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The role of functional monomers in bonding to enamel: Acid–base resistant zone and bonding performance
journal of dentistry 38 (2010) 722–730
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The role of functional monomers in bonding to enamel: Acid–base resistant zone and bonding performance Na Li a, Toru Nikaido b, Tomohiro Takagaki b, Alireza Sadr b,c, Patricia Makishi b,c, Jihua Chen a,*, Junji Tagami b,c a
Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, No. 145, Changlexi Road, 710032 Xi’an, China Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan c Global Center of Excellence (GCOE) Program; International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan b
article info
abstract
Article history:
Objectives: To investigate the effects of two functional monomers on caries-inhibition
Received 15 February 2010
potential and bond strength of two-step self-etching adhesive systems to enamel.
Received in revised form
Methods: Clearfil SE Bond and similar experimental formulations different in the functional
10 May 2010
monomer were used. Four combinations of primer and bonding agents were evaluated: (1)
Accepted 15 May 2010
Clearfil SE Bond which contains MDP in both primer and bonding (M–M); (2) Clearfil SE Bond primer and Phenyl-P in bonding (M–P); (3) Phenyl-P in primer and Clearfil SE Bond bonding (P–M); (4) Phenyl-P in primer and bonding (P–P). Ground buccal enamel surfaces of human
Keywords:
sound premolars were treated with one of the systems and the bonded interface was
Enamel
exposed to an artificial demineralising solution (pH 4.5) for 4.5 h, and then 5% NaOCl with
Functional monomer
ultrasonication for 30 min. After argon-ion etching, the interfacial ultrastructure was
Acid–base resistant zone
observed using SEM. Micro-shear bond strength to enamel was measured for all groups
Micro-shear bond strength
and results were analysed using one-way ANOVA and Turkey’s HSD, while failure modes
Two-step self-etching adhesive
were analysed by chi-square test. Results: An acid–base resistant zone (ABRZ) was found with all adhesive systems containing MDP either in primer or bond; however, ultramorphology and crystallite arrangement in the ABRZ were different among groups. P–P was the only group devoid of this protective zone. Micro-shear bond strength in M–M was significantly higher than those in M–P, P–M and P–P, while the latter three were not different from each other. Failure modes were significantly different ( p < 0.05). Conclusions: Functional monomers in two-step self-etching systems influence both the bonding performance and the formation of ABRZ on enamel. # 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
In 1955, Buonocore1 reported that the mechanical retention of an acrylic resin to enamel could be substantially increased by the phosphoric acid treatment. Since then, generations of dental adhesive materials have been developed and studied. More
recently, self-etching adhesive systems have been proposed.2,3 These systems eliminate the separate steps of etching and rinsing, therefore the application procedures and technique sensitivity could be reduced. The most widely used self-etching adhesive systems are the two-step self-etching adhesives, which involve two application steps: the conditioning of tooth
* Corresponding author. Tel.: +86 29 8477 6329; fax: +86 29 8477 6329. E-mail address: [email protected] (J. Chen). 0300-5712/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2010.05.015
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substrates with a self-etching primer, followed by the application of a bonding resin. Most self-etching adhesive systems contain specific functional monomers that, to a large extent, determine the adhesive performance. The functional monomers may serve for various functions, such as etching tooth substrates, enhancing monomer penetration,4 and also imparting the adhesives with chemical interaction potential to dental substrates.5 So far, the most commonly used functional monomers in commercial adhesives are phosphate monomers, such as MDP and Phenyl-P.6 Both of them have been used as etching monomers in self-etching primers,7,8 and used in bonding agents to promote resin diffusion and adhesion. It has also been proven that both of the two monomers could chemically interact with HAp. However, a different chemical bonding potential between MDP and Phenyl-P should be noticed. MDP, rated as one of the most promising monomers for chemical bonding to dental substrates,4,9 is capable of establishing an ionic bond with HAp most readily and intensively, even in the short exposure time of 30 s; in contrast, Phenyl-P, a functional monomer with very limited chemical bonding capacity, could only form weak ionic binding with HAp and produce highly soluble reaction product.9 Although the effects of these functional monomers were investigated in some studies, most of them tested commercial products, which only led to hypotheses concerning the properties of particular compositions.4 A few studies have focused on functional monomers alone or incorporated in one component of adhesive systems (either in primer, bond, or single-step self-etching agent)5,7–12; however, the influence of interchanging functional monomers has not been investigated for two-step self-etching adhesive systems, where the functional monomers are incorporated both into the primer and the bonding resin. In 2004, Tsuchiya et al.13 firstly observed a zone beneath the hybrid layer when dentin was treated with Clearfil SE Bond. It is characterized by a resistance to an acid and base challenge, and different from the conventional hybrid layer and cariesinhibition zone (fluoride-releasing material). Therefore, it was named as ‘‘acid–base resistant zone (ABRZ)’’. It is supposed to play an important role in the prevention of secondary caries, the sealing of restoration margins and the promotion of restoration durability. After that, the existence of dentin ABRZ has been confirmed with some other self-etching adhesive
systems.13–16 Various adhesive materials and evaluation methods were assessed with dentin14–21 in previous studies; however, it is still unknown whether or not an ABRZ forms when enamel is treated with self-etching adhesives. Based on these considerations, the purpose of this study was to investigate the role of two functional monomers (MDP and Phenyl-P) incorporated into two-step self-etching adhesives on the formation of ABRZ and enamel bonding performance by these adhesives. The null hypotheses to be tested were: (a) the functional monomers would not influence the formation of an ABRZ; (b) the functional monomers would not affect the micro-shear bond strength of the tested twostep self-etching adhesive systems to enamel.
2.
Materials and methods
A two-step self-etching primer adhesive system, Clearfil SE Bond (Kuraray Medical, Tokyo, Japan) served as a control. This system is composed of an MDP-containing primer and an MDP-containing bonding resin. An experimental primer and a bonding resin containing Phenyl-P as a substitution for MDP were prepared, in which the Phenyl-P concentrations were similar to those of MDP in Clearfil SE Bond. According to the combination of primer and bond as shown in Table 1, four experimental groups were formed: (1) Clearfil SE Bond as control (M–M); (2) Clearfil SE Bond primer and Phenyl-P in bonding (M–P); (3) Phenyl-P in primer and Clearfil SE Bond bonding (P–M); (4) Phenyl-P in primer and bonding (P–P). Twenty non-carious human third molars and 30 human premolars were collected after the individuals’ informed consent was obtained according to a protocol approved by the Institutional Review Board of Tokyo Medical and Dental University. The teeth were stored in water at 4 8C and used within 1 month after extraction.
