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Int Chin J Dent 2006; 6: 29-37.

Effect of acid etching time on nanoleakage and microtensile bondstrength of the adhesive-dentin bond interface
San-jun Zhao, DDS, PhD, Ji-hua Chen, DDS, PhD, Yu Xiong, DDS, PhD, and Hui Wang
DDS, PhD
Department of Prosthodontics, Stomatological College, The Fourth Military Medical University, Xi’an,
P. R. China
Purpose: To observe the effect of acid etching time on nanoleakage and microtensile bond strength (µTBS) of
the adhesive-dentin bond with different total-etch adhesive systems.
Materials and Methods Eighty human molars, whose occlusal enamel was removed to expose the dentin
surfaces, were conditioned with phosphoric acid for 0 (control), 15, 30 or 60 s (n=20). Resin-dentin bonded
specimens were produced using four one-bottle adhesives respectively; OptiBond Solo (OB), Single Bond (SB),One-Step (OS), and Prime & Bond NT (PB). Each specimen was then sectioned perpendicularly to the adhesiveinterface into eight beams. Four were used to measure the nanoleakage of adhesive-dentin interface bytransmission electron microscope and four to conduct the µTBS test. The data was collected and statisticallycompared with analysis of variance and post-hoc test.
Results: Nanoleakage increased with increasing etching time (0, 15, 30, and 60 s; p<0.05). For the OS and PBgroups, the highest µTBS were achieved after 15 s of etching, followed by 30, 60, and 0 s. For the OB and SB
groups, the µTBS between 15 and 30 s etching time subgroups were not significantly different between them but
significantly higher than those of 0 and 60 s etching time subgroups (p<0.05).
Conclusion: Increasing etching time demineralized the dentin surface to a depth greater than adhesives canpenetrate, resulting in more nanoleakage and lower µTBS. (Int Chin J Dent 2006; 6: 29-37.)
Key Words: dentin, etching time, nanoleakage, microtensile bond strength.

Introduction
There is agreement that dentin bonding is a more unpredictable and unreliable procedure than enamelbonding,1 what can be attributed to dentin’s complex histological structure, variable composition and greaterorganic content.2 Furthermore, an insufficient dentin bonding may frequently cause marginal discoloration,
recurrent caries, postoperative sensitivity and the loss of restorations.
In order to achieve high bond strengths and complete marginal seal with dentin, most modern adhesive
systems demineralize dentin with acidic conditioners, resulting in complex chemical and physical changes of thedentin matrix. Pashley et al.3 defined the purpose of acid etching dentin as follows: to remove the smear layer,
to expose the intertubular and peritubular dentin, to clean the dentin surface from debris, and more importantly,
to demineralize the superficial dentin matrix to allow resin infiltration into the dentin surface.
Furthermore, acid-etching dentin could create voids between the collagen fibers due to the loss ofhydroxyapatite crystals.4 These crystals support the collagen fibers and prevent its denaturation. And it isdifficult for adhesive monomers to penetrate a denaturated or collapsed collagen surface.5 The longevity ofcomposite fillings is, among other factors, dependent on the properties of the junction between the dentin matrix
and filling materials. Penetration of bacterial products, various acids and even oral fluids through the voids at
the interface between a restoration and tooth have been regarded as detrimental to the longevity of therestorations.6,7
To distinguish the leakage within demineralized dentin where disparities existed between the depths ofdemineralization and monomer diffusion, from the typical microleakage, Sano et al.8,9 introduced the term
“nanoleakage”, which was described as porosities located within/underneath the hybrid layer in a range less than
50 nm.10,11 These porosities features collagen-rich fibrous network created by dentin demineralization that29

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Int Chin J Dent 2006; 6: 29-37.

should be penetrated by resin to reinforce the demineralized dentin structures.12 It has been suggested that
improvement of the penetrating properties of the primer might facilitate the filling of porous demineralized
collagen zone. However, reducing the depth of demineralized dentin may also be equally effective in producing
a good bonding.13
The purpose of the present study was to observe the effect of different etching times on nanoleakage andmicrotensile bond strengths to dentin achieved with four adhesive systems. The null hypothesis to be tested wasthat the adhesive systems produce similar nanoleakage and bond strengths irrespective of etching time.

