INTRODUCTION
P
revious studies have shown that carbamide peroxide bleaching agents in the
range of 10-35% adversely affect the bond strength of composite to acid-
etched enamel when bonding is performed immediately after the bleaching
procedure (Titley et al., 1992; García-Godoy et al., 1993; Miles et al., 1994).
Reduction in bond strengths was reported to be more pronounced with the use
of acetone-based adhesives (Sung et al., 1999). This is a concern in cosmetic
dentistry and orthodontics, particularly with the popularity of “in-office”
bleaching techniques (Swift, 1997; Spyrides et al., 2000), since a period of up
to three weeks is required before resin-enamel bond strengths return to values
obtained for unbleached enamel (Cavalli et al., 2001).
Compromised bond strengths that were observed for some single-bottle
adhesives when dentin was treated with hydrogen peroxide before acid-
etching could be effectively reversed with an anti-oxidant such as sodium
ascorbate, when it was used for at least one-third of the time of application
of the oxidizing bleaching agent (Lai et al., 2001). We hypothesized that
compromised bonding to carbamide-peroxide-bleached enamel may be
similarly reversed with sodium ascorbate before resin bonding, since
polymerization inhibition of the adhesive resins is a likely mechanism for
the adverse effects of bleaching on enamel bonding (Dishman et al., 1994).
Since retention of surface and subsurface residual peroxide or peroxide-
related substances (Torneck et al., 1990) may be responsible for the time-
dependent reduction in the quality of resin-enamel bonds, we anticipated
that these subtle changes may be elucidated by transmission electron
microscopy examination of the distribution of nanoleakage patterns (Sano et
al., 1995) within the bonded interfaces of oxidized acid-etched enamel and
those that were subsequently neutralized with the anti-oxidant. Thus, the
null hypothesis of this study was that there is no difference in the
microtensile bond strengths and distribution of nanoleakage patterns of
single-bottle adhesives bonded to carbamide-peroxide-bleached, acid-etched
enamel and those that were further neutralized with sodium ascorbate.
MATERIALS & METHODS
Extracted human third molars were collected after the patients’ informed consent had
been obtained under a protocol reviewed and approved by the institutional review
board of the Medical College of Georgia, USA. The mesial and distal surfaces of
these teeth were cleaned with pumice and sandblasted with 50-ìm alumina to provide
bonding surfaces that were devoid of the surface aprismatic enamel.
Experimental Design
Single Bond (3M-ESPE, St. Paul, MN, USA), an ethanol-based, and
Prime&Bond NT (Dentsply DeTrey, Konstanz, Germany), an acetone-based
single-bottle adhesive were used, each consisting of 3 experimental groups with
5 teeth each. Four teeth were used for bond strength evaluation, and the fifth
Reversal of Compromised
Bonding in Bleached Enamel
S.C.N. Lai
1
, F.R. Tay
1*
, G.S.P. Cheung
1
,
Y.F. Mak
1
, R.M. Carvalho
2
, S.H.Y. Wei
1
,
M. Toledano
3
, R. Osorio
3
, and D.H. Pashley
4
1
Conservative Dentistry, Faculty of Dentistry, University of
Hong Kong, Prince Philip Dental Hospital, 34 Hospital
Road, Hong Kong, SAR, China;
2
Bauru School of
Dentistry, University of São Paulo, Brazil;
3
Department of
Dental Materials, University of Granada, Spain; and
4
Department of Oral Biology and Maxillofacial Pathology,
School of Dentistry, Medical College of Georgia, Augusta,
GA, USA; *corresponding author, kfctay@hknet.com
J Dent Res
81(7):477-481, 2002
477
RESEARCH REPORTS
Biomaterials & Bioengineering
ABSTRACT
Oxygen inhibits polymerization of resin-based
materials. We hypothesized that compromised
bonding to bleached enamel can be reversed with
sodium ascorbate, an anti-oxidant. Sandblasted
human enamel specimens were treated with
distilled water (control) and 10% carbamide
peroxide gel with or without further treatment
with 10% sodium ascorbate. They were bonded
with Single Bond (3M-ESPE) or Prime&Bond NT
(Dentsply DeTrey) and restored with a composite.
