Marginal adaptation after aging of a self-etching adhesive containing an antibacterial monomer

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T

he first ideas for incorporating antibacterial monomers into self-etching adhesive systems were expressed by Imazato et al in the early 90s.^14,15The main objectives of this development were to eliminate the problems associat- ed with residual bacteria that can remain in cavity preparations after restoration and to prevent secondary caries in case of bacteria-contaminated, fluid-filled gaps between the restorative material and the tooth substrate.^4,16

MDPB (12-methacryloyloxydodecylpyridinium bromide) is an antibacterial molecule based on a quaternary ammoni- um salt contained in the marketed adhesive system Clearfil Protect Bond (Kuraray Medical; Tokyo, Japan). This antibacterial agent is covalently bonded to the polymer matrix by copolymerization; its antibacterial effect is said to be due to cationic and hydrophobic bonding to cell wall components,

Marginal Adaptation After Aging of a Self-etching Adhesive Containing an Antibacterial Monomer

Tissiana Bortolotto

^a

/Wassila Doudou

^b

/Minos Stavridakis

^c

/Marco Ferrari

^d

/Ivo Krejci

^e

Purpose: To evaluate the marginal adaptation of mixed Class V cavities restored with Clearfil Protect Bond (Kuraray), Clearfil SE Bond (Kuraray), and two experimental combinations of both marketed adhesives, after fatigue and water storage.

Materials and Methods:Four groups (Clearfil Protect Bond, Clearfil SE Bond, Exp. 1 and Exp. 2) of Class V cavities were restored with a microhybrid restorative composite (Clearfil APX, Kuraray). The marginal quality of these restorations was quantified by evaluation of gold-coated epoxy replicas with scanning electron microscopy before loading, after loading, and after a 12-month period of water storage. Data from marginal adaptation along the total margin length, on enamel, and on dentin were analyzed with the Wilcoxon signed rank test for differences within a group and with Kruskal-Wal- lis in order to assess the differences between groups. The Bonferroni test was used for post-hoc comparisons, and the confidence level was set to 95%.

Results:The mean percentages (±SD) of “continuous margin” of the total marginal length ranged from 79.5% (±13.3) to 62.2% (±10.4) and from 70% (±11) to 61% (±15.1) after loading and after storage, respectively. No significant differences could be detected among the different groups. However, the marginal adaptation of Clearfil Protect Bond remained the most stable of all materials tested, as no significant differences were detected between the percentages of continuous margins before loading, after loading, or after storage.

Conclusions: The use of an antibacterial adhesive system was as effective as the conventional two-step self-etching adhesive in the marginal adaptation of Class V restorations.

Keywords: marginal adaptation, water storage, MDPB, antibacterial monomer, hydrolytic stability, enamel, dentin.

J Adhes Dent 2007; 9: 311-317. Submitted for publication: 10.08.06; accepted for publication: 11.01.07.

aPostdoctoral Assistant, Division of Cariology and Endodontology, School of Dentistry, University of Geneva, Geneva, Switzerland.

bDoctoral Assistant, Division of Cariology and Endodontology, School of Den- tistry, University of Geneva, Geneva, Switzerland.

cLecturer, Department of Operative Dentistry, Faculty of Dentistry, University of Athens, Athens, Greece.

dProfessor and Chairman, Department of Dental Materials and Restorative Dentistry, Policlinico ‘Le Scotte’, University of Siena, Siena, Italy.

eProfessor and Chairman, Division of Cariology and Endodontology, School of Dentistry, University of Geneva, Geneva, Switzerland.