2.1.
Observation of the acid–base resistant zone (ABRZ)
Sample preparation for SEM observation of the ABRZ is illustrated in Fig. 1. Premolars were used for this part of the study. Roots were removed and crowns were sectioned mesiodistally using a low-speed diamond saw (Isomet; Beuhler, Lake Buff, IL, USA). In order to prepare the midcoronal buccal enamel surfaces for bonding, crowns with buccal surfaces facing out were embedded in epoxy resin
Table 1 – Composition, pH and manufacturer’s instructions of the four experimental two-step self-etching adhesive system groups. Adhesive system
Functional monomer Primer
M–M (Clearfil SE Bond) M–P P–M P–P
Bond
MDP
MDP
MDP Phenyl-P Phenyl-P
Phenyl-P MDP Phenyl-P
Compositions in common Primer
Bond
Water, HEMA, hydrophilic dimethacrylate, CaM–phorquinone, N,N-diethanol p-toluidine
Bis-GMA, HEMA, hydrophobic dimethacrylate, CaM–phorquinone, N,N-diethanol p-toluidine, silanated colloidal silica
pH of primer
2.0
1.3
Application
Apply primer and leave for 20 s. Moderate air-dry for 10 s. Apply bond and leave for 10 s. Gently air-blow for 10 s. Light cure for 10 s
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Fig. 1 – Sample preparation for SEM observation of ABRZ on enamel.
(Epoxicure Resin, Buehler) overnight and then wet-polished with 600-grit SiC paper until flat enamel surfaces were exposed. The surfaces were examined under a stereo microscope (Nikon SMZ10, Tokyo, Japan) at 10 magnification to exclude any surface defects or decalcified areas. The unground enamel surfaces were covered by an adhesive tape to restrict the bonding area to the prepared enamel. Twenty-four of the prepared enamel discs were randomly divided into four groups. For each group, the enamel surfaces were treated with one of the four adhesive systems according to the manufacturer’s instructions for Clearfil SE Bond. A halogen light curing unit (Optilux 500; Demetron-Kerr, Danbury, CT, USA) was used to light cure the bonding agent for 10 s. A flowable composite Metafil Flow (shade A3, Sun Medical, Moriyama, Japan) was then applied on the top (approximately 1 mm in thickness) and cured for 20 s. After storage in distilled water at 37 8C for 24 h, each specimen was sectioned perpendicular to the bonding interface into two halves and embedded in the epoxy resin overnight again. They were wet-polished with SiC paper from 400-grit to 1200-grit to standardize the surface, and then subjected to an acid–base challenge. For the acid challenge, each specimen was stored in 100 ml buffered demineralising solution (pH 4.5, 2.2 mmol/l CaCl2, 2.2 mmol/ l NaH2PO4 and 50 mmol/l acetic acid) for 4.5 h to create artificial secondary caries.22,23 For the base challenge, the specimens were immersed in 5% NaOCl for 30 min with ultrasonic,24 in an attempt to remove any demineralised structures or enamel proteins, and rinsed with running water for 30 s. Following this, a 4-META/MMA-TBB resin (Super Bond C&B, Sun Medical, Moriyama, Japan) was applied without acid-etching of the treated surface, in order to prevent wear of the adhesive during polishing. Then the specimens were cut perpendicular to the enamel– adhesive interface, and polished with diamond pastes
(Struers A/S, Copenhagen, Denmark) down to a particle size of 0.25 mm. Argon-ion etching (EIS-IE, Elionix, Tokyo, Japan) was used for the polished surfaces to bring the resin– enamel interface into sharp relief, with an accelerating voltage of 1 kV and an ion current density of 0.2 mA/cm2 for 7 min.14 The specimens were then gold-sputter coated, and morphological attributes of the interface after the acid–base challenge were observed using SEM (JSM-5310LV, JOEL, Tokyo, Japan) at 2500, 10,000 and 60,000 magnifications with an accelerating voltage of 20 kV.
2.2. SEM observation of the enamel surface after primer treatment Buccal enamel surfaces of six premolars were wet-polished with 600-grit SiC paper until flat. The ground enamel surfaces on two teeth without any adhesive treatment were used as the control. The other four teeth were randomly divided into two groups. One group was conditioned using self-etching primer with MDP (Clearfil SE Bond primer) and the other with the Phenyl-P-containing primer for 20 s. Immediately after the primer treatment, the surface was rinsed with an ascending series of ethanol (30, 50, 70 and 95%) for 1 min each, and further ultrasonic cleaned in absolute acetone for 1 min in order to completely dissolve the primer and dehydrate the specimens for SEM observation. After air-drying, samples were coated with gold and examined using the SEM to observe surface texture and etching pattern.
2.3.
