DOI: 10.1111/ipd.12069

The effect of resin infiltration and oxidative pre-treatment on microshear bond strength of resin composite to hypomineralised enamel PUI LING CHAY1,2, DAVID J. MANTON1 & JOSEPH E.A. PALAMARA1 1

Melbourne Dental School, The University of Melbourne, Parkville, Vic., Australia, and 2KK Women’s and Children’s Hospital, Singapore, Singapore

International Journal of Paediatric Dentistry 2014; 24: 252–267 Background. Reduced bond strengths of resin

composites to hypomineralised enamel increase restorative failure. Aim. To investigate if the adhesion of resin composite to hypomineralised enamel can be improved by pre-treatments: resin infiltration, oxidative pre-treatment followed by a resin infiltration, or oxidative pre-treatment. Design. Twenty-one enamel specimens in each of five Groups: 1) Normal enamel; 2) Hypomineralised enamel; 3) Hypomineralised enamel pre-treated with a resin infiltrant, (Iconâ); 4) Hypomineralised enamel pre-treated with 5.25% sodium hypochlorite then treatment with resin infiltrant; 5) Hypomineralised enamel pre-treated with 5.25% sodium

Introduction

Molar incisor hypomineralisation (MIH) is defined as enamel hypomineralisation of systemic origin affecting one to four permanent first molars, frequently associated with affected incisors.1 It is a common condition with prevalence ranging from 2.4% to 40.2%, associated with a number of clinical problems including increased caries risk, increased sensitivity and post-eruptive breakdown.2 Most treatment need for MIH patients relates to the hypomineralised first permanent molar, with one study reporting that by 9 years of age, children affected by MIH had undergone treatment of their first permanent molars nearly ten times as often as children Correspondence to: Professor David J. Manton, Melbourne Dental School, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 720 Swanston Street, Melbourne, Vic. 3010, Australia. E-mail: [email protected]

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hypochlorite. A resin composite rod was bonded to each specimen using Clearfil™ SE bond as the adhesive (hereafter termed ‘routine bonding’), then subjected to microshear bond strength (MSBS) testing. Results. Overall, the mean MSBS between the five groups differed significantly (P = 0.001). Pre-treatment of hypomineralised enamel with 5.25% sodium hypochlorite with or without subsequent resin infiltration in Groups 4 and 5 prior to routine bonding resulted in increased mean MSBS compared to Groups 2 and 3, with mean MSBS values not differing significantly when compared to routine bonding to normal enamel. Conclusion. Increased bond strength of resin composite to hypomineralised enamel was obtained by pre-treatment of hypomineralised enamel specimens with 5.25% sodium hypochlorite with or without subsequent resin infiltration.

not affected by MIH, and on average, every affected tooth had been treated twice. This same cohort at 18 years of age had increased treatment need as compared to unaffected individuals, with the majority of the treatment being revision of restorations and extraction of the hypomineralised molars.3 Affected teeth are often difficult to restore adequately as the enamel is less mineralised, has greater porosity, and is poorly delineated from normal tissue; effective local analgesia is also often difficult to obtain.2 If the decision is made to restore affected teeth, the most appropriate intracoronal restorative material in hypomineralised molars is resin composite.4 There is a lack of evidence to indicate whether partial or complete removal of the defective enamel prior to restoration placement gives superior outcomes. Partial removal of the defective enamel is less invasive; defective enamel, however, tends to suffer from physical breakdown leading to poor restorative outcomes.2

© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Bonding to hypomineralised enamel

Published research regarding the adhesion of resin composite to hypomineralised enamel is limited. The results from an in vitro study indicated that the microshear bond strength (MSBS) of resin composite bonded to hypomineralised enamel was significantly lower than that of control enamel, and there was high frequency of cohesive failures within the defective enamel, indicating the inherent physical weakness of the hypomineralised lesion.5 The polycrystalline structure of hypomineralised enamel is more porous and disorganised than normal enamel, with a reported average 28% reduction in mineral content, 80% more carbonated apatite and 3- to 15fold elevation in protein content.6–8 The hardness of hypomineralised enamel is also significantly lower than sound enamel.6 A recently developed low viscosity triethyleneglycol dimethacrylate (TEGMA) resin with a high penetration coefficient has the purpose of occluding the highly porous structure of white spot lesions of enamel, inhibiting demineralisation by limiting diffusion of acid and also affording some mechanical support to the tissue.9 The adhesive performance of resin infiltrant has only been investigated on demineralised enamel in vitro, with the infiltrant increasing bond strength values to flowable resin composite.10 There was, however, a high percentage (65%) of cohesive failures-inenamel for the demineralised specimens treated with the infiltrant.10 It is postulated that the TEGMA resin may penetrate and seal the porous hypomineralised enamel, improving the subsequent bonding of resin composite to hypomineralised enamel and also affording mechanical support within the enamel tissue. The high level of protein in hypomineralised enamel may, however, inhibit infiltration. Sodium hypochlorite (NaOCl) has been utilised in dentistry for its non-specific antimicrobial affect, its ability to dissolve biofilms, capacity to solubilise necrotic tissue and for pulpotomies.11 With respect to the increased protein content in hypomineralised enamel which has been associated with impaired bonding, the proteolytic effect of NaOCl may be useful in removing this excess surface and intralesion protein.12–14 This may improve resin infiltration and subsequently increase

