journal of dentistry 42 (2014) 709–719

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Adjunctive application of chlorhexidine and ethanol-wet bonding on durability of bonds to sound and caries-affected dentine Manikandan Ekambaram a, Cynthia Kar Yung Yiu a,*, Jukka Pekka Matinlinna b, Nigel Martyn King c, Franklin Russell Tay d a

Faculty of Dentistry, The University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Pokfulam, Hong Kong SAR, China b Dental Materials Science, Faculty of Dentistry, The University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Pokfulam, Hong Kong SAR, China c School of Dentistry, Oral Health Centre of Western Australia, The University of Western Australia, Australia d Department of Endodontics, College of Dental Medicine, Georgia Health Sciences University, Augusta, GA, USA

article info

abstract

Article history:

Objectives: To examine the effect of adjunctive application of chlorhexidine (CHX) and

Received 28 August 2013

ethanol-wet bonding (EWB) on bond durability and nanoleakage of hydrophobic adhesive

Received in revised form

to sound (SD) and caries-affected dentine (CAD).

31 March 2014

Methods: Dentine surfaces of molars were etched after caries removal and randomly

Accepted 2 April 2014

allocated to four groups (n = 12). In Groups 1 and 2, dentine surfaces were saturated with either 2 ml of 100% ethanol or 2 ml of ethanol with 2% CHX for 60 s. In Groups 3 and 4, dentine surfaces were saturated with either 15 mL of distilled water or 15 mL of distilled water

Keywords:

with 2% CHX for 60 s. Two coats of primer, followed by neat resin were applied and light-

Ethanol-wet bonding

cured for 40 s. Resin composite build-ups were placed and bonded specimens were sec-

Chlorhexidine

tioned for bond strength testing after 24 h and 12 months’ storage in artificial saliva. Bond

Caries-affected dentine

strength data were analyzed using 3-way ANOVA and SNK tests. Interfacial nanoleakage

Hydrophobic adhesives

was evaluated after 24 h and 12 months using a field-emission scanning electron micros-

Dentine bonding

copy and data were analyzed using Kruskal–Wallis test.

Acid etching

Results: Significant differences were observed for the three factors: ‘‘substrate’’ ( p < 0.001), ‘‘rewetting agents’’ ( p < 0.001) and ‘‘time’’ ( p < 0.001) on bond strength. Incorporation of 2% CHX to EWB preserved bond strength to SD and CAD and reduced interfacial nanoleakage after 12 months. Incorporation of 2% CHX to WWB also preserved bond strength to SD after ageing. Conclusions: Incorporation of chlorhexidine to ethanol-wet bonding has an interaction effect on preservation of bond durability to sound and caries-affected dentine. Clinical significance: Incorporation of chlorhexidine to ethanol-wet bonding with hydrophobic adhesive enhances the success rate of aesthetic bonded restorations. # 2014 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +852 28590256; fax: +852 25593803. E-mail address: [email protected] (C.K.Y. Yiu). http://dx.doi.org/10.1016/j.jdent.2014.04.001 0300-5712/# 2014 Elsevier Ltd. All rights reserved.

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1.

