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Effect of solvent content on resin hybridization in wet dentin

bonding

Yong Wang1, Paulette Spencer1,2, Xiaomei Yao1, and Bohaty Brenda2

1Department of Oral Biology, University of Missouri-Kansas City School of Dentistry, Kansas City, Missouri

64108

2Department of Pediatric Dentistry, University of Missouri-Kansas City School of Dentistry, Kansas City,

Missouri 64108

Abstract

With wet bonding techniques, the channels between the demineralized dentin collagen fibrils are

filled with debris, solvent, and water. Commercial adhesives include solvents such as ethanol oracetone to facilitate resin-infiltration into this wet substrate. Under in vivoconditions, the solvent

may be diluted because of repeated exposure of the material to the atmosphere, or concentrated

because of separation of the bonding liquids into layers within the bottle. The purpose of this study

was to investigate the effect of different concentrations of ethanol (1050%) on infiltration of the

adhesive resin and collagen fibril encapsulation in the adhesive/dentin interface using light

microscopy, micro-Raman spectroscopy, and scanning electron microscopy. The results indicated

that under wet bonding conditions the hybridization process was highly sensitive to the initial solvent

concentration in the adhesive system. The staining and scanning electron microscopy results showed

that the quality of the interfacial hybrid layer was poor at the lower (10%) or higher (50%) ethanol

content. Micro-Raman analysis indicated that there was a distinct difference in the degree of adhesive

penetration among adhesives containing different concentrations of ethanol. Adhesives containing

10 or 50% ethanol did not realize effective penetration; the penetration of the adhesive monomers

increased dramatically when the initial ethanol content was 30%. The amount of solvents are essentialfor achieving effective bonding to dentin.

Keywords

dentin; adhesive; Raman; solvent; interface; hybridization

INTRODUCTION

Bonding of current one-bottle adhesive systems that acid-etch the dentin relies on resin-

infiltration and encapsulation of collagen fibrils in the wet demineralized dentin to form the

hybrid layer or resindentin interdiffusion zone. Ideally, this layer/zone is a structurally

integrated resincollagen biopolymer hybrid that provides a continuous and durable linkbetween the bulk adhesive and dentin substrate. However, upon removal of the dentin mineral

by acid, the demineralized collagen matrix is suspended in water. Under these conditions, the

quality of the resincollagen layer is highly sensitive to the specific wetting characteristics and/

or composition of the adhesive system.

Correspondence to:Y. Wang; e-mail: [email protected].

NIH Public AccessAuthor ManuscriptJ Biomed Mater Res A. Author manuscript; available in PMC 2008 November 4.

Published in final edited form as:

J Biomed Mater Res A. 2007 September 15; 82(4): 975983. doi:10.1002/jbm.a.31232.

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The basic ingredients of mostly one-bottle bonding agents are hydrophilic and hydrophobic

monomer mixtures and solvents. The most popular solvents in use today are acetone, ethanol,

and water. Acetone and ethanol are frequently used as the high vapor pressure solvents. The

presence of these solvents is essential for achieving effective bonding to demineralized dentin

substrates, since they can promote wetting of the dentin substrate and displace water that is

within the wet demineralized dentin matrix. Under wet bonding conditions, the type and

concentration of solvent are expected to have a big impact on the ability of the adhesive

components to tolerate water.

Previous studies suggest that solvents have important effects on the bond strength of one-bottle

dentin bonding systems that require separate acid etching.1-4These studies indicate that

varying the solvent content affects the resulting bond strength. In one study, it was suggested

that loss of solvent because of repeated opening of bottles might cause lower bond strength of

acetone-based one-bottle bonding agents.5However, in another study, when the acetone

content of the adhesive was increased, the microtensile bond strength decreased from the

highest value of 64 MPa (37% acetone) to 38 MPa (67% acetone).6The reasons for the

differences in bond strength as a function of solvent content of dentin bonding agents remain

unclear. The authors assigned a reduction in bond strength to interfacial cracks in specimens

with acetonerich bonding agents.6

It can be speculated that differences in composition and concentration of solvents could affectthe penetration of adhesive bonding agents and introduce differences in the structure of the

bond formed at the adhesive/dentin interface. In addition, it is agreed that the presence of