Reprint requests:Prof. Dr. Ivo Krejci, Division of Cariology and Endodontology, School of Dentistry, University of Geneva, 19, Rue Barthélemy-Menn, CH-1205 Geneva, Switzerland. Tel: +41-22-3794101, Fax: +41-22-379-4102.

e-mail: Ivo.Krejci@medecine.unige.ch

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thus disturbing membrane function and subsequently in- ducing leakage of the cytoplasmic material.^16,20

Recently, Imazato et al¹⁹evaluated in vitro/in vivo the bonding performance of this new adhesive to dentin and demonstrated that bond strength was not adversely affect- ed by the incorporation of the antibacterial monomer MDPB into the adhesive system. Apart from the pH of the primer, which is not modified by the incorporation of MDPB, the dif- fusivity of this monomer into dentin is similar to other effective acidic monomers, such as MDP (10-methacryloyloxydecyl dihydrogen phosphate).

It has been proposed that the antibacterial agent dode- cylpyridinium bromide is linked to the polymer network, and after polymerization, the immobilized bactericide can act on bacteria that contact its surface.^9,13However, it is not yet clear how the immobilized agent acts in respect to its affini- ty to water.¹⁷Previous investigations reported that the incorporation of MDPB raises the hydrophobicity of the adhesive, as MDPB is more hydrophobic than HEMA (2-hydroxyethyl

methacrylate), thus potentially improving the hydrolytic stability of the bonding interface.^19,20However, there is a lack of information about the effect of MDPB-containing primer/

bonding resin on the durability of resin-enamel and resin- dentin adhesion challenged in a humid environment that simulates the oral cavity.

It was the purpose of this study to evaluate the marginal adaptation by using scanning electron microscopic (SEM) analysis of two commercially available self-etching adhesives (Clearfil Protect Bond [PB] and Clearfil SE Bond [SE], Kuraray Medical; Tokyo, Japan) on enamel and dentin margins of Class V cavity restorations after thermomechanical stressing and after 12 months of water storage. To determine the distinct influence of the antibacterial primer on the results, two experimental combinations of these commercially available products were created by exchanging the primers of the two adhesives, ie, the primer of PB / bond of SE (Exp.

1) and primer of SE / bond of PB (Exp. 2). The null hypothe- ses tested were:

Table 1 Description of the groups tested

Groups

SE Bond

(batch # 00356A and 00469B)

Exp. 1

(batch # 00004A and 00469B)

Protect Bond (batch # 00004A and 00008A)

Exp. 2

(batch # 00356A and 00008A)

Application procedure a,b,c,b,d

a,b,c,b,d

a,b,c,b,d Adhesive systems

Clearfil SE Bond (SE)

Experimental combination

Clearfil Protect Bond (PB)

Experimental combination

Composition

Primer: MDP, HEMA, hydrophilic dimethacrylate, CQ, N,N-diethanol-p- toluidine, water

Bond:MDP, bis-GMA, HEMA.

Hydrophobic dimethacrylate,CQ, N,N-diethanol-p-toluidine, silanized colloidal silica

Primer from PB Bond from SE

Primer: MDP, MDPB, HEMA, hydrophilic dimethacrylate, water

Bond: MDP, bis-GMA, HEMA, hydrophobic dimethacrylate, dl-camphorquinone, N,N-diethanol-p-toluidine, silanated colloidal silica, surface treated sodium flu- oride

Primer from SE Bond from PB

Restorative composite used in all groups:

Clearfil APX (batch # 00359B)

Bis-GMA, TEG-DMA, fillers e

a: apply primer for 20 s, b: dry with compressed air, c: apply bond for 10 s, d: light cure for 20 s, e: apply one cervical layer, light cure, apply one occlusal layer and light cure. MDP: 10-methacryloyloxydecyl dihydrogen phosphate, HEMA: 2-hydroxyethyl methacrylate, MDPB: 12-methacryloyloxydodecylpyridinium bromide, bis-GMA: bisphenyl glycidyl methacrylate, TEG-DMA: triethylene glycol dimethacrylate, CQ: di-camphorquinone.

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1. There are no differences in the marginal adaptation to enamel and dentin margins in Class V cavities restored with a conventional and an antibacterial two-step self- etching adhesive.

2. There are no differences in degradation of enamel and dentin margins before loading, after loading, and after storage, for the adhesives tested.

