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Effects of Alpha-tocopherol Antioxidant on Dentin-composite Microtensile Bond Strength after Sodium Perborate Bleaching
CASE REPORT/CLINICAL TECHNIQUES M. Stephen Harrison, Jr., DDS, MS, Yong Wang, PhD, Kenneth J. Frick, DDS, MS, Jessica Moniz, BA, and Mary P. Walker, DDS, PhD
Effects of Alpha-tocopherol Antioxidant on Dentincomposite Microtensile Bond Strength after Sodium Perborate Bleaching ABSTRACT It has been reported that bond strength can be reversed to prebleached levels with the application of 10% alpha-tocopherol in a 2-hour time frame or by delaying bonding for 2 weeks. This study evaluated the effectiveness of a 5-minute application of 20% alphatocopherol to reverse the deleterious effects of nonvital bleaching on consequent bond strength. Thirty third molars were assigned to the following 3 groups: unbleached, bleached, and bleached followed by treatment with alpha-tocopherol. The bleached groups were exposed to sodium perborate (2 g/mL) for 7 days. The postbleach treatment group was subsequently treated with 20% alpha-tocopherol for 5 minutes, and then all groups were restored with composite resin. After 24 hours of storage at 37 C and 100% humidity, restored tooth specimens were sectioned into 1-mm2 dentin-composite beams. Six beams from each tooth were subjected to microtensile bond strength testing. Representative beams were further evaluated with Raman microspectroscopy and scanning electron microscopy. The mean bond strength values (MPa) for each group were as follows: unbleached control group 5 26.2, bleached control group 5 20.3, and post–bleach treatment group 5 18.5. A 1-factor analysis of variance and Tukey post hoc test (a 5 0.05) indicated that bleaching had a detrimental effect on bond strength and that short-term alpha-tocopherol treatments did not improve postbleach bond strength. Raman microspectroscopy and scanning electron microscopy revealed no noted improvement for the post–bleach treatment group.The application of 20% alpha-tocopherol in a clinically relevant time frame was not effective in counteracting the deleterious effect of bleaching on bond strength. Bonding procedures should be delayed after tooth bleaching. (J Endod 2019;-:1–7.)
SIGNIFICANCE The application of alphatocopherol in a clinically relevant time frame was not effective at reversing the deleterious effects of nonvital bleaching on dentincomposite bond strength. Based on the results, there is no evidence to change the recommended delay between bleaching and bonding procedures.
KEY WORDS Degree of conversion; microtensile bond strength; nonvital bleaching; sodium perborate treatment
Internal bleaching of nonvital teeth is a conservative and effective method to manage darkened root canal–treated teeth. However, after bleaching, the bond strength between dentin and composite resin is adversely affected1–4. It has been proposed that residual oxygen from the bleaching process remains in the tooth structure for up to 2 weeks and is believed to interfere with polymerization of the dentin adhesives5. Despite the immediate negative effect of bleaching on bond strength, multiple studies reported that after waiting at least 7 days, dentin bond strength values return to baseline6–8. Although bleaching effects on dentin are not long-term, antioxidants, such as sodium ascorbate, have been used to counteract the adverse effects of bleaching to allow immediate placement of a restoration9–11. However, when antioxidants such as sodium ascorbate and ascorbic acid are used for clinically relevant times rather than extended periods, the reversal of dentin bond strength reductions has not been successful12,13. Thus, a more effective antioxidant is needed to counteract bleaching adverse effects in a shorter clinically relevant time.
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From the University of Missouri-Kansas City, School of Dentistry, Kansas City, Missouri Address requests for reprints to Dr Mary P. Walker, 650 East 25th Street, Kansas City, MO 64108. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright © 2019 Published by Elsevier Inc. on behalf of American Association of Endodontists. https://doi.org/10.1016/ j.joen.2019.04.013
Effects of Alpha-tocopherol Antioxidant after Bleaching
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FIGURE 1 – (A) Teeth suspended in sodium perborate solution. (B ) A tooth serially sectioned into dentin-composite beams with a 1-mm2 cross-sectional area. (C ) A dentin-composite beam attached to a microtensile tester.
review board–reviewed protocol designated as not human subject research. The extracted teeth were stored at 0.4 C in 0.9% phosphatebuffered saline with 0.002% sodium azide to inhibit microbial growth. All procedures were performed under ambient temperature and humidity because clinical procedures are done with a rubber dam in place16. The teeth were mounted into acrylic resin, leaving the coronal third of the root structure and tooth crown exposed. The occlusal half of the crown was removed with a water-cooled diamond blade saw (Isomet 1000, blade 11-4246; Buehler, Lake Bluff, IL), leaving a flat, dentinal surface devoid of enamel. The dentin smear layer was then removed with a 3-minute application of 17% EDTA followed by a 15-second air/water spray to remove any residual EDTA. The mounted, prepared teeth were randomly assigned into 3 groups of 10 teeth each as follows:
MATERIALS AND METHODS
Prepared teeth assigned to group 1 were stored without bleach exposure, whereas teeth assigned to groups 2 and 3 were inverted and suspended on a plastic grate into a 5-mm layer of sodium perborate solution (Sultan Healthcare, York, PA) prepared with distilled water (2 g/mL), allowing for only the prepared surface to be in contact with the bleaching agent (Fig. 1A). All specimens were stored at 37 C and 100% humidity for 7 days.