solvents with other ingredients of one-bottle bonding agents must have an optimum

concentration. To date, questions regarding adhesive dentin bonding have mainly been

investigated using bond strength studies in combination with morphological analyses. Very

few techniques are available that can evaluate the effects of solvent content on the penetration

of resin monomers, and interfacial structure of the resin-infiltrated layer. Raman microscopy

has been shown to be a promising analytical technique for studying the composition and

structure of bonding of resin to dentin.7-14To understand better the relationship between the

solvent concentration and bonding, the morphology, quality, and chemistry of the interfaces

between dentin and Single Bond (SB) adhesives containing different ethanol concentrations

were studied using staining/light microscopy, scanning electron microscopy (SEM), and

micro-Raman spectroscopy. This study tested the hypothesis that varying the solvent contentof a one-bottle adhesive system would affect the penetration of resin and integrity of the

interface using wet bonding techniques.

MATERIALS AND METHODS

Adhesive/dentin specimen preparat ion

Extracted unerupted human third molars stored in 0.96% w/v phosphate buffered saline (PBS)

containing 0.002% sodium azide at 4C were used. The teeth were collected after the patients'

informed consent was obtained under a protocol approved by the University Adult Health

Sciences IRB. The occlusal one-third of the crown was removed by means of a water-cooled

low-speed diamond saw (Buehler, Lake Bluff, IL). A smear layer was created by abrading the

dentin with 600 grit SiC under water for 30 s. The prepared dentin specimens were selected

for treatment with Single Bond (SB) adhesive (3M ESPE, St Paul, MN) containing different

concentrations of solvents. The procedure to make SB adhesive system with different ethanol

contents is as follows: the adhesive resin was taken from SB adhesive bottle and allowed to

evaporate in a dark box. The weight loss was monitored until no loss was recorded. At the same

time, FTIR spectra of the above resins were detected by PerkinElmer Spectrum One

spectrometer in the ATR sampling mode, so that OH band of ethanol at 1040 cm1was

tracked until the peak intensity no longer decreased. After the solvent in SB adhesive was

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completely evaporated, the ethanol content of the original bottle was calculated to be 32.5%

(wt) based on weight loss method. Then 10, 30, and 50% (wt) solutions were prepared after

the appropriate amount of ethanol added and the mixtures were shaken overnight. These

adhesive mixtures were applied to the above prepared dentin specimens using wet bonding

technique according to manufacturer's instructions. The dentin was etched with 35%

phosphoric acid gel and rinsed with water; then blotted with absorbent paper to leave a visibly

moist surface. Two consecutive coats of a bonding agent were applied with a fully saturated

brush. The surface was gently dried for 5 s with an oil-free, moisture-free air spray (

18 psi).After air-drying, the adhesive coated dentin surfaces were light cured for 20 s using a

conventional halogen light unit (Spectrum light, Dentsply, Milford, DE). These specimens

were stored for 24 h in PBS at 25C before sectioning. The treated dentin surfaces were

sectioned perpendicular and parallel to the bonded surface.

Differential staining technique

The rectangular, 10 2 1.5 mm3, slabs of the three adhesives dentin interface specimens

were mounted on a methacrylate support and 5-m thick sections were cut from the face of the

slab using a tungsten carbide knife mounted on a Polycut S sledge microtome (Leica,

Germany). Following recovery of the microtomed sections, the remaining fraction of the

adhesive/dentin interface slabs was used for micro-Raman spectroscopic analysis and field

emission scanning electron microscopy. Thus, the same slab was used for light microscopic,

micro-Raman, and SEM analyses. Differential staining was accomplished with Goldner's

trichrome 15,16and the sections were examined and photographed at 100 magnification with

a Nikon E800 light microscope. The width of dentine demineralization and the exposed

collagen layer were determined by measuring directly from photomicrographs whose exact

magnification was established with a stage micrometer.

