assessment of the quality of resin_dentin bonded interfaces

8/12/2019 Assessment of the Quality of Resin_dentin Bonded Interfaces

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d ent a l ma te r ia l s 2 8 ( 2 0 1 2 ) 622631

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Assessmentof the quality of resindentinbonded

interfaces: An AFM nano-indentation, TBS and confocal

ultramorphology study

Salvatore Sauroa,b,, Raquel Osoriob, Timothy F. Watson a, Manuel Toledano b

a Biomaterials, Biomimetics and Biophotonics Research Group (B3), Kings College London Dental Institute, Guys Dental Hospital,

London, UKb Dental Materials, School of Dentistry, University of Granada, Colegio Mximo, Campus de Cartuja, Granada, Spain

a r t i c l e i n f o

Article history:

Received 3 June 2011

Received in revised form

4 November 2011

Accepted 16 February 2012

Keywords:

AFM/nano-indentation

Biomechanical properties

Confocal microscopy

EDTA

Resindentin interfaces

TBS

a b s t r a c t

Objective. The aim of this study was to assess by using confocal microscopy (CLSM), AFM

nano-indentation and microtensile bond strength test (TBS) thequality of theresindentin

interfaces created with selected bonding parameters.

Methods. Dentin conditioned with H3PO4or EDTA was bonded in ethanol- or water-wet condi-

tion using a HEMA-free or HEMA-containing adhesive. The resin-bonded teeth were stored

in distilled water (24 h) and sectioned as match-sticks (0.9mm2) for TBS. Further resin-

bonded teeth were sectioned and analyzed using CLSM, and AFM nano-indentation. The

AFM imaging and nano-indentation processes were undertaken using a Berkovich diamond

indenter. The modulus of elasticity (Ei) and hardness (Hi) across the interface were evalu-

ated with the specimens in a fully hydrated status. The AFM imaging was performed both

in dry and wet conditions for evaluating the shrinkage of the hybrid layer on dehydration.

Results. The HEMA-containing adhesive applied onto H3PO4-etched ethanol or water-wet

dentin created hybrid layers with the lowest biomechanicalnano-properties (p < 0.05);no sig-

nificant differences in TBS were found between the two wet-bonding techniques (p > 0.05).

However, the ethanol-wet bonding reduced the dye penetration into the adhesive layer cre-

ated with the HEMA-containingadhesive. Hybrid layers with high biomechanical properties,

low micropermeability and no shrinkagewere onlypossiblewhen usingHEMA-free adhesive

applied in ethanol wet-dentin. In particular, a significant increase in Ei and Hi was achieved

at the hybrid layer and underneath the resindentin interface of ethanol-wet EDTA-treated

dentin.

Significance. The use of HEMA-free adhesives applied onto ethanol-wet dentin may be con-

sidered as an alternative and suitable bonding strategy to achieve high quality resindentin

interfaces.

2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Corresponding author at: Biomaterials, Biomimetics and Biophotonics Research Group (B3), Kings College London Dental Institute, Floor17 Guys Tower, London SE1 9RT, England, UK. Tel.: +44 0207 188 3874; fax: +44 0207 188 1823.

E-mail address: [email protected] (S. Sauro).0109-5641/$ see front matter 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.dental.2012.02.005
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1. Introduction

The demineralization of dentin and the exposure of the colla-

gen matrix is a crucial step in adhesive dentistry to achieve a

micromechanical interlocking between resin monomers and

dentin [1,2]. The water-wet bonding technique is commonly

used in etch-and-rinse bonding procedures to suspend thedemineralized collagen fibrils and prevent shrinkage caused

by the electrostatic attraction (i.e. hydrogen bonds forma-

tion) of dentinal proteoglycans and glycosaminoglycans [3].

