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Citation: Llena, C.; Iglesias-Diaz, M.;
Ciscar-Muñoz, P.; Bataller-Martínez,
A.B.; Melo, M.; Sanz, J.L. Tooth
Bleaching of Discolorations Caused
by Hydraulic Cements in
Regenerative Endodontic Treatment:
A 3-Year In Vitro Study. Materials
2022,15, 7845. https://doi.org/
10.3390/ma15217845
Academic Editor: Francesco
Inchingolo
Received: 16 October 2022
Accepted: 4 November 2022
Published: 7 November 2022
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materials
Article
Tooth Bleaching of Discolorations Caused by Hydraulic
Cements in Regenerative Endodontic Treatment: A 3-Year
In Vitro Study
Carmen Llena , Manuel Iglesias-Diaz , Paula Ciscar-Muñoz, Ana Belén Bataller-Martínez, María Melo
and JoséLuis Sanz *
Department of Stomatology, Faculty of Medicine and Dentistry, Universitat de València, 46010 Valencia, Spain
*Correspondence: jsanzalex96@gmail.com
Abstract:
This study aimed to evaluate the color change caused by hydraulic cements after 3 years
in vitro
by simulating their use in regenerative endodontic treatment (RET) and to quantify the color
change after external bleaching with 40% hydrogen peroxide at 1, 6, and 12 months of follow-up.
Fifty teeth were treated simulating RET. Samples were distributed according to the hydraulic cement
to be used (n = 10 per group): negative control (no cement), ProRoot-MTA, MM-MTA, TotalFill
BC-RRM, or Biodentine. Three years after RET, two sessions of external bleaching with Opalescence
Boost were performed. The color was measured in the cervical and incisal halves of the teeth at
different time points: baseline, 3 years after performing RET, and after 1, 6, and 12 months after
bleaching. The
∆
L,
∆
a, and
∆
b were determined. A generalized linear model was used to compare
color considering group and time. The
∆
E
ab
and the
∆
E
00
were calculated and the acceptability in
color change was determined. Three years after RET, a reduction in lightness (negative
∆
L values)
was found in all groups. These values significantly increased 1 month after bleaching in all groups
(p< 0.05) and reversed at 6 months. One month after bleaching,
∆
E
00
values (color difference tolerance
(CIEDE2000)) ranged from very good (>3.6
≤
5.4) to excellent (>5.4). One year after bleaching, the
color reverted to values similar to those found 3 years after RET. All groups became darker after RET.
The color recovered and even improved compared with the baseline measurement after one month of
bleaching but did not remain stable over time.
Keywords: regenerative endodontic treatment; bleaching; hydraulic cements; in vitro; endodontics
1. Introduction
Regenerative endodontic treatment (RET) appeared as an alternative to traditional
apexification procedures for the treatment of necrotic immature permanent teeth, aiming to
achieve apical periodontitis healing, continued tooth root development, increased fracture
resistance, and improved survival [
1
]. The RET process consists of the removal of the
infected necrotic tissue, chemical disinfection and minor or instrumentation of the root
canal system, induction of bleeding into the root canal, and placement of a coronal barrier
with regenerative potential over the previously formed blood clot [2].
As a disinfection procedure, the use of tri-antibiotic pastes, which included minocy-
cline, was initially proposed. Due to the discoloration caused by minocycline, this com-
ponent was removed, leaving the antibiotic paste formed by the combination of cefaclor,
amoxicillin, or clindamycin. Recently, antibiotic pastes have been replaced by calcium
hydroxide for intracanal disinfection [3].
Hydraulic cements are a subgroup of bioceramic materials that contain ceramic crys-
tals, calcium silicate, calcium phosphate, hydroxyapatite, radiopacifiers, and other metal
oxides in their composition. They are biocompatible materials with osteoinductive poten-
tial, antimicrobial activity, and positive influence on cell proliferation and differentiation.
They are used in endodontic therapy as sealing cements, for the repair of perforations
Materials 2022,15, 7845. https://doi.org/10.3390/ma15217845 https://www.mdpi.com/journal/materials
Materials 2022,15, 7845 2 of 12
or resorptions, retrograde filling, as pulp cappers in vital pulp treatment, and as coronal
barriers in RET [4].
