Ethanol-wet Bonding and Chlorhexidine Improve

Resin-Dentin Bond Durability: Quantitative Analysis

Using Raman Spectroscopy
Supitcha Talungchita / Julie L.P. Jessopb / Deborah S. Cobbc / Fang Qiand / Saulo Geraldelie /
David H. Pashleyf / Steven R. Armstrongg

Purpose: To directly test the effectiveness of ethanol-wet bonding (EW) in improving monomer infiltration into
demineralized dentin through quantitative measurement of bis-GMA and TEG-DMA molar concentrations within
hybrid layers, and to comprehensively evaluate the effect of EW and chlorhexidine on durability of resin-dentin
bonds compared to conventional water-wet bonding (WW).
Materials and Methods: A three-step etch-and-rinse adhesive (70% bis-GMA/28.75%TEG-DMA) was applied to
coronal dentin using a clinically relevant ethanol-wet bonding protocol (EW) or the conventional water-wet bond-
ing (WW) technique. Bis-GMA and TEG-DMA molar concentrations at various positions across the resin/dentin in-
terfaces formed by EW and WW were measured using micro-Raman spectroscopy. The experiment was repeated
at the same positions after 7-month storage in phosphate buffer solution containing 0.1% sodium azide. The
μTBS and hybrid layer morphology (TEM) of bonding groups with and without chlorhexidine application were com-
pared immediately and after 1-year storage in terms of nanoleakage, collagen fibril diameter, collagen interfibril-
lar width, and hybrid layer thickness.
Results: Specimens bonded with EW showed significantly higher monomer molar concentrations and μTBS
throughout the hybrid layer immediately and after storage, providing direct evidence of superior infiltration of hy-
drophobic monomers in EW compared to WW. Microscopically, EW maintained interfibrillar width and hybrid layer
thickness for resin infiltration and retention. The application of chlorhexidine further preserved collagen integrity
and limited the degree of nanoleakage in EW after 1-year storage.
Conclusion: EW enhances infiltration of hydrophobic monomers into demineralized dentin. The results suggest
that a more durable resin-dentin bond may be achieved with combined usage of a clinically relevant EW and
chlorhexidine.
Keywords: bonding, collagen(s), matrix metalloproteinases (MMPs), ultrastructure, dentin, adhesives.

J Adhes Dent 2014; 16: 441–450. Submitted for publication: 23.10.13; accepted for publication: 17.4.14
doi: 10.3290/j.jad.a32695

a Full-time Faculty Member, Department of Conservative Dentistry, Prince of
Songkla University, Songkhla, Thailand. Hypotheses, experimental design,
performed the experiments in partial fulfillment of requirements for PhD,
wrote the manuscript, contributed substantially to discussion.
b Associate Professor, Department of Chemical and Biochemical Engineering,
College of Engineering, University of Iowa, Iowa City, IA, USA. Idea, consulted
on the experiment using Raman spectroscopy, proofread the manuscript,
contributed substantially to discussion.
c Associate Professor, Department of Operative Dentistry, College of Dentistry,
University of Iowa, Iowa City, IA, USA. Proofread the manuscript, contributed
substantially to discussion.
d Associate Research Scientist, Department of Preventive and Community
Dentistry, College of Dentistry, University of Iowa, Iowa City, IA, USA. Per-
formed statistical analyses, interpreted statistical findings, proofread the
manuscript.
e Clinical Associate Professor, Department of Restorative Dental Sciences,
College of Dentistry, University of Florida, Gainesville, FL, USA. Idea, con-
sulted on the experiment using TEM, proofread the manuscript, contributed
substantially to discussion.
f Emeritus Regents’ Professor of Oral Biology, Department of Oral Biology,
College of Dental Medicine, Georgia Regents University, Augusta, GA, USA.
Idea, consulted on ethanol-wet bonding and chlorhexidine, proofread the
manuscript, contributed substantially to discussion.
g Professor and Chair, Department of Operative Dentistry, College of Dentistry,
University of Iowa, Iowa City, IA, USA. Idea, hypotheses, experimental design,
proofread the manuscript, contributed substantially to discussion.
Correspondence: Professor Steven R. Armstrong, Department of Op-
erative Dentistry, College of Dentistry, University of Iowa, S229A DSB,
Iowa City, IA, USA 52242. Tel: +1-319-335-7211, Fax: +1-319-335-7267.
e-mail: steven-armstrong@uiowa.edu
Parts of this study were presented at the IADR 86th General Session and Ex-
hibition, Toronto, Canada, 2008, Abstract #0356 (Poster session), IADR 87th
General Session and Exhibition, Miami, FL, USA, 2009, Abstract #1517 (Oral
presentation session), and IADR 91st General Session and Exhibition, Seattle,
WA, USA, 2013, Abstract #1668 (Oral presentation session).

