Keywords bleaching - enamel - remineralization - scanning electron microscopy - energy dispersive
X-ray analysis - calcium silicate-sodium phosphate-fluoride - NovaMin
Introduction
Among the aesthetic management modalities of vital and nonvital discolored teeth,
dental bleaching has proven to be a highly conservative and simple treatment option.
At present, several bleaching agents are commercially available and are mainly based
on different concentrations of hydrogen peroxide (HP) or carbamide peroxide (CP).
These products are available for use at home or in the dental office, possibly aided
by various types of light sources to intensify the oxidation reaction.[1 ]
The process of bleaching teeth involves free radicals generation such as oxygen and
perhydroxyl because of the decomposition of HP. The presence of these radicals generates
an oxidation-reduction reaction.[2 ] Due to their exceedingly electrophilic nature, free radicals diffuse through the
matrices of enamel and dentin to bombard the pigment molecules and ultimately gain
stability, and this is possible due to the permeability of dental tissue. Following
this reaction, the organic macromolecules—of which the pigments are formed—are broken
down into less complex, smaller, and lighter molecules.[3 ] Due to the unspecific nature of this reaction, several studies have investigated
its undesirable effects on tooth structure such as mineral loss and alterations to
surface topography which can, in turn, alter the biomechanical properties of enamel.[4 ]
[5 ]
Morphological changes of enamel surface were reported, mainly due to the erosive nature
of the bleaching process.[6 ] Bleaching affects the organic components of enamel and produces changes in the mineral
phase, which ultimately creates visible morphological changes on the enamel surface.[7 ]
[8 ] A study by Ushigome et al described that HP selectively erodes rod sheath areas
while CP causes mineral dissolution and decalcification, which results in etch-like
erosion of surface and subsurface enamel.[9 ] The erosive effects are in view of the acidity of the bleaching agents, where some
bleaching products have been reported to have a pH as low as 2.4.[10 ]
The application of remineralizing agents was found to restore the morphological defects
caused by bleaching.[11 ] Currently, among the available remineralization promoting agents, fluoride-based
formulations remain the most commonly used anti-erosive materials. They enhance the
precipitation of calcium phosphates and in turn promote the formation of fluoro-hydroxyapatite
crystals in dental tissues.[12 ]
A dentifrice containing calcium silicate, sodium phosphate, and fluoride salts (CSSPF)
has been introduced. This technology was first proposed to supplement the natural
process of mineralization by human saliva, which was possible by providing auxiliary
calcium and phosphate that nucleates hydroxyapatite (HAP) formation. This mechanism
seems to promote the remineralization and repair of softened enamel and aids in protecting
enamel from acid challenges.[13 ] The CSSPF-based toothpaste was introduced with a boosting serum, which was claimed
to enhance the power of enamel remineralization.
Another class of commercially available agents that have been indicated in the treatment
of dental hypersensitivity and enamel remineralization is bioactive glass, specifically,
NovaMin. Primarily, NovaMin is a ceramic material that consists of amorphous sodium-calcium-phosphosilicate
as a powder of fine particle size that is highly reactive in water. NovaMin works
in two mechanisms. One mechanism is that the fine powder itself is capable of occluding
dentinal tubules,[14 ] and the second is by its reaction when exposed to the tooth’s aqueous environment.
The production of calcium and phosphate ions from the glass is brought about by the
rapid exchange of particles between the sodium ions in NovaMin and hydrogen cations
from the aqueous environment of the tooth.[15 ] The release of sodium at primary exposure of the material to water leads to a transitory
and localized increase in pH. This effect encourages the precipitation of the supplementary
calcium and phosphate ions delivered by the NovaMin ultimately forming a layer of
calcium phosphate. This layer then crystalizes into carbonate-enriched hydroxyapatite
(HCA).[16 ] The newly formed HCA combines with the residual NovaMin particles, remineralizing
the enamel surface and hinders further demineralization.[14 ]
The remineralization of bleached enamel has been scarcely tackled in the literature.
