Editorial Type:
Article Category: Research Article
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Online Publication Date: 01 Jul 2006

Morphological and Mechanical Characterization of the Acid-base Resistant Zone at the Adhesive-dentin Interface of Intact and Caries-affected Dentin

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Page Range: 466 – 472
DOI: 10.2341/05-62
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SUMMARY

This study examined the ultrastructure of both intact and caries affected dentin-adhesive interface after artificial secondary caries formation, using scanning electron microscopy and nanoindentation testing. Half of the prepared specimens were bonded with Clearfil SE Bond (Kuraray Medical, Japan) and a resin composite (Metafil Flo, Sun Medical, Japan) for the nanoindentation test. The other specimens were stored in a buffered demineralizing solution for 90 minutes, then observed using SEM. An acid-base resistant zone (ABRZ) was observed beneath the hybrid layer, distinguished by argon-ion etching. The ABRZ of caries-affected dentin was thicker than that of normal dentin, while its nanohardess was lower than normal dentin (p<0.05). It is suggested that the monomer of Clearfil SE Bond penetrated deeper than previously reported, creating a so-called “hybrid layer.” However, its physical properties depended on the condition of the dentin.

INTRODUCTION

Dentin bonding systems are used for the clinical placement of resin composite restorations. Adhesion is thought to enhance long-term bonding and sealing of the cavity margin, resulting in protection of the restoration against secondary caries. However, replacing restorations due to secondary caries is still an ongoing problem in restorative dentistry.

Several in vitro secondary caries tests and evaluation methods have been developed that assess demineralized lesions and inhibition zones of dentin due to acid challenge. These methods include polarized light microscopy (Pereira & others, 1998a), the nanoindentation test (Pereira & others, 1998b), microradiography (Torii & others, 2001), confocal laser-scanning microscopy and the x-ray analytical microscope (Okuda & others, 2003). However, the magnification of such methods is limited if more detailed information at the interface between the cavity and restoration is required.

Scanning electron microscopy (SEM) is widely used to analyze the ultrastructure of the interface. Tsuchiya and others (2004) observed artificial secondary caries inhibition around adhesive restorations to bovine root dentin using SEM. From the SEM examination, the ultrastructure of the cavity margins after acid-base challenge was found to be adhesive material-dependent. An acid-base resistant zone was found beneath the hybrid layer, distinguished by argon-ion etching. The mechanism of the acid-base resistant zone formation is still unclear; however, it is very different from the inhibition zone due to the release of fluoride. An acid-base resistant zone was formed in spite of the adhesive being fluoride-free. It was speculated that formation of the acid-base resistant zone should be related, in part, to the amount of fluoride release from the fluoride-containing adhesive; but it should also be related to the ability of adhesives infiltrating into dentin. However, there is still little information on the acid-base resistant zone.

The nanoindentation technique has been used in dentistry to measure the hardness of the resin-dentin bonded area (Van Meerbeek & others, 1993), the resin impregnated layer and its surrounding area (Akimoto & others, 1996), the dentin-enamel junction region (Urabe & others, 2000), caries affected dentin (Maneenut & others, 2003) and filler particles of dental restorative materials (Willems & others, 1993). The nanoindentation technique has several advantages for nanohardness determination, such as accurate positioning capability of the indenter to about 20 μm and a load resolution of approximately 0.2 μN.

Therefore, this study sought to establish a simple specimen preparation for application to human teeth and to characterize the acid-base resistant zone at the interface of human intact and caries-affected dentin specimens by scanning electron microscopy and nanoindentation testing. The null hypothesis was that the acid-base resistant zone created by a self-etching primer adhesive system is the same in both intact and caries-affected dentin.

METHODS AND MATERIALS

Materials Used

A 2-step self-etching primer adhesive system (Clearfil SE Bond, Kuraray Medical, Tokyo, Japan) and a flowable resin composite (Metafil Flo, shade A2, Sun Medical, Moriyama, Japan) were used in this study, both being fluoride-free materials. A conventional tungsten-halogen light-curing unit, XL3000 (600 mW/cm2, 3M-ESPE, St Paul, MN, USA) was used for curing the adhesive and resin composite.

Specimen Preparation

Sample preparation is illustrated in Figure 1. Six caries-free human third molars and 6 human third molars with dentin carious lesions, involving middle dentin on the occlusal surface, stored frozen after extraction, were used for this study. Verbal agreement was obtained for their use in research and education.

Figure 1. Schematic illustration of the methodology of specimen preparation.Figure 1. Schematic illustration of the methodology of specimen preparation.Figure 1. Schematic illustration of the methodology of specimen preparation.
Figure 1 Schematic illustration of the methodology of specimen preparation.

