INTRODUCTION

Aesthetic tooth-colored restoration has been widely accepted and commonly performed in daily dental treatment. As the basis of aesthetic restoration, the clinical manifestations of adhesive systems have improved since the release of the pioneer approach designed by Buonocore in 19551). Excellent sealing has been already obtained between bonding systems and enamel because of the superior structure of enamel. However, it is still a big challenge to obtain reliable and stable bonding strength and sealing effectiveness at adhesive-dentin interfaces2).

Adhesive-dentin bonding is a unique tissue engineering process; in particular, collagen fibers are exposed after the dentin surface is etched, and these collagen fibers function as a scaffold, allowing adhesive resin monomers to infiltrate dentinal tubules and the interspaces between collagens, thereby forming a resin-dentin interdiffusion zone, the so-called hybrid layer, initially defined by Nakabayashi et al.3). Thus far, the hybrid layer is imperfect because of inconsistencies between the depth of dentin demineralization and the depth of adhesive infiltration4,5). Furthermore, recent resin adhesives contain more acidic/hydrophilic monomers and larger amount of water to wet dentin substrate, resulting in incomplete polymerization of adhesive resin at the bonding interfaces2). As a result, microporous zones comprising 20 nm to 100 nm spaces formed6). This phenomenon is termed nanoleakage, which is different from traditional microleakage, in

which defects or marginal gaps are involved7,8).

Although nanoleakage may be very small in size, it could act as a path for water movement at the adhesive-dentin interfaces, and the water movement may extract unconverted monomers from adhesive resins over time, which can cause bonds to decrease9). Thus, nanoleakage is usually considered as an important indicator to evaluate the sealing capability and bond effectiveness of an adhesive system or an intervention8). Nanoleakage occurs within the hybrid layer and in the adhesive layer10). Therefore, a general evaluation along the bonding interface (covering composite-adhesive-dentin simultaneously) is necessary11).

Tay et al.12) developed a 50% ammoniacal silver nitrate solution to detect and analyze nanoleakage, because the diameter of silver ions is so small (0.059 nm) that they can easily infiltrate micro (even nano) gaps or water uptake at the adhesive-dentin interfaces. After a reduction reaction, nanoleakage was displayed by silver precipitation within water-filled channels and interaction between diamine silver ions and acidic/hydrophilic resin components, which can not be eliminated by cutting or washing. Depending on various high-resolving power microscopes, nanoleakage can be observed. Light microscope (LM)13-15), field-emission scanning electron microscope (FESEM)8,16,17), and transmission electron microscope (TEM)13,18) are the most commonly used qualitative or quantitative nanoleakage observation methods. However, under different approaches, whether the bonding interfaces from the same tooth could produce similar images and statistical results is still unknown.

Furthermore, confocal laser scanning microscope

Nanoleakage evaluation at adhesive-dentin interfaces by different observation methodsHongye YANG*, Jingmei GUO*, Jinxin GUO, Hongfei CHEN, Mirinal SOMAR, Jiaxi YUE and Cui HUANG

The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedical Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, ChinaCorresponding author, Cui HUANG; E-mail: huangcui@whu.edu.cn

The purpose of this study was to evaluate the capability and characteristics of different nanoleakage observation methods, including light microscope (LM), field-emission scanning electron microscope (FESEM), transmission electron microscope (TEM), and confocal laser scanning microscope (CLSM). Dentin specimens were bonded with either an etch-and-rinse adhesive (SBMP) or a self-etch adhesive (GB), and prepared for nanoleakge evaluation according to different observation methods. LM, FESEM and CLSM results demonstrated that the SBMP group showed more interfacial nanoleakage than the GB group (p<0.05); by contrast, no significant difference was found in TEM results (p>0.05), however, TEM illustrated concrete nanoleakage forms or patterns. The results suggested that different observation methods might exhibit distinct images and a certain degree of variations in nanoleakage statistical results. Researchers should carefully design and calculate the optimum assembly in combination with qualitative and quantitative approaches to obtain objective and accurate nanoleakage evaluation.