Vol 16, No 5, 2014 441

Talungchit et al
B onding effectiveness to dentin relies on resin infiltra-
tion and encapsulation of the demineralized collagen
fibril network, creating a hybrid layer.23 Deterioration of
the hybrid layer is a consequence of hydrolysis of the
polymer matrix and degradation of unencapsulated
demineralized collagen.2,20 Therefore, enhanced resin
infiltration and sustained integrity of both resin matrix
and collagen are crucial for a durable resin-dentin bond.
Hydrophobic monomers are preferable to hydrophilic
monomers to prevent water sorption and hydrolytic degrada-
tion of the resin matrix.16 However, hydrophobic monomers
fail to infiltrate hydrophilic water-saturated demineralized
dentin using the conventional water-wet bonding technique
(WW).1 The ethanol-wet bonding technique (EW) replaces
water in demineralized dentin with ethanol, thus increasing
miscibility and solubility of subsequently applied hydropho-
bic resin adhesives, yielding strong and durable bonds.28
Ethanol, a transitional solvent, maintains interfibrillar width
by preventing interpeptide H-bonding in collagen, thereby
preventing the collapse of the collagen matrix during sol-
vent evaporation and resin adhesive application.25 Various
EW protocols have been used in recent studies.15,26,29
However, the results were non-uniform, and there is no
study which quantitatively measured and compared the
amount of dimethacrylate monomers, ie, bis-GMA and TEG-
DMA, in hybrid layers formed by EW and WW.
Once activated, host-derived matrix metalloproteinases
(MMPs)22 and cysteine cathepsins24 slowly degrade un-
encapsulated collagen within hybrid layers, compromising
resin-dentin bond durability. Chlorhexidine (CHX) was in-
troduced in bonding to inhibit proteolytic degradation.10,32
The use of CHX with WW produced a more stable collagen
morphology and better bond strength compared to no CHX
both in vivo13 and in vitro.5,7 Although limited amounts of
denuded collagen are expected when using EW, CHX may
still be beneficial in preserving collagen integrity.
The purpose of this study was to evaluate the short-
and long-term (1-year) effects of EW and CHX on resin-
dentin microtensile bond strength (μTBS) and hybrid layer
quality as measured by nanoleakage, collagen fibril diam-
eter, interfibrillar width, hybrid layer thickness, and the
presence of collagen banding using TEM. Micro-Raman
spectroscopy was used to evaluate monomer infiltration
in both EW and WW specimens by measuring the bis-GMA
and TEG-DMA molar concentrations (CM) across hybrid
layers immediately and after 7-month storage in phos-
phate buffer solution. The null hypotheses were that EW
and CHX had no effect on resin-dentin μTBS nor on hybrid
layer quality and that EW had no effect on CM within hybrid
layers compared to WW.
MATERIALS AND METHODS
Microtensile Bond Strength Test
Institutional Review Board (IRB)-approved, sound human
molars were stored in 0.5% chloramine-T at 4°C and
used within 6 months of extraction. Teeth were rinsed
free of chloramine-T and stored in PBS before being
randomly divided into WW and EW groups (n = 32 per
442
group) and ground with a carbide bur (#55, Brasseler;
Savannah, GA, USA) in a computer numerical control
(CNC) specimen former (University of Iowa, Iowa City,
IA, USA) to expose a flat surface of coronal dentin. A
0.5-mm-deep groove was placed across the occlusal
surface to allow separate surface treatments on the
two halves. The three-step etch-and-rinse technique
was applied for WW. For EW, the acid-etched and water-
rinsed dentin was subsequently rinsed with absolute
ethanol for 15 s three times with blotting in between.
Before priming, half of each tooth in WW and EW was
treated for 30 s with 2% aqueous chlorhexidine diglu-
conate (Cavity Cleanser, Bisco; Schaumburg, IL, USA)
or 2% chlorhexidine diacetate in ethanol (Pashley Lab;
Augusta, GA, USA), respectively, creating 4 bonding
protocols: EW-no-CHX, EW-with-CHX, WW-no-CHX, and
WW-with-CHX. Then, primer (50% adhesive/50% ethanol)
was applied for 30 s. After solvent removal, experimen-
tal nonsolvated adhesive (70% bis-GMA/28.75% TEG-
DMA; Bisco) was applied and light cured (Demetron
501, irradiance 700-780 mW/cm2, Kerr; Orange, CA,
USA) for 20 s. Resin-based composite (Z100, 3M ESPE;
St Paul, MN, USA) was placed and light cured for 40 s in
three 1.5-mm-thick increments.
Four dumbbell-shaped specimens with a circular cross-
sectional area of 0.5 mm2 were formed per tooth using
the CNC specimen former. Any tooth failing to produce 4
testable specimens was replaced with a different tooth.
One beam from each side of the tooth, with and without
CHX, was immediately tensile tested to failure at 1 mm/
min in a passive gripping device3 (n = 32 per bonding
protocol). The remaining specimens were stored at 37°C
in phosphate buffer solution containing 0.1% sodium
azide (n = 32 per bonding protocol), which was changed
monthly until testing after 1-year storage.
Fractography
After the μTBS test, debonded specimens were glued to
aluminum stubs using a cyanoacrylate cement (Zapit,
Dental Ventures of America; Corona, CA, USA) and then
gold sputter coated for evaluation with SEM (Amray
1820-D, AMRAY; Bedford, MA, USA). Fracture pathways
were classified into 4 types: dentin cohesive failure,
resin-based composite cohesive failure, joint failure, and
mixed failure.
Hybrid Layer Morphology
Six additional teeth were randomly divided into WW and
EW groups (n = 3/per group). Within each group, the
teeth were bonded as described above, except flow-
able resin-based composite (Heliomolar Flow, Ivoclar
Vivadent; Amherst, NY, USA) was used to facilitate sec-
tioning for ultrastructural evaluations using TEM (JEOL
1230, 120 kV; Tokyo, Japan). Four 0.5 × 1 mm2 resin-
dentin beams from each half of each tooth were ran-
domly selected to evaluate nanoleakage and collagen
in the short term and after 1-year storage. For the short-
term evaluations, two beams were placed overnight in
Karnovsky’s fixative, one for nanoleakage and another
for collagen ultrastructure. The remaining beams were
The Journal of Adhesive Dentistry