Mineral trioxide aggregate (MTA) was introduced in the field of endodontics as the
first hydraulic material based on calcium silicates for root repair. However, although it
exhibits great regenerative potential, it causes tooth discoloration [
5
]. It has been suggested
that the bismuth oxide, which is used as a radiopacifier, together with other oxides within
its composition are the cause of this discoloration [
6
]. Also, the interaction of MTA with
blood and collagen from dentin may contribute to discoloration [
7
,
8
]. This manifests as a
grayish staining of the tooth [
9
,
10
]. Also, contact with irrigant solutions such as sodium
hypochlorite, which form sodium bismuthate and bismuth subcarbonate, as well as contact
with chlorhexidine contribute to discoloration caused by MTA [11].
Several authors have evaluated tooth discoloration
in vitro
by comparing different
MTA-based materials and other calcium silicate-based hydraulic cements in RET in the
presence and absence of blood, some of which conclude that the presence of blood did not
influence tooth discoloration [
12
]. However, other authors found greater discoloration in
the presence of blood [
8
,
13
]. In both circumstances, MTA caused greater discoloration than
the tricalcium silicate-based cement Biodentine.
Tooth discoloration can negatively affect quality of life, especially when treating ante-
rior teeth, and particularly because affected patients are usually young. Tooth whitening is
a simple and conservative procedure that has been shown to be effective for the treatment
of dental discolorations derived from RET. In a systematic review conducted in 2021 [
14
], a
series of
in vivo
studies were evaluated. Antibiotic pastes were used as intracanal medica-
tion, and MTA or Portland cement was used as the coronal barriers. All the
in vivo
studies
were single case reports or case series, in which bleaching was performed with hydrogen
peroxide, carbamide peroxide, or sodium perborate placed inside the pulp chamber, with
mixed results in terms of bleaching efficacy and duration over time. In a study comparing
Biodentine with MTA-based materials, a significant color improvement was found in the
Biodentine group immediately after bleaching [12].
Most of the studies available in the literature that evaluated the dental discoloration
caused by hydraulic cements used in RET were carried out with short follow-up times [
6
].
Likewise, the color change was immediately evaluated after bleaching and scarce infor-
mation is available in the medium and long term [
14
]. This justifies the present
in vitro
study, whose objectives were (1) to evaluate, using a spectrophotometer, the discoloration
potential of different hydraulic cements simulating their use in RET, three years after their
application; and (2) to quantify the color change after two sessions of external bleaching
with 40% hydrogen peroxide and the color variation after one, six, and twelve months
after bleaching.
2. Materials and Methods
For the present study, single-rooted human teeth extracted for periodontal reasons and
with an extraction period of no more than one year were used. The study was approved by
the Ethics Committee of the Universitat de València with reference number H152078413452.
The donors signed a written informed consent form beforehand.
2.1. Sample Selection and Preparation
All the teeth were visualized under 10
×
magnification, and two radiographs were
taken to confirm the radicular status. Teeth with caries, restorations, structural alterations
(cracks, fractures, and fissures), developmental anomalies, calcifications, staining, structural
color alterations, or previous root canal treatment were excluded. A sample of 50 intact
teeth was obtained. The teeth were kept in 0.1% thymol for 48 h and externally cleaned of
organic debris, stains, or calculus and then placed in a saline solution for preservation in
individual Eppendorf tubes for each specimen.
A transversal cut of the root apex was made with a diamond disk 10 mm from
the cementoenamel junction (CEJ) to simulate an open apex. Subsequently, an access
Materials 2022,15, 7845 3 of 12
cavity was opened using a diamond burr. Root canal shaping was then performed using
RECIPROC
®
blue R50 files (VDW, Frankfurt am Main, Germany) (25 mm, 8% taper) with
an endodontic motor (Reciproc Silver; VDW, Frankfurt am Main, Germany); following
the clinical sequence recommended by the manufacturer. An amount of 2.5% Sodium
hypochlorite (NaOCl) was used as an irrigant between each file. The final irrigation was
performed with 2.5% NaOCl, followed by 0.9% saline, 17% EDTA, and saline again. Finally,
the root canal was dried with paper points (#50). The apex was sealed with wax.
