Effect of air-blowing duration on the bond strenght of current one-step adhesives to dentin

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Effect of air-blowing duration on the bond strength of current one-step adhesives to dentin Jiale Fu a,∗ , Pipop Saikaew b , Shimpei Kawano c , Ricardo M. Carvalho d , Matthias Hannig e , Hidehiko Sano c , Denis Selimovic c a

General Dentistry, Department of Endodontics, School and Hospital of Stomatology, China Medical University, No. 117 Nanjing Street, Heping District, 110002 Shenyang, Liaoning Province, China b Department of Operative Dentistry and Endodontics, Faculty of Dentistry, Mahidol University, No. 6, Yothi Road, Ratchathewi District, Bangkok 10400, Thailand c Department of Restorative Dentistry, Division of Oral Health Science, Hokkaido University, Graduate School of Dental Medicine, Kita 13, Nishi 7, Kita-ku, Sapporo 060-8586, Japan d Department of Oral Biological and Medical Sciences, Division of Biomaterials, Faculty of Dentistry, University of British Columbia, Vancouver, BC V6T 1Z3, Canada e Clinic of Operative Dentistry, Periodontology and Preventive Dentistry, Saarland University, Homburg, Saar, Germany

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objectives. To evaluate the influence of different air-blowing durations on the micro-tensile

Received 2 January 2017

bond strength (␮TBS) of five current one-step adhesive systems to dentin.

Received in revised form

Methods. One hundred and five caries-free human molars and five current one-step adhe-

22 March 2017

sive systems were used: ABU (All Bond Universal, Bisco, Inc.), CUB (CLEARFILTM Universal

Accepted 23 March 2017

Bond, Kuraray), GPB (G-Premio BOND, GC), OBA (OptiBond All-in-one, Kerr) and SBU (Scotch-

Available online xxx

bond Universal, 3M ESPE). The adhesives were applied to 600 SiC paper-flat dentin surfaces

Keywords:

pressure of 0.25 MPa for either 0 s, 5 s, 15 s or 30 s before light-curing. Bond strength to dentin

Micro-tensile bond strength

was determined by using ␮TBS test after 24 h of water storage. The fracture pattern on the

according to each manufacturer’s instructions and were air-dried with standard, oil-free air

Universal system

dentin surface was analyzed by SEM. The resin–dentin interface of untested specimens was

Panoramic SEM

visualized by panoramic SEM image. Data from ␮TBS were analyzed using two-way ANOVA

Adhesion

(adhesive vs. air-blowing time), and Games-Howell (a = 0.05).

Air-blowing time

Results. Two-way ANOVA revealed a significant effect of materials (p = 0.000) and air-

Maximum bond strength

blowing time (p = 0.000) on bond strength to dentin. The interaction between factors was also significantly different (p = 0.000). Maximum bond strength for each system were recorded, OBA/15 s (76.34 ± 19.15 MPa), SBU/15 s (75.18 ± 12.83 MPa), CUB/15 s (68.23 ± 16.36 MPa), GPB/30 s (55.82 ± 12.99 MPa) and ABU/15 s (44.75 ± 8.95 MPa). The maximum bond strength of OBA and SUB were significantly higher than that of GPB and ABU (p < 0.05).

Significance. The bond strength of the current one-step adhesive systems is materialdependent (p = 0.000), and was influenced by air-blowing duration (p = 0.000). For the current

∗

Corresponding author.

E-mail address: fullers@126.com (J. Fu). http://dx.doi.org/10.1016/j.dental.2017.03.015 0109-5641/© 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Fu J, et al. Effect of air-blowing duration on the bond strength of current one-step adhesives to dentin. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.03.015

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one-step adhesive systems, higher bond strengths could be achieved with prolonged airblowing duration between 15–30 s. © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

One step self-etching systems, so-called ‘all-in-one’ system, have been initially advocated about 15 years ago [1]. They became increasingly popular and widely accepted for their distinguishing features; such as, handling convenience, timesaving and user-friendly properties [2]. However, it has been reported that the bond strength of 1-step self-etching systems is lower than that of 2-step and 3-step systems [3,4]. It appears that the simplified procedures of 1-step self-etching systems did not reduce the technique sensitivity of all-in-one systems, especially considering the air-blowing step [3–7].