Micro-Raman spectroscopy

The remaining fraction of above interface slabs was prepared for investigation using micro-

Raman spectroscopy. The micro-Raman spectrometer consisted of an argon ion laser beam

(514.5 nm) focused through a 60 Olympus water immersion objective (NA 1.2) to a 1.5

m beam diameter. Raman spectra were acquired at positions corresponding to 1 m intervals

across the adhesive/dentin interface using the computer controlledxyzstage with a minimum

step width of 50 nm. Two consecutive scans of spectra (with 90 s accumulation time each)were obtained from each site. Multiple sites across the interface of each specimen were

examined spectroscopically. The laser power was 7 mW. Since the micro-Raman technique

is nondestructive, these same specimens were available for analysis using SEM.

Scanning electron microscopy

Following micro-Raman analysis, the specimens described earlier were prepared for SEM

examination. To evaluate the presence of interface and resin tags, the specimens were subjected

to 30 s of 5NHCl, washed with water, followed by soaking in 5% NaOCl for 30 min. After

drying, the prepared specimens were mounted on aluminum stubs and sputter coated with

20 nm of gold-palladium. Specimens were examined at a variety of magnifications and tilt

angles in a Philips XL30 ESEM-FEG (Philips, Eindhoven, Netherlands) at 10 kV.

RESULTS

Staining microscopic technique

Representative light micrographs of Goldner's trichrome stained sections of the adhesive/

dentin interface are shown in Figures 1-3. Using these trichrome differential stains, mineralized

dentin collagen usually is stained green, unprotected demineralized collagen/protein stains red

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and pure adhesive is either stained pale beige or remains unstained. If the adhesive does not

penetrate the full depth of the demineralized layer or does not envelop the collagen/protein,

the Goldner's stains interact with the exposed collagen causing it to appear red. Representative

micrographs of stained adhesive/dentin specimens that were treated with the SB adhesive

containing 10% ethanol are shown in Figure 1. A spotted, discontinuous red layer is clearly

seen between the unstained adhesive and the green stained mineralized dentin. The width of

this red layer was about 3.1 m. It was also noticed that a thin green line was seen on the top

of the dentin surface [arrows, Fig. 1(B)]. The overall interface lacks structural integrity;separation was noted between the adhesive and dentin layer. Representative micrographs of

stained dentin sections treated with the SB adhesive containing 30 and 50% ethanol are shown

in Figures 2 and 3, respectively. For both cases, a uniform, continuous, red layer distinct from

either the adhesive or dentin is visible along the length of the adhesive/dentin interfaces. The

width of both the red layers was similar, about 6.5 m. The interfacial zone shows orange-red

color when it is treated with SB containing 30% ethanol. The color of the interfacial zone is

dark red when treated with SB containing 50% ethanol. The difference in color represents the

extent of exposed collagen at the interface. The dark red color indicates that the exposed

collagen remains unprotected and thus, is totally available for reaction with the stains. The

orange color indicates the resin-infiltrated layer where exposed collagen was slightly more

encapsulated with adhesive.15-17

Scanning electron microscopy

Representative SEM micrographs of the dentin interfaces with SB adhesive systems containing

10, 30, and 50% ethanol are shown in Figures 4-6. The common exposure technique, in which

the sectioned adhesive/dentin interface specimen was treated with 5NHCl (30 s) followed by

5% NaOCl (30 min), was used to reveal the adhesive penetration into the dentin. Representative

SEM micrographs of cross sections of adhesive/dentin specimens that were treated with SB

adhesive containing 10% ethanol after the acid/bleach treatments are shown in Figure 4. Short,

funnel-shaped resin tags were clearly observed in the 10% ethanol specimens by this SEM

evaluation. However, it was difficult to reveal the microscopic presence of an acid/bleach

resistant hybrid layer in the specimens. In addition, a separation between the adhesive layer

and the top of dentin was noted [Fig. 4(A)]. For the specimens treated with SB adhesive

containing 30% ethanol, the adhesive penetrated into the dentin and formed a well-defined

acid/bleach resistant hybrid layer (Fig. 5). Resin tags were longer, and also show small lateralbranches. For the specimens treated with SB adhesive containing 50% ethanol, a thick layer

was observed (Fig. 6). However, the surface of the hybrid layer was irregular, with a very

rough, porous appearance, with sites of incomplete adhesive penetration readily identified in

the micrographs. Adhesive tags were short and irregular (Fig. 6).