2.2. Regenerative Endodontic Treatment Simulation
To simulate RET, calcium hydroxide (Ultracal XS; Ultradent, South Jordan, UT, USA)
was placed inside the root canal, sealing the access cavity with a cotton pellet and a
temporary cement (Cavit; 3M Espe St Paul, MN, USA). Teeth were stored at 37
◦
C in a
100% humidity environment for 4 weeks. After the storage period, the temporary filling
was removed, and the root canal was washed with 10 mL of saline. Finally, the canal was
left filled with saline. At 6 mm from the CEJ, 1 mm of Teflon was placed, and 4 mm of
the hydraulic material under observation was placed over it, according to the distribution
shown in Table 1. Then, a humid cotton pellet and temporary cement (Cavit) were placed
over it, and the teeth were kept for 48 h in an environment of 100% humidity at 37
◦
C.
After this time, the temporary filling was removed and the access cavity was sealed with an
A1 shade Luna composite resin (SDI, Victoria, Australia) preceded by 37% orthophosphoric
acid etching and the application of a universal adhesive system (Zipbond Universal; SDI,
Victoria, Australia). The teeth were preserved in individual Eppendorf tubes with a saline
solution for the duration of the study, which was changed every month.
Table 1. Study groups and their composition and characteristics.
Composition Application
G1 Control negativo No cement. Cotton pellet. -
G2
ProRoot MTA White
(Dentsply Maillefer,
Ballaigues, Switzerland)
Powder: bismuth oxide, tricalcium silicate,
dicalcium silicate, calcium dialuminate, and
calcium sulfate dehydrated.
Liquid: distilled water
Manually mixed
powder/liquid (1:3 ratio)
G3
MM-MTA (Micro-Mega sur
MedicalExpo, BESANCON
Cedex, France).
Powder: tricalcium silicate, dicalcium silicate,
tricalcium aluminate, bismuth oxide, calcium
sulfate dehydrate, and magnesium oxide.
Liquid: calcium carbonate
Automixed
G4
TotalFill BC RRM (FKG
Dentaire, La Chaux-de-Fonds,
Switzerland).
Paste: Calcium silicate, zirconium oxide,
tantalum oxide, calcium phosphate
monobasic, and fillers
Pre-mixed material and ready
to apply
G5
Biodentine (Septodont,
Saint-Maur-des-Fossés,
France).
Powder: tricalcium silicate dicalcium silicate,
calcium carbonate, iron oxide,
and zirconium oxide.
Liquid: Water, calcium chloride, and soluble
polymer (polycarboxylate).
Automixed
2.3. Tooth Whitening Process Simulation
Three years after the RET simulation procedure, the coronal surfaces of the tooth
samples were polished with a prophylaxis brush and an abrasive paste. Subsequently, 40%
hydrogen peroxide in gel format was applied (Opalescense Boost; Ultradent, Madrid, Spain)
on the coronal surfaces of the tooth samples with a microbrush (three 20-min applications,
removing the whitening agent between each application with a thorough rinsing with
water). After one week, the application of the whitening agent was repeated following the
same protocol.
Materials 2022,15, 7845 4 of 12
2.4. Tooth Color Evaluation
To evaluate change in tooth color, a white silicone base was made for each group with
the individual imprint of each sample, which allowed the teeth to always be placed in the
same position. A 1-mm thick thermoplastic splint with two perforations was made for each
tooth, one in the coronal-cervical portion and the other in the coronal-incisal portion, both
5 mm in diameter, with the aid of a circular scalpel.
For the color measurement, a Vita Easyshade spectrophotometer (Germany) was used,
which provides the chromatic coordinates of the color in the CIELab space (L, a*, b*).
L stands for the amount of black or white in a color, a* stands for the amount of red or
green in a color, and b* stands for the amount of yellow or blue. The device was calibrated
after the evaluation of every 10 samples. Each measurement was repeated until the same
measurement values were obtained twice.
The color measurement was performed before performing RET simulation (baseline),
3 years after RET simulation, and 1, 6, and 12 months after the bleaching procedure.