dry the specimens. Group 1 (unbleached control) and group 2 (bleached control) received no antioxidant application. Group 3 (postbleach alpha-tocopherol) received a 5minute application of 20% alpha-tocopherol (Midwest Compounders Pharmacy, Lenexa, KS) that was reapplied with a microbrush every minute to the dentinal surface. In all groups, the dentinal surface was washed with a 15second air/water syringe spray and air dried. An approximately 4-mm layer of composite was added to all tooth specimens using the following protocol. After a 10-second total etch with 35% phosphoric acid gel (Ultraetch; Ultradent Products Inc, South Jordan, UT), adhesive (Prime & Bond NT; Dentsply Caulk, Milford, DE) was applied for 20 seconds, air thinned, and light cured (XL3000; 3M, Maplewood, MN) for 20 seconds. Restorative composite (TPH, Dentsply Caulk) was applied in increments of less than 2 mm, light curing for 40 seconds between layers. The average curing light output was 1210 mW/ cm2 as measured by a radiometer (3H, Nanjing, China). The restored teeth were stored for 24 hours in 100% humidity at 37 C and then serially sectioned into 1-mm-thick vertical dentin-composite slabs with the same watercooled diamond blade as used previously (Fig. 1B). The specimens were rotated 90 and sectioned again to obtain rectangular dentincomposite beams with a cross-sectional area of approximately 1 mm2. The surface area of the bonded interface was calculated for each specimen by measuring the narrowest portion with a digital caliper. Six to 10 beams were generated from each tooth.
Thirty previously extracted human third molar teeth were collected with no associated patient identifiers in accordance with an institutional
After 7 days of storage, prepared teeth were sprayed with the air/water syringe for 15 seconds to remove any residual bleach and/or
Six beams from each tooth were used for microtensile testing (Bisco, Schaumburg, IL). Beams were adhered to the tester fixture
Regarding potential antioxidant agents, a previous study evaluated the antioxidative capabilities of 15 compounds including ascorbic acid, sodium ascorbate, and alphatocopherol via the 2,2-diphenyl-1-picrylhydrazyl-hydrate free radical method. The antioxidant activity results were presented as a percentage decrease in the number of free radicals compared with control values, with ascorbic acid and alpha-tocopherol being the best at 95% and 92%, respectively. The next was sodium ascorbate gel at 76% and sodium bicarbonate at 74%. All other substances tested fell at 51% and lower for antioxidant activity14. Ten percent alpha-tocopherol was previously used as an alternate antioxidant and showed the potential to counteract the negative effects of bleaching on bond strength after a 2-hour application time15. However, to date, there has been no research using alphatocopherol to test its effect using a shorter exposure that would occur in a clinical setting. The purpose of this study was to evaluate the short-term effect of the antioxidant alpha-tocopherol on dentin adhesive microtensile bond strength immediately after a simulated nonvital bleaching procedure with sodium perborate. The research hypothesis was that alphatocopherol would counteract the effects of bleaching on dentin bond strength. In addition to microtensile bond strength, the quality of the dentin-adhesive interface was evaluated via Raman microspectroscopy and scanning electron microscopy.
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1. Group 1: no bleach (unbleached control) 2. Group 2: bleached for 7 days (bleached control) 3. Group 3: bleached for 7 days followed by alpha-tocopherol antioxidant treatment (postbeach alpha-tocopherol)
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FIGURE 2 – Representative Raman microspectroscopy line maps of the (A ) unbleached control, (B ) bleached control, and (C ) postbleach alpha-tocopherol groups. The peaks associated with the adhesive occur at 1720/cm (carbonyl), 1609/cm (phenyl C5C), 1453/cm (CH2 def), and 1187/cm (gem-dimethyl), whereas the peaks associated with dentin occur at 1667/cm (amide I), 1454/cm (CH2 def), 1245/cm (amide III), and 961/cm (PO432). JOE Volume -, Number -, - 2019
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FIGURE 3 – Representative spectra of the adhesive (0 mm) and dentin-adhesive interface (2 mm into the hybrid layer) in the range of 1580–1670/cm from the unbleached control, bleached control, and postbleach alpha-tocopherol groups. The peak at 1637/cm is associated with unpolymerized (or unconverted) C5C in the adhesive; the peak at 1609/cm is associated with phenyl C5C in the adhesive, which is used as an internal standard. The relative intensity of the 1637/cm peak in the unbleached control group is lower than in the bleached control and postbleach alpha-tocopherol groups. The higher the 1637/cm peak, the lower the DC.
with cyanoacrylate applied to the composite and dentin ends of the beams, ensuring the dentin adhesive interface is centered in the fixture (Fig. 1C). Adhered beam specimens were subjected to tensile forces at a crosshead speed of 1 mm/min. Microtensile bond strength (MPa) was calculated based on the force at debond (N) divided by the beam interface cross-sectional area (mm2). The bond strength values for all beams6 from a single tooth were averaged, providing a single bond strength measurement for that tooth. Ten tooth specimens were tested from each group. The dentin-adhesive interface of 2 additional beams from each tooth not used for microtensile testing tooth was evaluated with Raman microspectroscopy (LabRam HR 800; Horiba Scientific, Edison, NJ) using a He-Ne laser at a wavelength of 632.8 nm and an excitation power of 20 mW. The laser was focused through a 50! Olympus (Tokyo, Japan) objective. Data were collected in the
Raman spectral range between 800 and 1800/ cm with data acquired using a 30-second exposure time. Spectral line maps of the
DC 5
! Intensity Polymerized Intensity Polymerized 1637 cm21 1609 cm21 !100 12 Intensity Unpolymerized Intensity Unpolymerized 1637 cm21 1609 cm21
dentin-adhesive interface were collected at 2mm intervals starting at the composite interface edge at the midpoint of the beam interface on 3 sides of the beam with a total of 3 line maps per beam. Line maps were used to determine adhesive penetration into dentin; example line maps from the unbleached control, bleached control, and postbleach alphatocopherol groups are presented in Figure 2. Within line maps, the adhesive degree of conversion (DC) was also calculated using peak area ratios of 1637/cm (1625–1650/cm) and 1609/cm (1590–1625/cm) from the
TABLE 1 - Adhesive Resin Degree of Conversion (%) Means and Standard Deviations Group* Unbleached control Bleached control Postbleach alpha-tocopherol
Within adhesive only
2 mm into hybrid layer
82 (2.5) 64 (14.1) 67 (9.1)
79 (6.4) 53 (12.4) 61 (10.5)
*Because of testing of a representative sample only, no statistical evaluation was performed. However, based on numeric value comparisons, there is a trend for higher degree of conversion percentages in the unbleached control group compared with the bleached and postbleach alpha-tocopherol groups.