journal of dentistry 42 (2014) 709–719

Introduction

Dentine, unlike enamel, is a hydrated mineral tissue consisting of 50 vol% of inorganic crystalline matrix, 30 vol% of organic materials, of which Type I collagen predominates; while the remaining 20 vol% is fluid in nature. Furthermore, the structure of dentine is modified by physiological ageing and disease processes.1 Bond durability is essential for the longevity of aesthetic tooth-coloured restorations. However, resin-dentine bond deteriorates over time2 and bond failure occurs as a result of degradation of the adhesive resin, the collagen or both.3 The so called ‘‘wet bonding’’ technique was introduced to prevent the collapse of the demineralized dentine collagen fibrils after the acid etching process and to facilitate the infiltration of the resin adhesives.4 However, due to the presence of water in the current wet-bonding techniques, there is a potential risk for phase separation of the hydrophobic bis-GMA monomer from the hydrophilic resin monomers, which has limited water solubility.5 To avoid this problem, the wet-bonding technique involved the use of hydrophilic monomers, such as hydroxyethylmethacrylate (HEMA). However, these hydrophilic monomers increasingly absorb moisture after polymerization, which cause plasticization of the resin polymer chains and thereby decrease the resin-dentine bond strength with time.6 There is another important challenge to the dentine bond durability, which is degradation of collagen from the matrixbound proteases, namely matrix metalloproteinases (MMPs) and cysteine cathepsins.7 The MMPs are a class of zinc and calcium-dependent endopeptidases8 that are trapped within the mineralized dentine matrix during tooth development.9,10 These host-derived MMPs are activated by simplified etchand-rinse adhesives,11 and self-etch adhesives,12 which lead to slow degradation of the denuded collagen fibrils in the hybrid layer.7,13–15 Cysteine cathepsins are another group of endogenous proteases involved in the degradation of extracellular matrix components under various conditions of health and disease.16 There is evidence of cysteine cathepsins activities, especially cathepsin B in human pulp tissues and odontoblasts,17 as well as in carious dentine.18 The highly significant correlations between MMP and cysteine cathepsins activities strongly indicate that both proteases play an important role of collagen degradation in dental caries pathogenesis.18 Several experimental strategies to arrest the biodegradation of resin-dentine interfaces and to improve the longevity of bonded restorations have been conducted in recent years. These strategies have varied from stabilizing dentine collagen using collagen crosslinkers,19–22 pretreatment of the bonding substrate with agents that inhibit the activity of dentine matrix-bound endopeptidases,23–25 and ethanol-wet bonding of the demineralized dentine with hydrophobic resin.26–30 Chlorhexidine (CHX) is an anti-bacterial agent, which strongly inhibits the proteolytic activities of MMP-2, -8 and 9.31 Apart from MMPs, evidence of inhibition of dentinal cysteine cathepsins B, K and L by CHX has recently been demonstrated.32 The inhibitory effect of chlorhexidine on proteases has been shown to preserve collagen integrity

within the hybrid layers in both in vivo13,14,33 and in vitro studies.15,24,34,35 The ‘‘ethanol-wet bonding (EWB)’’ concept was developed to replace water with ethanol to support the demineralized dentine collagen fibrils.26 Ethanol-wet bonding was introduced as an experimental strategy to facilitate penetration of bis-GMA-based HEMA-free hydrophobic adhesive resin into ethanol-saturated demineralized dentine. Given this, the negative effects of moisture absorption and reduction in the mechanical properties of the dental adhesives could be minimized by this hydrophobic adhesion to demineralized dentine that has been saturated with ethanol.36 Recent studies have shown promising results with this ethanol-wet bonding technique.6,27–30 Bond strength testing with EWB has been carried out using extracted human teeth with sound dentine (SD). However, in the clinical scenario, when restoring a carious tooth, dental adhesives are placed on caries-affected dentine (CAD) after removal of the soft caries-infected dentine. Hence, it would appear to be more clinically relevant to test the bond strength and bond durability of this new bonding technique on CAD, so as to see the clinical relevance. Bonding to CAD obviously differs from SD. Besides the fact that the dentinal tubules are filled with mineral casts, the mechanical properties of CAD are also inferior to SD, as a result of the reduction in its mineral content, loss of the crystallinity of its remaining mineral phase and changes in the secondary structure of its organic components.37 Moreover, it is not known whether there are any interaction effects caused by simultaneous application of CHX and EWB on the durability of hydrophobic adhesive to SD and CAD. Hence, the aim of this study was to investigate the effect of adjunctive application of CHX and EWB on bond durability and nanoleakage of hydrophobic adhesive to SD and CAD. The null hypothesis tested was that the adjunctive application of CHX and EWB had (i) no effect on the bond strength of hydrophobic adhesive to SD and CAD and (ii) no effect on nanoleakage expression in the bonded interface formed by SD and CAD.

2.

Materials and methods

2.1.