2.5. Statistical Analysis
The L a* b* values at each time of the study were recorded in an Excel sheet for
analysis. The
∆
L (difference in lightness values between two time points),
∆
a (difference in
red or green values between two time points), and
∆
b (difference in yellow or blue values
between two time points) were calculated for each group between the baseline values and
the different times of the study, as schematized in Figure 1. The three variables were related
by calculating the
∆
E
ab
(chromatic distance in the CIELab space between two time points),
according to the formula:
∆Eab = ((La−Lb)2+ (aa−ab)2+ (ba−b*b)2)1/2
The ∆E00 (CIEDE2000) was also calculated, according to the formula:
∆E00 = [(∆L0/kLSL)2+ (∆C0/kcSc)2+ (∆H0/kHSH)2+ RT(∆C0/kcSc)(∆Hv0/kHSH)] 1/2
Materials 2022, 15, x FOR PEER REVIEW 4 of 12
rinsing with water). After one week, the application of the whitening agent was repeated
following the same protocol.
2.4. Tooth Color Evaluation
To evaluate change in tooth color, a white silicone base was made for each group
with the individual imprint of each sample, which allowed the teeth to always be placed
in the same position. A 1-mm thick thermoplastic splint with two perforations was made
for each tooth, one in the coronal-cervical portion and the other in the coronal-incisal por-
tion, both 5 mm in diameter, with the aid of a circular scalpel.
For the color measurement, a Vita Easyshade spectrophotometer (Germany) was
used, which provides the chromatic coordinates of the color in the CIELab space (L, a*,
b*). L stands for the amount of black or white in a color, a* stands for the amount of red
or green in a color, and b* stands for the amount of yellow or blue. The device was cali-
brated after the evaluation of every 10 samples. Each measurement was repeated until the
same measurement values were obtained twice.
The color measurement was performed before performing RET simulation (baseline),
3 years after RET simulation, and 1, 6, and 12 months after the bleaching procedure.
2.5. Statistical Analysis
The L a* b* values at each time of the study were recorded in an Excel sheet for anal-
ysis. The ΔL (difference in lightness values between two time points), Δa (difference in
red or green values between two time points), and Δb (difference in yellow or blue values
between two time points) were calculated for each group between the baseline values and
the different times of the study, as schematized in Figure 1. The three variables were re-
lated by calculating the ΔEab (chromatic distance in the CIELab space between two time
points), according to the formula:
ΔEab = ((La-Lb)2 + (aa-ab)2 + (ba-b*b)2) ½
The ΔE00 (CIEDE2000) was also calculated, according to the formula:
ΔE00 = [(ΔL′/kLSL)2 + (ΔC′/kcSc)2 + (ΔH′/kHSH)2 + RT(ΔC′/kcSc)(ΔHv′/kHSH)] ½
Figure 1. Study protocol flowchart.
Figure 1. Study protocol flowchart.
The CIEDE2000 formula allows a better correlation between color difference and visual
tolerance (perceptibility and acceptability) [15].
All calculations were performed for both the incisal and cervical halves of the teeth.
The SPSS 28.0 statistical package (IBM, Chicago, IL, USA) was used. Two-way repeated
measures ANOVA (analysis of variance) followed by Bonferroni posttest was performed
Materials 2022,15, 7845 5 of 12
to analyze the
∆
L,
∆
a,
∆
b,
∆
Eab, and
∆
E
00
, taking into account the treatment groups and
evaluation periods. Differences were considered significant when p< 0.05.
3. Results
3.1. Inicisal Half
In Figure 2A, a box plot is shown with the median and interquartile range (IQR) of
the
∆
L value between the baseline and each of the subsequent color measurements. In all
groups, there was a reduction in lightness after the three-year follow-up with no significant
differences between groups (p> 0.05). TotalFill and Biodentine groups showed the lowest
reduction, around 4 points in both groups, while in the rest it was around 6.
Materials 2022, 15, x FOR PEER REVIEW 5 of 12
The CIEDE2000 formula allows a better correlation between color difference and vis-
ual tolerance (perceptibility and acceptability) [15].
All calculations were performed for both the incisal and cervical halves of the teeth.
The SPSS 28.0 statistical package (IBM, Chicago, IL, USA) was used. Two-way repeated
measures ANOVA (analysis of variance) followed by Bonferroni posttest was performed
to analyze the ΔL, Δa, Δb, ΔEab, and ΔE00, taking into account the treatment groups and
evaluation periods. Differences were considered significant when p < 0.05.