Unfortunately this bonding technique does not allow a com-

plete resin infiltration of the demineralized dentin, leaving

unprotected collagen fibrils below and within the hybrid layer

[46]. The imperfect resin infiltration is mainly due to the

incomplete replacement of water from the demineralized col-

lagen network, especially when vital dental pulps perfuse

dentinal fluid [7,8]. Unprotected collagen fibrils within the

hybrid layer may be degraded by the action of endogenous

matrix metalloproteinases (MMPs) derived from the deminer-

alized dentin [9,10]. Moreover, poor infiltrated hybrid layershave an evident attitude to water-sorption that contributes

to the hydrolytic degradation of the resindentin interface

[11,12]. The ethanol-wet bonding technique has been shown

to increasethe longevityof resin-bondedH3PO4-etcheddentin

[3,13]. In this technique, absolute ethanol, a polar solvent with

less hydrogen bonding capacity than water [14], may be used

to chemically dehydrate the demineralized collagen matrix,

reduce the hydrophilicity of the collagen matrix and create

wider interfibrillar spaces for a better resin infiltration [15,16].

It has been demonstrated that poor-quality hybrid layers

are characterized by an excessive presence of water are also

affected by nano/micro porosities phase separation and low

monomer polymerization[15,18]. The assessment of thehard-nessand modules of elasticityalong the resindentin interface

[18] and the hybrid layer resistance to dry shrinkage may be

suitable to achieve further knowledge on the quality of hybrid

layers created using the etch-and-rinse technique. Neverthe-

less, there is still little information available regarding the

quality of formed resindentin interfaces with etch-&-rinse

adhesives applied with water or ethanol-wet bonding tech-

niques and with HEMA-free or HEMA-containing resins [20].

The aim of this study was to evaluate the bond

strength (TBS), the ultramorphology/micropermeability and

the biomechanical nano-properties of the resindentin inter-

face created with two experimental etch-&-rinse adhesives

applied onto 37%-H3PO4 or 0.5 M-EDTA conditioned dentinwhen using the water-wet or ethanol-wet bonding techniques

(5min.). The null hypotheses to be tested are that the use

of: (1) H3PO4 vs. EDTA to condition dentin, (2) water-wet vs.

ethanol-wet bonding techniques and (3) HEMA-free vs. HEMA-

containing adhesives do not affect the quality of the created

resindentin interfaces.

2. Materials and methods

2.1. Specimenpreparation

Caries-free human molars (age:1840 yr) extracted for surgical

reasons under an informed consent, reviewed and approved

by the Institutional Ethics Committee, were stored in 0.5%

chloramine-Tat 4 Cfornomore than 1 month.The teeth were

sectioned 1 mm beneath the cemento-enamel junction using

a diamond wafering blade (Isomet 11/1180, Buehler, Coventry,

UK). The occlusal enamel was removed to expose the middle

coronal dentin and a standard smear layer was created using

500 grit SiC paper (Struers LaboPol-4. Struers, Copenhagen,

Denmark).

2.2. Experimental adhesives and bondingprocedures

Two experimental etch-and-rinse bonding systems were pre-

pared: (i) a HEMA-containing resin blend (HEMA-containing)

was formulated using UDMA-60%/BisGMA-10%/TEGDMA-30%

(Esstech Essington, PA, USA). A hydrophilic monomer (2-

hydroxyethyl methacrylate, Aldrich Chemical Co, Gillingham,

UK) [2040%] and absoluteethanol (AldrichChemical), [70%for

the primer and 10% for the bond] were subsequently added to

the neat resin blend. (ii) a HEMA-free resin blend (HEMA-free)

was formulated using UDMA-60%/BisGMA-10%/TEGDMA-30%

(Esstech Essington, PA, USA) and dissolved in absolute ethanol(Aldrich Chemical), [70% for the primer and 10% for the

bond]. The experimental resin blends were finally mixed with

0.5wt% of camphoroquinone (Aldrich Chemical) and 1.0wt%

of ethyl 4-dimethylaminobenzoate (Aldrich Chemical). Forty-

eight dentin specimens were divided into two main groups.

Five-minute sonication (Model QS3, Ultrawave Ltd, Cardiff,

UK) and 2-day shaking (Orbital Shakers PSU-20i, Cole Fisher

Scientific Ltd, Loughborough, UK) were required to yield well-

mixed resin solutions. The specimens (n = 12) of the first group

were etched using a 37% phosphoric acid gel (H3PO4; Bisco,

Itasca, IL, USA) for 15s and the specimens of the second group

(n = 12)wereconditioned for60 s usinga 0.5M water solutionof

ethylenediaminetetraacetic acid (EDTA: 99.995%, Lot. 431788-Aldrich Chemical).