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polymerized adhesive compared with the unpolymerized adhesive reference. The following DC equation was used17:
The debonded interface surface from the dentin side of 2 representative beam specimens from each group was evaluated using a scanning electron microscope (XL30 ESEM-FEG; FEI-Philips, Amsterdam, Netherlands). The dentin interface surfaces were each sputter coated with approximately 20-nm-thick gold palladium and examined at varying magnifications at 15 kV for both secondary and backscattered electron emission (BSE) analysis. One-factor analysis of variance of microtensile strength measures was performed (SPSS v22; IBM Corp, Armonk, NY) with significance set at .05 and a Tukey post hoc test used if differences were detected. Data acquired from Raman microspectroscopy and scanning electron microscopic imaging were used to provide qualitative information regarding the dentinadhesive interface. Because of the use of a representative sample, no statistical analyses were provided for these evaluations.
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FIGURE 4 – Scanning electron microscopic imaging. (A–C ) Secondary electron emission images. (D–F ) BSE images. Magnifications across image rows were 10,000! and 20,000!. (B and E ) The presence of colloidal spheres (arrows) was noted on both the bleached control and (C and F ) the postbleach alpha-tocopherol experimental group. (A and D ) However, no spheres were apparent on the unbleached control specimen. In the 20,000! BSE images of a (E ) bleached and (F ) postbleach alpha-tocopherol specimen; the spheres demonstrate a darker color, suggesting they are composed of adhesive resin.
RESULTS The mean microtensile bond strength (MPa) was significantly higher (P .05) in the unbleached control group (26.2 6 11.2) compared with the bleached control (20.2 6 10.0) or the postbleach alpha-tocopherol group (18.5 6 9.9), which were not significantly different (P . .05) from each other (n 5 6 beams/tooth,10 teeth/group). Figure 2A–C shows representative line maps from the unbleached control, bleached control, and postbleach alpha-tocopherol groups. Line maps were used to determine the depth of adhesive penetration into dentin. The peaks associated with the adhesive occur at 1720/cm (carbonyl), 1609/cm (phenyl C5C), 1453/cm (CH2 def), and 1187/cm (gem-dimethyl), whereas the peaks associated with dentin occur at 1667/cm (amide I), 1454/cm (CH2 def), 1245/cm (amide III), and 961/cm (PO432). Peaks associated with the adhesive and dentin components are noted in the spectra collected from the interface. The mean depth of adhesive
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penetration (mm) into dentin was higher in the unbleached control group (6.86 6 3.2) compared with 4.00 6 0.00 and 4.33 6 1.90 in the bleached and postbleach alphatocopherol groups, respectively. Data for DC were reported at the point on the line map of pure adhesive closest to the hybrid layer and at 2 mm into the hybrid layer from the pure adhesive point. Figure 3 shows representative spectra of the adhesive (0 mm) and dentin-adhesive interface (2 mm) in the range of 1580–1670/cm from the unbleached control, bleached control, and postbleach alpha-tocopherol groups. Representative beams were typically selected based on their microtensile test value being near the group mean value. The peak at 1637/cm is associated with unpolymerized (or unconverted) C5C in the adhesive; the peak at 1609/cm is associated with phenyl C5C in the adhesive, which is usually used as an internal standard. As shown in Figure 3, the relative intensity of the 1637/cm peak in the unbleached control group is lower than those in the other 2 groups (the bleached control and
postbleach alpha-tocopherol group); the higher the 1637/cm peak, the lower the DC. The DC means and standard deviation values are presented in Table 1. Representative photomicrographs obtained via a scanning electron microscope using secondary electron and BSE imaging are presented in Figure 4. The most notable differences between groups were the colloidal spheres, approximately 200–500 nm in diameter, that were seen on the bleached control and postbleach alpha-tocopherol specimens (Fig. 4B, C, E, and F) but were not present on the unbleached control specimen (Fig. 4A and D). In addition, in the BSE images, it was noted that the spheres in the bleached control and bleach/alpha-tocopherol groups were a darker color, suggesting that the spheres were composed of adhesive resin.