3. Results
3.1. Inicisal Half
In Figure 2A, a box plot is shown with the median and interquartile range (IQR) of
the ΔL value between the baseline and each of the subsequent color measurements. In all
groups, there was a reduction in lightness after the three-year follow-up with no signifi-
cant differences between groups (p > 0.05). TotalFill and Biodentine groups showed the
lowest reduction, around 4 points in both groups, while in the rest it was around 6.
A
Materials 2022, 15, x FOR PEER REVIEW 6 of 12
B
C
Figure 2. (A) Variations in ΔL (lightness) in the incisal halves. Boxplots showing the median values
and IQR of the baseline and subsequent color measurements. * p < 0.05. (B) Variations in Δa (red–
green distance) in the incisal halves. Boxplots showing the median values and IQR of the baseline
and subsequent color measurements. * p < 0.05. (C) Variations in Δb (yellow–blue distance) in the
incisal halves. Boxplots showing the median values and IQR of the baseline and subsequent color
measurements. * p < 0.05.
Figure 2. Cont.
Materials 2022,15, 7845 6 of 12
Materials 2022, 15, x FOR PEER REVIEW 6 of 12
B
C
Figure 2. (A) Variations in ΔL (lightness) in the incisal halves. Boxplots showing the median values
and IQR of the baseline and subsequent color measurements. * p < 0.05. (B) Variations in Δa (red–
green distance) in the incisal halves. Boxplots showing the median values and IQR of the baseline
and subsequent color measurements. * p < 0.05. (C) Variations in Δb (yellow–blue distance) in the
incisal halves. Boxplots showing the median values and IQR of the baseline and subsequent color
measurements. * p < 0.05.
Figure 2.
(
A
) Variations in
∆
L (lightness) in the incisal halves. Boxplots showing the median values
and IQR of the baseline and subsequent color measurements. * p< 0.05. (
B
) Variations in
∆
a (red–
green distance) in the incisal halves. Boxplots showing the median values and IQR of the baseline
and subsequent color measurements. * p< 0.05. (
C
) Variations in
∆
b (yellow–blue distance) in the
incisal halves. Boxplots showing the median values and IQR of the baseline and subsequent color
measurements. * p< 0.05.
One month after bleaching was performed, a significant increase in lightness was
found in all groups (p< 0.05). In the ProRoot MTA group, the
∆
L was significantly higher
than all other groups (p< 0.05) at 9 units above the baseline value. Only the control group
and the MM-MTA group did not reach a lightness value equal to or higher than the baseline
value (the
∆
L had negative values, which means that the lightness value after bleaching
was lower than the baseline).
Six months after bleaching, lightness values decreased in all groups, with a significantly
higher
∆
L in the ProRoot MTA group compared with the MM-MTA and the control group
(p< 0.05). Twelve months after bleaching, the MM MTA group showed the greatest
reduction in lightness, around 11 units, showing a significant difference compared with the
ProRoot MTA group (p= 0.02).
Within each group, lightness values were similar 3 years after RET simulation and
12 months after bleaching (p> 0.05), and significantly lower than baseline values in all
groups (p< 0.05). Figure 2A shows that the
∆
L values were negative in all groups, indicating
that the value of L (lightness) was lower 12 months after bleaching compared with the
baseline value.
Variations in
∆
a (red–green distance) showed no significant differences (p> 0.01) either
between groups at the different times of the study, or within each group throughout the
follow-up period (Figure 2B). MM-MTA and Biodentine were the groups that showed the
greatest variability at all times of the study.
At all times of the study, a reduction of the b component (yellow-blue distance) was
found, as can be seen in Figure 2C, in which all the boxes are in negative values. No
significant differences were found between the groups at the different times of the study or
within each group during the follow-up period (p> 0.05). The greatest variability was found
in the MM MTA and Biodentine groups, especially at 6- and 12-months post bleaching.
Materials 2022,15, 7845 7 of 12
Regarding the
∆
E
00
and
∆
E
ab
, in all groups, three years after RET simulation, there
was a darkening above the acceptable range (
∆
E
00
> 1.8 and
∆
E
ab
> 2.7). One month
after bleaching, a very effective (
∆
E
00
> 3.6 and
∆
E
ab
> 5.4) to excellent (
∆
E
00
> 5.6 and
∆
E
ab
> 8.1) result was found in all groups primarily at the expense of an increase in
lightness (Figure 2A). At the end of the follow-up period, all groups showed a color
reversion, returning to
∆
E
00
values similar to those found three years after RET (
∆
E
00
in
the last column of Table 2). From the lowest to the highest level of acceptability, the groups
were ordered as follows: MM MTA< control < Biodentine < Totalfill < ProRoot.