Micro-shear bond test
The teeth were cut perpendicular to the tooth axis with the low-speed diamond saw to produce a 2-mm-thick cuspal enamel slice from each tooth. The slices were wet-polished with 600-grit SiC paper from the cuspal side until flat enamel surfaces were exposed. The surfaces were examined under an
journal of dentistry 38 (2010) 722–730
optical microscope at 10 magnification to exclude specimens with any defects. Then the specimens were randomly divided into four groups and treated with one of the four adhesive systems. Before light polymerisation of the adhesive resin, a Tygon tubing (R-3603, Norton Performance Plastic, Cleveland, USA), with an internal diameter and height of 0.7 mm and 0.5 mm, respectively, was placed on the enamel surface to restrict the bonding area.25 After the adhesive was light cured, a hybrid resin composite Clearfil AP-X (shade A3, Kuraray Medical) was injected into the tube and pressed gently, then light cured for 40 s. The specimens were kept at room temperature (23 8C) for 1 h, and then in 37 8C water for 24 h. After removing Tygon tubes carefully, each enamel slice with resin cylinders was attached to a jig (BencorMulti-T, Danville Engineering Co, San Ramon, CA, USA) using a cyanoacrylate glue (Zapit, Dental Ventures of America, Corona, CA, USA) and placed in a universal testing machine (EA-test-500N, Shimadzu, Kyoto, Japan). A thin wire with a diameter of 0.2 mm was looped around the resin cylinder and gently held flush against the resin/enamel interface. To insure the desired direction of shear force, the resin/enamel interface, wire loop, and centre of the load cell were aligned as straight as possible. A shear force was applied to each specimen at a crosshead speed of 1.0 mm/ min until failure occurred. The mean bond strengths were calculated then statistically analysed using one-way ANOVA and Turkey’s HSD test at the significance level of a = 0.05 (n = 10). All the de-bonded enamel surfaces after micro-
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shear bond test were observed under the lightscopy to identify the failure mode, and further gold-sputter-coated and confirmed by SEM. The distribution of failure mode was analysed by chi-square test at a significance level of a = 0.05.
3.
Results
3.1. SEM observations of ultrastructural features after acid–base challenge Typical interface morphologies after acid–base challenge were shown in Figs. 2–5. An outer lesion (OL), created by mineral loss due to the acid–base challenge, was observed in all groups. The depth of OL ranged from 10 to 15 mm in all groups. For the M–M, an electron dense zone (Fig. 2a and c), approximately 0.5 mm in thickness, was observed. At a higher magnification, the zone consisted of densely arranged grainlike crystallites (Fig. 2d). A right angle was observed at the junction of bonding, OL and enamel (Fig. 2b). For the M–P, an electron dense zone (Fig. 3a and c), less than 0.2 mm thick, was observed. Crystallite ultramorphology (Fig. 3d) was similar to that of the M–M, but the crystal distribution was sparser. A right angle was observed at the junction of bonding, OL and enamel (Fig. 3b). The electron dense zone formed in the P–M was approximately 1 mm thick, the thickest observed in this study (Fig. 4a and c). The edge of this zone towards the top OL showed a
Fig. 2 – Ultrastructure of enamel-adhesive interface after acid–base challenge in M–M group. OL: outer lesion; C: resin composite; B: bonding layer; E: enamel; ABRZ: acid–base resistant zone. (a) ABRZ was pointed between triangles. (b) A right angle (arrow) was observed at the junction of bonding, OL and enamel. (c) Thickness of the zone was approximately 0.5 mm. (d) Densely arranged grain-like crystallites in the zone.
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Fig. 3 – Ultrastructure of enamel-adhesive interface after acid–base challenge in M–P group. (a) ABRZ was pointed between triangles. (b) A right angle (arrow) was observed at the junction of bonding, OL and enamel. (c) Thickness of the zone was less than 0.2 mm. (d) Grain-like crystallites in the zone.
Fig. 4 – Ultrastructure of enamel-adhesive interface after acid–base challenge in P–M group. (a) ABRZ was pointed between triangles. (b) A right angle (arrow) was observed at the junction of bonding, OL and enamel. (c) Thickness of the zone was about 1 mm. The edge showed a wave-like shape. (d) Sparsely arranged large needle-like crystallites were observed in the zone.
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Fig. 5 – Ultrastructure of enamel–adhesive interface after acid–base challenge in P–P group. (a) ABRZ was absent at the corresponding region (triangles). (b) Funnel-shaped erosion (arrow) was present at the junction of bonding, enamel and OL. (c) In the corresponding region, a few grain-like structures were observed only at the bottom of OL. (d) A few remnant grainlike structures.
wave-like pattern. Sparsely arranged large needle-like crystallites (Fig. 4d) could be distinguished under high magnification (60,000). Compared with the crystallites in the M–M and M–P, these crystallites were obviously longer in length, but showed similar width. Intercrystalline spaces could be clearly observed. A right angle with no erosion was present at the junction of bonding, OL and enamel (Fig. 4b). The ABRZ-like structure was almost devoid in the P–P (Fig. 5a and c). Only at the bottom of OL, a very thin electron dense layer (less than 0.1 mm thick) was detected (Fig. 5b and c). Examination under 60,000 magnification revealed that this layer consisted of a few sparse grain-like structures (Fig. 5d). In addition, funnel-shaped erosion (arrow) at the junction of the bonding interface, enamel and OL was clearly observed (Fig. 5b).
3.2.
Enamel surface after primer treatment
Fig. 6a shows the enamel surface ground with 600-grit SiC paper, with a smear layer covering the entire surface. The MDP primer treated surface exhibited a shallow honeycomb etching pattern which is caused by preferential dissolution of enamel prism (Fig. 6b and c). The prism peripheries could be distinguished. On the other hand, when treated with PhenylP-containing primer, a deeper etching pattern with an overall increase in roughness was evident along the entire aprismatic enamel surface (Fig. 6d and e). Larger spaces appeared in the exposed crystalline structure.
3.3.
Micro-shear bond-strength test
The micro-shear bond-strength values and failure modes are shown in Table 2. M–M showed significantly higher bond strength compared to the other three groups (one-way ANOVA, p < 0.005). For groups M–P, P–M and P–P, the bond strength showed no significant difference ( p > 0.05). There was significant difference in the distribution of failure mode among groups ( p < 0.05). M–M, M–P and P–M exhibited adhesive and cohesive failure as the predominant failure mode, while the major failure mode in the P–P was adhesive failure.
Table 2 – Micro-shear bond strength and failure mode of the four adhesive systems to enamel. Group
M–M M–P P–M P–P
n
10 10 10 10
Micro-shear bond strength (MPa) 43.08 36.12 33.29 35.79
(3.73) a (6.61) b (5.29) b (5.42) b
Failure mode * A
C
AC
2 1 3 8
2 0 2 0
6 9 5 2
Values identified with different letters are significantly different at p < 0.005. A: adhesive failure; C: cohesive failure in enamel or composite; AC: adhesive and cohesive failure. * There was a significant difference in the distribution of failure modes (chi-square test, p < 0.05).