DISCUSSION This study sought to evaluate the effect of an antioxidant (ie, alpha-tocopherol) to allow for
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composite bonding at the bleaching agent removal appointment. Because of the presence of residual oxygen, the current recommendation is to remove the bleach, seal the tooth temporarily for 7 or more days, and have the patient return for a definitive access repair5,18. If the tooth is definitively restored with composite resin immediately after the removal of bleach, the strength of the microtensile bond and the quality of the hybrid layer at the restorative margin are diminished1–4,19. The week-long window during temporization allows for dissipation of the oxygen from dentinal tubules and the return of the subsequent microtensile bond strength and hybrid layer quality to prebleached levels7. In the current study, unbleached and bleached controls performed as expected, with the bleached control exhibiting significantly lower bond strengths. However, using postbleach treatment with 20% alphatocopherol for a clinically relevant time of 5 minutes, alpha-tocopherol failed to provide an improvement of microtensile bond strengths. The postbleach alpha-tocopherol group exhibited mean bond strengths statistically similar to the bleached control (18.5 and 20.2 MPa, respectively) and were significantly lower than the unbleached control (26.2 MPa); thus, the research hypothesis was rejected. The results of the current study are similar to a previous study using 35% sodium ascorbate for a clinically relevant time (10 minutes) in bleached human teeth with no improvement in bond strength12. One of the factors that may contribute to the decreased bond strength after bleach with or without alpha-tocopherol may be related to the lower penetration of adhesive resin in both the bleached control and the postbleach alpha-tocopherol groups compared with the unbleached control group. The depth of penetration of the unbleached group revealed a mean of 6.86mm. This is in line with a previously reported hybrid layer thicknesses of 6–10 mm for etch-and-rinse adhesive systems20–22. However, the bleached control and the alpha-tocopherol groups exhibited a mean depth of penetration of 4 mm and 4.33 mm, respectively. The mean depths for the bleached groups of less than two thirds that of the unbleached control suggest this is 1 mechanism leading to lower microtensile bond
strengths in the bleached groups. Little research could be located describing or evaluating this phenomenon. It has been hypothesized that less penetration may be caused by an interaction between resin and residual peroxide near the tooth surface23. It has been previously reported that there is a lower DC of adhesive resin when immediately bonding to a bleached tooth structure24. This is believed to be caused by the presence of residual oxygen in the tooth structure from the bleach treatment inhibiting monomer polymerization5. As with decreased resin penetration, decreased DC is likely another contributing factor to diminished bond strength with a bleached tooth structure. Despite postbleach treatment with alphatocopherol, the current study reported lower DC after bleach with or without the postbleach treatment compared with the unbleached control group. The unbleached control group displayed mean adhesive DC rates in pure adhesive and 2 mm into the hybrid layer at 82% and 79%, respectively, whereas the respective values for the bleached control group were 64% and 53% and for the alpha-tocopherol group 67% and 61%. Although no statistical analyses were completed because of the representative sample, the higher DC values for the unbleached control group compared with both bleached groups and the apparent similarities between the 2 bleached groups are worth noting. Scanning electron microscopic imaging of the hybrid layer after microtensile testing revealed colloidal, spherical particles throughout the bleached specimens. The spheres were noted in both the bleached control and postbleach treated specimens, suggesting the alpha-tocopherol treatment did not change this aspect. The lack of spheres in the unbleached specimen is suggestive that these particles are a product of the bleaching treatment. The 20,000! BSE scanning electron microscopic image reveals more than just the presence of spheres. The darker color of the object under BSE compared with the surrounding hybrid layer suggests that the spheres are largely composed of adhesive resin. The lack of continuity among the colloids could mean they are caused by less than optimal polymerization of the adhesive layer. This observation could be consistent with the reported lower DC.
Although a 10% concentration of alphatocopherol has been used previously15, 25, the 20% concentration was used in an attempt to garner the highest antioxidant effect possible without creating a solution that precipitates out of solution quickly. According to the pharmacy that compounded this material for our study, the solution needed to be used within 30 days of the compounding date. This short shelf life would make it difficult to adapt to clinical use because it would require dissolving the alphatocopherol into an alcohol solution very shortly before application. With this not being a commonly used procedure, the solution would likely have to be compounded by the pharmacy before each procedure. This creates another step that limits efficiency and works against the efforts to streamline clinical practice. Despite some of the practical limitations of using alpha-tocopherol, future research could evaluate whether varying additional application times of the 20% solution would counteract the effect of bleaching on dentin. Other future research could include evaluating other antioxidants, such as ascorbic acid, which has been reported to counteract the negative impact of bleach on bond strength using a 1-minute postbleach application on bovine teeth13. However, there are potential concerns about ascorbic acid’s low pH2 and possible overetching of dentin and enamel. Perhaps, further investigation into the management of this antioxidant could lead to its clinical usefulness.
CONCLUSIONS There was no significant difference in the microtensile bond strength or the qualitative assessment of the dentin adhesive layer as a function of bleach treatment before or after alpha-tocopherol treatment. Based on these results, this study provides no support to change the existing recommendation to wait the requisite 7 plus days between removing bleaching material and definitively bonding to the associated tooth structure.
ACKNOWLEDGMENTS The authors deny any conflicts of interest related to this study.
REFERENCES 1.
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Timpawat S, Nipattamanon C, Kijsamanmith K, Messer HH. Effect of bleaching agents on bonding to pulp chamber dentine. Int Endod J 2005;38:211–7.
Harrison Jr. et al.
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Titley KC, Torneck CD, Ruse ND, Krmec D. Adhesion of a resin composite to bleached and unbleached human enamel. J Endod 1993;19:112–5.
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Chng HK, Palamara JE, Messer HH. Effect of hydrogen peroxide and sodium perborate on biomechanical properties of human dentin. J Endod 2002;28:62–7.
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Crim GA. Post-operative bleaching: effect on microleakage. Am J Dent 1992;5:109–12.
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Dishman MV, Covey DA, Baughan LW. The effects of peroxide bleaching on composite to enamel bond strength. Dent Mater 1994;10:33–6.
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May LG, Salvia AC, Souza RO, et al. Effect of sodium ascorbate and the time lapse before cementation after internal bleaching on bond strength between dentin and ceramic. J Prosthodont 2010;19:374–80.
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Teixeira FB, Teixeira EC, Thompson J, et al. Dentinal bonding reaches the root canal system. J Esthet Restor Dent 2004;16:348–54. discussion 354.
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Turkun M, Kaya AD. Effect of 10% sodium ascorbate on the shear bond strength of composite resin to bleached bovine enamel. J Oral Rehabil 2004;31:1184–91.
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Kimyai S, Valizadeh H. The effect of hydrogel and solution of sodium ascorbate on bond strength in bleached enamel. Oper Dent 2006;31:496–9.
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Kimyai S, Valizadeh H. Comparison of the effect of hydrogel and a solution of sodium ascorbate on dentin-composite bond strength after bleaching. J Contemp Dent Pract 2008;9:105–12.
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Lai SC, Tay FR, Cheung GS, et al. Reversal of compromised bonding in bleached enamel. J Dent Res 2002;81:477–81.
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Hansen JR, Frick KJ, Walker MP. Effect of 35% sodium ascorbate treatment on microtensile bond strength after nonvital bleaching. J Endod 2014;40:1668–70.