Adhesive technology is continuously evolving by frequent replacement of commercial adhesive formulations [8]. Currently, as a new branch of 1-step self-etching system, the so-called ‘Universal System’ or ‘Multi-purpose System’ has become commercially available and regains attention from dental clinicians as ‘the eighth generation’ system [9]. These latest systems could not only be used in direct and indirect treatments following manufacturer’s instruction [9,10], but also seem to be adequate after short-term clinical evaluation [11,12] and medium-term clinical evaluation [13–15].

Taken together the mentioned advantages, universal systems might be a potential brand-new choice for the dentists in daily operative treatment.

In some of the current commercially available universal systems [10,11,16], 10-MDP is included as functional monomer, which has been mainly used as an etching monomer and proven very successful in promoting chemical adhesion to tooth tissue [17]. In previous studies, there were not so many 10-MDP containing 1-step self-etching systems included [2–5], because 10-MDP was originally synthesized and patented by Kuraray (Osaka, Japan) and hence was not widely available in the dental market [10–12,17].

Currently, an increased number of new systems, the so called ‘eighth generation’ adhesive system, have a common feature that they are prone to select 10-MDP as their functional monomer, but differ on application procedures, such as: coating manner, waiting time and air-blowing pressure.

To ensure the ideal bonding performance could be achieved, detailed information on direction should not be overlooked. In previous studies, it was investigated that bond strength could be influenced by both of air-blowing duration [5] and pressure [18], because the information on applying process was indefinite in some 1-step self-etching systems’ instruction.

Based on different chemical compositions, universal systems required differently on application process. So far, the influence of different air-blowing durations on bond strength of universal systems has barely been investigated. The aim of this study was to evaluate the influence of different airblowing durations on ␮TBS of current one-step adhesive systems. The null hypothesis tested in the present study was

that the bonding performance of current one-step adhesive systems is not affected by air-blowing duration.

2.

Materials and methods

2.1.

Teeth used

One hundred extracted caries-free human molars were used in this study to test 5 different current one-step adhesive systems. Each set consists of 20 teeth, which were further divided into 4 groups with 5 teeth in each. The teeth were collected under a protocol reviewed and approved by the institutional review board of China Medical University, Shenyang, China. The teeth were stored at 4 ◦ C in an aqueous solution of 0.5% Chloramine-T and used within 3 months after extraction.

Flat dentin surfaces were obtained by removing the coronal enamel of each tooth in a gypsum model trimmer with water coolant, leaving the surrounding enamel. After that, the dentin surfaces were ground with 600-grit SiC paper for 60 s under continuous water-cooling to produce a standardized smear layer prior to bonding.

2.2.

Adhesives

Five commercially available current one-step adhesive systems were applied in this experiment: ABU (All Bond Universal, Bisco, Inc.), CUB (CLEARFILTM Universal Bond, Kuraray), GPB (G-Premio BOND, GC), OBA (OptiBond All-in-one, Kerr) and SBU (Scotchbond Universal, 3M ESPE). Table 1 shows the chemical compositions and the respective manufacturer’s instructions for application of these 5 adhesives. Among these systems, only OBA does not contain 10-MDP, OBA was selected as a control group from former all-in-one systems. The adhesive procedures in the present study, except for the air-blowing duration, followed the respective manufacturer’s application guide. Dentin surface in the 4 subgroups consisting of 5 teeth per adhesive were air-blown for either 0 s, 5 s, 15 s, or 30 s, respectively, before light-curing. The maximum air-blowing pressure was adjusted to be 0.25 MPa, and the air syringe head was positioned vertically to the dentin surface at a distance of 15 mm. All systems selected in the present study were applied only as one-step self-etch materials. The same operator performed all steps.

All bonded surfaces were built-up with resin composite (Clearfil AP-X, Kuraray Medical Inc.; Okayama Japan, Shade A3, Lots: BH0052) in increments to a thickness of 5 mm. Each incremental layer was light cured (PENCURE 2000, J. MORITA MFG. CROP) for 20 s, the light output intensity was properly controlled to be more than 2000 mW/cm2 . The bonded teeth were stored in distilled water at 37 ◦ C for 24 h.