Specimens were prepared for microtensile bond
testing and transmission electron microscopy after
immersion in ammoniacal silver nitrate for
nanoleakage evaluation. Bond strengths of both
adhesives were reduced after bleaching but were
reversed following sodium ascorbate treatment (P
< 0.001). Resin-enamel interfaces in bleached
enamel exhibited more extensive nanoleakage in
the form of isolated silver grains and bubble-like
silver deposits. Reduction of resin-enamel bond
strength in bleached etched enamel is likely to be
caused by a delayed release of oxygen that affects
the polymerization of resin components.
KEY WORDS: carbamide peroxide, sodium
ascorbate, microtensile bond strength, ultrastructure.
Received December 27, 2001; Last revision April 17, 2002;
Accepted May 9, 2002
tooth was prepared for nanoleakage evaluation by transmission
electron microscopy. The 3 experimental groups were as follows:
(I) Control group. The teeth were placed in distilled water for
8 hrs. The bonding surfaces were etched with a 32% phosphoric
acid gel (Uni-Etch, Bisco, Inc., Schaumburg, IL, USA) for 15 sec
and rinsed with water for 20 sec before bonding.
(II) Bleached group. The teeth were bleached by the
placement of 10% carbamide peroxide (NuproGold, Dentsply
DeTrey; pH = 6.4) around the enamel at 100% relative humidity
for 8 hrs. They were rinsed and immersed in distilled water for 10
min and then etched with phosphoric acid as previously described.
(III) Ascorbate group. After the teeth were bleached and rinsed as
previously described, they were immersed in 10% sodium ascorbate
(Sigma Chemical Co., St. Louis, MO, USA) for 3 hrs (i.e., at least
one-third of the bleaching time) to neutralize the oxidizing effect of
carbamide peroxide, according to the method described in Lai et al.
(2001). Before being etched with phosphoric acid, the treated teeth
were immersed in distilled water for 10 min to dissolve the sodium
ascorbate crystals that were deposited on the bonding surfaces.
The treated teeth were bonded with two coats of either Single
Bond or Prime&Bond NT. Bonded surfaces were air-dried and
then light-cured for 10 sec. Composite buildups were performed in
5 1-mm increments with either a hybrid composite (Renamel
Sculpt, Cosmedent, Inc., Chicago, IL, USA) for bond strength
testing, or a microfilled lining composite (Protect Liner F, Kuraray
Medical Inc., Tokyo, Japan) for transmission electron microscopy.
The teeth were stored in distilled water at 37°C for 24 hrs.
Microtensile Bond Strength Evaluation
Bonded teeth were sectioned occluso-gingivally into serial slabs,
and further sectioned into 0.9 x 0.9 mm composite-enamel beams,
according to the “non-trimming” technique of the microtensile test
for enamel bond-testing reported by Pashley and Tay (2001).
Specimens were stressed to failure under tension in a Bencor
Multi-T device (Danville Engineering, San Ramon, CA, USA)
with the use of a universal testing machine, Model 4440 (Instron,
Inc., Canton, MA, USA) at a crosshead speed of 1 mm per min.
The results were analyzed by a two-way analysis of variance
(treatment regimen vs. adhesives), and multiple comparisons were
done by Tukey’s test at ␣ = 0.05.
Nanoleakage Evaluation and
Transmission Electron Microscopy
A modified silver staining technique (Pashley et al., 2002) was used
with basic 50 wt% ammoniacal silver nitrate (pH = 9.5) to avoid the
possibility of artifactual dissolution of enamel apatites. The solution
was prepared by the dissolution of 25 g of silver nitrate crystals
(Sigma) in 25 mL of distilled water. Concentrated (28%)
ammonium hydroxide (Sigma) was used to titrate the black solution
until it became clear as ammonium ions complexed the silver into
diamine silver ([Ag(NH
3
)
2
]
+
) ions. We diluted this solution to 50
mL with distilled water to achieve a 50 wt% solution.