MATERIALS AND METHODS

Two commercial products consisting of a self-etching primer and a bond, Clearfil SE Bond and Clearfil Protect Bond, were used in this investigation. Two additional experimental combinations were prepared by using the primer of PB with the adhesive of SE (Exp. 1) and the primer of SE with the bond of PB (Exp. 2). The description of the 4 experimental groups is detailed in Table 1.

Thirty-two intact, noncarious, nonrestored third human molars that had been collected according to local institution guidelines were selected for the present investigation within 1 month following extraction and stored in 0.1% thymol so- lution until use. A scaler and a rotating brush with a mixture of pumice and water were used to clean the teeth, which were subsequently randomly assigned to 4 experimental groups and prepared for the simulation of dentinal fluid.²⁴For this purpose, the apices were sealed with two coats of nail var- nish and the teeth were mounted on custom-made specimen holders. A hole was drilled into the pulpal chamber with an 80-μm cylindrical diamond bur (Coltène Whaledent; Altstät- ten, Switzerland) 1 mm above the cementoenamel junction of the molars’ distal surface, a metal tube was inserted and adhesively luted in this hole, then connected by a flexible sil- icone hose to an infusion bottle filled with horse serum diluted to a 1:3 ratio with 0.9% NaCl under a hydrostatic pressure of ca 3.3 kPa (25 mm Hg). One day before starting the cavity preparations, the pulp chambers were evacuated of the dentinal fluid and filled with the diluted horse serum. In- trapulpal pressure was maintained during cavity preparation, restoration placement, finishing, and stressing.

Cavity Preparation and Filling Procedures

Standardized v-shaped Class V cavities (3.0 to 3.5 mm in di- ameter, 2.5 to 3.0 mm in height and 1.5 mm in depth) were prepared on the buccal surface of each tooth with half of the margins in enamel and half of the margins in dentin. The cavities were prepared with 80-μm diamond burs (Coltène Whaledent) and finished using 15-μm finishing diamond burs (Coltène Whaledent) under continuous water spray.

Each bur was replaced by a new one after four cavity preparations. Enamel margins were bevelled to a crescent shape with a maximum width of 1.2 mm. The adhesive systems were applied following the manufacturer’s recommenda- tions (Table 1). Then a microhybrid restorative composite (Clearfil APX, Kuraray) was applied into the cavity and light cured in two incremental layers, one cervically up to one-half of the cavity and the other one occlusally, filling the other half of the cavity. Polymerization of the adhesive systems and the restorative composite was performed with a halogen light-curing unit (Optilux 501, Demetron/Kerr; Danbury, CT,

USA) with a monitored power output density of around 800 mW/cm²(Curing Radiometer Model 100, Demetron/Kerr).

After light curing of the restorative composite, polishing of the restoration margins was performed with flexible alu- minum oxide disks of decreasing grit sizes (Sof-Lex, PopOn, 3M ESPE; Seefeld, Germany) under a stereomicroscope at 12X magnification.

The restored teeth were simultaneously stressed under thermal and mechanical loading for ten days in a computer- controlled chewing machine. Thermocycling was performed by flushing water at two different temperatures (5°C and 50°C) 3000x with a dwell time of 2 min for each temperature and a transfer time of 10 s from temperature to temperature. Mechanical loading consisted of 1.2 million cycles at a frequency of 1.7 Hz with a maximum load of 49 N applied to the center of the previously restored molars’ occlusal surface with the lingual cusp of a natural, extracted molar as the antagonist.

After loading, the teeth were stored in water that contained 0.5% chloramine to prevent bacterial growth. Aging of the restorations occurred in an oven (Memmert; Schwabach, Germany) at a constant temperature of 37 °C in the dark for 12 months. After this period, the teeth were removed from water and cleaned for the evaluation of marginal adaptation.