Micro-Raman spectroscopy

Representative micro-Raman mapping spectra of the dentin interfaces with SB adhesives

containing 10, 30, 50% ethanol are shown in Figure 7(AC), respectively. All spectra were

recorded from 8751785 cm1, which spans the fingerprint region associated with adhesive,

collagen, and mineral. The peaks associated with the adhesive occur at 1720 cm1(carbonyl),

1609 cm1(phenyl C=C), 1113 cm1(COC); the major peaks associated with the collagen

appear at 1242 cm1(amide III), 1273 cm1(amide III), 1453 cm1(CH2), and 1667 cm1

(amide I). Those spectral features associated with the mineral occur at 961 (PO symmetricstretch) and 1072 cm1(carbonate).

As shown in Figure 7, the first three spectra were acquired from pure adhesive. Peaks associated

with the adhesive and collagen components of dentin were noted in the fourth spectrum. The

Raman peak of the PO group in the 11th or 12th spectrum suggested that this represented

the bottom of the demineralized dentin layer. Dentin was demineralized to a similar depth for

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the dentin specimens treated with adhesives containing 10, 30, 50% ethanol. Overall, the

intensity of the Raman bands associated with the adhesive (1113, 1609, 1720 cm1) decreased

as a function of depth, indicating the gradual decrease of adhesive penetration into the

demineralized dentin.In situspectra recorded at the second micrometer positions of the dentin

interfaces with SB adhesives containing 10, 30, 50% ethanol are shown in Figure 8. Major

spectral changes have been marked with arrows. In comparison of these spectra, we note that

the bands associated with collagen (1667 cm1) were dominant in the 10% ethanol specimens,

while the features associated with adhesive (1720 and 1609 cm1

) were dominant in the 30%ethanol specimens. The contribution from spectral features associated with the adhesive in the

50% ethanol specimens were decreased when compared with that in the 30% ethanol

specimens.

The ratios of the relative integrated intensities of the spectral features associated with the

adhesive and collagen were calculated, to determine differences in adhesive penetration as a

function of spatial position across the interfaces. The COC in BisGMA monomer (1113

cm1) and the CH2in HEMA/BisGMA (1453 cm1) were used to monitor the concentration

of the adhesive monomers and the amide I peak (1667 cm1) was selected for collagen. Because

of the overlapping of the collagen amide I peak with adhesive peaks, difference spectral method

was used for peak area measurement.12,18Figure 9 shows the ratios of 1113/1667 as a function

of spatial position across the dentin interfaces with adhesives containing 10, 30, 50% ethanol.

All showed a gradual decrease, while there were differences in the ratios of 1113/1667 as afunction of position. The ratios of 1113/1667 were the highest at each position for SB adhesive

containing 30% ethanol, and were the lowest for SB adhesive containing 10% ethanol.

Figure 10(AC) represent the adhesive penetration and degree of dentin demineralization as a

function of depth for SB adhesive containing different contents of ethanol. The ratios of the

relative integrated intensities of the spectral features from the mineral (961 cm1, PO) and

collagen (1453, CH2) (mineral/matrix ratios) were used to measure the extent of dentin

demineralization. Using this technique, the interfacial profile of adhesive penetration and

depth/degree of demineralization were observed clearly. As shown in Figure 10, dentin was

demineralized to a similar depth of 78 m, indicating well-controlled etching process. The

profiles of adhesive infiltration were totally different for these adhesives containing different

ethanol content. When adhesive contains only 10% ethanol, there was a very limited infiltration

of adhesive monomers (little BisGMA monomer) into the demineralized dentin. There was ademineralized zone with little contribution of both the adhesive monomers and mineral [Fig.

10(A)]; this zone of exposed collagen measures4m (from the 4th to 8th m). When adhesive

contains 30% ethanol, the penetration of HEMA/BisGMA resin and BisGMA monomer

increased dramatically when compared with adhesive containing 10% ethanol. The zone with

little contribution of the adhesive and mineral was narrowed to 12 m [Fig. 10(B)]. The

penetration of adhesive monomers decreased when the ethanol content in the adhesive

increased to 50% [Fig. 10(C)].