Tooth preparation

Forty-eight extracted human molars with coronal caries were used in this study. The teeth were collected after the patient’s informed consent was obtained under a protocol reviewed and approved by the Institutional Review Board of the University of Hong Kong (No: UW 11-159). The teeth were first stored in 1% chloramine T solution at 4 8C and used within 3 months’ following extraction. The occlusal enamel and superficial dentine of the teeth was removed horizontally, through the carious lesion mesio-distally, using a slow-speed diamond impregnated disc (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. Thus, a flat surface of middle to deep dentine became exposed, where the carious lesion was surrounded by SD. Exposed dentine surfaces were stained with Caries Detector solution (Kuraray Medical Inc., Tokyo, Japan) for 10 s and then rinsed with water. The carious lesion was then manually excavated until there was no bright red

journal of dentistry 42 (2014) 709–719

staining dentine. Caries-affected dentine was defined as dentine that was colourless to light pink, firm and opaque. The entire dentine surface was then ground flat with 600-grit SiC paper under running water for 15 s to create a standardized smear layer. The exposure of the CAD substrate, subsequent bonding and testing of all the specimens in this study were all performed by one operator to avoid interoperator variability.

2.2.

Preparation of the primer

A co-monomer resin blend, consisting of 70 wt% bisphenol A diglycidylether dimethacrylate (bis-GMA), 28.75 wt% tetraethylene glycol dimethacrylate (TEGDMA), 0.25 wt% camphorquinone (CQ) and 1 wt% ethyl N,N-dimethyl-4-aminobenzoate were used to formulate the experimental three-step etch-andrinse hydrophobic adhesive following the protocol by Sadek et al.38 The primer was prepared by mixing 50 wt% of this resin with 50 wt% ethanol. The neat resin was then used as the adhesive.

2.3.

Bonding procedures

All dentine surfaces were etched with a 35% phosphoric acid (Gluma etch 35 liquid, HeraeusKulzer, Armonk, NY, USA) for 15 s and then rinsed with de-ionized water for 15 s before bonding. The teeth were blot-dried and randomly assigned to 4 groups (n = 12) according to the rewetting agents:

2.4.

For all the EWB groups, the dentine surface was saturated with ethanol by inverting the dentine surface in 2 mL of 100% ethanol (Group 1) or 2 mL of 2% chlorhexidine diacetate (Sigma-Aldrich, St Louis, MO, USA) (Group 2) incorporated in ethanol for 60 s following the method of Hosaka et al.28 For the WWB groups, the dentine surface was rehydrated with either 15 mL of distilled water (Group 3) or 15 mL of distilled water incorporated with 2% CHX (Group 4) for 60 s and gently blotdried. Two thin coats of the hydrophobic primer, prepared by diluting the neat co-monomer blend with 50 wt% absolute ethanol, were applied to the ethanol-saturated dentine and gently agitated with a fine microbrush for 10 s. Excess ethanol solvent was evaporated from the primed dentine surface with a gentle stream of compressed air for 10 s. A layer of the same neat comonomer resin was then immediately applied as an adhesive and light-cured for 40 s using a quartz-halogen light-curing unit (Optilux, Demetron-Kerr, Orange, CA, USA) with a constant output intensity of 600 mW/cm2. Resin composite build-ups were created with a light-cured resin composite (Filtek Z250, 3M ESPE, ST Paul, MN, USA) in three increments, each with an average thickness of 1 mm that was individually light-cured for 40 s with the same light. The teeth were then subsequently stored in artificial saliva at 37 8C for 24 h.

Microtensile bond strength testing

Eight bonded teeth from each group were sectioned occlusogingivally into serial slabs. Four slabs (i.e. two with SD and the other two with CAD) were prepared from each tooth. The slabs were further sectioned into 0.9 mm  0.9 mm compositedentine beams, according to the ‘‘non-trimming’’ technique of the micro-test tensile bond strength test by Shono et al.39 Four beams of resin composite-SD and resin composite-CAD were obtained from each bonded tooth. The beams obtained from four teeth per group were tested immediately and the beams from the remaining four teeth were stored in sodium azide-containing artificial saliva at 37 8C to inhibit microbial growthand tested after 12 months. The artificial saliva contained (mmoles/L): CaCl2 (0.7), MgCl2
6H2O (0.2), KH2PO4 (4.0), KCl (30), NaN3 (0.3), and HEPES buffer.40 The storage medium was changed once every week. At each time point, the bonded beams were attached to the test apparatus with cyanoacrylate adhesive (Zapit, Dental Ventures of North America, Corona, CA, USA) and stressed to failure under tension in a Bencor Multi-T device (Danville Engineering, San Ramon, CA, USA) using a universal testing machine (Model 4440, Instron, Inc., Canton, MA, USA) at a constant crosshead speed of 1 mm/min. The fractured beams were removed from the testing apparatus and the crosssectional area of each specimen at the site of failure was measured with a pair of digital calipers (Model CD-6BS; Mitutoyo, Tokyo, Japan). Bond strength values were expressed in MPa.