Please cite this article in press as: Fu J, et al. Effect of air-blowing duration on the bond strength of current one-step adhesives to dentin. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.03.015

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Table 1 – Chemical composition of the universal systems used in present study and manufacturer’s instructions for application of the adhesive systems.

Materials & lot no.

All Bond Universal Lot no. 1300008729 (Bisco, Inc.)

Chemical formulation & pH

Manufactures’ instruction

10-MDP, Bis-GMA, HEMA, ethanol, water, initiators. pH = 3.2

1. Dispense 1–2 drops of bond into a clean well.

2. Apply two separate coats of bond, scrubbing the preparation with a micro-brush for 10–15 s per coat. Do not light cure between coats.

3. Evaporate excess solvent by thoroughly air-drying with an air syringe for at least 10s, there should be no visible movement of the adhesive.

4. Light cure for 10 s.

CLEARFIL Universal Bond Lot no. 1L0003 (Kuraray)

10-MDP, Bis-GMA, HEMA, ethanol, water, Silane coupling agent, fillers, Initiators. pH = 2.3

1. Dispense the bond into a dispensing dish

G-Premio BOND

10-MDP, Acetone, dimethacrylate component, photoinitiator, butylated hydroxytoluene. pH = 1.5

1. Prior to dispensing, shake the bottle of G-Premio BOND thoroughly. Dispense a few drops into a clean dispensing dish.

Lot no. 1411061G (GC)

OptiBond All-in-one

Lot no. 5538778 (Kerr)

Scotchbond Universal

Lot no. 572054 (3M ESPE)

2. Apply bond to the dentin surface and rub it in for 10 s.

3. Dry the dentin surface sufficiently by blowing mild air for more than 5 s until bond does not move.

4. Light cure for 10 s.

2. Immediately apply to the prepared enamel and dentin surfaces using the disposable applicator.

3. Leave undisturbed for 10 s after the end of application.

4. Dry thoroughly for 5 seconds with oil free air under maximum air pressure.

5. Light-cure for 10 s

GPDM, mono and dysfunctional methacrylate monomers, water, acetone, ethanol, CQ, filler sodium hexafluorosilicates and ytterbium fluoride. pH = 2.5

1. Shake bottle briefly

10-MDP, HEMA, ethanol, water, dimethacrylate resins, methacrylate-modified polyalkenoic acid copolymer, polyacrylic acid copolymer, silane, fillers, initiators. pH = 2.7

1. Apply the adhesive to the prepared tooth and rub it in for 20 s.

2. Dispense 2–3 drops into a clean well.

3. Apply the adhesive on dentin with applicator brush and scrub the surface for 20 s.

4. Repeat Step3 again.

5. Dry the adhesive with gentle air first and then medium air for at least 5 s with oil-free air.

6. Light cure for 10 s.

2. Gently air dry the adhesive for approximately 5 s to evaporate the solvent.

3. Light cure for 10 s.

10-MDP: 10-methacryloyloxydecyl dihydrogenphosphate; Bis-GMA: bisphenol A diglycidyl methacrylate; GPDM: Glycerol phosphate dimethacrylate, HEMA: 2-hydroxyethyl methacrylate; CQ: camphorquinone.

2.3.

Micro-tensile bond strength ( TBS) test

After storage in 37 ◦ C water for 24 h, each bonded tooth was sectioned into beams (cross-sectional area approximately 1 mm2 ) using an Isomet diamond saw (Isomet 1000, Buehler, Lake Bluff, Illinois, USA). For each tooth (n = 5), three beams from the central area were randomly selected for ␮TBS, therefore resulting in a total of 15 beams to be tested. The beams were fixed to a Ciucchi’s jig with cyanoacrylate glue (Model Repair 2 Blue, Dentsply-Sankin, Otahara, Japan) and subjected to a tensile force at a crosshead speed of 1 mm/min in a desktop testing apparatus (EZ test, Shimadzu, Kyoto, Japan). ␮TBS was expressed in MPa calculated by division of the applied

force (N) at the time of fracture by the bonded area (mm2 ), and data were analyzed by two-way ANOVA and Tukey’s test (a = 0.05).