Before loading, after loading, and after water storage, the teeth were cleaned with rotating nylon brushes impregnated with toothpaste, rinsed, and dried. Impressions of the cavities were taken with a polyvinylsiloxane material (President light body, Coltène Whaledent). Epoxy replicas (Epofix resin, Struers; Ballerup, Denmark) were generated from these impressions and subsequently gold coated for quantitative margin analysis using an SEM (XL20, Philips; Eindhoven, The Netherlands) at 200X magnification. The quality of the margins, expressed as percentages of “continuous margins”, was reported for the total margin length and for the enamel and dentin margins separately.

Data from the four groups on the total, enamel, and dentin margin lengths were not normally distributed (Kol- mogorov-Smirnov test). For this reason, a Wilcoxon Signed- Rank test was performed for pairwise comparisons before loading/after loading, after loading/after water storage, and before loading/after water storage. This statistical method was also used for assessing the differences between enamel and dentin margins at each interval, ie, enamel/dentin before loading, enamel/dentin after loading, and enamel/

dentin after 1 year of water storage. Differences among groups were evaluated using Kruskal-Wallis and Bonferroni tests at the 95% confidence level.

RESULTS

The mean values of “continuous margin” of all groups tested for total, enamel, and dentin marginal length are detailed in Table 2a. The comparison between Clearfil SE Bond and Clearfil Protect Bond revealed nonsignificant differences before loading (initial), after loading (terminal) and after water storage. The exchanging of the primers in the experimental combinations likewise did not reveal significant differences in marginal adaptation.

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Aside from the statistical evaluation, some trends towards a better performance of Protect Bond on enamel and Clearfil SE on dentin were detected. On enamel, Protect Bond presented higher percentages of continuous margins than did SE Bond, both after loading and after storage: 71.1% (±

23.5)/61% (± 19) and 54.2% (± 16.5)/41% (± 17.3), respectively. The opposite situation was observed in dentin: Clearfil SE Bond presented a higher percentage of continuous margins than did Protect Bond after loading and after storage, 99.3% (± 1)/89% (± 21.3) and 98.3% (±3)/89% (± 24), respectively. When considering each testing interval separately (initial, terminal or after storage), a significant difference of marginal adaptation in enamel margins vs dentin margins was observed in Clearfil SE Bond, Exp. 1 and Exp. 2, but not for Clearfil Protect Bond, where only nonsignificant differences between enamel and dentin marginal adaptation were found (Table 2b).

When comparing the differences among intervals (initial, terminal, after 1 year of water storage) within a group, a significant degradation of the total margin length and of enamel margins was observed between initial/terminal and initial/after water storage for SE Bond, Exp. 1 and Exp. 2, but not for Clearfil Protect Bond, which remained stable throughout the entire testing procedure. In dentin, no significant marginal degradation could be observed in SE Bond, Protect Bond, and Exp. 1; however, in Exp. 2, a significant marginal degradation was observed between the initial and 1 year water storage values. In all the groups tested, no significant differences were detected between the values terminal/after water storage.

Some representative SEM images of enamel and dentin bonded interfaces of the different restored groups are shown in Fig 1.

Total margin length SE Bond

Exp. 1 Protect Bond

Exp. 2 Enamel SE Bond

Exp. 1 Protect Bond

Exp. 2 Dentin SE Bond

Exp. 1 Protect Bond

Exp. 2

Initial 87.0 (6.5)â 80.0 (8.3)â 87.0 (15.2)â 81.5 (9.2)â 76.2 (13)â 68.0 (15)â 77.4 (29.3)â 70.0 (14.2)â 99.4 (1)â 99.5 (1.3)â 98.1 (4.5)â 100.0 (0.4)â

Terminal 78.0 (9.4)^{a, *} 62.2 (10.4)^{a, *} 79.5 (13.3)â 66.2 (14.3)^{a, *} 61.0 (19)^{a, *} 43.3 (15.3)^{a, *} 71.1 (23.5)â 49.0 (19)^{a *} 99.3 (1)â 98.2 (2)â 89.0 (21.3)â 98.0 (3.1)â