2.5. Group 1: ethanol-wet bonding (EWB) Group 2: ethanol-wet bonding + chlorhexidine (EWB + CHX) Group 3: water-wet bonding (WWB) Group 4: water-wet bonding + chlorhexidine (WWB + CHX)

711

Scanning electron microscopy

After bond strength testing, the fractured dentine sides of the bonded specimens were observed microscopically to evaluate the morphological differences between 24 h and 12 months debonded surfaces. The specimens were air-dried, sputtercoated with gold/palladium and examined using a SEM (Cambridge Stereoscan 440, Cambridge, UK) operated at 15 kV. The failure mode was classified as either: (i) cohesive failure in composite resin (CR), if the fracture occurred exclusively within the resin composite, resin adhesive or both; (ii) cohesive failure in dentine (CD); (iii) adhesive failure, if the fracture is located entirely between the adhesive and dentine; or (iv) mixed failure (M), if the fracture site continued from the adhesive into either the resin composite or dentine.41

2.6.

Nanoleakage evaluation

Four bonded teeth per group, two teeth per group for each time period tested, were used for nanoleakage analysis. The bonded teeth were sectioned occluso-gingivally into serial slabs as mentioned previously. Two slabs (one with SD and the other with CAD) were chosen from each tooth. Each slab was blotdried and was coated with two layers of nail varnish applied up to within 1 mm from the bonded interfaces. After rehydration in distilled water for 10 min, the varnish-coated tooth slabs were immersed in 50 wt% ammoniacal silver nitrate (AgNO3) solution in the dark for 24 h, followed by placing the slabs in a photodeveloping solution under a fluorescent light for 8 h to facilitate the reduction of silver nitrate ions into metallic silver grains.42 Next, the silver-stained bonded specimens were polished with

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journal of dentistry 42 (2014) 709–719

increasingly fine diamond pastes (6 mm, 3 mm and 1 mm; Pace Technologies, Tucson, AZ, USA). Then, the specimens were cleaned ultrasonically, air dried, mounted on aluminium stubs, placed in a desiccator for 24 h and then coated with carbon. The interfacial nanoleakage of the bonded composite-dentine slabs were examined with field-emission scanning electron microscope (Hitachi VP-SEM S-3400N; Hitachi High-Technologies Europe GmbH, Krefeld, Germany) using the backscattered electron mode at a voltage of 15 kV. At each time period of testing, 20 images of the bonded interfaces, at the same magnification, were obtained per group, of which ten were from those bonded to SD and the remaining ten to the CAD. The degree of interfacial nanoleakage was assessed based on the silver deposition along the bonded interface, which was independently scored by two examiners based on the classification of Saboia et al.43 The percentage of the bonded interface showing silver deposition was used to score the interfacial nanoleakage: 0, no nanoleakage; 1, 0.05, F = 0.639). The interaction among the three factors was also significant ( p < 0.001, F = 6.727), indicating that the changes in bond strength during storage were dependent on the substrates and rewetting agents. Irrespective of CHX application, EWB groups had significantly higher bond strengths than WWB groups after 24 h and 12 months ( p < 0.05). Conversely, irrespective of the rewetting agents, the bond strength of hydrophobic adhesive to SD was significantly higher than to CAD ( p < 0.05). The only exception was the WWB + CHX group, for which no significant difference in bond strength was found between SD and CAD after 24 h. The adjunctive application of CHX with WWB and EWB had no effect on the bond strength of hydrophobic adhesive to SD and CAD after 24 h ( p > 0.05), except WWB + CHX group to CAD, which showed a significantly higher bond strength ( p < 0.05). After 12 months of ageing in artificial saliva, the adjunctive application of CHX with EWB preserved the bond strength of hydrophobic adhesive to both SD and CAD. In the absence of CHX, significant bond strength reduction was observed in the EWB groups when bonding to SD (21.15%) and CAD (14.37%). Conversely, the adjunctive application of CHX with WWB similarly preserved the bond strength of hydrophobic adhesive to SD after 12 months; while significant drop in bond strength was observed in CAD after ageing (18.72%). In the absence of CHX, significant drop in bond strength was observed after 12 months in the WWB groups when bonding both to SD (27.61%) and CAD (60.87%).