Materials and Methods
Preparation of ammoniacal silver nitrate
Silver nitrate (25 g, AgNO3; XK13-201-0099-031, Jiaozuo Chemical, Henan, China) was dissolved in 25 mL
of 28% aqueous ammonium hydroxide (NH4OH; Dongda Chemical, KaiFeng, China) in the presence of ambientlaboratory light, creating a suspension of black silver particles. Additional 28% NH4OH was used to titrate theblack solution, with magnetic stirring at room temperature (25±2˚C), until the solution slowly became clear as
the ammonium ions combined the silver into diamine silver ions ([Ag(NH3)2]+). This solution was diluted to 50
mL with distilled water to achieve a 50 wt% solution (pH=9.5) that was transferred to an amber bottle forstorage.
Tooth preparation and bonding procedures
Eighty caries-free human molar teeth were stored in 0.5% chloramine-T trihydrate at 4˚C and used within six
months of extraction. The roots of teeth were mounted in self-curing acrylic resin (Quick Resin B, Shofu Inc.,
Kyoto, Japan) and placed in distilled water for 24 hours prior to the experiment. The occlusal enamel was
removed using a low-speed diamond disc (Isomet 11-1180, Buehler Ltd., Lake Bluff, IL, USA) under copious
air-water spray, and superficial occlusal dentin was finished with 600-grit silicon carbide paper under runningwater to create a smear layer of clinically relevant thickness.14
Table 1.

Single-bottle adhesives used for test.

Bonding system

Lot number

Composition

OptiBond Solo (OB)108D24

Bis-GMA, HEMA, GPDM, Photoinitators, Fillera, Ethanol

Single Bond (SB)1105

Bis-GMA, Polyalkenoic acid co-polymer, Dimethacrylates, HEMA,
Photoinitators, Ethanol, Water

One-Step (OS)0100011728

BIS-GMA, BPDM, HEMA, Photoinitators, Fillerb, Actone

Prime&Bond NT (PB)

0009000254

PENTA, UDMA, Resin R5-62-1, T-Resin, D-Resin, Nanofillers, Photoinitators,
Stabilizer, Cetylamine hydrofluoride, Actonea, A mixture of barium aluminum borosilicate glass, fumed silica, and disodium hexafluorosilicate; b, 8.5% filled, an average
particle size of 1 µm; Bis-GMA, bis-phenol A diglycidyl-methacrylate; PENTA, dipentaerythritol-pentaacrylate phosphoric acid
ester; HEMA, 2-hydroxylethyl methacrylate; GPDM, glycerolphosphate dimethacrylate; BPDM, biphenyl dimethacrylate.

The 80 specimens were randomly assigned to four groups (n=20) according to the four bonding agents;
OptiBond Solo (OB, Kerr Corp., Orange, CA, USA), Single Bond (SB, 3M-ESPE, St. Paul, MN, USA),
One-Step (OS, Bisco, Inc., Schaumburg, IL, USA), and Prime&Bond NT (PB, Dentsply/Caulk, Milford, DE,
USA). The composition of the adhesive systems used in this study is summarized in Table 1. Each group was
further randomly divided into four subgroups (n=5) accordingly to the different etching times: 0 (not-etched), 15,30

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Int Chin J Dent 2006; 6: 29-37.