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bond strength to resin composite and also may improve the physical characteristics of hypomineralised resin impregnated enamel; little research has, however, been undertaken in this area.12,13 Studies on enamel affected by hypocalcified amelogenesis imperfecta (HCAI) have found improved bonding by enamel pre-treatment with 5% NaOCl.13,14 Hypomineralised enamel has also been typified as a hypocalcification defect and hence may have similar bonding characteristics as HCAI enamel.7 The aims of this study are to investigate whether the adhesion of resin composite to hypomineralised enamel can be improved by the use of a resin infiltrant, the use of oxidative pre-treatment, or the use of oxidative pre-treatment followed by a resin infiltrant. Methods and materials

Ethics approval Ethical approval was obtained for the collection and use of extracted teeth from the Human Ethics Advisory Group (HEAG) of the University of Melbourne (Ethics ID 1034105) and the Human Research Ethics Committee (HREC) of the Department of Human Services, Victoria (Approval no.233). Tooth collection and study sample A pooled sample of 152 extracted, erupted, hypomineralised first permanent molars collected from children under 18 years of age who were patients of the Royal Dental Hospital of Melbourne, the private practices of paediatric dentists and general dental practices over an 18-month period. A parent/ guardian information sheet explaining the research project was provided, and informed consent was obtained for tooth collection. Collected teeth were extracted due to caries, post-eruptive breakdown, or for orthodontic purposes for these children with MIH, and often more than one first permanent molar was extracted per patient. Specimen bottles containing clear aqueous 0.02% chlorhexidine w/v (Baxter™, Toongabbie, NSW, Australia) were provided to dentists for

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tooth collection and temporary storage not exceeding 1 month. These teeth were then collected by the investigator and transferred to 1% chloramine-T hydrate (Sigma-Aldrich Co., St Louis, MO, USA) at room temperature for 2 weeks, after which the teeth were rinsed in distilled deionised water (DDW), blotted dry, and stored in a sealed containers at room temperature, with several drops of DDW to maintain a humidified environment. The collected teeth were inspected visually when wet and confirmed as teeth affected by MIH by the investigator, using the judgement criteria described by Weerheijm et al. in 2003.15 Teeth affected by other developmental or pathological conditions were excluded from the study. Areas of suitable ‘hypomineralised enamel’ and ‘normal enamel’ were identified. Hypomineralised enamel was defined as a visual defect with a demarcated white, yellow and/ or brown opacity in enamel, and of normal thickness. Normal enamel was defined as upon visual inspection free of discolouration, caries, and developmental defects and was obtained from the normal looking enamel of hypomineralised teeth. For normal enamel, specimens were obtained from the occlusal half of the tooth, avoiding the cervical half where the enamel was thinner. Only teeth with at least 2–3 mm diameter of normal or hypomineralised enamel were selected. Specimen preparation for microshear bond strength testing The roots of suitable first permanent molars were removed perpendicular to the long axis of the tooth with a 0.3-mm-thick diamond blade (Minitom, Struers A/S, Copenhagen, Denmark). The crowns were sectioned using the same blade perpendicular to the occlusal plane, in various planes as necessary per individual tooth to provide the specimens. In a few cases, more than one and up to four specimens were obtained from an individual tooth. Specimens were lapped manually with wet 600-grit silicon carbide paper (Struers A/S, Copenhagen, Denmark) to produce a flat surface 2–3 mm in diameter with even roughness. It was attempted to remove as little

enamel as possible; the process, however, removed the enamel surface layer if present. Specimens were rinsed in DDW, air-dried, then the prepared flat surface was placed in contact with a glass slide, and sticky wax (Associated Dental Products LTD, Wiltshire, UK) attached to the edges of the sample to stabilise it. A plastic ring of 15 mm internal diameter was placed over the specimen and filled with Type 3 dental stone (Yellowstone; Whip Mix, Louisville, KY, USA). Once set, the glass slide was removed, creating a supported flat enamel surface suitable for bonding. Specimen grouping based on colour The ground and mounted hypomineralised specimens were moistened with DDW then assessed for their colour by the investigator. Hypomineralised enamel specimens were divided into two groups based on creamywhite (CW) or yellow-brown (YB) colour. Pre-treatment, infiltrant application and bonding procedure A total of 105 enamel specimens comprised 21 specimens of normal enamel and 84 specimens of hypomineralised enamel. Of the hypomineralised specimens, 43 were determined to be CW and 41 YB. Enamel specimens were divided into five experimental groups: Group 1 – 21 normal enamel Group 2 – 21 hypomineralised enamel (11 CW, 10 YB) Group 3 – 21 hypomineralised enamel (11 CW, 10 YB) Group 4 – 21 hypomineralised enamel (11 CW, 10 YB) Group 5 – 21 hypomineralised enamel (10 CW, 11 YB) The application techniques and bonding procedures for the five experimental groups are described in Table 1. All five groups were acid-etched with Scotchbond™ Etchant (3M ESPE, St Paul, MN, USA). Clearfil SE Bond™ (SE; Kuraray Medical Inc., Tokyo, Japan) was used for all

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Table 1. Pre-treatments and bonding procedures for five groups. Control groups – Routine bonding

Test groups – Pre-treatments before bonding

Group 1

Group 2

Group 3

Group 4

Group 5

NE

HE

HE pre-treated with infiltrant

HE pre-treated with 5.25% NaOCl then infiltrant

HE pre-treated with 5.25% NaOCl

35% phosphoric acid etch (15 s) Water spray (15 s)

35% phosphoric acid etch (15 s) Water spray (15 s)

35% phosphoric acid etch (15 s) Water spray (15 s)

Air-dried (15 s)

Air-dried (15 s)

2 coats Clearfil™ SE Bond Air thinned Mounted in SDI™ rig for composite placement

2 coats Clearfil™ SE Bond Air thinned Mounted in SDI™ rig for composite placement