Table 2. Variations in incisal ∆E values.
Baseline/3 Years 3 Years/1 Month
Post Bleaching
3 Years/6 Months
Post Bleaching
3 Years/1 Year Post
Bleaching
Baseline/1 Year
Post Bleaching
∆E00 ∆Eab ∆E00 ∆Eab ∆E00
Control 4.23 7.73 4.57 6.82 4.63
ProRoot MTA 2.70 4.03 8.48 9.44 3.03
MM MTA 5.40 8.63 4.13 6.14 4.38
TotalFill 3.16 5.20 5.73 7.03 2.77
3.2. Cervical Half
In the cervical half, 3 years after RET simulation, lightness (
∆
L) remained similar to
baseline values in all groups (median
∆
L close to 0). After bleaching, lightness increased
significantly in all groups, compared with the baseline value and to the value 3 years after
RET simulation (p< 0.05), although without significant differences between them (p> 0.05).
6 months after bleaching, values were similar in all groups to those obtained after RET
simulation (p> 0.05) (Figure 3A).
∆
a values (red–green distance) remained without significant differences between the
different study groups and within each group at the different color measurement times
(p> 0.05) (Figure 3B).
The same was the case for the
∆
b values (blue–yellow distance), except for the TotalFill
group, where the value of b* was significantly reduced at 12 months after bleaching
compared with the value at three years after RET simulation (Figure 3C).
Using the
∆
E
00
and the
∆
E
ab
values, the level of acceptability in color change through-
out the study was determined. In all groups, the
∆
E
00
values obtained were within the
limits of very good or excellent acceptability 1 month after bleaching, fundamentally at
the expense of an increase in lightness (Figure 3A). Six and twelve months after bleaching,
∆
E
00
values reverted. Comparing the baseline values before RET with the values 1 year
after bleaching, from lowest to highest level of acceptability the groups were ordered as
follows: MM-MTA < Control < Biodentine < ProRoot < TotalFill.
∆
E
00
and
∆
E
ab
values
were similar to those obtained three years after RET simulation (Table 3).
Table 3. Variations in cervical ∆E values.
Baseline/3 Years 3 Years/1 Month
Post Bleaching
3 Years/6 Months
Post Bleaching
3 Years/1 Year Post
Bleaching
Baseline/1 Year
Post Bleaching
∆E00 ∆Eab ∆E00 ∆Eab ∆E00
Control 4.23 7.73 4.57 6.82 4.63
ProRoot MTA 2.70 4.03 8.48 9.44 3.03
MM MTA 5.40 8.63 4.13 6.14 4.38
TotalFill 3.16 5.20 5.73 7.03 2.77
Materials 2022,15, 7845 8 of 12
Materials 2022, 15, x FOR PEER REVIEW 8 of 12
A
B
Figure 3. Cont.
Materials 2022,15, 7845 9 of 12
Materials 2022, 15, x FOR PEER REVIEW 9 of 12
C
Figure 3. (A) Variations in ΔL (lightness) in the cervical halves. Boxplots showing the median values
and IQR of the baseline and subsequent color measurements. * p < 0.05. (B) Variations in Δa (red–
green distance) in the cervical halves. Boxplots showing the median values and IQR of the baseline
and subsequent color measurements. * p < 0.05. (C) Variations in Δb (yellow–blue distance) in the
cervical halves. Boxplots showing the median values and IQR of the baseline and subsequent color
measurements. * p < 0.05.
Δa values (red–green distance) remained without significant differences between the
different study groups and within each group at the different color measurement times (p
> 0.05) (Figure 3B).
The same was the case for the Δb values (blue–yellow distance), except for the Total-
Fill group, where the value of b* was significantly reduced at 12 months after bleaching
compared with the value at three years after RET simulation (Figure 3C).