2.4.

Failure mode analysis

The fractured specimens were mounted on an aluminum stub, then coated with Pt–Pd for 120 s. The fracture modes were determined using a scanning electron microscope (SEM, S4000, Hitachi, Tokyo, Japan) at an accelerating voltage of 10 kV. Fractured and specific features on dentin surfaces were further examined at lower magnification of 3000×. Fracture mode categories were classified into three groups: A: cohesive fail-

Please cite this article in press as: Fu J, et al. Effect of air-blowing duration on the bond strength of current one-step adhesives to dentin. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.03.015

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ure within dentin or composite resin; B: adhesive failure; C: failure at dentin or resin.

(p = 0.000; F = 342.000). The interaction between these two factors was also significant (p = 0.000; F = 13.900).

In general, increasing air-blowing duration from 0 s to 15 s resulted in a statistically significant higher mean ␮TBS for all 5 current one-step adhesive systems evaluated (Fig. 1; p = 0.000). Pre-test failures were calculated as 0 MPa in present study. Statistical lower bonding performance was observed with 0 s air-blowing duration for all adhesives. When evaporation duration was prolonged to 30 s, a slight increase in GPB, but mild decrease in the other systems was observed. Bond strength of OBA in 15 s and 30 s groups was higher than that of the other adhesives.

2.5.

Panoramic SEM observation of the resin–dentin interface In order to investigate the typical morphology of the resin–dentin interface of each system, 5 additional resinbonded specimens from maximum bond strength group of each system were sectioned perpendicularly to the adhesive interface with an Isomet saw to obtain two slabs of 2 mm thickness. The sectioned surfaces were sequentially polished with 600-, 800-, and 1000-grit silicon carbide papers under running water. This was followed by polishing sequentially with 6-, 3, 1-␮m diamond pastes (DP-Paste, Struers, Denmark), and by cleaning with an ultrasonic device after each diamond paste polish. After polishing, the specimens were immersed in 1 M hydrochloric acid for 30 s and 5% sodium hypochlorite for 5 min, followed by rinsing with water. After naturally drying in the laboratory over night, the specimens were sputter-coated with Pt–Pd for 120 s. The resin–dentin interfaces were then analyzed in a scanning electron microscope (SEM, S-4000, Hitachi, Tokyo, Japan). Panoramic SEM images were seamlessly connected by Adobe Photoshop CS4 (Version:11.0.27) from three independent sequential photographs from each resin-adhesive-dentin interface.

3.

Results

3.1.

TBS

3.2.

Fracture modes

The result of the failure modes was shown in Fig. 2. In general, the fracture modes were mainly categorized as cohesive failure and adhesive failure. There was a clear tendency that more cohesive failure occurred with air-blowing duration prolonged. High ␮TBS values were associated with a higher tendency to fail within dentin or composite resin, especially for SBU, CUB and OBA.

3.3.

SEM observation of fractured dentin surfaces after TBS testing Fig. 3 shows the SEM observations for the fractured surface of the dentin side. A large number of voids within the adhesive resin was noted in the no air-blowing specimens of OBA and GPB (Fig. 3-i and m) compared with that of ABU. Bubbles were observed on the top of adhesive layer of OBA, GPB and ABU in 5 s group. With prolonged air-blowing duration, both the size and the quantity of bubbles on the top of adhesive layer decreased (arrowed in Fig. 3).

Two-way ANOVA analysis of variance revealed that microtensile bond strength values were significantly affected by adhesive systems (p = 0.000; F = 39.926) and air-blowing time

Micro-tensile Bond Strength c,A b,A b

b,AB

b

b

c

b

b b

b b,BC b,C b

a

a

a

a

a a

Fig. 1 – Bond strength of five adhesives according to different air-blowing durations. Values are Mean (SD) in MPa. The same small and capital letters revealed no statistically differences (Games–Howell, p > 0.05). The same capital letter in maximum value of each system indicates that there is no statistical difference (Games–Howell, p > 0.05). The ␮TBS of specimen that failed before testing was set as 0 MPa. Please cite this article in press as: Fu J, et al. Effect of air-blowing duration on the bond strength of current one-step adhesives to dentin. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.03.015

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100% 90% 80%

70% 60% 50% 40% 30% 20% 10% 0% 0s

5s 15s 30s 0s SBU

5s 15s 30s 0s CUB

5s 15s 30s 0s OBA

5s 15s 30s 0s GPB

5s 15s 30s ABU

A:Cohesive failure within dentin or composite resin B: Adhesive failure C: Failure at dentin or resin

Fig. 2 – Fracture modes of the five adhesives bonded to dentin with different air-blowing durations.