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Fig. 6 – SEM observation of the enamel surface after treatment. (a) Without primer, the entire surface was covered by smear layer. (b and c) Enamel surface treated by MDP-containing primer. (d and e) Enamel surface treated by Phenyl-P-containing primer.
4.
Discussion
Secondary caries is considered as a major cause for the failure of restorations. Acidic bacterial byproducts may infiltrate not only the bonding interface, but also the tooth tissue at the periphery, creating a maginal demineralised zone, and thus rapidly promote caries occurrence. In this regard, it has been suggested that an increased resistance of the tooth–resin interface to acid may effectively retard the progression of secondary caries.21 With dentin substrate, the ABRZ beneath the hybrid layer has already been confirmed with some self-etching adhesives.14–21 Although the formative mechanism is still unclear, it was assumed that the penetration of the monomers into the tooth tissue beyond the hybrid layer and the chemical interaction between the functional monomer and hydroxyapatite (HAp) may contribute to the formation of ABRZ. The ABRZ observed on enamel in this study may be similar in nature to
those reported for dentin. However, differences between dentin and enamel should be noted; the self-etching primer removes the smear layer and demineralises the underlying dentin mildly, leaving some HAp crystallites partially attached to the dentin collagen.15 On the other hand, enamel has a higher mineral content with a matrix structure different from dentin collagen network. HAp crystals are distributed all over the demineralised layer. Therefore, for dentin, ABRZ forms under the hybrid layer; while for enamel, the ABRZ is not below the bonding interface, but it is rather at the interface, involving those parts of the interface which were not dissolved after the acid–base challenge. Sample preparation for SEM observation of dentin ABRZ has been established by Inoue14 and successfully applied in many studies.15–21 However, with regard to the structural differences between the two substrates, it is necessary to establish new experiment protocols for the observation of ABRZ on enamel. The optimal acid–base challenge conditions in this study were set up based on the
journal of dentistry 38 (2010) 722–730
results of pilot experiments. In the acid challenge, the same demineralising solution used for dentin was applied for enamel. The immersion period of 4.5 h was three times as long as the demineralising period for dentin, in order to create outer lesions with depths comparable to those created with dentin. For the base challenge, 5% NaOCl solution was applied for 30 min with ultrasonic vibration24 to completely remove the demineralised enamel. In this study, ABRZ could be clearly detected in the M–M, M– P and P–M groups (Figs. 2–4), in which MDP was included either in the primer or in the bonding agent. P–P was the only group in which the ABRZ could not be distinguished at the most parts of the corresponding region (Fig. 5). And a funnel-shaped erosion area was noted at the junction of ABRZ, OL and enamel in this group, indicating a weak area vulnerable to acid–base attack beneath the bonding interface. In self-etching adhesives, resin monomers penetrate into the demineralised enamel and envelop the HAp crystals upon curing, therefore protect these crystals from acid attack; however, when the adhesive system is not cured well, the crystals are less protected and thus, prone to faster dissolution by acid. Therefore, it is possible that the P–P group in this study had a lower curing efficacy. On the other hand, MDP has been found to interact chemically with HAp intensively and stably.9 It is capable of forming strong ionic bonds with calcium, producing MDP-calcium salt with a lower solubility. In contrast, the chemical bonding capacity of Phenyl-P is very limited. The resulting Ca-salts are susceptible to dissolution during the acid–base challenge. Further chemical analysis is necessary to prove these speculations. P–M group produced the thickest ABRZ with needle-like crystallites, possibly related to the more aggressive etching capacity of the Phenyl-P-containing primer; the pH value of this primer was lower than that of the MDP-containing primer (1.3 vs. 2.0 respectively, according to the manufacturer). In this regard, SEM examination showed that etching with Phenyl-Pcontaining primer produced a deeper and more porous etching pattern on enamel surface (Fig. 6d and e), in contrast to the shallow etching pattern produced by MDP-containing primer26 (Fig. 6b and c). When the bonding was applied to this more aggressively etched surface, monomers including MDP penetrated deeper. This may have contributed to the formation of a thicker ABRZ, and different morphological appearance involved. As far as this study was concerned, the second hypothesis that functional monomers would not affect the adhesive bonding performance was rejected. M–M showed significantly higher bond strength than the other three groups. Although the micromechanical adhesion, achieved by the diffusion and polymerisation of resin monomers into the pretreated enamel, is assumed to be the main bonding mechanism of dental adhesives to enamel,27 the chemical bonding is also supposed to influence the bonding performance of adhesive systems.5 As pointed previously, the higher and more stable chemical bonding performance of MDP compared to Phenyl-P may also have contributed to the higher bond strength to enamel. Besides, since the two monomers have different chemical structures, polymerisation efficacy and mechanical properties of the resulting polymers may be different. It has been shown that the polymerisation reactivity of functional monomers is
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affected by the length and flexibility of the alkyl spacer chains.28,29 MDP molecule has a long linear alkyl chain and phosphoric acid ester group, while Phenyl-P contains a shorter alkyl chain spacer and a phenyl group at the acidic moiety. The mode of failure in the micro-shear bond-strength technique employed in this study could be affected by the uneven stress distributions at the interface; however, it is noteworthy that a remarkable difference existed in the failure mode pattern of P–P compared to other groups; suggesting that the possibly lower polymerisation efficacy and the weak chemical bonding potential of Phenyl-P as discussed earlier may have resulted in the adhesive failure as its predominant fracture pattern. In conclusion, the null hypotheses of this study were both rejected. The findings suggested that the interface formed by MDP-containing adhesive systems remains after acid–base challenge, and may play an important role in improving bonding performance and resistance against the acid attack from the microorganisms in caries. Further study should be carried out to evaluate the long-term bonding efficacy of different functional monomers in dental adhesive systems. The ABRZ pattern obtained from each adhesive system may become a good indicator to predict its bonding durability to tooth substrates. Interchanging a single functional monomer in an adhesive system is an interesting model for investigating bonding performance and mechanisms. Future study and confirmatory experiments are necessary to interpret the mechanism of ABRZ formation and the bonding performance.
Acknowledgements This work was supported by the grant from the Chinese Government Graduate Student Overseas Study Program, the Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases at Tokyo Medical and Dental University and a Grantin Aid from the Japan Society for the Promotion of Science (JSPS No. 22592116).