Using the ΔE00 and the ΔEab values, the level of acceptability in color change through-
out the study was determined. In all groups, the ΔE00 values obtained were within the
limits of very good or excellent acceptability 1 month after bleaching, fundamentally at
the expense of an increase in lightness (Figure 3A). Six and twelve months after bleaching,
ΔE00 values reverted. Comparing the baseline values before RET with the values 1 year
after bleaching, from lowest to highest level of acceptability the groups were ordered as
follows: MM-MTA < Control < Biodentine < ProRoot < TotalFill. ΔE00 and ΔEab values were
similar to those obtained three years after RET simulation (Table 3).
Table 3. Variations in cervical ΔE values.
Baseline/3
Years
3 Years
/
1 Month Post
Bleaching
3 Years/6 Months Post
Bleaching
3 Years
/
1 Year Post
Bleaching
Baseline/1 Year Post
Bleaching
ΔE00 ΔEab ΔE00 ΔEab ΔE00
Control 4.23 7.73 4.57 6.82 4.63
ProRoot
MTA 2.70 4.03 8.48 9.44 3.03
MM MTA 5.40 8.63 4.13 6.14 4.38
TotalFill 3.16 5.20 5.73 7.03 2.77
Figure 3.
(
A
) Variations in
∆
L (lightness) in the cervical halves. Boxplots showing the median
values and IQR of the baseline and subsequent color measurements. * p< 0.05. (
B
) Variations in
∆
a (red–green distance) in the cervical halves. Boxplots showing the median values and IQR of the
baseline and subsequent color measurements. * p< 0.05. (
C
) Variations in
∆
b (yellow–blue distance)
in the cervical halves. Boxplots showing the median values and IQR of the baseline and subsequent
color measurements. * p< 0.05.
4. Discussion
Some authors report that the contact of the hydraulic materials used as a coronal
barrier with the intracanal blood clot, which is crucial in the RET process, is an essential
factor in tooth discoloration. This discoloration is often mainly attributed to the infiltration
of iron and other hemoglobin derivatives into the dentin and coronal barrier materials [
8
,
13
].
Other studies, however, found no difference in the level of discoloration with or without
contact with the blood of the hydraulic materials, especially when Biodentine was used [
12
].
In the present study, blood was not placed inside the root canal, since the intention was to
exclusively evaluate the effect of the hydraulic materials. In addition, a 3-year follow-up
was carried out, which would have caused a disintegration of the hematite that would have
interfered with the objective of the study.
As described in the literature, the use of antibiotic pastes as disinfectants, especially
if they contain tetracycline or synthetic derivatives of tetracycline such as minocycline or
doxycycline, results in a higher level of discoloration than if bi-antibiotic pastes, pastes
containing amoxicillin as a third antibiotic, or calcium hydroxide are used [
10
,
11
,
16
–
19
].
In the experimental model used in the present study, calcium hydroxide was used as
a disinfectant.
According to a recent systematic review in the present field of study, hydrogen perox-
ide, carbamide peroxide at generally high concentrations (above 35%), or sodium perborate
were used as bleaching agents in studies performing RET simulation. Bleaching products
were intracoronally applied (internal bleaching) or on the coronal surface of the teeth (exter-
nal bleaching). Follow-up times after bleaching ranged from 4 days to 3 weeks [
14
]. In the
present study, we opted for an external bleaching technique with 40% hydrogen peroxide
with two sessions of three applications each. We consider that if RET aims to form an
intracanal inflow of mesenchymal stem cells and other progenitor cells capable of forming
Materials 2022,15, 7845 10 of 12
a mineralized tissue that reinforces the dentinal root walls, [
20
,
21
] placing peroxides in the
pulp chamber of the tooth, even if there is a barrier material or a mineralized barrier has
been formed, could have an undesirable cytotoxic effect. Thus, we chose to test the effect of
an external bleaching technique [22].
To our knowledge, there are no studies that have evaluated the long-term effect
of hydraulic materials used as coronal barriers in RET on tooth color, nor is there any
information available on Totalfill regarding its potential discoloration effect and response
to tooth whitening procedures.
The outcome of tooth whitening in
in vitro
and
in vivo
studies with RET exhibits
different levels of effectiveness. Akbulut et al. [
14
] compared short-term discoloration
caused by ProRoot MTA, MM-MTA, and Biodentine. They found discoloration in all
groups after the application of the materials, with no significant differences between them.