stored at 37ºC in phosphate buffer solution containing
0.1% sodium azide for long-term study (1 year).
For nanoleakage evaluation, specimens were stained be-
fore sectioning with 50% ammoniacal silver nitrate for 24 h,
then placed in developer under fluorescent light for 8 h.
Specimen coding was used for blinded evaluation. Dehydra-
tion, resin embedding, and thin sectioning were performed.
For nanoleakage types, five 90-nm-thick sections from each
specimen were evaluated (n = 15 per bonding protocol).
After sectioning, for collagen ultrastructural evaluation,
5 non-demineralized 60-nm-thick sections from each
specimen were double-stained in 1% phosphotungstic
acid for 10 min and 5% uranyl acetate for 2 min. TEM
images were randomly taken and measurements were
performed using ImageJ (National Institutes of Health;
Bethesda, MD, USA). Hybrid layer thickness was randomly
measured at one spot per section (n = 15 per bonding
protocol). Collagen fibril diameter and interfibrillar width
within a 5 × 5 μm2 viewing area at upper, middle, and
lower hybrid-layer regions were measured at 10,000X
magnification in one area per section. Dentinal tubules
and their branches were avoided. In tangential sections
where the fibril profiles were oblong, the width of the fibril
was taken to be the diameter. Within each viewing area,
all cross-sectioned collagen fibrils were measured for col-
lagen fibril diameter (3 to 10 fibrils per viewing area), and
the same number of interfibrillar width measurements
were performed. The mean of each measurement within
each viewing area was calculated. Three-way ANOVA was
performed to assess the significance of hybrid layer region
on the mean collagen fibril diameter and mean interfibril-
lar width. Since no significant effect was found, mean
collagen fibril diameter and interfibrillar width within each
viewing area was assumed to be an independent statis-
tical unit (n = 45 per bonding protocol). The presence of
collagen banding was defined as 67 nm “collagen band-
ing” if more than 25% of fibril banding was present under
the TEM viewing area (n = 45 per bonding protocol).
Monomer Infiltration (Micro-Raman Spectroscopy)
Experimental co-monomers (Biomaterials lab, College
of Dentistry, University of Iowa) with different bis-GMA/
TEG-DMA concentrations (Table 1) were prepared to
construct calibration curves (Figs 1 and 2) for CM cal-
culation. Each co-monomer was placed in 3 aluminum
pans and polymerized for 60 s (Demetron Optilux 500,
irradiance 700 to 800 mW/cm2, Kerr) to create resin
blocks. Polymer samples were removed from aluminum
pans and kept in a vacuum desiccator until same-day
specimen pycnometer (Quanta Chrome, Model # MPY-2,
Quantachrome Instruments; Boynton Beach, FL, USA)
volumetric measurements were completed. Molar con-
centrations of bis-GMA and TEG-DMA (Cbis-GMA and CTEG-
DMA) in each co-monomer were calculated by formula (1):
CM = 1000(C% w/w × mspecimen)/(100 × mM × Vspecimen)
where CM is the molar concentration of a monomer
(mol/L), C%w/w is the mass percentage of a monomer
in a co-monomer mixture, m specimen is the specimen
mass (g), mM is molecular weight of a monomer (g/
mol), and Vspecimen is the volume of a specimen (m3).
Vol 16, No 5, 2014
Talungchit et al
Table 1 Resin co-monomer chemical composition (wt%)
Co-monomers Components
Co-monomer 1 98.65% TEG-DMA/ 1.1% EDMAB/ 0.25% CQ
Co-monomer 4 30.90% bis-GMA/ 67.77% TEG-DMA/
1.08% EDMAB/ 0.25% CQ
Co-monomer 8 71.04% bis-GMA/ 27.71% TEG-DMA/
(Resin 2) 1.01% EDMAB/ 0.24% CQ
Co-monomer 11 98.71% bis-GMA/ 1.00% EDMAB/ 0.29% CQ
Abbreviations: bis-GMA: 2,2-bis[4-(2-hydroxy-3-methacryloyloxypropoxy)]-
phenyl propane; TEG-DMA: triethylene glycol dimethacrylate; EDMAB:
2-ethyl dimethyl-4-aminobenzoate; CQ: camphorquinone (Sigma-Aldrich;
St Louis, MO, USA).
Note that 1000 is the conversion factor between vol-
ume units “L” and “m3”, and 100 is the conversion factor
between mass percentage and mass fraction.
To avoid the oxygen-inhibited layer, Raman spectra
were collected at 3 random spots on the bottom of each
resin block using a light microscope (RamanRxnl micro-
probe, Leica DMPL, Leica Microsystems; Buffalo Grove,
IL, USA) attached to a laser generator (Invictus 785-nm
NIR Laser, #0666135 single-mode excitation fiber, Kai-
ser Optical System; Ann Arbor, MI, USA) and a modular
research Raman spectrograph (HoloLab series 5000R,
#6000131 62.5-μm collection fiber, Kaiser Optical Sys-
tem). Calibration was performed on a silicon standard
following the manufacturer’s instruction using a calibra-
tion accessory (HCA) with a single mode excitation fiber
(#6000135) and 100-μm collection fiber (#6000113; all
Kaiser Optical System). The laser beam delivered through
a 10X objective to the sample had an intensity of 8 to
11 mW, and a spectral resolution of 4 cm-1 in the 100 to
3450 cm-1 region. Spectra were obtained through a 100X
objective with a 60-s exposure using auto-dark subtraction
and cosmic-ray correction via the manufacturer’s software
(HoloGRAMS, v. 4.1, Kaiser Optical System).
The peak area measurement at 1610 cm-1 (aromatic
ring on bis-GMA) and 605 cm-1 (C-C=O bend in meth-
acrylate groups on bis-GMA and TEG-DMA) was performed
using Raman data processing software (HoloMap, v. 7.3.0
[R2006b], Kaiser Optical System). The starting and end
points of each peak on an actual spectrum were selected
based on its second derivative spectrum. Mean peak ar-
eas at 605 and 1610 cm-1, bis-GMA and TEG-DMA molar
concentrations (Cbis-GMA and CTEG-DMA) were used to con-
struct calibration curves. Resin co-monomer 8 (Biomateri-
als lab, University of Iowa), with the same formula as resin
2 (Bisco), was used to fabricate resin-dentin bond speci-
mens, and thus the known Cbis-GMA in this co-monomer
was used as a reference to calculate short-term Cbis-GMA
and CTEG-DMA across hybrid layers.
Resin-Dentin Bond Specimen Preparation
Eight sound human molars were prepared as described
to expose coronal dentin. Sticky wax was used to build a
2-mm wall on top of the midline section to separate each
443
Talungchit et al