30 and 60 s. The 0 s subgroups served as a negative control group. All specimens were stored in distilled waterat room temperature for 24 hours. The bonding procedure for each group was carried out according to the
manufactures’ instructions (Table 2) except for the etching time, (15 s was the manufacturers’ recommended
etching time for the four test adhesives). After etching each tooth surface with a 32% phosphoric acid (Uni-Etch,
Lot: 0400008319, Bisco Inc.), a 2.0 mm increment of composite material (Charisma, Lot 081, Heraeus Kulzer,
GmbH & Co., Hanau, Germany) was applied to the bonded surfaces and light-cured for 40 s using a light curing
unit (QHL75 Curing Lite, Dentsply). A single operator, under identical room temperature conditions, performed
the etching and bonding procedures. After 24 hours in water at 37˚C, the specimens were thermocycled for 500
cycles in baths kept at 5˚C and 55˚C in order to simulate oral environment according to ISO/TS 11405, 2003.
Each specimen was sectioned perpendicularly to the bonded interface into eight 1.0×1.0×4.0 mm beams with alow-speed saw (KB-01, FMMU, Xi’an, China) under running water.
Table 2.

Instructions recommended by manufacturers.

Blot dry technique
Moist extent of dentin
Primer/Adhesive
Agitation
Air-drying
Light curing
Blot dry technique
Moist extent of dentin
Primer/Adhesive
Agitation
Air-drying
Light curing

OptiBond Solo (OB)Blot dry with cotton pellet
Dry lightly. Do not desiccate.
A coatLight brushing motion for 15 s
Lightly air-thin for maximum 3 s
20 s

Single Bond (SB)Blot excess water using a cotton pellet or mini-sponge
Appearing glistening without pooling of water
2-3 consecutive coats
Gentle agitation with a fully saturated applicator for 15 s
Gently air thin for 5 s
10 s

One-Step (OS)Remove excess water by blotting
the surface with a foam pellet,
alternatively, air dry for 2-3 s
Leaving visibly moist with ashiny surface
A minimum of two generous coats
Agitating slightly for 10-15 s
Gently, thoroughly
10 s

Prime&Bond NT (PB)
Blot dry conditioned areas with a moist cotton pellet
Leaving moist
A coat, remaining fully wet for 20 s
No
Gently, 5 s
10 s

Etching time, 15 s for all materials.

Silver staining techniqueFour of the eight beams sectioned from each specimen were coated with two layers of fast-setting nail varnish(2A075 Jet-Set, L'Oréal, Paris, France) applied 1 mm from the bonded interfaces. Without allowing these beams
to be dehydrated, they were immersed immediately in the ammoniacal silver nitrate solution in the dark. We did
not allow the tooth beams or varnish to dry completely so as to avoid the creation of artificial submicron hiati
beneath the resin-dentin interfaces that could be mistaken for nanoleakage.15
After 24 hours, the silver-stained beams were rinsed thoroughly in distilled water and placed in photodeveloping solution (Q/YYCX1-1999, Guanlong Photographic Equipment Co., Shanghai, China) for 8 hours
under a fluorescent light to facilitate reduction of the silver ions into metallic silver particles within potential
voids along the resin-dentin interface,16,17 and then fixed in fixative (2.5% glutaraldehyde and 2%
paraformaldehyde in 0.1 mol/L cacodylate buffer, pH=7.3) for 1 hour and rinsed thoroughly with 0.1 mol/L
sodium cacodylate buffer.
Transmission electron microscopic (TEM) observation
The silver-stained beams except for the ones showing microleakage (gap between dentin and composite
visible under optical microscope using 80x were demineralized in an aqueous solution of 0.1 mol/L ethylene

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Int Chin J Dent 2006; 6: 29-37.

diamine tetra-acetic acid (EDTA-Na2H2·2H2O; BG/T 1401-1998; Tianjing Chemical Reagent Co., Inc., Tianjing,
China), which has been buffered with sodium hydroxide to a pH of 7, for 48 hours.

Fig. 1.

Diagram detailing specimen preparation.

According to the TEM protocol of Tay et al.,18 the beams were dehydrated in an ascending acetone series(30% to 100%), immersed in propylene oxide as a transition fluid, and embedded in epoxy resin (E-20;
Hengyuan Chemical, Huangshan, China) at 60˚C for 48 hours. Each resin-embedded beam was trimmed into a
2×1 mm block that contained the silver-stained interface and re-embedded in epoxy resin. Dermineralized
sections with 90 nm thickness were prepared using a microtome (Ultrotome, LKB-2188, Nova, Sweden). The
sections were collected on single-slot, carbon and formvar-coated copper grids.