Therefore, this study was conducted to investigate the remineralizing capacity of
two anti-erosive/remineralizing agents (namely CSSPF and NovaMin bioactive glass)
on the bleached enamel, by assessing their effect on the enamel surface hardness and
roughness. The null hypothesis being that the tested remineralizing agents have no
effect on the surface hardness and surface roughness of the bleached enamel.
Materials and Methods
Samples Preparation
The compositions of the tested materials are listed in [Table 1 ]. Forty sound human premolars extracted for orthodontic purposes were used in this
study after ethical approval by the University of Sharjah research ethics committee
and after patient consent. The teeth were examined under a stereomicroscope for the
presence of cracks, decay, or any other defect, and teeth with defects were excluded
from the study. Any soft tissues remnants were removed by using an ultrasonic scaler.
The roots were sectioned at the cemento-enamel junction using a high-speed dental
handpiece and a diamond bur and then the pulp tissues were removed. The crown of each
tooth was sectioned mesio-distally into two halves using a diamond saw (Isomet 1000;
Buehler, Lake Bluff, Illinois, United States) to obtain 80 specimens. The specimens
were embedded in self-cured acrylic resin (Fastray, Harry J. Bosworth Co.; Skokie,
Illinois, United States) with the enamel surface facing upward. The enamel surfaces
were polished using a grinding/polishing machine (EcoMet 30; Buehler, Lake Bluff,
Illinois, United States) with 400, 600, and 1200 grit water-cooled sandpaper discs
to attain flat enamel surfaces and then polished using Synthetic Polishing Cloth (Super-Snap
Buff Disk, Shofu Dental Corp.; Kyoto, Japan) soaked with 1 µm diamond suspension (Buehler).
After polishing, the specimens were cleaned in an ultrasonic bath with deionized water
for 5 min.
Table 1
Materials tested in the study
Product
Manufacturer
Composition
pH value
Lot
Opalescence Boost
Ultradent Products, South Jordan, Utah, United States
38% hydrogen peroxide, 3% potassium nitrate, and 1.1% fluoride ions (10,000 ppm)
7.5230
B069
Sensodyne Repair & Protect
GSK, Middlesex, United Kingdom
Glycerin, PEG-8, silica, calcium sodium phosphosilicate (NovaMin), sodium lauryl sulfate,
aroma, titanium dioxide, carbomer, potassium acesulfame, limonene, contains sodium
monofluorophosphate 1.08% w/w (1,450 ppm fluoride)
8.6323
B7034603
Regenerate Enamel Science
Unilever; London, United Kingdom
Glycerin, calcium silicate, PEG-8, hydrated silica, trisodium phosphate, sodium phosphate,
aqua, PE-60, sodium lauryl sulfate, sodium monofluorophosphate, aroma flavour, synthetic
fluorphlogopite, sodium saccharin, polyacrylic acid, tin oxide, limonene
9.0223
4175CCC
Regenerate Enamel Science -Advanced Enamel Serum
Unilever; London, United Kingdom
NR-5 serum: glycerin, calcium silicate, peg-8, trisodium phosphate, sodium phosphate,
aqua, PEG-60, sodium lauryl sulfate, sodium monoflourophosphate, aroma/flavour, hydrated
silica, synthetic fluorphlogopite, sodium saccharin, polyacrylic acid, tin oxide,
CI 77891, Limonene
Activator gel: Aqaua, glycerin, cellulose, gum, sodium flouride, benzyl alchohol,
ethylexylglycerin, phenoxyethanol, CI 42090
Not available
42038CA
The enamel surface microhardness (baseline KHN) was determined by using a microhardness
tester (HMV-2; Shimadzu Corporation, Tokyo, Japan). Five indentations per sample were
performed at distances of 100 µm from each other using a microindentor (Knoop diamond,
100 g/15 sec. An average of five readings for each specimen was recorded as its KHN
value. According to KHN values at the baseline, data from 10 specimens (n = 10) were averaged so that the average baseline KHN in the eight experimental groups
was nearly equal ([Table 2 ]).
Table 2
Mean Knoop microhardness number (± standard deviation) of bleached and nonbleached
enamel surfaces after no treatment, treatment with sensodyne repair & protect, regenerate
enamel science, or regenerate enamel-advanced enamel serum
Treatment
Baseline (KHN)
Bleached (KHN)
Re-min (KHN)
Abbreviations: CS, regenerate enamel science; NR-5, regenerate enamel boosting serum;
KHN, Knoop hardness number; NM, sensodyne repair and protect; NT, no treatment.