3.2.

Failure mode distribution

The distribution of the failure modes of the debonded specimens in each group after 24 h and 12 months of ageing in artificial salivaare shown in Table 2, respectively. The EWB groups (with and without CHX) bonded to either SD or CAD exhibited predominantly mixed failures; whilst the WWB groups (with and without CHX) showed predominantly adhesive failures for both tested time periods. Cohesive

Table 1 – Microtensile bond strength of hydrophobic adhesive to sound and caries-affected dentine after 24 h and 12 months of ageing in artificial saliva. Groups

Sound dentine

Caries-affected dentine

24 h

12 m

24 h

12 m

EWB EWB + CHX WWB

51.78 (3.9) g 52.99 (4.4) g 25.24 (4.1)c,d

40.83 (5.7) f 50.37 (4.1) g 18.27 (4.0) b

33.61 (4.2) e 33.93 (3.3) e 20.52 (3.6) b

28.78 (3.6) d 31.76 (5.2) e 8.03 (3.1) a

WWB + CHX

26.64 (3.8)c,d

26.56 (1.9)c,d

23.45 (4.8) c

19.06 (3.5) b

Values are means  standard deviation in MPa Groups identified by different superscripts were significantly different at p < 0.05.

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journal of dentistry 42 (2014) 709–719

Table 2 – Distribution of failure mode after 24 h and 12 months of storage in artificial saliva. Groups

WWB-SD WWB-CAD WWB + CHX-SD WWB + CHX-CAD EWB-SD EWB-CAD EWB + CHX-SD EWB + CHX-CAD

A (%)

M (%)

CR (%)

CD (%)

24 h

12 m

24 h

12 m

24 h

12 m

24 h

12 m

68.8 25.0 62.5 43.8 25.0 13.0 12.5 6.3

75.0 43.7 50.0 50.0 12.5 6.3 12.5 6.2

25.0 31.3 37.5 6.2 75.0 62.5 81.2 62.5

25.0 18.8 50.0 50.0 56.3 56.3 62.5 75.0

0.0 0.0 0.0 0.0 0.0 18.7 6.3 0.0

0.0 0.0 0.0 0.0 0.0 6.2 0.0 0.0

6.2 43.7 0.0 50.0 0.0 6.2 0.0 31.2

0.0 37.5 0.0 0.0 31.2 31.2 25.0 18.8

Failure mode: A: adhesive failure; M: mixed failure; CR: cohesive failure in composite resin; CD: cohesive failure in dentine. Groups: WWB-SD: water-wet bonding to sound dentine; WWB + CHX-SD: water-wet bonding with 2% chlorhexidine to sound dentine; EWB-SD: ethanol-wet bonding to sound dentine; EWB + CHX-SD: ethanol-wet bonding with 2% chlorhexidine to sound dentine; WWB-CAD: water-wet bonding to caries-affected dentine; WWB + CHX-CAD: water-wet bonding with 2% chlorhexidine to caries-affected dentine; EWB-CAD: ethanol-wet bonding to caries-affected dentine; EWB + CHX-CAD: ethanol-wet bonding with 2% chlorhexidine to caries-affected dentine.

failures in the dentine were observed in the specimen beams generated from the CAD.

3.3.