After 1 yr water storage 67.2 (10.3)^{a, °} 61.0 (15.1)^{a, °} 70.0 (11)â 66.3 (14)^{a, °} 41.0 (17.3)^{a, °} 37.0 (22.2)^{a, °} 54.2 (16.5)â 48.3 (21.5)^{a, °} 98.3 (3)â 97.4 (4)â 89.0 (24)â 93.0 (7)^{a, °}

Differences among groups were statistically evaluated with Kruskal-Wallis and Bonferroni test (p < 0.05). Significant differences between initial/terminal percentages within a group are represented with *, and between initial/1 year with ° (Wilcoxon Signed-Rank Test). No significant differences were observed between the intervals terminal/1 year. Levels bearing the same letter or symbol are not statistically different.

Table 2a Numeric data and statistical evaluation of the groups tested. Percentages (± SD) of continuous margins of the total, enamel, and dentin margin length at the following intervals: before loading (initial), after loading (terminal) and after 1 year water storage

Table 2b Statistical evaluation of the differences between the marginal adaptation of enamel/dentin initial, enamel/dentin terminal, enamel/dentin after 1 year of water storage (Wilcoxon Signed-Rank Test, p < 0.05)

Groups Initial Terminal After 1y w.storage

SE Bond * * *

Exp. 1 * * *

Protect Bond ns ns ns

Exp. 2 * * *

Significant differences between the two tooth substrates were observed in all groups except for Clearfil Protect Bond. *: significant difference between the two values, ns: not significant.

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DISCUSSION

The present study evaluated the fatigue resistance of an antibacterial monomer-containing self-etching adhesive on enamel and dentin and compared it to a conventional two- step self-etching adhesive and two experimental combinations of both marketed products. Class V cavities in sound posterior teeth were adopted as an experimental model, as their preparation can be easily standardized, thus reducing practitioner variability.³⁰A chewing machine comprising thermocycling and cyclic occlusal mechanical loading together with the simulation of dentinal fluid was used for this purpose.23,24,26,27The rationale for this protocol was based on the work of Arola and Huang,³who found that the combination of thermal changes and occlusal forces comprised

almost 95% of the stresses applied to restorations. In addi- tion, long-term exposure to water is a known factor that de- grades tooth-composite bonds over time.^2,12To evaluate the restorations’ marginal adaptation, a computer-assisted quantitative SEM margin analysis on the replicas of these restorations was performed before loading, after loading, and after water storage. SEM evaluation based on replicas has several advantages.10,11,25,31Such a method can be used for evaluating cavities prepared both in vitro and in vivo. This method is also truly quantitative,⁵as the presence or absence of leakage is expressed as percentages of “continuous margin” along the entire tooth/restoration interface.

Furthermore, the method is nondestructive, allowing marginal qualities to be assessed before and after exposure to stressing, as well as highly discriminative, allowing the po- Fig 1a Representative SEM image of a bonded interface in

group 1 (SE) where gap-free margins in dentin can be observed after loading. Magnification: 200X. C: restorative composite, D:

dentin.

Fig 1c Representative SEM image of a bonded interface in group 3 (PB) after loading with no visible open margins in dentin.

Magnification: 100X. C: restorative composite, D: dentin.

Fig 1b Representative SEM image of a bonded interface in group 4 (primer of SE and bond of PB) with an evident marginal degradation after storage. Magnification: 100X. C: restorative composite, E: enamel.

Fig 1d Representative SEM image of a bonded interface in group 1 (SE) with open margins in enamel after water storage.

Magnification: 100X. C: restorative composite, E: enamel.