The specimens of each group were copiously rinsed with

water for 1 min and immediately immersed in absolute ethyl

alcohol (EtOH), (Aldrich Chemical) for 5 min (n =6) or in deion-

ized water (H2O) for 1min (n =6), [17,18]. The dentin surface

wasalways covered by ethanol to avoid surface tension forces,

keeping it visibly moist prior to the application of the resins.

The water-wet bonding substrate was achieved by water-

rinsing the dentin surfaces and gently blowing off the excess

water to leave a wet reflective surface [17]. The primer and

the bond were applied within a period of 20s and light-cured

for 30s using a halogen light-curing unit (Translux EC Kulzer

GmBh, Bereich Dental, Werheim, Germany). The output inten-sity was monitored with a Demetron Radiometer (Model 100,

Demetron Research, Danbury, CT, USA) to maintain a mini-

mal light output intensity of 600 mW/cm2 throughout all the

experiment. A flowable resin composite (X-FlowTM, Dentsply,

Caulk, UK) was placed incrementally in two 1 mm layers and

light-cured for 40 s (Demetron Research).

2.3. AFM imaging and nano-indentation

The resin-bonded specimens were left undisturbed in water

for 3h and then cut perpendicularly to the bonding zone

using a diamond saw (Isomet 11/1180) to obtain 3 resindentin

slabs with a thickness of 2 mm. The resindentin slabs were
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Table 1 Mean and standard deviation (SD) of the biomechanical nano-elasticity (Ei) and nano-hardness (Hi).

Ei HEMA-containing H2O HEMA-containing EtOH HEMA-free H2O HEMA-free EtOH

GPa PA EDTA PA EDTA PA EDTA PA EDTA

1st HL 2.2 0.8 A1 12.0 0.9 A2 2.2 0.8 A1 12.0 0.9 A2 6.4 0.6 A1 11.7 1.2 A2

2nd 10.9 1.1 B1 14.1 2.0 A2 10.9 1.1 B1 14.1 2.0 A2 11.7 1.4 B1 14.9 1.0A2

3rd 17.4 1.1 C1 16.8 1.7 B1 17.4 1.1 C1 16.8 1.7 B1 16.9 0.6 C1 18.9 1.0 B1

4th 18.1 1.1 C1 17.7 1.1 B1 18.1 1.1 C1 17.7 1.1 B1 18.2 1.4 C1 19.9 1.1 B15th 19.1 0.9 C1 18.2 1.2 B1 19.1 0.9 C1 18.2 1.2 B1 19.2 0.9 C1 20.1 0.7 B1

6th 19.8 1.1 C1 18.7 1.0 B1 19.8 1.1 C1 18.7 1.0 B1 19.2 1.5 C1 20.3 1.2 B1

Hi HEMA-containing H2O HEMA-containing EtOH HEMA-free H2O HEMA-free EtOH

GPa PA EDTA PA EDTA PA EDTA PA EDTA

1st HL 0.1 0.01 A1 0.3 0.03 A2 0.1 0.01 A1 0.3 0.12 A2 0.3 0.01 A2 0.3 0.04 A2

2nd 0.4 0.04 B1 0.6 0.05 B2 0.4 0.08 B1 0.6 0.07 B2 0.5 0.08 A2 0.6 0.03 B2

3rd 0.7 0.09 C1 0.7 0.04 C1 0.7 0.09 C1 0.7 0.03 C1 0.7 0.09 C1 0.8 0.05 C1

4th 0.6 0.16 C1 0.7 0.05 C1 0.7 0.10 C1 0.7 0.07 C1 0.7 0.10 C1 0.8 0.04 C1

5th 0.7 0.11 C1 0.7 0.08 C1 0.7 0.12 C1 0.7 010 C1 0.8 0.12 C1 0.9 0.09 C1

6th 0.7 0.06 C1 0.8 0.07 C1 0.8 0.08 C1 0.8 0.07 C1 0.8 0.08 C1 0.9 0.06 C1

Mean and standard deviation (SD) of the biomechanical properties (Ei and Hi) along the tested resindentin interfaces. Same letter indicatesno

differences in columns (p > 0.05). Same number indicates no differences in rows (p > 0.05).