Then, the silver-stainedinterfaces were examined by a transmission electron microscope (JEM-2000EX, JEOL, Tokyo, Japan). All
TEM sections were examined at 100 kV without further staining. The size and percentage distribution of silver
particles of the ten digitized micrograph from each of the 16 subgroups were determined as the amount of
nanoleakage using image analysis software (Scion Image 4.02, Scion Corp., Frederick, MD, USA). Means andstandard deviations of silver penetration areas were calculated for specimens from each time period (0, 15, 30,
and 60 s).
Microtensile test
Another four of the eight beams harvested from each specimen were further trimmed by an ultra-fine diamondbur mounted in a high speed hand-piece under water coolant to produce a cross-sectional surface area ofapproximately 1.0 mm2, measured using a digital caliper. The trimmed beams were attached to the grips of acustom-made holder with cyanoacrylate adhesive (Yuwang, Shandong Industrial & Commercial Co., Ltd., Jinan,
China) and stressed to failure at a crosshead speed of 1 mm/minute on a universal test machine (Autograph,32

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Int Chin J Dent 2006; 6: 29-37.

AGS210, Shimadzu, Kyoto, Japan). The micro tensile bond strength (µTBS) of each beam was calculated as theforce at failure divided by the bonded cross-sectional surface area and expressed in MPa. The µTBS data andnanoleakage amount of these materials were subjected to one-way ANOVA and Least Significant Difference(LSD) test at the 95% level of confidence. Fig. 1 summarizes the experimental design.

Results
Nanoleakage in the form of reticular silver deposits could be observed predominantly along the base of hybrid
layers of all adhesive systems under different etching times except for the 0 s subgroups (Fig. 2). The means andstandard deviations of the µTBS and nanoleakage are summarized in Table 3 (µTBS), Table 4 (nanoleakage),and Fig. 3.
Table 3.

Dentin microtensile bond strength (MPa) and standard deviations (SD).

Bonding system

OptiBond Solo (OB)Single Bond (SB)One-Step (OS)Prime&Bond NT (PB)

0s
µTBS

SD

15 s
µTBS

SD

30 s
µTBS

SD

60 s
µTBS

SD

(etching time)

8.4
8.4
9.3
8.3

1.7
1.6
2.7
1.5

25.4
24.3
28.7
27.1

4.2
4.0
4.9
4.1

26.4
24.8
25.0
23.6

5.2
3.5
3.7
3.9

21.1
21.2
21.3
20.2

4.0
3.1
3.9
3.7

For both OS and PB groups, the values of µTBS are significantly different among the four etching times. For OB and SB groups,
the values of µTBS between 15 s and 30 s etching subgroups were not significantly different, which were all significantly higher
than those of 0 and 60 s etching subgroups (p<0.05).

Table 4.

Dentin nanoleakage (%) and standard deviations (SD) for the different etching times.

Bonding system

OptiBond Solo (OB)Single Bond (SB)One-Step (OS)Prime&Bond NT (PB)

0s
%

SD

15 s
%

SD

30 s
%

SD

60 s
%

SD

(etching time)

0
0
0
0

0
0
0
0

14.3
12.9
11.7
11.5

2.1
1.8
2.0
2.2

17.7
17.3
13.9
13.6

2.4
2.2
1.9
2.2

21.2
22.3
18.5
17.5

3.3
3.1
2.4
2.3

For all groups, the values of nanoleakage are significantly different among the four etching times according to one-way ANOVA
analysis (p<0.05).