After bleaching, the Biodentine group obtained significantly better results. The authors
attributed this result to the fact that the particle size of Biodentine is smaller than the
other materials, as well as its porosity and to the difference in the radiopacifier (zirconium
oxide in Biodentine and bismuth oxide in the other two materials). In another study, the
discoloration produced by ProRoot MTA and MM-MTA two years after their application
was compared. Their response to intracoronal bleaching with sodium perborate with a
6-week follow-up was also compared. The authors found a discoloration mainly at the
expense of a reduction in lightness and an increase in the b* component, with a slight
improvement in color after bleaching at the expense of an increase in lightness and a
reduction in the b* component [
23
]. The present study found a slight improvement in color
at the expense of an increase in lightness and a reduction in the b* component.
In the present study, it was found that three years after RET simulation, there was a
darkening of all teeth in both the cervical and incisal halves. After bleaching, all groups
achieved a significant improvement in color that reverted after 6 months. These changes
were mainly produced by variations in lightness. Only the MM-MTA group showed
significantly lower levels of lightness in the incisal half than the rest of the groups at
the end of the follow-up period. The b* component (yellow–blue distance) showed a
reduction in both the cervical and coronal halves, showing a shift towards blue. This may
be due to the removal of the dentin needed to access the root canals and to the effect of the
composite material used for the restoration, which was an A1 shade composite. The trend
of negative
∆
b remained similar after bleaching and throughout the follow-up period, with
no significant differences between the groups. The red–green distance (a* component) was
reduced in all groups, except in the ProRoot MTA group, which showed no significant
changes within each group throughout the study. Therefore, it should be noted that it is the
lightness component that has shown the greatest influence on the color changes described
in this study.
The combination of the three parameters was obtained by calculating the
∆
Eab [
24
].
The
∆
E
00
was also used, which incorporates the chrome and hue parameters proposed by
Munsell and is more precise for determining tolerance to color changes [
15
]. However,
both parameters can establish the ranges of perceptibility and acceptability in color change.
However, they have the limitation of having no sign, i.e., the absolute value does not
guide us towards an improvement or worsening of the color; therefore, to determine the
directionality, they must be evaluated according to the change in their components. In
the present study, discoloration could be seen in all groups above the acceptable range;
three years after the regenerative therapy procedure (
∆
E00 > 1.8 and
∆
Eab > 2.7), however,
all groups experienced an improvement in color after bleaching in the very effective or
excellent range (
∆
E00 > 3.6 and
∆
Eab > 5.4). This improvement was not maintained over
time, as can be seen in Table 1, the
∆
E
00
and
∆
E
ab
values decreased at 6 and 12 months
after bleaching, returning to values similar to those found after RET simulation. Altogether,
this indicates that bleaching was effective in all groups, but the results were not maintained
in the medium or long term. These results agree with those obtained in the literature [
1
].
Materials 2022,15, 7845 11 of 12
The results of the present study indicate that bleaching was effective in all groups, but the
results were not maintained in the medium and long term.
5. Conclusions
Within the limitations of the present
in vitro
study, it can be concluded that discol-
oration was found in all groups, regardless of the hydraulic material used, and with no
differences with regard to the control group after three years of RET simulation. The color
improved after bleaching and was maintained in the short term but reverted in the medium
and long term. The lightness component had the greatest influence on both discoloration
and response to bleaching. Only the MM-MTA group showed significantly lower lightness
values than the other groups at the end of the follow-up period in the incisal half.
Author Contributions:
Conceptualization, C.L.; data curation, C.L.; formal analysis, C.L.; funding
acquisition, C.L.; investigation, M.I.-D., P.C.-M. and A.B.B.-M.; methodology, M.I.-D., P.C.-M. and
A.B.B.-M.; resources, C.L.; software, J.L.S.; supervision, C.L., M.M. and J.L.S.; validation, M.M.
and J.L.S.; visualization, M.M. and J.L.S.; writing—original draft, M.I.-D., P.C.-M. and A.B.B.-M.;
writing—review & editing, J.L.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement:
The study was conducted per the Declaration of Helsinki
and approved by the Institutional Review Board (or Ethics Committee) of the University of Valencia
(H152078413452). For studies involving humans.
Informed Consent Statement:
Informed consent was obtained from all subjects involved in
the study.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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