Nanoleakage evaluation

The inter-examiner reliability for scoring the nanoleakage as assessed by weighted Kappa test was over 0.80. The nanoleakage in the different groups after 24 h of bonding are shown in Fig. 1. Very sparse silver deposits were found along the bonded interfaces formed by the EWB/SD groups (Fig. 1A and C); while moderate silver deposits were observed along the bonded interfaces and in the final depths of the resin tags in the EWB/ CAD groups (Fig. 1B and D). Extensive silver deposits were found in bonded interfaces of the WWB/SD groups (Fig. 1E and G) and WWB/CAD (Fig. 1F and H) groups. The addition of CHX to EWB or WWB did not affect the nanoleakage expression at the bonded interfaces from both groups. The results for nanoleakage in the different groups after 12 months of ageing in artificial saliva are shown in Fig. 2. Among the specimens tested after 12 months of storage in artificial saliva, the EWB/CHX/SD (Fig. 2C) and EWB/CHX/CAD (Fig. 2D) groups showed less silver deposits along the bonded interfaces, when compared to the specimens from the EWB alone/ SD (Fig. 2A) and EWB alone/CAD groups (Fig. 2B). Table 3 shows the percentage distribution of nanoleakage scores among the tested groups both after 24 h and 12 months of storage in artificial saliva. Results of Kruskal–Wallis test with Dunnett’s post hoc test showed a significantly lower silver penetration at the bonded interfaces among the EWB/CHX groups at 12 months, when compared to the other groups ( p < 0.05).

4.

Discussion

The bond strength of dental adhesives to CAD has been shown in several studies to be less than that of SD.19,45 One major possible reason for such a reduction in bond strength could be attributed to the porous and weak hybrid layer formed in CAD. The loss of minerals due to the caries and the increased susceptibility of CAD to the effects of etching lead to an increased depth of demineralized intertubular dentine, which in turn may not be completely infiltrated by the adhesive.

Ethanol-wet bonding approach prevents the hydrolytic degradation associated with the contemporary simplified etch-and-rinse and self-etch adhesives and produces durable resin bonds with the dentine. Ethanol-wet bonding is ideally performed by the treatment of acid demineralized dentine with a series of increasing concentrations of ethanol, which requires a maximum time period of 3 min and 30 s.27 An experimental strategy that consumes a long time period would be very challenging to translate it into routine clinical practice; hence, its clinical applicability is questionable. Henceforth, an alternative protocol to treat demineralized dentine with 100% ethanol for 60 s followed by a bonding procedure with a hydrophobic adhesive was developed.28,46 Our current study examined the effects of adjunctive application of EWB with CHX on the bond durability of hydrophobic adhesive to SD and CAD. The results showed that the adjunctive application had an interaction effect on preserving the bond strength to both types of dentine substrates, compared to EWB alone. Conversely, the nanoleakage at the bonded interfaces of SD and CAD following the adjunctive application of CHX with EWB was also significantly lower than with EWB alone after 12 months of ageing. Therefore, the null hypotheses have to be rejected. For the EWB groups, CHX was incorporated directly into ethanol for EWB. The demineralized dentine was treated simultaneously with CHX and EWB in one step. This strategy eliminated an extra step needed for CHX pretreatment. A simplified ethanol dehydration protocol (100% ethanol for 60 s) was used in our study. Although EWB allowed better infiltration of hydrophobic resin into demineralized dentine as a result of less mismatch in Hoy’s solubility parameter with ethanol than with water,6 microvoids were evident within the hybrid layers created with the hydrophobic adhesive applied on ethanolsaturated dentine.47 The presence of microvoids could be attributed to the incomplete removal of water from the hybrid layer. In the present study, complete removal of water was more difficult to achieve in CAD due to the increased water content because the mineral loss was replaced by water.48 Conversely, it is also unlikely for the hydrophobic resin to completely infiltrate the full depth of demineralized dentine in CAD. Hence, the proteases-bound to the unprotected collagen fibrils may hydrolyze and initiate cleavage of the surrounding

