Fig. 4. Well-defined hybrid layers
Dry dry dry
ranging from 1.1 to 3.58 µm were identified for all groups. Two-way
Simplified E&R 3.23Aa 1.02Ba 3.06Aa 3.27Aa ANOVA revealed that “bonding resin” had no significant effect on
(SB) (±0.38) (±0.11) (±0.34) (±0.32)
hybrid layer thickness. Differently, “bonding protocol” (p < 0.001;
Multistep E&R 3.58Aa 1.10Ba 3.14Aa 3.39Aa
(SBMP) (±0.45) (±0.13) (±0.3) (±0.32) η2 = 0.922) significantly affect hybrid layer formation. Dry bonding
(Control Dry) produced the lowest thickness values. Wet bonding pro­
Different upper-case letters indicate significant differences between dentin duced a significantly 3-fold increase in thicker hybrid layer thickness
pretreatments (row). Different lower-case letters indicate significant differences
compared to dry bonding (Control Dry) regardless of bonding resin
between adhesives (column). Statistical comparisons were performed by the
(p < 0.05). No significant differences were observed between DMSO-dry
Tukey test (α = 0.05).
bonded samples (5 or 50 % DMSO) and wet-bonded samples (Control
Wet) regardless of bonding resin (p > 0.05). Dry-bonded samples
ageing, wet-bonded samples presented adhesive failures as the pre­
(Control Dry) were characterized by hybrid layers with shorter (8 –
dominant pattern and DMSO-dry bonded samples did not present sub­
21 µm) and thinner resin tags sparsely distributed along irregularly
stantial changes in fracture patterns.
bonded interfaces for both simplified and multistep etch-and-rinse ad­
hesives. The two-step etch-and-rinse adhesive displayed silica-filler ag­
3.2. Interfacial nanoleakage evaluation (SEM)
glomerates mostly across the adhesive layer, which were not affected by
DMSO-dry bonding protocols. No substantial differences in hybrid layer
Representative backscattered SEM micrographs showing silver
morphology were observed between conventional wet bonding (Control
infiltration across hybrid layers are shown in Fig. 3. All bonding pro­
Wet) and DMSO-dry bonding (5 or 50 % DMSO). Resin-impregnated
tocols presented silver deposits in the peritubular region of dentin tu­
intertubular dentin continuously extended into deep-penetrating resin
bules surrounding resin tags. Dry bonding (Control Dry) presented the
tags (16 – 38 µm) uniformly distributed along the entire extension of
highest levels of silver uptake showing hybrid layers heavily impreg­
bonded interfaces.
nated by heavy silver deposits. Such high nanoleakage levels were
characterized by heavy reticular silver deposits depicting large exten­
sions of water-filled porous zones. The conventional wet-bonding tech­ 4. Discussion
nique (Control Wet) produced considerably lower silver uptake than dry
bonding (Control Dry). The two-step etch-and-rinse adhesive (SB) pre­ Since lower DMSO fractions had a positive effect on etch-and-rinse
sented higher nanoleakage levels than the three-step etch-and-rinse bonding to extensively air-dried dentin (i.e., 30 s), the first null hy­
adhesive (SBMP) under wet bonding. Nanoleakage patterns were mostly pothesis was rejected. Bonding methacrylate monomers to dry collagen
reticular silver deposits located at the bottom of hybrid layers, albeit is not an easy task [10]. Although wet bonding benefits from low DMSO
spotted patterns were frequently observed for both adhesives. DMSO- concentrations [29–32], DMSO-dry bonding has otherwise relied on
dry bonded samples presented lower nanoleakage extensions than wet relatively higher DMSO concentrations (i.e., 50 % w/w) to bond resins
bonding, characterized by spotted silver deposits lightly distributed under dry conditions [18–21]. The rationale for originally using high
along hybrid layers for both adhesives. No substantial differences were DMSO fractions was to benefit from DMSO’s concentration-dependent
detected between 5 % and 50 % DMSO-dry bonded samples considering ability to dissociate highly cross-linked collagen into a sparser
the same bonding resin at the same storage period. network of fibrils [33] and increase the wettability of dry collagen [19,
After ageing, higher nanoleakage levels were identified for all 34]. It has been shown that high DMSO concentrations can eliminate the
bonding protocols: Control Dry > Control Wet > 5 % DMSO-dry = 50 % negative impact of air-drying on collagen wetting by commonly
DMSO-dry regardless of adhesive type. Under wet bonding (Control employed methacrylate-based hydrophilic resin blends [19]. Findings
Wet), the three-step etch-and-rinse adhesive (SBMP) presented consid­ presented here confirm the ability of 50 % DMSO pretreatments to
erably less silver uptake than the two-step adhesive (SB). Silver uptake effectively bond multistep [18–21], as well as simplified etch-and-rinse
consisted of mostly reticular deposits with varying extensions along the adhesives, to dry dentin, improving long-term in vitro performance
hybrid layers. DMSO-dry bonding reduced nanoleakage at bonded in­ [18–21]. Nonetheless, this study provides evidence that high DMSO
terfaces for both etch-and-rinse adhesives regardless of DMSO concen­ concentrations are not necessarily mandatory to achieve long-lasting
tration. Areas with complete-dense hybrid layer impregnation by silver bonding to dry dentin.
were hardly identified on DMSO-dry treated samples, characterized Water-free DMSO solutions as low as 5 % (v/v) not only restored
mostly by spotted silver patterns. immediate bond strengths to dry collagen, but also prevented bond
strength degradation over time. The selection of 5 % DMSO was based
on DMSO’s low toxicity at concentrations <10 % (v/v) [35,36].