Normalized 1610 Peak Area 1.2

1.0
y = 0.3727x + 0.1134
R2 = 0.9939
0.8

0.6

0.4 Fig 1 Calibration curve I constructed

from bis-GMA molar concentration
0.2 (C bis-GMA ) and normalized peak area
of experimental co-monomer resin
0.0 blocks obtained by dividing the mean
0.0 0.5 1.0 1.5 2.0 2.5 1610 cm-1 peak area of each co-mono-
Bis-GMA Molar Concentration [mol/L] mer by the mean 1610 cm-1 peak area
of co-monomer 11.

Bis-GMA Molar Concentration [mol/L]

TEG-DMA Molar Concentration [mol/L]

4.5 2.5
4.0
y = –0.2911x2 + 1.7715x – 0.3201 2.0
3.5
R2 = 1
3.0
1.5
2.5
2.0
1.0
1.5
y = –0.5096x2 – 3.0955x + 4.6958
1.0 0.5
R2 = 1
Fig 2 Calibration curve II constructed
0.5
from bis-GMA and TEG-DMA molar
0.0 0.0 concentrations (Cbis-GMA and CTEG-DMA)
0 2 4 6 and 1610:605 peak area ratio obtained
Ratio 1610/605 TEG-DMA Bis-GMA from resin blocks of the experimental
co-monomers.

tooth into halves that were randomly treated with EW and Absolute bis-GMA molar concentration
WW. The same EW and WW bonding protocols were fol- For the short-term data, Cbis-GMA in the adhesive resin
lowed as previously described, with the exception that (AR) layer (Position 1, Table 4) was assumed to be equal
CHX treatment was not included to avoid Raman spectral to that in co-monomer 8 (1.689 mol/L) and was used as
interference from CHX at 1610 cm-1.18 Two 1-mm-thick a reference. The peak area at 1610 cm-1 from each spot
resin-dentin slabs containing both EW- and WW-treated within a scanned location was divided by that at Position
surfaces were formed per tooth and polished. On each 1 in the same location to obtain a normalized peak area
side of the slabs, two locations with 3-μm-thick hybrid at 1610 cm-1. From Calibration Curve I (Fig 1), the equa-
layers were randomly selected on EW- and WW-treated tion for the best fit line can be written for any position in
surfaces. At each location, Raman spectra were obtained the line scan (formula 2), as well as the reference point
at 5 positions (Table 4) across resin/dentin interfaces at Position 1 (formula 3). By dividing formula 2 by formula
(n = 64 per group) through a 100X objective lens using 3, formula 4 results (see below). Since Cbis-GMA in the
8 to 11 mW from the 785-nm laser (1.5 μm spot size) resin adhesive layer (Position 1) was assumed equal to
for 60 s (Raman microprobe, Kaiser Optical System). 1.689 mol/L (x2), the normalized 1610 cm-1 peak area
An extra resin-dentin slab was used to specify the time at Position 1 was equal to 1 (y2). The absolute bis-GMA
for re-collecting data based on degree of surface deposi- molar concentration ([bis-GMA]abs) at each spot (x1) could
tion and Raman signal strength. Resin-dentin slabs were be obtained from formula 5 using these values along with
stored for 7 months as described above, before re-col- the normalized 1610 cm-1 peak area at each spot (y1).
lecting data at the same positions. y1 = 0.3727x1 + 0.1134 (2)
y2 = 0.3727x2 + 0.1134 (3)
Monomer Molar Concentration Calculation
Absolute molar concentration of each co-monomer was From formula (2)/(3),
determined using an adaptation of the method reported y1/y2 = (0.3727x1 + 0.1134)/(0.3727x2 + 0.1134) (4)
by Zou et al.37 x2 = 1.689 and y2 = 1; therefore,