Two 0.9-mm slabs from each group were prepared from the
bonded teeth overlaid with the lining composite. They were coated
with two layers of fast-setting nail varnish applied 1 mm from the
bonded interfaces. They were immersed in ammoniacal silver
nitrate for 24 hrs. The silver-stained slabs were rinsed thoroughly
in distilled water and placed in photodeveloping solution for 8 hrs
under a fluorescent light to reduce the diamine silver ions into
metallic silver grains within potential voids along the bonded
interfaces. Undemineralized, epoxy-resin-embedded, 90-nm-thick
ultrathin sections were prepared according to the transmission
electron microscopy protocol of Tay et al. (1999). The unstained
sections were examined by means of a transmission electron
microscope (Philips EM208S, Philips, Eindhoven, The
Netherlands) operating at 80 kV.
RESULTS
Microtensile bond strength results are shown in the Table. There
were significant differences among the three treatment regimes
(P < 0.001) but not between the adhesives (P = 0.196). There was
no significant interaction between the two factors (P = 0.822).
For both adhesives, bond strengths were reduced by about 25%
when bonding to carbamide-peroxide-bleached enamel. The
compromised bond strengths were effectively reversed when the
bleached enamel was treated with 10% sodium ascorbate prior to
being acid-etched and adhesive application.
Transmission electron microscopy revealed that acid-etching
of the sandblasted enamel resulted in the complete removal of
the superficial layer of aprismatic enamel. The etching pattern
was mild in some regions of the control group (Fig. 1A). A
baseline nanoleakage pattern consisting of isolated silver grains
could be seen (Figs. 1B, 1C). In the bleached enamel, a more
extensive etching pattern was seen (Fig. 2A). Dense aggregation
of silver grains could be observed along the resin-enamel
interface as well as within the basal part of the adhesive layer
(Figs. 2B, 2C). In addition, bubble-like structures with peripheral
silver deposits were ubiquitously identified (Figs. 2B, 2C). In the
bleached but ascorbate-treated group, a mild etching pattern
similar to the control was observed (Fig. 3A). The abnormal
bubble-like structures were absent. However, the baseline silver
grain deposition could still be seen within the
acid-etched prismatic enamel (Fig. 3B).
The distribution of nanoleakage patterns in
Single Bond in the 3 experimental groups was
similar to that in Prime&Bond NT (not shown).
DISCUSSION
Microtensile bond strengths and nanoleakage
distribution of both the ethanol-based and the
acetone-based single-bottle adhesives were
different when the adhesives were bonded to
carbamide-peroxide-bleached, acid-etched
enamel, compared with those that were further
neutralized with sodium ascorbate. Hence, the
null hypothesis is rejected.
Table. Microtensile Bond Strengths of Adhesives to Acid-etched, Bleached Enamel
Single Bond Prime & Bond NT
Treatment Regime Bond Strength
a
(MPa) Bond Strength
a
(MPa)
Distilled water (control) 32.0 ± 6.0 (18)
1
33.1 ± 4.2 (20)
1
10% carbamide peroxide 24.0 ± 5.1 (16)
2
23.7 ± 7.9 (21)
2
10% carbamide peroxide,
then 10% sodium ascorbate 33.5 ± 8.7 (20)
1
36.2 ± 7.3 (20)
1
a
Values are means ± standard deviation. The number of specimens tested is included
in brackets. Bond strength results were analyzed by two-way analysis of variance and
Tukey’s multiple-comparison tests. Groups identified by different superscript numbers
are significantly different (P < 0.05).
478 Lai
et al. J Dent Res
81(7) 2002
We understand that hybrid-
ization of aprismatic enamel can
occur via resin penetration into
subsurface microporosities
created by phosphoric-acid-
etching (Pashley and Tay, 2001)
and do not advocate the removal
of this surface layer clinically. In
this study, we removed the
surface aprismatic enamel layer
by sandblasting only to create a
more uniform surface for
comparison of etching effects.
This is based on our previous
study that the aprismatic layer
was inconsistently observed and
was present only in some regions
of the enamel surface after acid-
etching (Pashley and Tay, 2001),
making it difficult for com-
parisons to be made between
bleached and unbleached etched
enamel. Using this protocol, we
did not find any difference in the
etching effect between bleached
and unbleached etched enamel at
the ultrastructural level.