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Fig. 3. Representative SEM micrographs showing nanoleakage extensions of resin-dentin interfaces bonded to dry dentin using 5 % or 50 % DMSO/EtOH solutions as
pretreatments. A two-step (SB) or a three-step (SBMP) etch-and-rinse adhesive were used for bonding. Resin-dentin beams were tested at 24 h or after 1 year of ageing
in artificial saliva at 37 ℃.

Although substantially higher DMSO concentrations have been protocols may not require such high DMSO concentrations as initially
employed in the medical field, DMSO can induce cell apoptosis due to thought. Using ethanol-based pretreatments containing low DMSO
pore formation on plasma membranes [35,36]. Hence, attempts to fractions invariably opens new possibilities to dry bonding. It is inter­
minimize potential cytotoxic effects for dental applications remain esting how the incorporation of relatively low DMSO fractions modified
valid. Using 5 % DMSO (v/v) as a threshold further increased safety ethanol-collagen interactions allowing effective bonding under dry
margins still resulting in improved bonding to dry dentin. DMSO’s conditions. The 5 % DMSO pretreatment was composed mostly by
ability to bond methacrylate-based resins to dry dentin and its protective ethanol (i.e., 95 % v/v), which was air-dried for 30 s before adhesive
effect on long-term bonding were not concentration dependent between application. Normally, bonding resins to chemically dehydrated
5 % and 50 % (v/v). This clearly shows that DMSO-dry bonding ethanol-saturated dentin (i.e., ethanol-wet bonding [10]) must be

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Fig. 4. Representative SEM micrographs showing hybrid layers of resin-dentin interfaces bonded to dry dentin using 5 % or 50 % DMSO/EtOH solutions as pre­
treatments. A two-step (SB) or a three-step (SBMP) etch-and-rinse adhesive were used for bonding.