Fig. 2. Transmission electron micrographs of resin-interface achieved with Single Bond (SB).
There was no nanoleakage observed along the resin-dentin interface with 0 s etching time (not etched) (A). Nanoleakage (silver
particles) in the form of reticular silver deposits could be observed predominantly along the base of hybrid layers with 15 (B), 30(C), and 60 s (D) etching times. Open arrowhead: nanoleakage, in the form of interconnecting dendrite silver deposits, could be
identified within the resin/dentin interface. A, adhesive layer; B, air bubble; C, composite resin; D, intertubular dentin; and T,
dentin tubule.

To explore the interaction term, one-way ANOVA test was performed for each of the four different adhesives

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Int Chin J Dent 2006; 6: 29-37.

as a function of etching time. For all groups, nanoleakage gradually increased in accord with increasing etchingtime. For both OS and PB groups, one-way ANOVA revealed significant differences among the four etchingtime and LSD test indicated that etching for 15 s resulted in the hightest µTBS (28.7 MPa for OS, 27.1 MPa for
PB) following by 30, 60 and 0 s. For OB and SB groups, the µTBS between 15 s and 30 s etching subgroups
were not significantly different, while the µTBS of 15 s and 30 s etching subgroups were significantly higher
than those of 0 and 60 s etching subgroups (p<0.05).

Fig. 3.

OB

SB

OS

PB

Relationship between microtensile bond strengths (µTBS) and nanoleakage (silver penetration area) at each etchingtime period for OptiBond Solo (OB), Single Bond (SB), One-Step (OS), and Prime&Bond NT (PB).

Discussion
The bonding mechanism of resin to dentin is described as micromechanical, and is generated by monomer
impregnation of exposed collagen of demineralized superficial dentin.

Theoretically, in order to promote

optimal bonding and sealing, the adhesive should completely diffuse across the full thickness of the etcheddentin. This would encapsulate and protect the collagen fibril, fill interfibrillar spaces and permit physicalinteraction with the unaltered underlying dentin.19-21 The diffusion and curing of adhesive resins throughdemineralized dentin are of crucial importance to create strong adhesion. However, it might be difficult todiffuse adhesive resin through demineralized dentin because of the presence of water around collagen fibers, thedepth of the demineralized zone, the time allowed for the primer to penetrate, and nonuniform distribution ofmonomers (such as the viscosity).20,22-24
Most contemporary adhesives use an acid etching gel (32-37% phosphoric acid) to demineralize dentin
surface. It is easier for the monomer to infiltrate a thin demineralized zone than a deeper zone. Immoderate

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Int Chin J Dent 2006; 6: 29-37.