Note: Within a column, different superscript capital letters indicate a significant
difference (p < 0.05). Within a row, different superscript small letters indicate a significant
difference (p < 0.05).
Nonbleached
NT
342.7 ± 34.2
321.4 ± 33.4A
NM
341.4 ± 53.6
332.5 ± 22.5A
CS
346.6 ± 35.4
338.5 ± 23.9A
CS+NR-5
347.3 ± 42.1
328.8 ± 43.2A
Bleached
NT
346.1 ± 29.9a
281.1 ± 47.3b
272.1 ± 33.7B, b
NM
348.5 ± 21.0a
274.9 ± 25.4b
293.6 ± 36.2B, b
CS
343.0 ± 42.8a
272.2 ± 37.6b
324.2 ± 42.6A, a
CS+NR-5
349.1 ± 46.3a
276.5 ± 43.5b
335.0 ± 31.5A, a
Bleaching of Enamel
Four groups (nonbleached groups) were randomly selected and did not receive any bleaching
treatment. In contrast, the other four groups (bleached groups) were treated with
a 38% HP bleaching gel (Opalescence R Boost, Ultradent Products, Inc.; South Jordan,
Utah, United States). Bleaching of the enamel samples was done following the manufacturer’s
instructions, and each sample was subjected to three consecutive 20-min applications.
Following each bleaching gel application, the sample surface was washed with a strong
stream of air and water for 20 sec. After the third session, the bleached surfaces
were resubmitted to KHN microhardness test to obtain the degree of softening.
Remineralization of Enamel
The nonbleached groups (1–4) and bleached groups (5–8) were randomly assigned to four
different remineralization protocols. Group 1 (nonbleached [NT]) served as a negative
control (NT), and Group 5 served as a bleached positive control. Both groups were
kept in artificial saliva for 24 h and did not receive any remineralization treatment.
Groups 2 and 6 were subjected to remineralization treatment with NovaMin-based toothpaste
(NM), and groups 3, 4, 7, and 8 were subjected to CSSPF-based toothpaste (CS). Group
4 and 8 received additional treatment with NR-5 boosting serum (CS+NR-5).
The remineralization procedure in groups 2, 3, 4, 6, 7, and 8, consisted of 3-min
application of a solution of toothpaste/water in 1:2 ratio two times a day for 7 days.
The toothpaste solution was applied by using a soft brush and minimal pressure. In
Groups 4 and 8, after treatment with the toothpaste, the remineralizing NR-5 gel was
prepared by mixing equal amounts of NR-5 serum with the activator gel (1:1 ratio)
on a glass slab with a plastic mixing stick. This mixture was applied to the specimens’
surfaces and kept undisturbed for 3 minutes once daily for 3 consecutive days.
After each application of toothpaste or serum gel, the specimens were rinsed in distilled
water for 5 sec and kept in the freshly prepared artificial saliva in an incubator
at 37°C. The artificial saliva was prepared following Holland’s protocol,[17 ] (composition: 0.4 g NaCl, 0.4 g KCl, 0.795 g CaCl2 ·2H2 O, 0.78 g NaH2 PO4 ·2H2 O, 0.005 g Na2S · 9H2 O, 1 g urea, and 1,000 mL distilled water), and the pH of the saliva was adjusted
at 7 ± 0.2.
Knoop Surface Hardness Test
As mentioned earlier, the Knoop surface microhardness (KHN) of the enamel surfaces
before and after bleaching was recorded. Subsequently, the test was repeated following
each remineralization treatment. The number of indentions per sample and the testing
procedures were standardized throughout the study. At each testing point, data of
each experimental group from 10 specimens were averaged.
Surface Roughness Test
Forty additional enamel samples were prepared and polished, as mentioned above. The
baseline surface roughness of each specimen was evaluated under an atomic force microscope
(AFM; Flex-Axiom, Nanosurf AG, Liestal, Switzerland). AFM was used in a noncontact
mode by using an AFM cantilever with magneto-resistive sensors integrated into its
tip (7 µm thickness, 225 µm length, and 38 µm width) under a constant force of 48
N/m.
On each polished surface, four standardized rectangular spots (25 × 25 µm each) were
scanned. The changes in the vertical position provide the height of the images, registered
as bright and dark regions. The tip-specimens distance was maintained stable through
constant oscillation amplitude (set-point amplitude). AFM micrographs were analyzed
by using a scanning probe microscopy data analysis software (C3000 control software;
version 3.7.2.8, Nanosurf AG, Liestal, Switzerland). The average surface roughness
(Sa), valley depth (lowest value - Sv), and peak height (highest value - Sp) of the
unbleached enamel surfaces were recorded as the baseline readings and were expressed
as numeric values in nanometers.
Then the samples were divided into eight equal groups (n = 5) according to the experimental design mentioned above. The surface roughness
measurements were repeated after each allocated treatment for each experimental group.
Micromorphological and Energy Dispersive X-Ray Analysis
Three representative samples from each experimental group were dehydrated in a desiccator
containing dehydrated silica gel at room temperature for 24 h. The micromorphological
analysis was performed after sputter coating with 100 Å Gold-Palladium (EMS 7620 Mini
Sputter Coater; Electron Microscopy Sciences, Hatfield, Pennsylvania, United States)
to create a conductive specimen surface and to reduce electron charging, which may
reduce the quality of the image. Observations were performed under different magnifications
up to ×5000 by using a scanning electron microscope (SEM) (VEGA3 XM–TESCAN; Kohoutovice,
Czech Republic) operating at 20 kV acceleration voltage and 10 to 25 mm working distance.
SEM-coupled energy-dispersive X-ray spectroscopy (AZtecLive, Oxford Instruments; Abingdon,
United Kingdom) operating at a take-off angle of 35 degrees with map mode was conducted
at the same operating voltage. The average main elements % weight content of the experimental
groups (calcium and phosphorus), and the calcium to phosphorus ration (Ca/P) per each
group was calculated.
Statistical Analysis
Results were analyzed with statistical software (SPSS software; version 15.0, SPSS
Inc., Chicago, Illinois, United States) by using a two-way analysis of variance and
Bonferroni post hoc multiple comparison tests (α = 0.05) to determine the effect of
the variables (bleaching and the type of the remineralizing agent) on the surface
hardness and surface roughness of enamel, and to determine the effect of bleaching
and type of the remineralizing agent on the surface hardness and surface roughness
of enamel
Results
The results of the Knoop microhardness test of the experimental groups are listed
in [Table 2 ]. The post hoc test revealed a statistically significant difference (p < 0.05) between the surface hardness of the enamel specimens before and after bleaching,
with a significant reduction in the enamel surface hardness by 18 to 21%. NM showed
inferior ability to restore the reduced surface hardness of enamel, compared with
CS and CS+NR-5 groups, which showed no significant difference (p > 0.05) between the
KHN of enamel at baseline and after remineralization with these two groups.
The results of the AFM surface roughness test of the experimental groups are listed
in [Table 3 ] and illustrated in [Fig. 1 ]. A statistically significant difference (p < 0.05) between the surface roughness of the enamel specimens before and after bleaching
was evident, with more than threefold increase in the enamel surface roughness. Treatment
of enamel with NM significantly increased the surface roughness of both bleached and
nonbleached enamel (p < 0.05). Meanwhile, the CS and CS+NR-5 groups showed a significant decrease in surface
roughness of the bleached enamel (p < 0.05).
Table 3
Mean area surface roughness in nm (± standard deviation) of bleached and nonbleached
enamel surfaces after no treatment, treatment with sensodyne repair & protect, regenerate
enamel science, or regenerate enamel-advanced enamel serum
Treatment
Baseline (nm)
Bleached (nm)
Re-min (nm)
Abbreviations: CS, regenerate enamel science; NR-5, regenerate enamel boosting serum;
NM, sensodyne repair and protect; NT, no treatment.
Note: Within a column, different superscript capital letters indicate a significant
difference (p < 0.05). Within a row, different superscript small letters indicate a significant
difference (p < 0.05).
Nonbleached
NT
42.6 ± 8.6
42.4 ± 5.5A
NM
46.7 ± 7.4a
84.0 ± 7.6B, b
CS
44.7 ± 5.9
51.6 ± 7.4A
CS+NR-5
42.9 ± 6.8
54.7 ± 8.4A
Bleached
NT
44.3 ± 5.1a
136 ± 9.6b
144.7 ± 9.4C, b
NM
40.8 ± 8.3a
130 ± 7.6b
173.6 ± 11.3D, c
CS
44.2 ± 9.2a
140 ± 8.2b
89.7 ± 8.2B, c
CS+NR-5
46.3 ± 8.4a
142 ± 9.3b
82.4 ± 9.1B, c
Fig. 1 Scanning electron microscope and atomic force microscope microphotographs of bleached
and nonbleached enamel surface after no treatment, or treatment with sensodyne repair
and protect (NM), regenerate enamel science (CS) or CS followed by regenerate enamel
boosting serum (CS+NR-5).
The average element content (wt%) of calcium, and phosphorus, and Ca/P ratio of the
experimental groups are listed in [Table 4 ]. Within the nonbleached groups, there was no significant difference in the main
element wt% and the Ca/P ratio between NT and the treated groups. A significant reduction
in the Ca/P ratio was recorded after bleaching (p < 0.05). In comparison to the PC group, an increase in the Ca/P ratios was measured
for the bleached groups after treatment with NM, CS, and CS+NR-5.
Table 4
Energy dispersive X-ray analysis elemental analysis of Ca and P (wt%) and Ca/P ratio
of bleached and nonbleached enamel surfaces after no treatment, treatment with sensodyne
repair & protect, regenerate enamel science, or regenerate enamel-advanced enamel
serum
Treatment
Element composition (wt %)
Ca
P
Ca/P
Abbreviations: CS, regenerate enamel science; CS+NR-5, regenerate enamel boosting
serum; NM, sensodyne repair and protect; NT, no treatment.
Note: Within a column, different superscript letters indicate a significant difference
(p < 0.05).
Nonbleached
NT
37.80 ± 1.3a
17.20 ± 0.7a
2.20 ± 0.2a
NM
36.20 ± 1.1a
17.60 ± 0.9a
2.06 ± 0.1b
CS
36.10 ± 1.1a
17.10 ± 1.3a
2.11 ± 0.1b
CS+NR-5
35.30 ± 0.5a
16.40 ± 0.8a
2.15 ± 0.1b
Bleached
NT
20.00 ± 1.2b
11.70 ± 1.0b
1.71 ± 0.1c
NM
25.00 ± 1.0c
11.50 ± 1.1b
2.17 ± 0.1b
CS
25.80 ± 0.8c
11.90 ± 0.9b
2.20 ± 0.1a
CS+NR-5
24.50 ± 1.1c
11.00 ± 0.8b
2.23 ± 0.1a
Sample SEM microphotographs of the investigated groups are presented in [Fig. 1 ]. The surface morphological analysis showed a typical honeycomb appearance in all
the bleached groups, the pattern is similar to that of the acid-etched enamel. Compared
with the NT bleached group, this pattern was less evident after treatment with the
tested remineralizing agents. None of these treatments were able to completely reverse
the effect of bleaching, compared with the nonbleached treated groups.
Discussion
Teeth whitening, although comparatively prevalent, is still considered controversial
regarding the morphological changes brought upon enamel, accompanying the procedure.
The significance of this concern has manifested as the recommendation of using a remineralizing
agent after bleaching. The effectiveness of the various remineralizing agents remains
in question. In this study, the effectiveness of CSSPF and NovaMin-based dentifrice
to remineralize bleached human enamel were tested, using the Knoop microhardness test
and analyzing mean surface roughness values with an AFM. This study has observed that
the tested remineralizing products have positively affected the microhardness and
surface roughness of the bleached enamel; thus, the null hypothesis tested should
be rejected.
Conflicting results were reported in the literature regarding the effect of bleaching
on enamel. Cadenaro et al, in an in vivo study, found no significant effect of 38%
HP in-office bleaching on the roughness of enamel.[18 ] In contrast, Hosoya et al[19 ] reported morphological changes to enamel following bleaching with a high concentration
of HP, and concluded a strong association between bleaching and the increased enamel
roughness and Streptococcus mutans adhesion. While Basting et al[20 ] reported a significant reduction by 10 to 23% KHN of bleached enamel, Rodrigues
et al[4 ] reported a 47% reduction in KHN of bleached enamel. These inconsistencies may be
due to differences in study design, namely, the magnitude and time of loading used
in the hardness testing or the different concentration and time of application of
the bleaching agent.
In the current study, a significant reduction in surface hardness and an increase
in surface roughness of enamel was found after bleaching, and these results were confirmed
by the SEM morphological analysis and the AFM microradiographs ([Fig. 1 ]). Our findings are in agreement with a study by Lewinstein et al,[21 ] who reported a reduction of 13% KHN for home bleaching and up to 25% for in-office
bleaching.
These changes in surface roughness and hardness after enamel bleaching can be attributed
to either the low pH or high concentration of the peroxides used with the in-office
bleaching. The bleaching material used in this study has a pH value of 7.52, which
is considered neutral or slightly alkaline. Therefore, the increased porosity of enamel
can be attributed to the effect of the nascent oxygen of the high concentration HP
rather than the acidic erosion of the hydroxyapatite.[22 ]
[23 ]
The EDX elemental analysis ([Table 4 ]) revealed a significant reduction in the Ca/P ratio of the bleached enamel in comparison
to the baseline readings, which in agreement with the findings of several previous
studies.[24 ]
[25 ]
The application of CSSPF showed a significant increase in microhardness compared with
values obtained after bleaching. This finding is consistent with other investigations;
an in situ study by Joiner et al placed acid-challenged tooth enamel inserts onto
patients’ partial dentures. The inserts were then treated with four different agents.
The study found that the combination of calcium, phosphate, and fluoride ions from
the sodium fluoride and mono-fluoro-phosphate resulted in an increase in microhardness,
which was consistent along with all evaluation intervals.[26 ] Moreover, another experiment conducted by Hornby et al studied the effectiveness
of CSSPF versus other agents under conditions of pH cycling within the normal range
of demineralization and remineralization, and they concluded that CSSPF proved significant
effectiveness versus different formulations included in their study.[27 ]
The superior effects of CSSPF can be attributed to its mode of action, which includes
multiple mechanisms. Calcium silicate has the ability to release calcium ions in an
acidic environment, which deprives the area of protons while simultaneously enriching
the local calcium concentration. This mechanism increases the degree of saturation
and theoretically inhibits demineralization. Moreover, in the presence of phosphate
salts, calcium silicate has been shown to deposit directly onto the enamel surface
and maintain its affinity even after rinsing. This affinity, aided by the presence
of phosphate ions, results in the nucleation and eventual precipitation of HAP on
the surface of the calcium silicate particles. This nucleation takes place after only
one exposure and at pH values as low as 4.[13 ] Moreover, the newly formed HAP may act as the first defense against future acid
attacks, keeping the underlying enamel protected. These explanations were supported
by the findings of the EDX elemental analysis, where an increase in the calcium and
phosphate wt% was recorded after treatment with CS. Finally, the efficacy of fluoride
alone, in remineralization, is directly dependent on the availability of calcium and
phosphate at the site of action.[28 ] This dependency is absent in calcium silicate and phosphate technologies.
It is worth noting that none of the tested materials was able to reverse the effect
of bleaching on enamel roughness completely. This may be attributed to the negative
effect of the abrasive particles that are added to them to increase their mechanical
ability to remove plaque.[29 ]
[30 ]
Conclusion
Physical and chemical structural changes brought upon enamel must be a matter of serious
concern to any practitioner utilizing bleaching procedures for improved aesthetics.
Also, knowledge of the different remineralizing agents and their efficacy is useful
in enhancing treatment outcome and long-term patient satisfaction. The application
of CSSPF for 3 min, two times a day and for 7 days was able to significantly re-harden
acid-eroded enamel and greatly decrease its surface roughness.