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Muraguchi K, Shigenobu S, Suzuki S, Tanaka T. Improvement of bonding to bleached bovine tooth surfaces by ascorbic acid treatment. Dent Mater J 2007;26:875–81.
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Garcia EJ, Oldoni TL, Alencar SM, et al. Antioxidant activity by DPPH assay of potential solutions to be applied on bleached teeth. Braz Dent J 2012;23:22–7.
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Sasaki RT, Florio FM, Basting RT. Effect of 10% sodium ascorbate and 10% alpha-tocopherol in different formulations on the shear bond strength of enamel and dentin submitted to a home-use bleaching treatment. Oper Dent 2009;34:746–52.
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Plasmans PJ, Creugers NH, Hermsen RJ, Vrijhoef MM. Intraoral humidity during operative procedures. J Dent 1994;22:89–91.
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Wu WC, Shrestha D, Wei X, et al. Degree of conversion of a methacrylate-based endodontic sealer: a micro-Raman spectroscopic study. J Endod 2010;36:329–33.
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Plotino G, Buono L, Grande NM, et al. Nonvital tooth bleaching: a review of the literature and clinical procedures. J Endod 2008;34:394–407.
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Garcia-Godoy F, Dodge WW, Donohue M, O’Quinn JA. Composite resin bond strength after enamel bleaching. Oper Dent 1993;18:144–7.
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Hashimoto M, Ohno H, Endo K, et al. The effect of hybrid layer thickness on bond strength: demineralized dentin zone of the hybrid layer. Dent Mater 2000;16:406–11.
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Kenshima S, Francci C, Reis A, et al. Conditioning effect on dentin, resin tags and hybrid layer of different acidity self-etch adhesives applied to thick and thin smear layer. J Dent 2006;34:775–83.
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Perdigao J, May Jr KN, Wilder Jr AD, Lopes M. The effect of depth of dentin demineralization on bond strengths and morphology of the hybrid layer. Oper Dent 2000;25:186–94.
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Titley KC, Torneck CD, Smith DC, et al. Scanning electron microscopy observations on the penetration and structure of resin tags in bleached and unbleached bovine enamel. J Endod 1991;17:72–5.
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Cadenaro M, Breschi L, Antoniolli F, et al. Influence of whitening on the degree of conversion of dental adhesives on dentin. Eur J Oral Sci 2006;114:257–62.
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Garcia EJ, Mena-Serrano A, de Andrade AM, et al. Immediate bonding to bleached enamel treated with 10% sodium ascorbate gel: a case report with one-year follow-up. Eur J Esthet Dent 2012;7:154–62.
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Effects of Alpha-tocopherol Antioxidant on Dentincomposite Microtensile Bond Strength after Sodium Perborate Bleaching ABSTRACT It has been reported that bond strength can be reversed to prebleached levels with the application of 10% alpha-tocopherol in a 2-hour time frame or by delaying bonding for 2 weeks. This study evaluated the effectiveness of a 5-minute application of 20% alphatocopherol to reverse the deleterious effects of nonvital bleaching on consequent bond strength. Thirty third molars were assigned to the following 3 groups: unbleached, bleached, and bleached followed by treatment with alpha-tocopherol. The bleached groups were exposed to sodium perborate (2 g/mL) for 7 days. The postbleach treatment group was subsequently treated with 20% alpha-tocopherol for 5 minutes, and then all groups were restored with composite resin. After 24 hours of storage at 37 C and 100% humidity, restored tooth specimens were sectioned into 1-mm2 dentin-composite beams. Six beams from each tooth were subjected to microtensile bond strength testing. Representative beams were further evaluated with Raman microspectroscopy and scanning electron microscopy. The mean bond strength values (MPa) for each group were as follows: unbleached control group 5 26.2, bleached control group 5 20.3, and post–bleach treatment group 5 18.5. A 1-factor analysis of variance and Tukey post hoc test (a 5 0.05) indicated that bleaching had a detrimental effect on bond strength and that short-term alpha-tocopherol treatments did not improve postbleach bond strength. Raman microspectroscopy and scanning electron microscopy revealed no noted improvement for the post–bleach treatment group.The application of 20% alpha-tocopherol in a clinically relevant time frame was not effective in counteracting the deleterious effect of bleaching on bond strength. Bonding procedures should be delayed after tooth bleaching. (J Endod 2019;-:1–7.)
SIGNIFICANCE The application of alphatocopherol in a clinically relevant time frame was not effective at reversing the deleterious effects of nonvital bleaching on dentincomposite bond strength. Based on the results, there is no evidence to change the recommended delay between bleaching and bonding procedures.
KEY WORDS Degree of conversion; microtensile bond strength; nonvital bleaching; sodium perborate treatment
Internal bleaching of nonvital teeth is a conservative and effective method to manage darkened root canal–treated teeth. However, after bleaching, the bond strength between dentin and composite resin is adversely affected1–4. It has been proposed that residual oxygen from the bleaching process remains in the tooth structure for up to 2 weeks and is believed to interfere with polymerization of the dentin adhesives5. Despite the immediate negative effect of bleaching on bond strength, multiple studies reported that after waiting at least 7 days, dentin bond strength values return to baseline6–8. Although bleaching effects on dentin are not long-term, antioxidants, such as sodium ascorbate, have been used to counteract the adverse effects of bleaching to allow immediate placement of a restoration9–11. However, when antioxidants such as sodium ascorbate and ascorbic acid are used for clinically relevant times rather than extended periods, the reversal of dentin bond strength reductions has not been successful12,13. Thus, a more effective antioxidant is needed to counteract bleaching adverse effects in a shorter clinically relevant time.