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The role of functional monomers in bonding to enamel: Acid–base resistant zone and bonding performance Na Li a, Toru Nikaido b, Tomohiro Takagaki b, Alireza Sadr b,c, Patricia Makishi b,c, Jihua Chen a,*, Junji Tagami b,c a
Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, No. 145, Changlexi Road, 710032 Xi’an, China Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan c Global Center of Excellence (GCOE) Program; International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan b
article info
abstract
Article history:
Objectives: To investigate the effects of two functional monomers on caries-inhibition
Received 15 February 2010
potential and bond strength of two-step self-etching adhesive systems to enamel.
Received in revised form
Methods: Clearfil SE Bond and similar experimental formulations different in the functional
10 May 2010
monomer were used. Four combinations of primer and bonding agents were evaluated: (1)
Accepted 15 May 2010
Clearfil SE Bond which contains MDP in both primer and bonding (M–M); (2) Clearfil SE Bond primer and Phenyl-P in bonding (M–P); (3) Phenyl-P in primer and Clearfil SE Bond bonding (P–M); (4) Phenyl-P in primer and bonding (P–P). Ground buccal enamel surfaces of human
Keywords:
sound premolars were treated with one of the systems and the bonded interface was
Enamel
exposed to an artificial demineralising solution (pH 4.5) for 4.5 h, and then 5% NaOCl with
Functional monomer
ultrasonication for 30 min. After argon-ion etching, the interfacial ultrastructure was
Acid–base resistant zone
observed using SEM. Micro-shear bond strength to enamel was measured for all groups
Micro-shear bond strength
and results were analysed using one-way ANOVA and Turkey’s HSD, while failure modes
Two-step self-etching adhesive
were analysed by chi-square test. Results: An acid–base resistant zone (ABRZ) was found with all adhesive systems containing MDP either in primer or bond; however, ultramorphology and crystallite arrangement in the ABRZ were different among groups. P–P was the only group devoid of this protective zone. Micro-shear bond strength in M–M was significantly higher than those in M–P, P–M and P–P, while the latter three were not different from each other. Failure modes were significantly different ( p < 0.05). Conclusions: Functional monomers in two-step self-etching systems influence both the bonding performance and the formation of ABRZ on enamel. # 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
In 1955, Buonocore1 reported that the mechanical retention of an acrylic resin to enamel could be substantially increased by the phosphoric acid treatment. Since then, generations of dental adhesive materials have been developed and studied. More
recently, self-etching adhesive systems have been proposed.2,3 These systems eliminate the separate steps of etching and rinsing, therefore the application procedures and technique sensitivity could be reduced. The most widely used self-etching adhesive systems are the two-step self-etching adhesives, which involve two application steps: the conditioning of tooth
* Corresponding author. Tel.: +86 29 8477 6329; fax: +86 29 8477 6329. E-mail address: [email protected] (J. Chen). 0300-5712/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2010.05.015
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substrates with a self-etching primer, followed by the application of a bonding resin. Most self-etching adhesive systems contain specific functional monomers that, to a large extent, determine the adhesive performance. The functional monomers may serve for various functions, such as etching tooth substrates, enhancing monomer penetration,4 and also imparting the adhesives with chemical interaction potential to dental substrates.5 So far, the most commonly used functional monomers in commercial adhesives are phosphate monomers, such as MDP and Phenyl-P.6 Both of them have been used as etching monomers in self-etching primers,7,8 and used in bonding agents to promote resin diffusion and adhesion. It has also been proven that both of the two monomers could chemically interact with HAp. However, a different chemical bonding potential between MDP and Phenyl-P should be noticed. MDP, rated as one of the most promising monomers for chemical bonding to dental substrates,4,9 is capable of establishing an ionic bond with HAp most readily and intensively, even in the short exposure time of 30 s; in contrast, Phenyl-P, a functional monomer with very limited chemical bonding capacity, could only form weak ionic binding with HAp and produce highly soluble reaction product.9 Although the effects of these functional monomers were investigated in some studies, most of them tested commercial products, which only led to hypotheses concerning the properties of particular compositions.4 A few studies have focused on functional monomers alone or incorporated in one component of adhesive systems (either in primer, bond, or single-step self-etching agent)5,7–12; however, the influence of interchanging functional monomers has not been investigated for two-step self-etching adhesive systems, where the functional monomers are incorporated both into the primer and the bonding resin. In 2004, Tsuchiya et al.13 firstly observed a zone beneath the hybrid layer when dentin was treated with Clearfil SE Bond. It is characterized by a resistance to an acid and base challenge, and different from the conventional hybrid layer and cariesinhibition zone (fluoride-releasing material). Therefore, it was named as ‘‘acid–base resistant zone (ABRZ)’’. It is supposed to play an important role in the prevention of secondary caries, the sealing of restoration margins and the promotion of restoration durability. After that, the existence of dentin ABRZ has been confirmed with some other self-etching adhesive
systems.13–16 Various adhesive materials and evaluation methods were assessed with dentin14–21 in previous studies; however, it is still unknown whether or not an ABRZ forms when enamel is treated with self-etching adhesives. Based on these considerations, the purpose of this study was to investigate the role of two functional monomers (MDP and Phenyl-P) incorporated into two-step self-etching adhesives on the formation of ABRZ and enamel bonding performance by these adhesives. The null hypotheses to be tested were: (a) the functional monomers would not influence the formation of an ABRZ; (b) the functional monomers would not affect the micro-shear bond strength of the tested twostep self-etching adhesive systems to enamel.
2.
Materials and methods
A two-step self-etching primer adhesive system, Clearfil SE Bond (Kuraray Medical, Tokyo, Japan) served as a control. This system is composed of an MDP-containing primer and an MDP-containing bonding resin. An experimental primer and a bonding resin containing Phenyl-P as a substitution for MDP were prepared, in which the Phenyl-P concentrations were similar to those of MDP in Clearfil SE Bond. According to the combination of primer and bond as shown in Table 1, four experimental groups were formed: (1) Clearfil SE Bond as control (M–M); (2) Clearfil SE Bond primer and Phenyl-P in bonding (M–P); (3) Phenyl-P in primer and Clearfil SE Bond bonding (P–M); (4) Phenyl-P in primer and bonding (P–P). Twenty non-carious human third molars and 30 human premolars were collected after the individuals’ informed consent was obtained according to a protocol approved by the Institutional Review Board of Tokyo Medical and Dental University. The teeth were stored in water at 4 8C and used within 1 month after extraction.