Keywords: Nanoleakage, Silver nitrate, Dentin bonding, Adhesive, Dentin

*Authors who contributed equally to this work.Color figures can be viewed in the online issue, which is avail-able at J-STAGE.Received Feb 6, 2015: Accepted May 2, 2015doi:10.4012/dmj.2015-051 JOI JST.JSTAGE/dmj/2015-051

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Table 1 Adhesive systems, manufactures, compositions and application modes in this study

Materials Classification Main components Application modes

Adper ScotchBond Multi-Purpose(3M ESPE, St. Paul, USA)

Three-step etch-and-rinse

adhesive system

Etchant: 35% phosphoric acid solution, water, synthetic amorphous silica, polyethylene glycol,

aluminium oxide.Primer: HEMA, polyalkenoic acid copolymer, water. Bonding agent: Bis-GMA, HEMA, tertiary amines,

photo-initiator.

1. Apply etchant 15 s, rinse and blot dry;

2. Apply one coat of primer, blow gently for 5 s;

3. Apply a layer of bonding agent and light-cured for 10 s.

G-Bond(GC, Tokyo, Japan)

One-step self-etch

adhesive system

4-MET, UDMA, silanated colloidal silica, initiators, water, and acetone.

Apply sufficient amount of adhesive for 10 s, strong air dry

for 5 s, light-cure for 10 s.

HEMA, 2-hydroxyethyl methacrylate; Bis-GMA, bisphenol A and glycidyl methacrylate; 4-MET, 4-methacryloxyethyl trimellitic acid; UDMA, urethane dimethacrylate.

Fig. 1 The diagram of the test design used in the study.

(CLSM) is a newly developed nanoleakage observation method that does not depend on silver nitrate staining19). It is inevitable to add some fluorescent dye (e.g., Rhodamine B) into adhesives to produce distinct image for CLSM observation, however, whether the nanoleakage results from CLSM would be consistent with other observation methods and whether Rhodamine B mixed in primer and adhesive would affect bonding performance are still not sure.

Three-step etch-and-rinse adhesive is considered as the gold standard of dentin bonding and nanoleakage observation because of its consistent good laboratory and clinical performance20). The unmatched depths of dentin demineralization and adhesive penetration always lead to some obvious nanoleakage4,5). But for self-etch adhesive, the dissolution of dentine inorganic constituents and the infiltration of adhesive resin around the collagen network simultaneously develop to form a uniform hybrid layer21). In general, the etch-and- rinse adhesives should present more nanoleakage than the self-etch adhesives. However, whether the same conclusion could be obtained through different observation methods is questionable.

Therefore, this study aimed to evaluate the capability and characteristics of LM, FESEM, TEM, and CLSM for nanoleakage expression. We also conducted this study to determine whether there are some differences on nanoleakage expression between etch-and-rinse adhesives and self-etch adhesives under each observation method.

MATERIALS AND METHODS

The diagram of the test design used in the study is shown in Fig 1. Twenty-four freshly extracted caries-free human third molars were used. Donors submitted and signed written informed consents, and the protocol used in this study was approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University, China. The teeth were sectioned parallel to the occlusal surface by a low-speed water-cooled diamond saw under water irrigation (Isomet, Buehler, Evanston,

IL, USA). The exposed dentin surfaces were ground with 600-grit silicon carbide (SiC) paper under running water for 60 s to create a standardized smear layer.

Dentin bondingTwo adhesive systems, particularly a three-step etch-and-rinse adhesive Adper ScotchBond Multi-Purpose (SBMP) and an one-step self-etch adhesive G-Bond (GB), were evaluated. The materials, manufacturers, classifications, compositions, and application modes used in the present study are listed in Table 1. All the bonding procedures were conducted by the same researcher. Each adhesive group (n=12) was further divided into two subgroups (n=6) according to whether the Rhodamine B was doped into the adhesives or not before bonding.