ICONâ-dry (30 s) Air-dried (30 s) ICONâ-infiltrant (3 min) Light-cured (40 s) ICONâ-infiltrant (1 min) Excess removed with cotton roll and light-cured (40 s) 1 coat Clearfil™ SE Bond Air thinned Mounted in SDI™ rig for composite placement

35% phosphoric acid etch (15 s) Water spray (15 s) 5.25% NaOCl applied with microbrush in back and forth rubbing motion (1 min) Water spray (30 s) ICONâ-dry (30 s) Air-dried (30 s) ICONâ-infiltrant (3 min) Light-cured (40 s) ICONâ-infiltrant (1 min) Excess removed with cotton roll and light-cured (40 s)

35% phosphoric acid etch (15 s) Water spray (15 s) 5.25% NaOCl applied with microbrush in back and forth rubbing motion (1 min) Water spray (30 s) ICONâ-dry (30 s) Air-dried (30 s)

1 coat Clearfil™ SE Bond

2 coats Clearfil™ SE Bond

Air thinned Mounted in SDI™ rig for composite placement

Air thinned Mounted in SDI™ rig for composite placement

NE, normal enamel; HE, hypomineralised enamel.

five groups without use of a primer, as the hydrophilic component of a priming agent was postulated to interfere with the subsequent infiltration of the hydrophobic low viscosity resin infiltrant. Two coats of SE were applied to normal enamel in Group 1 and hypomineralised enamel in Group 2. The second application of SE was necessary due to the absorption of the first coat of bond into the porous hypomineralised enamel. Two coats of SE were also applied to the normal enamel in Group 1 for consistency. In Group 3, ICONâ infiltrant (DMG, Hamburg, Germany) was applied to hypomineralised enamel according to manufacturer’s instructions; the acid etching step with 15% hydrochloric acid gel (ICONâ-etch, ICONâ; DMG, Hamburg, Germany), however, was substituted with acid etching with 35% phosphoric acid (Scotchbond™ Etchant), as the surface layer had been removed during the specimen preparation. After application of

(ethanol; DMG, Hamburg, ICONâ-dry â Germany) and ICON -infiltrant (DMG, Hamburg, Germany) according to manufacturer’s instructions, only one coat of SE was applied as the resinous TEGMA infiltrant material was assumed to have penetrated into and occluded the spaces in the porous enamel. In Group 4, after acid etching, 5.25% aqueous NaOCl (Chem-Supply Pty Ltd, Gillman, SA, Australia) was applied to the hypomineralised enamel surface for 1 min. Thereafter, ICONâ-dry and ICONâ-infiltrant were applied in a similar manner to Group 3. Compared to Group 4, the resin infiltration procedure with ICONâ-infiltrant was eliminated in Group 5 after pre-treating the hypomineralised enamel with 5.25% aqueous NaOCl, and prior to the application of SE. ICONâ-dry was applied after oxidative pretreatment to help remove remnant moisture and residual NaOCl which may interfere with polymerisation of bonding resin.

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Before light curing the adhesive (Clearfil SE Bond™) in all five groups, each mounted enamel specimen was placed into the SDI™ rig (SDI, Bayswater, Vic., Australia), with the enamel specimen centred over the middle of the base plate. A milled hollow cylindrical standard metal mould of internal diameter 1.13 mm and height 2.00 mm was placed on the enamel specimen and stabilised in place by the compression plate of the SDI rig (Fig. 1). The adhesive was then light-cured for 10 s. The mould was filled with resin composite (Gradiaâ Direct; X-A2, Lot 1010191, GC Corporation, Tokyo, Japan) packed with a periodontal probe and light-cured for 40 s. The metal mould was not removed prior to the MSBS test. To ensure appropriate hydration of the enamel, specimens were placed in DDW at 37°C for 12 h prior to testing. Microshear bond strength test The bonded surfaces were subjected to a MSBS test in a Mecmesin testing machine (Imperial 1000 S1 instrument, Mecmesin, UK). A loop of ligature wire delivered a force parallel to the bonded surface at a cross-speed of 1.0 mm/min until fracture and MSBS values recorded. The

Fig. 1. Use of the SDITM rig. Mounted enamel specimen placed into the SDITM rig, with the enamel specimen centred over the middle of base plate. A milled standard hollow cylindrical metal mould is placed on the enamel specimen and stabilised in place by the compression plate of the SDITM rig, facilitating the packing of resin composite.

sheared cylindrical resin composite rods were retrieved for examination under light microscopy. Modes of failure Fractured surfaces were examined with an optical microscope (Leica DML; Ernst-LeitzStrasse, Wetzler, Germany) at 409 magnification and digital images obtained. Failures were categorised as one of the following: adhesive failure at the enamel–resin adhesive interface, cohesive failure-in-composite, cohesive failure-in-enamel, or a mixed failure that includes a partial cohesive failure and partial adhesive failure. A specimen was categorised as an adhesive failure, cohesive failure-in-composite, or cohesive failure-in-enamel when more than 80% of the fractured surface area examined consisted of that particular failure mode. If less than 80% of the fractured surface area had one particular mode of failure, it was classified as a mixed failure. Preparation for visualisation using scanning electron microscope (SEM) The digital images of all specimens were examined, and representative specimens were selected for examination under SEM. The enamel specimens were retrieved and airdried for two days on filter paper. The specimens were then mounted on aluminium stubs with conductive silver liquid (Pro Sci Tech, Townsville, Qld, Australia), gold sputtercoated (Gold Sputter Coater S150B, BOC Edwards Vacuum Ltd, Crawley, UK) and examined under a field-emission SEM (Philips XL 30 FEG, Eindhoven, the Netherlands) for verification of type of failure. Sheared enamel interfaces and the corresponding sheared cylindrical composite rod surfaces from the representative samples were also examined using magnifications of up to 40009 for analysis of surface morphology, with emphasis on areas of adhesive failure or areas of cohesive failure-in-enamel. Statistical analysis All statistical tests were performed using SPSS 20.0 statistical software (IBM Corporation,