444 The Journal of Adhesive Dentistry


Table 2 Bond strength and fractography by storage time, substrate, and CHX treatment
Acid-etched dentin sur- No. of teeth re- Immediate result
face condition (n = 32) done due to PTFs mean (SD) (MPa) [CV]
WW-with-CHX 17 32.8(11.2)a
[34%]
WW-no-CHX 29.9(15.1)a
[51%]
EW-with-CHX 1 47.5(14.0)a
[30%]
EW-no-CHX 51.5(10.2)a
[20%]
Within a row, groups with the same lowercase letters are not significantly different using a paired-sample t-test. Solid brackets show significant difference
using two-sample t-test. Dotted brackets show no significant difference using a paired-sample t-test. Abbreviation: CHX: chlorhexidine; CV: coefficient of
variation; PTFs: pre-test failures; M: mixed failure; J: joint failure; CR: resin-based composite cohesive failure; CD: dentin cohesive failure; ¥percentage of
specimens fractured solely at the top of hybrid layers (short-term fractography followed the same trend).
y1 = (0.3727x1 + 0.1134)/((0.3727x1.689)+ 0.1134)
x1 = (0.743y1 - 0.1134)/0.3727 (5)
Since monomer dissolution was expected during stor-
age, the assumption of equal C bis-GMA between that
in the resin block and in the resin adhesive layer of
resin-dentin bond specimens was no longer valid for the
long-term experiment. Therefore, a known Cbis-GMA from
a resin block was used as a reference for the long term.
The normalized 1610 cm-1 peak area was calculated by
dividing the 1610 cm-1 peak area at each scanned spot
by the mean 1610 cm-1 peak area obtained from the
resin block at the beginning and the end of the experi-
ment on that day. For the long-term experiment, x2 and
y2 in formula 4 referred to Cbis-GMA in the resin block,
which was equal to 1.689 mol/L, and the normalized
1610 cm-1 peak area of the resin block, which was
equal to 1, respectively. Since x2 and y2 in formula 4
were the same value as those at short-term, the rest of
the calculation was the same as in the short-term. The
long-term absolute bis-GMA molar concentration (x1) at
each scanned spot was obtained by using the normal-
ized 1610 cm-1 peak area at each spot (y1) in formula 4.
Absolute TEG-DMA molar concentration calculation
For both short- and long-term data, the 1610:605 ratio
at each spot (x3) was used in formulas 6 and 7 (the
best fit lines from Calibration Curve II, Fig 2) to obtain
relative bis-GMA molar concentration (y3 in formula 6)
and relative TEG-DMA molar concentration (y4 in for-
mula 7).
y3 = -0.2911(x3)2 + 1.7715x3 - 0.3201 (6)
y4 = 0.5096(x3)2 - 3.0955x3 + 4.6958 (7)
Then, the absolute bis-GMA molar concentration (x 1)
from formula 5 and relative bis-GMA (y3) and TEG-DMA
(y4) molar concentration from formulas 6 and 7, respect-
ively, were used in formula 8 to obtain absolute TEG-
DMA molar concentration (x4).
x4 = x1y4/y3 (8)
Vol 16, No 5, 2014
Talungchit et al
1-year result Fractography (%) at 1 year
mean (SD) (MPa)
[CV] M J CR CD
29.8(11.8)a [40%] 3.1 90.6 0 6.3
(¥81.3)
26.2(11.0)a [42%] 6.3 93.8 0 0
(¥84.4)
45.1(12.4)a [28%] 12.5 62.5 0 25.0
(¥18.8)
46.0(11.3)a [49%] 31.6 46.9 3.1 18.8
(¥28.1)
Statistical Analyses
Statistical methods are described in Tables 2 to 4.
Additionally, the Weibull regression model was used
to determine effects of storage time, type of bonding
protocol, and their interaction on μTBS. Statistical sig-
nificance was set at p < 0.05, and marginal significance
was defined as 0.05<p<0.01. SAS for Windows (v9.3,
SAS Institute; Cary, NC, USA) was used for all analyses.
RESULTS
Comparisons of resin-dentin μTBS and fractography be-
tween groups are reported in Table 2. Specimens bonded
using ethanol rinsing (EW) had a significantly higher μTBS
than WW groups with or without CHX treatment, both im-
mediately and after 1-year storage (p < 0.0001). Consis-
tent with the results of the paired-sample t-test, Weibull
regression model analysis (Fig 3) revealed no significant
difference in μTBS between EW-with-CHX and EW-no-CHX,
nor between WW-with-CHX and WW-no-CHX. Within each
bonding protocol, there was no significant difference
between short- and long-term μTBS, although there was
a marginally significant reduction in μTBS found in EW-
no-CHX (p = 0.0558). Nanoleakage was observed in all
specimens and increased with storage (Fig 4). WW groups
showed higher percentages (33% to 100%) of spotted na-
noleakage with generalized distribution throughout hybrid
layers, while most sections (73% to 100%) from EW pre-
sented localized reticular nanoleakage.
Table 3 summarizes differences in hybrid layer mor-
phology among groups. Non-significantly smaller collagen
fibril diameter was observed in EW vs to WW. Only WW-
with-CHX treated specimens showed a significantly larger
interfibrillar width compared to other protocols after short-
and long-term storage times. After storage, mean fibril
diameter was significantly decreased in all groups, while
interfibrillar width was increased in all groups, with a sig-
nificant difference in WW-with-CHX. In general, the hybrid
layer thickness in EW was not different from that in WW.
445
Talungchit et al

100 95% 100 95%

Comulative Probability of Failure (%)

Comulative Probability of Failure (%)

80 80

60 60
EW-CHX 1 year EW-CHX
EW 1 year EW
EW-CHX same day WW-CHX
40 EW same day 40 WW

20 20

0
5% 0 5%
0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100
a TBS (MPa) b TBS (MPa)

Fig 3 a: Weibull curves of ethanol saturated groups with and without chlorhexidine application on same day and after 1 year. b:
Weibull curves of ethanol and water saturated groups with and without chlorhexidine application at 1 year. Weibull regression model
showed that the survival strength for EW-with-CHX was 1.42 times of that for WW-with-CHX when μTBS of WW-with-CHX was used as
the reference level. The survival strength for EW-no-CHX was 1.52 times of that for WW-no-CHX when μTBS of WW-no-CHX was used
as the reference level.

μTBS 51.45 Fig 4 Nanoleak-

47.49 45.14 46.01 age classification
(MPa) 32.77
29.80 29.91 and its percent-
26.18
age according to
100%
Heavy reticular treatment groups.
90% Reticular type: a
Percentage of nanoleakage (%)

discontinuous silver
80% deposition within
70% the hybrid layer.
Spotted type: iso-
60% lated spots within
Small reticular the hybrid layer or
50%
resin adhesive.
40% The spotted type
30% was observed ex-
clusively within the
20% hybrid layer. CHX
10% Heavy spotted reduced nanoleak-
age severity in the
0% long-term EW group,
WW-CHX WW-CHX WW-no-CHX WW-no-CHX EW-CHX EW-CHX EW-no-CHX EW-no-CHX
short term long term short term long term short term long term short term long term which was consis-
tent with the μTBS
Treatment groups result. *Marginally
significant differ-
Small spotted
ence in μTBS.