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From the University of Missouri-Kansas City, School of Dentistry, Kansas City, Missouri Address requests for reprints to Dr Mary P. Walker, 650 East 25th Street, Kansas City, MO 64108. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright © 2019 Published by Elsevier Inc. on behalf of American Association of Endodontists. https://doi.org/10.1016/ j.joen.2019.04.013
Effects of Alpha-tocopherol Antioxidant after Bleaching
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FIGURE 1 – (A) Teeth suspended in sodium perborate solution. (B ) A tooth serially sectioned into dentin-composite beams with a 1-mm2 cross-sectional area. (C ) A dentin-composite beam attached to a microtensile tester.
review board–reviewed protocol designated as not human subject research. The extracted teeth were stored at 0.4 C in 0.9% phosphatebuffered saline with 0.002% sodium azide to inhibit microbial growth. All procedures were performed under ambient temperature and humidity because clinical procedures are done with a rubber dam in place16. The teeth were mounted into acrylic resin, leaving the coronal third of the root structure and tooth crown exposed. The occlusal half of the crown was removed with a water-cooled diamond blade saw (Isomet 1000, blade 11-4246; Buehler, Lake Bluff, IL), leaving a flat, dentinal surface devoid of enamel. The dentin smear layer was then removed with a 3-minute application of 17% EDTA followed by a 15-second air/water spray to remove any residual EDTA. The mounted, prepared teeth were randomly assigned into 3 groups of 10 teeth each as follows:
MATERIALS AND METHODS
Prepared teeth assigned to group 1 were stored without bleach exposure, whereas teeth assigned to groups 2 and 3 were inverted and suspended on a plastic grate into a 5-mm layer of sodium perborate solution (Sultan Healthcare, York, PA) prepared with distilled water (2 g/mL), allowing for only the prepared surface to be in contact with the bleaching agent (Fig. 1A). All specimens were stored at 37 C and 100% humidity for 7 days.
dry the specimens. Group 1 (unbleached control) and group 2 (bleached control) received no antioxidant application. Group 3 (postbleach alpha-tocopherol) received a 5minute application of 20% alpha-tocopherol (Midwest Compounders Pharmacy, Lenexa, KS) that was reapplied with a microbrush every minute to the dentinal surface. In all groups, the dentinal surface was washed with a 15second air/water syringe spray and air dried. An approximately 4-mm layer of composite was added to all tooth specimens using the following protocol. After a 10-second total etch with 35% phosphoric acid gel (Ultraetch; Ultradent Products Inc, South Jordan, UT), adhesive (Prime & Bond NT; Dentsply Caulk, Milford, DE) was applied for 20 seconds, air thinned, and light cured (XL3000; 3M, Maplewood, MN) for 20 seconds. Restorative composite (TPH, Dentsply Caulk) was applied in increments of less than 2 mm, light curing for 40 seconds between layers. The average curing light output was 1210 mW/ cm2 as measured by a radiometer (3H, Nanjing, China). The restored teeth were stored for 24 hours in 100% humidity at 37 C and then serially sectioned into 1-mm-thick vertical dentin-composite slabs with the same watercooled diamond blade as used previously (Fig. 1B). The specimens were rotated 90 and sectioned again to obtain rectangular dentincomposite beams with a cross-sectional area of approximately 1 mm2. The surface area of the bonded interface was calculated for each specimen by measuring the narrowest portion with a digital caliper. Six to 10 beams were generated from each tooth.
Thirty previously extracted human third molar teeth were collected with no associated patient identifiers in accordance with an institutional
After 7 days of storage, prepared teeth were sprayed with the air/water syringe for 15 seconds to remove any residual bleach and/or
Six beams from each tooth were used for microtensile testing (Bisco, Schaumburg, IL). Beams were adhered to the tester fixture
Regarding potential antioxidant agents, a previous study evaluated the antioxidative capabilities of 15 compounds including ascorbic acid, sodium ascorbate, and alphatocopherol via the 2,2-diphenyl-1-picrylhydrazyl-hydrate free radical method. The antioxidant activity results were presented as a percentage decrease in the number of free radicals compared with control values, with ascorbic acid and alpha-tocopherol being the best at 95% and 92%, respectively. The next was sodium ascorbate gel at 76% and sodium bicarbonate at 74%. All other substances tested fell at 51% and lower for antioxidant activity14. Ten percent alpha-tocopherol was previously used as an alternate antioxidant and showed the potential to counteract the negative effects of bleaching on bond strength after a 2-hour application time15. However, to date, there has been no research using alphatocopherol to test its effect using a shorter exposure that would occur in a clinical setting. The purpose of this study was to evaluate the short-term effect of the antioxidant alpha-tocopherol on dentin adhesive microtensile bond strength immediately after a simulated nonvital bleaching procedure with sodium perborate. The research hypothesis was that alphatocopherol would counteract the effects of bleaching on dentin bond strength. In addition to microtensile bond strength, the quality of the dentin-adhesive interface was evaluated via Raman microspectroscopy and scanning electron microscopy.
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1. Group 1: no bleach (unbleached control) 2. Group 2: bleached for 7 days (bleached control) 3. Group 3: bleached for 7 days followed by alpha-tocopherol antioxidant treatment (postbeach alpha-tocopherol)
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FIGURE 2 – Representative Raman microspectroscopy line maps of the (A ) unbleached control, (B ) bleached control, and (C ) postbleach alpha-tocopherol groups. The peaks associated with the adhesive occur at 1720/cm (carbonyl), 1609/cm (phenyl C5C), 1453/cm (CH2 def), and 1187/cm (gem-dimethyl), whereas the peaks associated with dentin occur at 1667/cm (amide I), 1454/cm (CH2 def), 1245/cm (amide III), and 961/cm (PO432). JOE Volume -, Number -, - 2019
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FIGURE 3 – Representative spectra of the adhesive (0 mm) and dentin-adhesive interface (2 mm into the hybrid layer) in the range of 1580–1670/cm from the unbleached control, bleached control, and postbleach alpha-tocopherol groups. The peak at 1637/cm is associated with unpolymerized (or unconverted) C5C in the adhesive; the peak at 1609/cm is associated with phenyl C5C in the adhesive, which is used as an internal standard. The relative intensity of the 1637/cm peak in the unbleached control group is lower than in the bleached control and postbleach alpha-tocopherol groups. The higher the 1637/cm peak, the lower the DC.