Fig. 3 – SEM analysis of fractured surfaces after ␮TBS testing.

3.4.

Panoramic SEM observation of the resin–dentin interface The Panoramic SEM observations of the maximal ␮TBS of each system are shown in Fig. 4. The adhesive layer showed a wave shape in OBA, and smaller voids could be observed on top of (or

within) the adhesive layer of OBA and GPB. The adhesive layer of SBU and CUB is flat and smooth without bubbles. ABU produces the thinnest adhesive layer, which was approximately 5 ␮m on average.

In present study, different current one-step adhesive systems revealed different features on dentin surface before

Please cite this article in press as: Fu J, et al. Effect of air-blowing duration on the bond strength of current one-step adhesives to dentin. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.03.015

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Fig. 4 – Panoramic SEM figures of the resin–dentin interface in specimen revealing the maximal ␮TBS value of each adhesive systems.

Please cite this article in press as: Fu J, et al. Effect of air-blowing duration on the bond strength of current one-step adhesives to dentin. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.03.015

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Fig. 5 – Dentin surface after air blowing.

light-curing. A lumpy adhesive layer of GPB (Fig. 5-a) and a smooth adhesive layer of ABU (Fig. 5-b) was observed respectively.

4.

Discussion

Recently, the term of ‘Universal System’ has been frequently mentioned, but still, neither officially accepted by academic dental associations nor world-widely used by dental clinicians. ‘Universal System’ has been described as ideally a single-bottle, with no-mixture, and it could not only be used in the total-etching, self-etching and selective-etching mode in specific dentin/enamel clinical treatments [19], but it is also qualified to be used as primer to zirconia [20], and various ceramics [21,22]. In the present study, 5 self-etching one-step systems were consistent with the descriptions mentioned above and thus, were selected for this study.

In previous studies, evaporation of solvents and water with compressed air was considered as a technique-sensitive step to both all-in-one systems [5] and universal systems [16].

Undoubtedly, reducing the operational technique-sensitivity to achieve the optimal bonding performance in different specific clinical situations seems to be another bottleneck beyond that of keeping balance with complex chemical formulation [23]. Somehow, reducing the application step or time seems to be a most direct and reliable way to reduce technique-sensitivity [8], such as ‘0 s waiting’ claimed by GBP in the present study, however, the manufacturers’ opinions are inconsistent. According to the manufacturer’s instructions, the total application time of ABU and OBA is longer than that of GPB, CUB and SBU, because the former adhesives required double layering, which increased the clinical application time to 50 s and 55 s respectively. In present study, the two-way ANOVA analysis indicated that the interaction between adhesive system and air-blowing duration was significant on micro-tensile bond strength (p = 0.000; F = 13.900).

Therefore, null hypothesis that the bonding performance of current one-step adhesive system is not affected by airblowing duration has to be rejected.

In present study, the bond strengths of each system revealed significant increased tendency when air-blowing duration prolonged from 0 s to 15s. The poor bond strength of 0 s in each group could be attributed to the residual water and solvents within the adhesive layer, which should be completely evaporated before light-curing. Residual water and solvents through extremely insufficient air-blowing application may interfere with polymerization of adhesive monomers [24,25], lowering the quality of the hybrid layer [26], and result in ‘phase-separation’ [27], thereby decreasing bond strength.

Since Kuraray’s patent of 10-MDP expired, 10-MDP containing systems were becoming commercially available in dental market, meanwhile, 10-MDP was also esteemed as a symbol to tell the difference between old and new one-step systems for its superior chemical bonding feature between teeth tissue and restorations. Without evaporation, solvents and water mixed in adhesive layer caused 100 percentage of adhesive failure in each adhesive, therefore, the effect of 10-MDP on bond strength to dentin could not be evaluated.