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tential of different operative techniques to be quantified in terms of the percentages of continuous margin. Therefore, thermomechanical fatigue tests and SEM marginal assess- ments used together may provide relevant information when in vivo behavior of dentin bonding agents is to be predicted on the basis of an accelerated in vitro test.1,6,8,22,28

The results of the present investigation revealed no significant differences in marginal adaptation between Clearfil SE Bond, the antibacterial adhesive Clearfil Protect Bond, and between the experimental combinations. According to the authors’ knowledge, this is the only study available at present comparing the marginal performance of a two-step self-etching adhesive with and without antibacterial properties after artificial aging. Only one recent clinical trial on cervical restorations of human teeth reported excellent results with Clearfil Protect Bond after 1 year in oral function, but in that study, the antibacterial adhesive was compared with the one-step self-etching adhesive Xeno III (Dentsply/De- Trey; Konstanz, Germany).³²

When enamel and dentin were evaluated separately, both adhesives showed distinct performances on these substrates; Protect Bond tended to perform better than SE Bond on enamel (a hydrophobic substrate in respect to dentin) and the opposite situation was observed in dentin. MDPB is described as a more hydrophobic monomer than HEMA.

Therefore, by incorporating MDPB into the formulation, the hydrophobicity of the adhesive system is increased.^19,20In terms of marginal adaptation, this could result in a greater stability of the bonding interface, which was confirmed in the present study by the performance of Protect Bond throughout the testing conditions, as no significant marginal degradation was observed after loading or after 1 year of water storage. Assuming that PB is more hydrophobic than SE, the bonding ability of this adhesive to dry enamel could be ex- pected to be enhanced.²¹ The present results confirmed these findings; the comparison of the results of enamel/

dentin within the groups at each interval (enamel/dentin initial, enamel/dentin terminal, enamel/dentin after storage) revealed that significant differences did not exist between the marginal adaptation of Protect Bond on either enamel or dentin substrates. This was not the case in the rest of the groups, as significant differences in the results between enamel and dentin were observed at all intervals (Table 2b).

These results could only be due to a more stable adhesion of the antibacterial adhesive to enamel. In respect to dentin, no significant differences were observed between the materials, confirming other research reports that effective bonding to dentin can be achieved with this antibacterial adhesive system.^7,18,19

The MDPB contained in Clearfil Protect Bond shows bac- tericidal effects without releasing any antibacterial components after polymerization.⁷From the clinical point of view, until further research demonstrates a real cariostatic effect of antibacterial monomers in fluid-filled gaps, perfect adhesion between the tooth structure and the restoration is still one of the most important factors determining the success of a restoration in the long run. In this study, completely gap- free margins could not be achieved with any of the formulations tested. Although most of the adhesives bonded rela- tively well to bur-cut enamel and dentin prior to thermome-

chanical loading and water storage, some of them were significantly less effective after fatigue. However, the margins of the antibacterial formulation were not significantly different from the ones of the restorations bonded with SE Bond.

With Protect Bond, there was a tendency to a greater stability after storage compared to the other formulations tested, presumably due to the effect of the antibacterial monomer.

Hence, minimally invasive restorative techniques,²⁹where incomplete removal of the carious tissues can inadvertent- ly occur, may already profit from the use of antibacterial monomer-containing adhesive systems. Apart from reducing microbial contamination,^9,18they do not decrease marginal quality, contributing to the restoration’s longevity.

CONCLUSION

There were no differences in the marginal adaptation of enamel and dentin margins in Class V cavities bonded either with Clearfil SE Bond, Clearfil Protect Bond, or the two experimental combinations of both marketed products. There- fore, the first null hypothesis is accepted. The second null hypothesis has to be partially rejected; no significant degradation of the dentinal margins occurred (with the exception of Exp. 2) throughout the testing intervals for the rest of the groups. On enamel, a significant degradation of the margins was observed after loading and water storage for all groups tested, with the exception of the antibacterial self-etching adhesive. Clearfil Protect Bond improved the stability of the resin/enamel interface not only after thermomechanical stressing, but also after additional long-term storage in water.

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Clinical relevance:A similar quality of marginal adaptation in enamel and dentin of Class V restorations can be achieved with a self-etching adhesive with and without an antibacterial monomer under simulated clinical conditions.