Although carbamide peroxide
bleaching produced enamel
surface morphological alter-
ations (Perdigão et al., 1998;
Cimilli and Pameijer, 2001),
these changes were slight with
the use of 10-16% compared
with 35% carbamide peroxide
(Oltu and Gurgan, 2000). More-
over, the etching effect of 10%
carbamide peroxide is system-
specific (Rodrigues et al., 2001)
and is likely to be pH-dependent
(Shannon et al., 1993). Thus, it
can be expected that the demineralization effect of NuproGold,
with a pH value of 6.4, is relatively mild (McCracken and
Haywood, 1996), and any surface and subsurface alterations
would probably have been masked by the more aggressive
phosphoric-acid-etching (Ernst et al., 1996; Potocnik et al.,
2000).
To date, all nanoleakage studies were performed on resin-
dentin bonds, with the assumption that the high-energy enamel
surfaces created by acid-etching are optimized for resin
infiltration (Pioch et al., 2001). In this study, a baseline
nanoleakage pattern, in the form of isolated silver grains, could
be observed within the etched enamel in all treatment groups.
Since a basic version of ammoniacal silver nitrate was used (pH
= 9.5), it is unlikely that the observed results were artifacts
produced by laboratory demineralization of enamel apatites that
can occur with the use of acidic, conventional 50 wt% silver
nitrate solutions (pH = 3.4). Without the use of demineralized
sections with special staining for enamel proteins (Pashley and
Tay, 2001), we could not see the extent of resin-infiltration
within the hybridized enamel. The electron-dense, almost
Figure 1. Transmission electron micrographs showing the nanoleakage in phosphoric-acid-etched
enamel (control) that was bonded with Prime&Bond NT. (A) A low-magnification view of the resin-
enamel interface. C, resin composite; A, adhesive containing nanofiller particles; E, prismatic enamel;
arrow, interprismatic sheath. (B) A high-magnification view showing the presence of isolated, electron-
dense silver grains (open arrowheads) within the etched, resin-infiltrated enamel (E). Apatite crystallites
were partially dissolved and exhibit central hole regions (pointer). Arrow: nanofiller clusters within the
adhesive layer (A). (C) A very high magnification of (B), showing the presence of the central dark line
(pointers) within a partially dissolved apatite crystallite.
Figure 2. Transmission electron micrographs showing the nanoleakage in carbamide-peroxide-bleached,
acid-etched enamel that was bonded with Prime&Bond NT. (A) A low-magnification view of the resin-
enamel interface. C, resin composite; A, adhesive containing nanofiller particles; E, prismatic enamel. A
region with more extensive etching is depicted, although areas with etching effect similar to that in Fig. 1A
were commonly observed. (B) A high-magnification view of the resin-enamel interface, showing more
extensive silver grain deposition (open arrowheads) within the etched enamel (E) as well as the adhesive
layer (A). Additional bubble-like structures with peripheral silver deposits (pointers) were also evident.
Arrow: less-electron-dense nanofiller clusters in the adhesive. (C) A high-magnification view showing a
dense aggregation of isolated silver grains (open arrowheads) and almost spherical, bubble-like structures
with incomplete peripheral silver deposits (pointers) in the adhesive layer. Arrow: nanofiller clusters.
Figure 3. Transmission electron micrographs showing the nanoleakage
in carbamide-peroxide-bleached enamel that was treated with sodium
ascorbate prior to being acid-etched and the application of the
Prime&Bond NT adhesive. (A) A low-magnification view of the resin-
enamel interface. C, resin composite; A, adhesive containing nanofiller
particles; E, prismatic enamel. (B) A high-magnification view showing
the presence of isolated, electron-dense silver grains (open arrowheads)
within the etched, resin-infiltrated enamel (E). Bubble-like structures that
were previously observed in the bleached enamel were absent after
sodium ascorbate treatment.
J Dent Res
81(7) 2002 Improved Bonding to Bleached Enamel 479
ABC
ABC
AB
480 Lai
et al. J Dent Res
81(7) 2002
spherical isolated silver grains within the etched enamel could be
easily differentiated from the adjacent angular apatite crystallites
or the less electron-dense nanofiller clusters in the adhesive (Tay
et al., 1999). Their presence suggested that there is a possibility
of over-etching and incomplete resin infiltration at the base of
phosphoric-acid-etched enamel, unlike the use of self-etch
adhesives (Shimada and Tagami, personal communication).