Similar to fibril diameter, the long-term hybrid layer thick- Bis-GMA and TEG-DMA molar concentrations (Cbis-GMA
ness was lower than in the short-term in all protocols, with and CTEG-DMA) across resin/dentin interfaces are pre-
significant differences in protocols without CHX. sented in Table 4. Both Cbis-GMA and CTEG-DMA decreased
Ethanol-wet bonded specimens showed significantly as a function of depth into hybrid layers. Short- and
more distinct collagen banding after long-term storage com- long-term Cbis-GMA in EW was higher than that in WW at
pared to WW regardless of CHX treatment (p < 0.0001) all positions within hybrid layers. Similar results were
(Fig 5). Significantly less collagen banding after 1-year stor- observed in short-term CTEG-DMA, while the long-term re-
age was observed in all bonding protocols. However, CHX sults showed higher CTEG-DMA in EW as compared to WW
seemed to preserve collagen banding in both WW and EW only at the lowest two positions of hybrid layers. In both
groups, as demonstrated by a lower reduction in percent- groups, Cbis-GMA and CTEG-DMA decreased after 7-month
age of collagen banding in CHX-treated groups (Table 3). storage at all positions.

446 The Journal of Adhesive Dentistry

 Talungchit et al

Table 3 Fibril diameter, interfibrillar width, hybrid layer thickness, and collagen banding according to treatment
groups

Acid-etched Mean fibril diameter Mean interfibrillar width Hybrid layer thickness Collagen banding (%)
dentin surface (SD) (nm) (SD) (nm) (μm)
condition
Short-term Long-term Short-term Long-term Short-term Long-term Short-term Long-term
WW-with-CHX 50.95b A 45.23a,B 29.46a,B 33.74a,A 4.67b,A 4.19b,c,A 86.67a,A 33.33a,B
(10.11) (11.27) (3.35) (3.48) (0.55) (1.03)

WW-no-CHX 58.23a,A 43.0a,b,B 23.59b,A 26.66b,A 6.17a,A 4.85a,B 100b,A 33.33a,B

(10.38) (8.65) (3.11) (3.22) (0.82) (0.97)

EW-with-CHX 47.81b,A 39.42b,B 23.76b,A 24.61b,A 4.84b,A 4.45a,b,A 100b,A 86.67b,B

(7.81) (5.58) (3.14) (3.17) (0.92) (0.60)

EW-no-CHX 49.62b,A 45.43a,B 24.24b,A 23.46b,A 4.50b,A 3.82c,B 100b,A 66.67b,B

(9.07) (7.17) (3.16) (3.14) (0.46) (0.47)

Within a row, groups with the same upper case letter are not significantly different using a two-sample t-test for the mean collagen diameter and mean colla-
gen interfibrillar width, nonparametric Wilcoxon rank-sum test for mean hybrid layer thickness, and chi-square test and Fisher’s exact test for the presence of
collagen banding. Within a column, groups with the same lower case letter are not significantly different using one-way ANOVA with post-hoc Tukey-Kramer’s
test or Tukey’s Studentized Range test for fibril diameter, short-term hybrid layer thickness, and log transformed data of interfibrillar width (normally distrib-
uted data), ANOVA was used for the ranked data followed by the post-hoc Bonferroni multiple comparison test for long-term hybrid layer thickness data (lack
of normality), and Fisher’s exact test and the Cochran-Mantel-Haenszel chi-square test for the presence of collagen banding.

a 0.2 μm b 0.2 μm c 0.2 μm d 0.2 μm

h AR AR
h
h
t d
d t t
h
d t
d
e 1μm f 1μm g 1μm h 1μm

Fig 5 A to D show representative ultrastructural TEM micrographs after 1-year storage. A: WW-with-CHX; B: EW-with-CHX; C: WW-no-
CHX; D: EW-no-CHX. EW showed smaller fibril diameter and interfibrillar width compared to WW. TEM section with no collagen band-
ing was demonstrated in WW. E to H show nanoleakage classification. E: small spotted (WW-no-CHX short-term specimen); F: heavy
spotted (WW-with-CHX short-term specimen); G: small reticular (EW-with-CHX long-term specimen); H: heavy reticular (EW-no-CHX
long-term specimen) nanoleakage. Abbreviations: h: hybrid layer; d: underlying sound dentin; t: dentinal tubule; AR: adhesive resin.

DISCUSSION to its superior performance over other resin monomer

A relatively hydrophobic 70% bis-GMA/28.75% TEG-DMA
co-monomer blend was used to form hydrolytically resis-
tant hybrid layers. Resin primer (50% resin/50% ethanol)
was formulated in the 3-step etch-and-rinse adhesive due
mixtures with different resin:ethanol ratios.30 The moist
dentin bonding technique was applied in all groups to pre-
vent collagen collapse. In EW, demineralized dentin was
blotted before and between each ethanol rinsing to ac-
celerate evaporation of water via low-boiling water-ethanol

Vol 16, No 5, 2014 447

Talungchit et al

Table 4 Immediate and 7-month bis-GMA and TEG-DMA molar concentrations (Cbis-GMA and CTEG-DMA) across the
resin/dentin interfaces formed by WW and EW (n = 64)

Positions Immediate results Long-term result (7-month storage)