DISCUSSION

The hybridization process is very complex and affected by many factors during dentin bonding.19,20Thus, it is desirable that multiple structural and chemical characterizations of an interface

can be done on the same specimen. In this study, direct and comprehensive informationregarding morphology, quality, and chemistry of the interfaces between dentin and adhesives

containing different ethanol concentrations was obtained using staining/light microscopy,

SEM and micro-Raman spectroscopy. This characterization protocol allows us to complete

complementary, physicochemical analyses on the same interface specimens. The results of this

investigation indicated that under wet bonding conditions the hybridization process was highly

sensitive to the solvent concentration in the adhesive system.

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The morphology and properties of the adhesive/dentin interfacial layer are highly affected by

the solvent content in the adhesive. At the lower initial ethanol content (10%), the quality of

the hybrid layer was very poor (Fig. 1), and the hybrid layer formed with this adhesive system

was not resistant to the acid/bleach treatments (Fig. 4). Some areas of de-bonding occurred at

the interface between the hybrid layer and the adhesive. The gaps or separations were possibly

related to water that had penetrated from the underlying dentin. Previous authors have similarly

reported reduced bond strength values with reduced solvent content, that is, Reis et al. reported

that the micro-tensile bond strength values of resindentin specimens were significantlyreduced when adhesives were applied to moist dentin without their solvents.3

The quality of the hybrid layer was improved at the higher initial ethanol content (30 and 50%).

A uniform, acid-resistant layer was observed for these specimens (Figs. 2,3,5,6). However, the

quality of the hybrid layer in 30% ethanol specimens was better than that of 50% ethanol

specimens. This difference may be related to a variety of factors including evaporation of the

high initial content of solvent that leads to porous hybrid layers. Results from previous studies

have indicated that increasing the initial content of acetone in one-bottle bonding agents

decreased their micro-tensile bond strength.6The higher initial solvent content (67%) resulted

in thinner adhesive layers and might leave residual solvent in the adhesive resin, which in turn

lead to pores in the cured adhesive and interfacial layers.6,21Our morphologic results were

consistent with these previous bond strength studies.

Micro-Raman results indicated that there was a distinct difference in the degree of adhesive

penetration among Single Bond (SB) adhesives containing three different concentrations of

ethanol. Adhesive monomers containing 10% ethanol resisted penetration into the wet

demineralized dentin matrices. The penetration of these adhesives monomers increased

dramatically when the initial ethanol content was increased to 30%. It is postulated that the

inclusion of ethanol decreased the viscosity of the adhesive solution, allowing better

penetration. In addition, because ethanol has a water-displacing effect, the impact of water on

the penetration of relatively hydrophobic adhesive components into the wet demineralized

dentin matrix is likely reduced. When the adhesive only contains 10% ethanol, the relatively

high viscosity of the adhesive solution resulted in poor penetration of adhesive monomers into

the wet demineralized dentin layer. Lower ethanol content will also cause thicker adhesive

layers. Apparently the ethanol content is not adequate to displace the residual water within the

demineralized dentin matrix and thus, the penetration of the hydrophobic components isimpeded. With increasing ethanol content, the viscosity of the adhesive solution decreases and

the penetration of the adhesive monomers into the wet demineralized dentin layer is enhanced.

However, when the ethanol content was higher (50%), the penetration of adhesive monomers

decreased again; which may be due to component dilution, or also due to greater chemical

dehydration with higher concentrations of ethanol that could have partially collapsed the

nanochannels between fibrils.22

The differences in solvent concentrations of bonding systems determine their wetting

capability. There must be an optimum concentration of solvents with other ingredients of the

bonding agent. Based on the above morphologic and Raman spectroscopic results, 30% ethanol

appears to be the optimum concentration for the adhesive formulation used in this study. Our

morphological analyses in combination micro-Raman studies provide promising

characterization techniques for evaluating the dentin bonding performance of adhesivesystems. Most current bonding agents are mixtures of hydrophilic/hydrophobic monomers and

solvents. The selection and composition of these ingredients have dramatic effects on the

structure and durability of the bond formed at the adhesive/dentin interface. The formulation

of adhesive systems has mainly been determined based on the results of bond strength studies

in combination with SEM morphologic analyses. This protocol only provides a single measure

at one point; it provides a gross overview as opposed to specific identification of the site that

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experiences breakdown. In addition, many factors from dentin substrates and adhesives could

affect the final bond strength values. The research protocol used in this study could provide a

remarkable tool for the development of dentin bonding agents. The differences in quality and

adhesive penetration of the interfacial zone were clearly observed for adhesives with difference

concentrations of solvent. Direct measurements of the complete chemical profile and

monomer/mineral distribution across the adhesive/dentin interfaces will offer critical data that

is integral to the appropriate selection and determination of adhesive composition.