2.1.
Observation of the acid–base resistant zone (ABRZ)
Sample preparation for SEM observation of the ABRZ is illustrated in Fig. 1. Premolars were used for this part of the study. Roots were removed and crowns were sectioned mesiodistally using a low-speed diamond saw (Isomet; Beuhler, Lake Buff, IL, USA). In order to prepare the midcoronal buccal enamel surfaces for bonding, crowns with buccal surfaces facing out were embedded in epoxy resin
Table 1 – Composition, pH and manufacturer’s instructions of the four experimental two-step self-etching adhesive system groups. Adhesive system
Functional monomer Primer
M–M (Clearfil SE Bond) M–P P–M P–P
Bond
MDP
MDP
MDP Phenyl-P Phenyl-P
Phenyl-P MDP Phenyl-P
Compositions in common Primer
Bond
Water, HEMA, hydrophilic dimethacrylate, CaM–phorquinone, N,N-diethanol p-toluidine
Bis-GMA, HEMA, hydrophobic dimethacrylate, CaM–phorquinone, N,N-diethanol p-toluidine, silanated colloidal silica
pH of primer
2.0
1.3
Application
Apply primer and leave for 20 s. Moderate air-dry for 10 s. Apply bond and leave for 10 s. Gently air-blow for 10 s. Light cure for 10 s
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Fig. 1 – Sample preparation for SEM observation of ABRZ on enamel.
(Epoxicure Resin, Buehler) overnight and then wet-polished with 600-grit SiC paper until flat enamel surfaces were exposed. The surfaces were examined under a stereo microscope (Nikon SMZ10, Tokyo, Japan) at 10 magnification to exclude any surface defects or decalcified areas. The unground enamel surfaces were covered by an adhesive tape to restrict the bonding area to the prepared enamel. Twenty-four of the prepared enamel discs were randomly divided into four groups. For each group, the enamel surfaces were treated with one of the four adhesive systems according to the manufacturer’s instructions for Clearfil SE Bond. A halogen light curing unit (Optilux 500; Demetron-Kerr, Danbury, CT, USA) was used to light cure the bonding agent for 10 s. A flowable composite Metafil Flow (shade A3, Sun Medical, Moriyama, Japan) was then applied on the top (approximately 1 mm in thickness) and cured for 20 s. After storage in distilled water at 37 8C for 24 h, each specimen was sectioned perpendicular to the bonding interface into two halves and embedded in the epoxy resin overnight again. They were wet-polished with SiC paper from 400-grit to 1200-grit to standardize the surface, and then subjected to an acid–base challenge. For the acid challenge, each specimen was stored in 100 ml buffered demineralising solution (pH 4.5, 2.2 mmol/l CaCl2, 2.2 mmol/ l NaH2PO4 and 50 mmol/l acetic acid) for 4.5 h to create artificial secondary caries.22,23 For the base challenge, the specimens were immersed in 5% NaOCl for 30 min with ultrasonic,24 in an attempt to remove any demineralised structures or enamel proteins, and rinsed with running water for 30 s. Following this, a 4-META/MMA-TBB resin (Super Bond C&B, Sun Medical, Moriyama, Japan) was applied without acid-etching of the treated surface, in order to prevent wear of the adhesive during polishing. Then the specimens were cut perpendicular to the enamel– adhesive interface, and polished with diamond pastes
(Struers A/S, Copenhagen, Denmark) down to a particle size of 0.25 mm. Argon-ion etching (EIS-IE, Elionix, Tokyo, Japan) was used for the polished surfaces to bring the resin– enamel interface into sharp relief, with an accelerating voltage of 1 kV and an ion current density of 0.2 mA/cm2 for 7 min.14 The specimens were then gold-sputter coated, and morphological attributes of the interface after the acid–base challenge were observed using SEM (JSM-5310LV, JOEL, Tokyo, Japan) at 2500, 10,000 and 60,000 magnifications with an accelerating voltage of 20 kV.
2.2. SEM observation of the enamel surface after primer treatment Buccal enamel surfaces of six premolars were wet-polished with 600-grit SiC paper until flat. The ground enamel surfaces on two teeth without any adhesive treatment were used as the control. The other four teeth were randomly divided into two groups. One group was conditioned using self-etching primer with MDP (Clearfil SE Bond primer) and the other with the Phenyl-P-containing primer for 20 s. Immediately after the primer treatment, the surface was rinsed with an ascending series of ethanol (30, 50, 70 and 95%) for 1 min each, and further ultrasonic cleaned in absolute acetone for 1 min in order to completely dissolve the primer and dehydrate the specimens for SEM observation. After air-drying, samples were coated with gold and examined using the SEM to observe surface texture and etching pattern.
2.3.
Micro-shear bond test
The teeth were cut perpendicular to the tooth axis with the low-speed diamond saw to produce a 2-mm-thick cuspal enamel slice from each tooth. The slices were wet-polished with 600-grit SiC paper from the cuspal side until flat enamel surfaces were exposed. The surfaces were examined under an
journal of dentistry 38 (2010) 722–730
optical microscope at 10 magnification to exclude specimens with any defects. Then the specimens were randomly divided into four groups and treated with one of the four adhesive systems. Before light polymerisation of the adhesive resin, a Tygon tubing (R-3603, Norton Performance Plastic, Cleveland, USA), with an internal diameter and height of 0.7 mm and 0.5 mm, respectively, was placed on the enamel surface to restrict the bonding area.25 After the adhesive was light cured, a hybrid resin composite Clearfil AP-X (shade A3, Kuraray Medical) was injected into the tube and pressed gently, then light cured for 40 s. The specimens were kept at room temperature (23 8C) for 1 h, and then in 37 8C water for 24 h. After removing Tygon tubes carefully, each enamel slice with resin cylinders was attached to a jig (BencorMulti-T, Danville Engineering Co, San Ramon, CA, USA) using a cyanoacrylate glue (Zapit, Dental Ventures of America, Corona, CA, USA) and placed in a universal testing machine (EA-test-500N, Shimadzu, Kyoto, Japan). A thin wire with a diameter of 0.2 mm was looped around the resin cylinder and gently held flush against the resin/enamel interface. To insure the desired direction of shear force, the resin/enamel interface, wire loop, and centre of the load cell were aligned as straight as possible. A shear force was applied to each specimen at a crosshead speed of 1.0 mm/ min until failure occurred. The mean bond strengths were calculated then statistically analysed using one-way ANOVA and Turkey’s HSD test at the significance level of a = 0.05 (n = 10). All the de-bonded enamel surfaces after micro-
725
shear bond test were observed under the lightscopy to identify the failure mode, and further gold-sputter-coated and confirmed by SEM. The distribution of failure mode was analysed by chi-square test at a significance level of a = 0.05.