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Bonding to hypomineralised enamel

New York, NY, USA). Descriptive statistics were calculated for MSBS values and modes of failure in each group. Assumptions of normality and equal variance were verified for MSBS values obtained, with Kolmogorov–Smirnov test (P = 0.2) and plotting of residuals respectively. One-way analysis of variance (ANOVA) tests were used to compare the mean MSBS values between the five groups, followed by post hoc Tukey’s HSD tests. The association between modes of failures in each group was examined with Pearson’s chi-square test. Mean MSBS values between groups were also tested for any associations with lesion colour, use and non-use of oxidative pre-treatment, and use and non-use of resin infiltration, using ANOVA tests. The level of significance was set at P ≤ 0.05 for all statistical tests performed. Results

No samples failed during preparation. During microshear bond strength testing, one of the sheared cylindrical composite rods from a specimen in Group 1 was not captured; hence, examination of the sheared composite surface was not possible for that specimen. The mode of failure for that specimen could, however, still be discerned from examination of the sheared enamel surface, hence not affecting the collection of results. A total of 14 representative specimens from the five groups were selected for examination under SEM to verify the mode of failure

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interpreted under light microscopy. Only one specimen was reclassified after examination under SEM; this specimen from Group 5 was reclassified from a mixed failure to an adhesive failure, as the area of cohesive failure-in-composite was less than 20% of the whole surface area, when it was previously thought to be greater than 20% when viewed under light microscopy. This observation was corrected in the data set before statistical analysis was undertaken. MSBS values The mean MSBS (MPa) values ( SD) and distributions of values are shown in Table 2. Amongst all five groups, the mean MSBS of resin composite to normal enamel (Group 1) was the greatest. When compared to the mean MSBS to hypomineralised enamel in the control group (Group 2), the use of resin infiltration in Group 3 decreased the mean MSBS value, whereas the use of oxidative pre-treatment followed by resin infiltration, as well as the use of oxidative pre-treatment in Group 4 and 5, respectively, increased the mean MSBS value. Overall, the mean MSBS (MPa) values ( SD) of the five groups differed significantly (one-way ANOVA, F ratio = 5.02, df = 104, P = 0.001). Tukey’s HSD post hoc tests revealed significant differences in the mean MSBS values between normal enamel (Group 1) and hypomineralised enamel (Group 2):

Table 2. Mean microshear bond strength of resin composite bonded to normal enamel, hypomineralised enamel and pretreated hypomineralised enamel.

No. of specimens Mean MSBS (MPa)  SD 95% CI (Upper) (MPa) 95% CI (Lower) (MPa)

Control groups

Test groups

Group 1: Bond to normal enamel

Group 2: Bond to HE

Group 3: Resin infiltration, bond to HE

Group 4: Oxidative pre-treatment, resin infiltration, bond to HE

Group 5: Oxidative pre-treatment, bond to HE

21 29.03  6.75*,a,b 32.10 25.95

21 22.05  5.14*,a 24.38 19.71

21 19.36  8.43*,b,c 23.20 15.53

21 25.81  8.87*,c 29.84 21.77

21 24.61  7.82* 28.17 21.05

MSBS, microshear bond strength; HE, hypomineralised enamel; SD, standard deviation; 95% CI (Upper) = 95% confidence interval for mean, upper bound; 95% CI (Lower) = 95% confidence interval for mean, lower bound. *Significant difference (P = 0.001) between mean MSBS of five groups (one-way ANOVA). a,b,c Similarly labelled values had significant differences when tested using post hoc Tukey’s HSD tests. © 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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29.03  6.75 vs 22.05  5.14, respectively (P = 0.027); normal enamel (Group 1) and hypomineralised enamel pre-treated with resin infiltration (Group 3): 29.03  6.75 vs 19.36  8.43, respectively (P = 0.001); hypomineralised enamel pre-treated with resin infiltration (Group 3) and hypomineralised enamel pre-treated with oxidative pre-treatment followed by resin infiltration (Group 4): 19.36  8.43 vs 25.81  8.87, respectively (P = 0.05) (Table 2). There were no statistically significant differences between the seven other configurations of pairwise comparisons. Overall, within the groups with hypomineralised enamel, white hypomineralised enamel gave consistently higher mean MSBS compared to yellow-brown hypomineralised enamel, although this was not statistically significant for all groups (one-way ANOVA, F ratio = 3.19, df = 83, P = 0.078) (Table 3). When hypomineralised enamel was grouped based on the use (N = 42) or non-use (N = 42) of oxidative pre-treatment, the use of oxidative

pre-treatment gave significantly higher MSBS (MPa) values ( SD): 25.21  8.28 vs 20.71  7.03, respectively (one-way ANOVA, F ratio = 7.23, df = 83, P = 0.009). When hypomineralised enamel was grouped based on the use (N = 42) or non-use (N = 42) of resin infiltration, the use of resin infiltration showed no significant difference in mean MSBS (MPa) values ( SD): 22.59  9.14 vs 23.33  6.66, respectively (one-way ANOVA, F ratio = 0.18, df = 83, P = 0.67). Modes of failure The distributions of the modes of failure within groups are shown in Table 4. Overall, adhesive failures were the most common (54.3% of all failures), followed by mixed failures (40.0% of all failures). From 105 specimens, only five specimens (4.8%) had cohesive failures-inenamel, all of which involved hypomineralised enamel. As the total number of cohesive failures-in-enamel and the total number of cohe-

Table 3. Mean microshear bond strength of resin composite bonded to white and yellow-brown hypomineralised enamel within groups.