For non-Rhodamine group (n=6), the adhesives were directly applied on dentin surfaces according

655Dent Mater J 2015; 34(5): 654–662

to the manufacturer’s instructions and polymerized using a LED light-curing unit (Bisco, Schaumburg, IL, USA) with approximately 700 mW/cm2 irradiance. A resin composite (Charisma, Heraeus Kulzer, Hanau, Germany) was formed at the interface in four increments (thickness of 3 mm to 4 mm), and each increment was light cured for 20 s. The teeth were then stored in water for 24 h at 37°C. Afterwards, one tooth was vertically sectioned to produce slabs with thickness of approximately 1 mm. Light microscopy (DP72, Olympus,Tokyo, Japan) was used to observe if there are some contraction gaps at the bonding interfaces of slabs. Four gap-free slabs from the tooth were chosen and further prepared for nanoleakage observation through LM (two slabs), FESEM (one slab) and TEM (one slab), separately. The slabs were coated with two layers of nail varnish, applied up to 1 mm from the interface to allow contact between the tracing agent and the adhesive-dentin interface. The slabs were immersed in 50 wt% ammoniacal silver nitrate solution (pH 9.5) in the dark for 24 h and then in a photo-developing solution for 8 h under a fluorescent light12). All slabs were wet-polished with 600-, 800- and 1200- grit SiC papers and 0.25 um diamond paste using a polishing cloth.

For Rhodamine-containing group (n=6), the primer and adhesives were doped with fluorescent dye Rhodamine B at approximately 0.1%. After bonding and composite building, we exposed the pulp chamber of each tooth and filled it with 0.1 wt% sodium fluorescein for 3 h. One tooth was vertically sectioned to produce slabs with a thickness of 1 mm. The interfaces were also checked by LM, one gap-free slab was selected for nanoleakage observation, it was ground with SiC paper grit up to 2500-grit, and ultrasonicated for 2 min per step.

LM evaluationThe silver-stained bonded slabs for LM observation were fixed on glass slides, polished with SiC paper, stained using 0.5% acid fuchsin, and then analyzed under LM (DP72, Olympus). Ten interface images from two slabs (five from each slab) were obtained at 100× magnification for each adhesive group. The amount of nanoleakage expression was scored by two observers on the basis of the percentage of silver uptake along the bonding interface14): 0, no nanoleakage; 1, <25% with nanoleakage; 2, 25% to 50% with nanoleakage; 3, 50% to 75% with nanoleakage; and 4, >75% with nanoleakage. Inter-observer agreement was measured by the Kappa test (K=0.85).

FESEM evaluationThe silver-stained specimens for FESEM observation were mounted in stubs, sputter-coated with carbon, and observed under FESEM (S4800, Hitachi, Tokyo, Japan) in a backscattered electron mode. Ten fields-of-view along the interfaces of one slab were randomly captured. The elemental distribution of silver in each image was subsequently presented and calculated in an energy dispersive X-ray (EDX) mapping mode.

TEM evaluationThe silver-stained bonded slabs for TEM observation were embedded in epoxy resin at 60°C for 120 h. Representative ultrathin sections (90 nm to 100 nm) were produced using an ultramicrotome and accumulated on copper grids (100 mesh). The sections were observed with TEM (7700-SS, Hitachi) at 80 kV. Ten interfaces images from one disc were randomly captured. The Image J (NIH, Frederick, MD, USA) was used to compute the percentage distribution of silver deposits along bonding interfaces in each image9).

CLSM evaluationCLSM (LSM 510, Carl Zeiss, Jena, Germany) equipped with a 63×/1.4 numerical aperture oil immersion lens was used. Emission fluorescence was recorded at 488–510 nm and 570–590 nm, respectively. Topographic single projection was created from the serial images captured to a depth of 20 μm (0.3 μm per layer) along a fixed axis. The configuration of the system was standardized and used throughout the investigation. Ten interface images from one slab were randomly captured. Zeiss LSM Image Examiner (Carl Zeiss) was used to analyze the obtained images, specifically, the percentage distribution of green pixels along bonding interfaces in each image can be obtained.

Microtensile bond strength testOther five bonded teeth in non-Rhodamine or Rhodamine-containing group were sectioned vertically into beams with a dimension of 0.9×0.9 mm. Six beams were chose from each tooth after external beams were excluded, resulting in a total of 30 beams for each group. The prepared beams were forced to fail under a universal testing apparatus (Microtensile Tester, Bisco) at 1 mm/min crosshead speed. Cross-sectional interface area was measured, and microtensile bond strength (MTBS) was obtained in MPa.