Citation: Operative Dentistry 31, 4; 10.2341/05-62

Intact molars were sectioned at the mid-coronal portion, horizontal to the tooth axis, using a low-speed diamond saw (ISOMET, Buehler, Lake Bluff, IL, USA) to obtain 1-mm thick dentin disks. The dentin surfaces were ground with wet #600-grit silicon carbide paper under running water. SE Primer was applied to one surface of each disk for 20 seconds and SE Bond was then applied and light-cured for 10 seconds. Next, a resin composite was placed between pairs of prepared dentin disks and light-cured for 40 seconds from the top and bottom surfaces to make a dentin disk sandwich (Inokoshi & others, 1993).

Caries-affected dentin was obtained using the combined criteria, including visual examination, surface hardness using a dental explorer and staining by caries detector solution. The relatively hard, caries-affected, lightly stained dentin was left on the experimental side (Nakajima & others, 1995). For molars with dentin caries lesions, coronal enamel was completely removed by a diamond bur (ISO #106), then the outer carious dentin was carefully removed with a slow speed steel round bur (ISO#010) using caries detector solution (Caries Detector, Kuraray Medical, Tokyo, Japan). Restoration of the caries-removed dentin surface was accomplished using Clearfil SE Bond and a resin composite.

Each prepared specimen of intact dentin and caries-affected dentin was sectioned perpendicular to the dentin-adhesive interface and fixed with epoxy resin (Epoxicure Resin, Buehler).

Half of the fixed specimens (3 for each group) were subjected to the following acid-base challenge, while the remaining specimens were used for the nanoindentation test.

SEM Observation of the Specimen After Acid-base Challenge

For SEM, each specimen was first stored in 100 ml of a buffered demineralizing solution containing 2.2 mmol/L CaCl2, 2.2 mmol/L NaH2PO4 and 50 mmol/L acetic acid adjusted at pH 4.5 for 90 minutes to create artificial secondary caries (Wefel, Heilman & Johdan, 1995). In a pilot study, the time for acid challenge was decided by SEM observation of the artificial caries lesion being approximately 10 mm in depth. The specimens were then immersed in 5% NaClO for 20 minutes in an attempt to remove any demineralized dentin collagen fibrils and they were rinsed with running water for 30 seconds. Following this, 4-META/MMA-TBB resin (Super Bond C&B, Sun Medical, Moriyama, Japan) was applied without acid-etching of the treated surface to protect the surface against wear during subsequent polishing. After curing the 4-META/MMA-TBB resin, the specimens were sectioned perpendicular to the dentin-adhesive interface and reduced to approximately 1 mm in thickness, then polished with diamond pastes (Struers A/S, Copenhagen , Denmark) down to 0.25 μm. The polished surfaces were etched with a beam of argon-ions (EIS-IE, Elionix, Tokyo, Japan) for 7 minutes to bring the hybrid layer into sharp relief. The operating conditions for the argon ion beam etching were an accelerating voltage of 1 kV and an ion current density of 0.2 mA/cm2, with the ion beam directed perpendicular to the polished surface (Okuda & others, 2003). The specimens were then gold-sputter coated, and morphological changes to the dentin-adhesive interface due to acid-base challenge were observed using SEM (JSM-5310LV, JOEL, Tokyo, Japan).

Nanohardness Measurement

The nanohardness of the dentin-adhesive interface of the polished specimens was measured from the dentin side to the resin composite side using a nanoindentation tester (ENT-1100, Elionix Corp, Tokyo, Japan). Figure 2 illustrates simulated indentation areas of the bonded specimens. The indentations were made as follows: in deep dentin, they were made at 4 points at 5-μm intervals. In dentin 2 μm below the bottom of the hybrid layer, the 4 points were lined up obliquely to the HA/dentin-adhesive interface. As the interval of each indentation should be 5 times the size of the indentation, each measurement was taken at 5-mm intervals in order to avoid corruption of the abutment (Urabe & others, 2000). One point at the middle of the hybrid layer and 3 points equally spaced 5 mm to the bonding layer and resin composite were selected. The load was 5 gf. After indentation, mean nanohardness at each point was calculated. The data were compared by 2-way ANOVA. The 2 factors were type of dentin and position of measurement. After that, post-hoc comparison was performed using Scheffe's test at p<0.05.