Group 2 3 4 5

Hypomineralised enamel lesion colour

No. of specimens

Mean MSBS (MPa)  SD

White Yellow-brown White Yellow-brown White Yellow-brown White Yellow-brown

11 10 11 10 11 10 10 11

23.68 20.25 21.40 17.12 26.63 24.91 26.27 23.11

       

P-value (within groups)

4.57 5.34 8.11 8.61 9.43 8.61 9.37 6.17

0.129* 0.255* 0.669* 0.369*

MSBS, microshear bond strength; SD, standard deviation. *No significant difference within each group for mean MSBS of white and yellow-brown lesions (one-way ANOVA). No significant difference (P = 0.078) for mean MSBS values of white and yellow-brown lesions between groups (one-way

ANOVA).

Table 4. Mode of failure of specimens. Control groups Group 1

Test groups Group 2

Group 3

Group 4

Group 5

Failure type

N = 21

(%)

N = 21

(%)

N = 21

(%)

N = 21

(%)

N = 21

(%)

Adhesive Mixed Cohesive-in-enamel Cohesive-in-composite

12 9 0 0

(57.1) (42.9) (0) (0)

7 11 2 1

(33.3) (52.4) (9.5) (4.8)

14 7 0 0

(66.7) (33.3) (0) (0)

9 9 3 0

(42.9) (42.9) (14.3) (0)

15 6 0 0

(71.4) (28.6) (0) (0)

No significant association (P = 0.252) for modes of failure between groups (Pearson’s chi-square test). © 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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sive failures-in-composite were both less than five, these two modes of failure were excluded from the Pearson’s chi-square test in the analysis between the groups. Subsequently, no association was seen for the modes of failure between groups (v2 = 5.36, df = 4, P = 0.25). SEM findings In two specimens from each of the control groups (Groups 1 and 2), a surface morphology resembling an etching pattern was observed consistently in the sheared enamel interfaces of normal enamel or hypomineralised enamel that exhibited adhesive failure and where the adhesive was adhered to the sheared composite rod rather than the sheared enamel surface so that the etched surface could be visualised. Specimens from Groups 1 and 2 showed a sheared enamel surface with surface morphology similar to etched enamel, with increased intrarod space (Figs 2 and 3), and the corresponding surfaces on the associated composite rods from the same specimen demonstrated projections of adhesive or composite, which correlated to a corresponding imprint of the ‘positive composite surface’ and ‘negative enamel surface’ (Figs 4 and 5). It was not possible to make an objective comparison of the surface patterns observed between the control normal enamel and control hypomineralised enamel due to differences in the orientation of the enamel rods between every specimen. In two representative specimens from Group 3 (hypomineralised enamel specimens pre-treated with resin infiltration prior to adhesive bonding), differences in sheared surface morphology were seen between specimens when areas of adhesive failure were examined. One specimen from Group 3 had relatively smooth surface noted on the sheared hypomineralised enamel surface, with no evidence of any etched and exposed enamel rods, and likely the enamel rods were covered by a resinous material (Fig. 6). In the corresponding surface morphology on the associated composite rod, scarce, small ‘positive’ imprints of adhesive or composite were seen (Fig. 7). The other specimen from Group 3 had a sheared enamel surface which

Fig. 2. Scanning electron microscope image of a sheared normal enamel surface from a specimen in Group 1, illustrating intrarod spaces (yellow arrow).

Fig. 3. Scanning electron microscope image of a sheared hypomineralised enamel surface from a specimen in Group 2, showing intrarod enamel space (yellow arrow).

did not show an etching pattern, but was rough, with tiny projections protruding out of the surface (Fig. 8). A corresponding rough surface is also noted on the associated composite rod, with some evidence of projections protruding out of the composite surface (Fig. 9). Similar observations were noted in the adhesive interfaces in two representative samples from Group 4 (hypomineralised enamel specimens subject to oxidative pre-treatment,

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Fig. 4. Scanning electron microscope image of the corresponding surface on the opposing composite rod, belonging to the specimen in Fig. 2, demonstrating projections of adhesive or composite (black arrow), which correlates to a corresponding imprint of the ‘positive composite surface’ and ‘negative enamel surface’.

Fig. 6. Scanning electron microscope image of a sheared hypomineralised enamel surface from Group 3, which shows a relatively smooth sheared hypomineralised enamel.

*

Fig. 5. Scanning electron microscope image of the corresponding surface on the opposing composite rod, belonging to the specimen in Fig. 4, demonstrating projections of adhesive or composite (black arrow), similarly correlating to the surface in Fig. 4.

followed by resin infiltration prior to adhesive bonding), when compared to Group 3. One sample had a fairly smooth sheared enamel surface with no obvious etched enamel pattern. In the other specimen from Group 4, the sheared enamel surface was rough and

*

Fig. 7. Scanning electron microscope image of the corresponding sheared surface on the opposing composite rod belonging to the specimen in Fig. 6. Scarce, small ‘positive’ imprints of adhesive or composite are seen (white asterisk). Note that this image is at a higher magnification of 80009.

showed tiny projections protruding out of the surface and without an etching pattern. In the two representative specimens from Group 5 (hypomineralised enamel specimens subject to oxidative pre-treatment prior to adhesive bonding) where areas of adhesive failure were examined under SEM, the surface morphology of the sheared enamel and

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Bonding to hypomineralised enamel

Fig. 8. Scanning electron microscope image shows another specimen from Group 3, where the sheared enamel surface is rough and has tiny projections protruding out of the surface (yellow arrow).