Each debonded specimen was assessed under a stereomicroscope (Stemi 2000-C, Carl Zeiss) at 50× magnification to determine the failure mode classified as13): adhesive failure (A); cohesive failure in dentin (CD); cohesive failure in composite (CC); and mixed failure (M). The number of premature failed beams per group during specimen preparation was also recorded.

Statistical analysisSPSS 16.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. For LM observation, the Chi-square test was used to evaluate the differences among score groups. After normal distribution was confirmed, t-test was performed to evaluate the quantity of nanoleakage expression in the two adhesive groups using FESEM, TEM, or CLSM data, respectively. For MTBS results, the correlation between adhesive systems and Rhodamine B was evaluated by two-way ANOVA factorial analysis, followed by Tukey’s post-hoc multiple comparison test (a=0.05).

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Table 2 Microtensile bond strength, failure frequency and nanoleakage expression under different observation methods

GroupMTBS and failure

frequency (%)

Nanoleakage expression

LM observation FESEM/EDX observation

(%)

TEM observation

(%)

CLSM observation

(%)Score 0–4

N of Images (total images)

Percentage (%)

Statistical Difference

Group1:Adper ScotchBond Multi-Purpose

Non-Rhodamine group44.6±8.1a MPa [1/30]A:15;CC:4;CD;6;M:4

Rhodamine-containing group

43.7±6.9a MPa [2/30]A:13;CC:3;CD;7;M:5

01234

2(10)5(10)2(10)0(10)1(10)

20.050.020.0

010.0

A1 4.5(0.9) A2 9.2(2.9) A3 14.6 (2.3) A4

Group2:G-Bond

Non-Rhodamine group41.8±9.2 a MPa [2/30]A:11;CC:6;CD;3;M:8

Rhodamine-containing group

42.2±7.3 a MPa [3/30]A:15;CC:0;CD;1;M:11

01234

6(10)2(10)1(10)1(10)0(10)

60.020.010.010.0

0

B1 2.2(0.7) B2 9.0(3.3) A3 8.5 (1.9) B4

Values of MTBS are mean±standard deviation [number of premature failed beams/number of intact beams tested]. Percentages of the failure modes were classified as: A, adhesive failure; CC, cohesive failure in composite; CD, cohesive failure in dentine; M, mixed failure. For interfacial nanoleakage expression under LM obervation, the score was based on the percentages of the adhesive surface showing silver nitrate depositon:14 0, no nanoleakage; 1, <25% with nanoleakage; 2, 25–50% with nanoleakage; 3, 50–75% with nanoleakage; 4, >75% with nanoleakage. Groups with the same superscripts are not statistically significant (p>0.05).

Fig. 2 Light microscope (LM) images showing interfacial nanoleakage expression (arrow). (A) Representative image of deposits of silver nitrate (score=2) within the adhesive

interface created by adhesive SBMP. (B) Representative image of score=1 of GB adhesive-dentin interface. c, composite resin; hl, hybrid layer; d, dentin.

RESULTS

Table 2 summarizes the nanoleakage data of different observation methods, MTBS (means±standard deviations), and failure frequency of each subgroup. LM statistical results of the number of sections assigned to the same score revealed that the SBMP adhesive group presented a higher score of nanoleakage expression than the GB group (p<0.05). FESEM and CLSM results indicated that the SBMP group exhibited greater interfacial silver uptake than the GB group (p<0.05).

However, no significant difference was found between two types of adhesives in the TEM data (p>0.05). There was no significant difference in MTBS between the two adhesive systems (p>0.05), while Rhodamine B did not affect the MTBS significantly (p>0.05). The interaction of adhesive systems and Rhodamine B was not significant (p>0.05). The predominant failure mode was adhesive failure regardless of adhesive type.