Fig. 2 Topographic AFM images obtained in dry and wetconditions from the resindentin interfaces created using the

HEMA-containing experimental adhesives applied onto H3PO4-etched water-wet dentin. [A] In this image it is possible to

observe how the hybrid layer (HL) collapsed and formed a wide gap between the dentin (d) and the adhesive (a)

characterized by the presence of uncollapsed resin tags (t). [B] The re-hydration of the specimen induced a complete

expansion of the hybrid layer (hl), leaving only a thin gap between the adhesive (a) and the hybrid layer.

Table 2 Microtensile bond strength values (MPa) to dentin when resin adhesives were applied with the ethanol- orwater-wet bonding in EDTA or H3PO4 treated dentin.

EDTA PA: H3PO4

HEMA-containing H2O-wet 41.3 (13.1) a1 (5%) [0/72/28] 38.3 (12.2)a1 (12%) [0/69/31]

HEMA-containing EtOH-wet 43.8 (11.1) a1 (0%) [0/84/16] 45.2 (10.9)a1 (2%) [0/78/22]

HEMA-free H2O-wet

HEMA-free EtOH-wet 44.9 (10.1) a1 (8%) [2/68/30] 36.1 (11.5)a1 (16%) [12/71/17]

Mean (standard deviation) microtensile bond strength (MPa) to dentin. Same superscripts letters indicate no differences (p > 0.05) in columns.

Same superscript numbers indicate no differences (p > 0.05) in rows. Percentage of premature failures is indicated in parentheses. The modes

of failure are also expressed in percentage into brackets as [adhesive/mix/cohesive].


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Fig. 3 Topographic AFM images obtained in dry and fully hydrated conditions from the resindentin interfaces created

using the two experimental etch & rinse adhesives applied onto the EDTA or H3PO4 treated dentin in ethanol or water-wet

bonding. [A] A resindentin interface created using the HEMA-containing adhesive that was applied onto H3PO4-etched

water-wet dentin is presented. The imaging process performed in dry conditions caused the shrinkage of the hybrid layer

(HL) and the formation of a wide gap (78m)between the dentin (d) and the adhesive layer (a). [B] The experimental

HEMA-containing adhesive applied onto EDTA treated dentin with the water-wet bonding technique was also characterized

by the presence of a wide gap (78m)between the dentin (d) and the composite (c) due to the shrinkage of the hybrid (hl)

and adhesive layer (a). [C] The hybrid layer of the resindentin interface created using the HEMA-containing adhesive

applied onto ethanol-saturated H3PO4-etched dentin showed only a slender gap/shrinkage (34m) of the hybrid layer (a) in

the proximity of the dentin (d); uncollapsed resin tags (t) are still present within the gap. [D] The resindentin interface

created using the HEMA-containing adhesive applied onto ethanol-wet EDTA-treated dentin showed a well extended


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sub-dived (n = 5)according to the wet-bonding technique (EtOH

or H2O). The specimens were sectioned with a diamond blade

(Accutom-50, Struers, Copenhage, Denmark) using a hard tis-

sue saw (330-CA RS-70300, Struers, Copenhage, Denmark) in

both x andy directions across the adhesive interface to obtain

match-sticks with cross-sectional areas of 0.9mm2.

Eachbeam was attached to a modified Bencor Multi-testing

apparatus (Danville Engineering Co., Danville, CA, USA) withcyanoacrylate adhesive (Zapit, Dental Ventures of America

Inc., Corona, CA, USA) and tested using a microtensile bond

strength testing machine (Instron 4411, Instron Corporation,

Canton, MA, USA) at a crosshead speed of 0.5 mm/min. Bond

strength datawere calculated in MPa. Pre-testing failures were

arbitrarily assigned with a value 0 and included in the sta-

tistical analysis. Two-way ANOVA including interactions and

Student-Newman-Keuls multiple comparisons were used for

the statistical analysis. Adhesive systems and dentin sur-

face treatment were considered as independent variables and

TBS as the dependent variable. Statistical significance level

was set in advance at =0.05.