performed following a quite laborious-sensitive technique [37]. The obtained low bond strengths to untreated-dry dentin (Control Dry)
Collagen must be initially moist to prevent interpeptide hydrogen corroborates the inefficiency of water-based resins to re-expand [10]
bonding for subsequent dehydration with a series of ascending ethanol and properly bond to air-dried collagen [26,38]. Considering the
solutions [10]. It is crucial for collagen to be fully embedded in ethanol extreme dry-bonding conditions, it seemed reasonable to select
before adhesive application [10]. Failure to prevent initial collapse of water-based adhesives to complement collagen re-expansion produced
collagen compromises bonding efficacy [10]. This was not impeditive by water-free DMSO pretreatments as in previous studies [18–21]. In
for high-, as previously shown [18–21], nor now for low-percentage such manner, DMSO breaks self-associative tendencies of water [39] and
DMSO-dry bonding. No significant differences on bond strengths acts as a surfactant, facilitating the diffusion of adhesive components
occurred between low or high DMSO concentrations. Conceptually, into collagen [40]. The combined collagen re-expansion produced by
DMSO-dry bonding differs from ethanol-wet bonding in the sense that DMSO pretreatments (i.e., high or low content) and water-based adhe­
collagen does not need to be fully immersed in ethanol before hybridi­ sives seemed sufficient for adequate bonding. SEM analyses of hybrid
zation [18]. Air-drying high- or low-content DMSO before hybridization layers provided indirect evidence that resin infiltration of collagen
did not hinder bonding efficiency. It is clear that even at lower con­ matrices was not hampered by lower DMSO concentrations of pre­
centrations, DMSO broadened the moisture spectrum for resin bonding treatments nor by reducing water content of adhesives. While conven­
[19] bringing more flexibility and long-term stability to dry bonding tional dry bonding showed clear limitations in adhesive diffusion, due to
protocols. severe collagen collapse, no significant differences were observed be­
The central aim of this study was to investigate clinically feasible tween hybrid layer thicknesses or bond strengths between wet (Control
dry-bonding approaches to ultimately reduce the overall water entrap­ Wet) and DMSO-dry bonded protocols. This indicates that the water
ment within hybrid layers. However, two water-based adhesives (i.e., SB content of bonding resins and DMSO concentrations can be reduced,
<10 and SBMP >40 % water content) were chosen. Bonding with water- without negative impacts on resin-dentin interactions. Future studies
free adhesives would indeed lower water entrapment within resin- should focus on determining the lowest water content of bonding resins
dentin interfaces. However, full re-expansion of air-dried collagen is to effectively bond hydrophilic resins using the DMSO-dry bonding
not easily accomplished by commonly used adhesive solvents applied approach. It is tempting to speculate the possibility of bonding
under clinically feasible times [10]. The higher the Hoy’s solubility water-free resins to extensively air-dried etched dentin employing pro­
parameters for hydrogen bonding forces (δh) of a polar solvent above tocols that are more technique forgiving.
14.8, the greater is the degree of collagen re-expansion [10]. Water is Successful resin-dentin bonding is strongly related to the stability of
indeed the strongest known hydrogen bonding solvent (δh 37.3 bonded interfaces over time. Adhesive composition plays a major role on
(J/cm3)1/2). As such, it effectively breaks interpeptide bonds of air-dried handling and dentin bonding performance [1,5]. While the wet bonded
collagen (δh 14.8 (J/cm3)1/2), promptly re-expanding and increasing (Control Wet) simplified etch-and-rinse adhesive (SB) presented signif­
interfibrillar spaces [10]. Previously used aqueous solutions containing icant bond strength reductions after ageing, the tested multistep
50 % DMSO (δh 26.8 (J/cm3)1/2) result in modest re-expansion (roughly etch-and-rinse adhesive (SBMP) did not. The superiority of multistep
70 %) of dry collagen [34]. The tested water-free DMSO pretreatments over simplified adhesives has been well stablished [1,6,41,42]. Multi­
present even lower δh, which further reduces their ability to re-expand step adhesives make use of a separate more hydrophobic resin that
dry collagen. Incomplete collagen re-expansion before hybridization improves the mechanical stability of hybrid layers [6,43] and reduces
impairs resin infiltration and compromise resin-dentin bonding [6,10]. hydrolytic degradation of polymers over time [42]. Bonding resins with

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higher ratios of hydrophobic dimethacrylate monomers tend to result in [3] Tjäderhane L, Nascimento FD, Breschi L, Mazzoni A, Tersariol ILS, Geraldeli S,
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Disclosure statement [21] Stape THS, Seseogullari-Dirihan R, Tjäderhane L, Abuna G, Martins LRM,
Tezvergil-Mutluay A. A novel dry-bonding approach to reduce collagen
degradation and optimize resin-dentin interfaces. Sci Rep 2018;8:16890. https://
The authors declare no potential conflicts of interest with respect to doi.org/10.1038/s41598-018-34726-8.
the authorship and/or publication of this article. [22] Mai S, Wei CC, Gu L, sha, Tian F, Arola cong, Chen DD, hua J, et al. Extrafibrillar
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between wet and dry dentin bonding. Acta Biomater 2017;57:435–48. https://doi.
Acknowledgments org/10.1016/j.actbio.2017.05.017.
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