with cyanoacrylate applied to the composite and dentin ends of the beams, ensuring the dentin adhesive interface is centered in the fixture (Fig. 1C). Adhered beam specimens were subjected to tensile forces at a crosshead speed of 1 mm/min. Microtensile bond strength (MPa) was calculated based on the force at debond (N) divided by the beam interface cross-sectional area (mm2). The bond strength values for all beams6 from a single tooth were averaged, providing a single bond strength measurement for that tooth. Ten tooth specimens were tested from each group. The dentin-adhesive interface of 2 additional beams from each tooth not used for microtensile testing tooth was evaluated with Raman microspectroscopy (LabRam HR 800; Horiba Scientific, Edison, NJ) using a He-Ne laser at a wavelength of 632.8 nm and an excitation power of 20 mW. The laser was focused through a 50! Olympus (Tokyo, Japan) objective. Data were collected in the
Raman spectral range between 800 and 1800/ cm with data acquired using a 30-second exposure time. Spectral line maps of the
DC 5
! Intensity Polymerized Intensity Polymerized 1637 cm21 1609 cm21 !100 12 Intensity Unpolymerized Intensity Unpolymerized 1637 cm21 1609 cm21
dentin-adhesive interface were collected at 2mm intervals starting at the composite interface edge at the midpoint of the beam interface on 3 sides of the beam with a total of 3 line maps per beam. Line maps were used to determine adhesive penetration into dentin; example line maps from the unbleached control, bleached control, and postbleach alphatocopherol groups are presented in Figure 2. Within line maps, the adhesive degree of conversion (DC) was also calculated using peak area ratios of 1637/cm (1625–1650/cm) and 1609/cm (1590–1625/cm) from the
TABLE 1 - Adhesive Resin Degree of Conversion (%) Means and Standard Deviations Group* Unbleached control Bleached control Postbleach alpha-tocopherol
Within adhesive only
2 mm into hybrid layer
82 (2.5) 64 (14.1) 67 (9.1)
79 (6.4) 53 (12.4) 61 (10.5)
*Because of testing of a representative sample only, no statistical evaluation was performed. However, based on numeric value comparisons, there is a trend for higher degree of conversion percentages in the unbleached control group compared with the bleached and postbleach alpha-tocopherol groups.
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polymerized adhesive compared with the unpolymerized adhesive reference. The following DC equation was used17:
The debonded interface surface from the dentin side of 2 representative beam specimens from each group was evaluated using a scanning electron microscope (XL30 ESEM-FEG; FEI-Philips, Amsterdam, Netherlands). The dentin interface surfaces were each sputter coated with approximately 20-nm-thick gold palladium and examined at varying magnifications at 15 kV for both secondary and backscattered electron emission (BSE) analysis. One-factor analysis of variance of microtensile strength measures was performed (SPSS v22; IBM Corp, Armonk, NY) with significance set at .05 and a Tukey post hoc test used if differences were detected. Data acquired from Raman microspectroscopy and scanning electron microscopic imaging were used to provide qualitative information regarding the dentinadhesive interface. Because of the use of a representative sample, no statistical analyses were provided for these evaluations.
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FIGURE 4 – Scanning electron microscopic imaging. (A–C ) Secondary electron emission images. (D–F ) BSE images. Magnifications across image rows were 10,000! and 20,000!. (B and E ) The presence of colloidal spheres (arrows) was noted on both the bleached control and (C and F ) the postbleach alpha-tocopherol experimental group. (A and D ) However, no spheres were apparent on the unbleached control specimen. In the 20,000! BSE images of a (E ) bleached and (F ) postbleach alpha-tocopherol specimen; the spheres demonstrate a darker color, suggesting they are composed of adhesive resin.
RESULTS The mean microtensile bond strength (MPa) was significantly higher (P .05) in the unbleached control group (26.2 6 11.2) compared with the bleached control (20.2 6 10.0) or the postbleach alpha-tocopherol group (18.5 6 9.9), which were not significantly different (P . .05) from each other (n 5 6 beams/tooth,10 teeth/group). Figure 2A–C shows representative line maps from the unbleached control, bleached control, and postbleach alpha-tocopherol groups. Line maps were used to determine the depth of adhesive penetration into dentin. The peaks associated with the adhesive occur at 1720/cm (carbonyl), 1609/cm (phenyl C5C), 1453/cm (CH2 def), and 1187/cm (gem-dimethyl), whereas the peaks associated with dentin occur at 1667/cm (amide I), 1454/cm (CH2 def), 1245/cm (amide III), and 961/cm (PO432). Peaks associated with the adhesive and dentin components are noted in the spectra collected from the interface. The mean depth of adhesive
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penetration (mm) into dentin was higher in the unbleached control group (6.86 6 3.2) compared with 4.00 6 0.00 and 4.33 6 1.90 in the bleached and postbleach alphatocopherol groups, respectively. Data for DC were reported at the point on the line map of pure adhesive closest to the hybrid layer and at 2 mm into the hybrid layer from the pure adhesive point. Figure 3 shows representative spectra of the adhesive (0 mm) and dentin-adhesive interface (2 mm) in the range of 1580–1670/cm from the unbleached control, bleached control, and postbleach alpha-tocopherol groups. Representative beams were typically selected based on their microtensile test value being near the group mean value. The peak at 1637/cm is associated with unpolymerized (or unconverted) C5C in the adhesive; the peak at 1609/cm is associated with phenyl C5C in the adhesive, which is usually used as an internal standard. As shown in Figure 3, the relative intensity of the 1637/cm peak in the unbleached control group is lower than those in the other 2 groups (the bleached control and
postbleach alpha-tocopherol group); the higher the 1637/cm peak, the lower the DC. The DC means and standard deviation values are presented in Table 1. Representative photomicrographs obtained via a scanning electron microscope using secondary electron and BSE imaging are presented in Figure 4. The most notable differences between groups were the colloidal spheres, approximately 200–500 nm in diameter, that were seen on the bleached control and postbleach alpha-tocopherol specimens (Fig. 4B, C, E, and F) but were not present on the unbleached control specimen (Fig. 4A and D). In addition, in the BSE images, it was noted that the spheres in the bleached control and bleach/alpha-tocopherol groups were a darker color, suggesting that the spheres were composed of adhesive resin.