Figure 2. Schematic illustration of the methodology of the nanohardness measurement using a nanoindentation testing machine (ENT-1100).Figure 2. Schematic illustration of the methodology of the nanohardness measurement using a nanoindentation testing machine (ENT-1100).Figure 2. Schematic illustration of the methodology of the nanohardness measurement using a nanoindentation testing machine (ENT-1100).
Figure 2 Schematic illustration of the methodology of the nanohardness measurement using a nanoindentation testing machine (ENT-1100).

Citation: Operative Dentistry 31, 4; 10.2341/05-62

RESULTS

SEM Observation

The ultrastructures around the dentin-adhesive interfaces after the acid-base challenge are shown in Figures 3a–d. The outer lesion, which is the dentin surface demineralized after acid-base challenge, was observed in both intact and caries-affected dentin, respectively (Figures 3a and 3c). The depth of the outer lesion ranged from 10 mm to 15 mm, in which there was no difference between intact and caries-affected dentin.

Figure 3. a. For intact teeth under 2000× magnification, the depth of outer lesion was approximately 10 μm. An acid-base resistant zone (white arrowheads) was clearly observed beneath the hybrid layer (HA). / b. Close-up of top surface under 7500× magnification, an approximately 1-mm thick acid-base resistant zone was observed (HA). / c. For caries-affected teeth under 2000× magnification, the depth of outer lesion was almost the same as intact teeth. Also, an acid-base resistant zone (white arrowheads) was observed clearly beneath the hybrid layer (HA). / d. Close-up of the top surface under 7500× magnification, a more than 1-mm thick acid-base resistant zone was observed. It was thicker than that of intact teeth (HA).Figure 3. a. For intact teeth under 2000× magnification, the depth of outer lesion was approximately 10 μm. An acid-base resistant zone (white arrowheads) was clearly observed beneath the hybrid layer (HA). / b. Close-up of top surface under 7500× magnification, an approximately 1-mm thick acid-base resistant zone was observed (HA). / c. For caries-affected teeth under 2000× magnification, the depth of outer lesion was almost the same as intact teeth. Also, an acid-base resistant zone (white arrowheads) was observed clearly beneath the hybrid layer (HA). / d. Close-up of the top surface under 7500× magnification, a more than 1-mm thick acid-base resistant zone was observed. It was thicker than that of intact teeth (HA).Figure 3. a. For intact teeth under 2000× magnification, the depth of outer lesion was approximately 10 μm. An acid-base resistant zone (white arrowheads) was clearly observed beneath the hybrid layer (HA). / b. Close-up of top surface under 7500× magnification, an approximately 1-mm thick acid-base resistant zone was observed (HA). / c. For caries-affected teeth under 2000× magnification, the depth of outer lesion was almost the same as intact teeth. Also, an acid-base resistant zone (white arrowheads) was observed clearly beneath the hybrid layer (HA). / d. Close-up of the top surface under 7500× magnification, a more than 1-mm thick acid-base resistant zone was observed. It was thicker than that of intact teeth (HA).
Figure 3 a. For intact teeth under 2000× magnification, the depth of outer lesion was approximately 10 μm. An acid-base resistant zone (white arrowheads) was clearly observed beneath the hybrid layer (HA). b. Close-up of top surface under 7500× magnification, an approximately 1-mm thick acid-base resistant zone was observed (HA). c. For caries-affected teeth under 2000× magnification, the depth of outer lesion was almost the same as intact teeth. Also, an acid-base resistant zone (white arrowheads) was observed clearly beneath the hybrid layer (HA). d. Close-up of the top surface under 7500× magnification, a more than 1-mm thick acid-base resistant zone was observed. It was thicker than that of intact teeth (HA).

Citation: Operative Dentistry 31, 4; 10.2341/05-62

For the intact dentin specimen, the top surfaces of the resin composite, Metafil Flo (C) and the adhesive, Clearfil SE Bond (B), were not changed by the acid-base challenge (Figure 3a). The polished surface of the adhesive was approximately 10-mm thick. A hybrid layer distinguished by argon ion beam etching (HA) was observed at the interface. In the SEM picture under 7500× magnification (Figure 3b), the thickness of the hybrid layer (HA) is approximately 1 mm. The top surface of the hybrid layer (HA) was not damaged by the acid-base challenge. An acid-base resistant zone (ABRZ) was recognized beneath the hybrid layer (HA), which was approximately 1-mm thick in the intact dentin specimen.

For the caries-affected dentin specimen, the top surfaces of the resin composite (C) and the adhesive (B) were not changed by the acid-base challenge and were found to be quite similar to those of the intact dentin specimens (Figure 3c). A hybrid layer distinguished by argon-ion beam etching (HA) was recognized, which was approximately 1-mm thick under 7500× magnification (Figure 3d). The top surface of the hybrid layer (HA) was not damaged by the acid-base challenge. An acid-base resistant zone (ABRZ) was observed beneath the hybrid layer (HA). The acid-base resistant zone of the caries-affected dentin specimen was approximately 2-mm thick, which was thicker than that of the intact dentin specimen.