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Fig. 1 – Representative backscattered SEM images of the resin-dentine interfaces after 24 h of ethanol-wet bonding, with (A) or without chlorhexidine (C) to the sound dentine showing sparse silver uptake; ethanol-wet bonding, with (B) or without chlorhexidine (D) to the caries-affected dentine showing moderate silver uptake along the hybrid layer and in the final length of the resin tags. Water-wet bonding with (E) or without chlorhexidine (G) to sound dentine and water-wet bonding with (F) or without chlorhexidine (H) to caries-affected dentine respectively, showing extensive silver uptake. (Co: Composite; AL: adhesive layer; HL: hybrid layer; De: dentine.)

collagen molecule in the presence of residual water in the interfibrillar spaces. Therefore, the adjunctive application of CHX with EWB, especially when a simplified dehydration protocol is used, played a significant role in inhibiting

collagenolytic activities of these proteases and extending the longevity of resin-dentine bonds. This study demonstrated that the interaction effect of CHX and EWB was found in both sound and clinically relevant

journal of dentistry 42 (2014) 709–719

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Fig. 2 – Representative backscattered SEM images of the resin-dentine interface after 12 months of bonding: ethanol-wet bonding without chlorhexidine to sound dentine (A) and to caries-affected dentine (B) show moderate silver uptake along the hybrid layer; ethanol-wet bonding with chlorhexidine to sound dentine (C) and to caries-affected dentine (D) show sparse silver uptake; water-wet bonding with or without chlorhexidine to both the sound (E, G), and caries-affected dentine (F, H) show extensive silver uptake along the hybrid layer. (Co: Composite; AL: adhesive layer; HL: hybrid layer; De: dentine).

caries-affected dentine substrates. EWB is a suitable bonding protocol for deep CAD. The mineral casts in the dentinal tubules of CAD substrate have dual benefits. It would protect the seepage of the solvent into the pulp and prevent any potential pulpal complications; whilst from the pulpal side it

would block the fluid movement and thereby prevent further moisture contamination of the relatively hydrophobic bonding substrate. Furthermore, CHX would act on the dentinal fluids that have been shown to contain proteolytic enzymes,49 especially dentinal fluids from the carious dentine.50,51

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journal of dentistry 42 (2014) 709–719

Table 3 – Percentage distribution of nanoleakage scores among the tested groups after 24 hrs and 12 months of ageing in artificial saliva. Group

Age

24 h

12 m

WWB-CAD

24 h

12 m

WWB + CHX-SD

24 h

12 m

WWB + CHX-CAD

24 h

12 m

EWB-SD

24 h

12 m

EWB-CAD

24 h

12 m

Group

Age

Nanoleakage expression Score 0–4

WWB-SD

Table 3 (Continued )

%

Statistical difference

0 1 2 3 4 0 1 2 3 4

0 0 20 50 30 0 0 0 0 100

B

0 1 2 3 4 0 1 2 3 4

0 0 0 30 70 0 0 0 0 100

A

0 1 2 3 4 0 1 2 3 4

0 0 10 70 20 0 0 10 20 70

B

0 1 2 3 4 0 1 2 3 4

0 0 0 20 80 0 0 0 30 70

A

0 1 2 3 4 0 1 2 3 4

20 60 20 0 0 0 0 40 40 20

D

0 1 2 3 4 0 1 2 3 4

0 30 50 20 0 0 0 20 50 30

C

EWB + CHX-SD

24 h

12 m A

EWB + CHX-CAD

24 h

12 m A

Nanoleakage expression Score 0–4

%

Statistical difference

0 1 2 3 4 0 1 2 3 4

10 60 30 0 0 50 30 20 0 0

D

0 1 2 3 4 0 1 2 3 4

0 40 40 20 0 0 20 60 20 0

C

D

C

Groups identified by different alphabets were significantly different at p < 0.05.