2.5. Confocal laser scanning microscopy evaluation

(CLSM)

Further dentin specimens (n =12) for each principal group

(Hema-free or Hema-containing) were first dived in 2 sub-

groups (n =6) according to the dentin conditioning method

(EDTA or H3PO4) and subsequentlysub-dived (n = 3) according

to thewet-bonding technique (EtOH or H2O).The dentinspeci-

mens were bonded as previously described with the adhesives

doped with 0.1wt% Rhodamine B (Sigma Chemicals, St. Louis,

MO, USA). The pulp chamber of the specimens was exposed

by gently removing the pulpal tissue using a thin tissue pin-

cers. The specimens were immediately filled with 0.1wt%water solution of fluorescein (FL: Sigma Chemicals) for 3h

[17,18]. The specimens were finally rinsed in a H2O ultrasonic

bath for 2min and sectioned into 1mm mesio-distal slabs

using a slow-speed water-cooled diamond saw (Labcut, Agar

Scientific, Stansted, UK). The resindentin slabs were finally

polished using 1200-grit SiC paper for 30s followed by H2O

ultrasonic bath (1m). Each resin-dentin interface was com-

pletely investigated and five images representing the most

common features of micropermeability were randomly cap-

tured and recorder. The imaging procedures were performed

using a confocal laser scanning microscope (Leica SP2 CLSM,

Heidelberg, Germany) equipped with a 63/1.4 NA oil immer-

sion lens using488 nm argon/heliumand a 633nm krypton ion

laser illumination. CLSM fluorescence images were obtained

from 20m optical sections using a 1m z-step below the sur-

face. The z-axis scan of the interface surface were converted

into pseudo-color for better visualization, and compiled into

both single and topographic projections using Leica SP2 CLSM

image-processing software (Leica, Heidelberg, Germany). The

configuration of the system was standardized and used at the

same level for the entire investigation.

3. Results

3.1. AFM imaging and nano-indentation

Ei and Hi were both affected by dentin surface treat-

ment (EDTA/H3PO4), (p 0.05). Mean andstandard deviations of Ei andHi measured

along the resindentin interfaces are shown in Table 1.

The H3PO4-etched water-wet dentin exhibited the lowest

(p


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Fig. 4 Confocal fluorescence images of the resindentin interfaces created with the two experimental etch-&-rinse

adhesives (i.e. HEMA-free and HEMA-containing) applied onto H3PO4 or EDTA-treated dentin in water-wet or ethanol-wet

bonding conditions. The scale bar is 20m. [A] CLSM fluorescence 3D-single projection of resin-bonded dentin interfaces

created with HEMA-containing resin applied onto H3PO4etched water-wet dentin. This image shows how the water-wet

bonding induces severe micropermeability within the interface. The fluorescein dye penetrated throughout the dentinal


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type (EtOH/H2O), (F = 84.11; p 0.05). Mean bond strengths in MPa and

mode of failures obtained for each group are shown in Table 2.

The HEMA-containing adhesive produced no statistical dif-

ferences in bond strengths when applied onto H3PO4 acid

or EDTA-treated water-wet dentin. The failure mode waspredominantly cohesive or mixedin both groups. The applica-

tion of the HEMA-containing adhesive induced no statistical

differences when applied both onto H3PO4 acid etched or

EDTA-treated ethanol-wet dentin. Also in this case the failure

mode was predominantly cohesive or mixed in both groups.

No statistical difference was observed when the HEMA-

containing adhesive was applied onto H3PO4 acid etched or

EDTA-treated water-wet dentin.

The HEMA-free adhesive applied onto water-wet H3PO4acid etched or EDTA-treated dentin failed completely dur-

ing the cutting procedures. Conversely, when the HEMA-free

adhesive was applied onto ethanol-wet H3PO4 acid etched or

EDTA-treated dentin it was possible to obtain values (MPa)comparable to those attained with HEMA-containing adhe-

sive; no statistical differences were observed between H3PO4acid etched and EDTA-treated ethanol-wet dentin.

3.3. Confocal laser scanning microscopy evaluation

(CLSM)

The CLSM images of the resindentin interfaces created with

the two experimental etch-&-rinse adhesives applied onto

H3PO4 or EDTA-treated water/ethanol-wet dentin are shown

in Fig. 4.