etching can expose collagen to depths that current dentin adhesives may not be able to penetrate, resulting in a
porous collagen layer susceptible to degradation,5,25 which results in nanoleakage. The term “nanoleakage” hasbeen suggested to distinguish silver uptake into gap-free resin bonded interfaces from microleakage.8
Nanoleakage was defined as the diffusion of silver ions through nanometer-sized channels that has not been fully
penetrated by the adhesive, leaveing spaces for fluid penetration under the hybrid layer.
For measuring the effect of different etching time on nanoleakage, in the present study, we etched the dentin
surface with 32% phosphoric acid for 0 (not etched), 15, 30 and 60 s and proceeded to bond with OB, SB, OS,
and PB respectively. The statistically significant increase in the amount of penetration of silver ions in accord
with increasing in etching time (0, 15, 30 and 60 s) supports that nanoleakage is located in the partiallydemineralized dentin, which is not completely penetrated by the adhesive systems. The longer the etching time,the deeper the expected layer of partially demineralized dentin would be. This would also explain why there
were differences between adhesive systems with different compositions, as the degree of silver particles
penetration not only depends on the depth of the partially demineralized dentin but also on the ability of theindividual adhesive system to penetrate and reinforce this zone.
Except for nanoleakage reported recently, to a certain extent, conditioning with etching gel could considerably
improve the bond strength of adhesive resins to dentin, which has been already accepted by clinicians today.
Methacrylates with both hydrophobic and hydrophilic groups promoted monomer penetration and impregnation
into the etched dentin, becoming entangled with the collagen fibrils, creating a hybrid layer after theirpolymerization,26-30 which is essential to obtain proper adhesion of resinous materials to dentin. When etchingtime is increased, the hybrid layer thickness increases as a result of deeper demineralization.31 Although thedepth of demineralization needed for optimal adhesion has not been determined, Nakabayashi et al. suggested
that a demineralization depth into dentin of 1.0-2.0 µm is sufficient for optimal bond strength.21 Paul et al.
reported that increasing the etching time had a negligible effect on bond strength of Single Bond, however, thesilver uptake increased upon prolonged etching times.32 In contrast, Abu-Hanna et al. correlated dentin bondstrength to etching time and reported a significant decrease in bond strength values for Syntac as a result of theincreased etching time.33
The current study evaluated the effect on dentin nanoleakage and bond strength of reducing the etching timeto 0 s or increasing it to 60 s from the recommended 15 s etching time. In order to avoid the possibility of theminerals such as amorphous calcium phosphates may be dissolved in the acidic conventional silver nitrate(AgNO3) solution (pH=3.4),34 which may produce artefactual microporosities that give rise to false positive
results,35 a modified silver-staining techinique, ammoniacal silver nitrate ([Ag(NH3)2]+) solution (pH=9.5), wasused in this study.36 For both acetone-based adhesives, OS and PB groups, the highest µTBS were achieved
after 15 s of etching, followed by 30, 60 and 0 s (not-etched). For both the ethanol and water-based adhesives,
OB and SB groups, the µTBS between 15 and 30 s etching subgroups were not significantly different, while theµTBS of 15 and 30 s etching subgroups were significantly higher than those of 0 and 60 s etching subgroups.
These differences may indicate that the solvents used in adhesives influence their performances and thatacetone-based adhesives are more technique-sensitive than the ethanol and water-based adhesives, probably
because the different ability of acetone and ethanol to chase water as well as the different vapor pressure.33,37,38
More studies are needed to elucidate the effect of different solvents on morphology and bond strength ofadhesive-dentin bonding.

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Int Chin J Dent 2006; 6: 29-37.

Undoubtedly, the existence of nanoleakage may have potential long-term consequences in dentine bonding
procedures. Indeed, this kind of leakage may allow for the penetration of bacterial products and dentinal or oral
fluid along the interface, which may result in hydrolytic degradation of either the adhesive resin or the exposed
collagen, thereby compromising the stability of the bond,39,40 ultimately, decreasing the longevity of thecomposite restoration. Consequently, from a clinical perspective, it would be advantageous to optimize dentinal
bonding during clinical operation (concentration of acidic conditioners, application technique, etching time, etc.),
and further studies should be performed to develop adhesive systems that could minimize the nanoleakage andachieve high bond strength on the resin-dentin interface.
Within the limitations of this in vitro study, it was concluded that different etching time resulted insignificantly different dentin µTBS and nanoleakage. In addition, increased etching times demineralized thedentin surface to a depth greater than adhesives could penetrate, resulting in more nanoleakage and lower µTBS.
The 15 s etching dentin (the manufacturers’ recommendation) produced the highest µTBS and a moderateamount of nanoleakage for all adhesive systems tested. The results reject the null hypothesis that the adhesive
systems produce similar nanoleakage and bond strengths irrespective of etching time.
Acknowledgment
This adhesives used in this study were generously sponsored by 3M-ESPE, Bisco Inc., and Kerr Co. The authors are
grateful to Biao Kang for his technical support and Dr. Kai-cheng Li for his assistance in preparing this manuscript.

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Correspondence to:
Dr. Ji-hua Chen
Department of Prosthodontics, Stomatological College, The Fourth Military Medical University
Xi’an, 710032, P. R. China
Fax: +86-29-8253-4510 E-mail: jhchen@fmmu.edu.cn
Received November 29, 2005. Revised January 5, 2006. Accepted February 21, 2006.
Copyright ©2006 by the International Chinese Journal of Dentistry.

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