A

A

B

B

Sadek et al.52 reported that bonds made to ethanolsaturated dentine with hydrophobic adhesive were preserved regardless of CHX pretreatment. This can be explained by the difference in the ethanol dehydration protocol and the CHX preparation used. In our study, the ethanol wet bonding protocol involved with a more clinically relevant time period (60 s); while a progressive dehydration with ascending ethanol concentrations (50%, 70%, 80%, 95% and 3  100% ethanol applications for 30 s) was employed by Sadek et al.52 The prolonged dehydration protocol is less technique sensitive, when compared to direct rinsing with absolute alcohol. The longer dehydration protocol allows more complete removal of water from the demineralized collagen matrix52 and it prevents collapse of the collagen fibrils either by evaporation of water or absolute ethanol.53 However, the progressive dehydration protocol is more time consuming and impractical for clinical use. A 2% aqueous CHX diacetate was used as the MMP inhibitor with EWB in the study by Sadek et al.38; while a 2% CHX diacetate dissolved in ethanol was used in the present study. The CHX dissolved in ethanol is more compatible with the EWB technique and allows a maximum concentration of CHX in the hybrid layer. The EWB protocol was found to be sensitive in the presence of water as hydrophobic monomers are immiscible with water.54 Furthermore, a higher uptake of CHX by ethanol-solvated adhesive is expected with CHX dissolved in ethanol than CHX dissolved in water. For the WWB groups, the adjunctive application of CHX and WWB also preserved the bond strength to SD. Though the bond strength of WWB with CHX group was lower in CAD than SD after 12 months, it was significantly higher than the WWB only group. As discussed previously, caries-affected dentine was found to have more water than normal dentine.48 Application of

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acid etchant to caries-affected dentine resulted in deeper demineralization and more residual water after rinsing the acid-etched dentine. Debinding of CHX from demineralized dentine is found to be higher for water than ethanol.55 Hence, the concentration of CHX in the hybrid layer decreased over time, resulting in reduced effectiveness of CHX in preventing collagen degradation in the hybrid layer over time. In the current study, a mixed failure pattern was found predominantly in the EWB groups. This failure mode could be due to the relatively higher bond strength to SD and CAD; whilst the WWB groups were not a compatible substrate with our hydrophobic adhesive, which exhibited predominantly an adhesive failure pattern. Moreover, the cohesive failure pattern observed in the groups bonded to the CAD could have been due to the inherent weakness of the dentine as a result of the caries process. However, the WWB with CHX group demonstrated a reduced percentage of cohesive failure pattern in the dentine after 12 months and this indicates the improved preservation of the dentinal mechanical strength by CHX. After 24 h of bonding, the nanoleakage evident by sparse silver deposits along the bonded interface was found in the EWB/SD group, which indicated a good hybridization of demineralized dentine has been achieved. The extensive silver deposits found in the WWB/SD and WWB/CAD groups indicated the incompatibility of the water present in the etched dentine surface and the hydrophobic adhesive applied. Given this, the moderate silver deposits found along the bonded interface and in the extremity of the resin tags in the EWB/CAD indicated that a porous hybrid layer had formed and that there was incomplete penetration of the adhesives into demineralized CAD. After 12 months of storage in artificial saliva, the nanoleakage along the bonded interfaces created with adjunctive application of EWB and CHX showed less silver deposits than the specimens bonded with EWB without CHX incorporation. The preservation of the hybrid layer from the endogenous proteases by the application of CHX and from the hydrolysis of resin adhesive by the use of hydrophobic adhesive could be the possible explanation for such an observation. In our study, the two bonding strategies (EWB or WWB) were used with hydrophobic adhesive and therefore the results may not be extrapolated to other adhesives. However, CHX application should be encouraged in adhesive dentistry, where bonding to dentine is indicated, as it preserves the resin-dentine bonded interfaces. In conclusion, the current study demonstrated the interaction effect of adjunctive application of CHX and EWB on bond durability and nanoleakage of hydrophobic adhesive to SD and CAD. Nevertheless, further studies are required to find a clinically relevant EWB dehydration protocol to improve the bond durability to caries-affected dentine.

5.

Conclusions

(1) The adjunctive application of CHX and EWB preserved the bond strength of the hydrophobic adhesive to both SD and CAD after 12 months.

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(2) The bonded interfaces formed with hydrophobic adhesive using the adjunctive application of CHX and EWB showed less nanoleakage after 12 months.

Acknowledgements The comonomer blend used in this study was generously sponsored by Bisco Inc. This work was supported by GRF HKU 784410M, Faculty of Dentistry, The University of Hong Kong. The authors thank Frankie Chan of the Electron Microscopy Unit, the University of Hong Kong for technical support.

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