The HEMA-containing adhesive applied onto H3PO4

etched or EDTA-treated water wet dentin was character-ized by severe micropermeability throughout the dentinal

tubules, hybrid layer and adhesive layer (Fig. 4A and B). On

the contrary, the resindentin interfaces created with HEMA-

containing adhesive applied onto H3PO4 etched ethanol-wet

dentin was characterized by long resin tags and an opti-

mal sealing ability; hybrid and adhesive layers were devoid

of any FL penetration (Fig. 4C). The resin-bonded dentin

interfaces created with HEMA-containing adhesive applied

onto EDTA-conditioned ethanol-wet dentin showed no FL dif-

fusion both into the adhesive layer and hybrid layer (Fig. 4D).

The HEMA-free adhesive applied onto H3PO4-etched

ethanol-wet dentin presented modest micropermeability (FL)

localized at the bottom of a hybrid layer (Fig. 4E and F).

Likewise, the micropermeability of the resindentin inter-face created with the HEMA-free adhesive applied onto

EDTA-conditioned ethanol-wet dentin was only detected

throughout the dentinal tubules and on the bottom of the

hybrid layer (Fig. 4G and H).

4. Discussion

The null hypotheses have to be rejected as the

biomechanical nano-properties, TBS and microperme-

ability/ultramorphology were influenced by the dentin

conditioning approach (H3PO4 or EDTA), bonding technique

(ethanol-wet or water-wet) and adhesive type (HEMA-free orHEMA-containing) selected in this study.

The results of this study have shown that the resindentin

interfaces created using the HEMA-containing adhesive

applied onto EDTA-treated ethanol-wet dentin were charac-

terized by a slender shrinkage of the hybrid layer (when

imaged in dry condition), (Fig. 3D). However, the biomechani-

cal nano-properties of the hybrid layer and the dentin region

immediately underneath the hybrid layer resulted higher than

those observed along the resindentin interfaces created in

H3PO4-etched dentin (Table 1). The demineralized zone at the

bottom of the hybrid layer is a critical part of the resin-dentin

interfaces created when using the etch-and-rinse bonding

approach [18,19]. In this particular area of the resin-dentininterface the resin probably remains partially polymerized

[22,23] and more susceptible to enzymatic and hydrolytic

degradation [24,25].

The H3PO4-etched water-wet dentin bonded using the

HEMA-containing adhesive exhibited the lowest (p


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ultramorphology analysis showed that the hybrid layer com-

pletely collapsed in dry conditions forming a gap within the

interface (Figs. 2A, 3A and B). Conversely, the resindentin

interface created with the HEMA-containing adhesive applied

onto H3PO4-etched ethanol-wet dentin showed hybrid layer

shrinkage of only a few microns in the proximity of

its underside (Fig. 3C). The Confocal micropermeabil-

ity/ultramorphology evaluation showed that the HEMA-containing resin applied onto water-wet H3PO4-etched or

EDTA-treated dentin was characterized by severe FL penetra-

tion throughout the hybrid layer and adhesive layer (Fig. 4A

and B) whereas, the ethanol-wet dentin allowed the formation

of a resindentininterface which prevented the FL penetration

throughout the hybrid and adhesive layer (Fig. 4C and D). In

terms of bond strength, this study confirmed that the appli-

cation of a HEMA-containing resin applied onto H3PO4 acid

or EDTA-treated dentin results in comparable TBS results

both when using the water-wet or the ethanol-wet bond-

ing technique (Table 2). These results are in accordance with

those reported by Sauro et al. [16,26] who showed that the

resindentin interface created in EDTA-treated dentin withHEMA-containing adhesives presented bond strength val-

ues comparable to those of H3PO4-etched dentin and higher

resistance to chemical degradation, possibly due to a higher

amount of residual apatite crystallites left within the collagen

matrix which prevented the denaturation of collagen [27].

The ethanol-wet bonding technique employed during the

bonding procedures of the HEMA-free adhesive favored the

formation of hybrid layers with higher biomechanical proper-

ties compared to those obtained with the HEMA-free adhesive

applied onto water-wet dentin (Table 1) which could not be

tested due to premature failure of the resindentin inter-

faces caused by a lack of diffusion of the hydrophobic resin

comonomers into the water-rich demineralized dentin [3,15]The AFM analysis performed along the resindentin inter-

faces createdwith HEMA-free resin appliedon theethanol-wet

dentin showed a high quality hybrid layer with no sign

of shrinkage in dry conditions (Fig. 3E). Furthermore, the

HEMA-free resin applied onto H3PO4 acid or EDTA-treated

ethanol-wet dentin showed modest micropermeability local-

ized at the bottom of the hybrid layer (Fig. 4EH).