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Fig. 10. Scanning electron microscope image showing a sheared hypomineralised enamel surface from Group 5, with interrod spacing (yellow arrow).

Fig. 9. Scanning electron microscope image of the corresponding sheared surface on the opposing composite rod belonging to the specimen in Fig. 8. Projections, likely to be resin infiltration penetration tags, protruding from the composite surface (yellow arrow).

Fig. 11. Scanning electron microscope image of a hypomineralised enamel specimen with cohesive failure-inenamel, where the enamel rods seem to have been ‘pulled off’ and fractured along rod boundaries.

the associated composite rod was similar to that observed for the specimens in Group 2 (control group with hypomineralised enamel) (Fig. 10). Cohesive failures-in-enamel were few and occurred in five specimens belonging to Groups 2 and 4. When cohesive failure surfaces were viewed under SEM, the picture was particularly characteristic. On the

sheared enamel surface, the enamel rods seemed to have been ‘pulled off’, with failure occurring in the interrod areas (Fig. 11). The enamel that had broken off the enamel specimen and consequently adhered on the associated composite rod had exposed enamel rods and visible enamel rod cores. The orientation of the enamel rods was non-uniform (Fig. 12).

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cally relevant, as one of the clinical issues at hand was whether the resin composite should be bonded to the visibly affected but clinically hard enamel or to the normal looking enamel on hypomineralised molars. Microshear bond strengths

Fig. 12. Scanning electron microscope image of enamel that has broken off from the specimen in Fig. 11. The enamel rods are exposed and the cores of the rods are exposed.

Discussion

It has been reported that the bond strength of resin composite to hypomineralised enamel is reduced when compared to that of control enamel, irrespective of the adhesive agent used.5 Although there is a general consensus that the most suitable material at present for restoration of hypomineralised molars is resin composite, whether partial or total removal of the defective enamel prior to restoration would be best for the patient is still unclear.2,4 Hence, any means to increase the bond strength of composite resin to hypomineralised enamel or to afford the weakened enamel more mechanical strength may be beneficial and will also allow for preservation of more tooth structure. The control enamel specimens from this study, hereafter termed ‘normal enamel’, were obtained from ‘normal looking enamel of hypomineralised teeth’. A recent study has found that the hardness, mineral content, and porosity of control enamel are consistent with normal enamel from patients not affected by MIH.8 Moreover, the structure of control enamel has been reported to have a similar appearance to normal enamel, with welldefined enamel rods, narrow interrod zones, tightly packed crystals and uniform orientation.16 Therefore, the use of the control enamel in this study was considered to be clini-

This study demonstrated significantly higher mean MSBS of resin composite, bonded with Clearfil™ SE Bond, to normal enamel compared to hypomineralised enamel. A previous study by William et al. also reported significantly higher mean MSBS of resin composite bonded to normal enamel using Single Bond™ or Clearfil™ SE Bond as compared to bonding to hypomineralised enamel with the same.5 Lower MSBS values in hypomineralised enamel specimens could be attributed to poor microtag formation within the prism rods, aberrant etching patterns, intercrystalline porosity, moisture retention within the larger interrod spaces and higher protein content in hypomineralised enamel.5,7 It is difficult to compare values obtained from this study and the study by William et al.5 due to the differences in storage media, specimen preparation, dental materials used, shearing method, and operator variability. Less distinct differences in MSBS values between normal and hypomineralised enamel observed in this study compared to William et al.’s study could be due to the variation in severity of the hypomineralisation in the specimens used. In this study Clearfil™ SE Bond was selected as the bonding agent without the use of Clearfil™ SE Primer as the use of a hydrophilic component in a priming agent before infiltration was postulated to interfere with the subsequent infiltration of the hydrophobic low viscosity resin infiltrant. Different forms of pre-treatment of hypomineralised enamel lesions prior to bonding had varying effects on MSBS values. The use of resin infiltration alone prior to bonding decreased MSBS values and also resulted in significantly lower MSBS compared to normal enamel. On the other hand, the use of oxidative pre-treatment (NaOCl) alone and followed by resin infiltration resulted in higher MSBS compared to the control hyp-

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Bonding to hypomineralised enamel

omineralised enamel group, with no significant differences in MSBS when compared to normal enamel. This implies that these two forms of pre-treatment improved the bond to hypomineralised enamel. To the authors’ knowledge, there are no published comparable laboratory studies investigating the adhesion of resin composite to hypomineralised MIH enamel lesions pretreated with resin infiltration, oxidative pre-treatment, or a combination of both. Resin infiltrant was designed for and has been shown to penetrate deep into the enamel of non-cavitated natural carious lesions, and slow carious lesion progression in cariogenic conditions by the sealing of porous channels and blocking the diffusion pathways for acid penetration and ionic movement.17 Similar to carious lesions, hypomineralised enamel has increased porosities and may be amenable to resin infiltration, which could help increase the surface area for micromechanical retention of resin tags and possibly improve bonding, as the hypomineralised enamel has poor etching properties and decreased bond strengths.5,6 This form of pre-treatment appeared promising, as a laboratory shear bond strength study found better bonding to demineralised enamel with the use of resin infiltration pre-treatment (including hydrochloric acid etching rather than conventional phosphoric acid etching).10 In this study, however, the resin infiltration pre-treatment group had the lowest MSBS values, performing worse than ‘routine’ bonding to hypomineralised enamel. Possibly the increased protein content in hypomineralised enamel acted as a physical barrier or chemical barrier (potentially having hydrophilic constituents) that prevented adequate penetration of resin infiltrant, or Clearfil™ SE Bond was not bonding to the resin infiltrant predictably. In this study, the resin infiltration procedure was modified by the substitution of 15% hydrochloric acid etching with 35% phosphoric acid etching as specimen preparation (i.e., lapping) had removed the surface layer (the intent of the hydrochloric acid etchant), and the hydrochloric acid was deemed too destructive to the body of the hypomineralised lesion. Developmentally hypomineralised enamel in MIH being a hypocalcification defect may