Nanoindentation

The mean values of the nanoindentation test are shown in Figure 4. Two-way ANOVA revealed that the nanohardness was influenced by the type of dentin (F=61.80, p<0.05) and the position of measurement (F=28.01, p<0.05). The mean nanohardness of normal dentin was 57 MPa and the caries-affected dentin was 36 MPa. At a width of 2-mm deep in dentin, under the hybrid layer, it was suggested that there was an acid-base resistant zone. The intact dentin group was statistically higher than the caries-affected dentin group (p<0.05). At the hybrid layer, there was no significant difference between the intact dentin and the caries-affected dentin (p>0.05). The nanohardness of the resin composite Metafile Flo in the intact dentin group and the caries-affected dentin group was 35 MPa and 37 MPa, with no significant differences between them (p>0.05), respectively. The bonding agent demonstrated 29 MPa (intact) and 25 MPa (caries-affected), which were also not statistically different from intact dentin and caries-affected dentin (p>0.05).

Figure 4. Mean value of the nanohardness test (n=10).Figure 4. Mean value of the nanohardness test (n=10).Figure 4. Mean value of the nanohardness test (n=10).
Figure 4 Mean value of the nanohardness test (n=10).

Citation: Operative Dentistry 31, 4; 10.2341/05-62

DISCUSSION

It is essential to create a hybrid layer at the resin-dentin interface in order to obtain proper adhesion. The hybrid layer is created by penetration and polymerization of adhesive monomers after removal and/or modification of the smear layer and superficial demineralization of the dentin (Nakabayashi, 1985).

Argon-ion beam etching has been used to clearly reveal the hybrid layer at the resin-dentin interface (Inokoshi & others, 1993). Roughening of the hybrid layer through argon-ion beam etching seems to be produced by selective removal of the impregnated resin component in demineralized dentin. As a result of the edge effect of the roughened surface, this layer was clearly distinguished through the secondary electron image of the roughened surface (Inokoshi & others, 1993). However, it was demonstrated that resin monomer of some adhesive systems penetrated deeper than the hybrid layer distinguished by argon-ion etching (Miyazaki & others, 2003; Nakajima & others, 2005).

During sample preparation, a demineralizing solution was used to create artificial secondary caries. Thereafter, a 5% sodium hypochlorite solution was used to remove the demineralized dentin collagen, which makes observation of the ultrastructure of the caries lesion easier. The depth of the outer lesion created in this study ranged from 10 to 15 mm. Variation in the depth of the outer lesion might be due to dentinal tubule orientation, mineralization of dentin and inclination of the specimen's surface during SEM observation. In a previous study by Tsuchiya and others (2004), the edge of the adhesive was torn away during specimen polishing. In order to prevent wear of the adhesive during polishing, Super Bond C&B was placed on the surface after the acid-base treatments. As a result, the interface and surrounding area were clearly observed in this study.

Clearfil SE Bond is a fluoride-free 2-step self-etching primer system. An acidic monomer, 10-methacryloxydecyl dihydrogenphosphate (MDP), in the primer, dissolves the smear layer and demineralizes the underlying dentin, resulting in mild surface etching. Good dentin bonding with a self-etching primer system has already been demonstrated in numerous laboratory studies (Inoue & others, 2001; Yoshida & others, 2004). However, the tensile bond strength of a self-etching primer adhesive system to caries-affected dentin was lower than to normal dentin (Doi & others, 2002; Nakajima & others, 1999). From SEM observations, the SE Bond adhesive demonstrated good resistance to the acid-base challenge in both the intact and caries-affected dentin specimens, which could be due to the properties of the adhesive and the ingredients, such as the multi-functional methacrylates and microfillers (Tsuchiya & others, 2004).

After argon-ion etching, the hybrid layer (HA) detected was approximately 1-mm thick for intact dentin, while a slightly thicker hybrid layer (HA) was observed for caries-affected dentin. These measurements are similar to previous studies (Arrais & Giannini, 2002; Doi & others, 2002). An acid-base resistant zone, approximately 1-mm thick, was observed beneath the hybrid layer (HA) for intact dentin, while a thicker acid-base resistant zone, approximately 1.5-mm thick, was created in the caries-affected dentin.