Since there was no statistical differences between the

3rd, 4th, 5th and 6th indentation along any of the tested

resindentin interface, it is possible to affirm that both the

etching procedures used in this study may considerably influ-

ence the bio-mechanical nano-properties of the dentin within

the hybrid layer and at the bottom of the hybrid layer.The maintenance of a wet demineralized dentin sur-

face to prevent the collapse of the acid-etched dentin is

remarkably essential during the bonding procedure [28,29].

Although the water-wet-bonding is the most common tech-

nique used to prevent the collapse of the demineralized

collagen matrix, it remains a sensitive technique which may

influence substantially the formation of the hybrid layer

[3,30]. An excessive presence of water may be responsible

for the formation of a poor-quality hybrid layer character-

ized by micro-porosities and phase separation within the

resindentin interface [15,17,18,32,31]. Moreover, water may

compete with a hydrophilic resin monomers (i.e. HEMA) for

space in the demineralized zone, decreasing the monomer

density within the collagen network, and probably inter-

fering with its polymerization [32]. The reversible hybrid

layers shrinkage observed within the interface of the HEMA-

containing bonded dentin (Fig. 2A and B) and the FL

penetration within thehybrid andadhesive layers (Fig. 4A and

B) was probably due to the high water content rather than

polymerized resin. The ethanol-wet bonding technique may

indeed offer the possibility to replace water by an excess ofabsolute ethanol, achieving an enhanced coating of the col-

lagen fibrils and sealing ability (Fig. 4CH) that allow more

hydrophobic resin comonomers to diffuse into the demineral-

ized dentin [3,15]. A higherdegree of polymerization wasmost

likely achieved due to the hydrogen bond formation between

comonomers containing hydroxyl groups, such as BisGMA,

and ethanol [25].

The advantage of using HEMA-free rather than HEMA-

containing adhesives in clinical practice is that restoration

performed with more hydrophobic adhesives absorb much

less water over time, increasing bond longevity, if compared

to those performed with adhesivesbased on more hydrophilic

monomers [3335].In conclusion, this study showed an improvement of qual-

ity of the hybrid layer within the resindentin interfaces

created using both the HEMA-free and the HEMA-containing

adhesives with the ethanol-wet-bonding technique. Further

investigations regarding the ability of this experimental bond-

ing technique to improve the quality of the resindentin

interface via remineralizationof the hybrid layer are at present

ongoing.

Acknowledgments

This work was supported by grants CICYT/FEDER MAT2008-

02347, CICYT/FEDER MAT2011-24551, JA-P07-CTS2568 and

JA-P08-CTS-3944. This article presents independent research

commissioned by the National Institute for Health Research

(NIHR) under the Comprehensive Biomedical Research Cen-

tre at Guys & St. Thomas Trust. The views expressed in this

publication are those of the author(s) and not necessarily

those of the NHS, the NIHR or the Department of Health. The

authors also acknowledge support from the Centre of Excel-

lence in Medical Engineering funded by the Wellcome Trust.

The authors have no financial affiliation or involvement with

any commercial organization with direct financial interest in

the materials discussed in this manuscript. Any other poten-

tial conflict of interest is disclosed.

r e f e r ence s

[1] Marshall GW, Marshall SJ, Kinney JH, Balooch M. The dentinsubstrate: structure and properties related to bonding. JDent 1977;25:44158.

[2] Nakabayashi N, Pashley DH. Hybridization of dental hardtissues. Tokyo: Quintessence Publishing Co. Ltd.; 1998.

[3] Pashley DH, Tay FR, Carvalho RM, Rueggeberg FA, Agee KA,Carrilho M, Donnelly A, Garca-Godoy F. From dry bonding towater-wet bonding to ethanol-wet bonding. A review of the

interactions between dentin matrix and solvated resins


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assessment of the quality of resin_dentin bonded interfaces

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