When air-blowing step was taken, the strong intervention form phase-separation was significantly reduced and thus, as a feature of 10-MDP, strongly ionic bond with calcium on dentin [28] could be able to emerge. The distinction on bond strength observed in this study was probably related to the various concentration or purity of 10-MDP contained in different systems [29] and thus yielded different bonding performances [30]. One-step self-etching systems are very complex chemical productions, even though most adhesive systems contain the same components, they may differ significantly [23]. As a consequence, particular shortcomings related to the specific composition of the tested adhesive systems might be considered in order to explain the different bonding effectiveness obtained with these adhesives in present study.

As one of the benefits from the simplified application procedure, relatively shorter clinical application time was achieved by one-step self-etching system [2]. In the present study, the information form ‘manufactures’ instruction’ represent different designs on application modes (scrubbing or not), chair-side time management (time-saving or not) and air syringe control (gentle, medium or maximum air pressure). Duplicating the layering or doubling the application

Please cite this article in press as: Fu J, et al. Effect of air-blowing duration on the bond strength of current one-step adhesives to dentin. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.03.015

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time significantly increased bond strength values of selfetching systems [31], because increasing the application time may facilitate solvent evaporation and adhesive infiltration [32], the result of OBA is consistent with the earlier report.

Inversely, due to the pH (pH = 2.5–3.5, claimed by manufacturer’s Safety Data Sheet), ABU should be classified as the ultra-mild category as well as SBU [4,33]. However, relatively higher bond strength of ABU was achieved in 0 s group, but the maximum bond strength of ABU was significantly lower than that of SBU. HEMA containing ABU behaved like hydrophobic adhesives of GPB and OBA (Fig. 3-r). To ABU, both relatively lower concentration of HEMA and double scrubbing application might win time for solvents and water evaporation, thus to achieve higher bond strength when air-blowing time was limited; however, compact adhesive layer (Fig. 4-e) did not contribute higher bond strength to ABU for its limited demineralized ability, which consistent with its higher adhesive failure ratio in both 15 s and 30 s group. The effectiveness of double scrubbing on dentin surface has been confirmed by clinical studies [34–37], however, the exact long-term clinical treatment effect of universal system was rarely reported, therefore, further clinical investigations are necessary.

In our previous study, visible voids remaining in the adhesive layers could be observed even after air-blowing for 35 s [5]. In the present study, a great number of voids could be observed inside the adhesive layer of OBA and GPB (Fig. 3-i, m).

The reason might be that there is an immediate phase separation once the adhesive was applied to the surface and acetone droplets rapidly migrate to the surface, burst and evaporate, thus leaving the voids behind upon curing the adhesive.

Changing chemical composition could affect the bond strength significantly [9]. As one of the effective hydrophilic monomer, HEMA was added to self-etching system to accelerate water transport from the adhesive into the interface to be removed simply [3–5,17,38] therefore, bubbles could be observed in HEMA-free universal systems (Fig. 3-k, o) or under severe insufficient evaporation conditions (Fig. 3-i, m, q). Consequently, for acetone-based HEMA-free self-etching systems such as OBA and GPB, prolonged [5] or strong air-blowing [18] application may contribute to improve bond strengths [3].

In the present study, higher bond strengths were achieved by prolonged air-blowing durations from 15 s to 30 s. Therefore, at least 5 s, but no more than 30 s air-blowing application should be accomplished when universal system is selected to bond to dentin. The optional air-blowing durations of different universal systems should be investigated further under clinical conditions.

5.

Conclusion

Within the limits of this in-vitro study, considering air-blowing duration and the related bond strength of current one-step adhesive, it can be concluded that 1. The bond strength of the current one-step adhesive systems is material-dependent and could be significantly influenced by air-blowing duration.

2. Relatively higher bond strength could be achieved by 15 s air-blowing duration.

Acknowledgements This experiment was supported by the New Teacher Fund (XZR20160015) of China Medical University and UBC-Dentistry, Start-up grant to RMC. This experiment was performed in the laboratory of the Department of Restorative Dentistry, Division of Oral Health Science, Hokkaido University, Graduate School of Dental Medicine.

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