The representative views of interfacial nanoleakage expression under LM, FESEM, TEM, and CLSM observations were shown in Figs. 2, 3, 4, and 5,

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Fig. 3 Representative FESEM images showing the interfacial silver uptake. Images (1) indicate the silver uptake (hollow arrow) in the backscattered electron mode of

FESEM, images (2) indicate the elemental distribution of silver (solid arrow) on the whole field-of-view through the corresponding EDX mapping mode, and images (3) indicate the amount of several dominant element, especially metallic silver (black arrow), through the elemental energy spectra. (A) SBMP group shows massive line-like homogeneous silver deposits through the whole adhesive layer, even in dentin tubules (circle). (B) GB group shows large size of discontinuous silver uptake located within the adhesive layer. c, composite resin; d, dentin.

Fig. 4 Representative TEM images showing the silver deposition patterns at the adhesive-dentin interface. From image (1) to (3), the magnification increased from 1,000×, 2,500× to 5,000×.

(A) SBMP group shows massive silver deposition (white arrow) along the hybrid layer and within the dentinal tubules (triangle), indicating extensive nanoleakage expression. (B) G group shows a relatively small amount of silver uptake (white arrow) located at the one side of the dentinal tubule (triangle) wall underneath the adhesive layer, indicating discontinuous nanoleakage expression. d, dentin; ad, adhesive layer.

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Fig. 5 Representative CLSM single-projection images showing the interfacial characterization and nanoleakage.

Images (1) indicate the nanoleakage (sodium fluorescein dye) with the adhesive-dentin interface, images (2) present the diffusion (Rhodamine B) of the adhesive systems within the dentin, and images (3) are the combined projections of both dyes. (A) The adhesive-dentin interface created by SBMP shows severe fluorescein (nanoleakage) penetration (white arrow) within the entire interface, a clear hybrid layer (hl) with long resin tags (rt) penetrating the dentinal tubules under the adhesive layer (ad) is observed. (B) GB group shows a weak-to-moderate nanoleakage (white arrow) and comparatively short resin tags (rt) underneath the adhesive layer (ad), and the hybrid layer is well defined. ad, adhesive layer; rt, resin tags; hl, hybrid layer.

respectively. In LM images (Fig. 2), it was found that massive black silver accumulations could be identified at 100× magnification under a relatively wide visual field, but it is hard to observe details. In FESEM images (Fig. 3), white metallic silver was distinct along the interface, while some dentinal tubules were filled with silver patches, especially in SBMP group. Line-like homogeneous silver deposits (Fig. 3A) and large size of discontinuous silver uptakes (Fig. 3B) were found in SBMP and GB groups, respectively. TEM images (Fig. 4) showed that numerous very small deposits of silver deposited along the dentinal tube wall. Representative CLSM images (Fig. 5) showed impressive views; long resin tags with severe sodium fluorescein dye were found in SBMP group (Fig. 5A), while weak-to-moderate fluorescein and short resin tags were observed in GB group (Fig. 5B).

DISCUSSION

Although nanoleakage is the main marker of structural imperfections and degradation mechanism of adhesive-dentin interfaces, reports on nanoleakage are largely

inconsistent22,23). LM, FESEM and TEM are three most commonly used observation methods for nanoleakage expression, researchers usually choose one of them by their convenience when they study dentin bond durability. However, whether different nanoleakage observation methods will produce different statistical results for the same tooth was unknown and barely studied. The present study showed the differences and similarities among them, indicated that the etch-and-rinse adhesive could produce more nanoleakage expression than self-etch adhesive, and revealed that CLSM could also be used to observe and calculate nanoleakage expression objectively and accurately.

Due to variation of dentin substrate, specimens from different tooth may produce different nanoleakage expression, as a result, the nanoleakge among different observation methods may not be fairly compared. Actually, the specimen from the same tooth could be evaluated under LM, FESEM, and TEM. Thus, we adopted this approach in this study in order to reduce the tooth effect and provide more valid results.