Nevertheless, such a phenomenon was extremely mild in view of
the very low density of the silver grains observed.
This nanoleakage pattern became more dense along the
resin-enamel interface of carbamide-peroxide-bleached enamel.
In addition, bubble-like structures with incomplete peripheral
silver deposits were also observed. These features were present
even after the bleached enamel was rinsed and immersed in
water for 10 min prior to being acid-etched and the adhesive
application. It is known that hydrogen peroxide released from
carbamide peroxide, due to its low molecular weight, can
penetrate enamel to reach the dental pulp (Gokay et al., 2000),
and that there is a continuous leaching of the hydrogen
peroxide that is retained in the bleached enamel (Adibfar et al.,
1992). Since dental adhesives polymerize by a free radical
polymerization mechanism that involves the generation of free
radicals through light-activated redox initiators (Monroe et al.,
1968), the hydrogen peroxide may break down to release
oxygen that is trapped within the adhesive during light-
activation. This may account for the preponderance of the
almost spherical bubble-like structures along the resin-enamel
junction and close to the base of the adhesive layer (Figs. 2B,
2C). Release of oxygen from the bleached enamel probably
results in incomplete polymerization of the adhesive in these
regions (Torneck et al., 1990; Dishman et al., 1994). This could
account for the observation of an increased density of voids
along the acid-etched, bleached enamel interface (McGuckin et
al., 1992). Similar to previous studies (Torneck et al., 1990;
McGuckin et al., 1992; García-Godoy et al., 1993), our failure
mode results (not shown) indicated predominant adhesive
failures along the resin-enamel interface in bonded bleached
enamel, compared with more mixed and cohesive failures in
the other two treatment regimes.
It is interesting that compromised bonding to acid-etched
bleached enamel was reversed with sodium ascorbate, an anti-
oxidant. Previous studies suggested the subsurface enamel
organic matrix was altered by the oxidizing effect of hydrogen
peroxide (Seghi and Denry, 1992; Hegedus et al., 1999). Based
on our present findings, it is possible that these are not permanent
structural alterations, but reversible changes in redox potential of
the organic components. It is also speculated that the peroxide
ions may have temporarily substituted the hydroxy radicals in the
apatite lattice (Zhao et al., 2000). Since these lattice substitutions
are thermodynamically unfavorable, such a process may be
reversed by an anti-oxidant. Such a hypothesis remains
speculative and has to be further investigated by chemico-
analytical methods. Unlike our previous study, we immersed our
sodium-ascorbate-treated specimens in water for an additional 10
min to dissolve completely the rhombohedral crystal depositions
that are present after sodium ascorbate treatment (Lai et al.,
2001). This may prevent the dissolution of the crystalline deposits
and the generation of voids along the resin-enamel interfaces as
resins eventually absorb water and leach. Understandably, the use
of sodium ascorbate to reverse the oxidizing effect of a bleaching
agent involves a substantially lengthy period, which may not be
clinically acceptable. However, in light of the fact that a post-
bleaching period of 2-3 wks is required for enamel bonding to
return to normal, it may be possible to incorporate the sodium
ascorbate into a gel to be placed by patients themselves in the
bleaching tray before bonding. Since vitamin C and its salts are
non-toxic and are widely used in the food industry as anti-
oxidants, it is unlikely that their intra-oral use will create any
adverse biological effect or clinical hazard. The potential clinical
use of a sodium ascorbate gel to reverse the oxidizing effect of
home and “in-office” bleaching with carbamide peroxide must be
further investigated.
ACKNOWLEDGMENTS
This study was based on the work performed by Shirley Lai for
partial fulfillment of the Advanced Diploma in Endodontics,
the University of Hong Kong. We thank Amy Wong of the
Electron Microscopy Unit, The University of Hong Kong, for
technical support, and Michelle Barnes of the Medical College
of Georgia for secretarial support. This study was supported by
grant DE 06427 from the National Institute of Dental and
Craniofacial Research, USA, grant number CNPq300481/95-0
from Brazil, and CICYT grant number MAT98-0937-C02-02
from Spain. The materials used in this study were generously
sponsored by 3M-ESPE and Dentsply DeTrey.
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