Groups

Mean Mean Cbis-GMA CTEG-DMA

Cbis-GMA CTEG-DMA
(SD) in (SD) in Mean (SD) in % mean de- Mean (SD) in % mean de-
mol/L mol/L mol/L crease from mol/L crease from
immediate immediate
1 AR 1.689 (0)A 1.428 1.358 (0.330)A* 19.61 1.136 (0.645)A* 20.44
(0.895)A

2 UHL 0.997 0.806 0.655 (0.287)A* 34.29 0.685 (0.492)A 15.03

(0.401)B (0.571)A

3 In hybrid layer: 1 0.588 0.565 0.369 (0.242)B* 37.28 0.477 (0.426)A 15.49
WW

ȝm below UHL (0.349)B (0.465)A

4 In hybrid layer: 2 0.334 0.391 0.161 (0.215)B* 51.89 0.150 (0.204)B* 61.73
ȝm below UHL (0.238)B (0.445)A

5 LHL 0.201 0.170 0.067 (0.120)B* 66.72 0.097 (0.207)B* 43.07

(0.225)B (0.223)B

1 AR 1.689 (0)A 1.188 1.354 (0.284)A* 19.81 1.117 (0.609)A 5.97

(0.621)A

2 UHL 1.170 0.973 0.749 (0.297)A* 35.97 0.654 (0.455)A* 32.77

(0.339)A (0.624)A

3 In hybrid layer: 1 0.818 0.670 0.495 (0.239)A 39.46 0.410 (0.292)A* 38.76
EW

ȝm below UHL (0.304)A (0.488)A

4 In hybrid layer: 2 0.559 0.417 0.272 (0.200)A* 51.38 0.268 (0.278)A* 35.77
ȝm below UHL (0.316)A (0.259)A

5 LHL: 3 ȝm below 0.354 0.301 0.142 (0.141)A* 59.85 0.156 (0.202)A* 48.33
UHL (0.252)A (0.419)A

Mean Cbis-GMA and CTEG-DMA of pure resin adhesive were 1.689 and 1.179, respectively. Within each storage time, means with the same upper case letters
showed no significant difference of Cbis-GMA or CTEG-DMA between WW and EW groups at each position using a two-sample z-test or a nonparametric Wilcoxon
rank-sum test. (*) indicates significant reduction of the mean Cbis-GMA or CTEG-DMA after 7-month storage as compared to that at short-term at each position
within each bonding group using a paired-sample t-test or a nonparametric Wilcoxon signed-rank test (p < 0.05 in each instance). Abbreviations: WW: water-
wet bonding group; EW: ethanol-wet bonding group; Cbis-GMA: bis-GMA molar concentration; CTEG-DMA: TEG-DMA molar concentration; SD: standard deviations;
AR: adhesive resin layer (5 μm from adhesive-hybrid layer interface); UHL: upper hybrid layer (adhesive/hybrid layer interface); LHL: lower hybrid layer (hybrid
layer/sound dentin interface).

azeotropes. Chlorhexidine digluconate aqueous solution
and ethanol-solvated chlorhexidine diacetate were used
in WW and EW due to their miscibility in water and etha-
nol, respectively. Although the substantivity of CHX diglu-
conate and diacetate to mineralized and demineralized
dentin was reported,8,19 differences in the binding perfor-
mance of these CHX compounds within 30-s application
time are unknown. However, an application of a miscible
CHX solution to water-saturated and ethanol-saturated
dentin should maximize CHX concentration within hybrid
layers. Without rinsing, excess CHX may be incorporated
into the primer and be released slowly over time.8 The
high μTBS values achieved indicate that CHX may not
interfere with resin infiltration and polymerization.12 It
was reported that although 1% chlorhexidine diacetate
powder did not alter the degree of conversion of 70% bis-
GMA/28.75% TEG-DMA resin, it did increase the modulus
of elasticity.6
Within the limits of this study, all null hypotheses were
rejected. The improvement in μTBS of hydrophobic bis-
GMA/TEG-DMA resin adhesive to dentin under EW may
be explained by Hoy’s solubility parameters. By replacing
water within acid-etched dentin with ethanol, the dentin
matrix was made more hydrophobic. Hoy’s solubility par-
ameter for the total cohesive forces (δt) shifted from
30.5 (J/cm3)1/2 to 25.1 (J/cm3)1/2, allowing miscibility
of the hydrophobic bis-GMA/TEG-DMA resin adhesive
(δt = 21.2 [J/cm3]1/2) in ethanol-saturated dentin.28
This speculation was supported by Raman results. EW
had higher CM at all positions in hybrid layers in the
short term, indicating more efficient infiltration of a hy-
drophobic bis-GMA/TEG-DMA resin into demineralized
dentin as compared to WW.33 Nevertheless, significant
reduction of CM at nearly all positions in EW after stor-
age supports the need to further improve resin-dentin
bonding durability.