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have similarities to the enamel in hypocalcified amelogenesis imperfecta (AI).7,18 Enhanced macroshear bond strength of resin composite to enamel of hypocalcified AI affected primary teeth has been reported when 5% NaOCl application was applied for 1 min after acid etching and prior to adhesive application (post-etching); mean shear bond strengths improved from 13.9 to 27.4 MPa and approached bond strengths to normal primary enamel.14 Contrary findings were noted by Faria-e-Silva et al., who reported no significant effect on MSBS of resin composite to hypocalcified AI affected permanent enamel, when enamel specimens were soaked in 5% NaOCl solution prior to etching and bonding (pre-etching). Faria-e-Silva et al. attributed the differing findings from Saroglu et al.’s study to the different test specimens used (permanent versus primary enamel) which may affect bond strengths.19 The timing of the use of 5% NaOCl, whether it was applied pre- or post-etching, also differed between the studies, however, and could be critical in influencing bond strength values. An in vivo split mouth study compared the clinical success of resin composite restorations in hypocalcified AI teeth when the use or non-use of 5% NaOCl was carried out post-etching, similar to the application protocol in Saroglu et al.’s study. No difference in clinical success was noted between the two groups at 36 months, although cervical discolouration was found to be significantly lower in the group with 5% NaOCl use post-etching, possibly indicating less leakage. The study, however, involved only four children, had no mention of intraexaminer or interexaminer reliability, and it was unclear whether restorations were bonded to enamel or dentine, so results should be interpreted with caution.20 It would appear that the use of NaOCl postetching results in increased MSBS. It is postulated that etching helps to increase access into the enamel to facilitate protein breakdown by NaOCl, putatively allowing better infiltration of the bonding agent and improved bonding to hypomineralised enamel. A recent study on hypomineralised enamel found a high predictability of obtaining ‘poor’ fissure sealant resin sealant tags and no difference in tag

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quality when 5% NaOCl was added to preetching and sealing.21 The use of NaOCl postetching rather than pre-etching and the additional use of a bonding agent prior to sealing could possibly have improved tag quality in this study – further research is warranted.21,22 In this study, it was found that the greatest bond strengths to hypomineralised enamel occurred when the enamel was pre-treated with NaOCl followed by resin infiltration. Interestingly, although there were more adhesive interfaces in this group (between enamel and infiltrant, infiltrant and bond, bond and composite resin), which may all be susceptible to adhesive failure, bond strength was not adversely affected. Although MSBS values did not reach those of normal enamel, the values were not significantly different. Significant differences in MSBS existed when specimens were grouped based on the use or non-use of oxidative pre-treatment, but not when specimens were grouped based on the use or nonuse of resin infiltration, indicating that the use of NaOCl post-etching is the more critical factor contributing to the improved bond strength. Also interesting is the greater variability of the MSBS values, with slightly larger standard deviations when the use of resin infiltrant was involved, possibly indicating inconsistent penetration of the resin infiltrant amongst specimens. In this study, specimens were surfacelapped so that the bonding surface involved the ends of the enamel prism rods rather than their length. In a cavity preparation, resin composite will bond to both the ends as well as the length of the prism rods in different areas of the cavity, so the results of this study should be seen in this context. It is acknowledged that the distinction of lesion colour in this study was not objective, and reliability and consistency were not tested. Although not statistically significant, however, the consistent finding in all groups was that yellow-brown lesions had lower MSBS values of similar magnitudes compared to white lesions. This finding is similar to previous studies which have found lower hardness in darker lesions and also associated with increased porosity and lower mineral density.23,24 The colour of

the hypomineralised lesion seems to be a good clinical indicator of its mechanical properties and consequent clinical behaviour. Apart from lesion colour, lesion location, particularly cuspal regions, is at greater risk of post-eruptive breakdown.25 Modes of failure The majority of the failures were adhesive and mixed in nature amongst all of the five groups. The distribution of failures was fairly consistent with previous studies investigating the adhesive bond strength to ground normal enamel.26,27 In contrast to a previous study which reported that half of the hypomineralised enamel specimens exhibited cohesive failures-in-enamel,5 only five of 84 hypomineralised enamel specimens in this study exhibited cohesive failure-in-enamel. We attribute this difference to the possible variability in the severity of the hypomineralised enamel specimens used in both studies (it is possible that the previous study involved a higher proportion of yellow-brown lesions5), the possible difference in lapping depths into the body of the lesion, and the slight difference in the MSBS test method used. Four of the five cohesive failurein-enamel specimens in this study involved yellow-brown lesions, confirming previous research.23,24 Given the low number of specimens with cohesive failure-in-enamel in this study, it was not possible to analyse statistically whether the mode of failure was associated with lesion colour. It is not apparent from this study whether pre-treatment of the enamel may afford any increase in cohesive strength of the enamel which could also contribute to fewer cohesive failures-in-enamel, as the specimens which had cohesive failure-in-enamel were evenly distributed between the control (two specimens) and pre-treatment groups (three specimens). Due to the small number of cohesive failures-in-enamel, it is difficult to determine whether cohesive failures-in-enamel occurred in Group 4 but not in Groups 3 and 5 by chance or not. Increasing amounts of TEGMA in BisGMA/TEGMA composites have been shown to increase polymerisation shrinkage and polymerisation stress.28 If oxidative