3.
Results
3.1. SEM observations of ultrastructural features after acid–base challenge Typical interface morphologies after acid–base challenge were shown in Figs. 2–5. An outer lesion (OL), created by mineral loss due to the acid–base challenge, was observed in all groups. The depth of OL ranged from 10 to 15 mm in all groups. For the M–M, an electron dense zone (Fig. 2a and c), approximately 0.5 mm in thickness, was observed. At a higher magnification, the zone consisted of densely arranged grainlike crystallites (Fig. 2d). A right angle was observed at the junction of bonding, OL and enamel (Fig. 2b). For the M–P, an electron dense zone (Fig. 3a and c), less than 0.2 mm thick, was observed. Crystallite ultramorphology (Fig. 3d) was similar to that of the M–M, but the crystal distribution was sparser. A right angle was observed at the junction of bonding, OL and enamel (Fig. 3b). The electron dense zone formed in the P–M was approximately 1 mm thick, the thickest observed in this study (Fig. 4a and c). The edge of this zone towards the top OL showed a
Fig. 2 – Ultrastructure of enamel-adhesive interface after acid–base challenge in M–M group. OL: outer lesion; C: resin composite; B: bonding layer; E: enamel; ABRZ: acid–base resistant zone. (a) ABRZ was pointed between triangles. (b) A right angle (arrow) was observed at the junction of bonding, OL and enamel. (c) Thickness of the zone was approximately 0.5 mm. (d) Densely arranged grain-like crystallites in the zone.
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Fig. 3 – Ultrastructure of enamel-adhesive interface after acid–base challenge in M–P group. (a) ABRZ was pointed between triangles. (b) A right angle (arrow) was observed at the junction of bonding, OL and enamel. (c) Thickness of the zone was less than 0.2 mm. (d) Grain-like crystallites in the zone.
Fig. 4 – Ultrastructure of enamel-adhesive interface after acid–base challenge in P–M group. (a) ABRZ was pointed between triangles. (b) A right angle (arrow) was observed at the junction of bonding, OL and enamel. (c) Thickness of the zone was about 1 mm. The edge showed a wave-like shape. (d) Sparsely arranged large needle-like crystallites were observed in the zone.
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Fig. 5 – Ultrastructure of enamel–adhesive interface after acid–base challenge in P–P group. (a) ABRZ was absent at the corresponding region (triangles). (b) Funnel-shaped erosion (arrow) was present at the junction of bonding, enamel and OL. (c) In the corresponding region, a few grain-like structures were observed only at the bottom of OL. (d) A few remnant grainlike structures.
wave-like pattern. Sparsely arranged large needle-like crystallites (Fig. 4d) could be distinguished under high magnification (60,000). Compared with the crystallites in the M–M and M–P, these crystallites were obviously longer in length, but showed similar width. Intercrystalline spaces could be clearly observed. A right angle with no erosion was present at the junction of bonding, OL and enamel (Fig. 4b). The ABRZ-like structure was almost devoid in the P–P (Fig. 5a and c). Only at the bottom of OL, a very thin electron dense layer (less than 0.1 mm thick) was detected (Fig. 5b and c). Examination under 60,000 magnification revealed that this layer consisted of a few sparse grain-like structures (Fig. 5d). In addition, funnel-shaped erosion (arrow) at the junction of the bonding interface, enamel and OL was clearly observed (Fig. 5b).
3.2.
Enamel surface after primer treatment
Fig. 6a shows the enamel surface ground with 600-grit SiC paper, with a smear layer covering the entire surface. The MDP primer treated surface exhibited a shallow honeycomb etching pattern which is caused by preferential dissolution of enamel prism (Fig. 6b and c). The prism peripheries could be distinguished. On the other hand, when treated with PhenylP-containing primer, a deeper etching pattern with an overall increase in roughness was evident along the entire aprismatic enamel surface (Fig. 6d and e). Larger spaces appeared in the exposed crystalline structure.
3.3.
Micro-shear bond-strength test
The micro-shear bond-strength values and failure modes are shown in Table 2. M–M showed significantly higher bond strength compared to the other three groups (one-way ANOVA, p < 0.005). For groups M–P, P–M and P–P, the bond strength showed no significant difference ( p > 0.05). There was significant difference in the distribution of failure mode among groups ( p < 0.05). M–M, M–P and P–M exhibited adhesive and cohesive failure as the predominant failure mode, while the major failure mode in the P–P was adhesive failure.
Table 2 – Micro-shear bond strength and failure mode of the four adhesive systems to enamel. Group
M–M M–P P–M P–P
n
10 10 10 10
Micro-shear bond strength (MPa) 43.08 36.12 33.29 35.79
(3.73) a (6.61) b (5.29) b (5.42) b
Failure mode * A
C
AC
2 1 3 8
2 0 2 0
6 9 5 2
Values identified with different letters are significantly different at p < 0.005. A: adhesive failure; C: cohesive failure in enamel or composite; AC: adhesive and cohesive failure. * There was a significant difference in the distribution of failure modes (chi-square test, p < 0.05).
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Fig. 6 – SEM observation of the enamel surface after treatment. (a) Without primer, the entire surface was covered by smear layer. (b and c) Enamel surface treated by MDP-containing primer. (d and e) Enamel surface treated by Phenyl-P-containing primer.