The acid-base resistant zone and surrounding lesion were also characterized by the nanoindentation technique. The results demonstrated differences in nanohardness of the intact and caries-affected dentin specimens. The nanohardness of the intact dentin area was significantly higher than that of the caries-affected dentin (Nakajima & others, 1995; Hosoya & Marshall, 2004) and did not change at different measuring points on the dentin surface. For caries-affected dentin, the area 2 mm beneath the hybrid layer indicated higher nanohardness compared to the other caries-affected dentin surface, which was coincident with the acid-base resistant zone in the SEM observation. On the other hand, nanohardness around the hybrid layer was approximately 35 MPa in both the intact and caries-affected dentin. In addition, nanohardness of the adhesive and resin composite was approximately 27 MPa and 36 MPa, respectively, in both the intact and caries-affected dentin.

Since the caries-affected intertubular dentin is already partially demineralized and more porous, caries-affected dentin is softer than normal dentin (Ogawa & others, 1983; Hamid & Hume, 1997), and this was confirmed in this study. The intertubular dentin of caries-affected dentin is more permeable to primer than that of normal dentin. Also, the smear layer in caries-affected dentin might be more porous than normal dentin (Nakajima & others, 1999). Therefore, resin monomer could penetrate caries-affected dentin deeper than intact dentin, resulting in a thicker hybrid layer (HA) in caries-affected dentin.

An acid-base resistant zone was observed beneath the hybrid layer (HA) in both intact and caries-affected dentin specimens that used SEM. Formation of an acid-base resistant zone created by Clearfil SE Bond would be related to penetration of the adhesive, but also to the quality of the hybrid layer (HA). Watanabe, Takarada and Nakabayashi (1991) observed a self-etching primer adhesive system—dentin interface after mild acidic treatment by TEM. They reported hydroxyapatite particles wrapped with resin monomer remaining below the hybrid layer. Miyazaki and others (2003) performed a micro laser Raman spectroscopic study of the interface between a self-etching primer system and dentin, in which the authors demonstrated that a resin monomer penetrates deeper than the hybrid layer identified by an argon-ion beam etching (HA). The creation of an acid-base resistant zone may depend on the adhesive system, fluoride-release, monomer penetration, polymerizability and caries-affected mineralization of dentin.

An acid-base resistant zone was identified as part of the hybrid layer in this study. Higher nanohardness of caries-affected dentin 2 mm beneath the hybrid layer supported the theory that the acid-base resistant zone is composed of penetrated monomer and dentin. Tsuchiya and others (2004) suggested that the formation of an acid-base resistant zone was adhesive-material dependent. Also, a fluoride-releasing adhesive created a thick acid-base resistant zone.

Secondary caries begins at the margin between dentin and the restorative material. However, the Clearfil SE Bond-dentin interface demonstrated resistance against the acid-base challenge more often than both the intact and caries-affected dentin. The formation of an outer lesion around the restoration should not be referred to as secondary caries, but primary caries. Therefore, formation of an acid-base resistant zone is important in the prevention of secondary caries around a restoration. Further studies should be performed to characterize the acid-base resistant zone in the various adhesive materials.

CONCLUSIONS

An acid-base resistant zone (ABRZ) created by Clearfil SE Bond was clearly observed beneath the hybrid layer (HA) in both intact and caries-affected dentin.

  • The ABRZ of caries-affected dentin was thicker than that of normal dentin, while its nanohardness was lower than normal dentin.

  • A modified specimen preparation for SEM is useful for observing the hybrid layer (HA) and acid-base resistant zone around the dentin-adhesive interface.

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Copyright: Copyright: © 2006 This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. 2006
Figure 1
Figure 1

Schematic illustration of the methodology of specimen preparation.

Figure 2
Figure 2

Schematic illustration of the methodology of the nanohardness measurement using a nanoindentation testing machine (ENT-1100).

Figure 3
Figure 3

a. For intact teeth under 2000× magnification, the depth of outer lesion was approximately 10 μm. An acid-base resistant zone (white arrowheads) was clearly observed beneath the hybrid layer (HA).

b. Close-up of top surface under 7500× magnification, an approximately 1-mm thick acid-base resistant zone was observed (HA).

c. For caries-affected teeth under 2000× magnification, the depth of outer lesion was almost the same as intact teeth. Also, an acid-base resistant zone (white arrowheads) was observed clearly beneath the hybrid layer (HA).

d. Close-up of the top surface under 7500× magnification, a more than 1-mm thick acid-base resistant zone was observed. It was thicker than that of intact teeth (HA).

Figure 4
Figure 4

Mean value of the nanohardness test (n=10).

Contributor Notes

*Reprint request: 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan; e-mail: inoue.ope@tmd.ac.jp
Received: 25 Apr 2005
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