LM has been used to observe the microleakage of a staining sample before the concept of nanoleakage

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was introduced. However, the concrete structure of the hybrid layer is difficult to show because of limited lateral resolution and focal depth; thus, the difference between microleakage and nanoleakage is difficult to elucidate24). As such, LM was gradually replaced with advanced instruments, such as SEM and TEM, with high lateral resolution. Despite these shortcomings, LM remains the preferred instrument by some researchers13-15) to observe silver infiltration. With LM, each representative field-of-view shows a considerably wide adhesive-dentin interface, which may prevent high regional variability; thus, comprehensive and objective results can be obtained. In LM assessment, a scoring system from 0 to 4 is used, firstly described by Saboia et al.14) LM results in our study showed a score of 1 was the most common among all of the images of the SBMP group; by contrast, a score of 0 was mainly obtained in the images of the GB group. The scoring system provided an intuitive sense of the extent of nanoleakage actually developed in two different adhesive groups.

FESEM has been widely used to observe and locate nanoleakage expression in adhesive-dentin interfaces10). It is worth noting that the backscattered electron mode of FESEM is more suitable for nanoleakage observation than the secondary electron mode. The reason is that the backscattered mode can present supplementary information and produce a high contrast image because of its element atomic number-dependent characteristics25). For example, Ag used in this study exhibits a high atomic number, it can reflect a higher number of electrons and display a brighter image than the background. Thus, electron microscopic edge effects that usually occur in a secondary electron mode could be effectively avoided, and erroneous interpretations could be reduced.

Combined with energy dispersive X-ray mapping mode, an accurate and sensitive chemical component detection method, the presence and distribution of various elements (such as Ag) could be displayed vividly and calculated objectively, as shown in our study. Therefore, the occurrence of false positive/negative in one field of vision could be reduced and the credibility of nanoleakage results is likely increased8). With all advantages the FESEM offers, its disadvantages are also obvious. In FESEM, drying should be performed, but this process may produce artificial artifacts, such as shrinkage, thereby causing specimens to crack26). Another disadvantage is that the FESEM method could only be applied to analyze surfaces. Therefore, a slight deficiency possibly occurs if FESEM was used to observe internal nanoleakage expression, or concreted nanoleakage mode.

Using TEM, Tay et al.12) defined two nanoleakage modes, namely, spotted and reticular. A “water-tree” could be observed when reticular nanoleakage is perpendicular to the hybrid layer12,23). In this study, the TEM image showed relative distinct gradations among composite, adhesive layer, hybrid layer, and dentin. Silver ions blocked incident electrons, and thus, black spotted particles were found in the collagen fiber

network or adhesive resin. Water trees were also observed in our study. Our finding was in agreement with that of Tay et al.27), who indicated that water trees could be observed in immediate bonding interfaces of water-containing adhesives or aged interfaces of water-free adhesives. Here, both of resin adhesives used in this study contain water which is extremely difficult to be completely removed from the bonded interfaces.

A fact should be noted that ultrathin sections of hard dental tissue are relatively difficult to produce and the sample preparation is complex. TEM specimens should be embedded; in this process, water in dentin is replaced with epoxy resin, thereby affecting illiquidity tracers28). The ability of TEM technology to quantitatively analyze nanoleakage expression may also be limited. One reason is that the percentage of Ag black pixels from background colors in TEM images is difficult to determine when conventional image processing software is used. Another reason is that high magnification, which is necessary to observe a nanoleakage mode, can produce high regional variability. These findings may explain the difference between the statistical results of nanoleakage expression using TEM images and those obtained using other observation methods (LM, FESEM, and CLSM). Therefore, TEM technique is definitely a good choice to illustrate nanoleakage forms or patterns, but it should be cautiously applied when used for quantitative analysis.