448 The Journal of Adhesive Dentistry


Besides minimizing miscibility mismatch, preservation
of demineralized collagen morphology plays an important
role in monomer infiltration into demineralized dentin.
Based on this clinically relevant protocol, EW showed
smaller interfibrillar widths and collagen fibril diameters
than did WW at both storage times. These results may
be explained by the lower H-bonding capacity of ethanol
(δh = 20 [J/cm3]1/2) vs water (δh = 40 [J/cm3]1/2); there-
fore, ethanol allows more collagen interpeptide (dry col-
lagen δh = 14.8 [J/cm3]1/2) H-bond formation. Increased
interpeptide H-bonding between collagen peptides may
slightly reduce collagen interfibrillar width. In turn, this re-
duction increases stiffness of the collagen matrix,21 allow-
ing effective resin infiltration with minimal further collagen
collapse during resin adhesive application and solvent
evaporation.9 As a result, interfibrillar spaces in EW are
preserved for effective resin infiltration.21,28 Application
of 2% CHX digluconate may cause shrinkage of collagen
fibrils, since short-term fibril diameter in WW-with-CHX was
significantly smaller than that in WW-no-CHX.
In our study, only 3-μm-thick hybrid layers were used for
monomer molar concentration measurement in both EW
and WW. Within this thickness, monomers may occupy
a smaller unit volume of the hybrid layers formed by EW
than that formed by WW. Nevertheless, monomer molar
concentrations in hybrid layers formed by EW were higher
than that formed by WW. Therefore, besides better mono-
mer infiltration, it is assumed that there was less volume
of EW hybrid layers occupied by water and residual solvent
compared to that of WW.
Despite enabling better monomer infiltration, both WW
and EW showed nanoleakage. Reticular nanoleakage ob-
served in EW might be attributed to isolated incomplete
resin infiltration within hybrid layers29 rather than incom-
plete removal of water,35 since this reticular form of na-
noleakage was not present in a majority of short-term WW
specimens.
Reduction in μTBS and CM, as well as signs of ultras-
tructural degradation (eg, increasing of interfibrillar width,
reduction of collagen fibril diameter and hybrid layer thick-
ness, loss of collagen banding), affirmed the deterioration
of resin/dentin interfaces after storage. Entrapment of
water within the resin/dentin interface causes phase sep-
aration,34 poor polymerization,17 and low bond strength.
After storage, unpolymerized monomer may leach out,
reducing monomer concentration in the polymer matrix.
Water may penetrate between loosely bound polymer
chains, causing hydrolysis and swelling of the polymer
matrix, thereby increasing interfibrillar width and decreas-
ing collagen fibril diameter.
This ultrastructural degradation of collagen fibrils was
pronounced in WW-treated specimens. Reduction in μTBS
was observed after storage. However, due to high coef-
ficients of variation, the statistical analysis could not dis-
tinguish the difference between the short-term and 1-year
μTBS results. Consistent with previous studies, CHX had
no detrimental effect on resin-dentin bond strength.7,31
However, the combined usage of chlorhexidine with WW
did not improve bond durability. Besides residual water,
elution of water-soluble chlorhexidine digluconate from
Vol 16, No 5, 2014
Talungchit et al
hybrid layers may induce water sorption and elution of
unreacted monomers, resulting in a significant increase
in collagen interfibrillar width in WW-with-CHX after 1-year
storage.11 CHX may also leach out rapidly through swelled
resin matrix in WW,14 permitting enzymatic degradation
of a large amount of uninfiltrated collagen matrix by en-
dogenous dentin proteases. Both hydrolytic degradation
of resin and enzymatic degradation of collagen within WW
may reduce μTBS over time and mask the enzyme inhibi-
tion of chlorhexidine.
Deterioration of hybrid layers was less pronounced in
EW. Mean μTBS and CM in EW after storage remained
higher than that in WW. Moreover, EW showed signifi-
cantly more distinct collagen banding after long-term stor-
age compared to WW regardless of CHX treatment. In EW,
collagen and endogenous collagenolytic enzymes may be
impregnated by the hydrophobic resin matrix. Although
small amounts of water may persist within hybrid layers
formed by EW, the highly polymerized hydrophobic resin
matrix limits water sorption and prevents the formation of
water channels through the swelled resin polymer matrix.
Without water, proteolytic enzymes lose their collagen-
cleaving function.27 Besides a direct inhibition effect of
CHX,5,32 we speculated that by entrapping endogenous
proteases and eliminating water, ethanol indirectly dimin-
ishes collagenolytic activities of these enzymes. CHX may
only inhibit endogenous dentin proteases that are not
resin infiltrated. Since CHX diacetate has low solubility in
water, it may remain in hybrid layers to effectively inhibit
the collagenolytic activity of MMPs for a long period of
time. In this study, EW-with-CHX showed no significant
reduction in μTBS after storage (p = 0.4593), while mar-
ginally significant reduction was observed in EW-no-CHX
(p = 0.0558). After water storage, a higher percentage
of specimens in EW-with-CHX (20%) showed distinct col-
lagen banding compared to that in EW-no-CHX. Therefore,
the combined usage of CHX tended to further improve the
durability of the resin-dentin bond formed by EW.
A higher percentage of joint failure at the top of the
hybrid layer in WW (81% to 84%) compared to that in
EW (19% to 28%) (Table 2) may be partially explained by
higher CTEG-DMA in the resin adhesive layer of WW. Fracture
at this junction may result from the abrupt change be-
tween low-modulus resin matrix formed by high CTEG-DMA4
and a higher modulus hybrid layer.36
CONCLUSION
The results of this study provide direct evidence of the
efficacy of a clinically relevant EW on facilitating infiltra-
tion of a hydrophobic resin adhesive for durable resin-
dentin bonds. The combined usage of 2% chlorhexidine
diacetate and EW improved the longevity of resin-dentin
bonds. Further study on developing resin monomers
with CHX functionality to be incorporated in the polymer
matrix and improving polymerization of resin adhesive
may prolong enzymatic inhibition and prevent monomer
elution in the hybrid layer for durable resin-dentin bonds
under EW.
449
Talungchit et al
ACKNOWLEDGMENTS
This study was supported by the Ministry of Science and Technol-
ogy, Thailand, and the Biomaterials Lab at the University of Iowa’s
College of Dentistry. The authors are grateful to Bisco Inc. and
Pashley Lab for providing experimental resin and chlorhexidine
diacetate, respectively. We greatly appreciate the support of Dr.
Marcos Vargas and EMRB, University of Iowa, for guidance on the
TEM technique.
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The Journal of Adhesive Dentistry