DISCUSSION This study sought to evaluate the effect of an antioxidant (ie, alpha-tocopherol) to allow for
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composite bonding at the bleaching agent removal appointment. Because of the presence of residual oxygen, the current recommendation is to remove the bleach, seal the tooth temporarily for 7 or more days, and have the patient return for a definitive access repair5,18. If the tooth is definitively restored with composite resin immediately after the removal of bleach, the strength of the microtensile bond and the quality of the hybrid layer at the restorative margin are diminished1–4,19. The week-long window during temporization allows for dissipation of the oxygen from dentinal tubules and the return of the subsequent microtensile bond strength and hybrid layer quality to prebleached levels7. In the current study, unbleached and bleached controls performed as expected, with the bleached control exhibiting significantly lower bond strengths. However, using postbleach treatment with 20% alphatocopherol for a clinically relevant time of 5 minutes, alpha-tocopherol failed to provide an improvement of microtensile bond strengths. The postbleach alpha-tocopherol group exhibited mean bond strengths statistically similar to the bleached control (18.5 and 20.2 MPa, respectively) and were significantly lower than the unbleached control (26.2 MPa); thus, the research hypothesis was rejected. The results of the current study are similar to a previous study using 35% sodium ascorbate for a clinically relevant time (10 minutes) in bleached human teeth with no improvement in bond strength12. One of the factors that may contribute to the decreased bond strength after bleach with or without alpha-tocopherol may be related to the lower penetration of adhesive resin in both the bleached control and the postbleach alpha-tocopherol groups compared with the unbleached control group. The depth of penetration of the unbleached group revealed a mean of 6.86mm. This is in line with a previously reported hybrid layer thicknesses of 6–10 mm for etch-and-rinse adhesive systems20–22. However, the bleached control and the alpha-tocopherol groups exhibited a mean depth of penetration of 4 mm and 4.33 mm, respectively. The mean depths for the bleached groups of less than two thirds that of the unbleached control suggest this is 1 mechanism leading to lower microtensile bond
strengths in the bleached groups. Little research could be located describing or evaluating this phenomenon. It has been hypothesized that less penetration may be caused by an interaction between resin and residual peroxide near the tooth surface23. It has been previously reported that there is a lower DC of adhesive resin when immediately bonding to a bleached tooth structure24. This is believed to be caused by the presence of residual oxygen in the tooth structure from the bleach treatment inhibiting monomer polymerization5. As with decreased resin penetration, decreased DC is likely another contributing factor to diminished bond strength with a bleached tooth structure. Despite postbleach treatment with alphatocopherol, the current study reported lower DC after bleach with or without the postbleach treatment compared with the unbleached control group. The unbleached control group displayed mean adhesive DC rates in pure adhesive and 2 mm into the hybrid layer at 82% and 79%, respectively, whereas the respective values for the bleached control group were 64% and 53% and for the alpha-tocopherol group 67% and 61%. Although no statistical analyses were completed because of the representative sample, the higher DC values for the unbleached control group compared with both bleached groups and the apparent similarities between the 2 bleached groups are worth noting. Scanning electron microscopic imaging of the hybrid layer after microtensile testing revealed colloidal, spherical particles throughout the bleached specimens. The spheres were noted in both the bleached control and postbleach treated specimens, suggesting the alpha-tocopherol treatment did not change this aspect. The lack of spheres in the unbleached specimen is suggestive that these particles are a product of the bleaching treatment. The 20,000! BSE scanning electron microscopic image reveals more than just the presence of spheres. The darker color of the object under BSE compared with the surrounding hybrid layer suggests that the spheres are largely composed of adhesive resin. The lack of continuity among the colloids could mean they are caused by less than optimal polymerization of the adhesive layer. This observation could be consistent with the reported lower DC.
Although a 10% concentration of alphatocopherol has been used previously15, 25, the 20% concentration was used in an attempt to garner the highest antioxidant effect possible without creating a solution that precipitates out of solution quickly. According to the pharmacy that compounded this material for our study, the solution needed to be used within 30 days of the compounding date. This short shelf life would make it difficult to adapt to clinical use because it would require dissolving the alphatocopherol into an alcohol solution very shortly before application. With this not being a commonly used procedure, the solution would likely have to be compounded by the pharmacy before each procedure. This creates another step that limits efficiency and works against the efforts to streamline clinical practice. Despite some of the practical limitations of using alpha-tocopherol, future research could evaluate whether varying additional application times of the 20% solution would counteract the effect of bleaching on dentin. Other future research could include evaluating other antioxidants, such as ascorbic acid, which has been reported to counteract the negative impact of bleach on bond strength using a 1-minute postbleach application on bovine teeth13. However, there are potential concerns about ascorbic acid’s low pH2 and possible overetching of dentin and enamel. Perhaps, further investigation into the management of this antioxidant could lead to its clinical usefulness.
CONCLUSIONS There was no significant difference in the microtensile bond strength or the qualitative assessment of the dentin adhesive layer as a function of bleach treatment before or after alpha-tocopherol treatment. Based on these results, this study provides no support to change the existing recommendation to wait the requisite 7 plus days between removing bleaching material and definitively bonding to the associated tooth structure.
ACKNOWLEDGMENTS The authors deny any conflicts of interest related to this study.
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