CLSM is high-technology equipment that functions in combination with optical microscope, laser scanning technology, and computer image processing technology. CLSM exhibits many advantages when adhesive-dentin interfaces are observed29). For example, CLSM is a non-destructive method, which can be used directly to observe fresh samples and avoid dehydration interference. Another advantage is that CLSM can present a 2D maximum projection of a series of layers instead of dentin surface only. The lateral resolution of CLSM is also higher than that of an optical microscope. Moreover, different structures around the bonding interface can be marked clearly with fluorescent markers (such as Rhodamine B in this study), and images are usually beautiful and impressive. In this study, additional details, such as the thickness of hybrid layer and adhesive layer, adhesive infiltrating conditions, formation of resin tags, and nanoleakage expression along the adhesive-dentin interface, could be obtained. Furthermore, the present study showed that Rhodamine B mixed in primer and adhesive did not affect bonding performance of adhesives to dentin. The findings also echoed previous research of D’Alpino et al.30). Therefore, CLSM is a relatively new technology in nanoleakage observation and can be widely used for qualitative and quantitative statistical analyses. However, artificial artifacts were still presented, and some researchers thought that the phenomenon obtained by CLSM was micro-permeability rather than nanoleakage31). Therefore, future studies should be conducted to distinguish these two concepts under CLSM, and the possible problems of dye mixing, leaching or cross talk

660 Dent Mater J 2015; 34(5): 654–662

phenomena should be further studied in detail. It should be noted that nanoleakage expression

depends on the adhesive system tested10). The demineralized dentin at the bottom of the hybrid layer significantly increased nanoleakage mainly due to the inconsistencies between the depth of dentin demineralization and the depth of adhesive infiltration4,5). There was no such depth difference in self-etch adhesive, thus, a uniform hybrid layer could be formed21). Furthermore, an adhesive monomer present in self-etch adhesive, such as 4-MET in GB in this study, could form an intense calcium salt with residual hydroxyapatite, which likely contributed to a decrease in nanoleakage expression32). This finding was corroborated by the LM, FESEM and CLSM data in this study.

Researchers proposed a relationship between resin-dentin bond strength and quantity of nanoleakage expression because high bonding strength corresponds to low nanoleakage with the following characteristics: adequate adhesive infiltration; few residual solvent; and complete polymerization of resin monomers16). However, this relationship is complex and remains controversial. Using LM, Guzmán-Armstrong et al.33) found a weak-to-moderate negative relationship between dentin bond strength and nanoleakage in a two-step etch-and-rinse adhesive system. By contrast, Hashimoto et al.18) obtained FESEM/EDX results showing that bond strength was not correlated with nanoleakage expression among different adhesives (self-etch adhesives or etch-and-rinse adhesive under different conditions). Reis et al.34) applied TEM to quantitatively analyze nanoleakage changes under different bonding strategies instead of conventional qualitative analysis. Ding et al.35) found that the degree of nanoleakage failed to affect the bond strength of two material combinations under CLSM. In the present study, MTBS were not significantly different between the two adhesive systems. However, the degrees of nanoleakage expression differed between the two adhesive groups using statistical data from LM, FESEM, or CLSM, except TEM. Considering these differences, we presumed that different observation methods may be partly accounted for different conclusions regarding the relationship between bonding strength and amount of nanoleakage.

In conclusion, LM is a simple and low-cost method to quantitatively analyze nanoleakage, and high regional variability can be prevented because the area of one field-of-view of LM is relatively large. Through FESEM, clear images can be obtained, accurate and sensitive quantitative analysis can be performed when the backscattered electron mode is used in combination with EDX, but the size of evaluation area should be wide enough, because more images taken from high resolution FESEM may be added up to meet the same size of evaluation area taken from lower resolution LM. TEM is an efficient technique to observe concrete distribution characteristics, such as spotted modes, reticular modes, or water tree. However, the contrast and magnification of TEM image, and the inclination of field-of-view may limit the application for quantitative

analysis of interfacial nanoleakage expression. CLSM can be used directly to observe fresh samples and avoid dehydration interference. Impressive images can be obtained, and thus concreted structures of the bonding interface can be revealed; meanwhile, a 2D maximum projection of sufficient thickness could improve the accuracy of statistical results.

CONCLUSION

Each observation method (LM, FESEM, TEM, or CLSM) exhibited unique mechanisms, characteristics, application scope, advantages, and disadvantages in evaluating nanoleakage. In order to obtain objective and accurate nanoleakage results, researchers should carefully select appropriate observation methods in combination with qualitative and quantitative techniques.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

ACKNOWLEDGMENTS

This work was financially supported by National Nature Science Foundation of China (No. 81371191) and the Fundamental Research Funds for the Central Universities.

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