The results shown in this study also have important clinical relevance. Solvents such as acetone

and/or ethanol are volatile solvents that could easily evaporate from bottles during use of

adhesive systems in the clinical environment. The concentration of solvents may change as a

function of time. Several authors have raised concerns as to the effects of solvent evaporation

in one-bottle adhesives.5,23In an in vitrostudy, the effects of repeatedly opening of bottles

on dentin shear bond strength were evaluated. The acetone-based adhesive had significantly

lower mean bond strength because of the evaporation of the acetone after 3 weeks of simulated

use.5In another study, the four one-bottle bonding agents were tested for phase separation of

the liquids into layers within the bottle.24Since the liquids (resin monomers and solvents) in

the bottles have different densities, phase separation can easily occurs within 12 h, which can

even be visualized by the naked eye. In the clinic, if dentists do not shake the bottles before

each use, agents containing mostly solvent will be applied to the dentin surface; after such

several applications, then primarily resin with less solvent will be applied to the dental cavity.Our results indicated that the amounts of solvent were essential for achieving effective bonding

to dentin. Solvent content has a substantial effect on resin infiltration and the interfacial

structure of the hybrid layer. Adhesives containing higher or lower initial solvent content did

not realize effective penetration and the integrity of the bond formed at the adhesive/dentin

interface was severely compromised. Dentists must pay careful attention to the volatile

characteristics and the differences in density of the ingredients of bonding agents. All bottles

must be shaken thoroughly to obtain a uniform mix before applying to the tooth structure. In

addition, attention should be paid to minimize solvent loss during clinical use by immediately

replacing caps on solvent-based dentin bonding agents.

Acknowledgements

This work is a contribution from the UMKC Center for Research on Interfacial Structure and Properties (UMKC-CRISP).

Contract grant sponsor: National Institute of Dental and Craniofacial Research, National Institutes of Health; contract

grant number: R01DE14392, K25DE015281, K23DE/HD00468

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Figure 1.

Representative light micrographs of the dentin interfaces with SB adhesive containing 10%

ethanol. The overall interface lacks structural integrity; a spotted, discontinuous red layer is

clearly seen between the adhesive and dentin (A), separation is also noted between the adhesive

and dentin layer (B). [Color figure can be viewed in the online issue, which is available at

www.interscience.wiley.com.]

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Figure 2.

Representative light micrograph of the dentin interfaces with SB adhesive containing 30%

ethanol. [Color figure can be viewed in the online issue, which is available at


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Figure 3.

Representative light micrograph of the dentin interfaces with SB adhesive containing 50%

ethanol. [Color figure can be viewed in the online issue, which is available at


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Figure 4.

Representative SEM micrographs of acid-bleach treated dentin interfaces with SB adhesive

containing 10% ethanol. (A) 2000; (B) 4000.

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Figure 5.



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Figure 6.



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Figure 7.Representative micro-Raman mapping spectra of the dentin interfaces with SB adhesives

containing 10% (A), 30% (B), 50% (C) ethanol. [Color figure can be viewed in the online issue,

which is available at www.interscience.wiley.com.]

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Figure 8.

Raman spectra recorded at the second micrometer positions of the dentin interfaces with SB

adhesives containing 10% (A), 30% (B), 50% (C) ethanol. [Color figure can be viewed in the

online issue, which is available at www.interscience.wiley.com.]

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Figure 9.

Adhesive penetration as a function of depth across the dentin interfaces with adhesives

containing 10, 30, 50% ethanol. [Color figure can be viewed in the online issue, which is

available at www.interscience.wiley.com.]

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Figure 10.

Adhesive penetration and degree of dentin demineralization as a function of depth for SB

adhesive containing different contents of ethanol. [Color figure can be viewed in the onlineissue, which is available at www. interscience.wiley.com.]

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