4.
Discussion
Secondary caries is considered as a major cause for the failure of restorations. Acidic bacterial byproducts may infiltrate not only the bonding interface, but also the tooth tissue at the periphery, creating a maginal demineralised zone, and thus rapidly promote caries occurrence. In this regard, it has been suggested that an increased resistance of the tooth–resin interface to acid may effectively retard the progression of secondary caries.21 With dentin substrate, the ABRZ beneath the hybrid layer has already been confirmed with some self-etching adhesives.14–21 Although the formative mechanism is still unclear, it was assumed that the penetration of the monomers into the tooth tissue beyond the hybrid layer and the chemical interaction between the functional monomer and hydroxyapatite (HAp) may contribute to the formation of ABRZ. The ABRZ observed on enamel in this study may be similar in nature to
those reported for dentin. However, differences between dentin and enamel should be noted; the self-etching primer removes the smear layer and demineralises the underlying dentin mildly, leaving some HAp crystallites partially attached to the dentin collagen.15 On the other hand, enamel has a higher mineral content with a matrix structure different from dentin collagen network. HAp crystals are distributed all over the demineralised layer. Therefore, for dentin, ABRZ forms under the hybrid layer; while for enamel, the ABRZ is not below the bonding interface, but it is rather at the interface, involving those parts of the interface which were not dissolved after the acid–base challenge. Sample preparation for SEM observation of dentin ABRZ has been established by Inoue14 and successfully applied in many studies.15–21 However, with regard to the structural differences between the two substrates, it is necessary to establish new experiment protocols for the observation of ABRZ on enamel. The optimal acid–base challenge conditions in this study were set up based on the
journal of dentistry 38 (2010) 722–730
results of pilot experiments. In the acid challenge, the same demineralising solution used for dentin was applied for enamel. The immersion period of 4.5 h was three times as long as the demineralising period for dentin, in order to create outer lesions with depths comparable to those created with dentin. For the base challenge, 5% NaOCl solution was applied for 30 min with ultrasonic vibration24 to completely remove the demineralised enamel. In this study, ABRZ could be clearly detected in the M–M, M– P and P–M groups (Figs. 2–4), in which MDP was included either in the primer or in the bonding agent. P–P was the only group in which the ABRZ could not be distinguished at the most parts of the corresponding region (Fig. 5). And a funnel-shaped erosion area was noted at the junction of ABRZ, OL and enamel in this group, indicating a weak area vulnerable to acid–base attack beneath the bonding interface. In self-etching adhesives, resin monomers penetrate into the demineralised enamel and envelop the HAp crystals upon curing, therefore protect these crystals from acid attack; however, when the adhesive system is not cured well, the crystals are less protected and thus, prone to faster dissolution by acid. Therefore, it is possible that the P–P group in this study had a lower curing efficacy. On the other hand, MDP has been found to interact chemically with HAp intensively and stably.9 It is capable of forming strong ionic bonds with calcium, producing MDP-calcium salt with a lower solubility. In contrast, the chemical bonding capacity of Phenyl-P is very limited. The resulting Ca-salts are susceptible to dissolution during the acid–base challenge. Further chemical analysis is necessary to prove these speculations. P–M group produced the thickest ABRZ with needle-like crystallites, possibly related to the more aggressive etching capacity of the Phenyl-P-containing primer; the pH value of this primer was lower than that of the MDP-containing primer (1.3 vs. 2.0 respectively, according to the manufacturer). In this regard, SEM examination showed that etching with Phenyl-Pcontaining primer produced a deeper and more porous etching pattern on enamel surface (Fig. 6d and e), in contrast to the shallow etching pattern produced by MDP-containing primer26 (Fig. 6b and c). When the bonding was applied to this more aggressively etched surface, monomers including MDP penetrated deeper. This may have contributed to the formation of a thicker ABRZ, and different morphological appearance involved. As far as this study was concerned, the second hypothesis that functional monomers would not affect the adhesive bonding performance was rejected. M–M showed significantly higher bond strength than the other three groups. Although the micromechanical adhesion, achieved by the diffusion and polymerisation of resin monomers into the pretreated enamel, is assumed to be the main bonding mechanism of dental adhesives to enamel,27 the chemical bonding is also supposed to influence the bonding performance of adhesive systems.5 As pointed previously, the higher and more stable chemical bonding performance of MDP compared to Phenyl-P may also have contributed to the higher bond strength to enamel. Besides, since the two monomers have different chemical structures, polymerisation efficacy and mechanical properties of the resulting polymers may be different. It has been shown that the polymerisation reactivity of functional monomers is
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affected by the length and flexibility of the alkyl spacer chains.28,29 MDP molecule has a long linear alkyl chain and phosphoric acid ester group, while Phenyl-P contains a shorter alkyl chain spacer and a phenyl group at the acidic moiety. The mode of failure in the micro-shear bond-strength technique employed in this study could be affected by the uneven stress distributions at the interface; however, it is noteworthy that a remarkable difference existed in the failure mode pattern of P–P compared to other groups; suggesting that the possibly lower polymerisation efficacy and the weak chemical bonding potential of Phenyl-P as discussed earlier may have resulted in the adhesive failure as its predominant fracture pattern. In conclusion, the null hypotheses of this study were both rejected. The findings suggested that the interface formed by MDP-containing adhesive systems remains after acid–base challenge, and may play an important role in improving bonding performance and resistance against the acid attack from the microorganisms in caries. Further study should be carried out to evaluate the long-term bonding efficacy of different functional monomers in dental adhesive systems. The ABRZ pattern obtained from each adhesive system may become a good indicator to predict its bonding durability to tooth substrates. Interchanging a single functional monomer in an adhesive system is an interesting model for investigating bonding performance and mechanisms. Future study and confirmatory experiments are necessary to interpret the mechanism of ABRZ formation and the bonding performance.
Acknowledgements This work was supported by the grant from the Chinese Government Graduate Student Overseas Study Program, the Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases at Tokyo Medical and Dental University and a Grantin Aid from the Japan Society for the Promotion of Science (JSPS No. 22592116).
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