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pre-treatment in Group 4 was indeed effective in removing the proteins and resulted in improved penetration of the resin infiltrant, polymerisation shrinkage of the increased amount of infiltrant could have resulted in stresses in the already mechanically weak hypomineralised enamel, precipitating cohesive failures-in-enamel when the shear force was applied. SEM findings Smooth fractured enamel surfaces without evidence of an etching pattern only occurred in Groups 3 and 4 which involved resin infiltration in the pre-treatment protocol. This indicated that resin infiltrant was able to penetrate into the enamel porosities; the depth and consistency of infiltration, however, were not part of this study. This question deserves further research. SEM images from specimens in Groups 3 and 4 illustrated that the weakest link in the adhesion of the hypomineralised enamel to resin composite in these groups seemed to involve the resin infiltrant layer. The relatively smooth sheared enamel surfaces in Figs 6 and 7 suggested that failure could have been cohesive within the infiltrant layer which could have been present over the enamel surface; adhesive between the infiltrant layer and the bond; or adhesive between the bond and the composite. The SEM images of projections protruding out of the enamel surface in Figs 8 and 9 resemble infiltrant penetration tags. The resin infiltrant penetration tags may have previously penetrated into the porous and etched hypomineralised enamel; when the specimen was subjected to a shear force, however, the resin infiltration penetration tags were pulled out. In this case, adhesive failure occurred between the enamel and the infiltrant. Again this indicates that at least superficial penetration of the infiltrant into the porous enamel is possible. As the depth of the resin penetration is not known, however, the protruding projections suggest that there has been cohesive failure within the resin infiltrant tags, or resin infiltration has only occurred superficially and cannot confer enough micromechanical retention to resist adhesive failure.

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Scanning electron microscope images of specimens from Group 5 which had oxidative pre-treatment with 5.25% NaOCl were similar to the images observed in Group 2. We postulated that protein degradation by NaOCl would allow better infiltration of the bonding agent and improved bonding. Unfortunately, the SEM images did not reveal why the improved bonding in Group 5 was observed. Microscopic examination of the adhesive interface in crosssection would be valuable in future studies to compare resin tag penetration with and without oxidative pre-treatment. The sheared enamel rods observed in SEM images of cohesive failure-in-enamel affirmed findings of previous studies on hypomineralised enamel which reported less dense prismatic structure, wider sheath regions, and non-uniform orientation of enamel rods.6,29 Clinical considerations This study has demonstrated that partial rather than complete removal of the hypomineralised area of enamel may be considered, as improved bond strengths to resin composite can be obtained when the hypomineralised lesion is pre-treated with NaOCl, with or without subsequent resin infiltration, prior to routine bonding. Other aspects such as the effect of either pre-treatments (i.e., NaOCl pre-treatment alone, or NaOCl followed by resin infiltration pre-treatment) on hypomineralised enamel appearance, hardness, fracture resistance, and solubility are worthy of further investigation. These would help determine whether pre-treatment of hypomineralised enamel prior to bonding also has an effect on the mechanical properties of the lesion, apart from its influence on adhesive strength, as these also influence the longevity of resin composite restorations in hypomineralised teeth, especially because the weaker hypomineralised enamel tends to break down around the margins of the restoration. Further research is needed to justify the use of resin infiltration as a step in pre-treatment for hypomineralised enamel prior to bonding. Although hypomineralised enamel also has increased porosities with similarity to non-cavi-

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tated white spot lesions, currently resin infiltration into hypomineralised enamel is inconsistent and needs to be investigated further.30 At present, the application of NaOCl for 1 min post-etching prior to bonding would seem to be a simple and effective step to improve the adhesion of resin composite to hypomineralised enamel. It would be useful to investigate further the effect on bond strength with decreased concentrations of NaOCl or altered application time. Whether 15% hydrochloric acid etching or 35% phosphoric acid etching should be used prior to NaOCl warrants further investigation. In resin composite restorations where the surface enamel is bevelled prior to restoration, thereby likely removing the hypermineralised surface layer, however, it is postulated that aggressive etching with 15% hydrochloric acid may not be essential. Conclusions

In this study, increased mean MSBS of resin composite to hypomineralised enamel was obtained by pre-treatment of hypomineralised enamel with 5.25% NaOCl with or without subsequent infiltration. Pre-treatment with resin infiltration alone decreased mean MSBS of resin composite to hypomineralised enamel. The highest mean MSBS of hypomineralised enamel to resin composite was obtained when the hypomineralised enamel was pre-treated with 5.25% NaOCl followed by resin infiltration. The predominant modes of bond failures were adhesive and mixed failures. All specimens not subject to resin infiltration pre-treatment had a sheared surface morphology resembling an etching pattern in areas of adhesive failure.

Why this manuscript is important to paediatric dentists ● Hypomineralised molars are often difficult to restore adequately as the enamel is less mineralised, porous and poorly delineated from normal tissue. ● The microshear bond strength (MSBS) of resin composite bonded to hypomineralised enamel was significantly lower than that of control enamel. ● The application of 5.25% NaOCl for 1 min post-etching prior to bonding would seem to be a simple and effective step to improve the adhesion of resin composite to hypomineralised enamel. ● The use of resin infiltration as a pre-treatment may be beneficial.

Acknowledgements

The authors would like to acknowledge the Melbourne Dental School Research Committee and Dentsply Research Fund for providing financial assistance and SDI™ for providing the rig and metal moulds. Conflict of interest

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© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd