Keywords
in-office bleaching - home bleaching - artificial caries lesions - microhardness -
SEM
Introduction
Early enamel caries lesions on the labial surface of anterior teeth are considered
an oral health and aesthetic problem. These lesions are mostly seen following fixed
orthodontic treatment or as chronic lesions seen in high caries risk patients. The
treatment decision and the appropriate clinical management of these lesions depend
on caries activity assessment mainly and many other factors such as cavitation, cleansability,
caries risk, behavioral adherence, age, and dentition.[1]
[2] Active early enamel caries lesions are characterized by subsurface demineralization
at 200 to 300 µm depth, and enamel surface roughness with loss of shine. Regarding
the minimal intervention dentistry the first strategy of treatment focuses on diet
management and oral hygiene, while the second strategy focuses on reversing the lesions
by applying remineralizing agents such as fluorides and phosphopeptide compounds.
Both these strategies aim to prevent the caries lesions progression and to improve
their mechanical properties. However, the third strategy aims to mask or enhance the
lesions aesthetic appearance by dental bleaching, microabrasion, or the recently developed
resin infiltration technique.[2]
[3]
[4]
[5] On the other hand, the inactive early enamel carious lesions characterized by the
intact, remineralized smooth surfaces and high mechanical properties are frequently
associated with discolorations. This unesthetic appearance is commonly managed by
dental bleaching or microabrasion technique.[1]
[6]
Dental bleaching is an oxidation process, the bleaching agents generate free radicals
that penetrate through enamel into the dentin and convert the high molecular chromophores
into smaller molecules leading to whiter appearance.[7]
[8] Dental bleaching procedures are applied using two techniques; in-office bleaching
and home bleaching using various concentrations of hydrogen peroxide (HP) and carbamide
peroxide gels (CP).[9]
Since bleaching agents may change the value of the enamel visibly, many studies showed
a satisfactory camouflage effect of bleaching agents on post orthodontic white spot
lesions and artificial caries lesions as well.[10]
[11]
[12]
[13] However, the application of bleaching agents to an already demineralized enamel
tissue is a controversial issue for researchers and dental practitioners. Some studies
reported a significant reduction in the microhardness of demineralized enamel lesions
after bleaching, while others demonstrated no significant effect or even an increase
in the microhardness values. But most of these studies were conducted under in vitro conditions with different study designs. and low concentrations of bleaching agents
were used, claiming that high concentration may cause further demineralization and
compromise the mechanical properties of these mineral-depleted lesions.[5]
[14]
[15]
[16]
[17]
To the best of our knowledge, the effect of different concentrations of both in-office
and home bleaching agents on the microhardness and surface morphology of the demineralized
enamel lesions has not been published. Therefore, in the present in vitro study we aimed to evaluate both the high and low concentrations of HP and CP bleaching
agents effects effect on the microhardness of artificially demineralized enamel under
intra-oral simulating conditions. Also, we aimed to qualitatively evaluate the enamel
surface morphological micro-changes of the demineralized enamel after bleaching. The
null hypotheses are: (1) bleaching agents have no significant effect on the microhardness
of the demineralized enamel lesions, and (2) the the bleaching agents type and concentration
have no impact on the surface morphology.
Materials and Methods
Ethical approval of this study was obtained from Istanbul University Animal Experiments
Local Ethics Committee under the number of 2016/08. The bovine teeth were obtained
from animals cut independently from this research.
Specimens Preparation
A total of 224 sound bovine permanent incisors were stored in 0.1% Thymol solution
for 1 week before processing to cut enamel slabs 5× 4 × 4 mm by a Precision Saw (IsoMet
1000, Buehler, Lake Bluff, Illinois, United States) under continuous water irrigation.
The prepared enamel specimens were embedded in methyl methacrylate resin blocks. The
surface of each specimen was grounded and polished using waterproof silicon carbide
papers (1,200–2,400 grit; Allied High-Tech Product Inc, Compton, California, United
States) attached to the polishing machine (Buehler MetaServ 250, Düsseldorf, Germany).
Subsequently, acid-resistant nail varnish was used to create enamel windows 4 mm ×
4 mm.
Artificial Caries Lesions Formation
In the present study, the chemical protocol was used to create subsurface carious
lesions artificially because it is a rapid, reliable, and low-costing test. The demineralization
solution used was composed of 50 mM acetate buffer solution; it contains 1.28 mM Ca
(NO3)2 × 4H2O, 0.74 mM NaH2PO4 × 2H2O, and 0.03 ppm F at pH 5.0 for 16 hours (Sigma Aldrich, Steinheim, Germany).[18] Each sample was immersed in 20 mL of the solution at 37°C. After the demineralization
process, the specimens were washed thoroughly with distilled water and air-dried using
the triple syringe.
Experimental Design
The demineralized enamel specimens were divided according to the bleaching agents
type and concentration (n = 56). Group I: office bleaching 35% hydrogen peroxide (Whiteness HP Maxx, FGM Dental
Products, Joinville, Santa Catarina, Brazil); Group II: home bleaching 16% carbamide
peroxide (Whiteness Perfect, FGM Dental Products, Joinville, Santa Catarina, Brazil)
and Group III: home bleaching 22% carbamide peroxide (Whiteness Perfect; Group IV:
nonbleached demineralized enamel ([Table 1]).
Table 1
The materials used in the study
|
Material
|
Composition
|
Batch number
|
Manufacture
|
|
Whiteness HP Maxx 35%
|
35% hydrogen peroxide, dye, glycol, thickening agent, and distilled water
|
110116
|
FGM Dental Products, Joinville, Brazil
|
|
Whiteness Perfect 16%
|
16% carbamide peroxide, water, potassium nitrate, sodium fluoride, and glycol.
|
080316
|
FGM Dental Products, Joinville, Brazil
|
|
Whiteness Perfect 22%
|
22% carbamide peroxide, water, potassium nitrate, sodium fluoride, and glycol
|
120116
|
FGM Dental Products, Joinville, Brazil
|
Bleaching
Group I (In-office Bleaching 35% HP)
The bovine enamel samples with artificial carious lesions in GI were treated by in-office
bleaching technique. 35% HP bleaching gel (Whiteness HP Maxx, FGM Dental Products,
Joinville, Santa Catarina, Brazil) was applied on the demineralized enamel surface
in a 2-mm thickness layer. To mimic the clinical applications the bleaching gel was
applied twice, each session lasts for 20 minutes. bleaching gel was activated by LED
light curing unit until the color has changed. At the end of each session the specimens
were thoroughly rinsed with distilled water for 30 seconds before being immersed in
an artificial saliva solution for 14 days.
Group II (Home Bleaching 16% CP)
The enamel samples in this group were treated by at-home bleaching technique for 14
days to imitate the clinical indications. 16% CP (Whiteness Perfect) was applied on
the demineralized enamel samples for 4 h/day. Approximately 1-2 mm thickness layer
of the bleaching gel was applied in the acrylic bleaching trays and adapted well over
the models. During bleaching period, the models were kept in a wet enviornment at
37°C in the incubator. After each bleaching session, the specimens were washed, dried,
and stored in the artificial saliva solution until the next day.
Group III (Home Bleaching 22% CP)
For this group, the same methodology applied in GII was followed. 22% CP (Whiteness
Perfect) bleaching gel was used in the trays for 1 h/day for a period of 14 days.
After each bleaching session, the specimens were washed, dried, and stored in an artificial
saliva solution until the next session.
Artificial Saliva Preparation
To simulate the oral environment and standardize the study conditions in our protocol,
all specimens were immersed in the artificial saliva solution at 37°C in the incubator
for 14 days. The samples were stored in distilled water during the microhardness test.
The prepared artificial saliva solution contains 42 mMol C8H16NaO8, 4.6 mMol HK2O4P, 16 mMol NaCl, 564 µMol Cl2Mg, and 29 µg C14H18N2O5 at pH = 7.[19]
Enamel Microhardness Measurement (Vickers Test)
The enamel surface microhardness was measured by the Vickers microhardness (Innovatest,
Manual Impression XT Hardness Testing Instrument, Software Version 1.07, Hollanda).
The elongated pyramid indentor with a specific constant load of 300 g was applied
over the enamel surface for 15 seconds.[20] The microhardness measurements were calculated for each sample at sound bovine enamel
(baseline), artificial caries lesion (demineralized), and after bleaching successively.
The built-in scaled microscope measured the indentation’s diagonal length, and Vickers
test values were calculated. Three indentations were placed in the enamel surface
and the distance between the indentations was approximately 50 µm. The average value
was counted for each specimen.
Scanning Electron Microscope
The enamel surface microtopographic features and changes during the current study
were analyzed by the scanning electron microscope (Quanta FEG 450 SEM; FEG company,
Hillsboro, Oregon, United States). Three samples from each group were analyzed at
the sound bovine enamel (baseline), artificial caries (demineralized), and after bleaching.
The randomly chosen samples were dehydrated using the direct technique recommended
by Janda et al.[21] First, all specimens were immersed in 50, 60, 70, and 99% ethyl alcohol solutions
subsequently for 20 minutes, then in 99% ethyl alcohol for 1 hour.[22] The presence of a thick layer of dentin underlying the enamel made the samples more
difficult to dry out. Therefore, samples were again immersed in 99% ethyl alcohol
for 24 hours. After that, gold sputtering at 100 Angstrom (A°) thickness was performed
and the samples were scanned at 2,000X to 20,000X magnification. Scanning electron
microscope (SEM) images were evaluated qualitatively.
Statistical Analysis
Data were analyzed by the Statistical Package for the Social Sciences (SPSS 21.0,
IBM Corporation, Armonk, New York, United States) software program. Mean and standard
deviation (SD) were calculated for continuous variables. The normality of the variables
was analyzed using the Shapiro-Wilk test. For the parametric groups, the one-way analysis
of variance (ANOVA) test followed by Post Hoc Tukey’s and Fisher’s least significant
difference (LSD). The variables distributed categorically or discretely were analyzed
by using Wilcoxon for the Paired test and Kruskal-Wallis test for multiple groups.
Tow-sided p-values were considered statistically significant at p ≤ 0.05.
Results
Enamel Microhardness Test
[Table 2] shows the mean and SD of Vickers microhardness values. All study groups showed a
statistically significant reduction in the mean microhardness values VHN of the sound
bovine enamel demineralization and artificial caries creation (p < 0.05). The evaluation of microhardness revealed a reduction of 78% for GI, 71%
for GII, 76% for GIII, and 75% for the control group. However, no significant difference
had been noticed between the microhardness value of the control group and the demineralized
enamel after bleaching (p > 0.05). On the contrary, a slight increase in the microhardness values had been
noticed after bleaching. The increasing percentage of the microhardness values was
13% for GI, 6% for GII, and 2% for GIII. GI presented the highest microhardness increase
with the statistically non-significant difference compared with GII and GIII (p > 0.05).
Table 2
The mean and standard deviation (mean ± SD) of the Vickers microhardness values of
all groups at the different stages (sound enamel, demineralized enamel, and bleached
enamel)
|
Group
|
|
Sound enamel
Mean ± SD
|
Demineralized enamel
Mean ± SD
|
Bleached enamel
Mean ± SD
|
|
Abbreviations: CP, carbamide peroxide; GI, group I; GII, group II; GIII, group III;
GIV, group IV; H2O2, hydrogen peroxide; SD, standard deviation.
Note: One-way ANOVA p < 0.05 significant.
|
|
GI = 35% H2O2
|
n = 56
|
304.63 ± 34.19
|
68.39 ± 29.12
|
77.42 ± 31.07
|
|
GII = 16% CP
|
n = 56
|
312.67 ± 38.84
|
73.85 ± 29.02
|
74.82 ± 29.31
|
|
GIII = 22% CP
|
n = 56
|
316.84 ± 35.96
|
89.73 ± 23.91
|
91.57 ± 30.75
|
|
GIV = control group
|
n = 56
|
313.92 ± 36.77
|
77.54 ± 39.49
|
|
|
p-Value between groups
|
|
0.633
|
0.072
|
0.092
|
SEM Analysis
The enamel surface microchanges representative micrographs are illustrated in
[Fig. 1]
. The intact bovine enamel specimens viewed under 2,000X magnification revealed homogeneous
and slightly rough surface integrity following the enamel natural crystal structure
and the study methodology ([Fig. 1A]). Considering the enamel surface quantitative evaluations after demineralization,
SEM images viewed at 20,000X magnification presented a rougher surface and enlargement
in the interprismatic area along with the dissolution of the peripheries of the prism
sheath (type III enamel etching pattern;
[Fig. 1B]
). However, after bleaching, the SEM images under 20,000X magnification demonstrated
dissolution in the periphery of the prism sheath, rough surfaces, and a wide interfractional
area, representing the same etching pattern of demineralized enamel but in a more
prominent manner (
[Figs. 1C–E]
). The high and low concentrations of the bleaching agents showed the same demineralization
pattern.
Fig. 1 SEM images analysis. (A) Sound enamel, a homogeneous and slightly rough surface is observed under 2000X magnification.
(B) Demineralized enamel; the porous surface represents partial dissolution of the aprismatic
layer and enlargement of the interprismatic spaces. Slight dissolution of the prismatic
core is observed under (20,000X) magnification. (C) GI; substantial dissolution of the aprismatic layer mainly in the interprismatic
spaces with moderate dissolutions of prismatic core (20,000X). (D) GII; considerable dissolution of the aprismatic layer associated with increased
porosity and moderate dissolution of the interprismatic area (20,000X). (E) GIII; mild dissolution of prismatic core and peripheries areas with enlargement
of the interprismatic spaces (20,000X). SEM, scanning electron microscope.
Discussion
Bleaching early enamel caries lesions manifested as WSLs is broadly evaluated in research,[5]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17] but the dilemma about its efficacy and safety is still present. The surface microhardness
which represents the mechanical properties of the dental hard tissues and their mineral
content,[15] is the most commonly used test. Since detecting a square surface reveals more clear
traces, Vickers test was chosen instead of the Knoop test in our protocol to measure
the microhardness.[23] In the present study, the effects of two types of bleaching agents applied in different
concentrations and techniques on the microhardness of artificial caries lesions were
evaluated.
The bleaching agents used in the this study are; in-office bleaching 35% HP and at-home
bleaching 16% CP and 22% CP. The chosen products have a neutral pH value which is
necessary for the whitening safety. Home bleaching products contain remineralizing
agents which are missing in the office bleaching agents. Both types showed no significant
effect on the microhardness. However, a slight increase in the mean VNH had been investigated
in all bleaching groups (GI, GII and GIII) with no significant difference between
study groups. Thus, our first hypothesis was accepted.
These findings agree with many studies providing evidence that bleaching could camouflage
WSLs without affecting their mineral content and microhardness values.[16] Furthermore, significant remineralization and improved mineral content were obtained
especially when fluoridated bleaching gel was applied.[15]
[24] These outcomes contrast with results showing significant microhardness reduction
obtained following at-home and laser-assisted office bleaching of WSLs.[25]
The slight increase of the microhardness of artificial caries lesions after bleaching
in the present study is thought to be an accumulative result of many factors, including
the bleaching agent’s content, pH value, and study design. The nonsignificant increase
in the microhardness had been noticed in all study groups. It is suggested that in
GII and GIII (16 and 22% Whiteness Perfect), the potassium nitrate and sodium fluoride
content of the bleaching gels could have attributed to microhardness increase. It
was already demonstrated that adding remineralizing agents such as CPP-ACP paste,
fluoride, and nano-biomaterials to bleaching gels enhances the mineral deposition,
induces fluoride acquisition and prevents irreversible changes on enamel without altering
the whitening efficacy.[16]
[24]
[26]
[27]
[28]
[29]
[30] While, in GI (35% HP Whiteness HP Maxx) the microhardness increase is thought to
be related to the short application time of the bleaching gel and the early contact
with saliva. Artificial saliva, following the literature, can boost the remineralization
and increase enamel microhardness subsequently.[15]
[25]
[31] Another reason to support these findings is supposed to be the neutral pH value
of the bleaching gels. The literature had shown less demineralization, microhardness
reduction, and enamel surface roughness in neutral pH agents compared with those with
lower pH values.[16]
[27]
The qualitative evaluation of SEM images revealed surface porosities, roughness, dissolution
in the prism sheath periphery, and wide interprismatic area after enamel demineralization.
More porous surfaces, wider interprismatic area, and further dissolutions in the aprismatic
surfaces with grooves are identified after the bleaching regimen. These findings display
that HP and CP bleaching agents in different concentrations showed a similar demineralization
pattern to the artificial caries (type III) but in a more prominent form. Thus, the
second hypothesis was accepted. These results agree with the previous study presenting
enamel demineralization up to 50-µm depth after 10% CP bleaching gel application evaluated
by a computerized tomography technique.[32] However, it contrasts with CLSM study which reported that the increase in the demineralization
depth noticed after bleaching was not significant.[14]
The revealed structural changes of enamel and caries susceptibility associated with
bleaching in some studies were considered clinically negligible,[15]
[33] while, in other studies it is thought to be a consequence of saliva remineralization
property.[34]
[35] In the present study, the use of artificial saliva solutions during and after bleaching
might promoted mineral deposition and subsequently increased the demineralized enamel
microhardness after bleaching without altering the surface characteristics.
The clinical bleaching outcomes depend on several factors such as the bleaching agent
type, concentration, content, pH value, and the application technique. In the case
of in vitro studies, the enamel characteristics and structural discrepancies between
the natural and artificially prepared lesions may also contribute to the variation
of the results. Accordingly, in vitro studies limitations make these results confined, and emphasize the need of more in vivo and in situ studies.
Within the limitations of the present study, low and high concentrations of HP and
CP bleaching agents can be used safely for whitening early enamel caries lesions without
further compromisation of their mechanical and surface properties. However, taking
into considerations the hypomineralization nature and low mechanical properties of
these lesions compared to sound enamel, various contemporary minimally invasive treatment
options combined with bleaching can be recommended to enhance the microhardness recovery
and prevent further demineralization.
