TITLE: Spectrophotometric analysis of crown discoloration induced by MTA- and
ZnOE-based sealers

  ABSTRACT:
Crown discoloration can be induced by root canal sealer remnants following root canal
treatment.

ABSTRACT.OBJECTIVE:
The aim of this study was to evaluate chromatic alterations in human tooth crowns
induced by a Mineral Trioxide Aggregate-based sealer (MTA Fillapex®
and a commonly used ZnOE-based sealer (Roth-811). The tested null hypothesis was
that the application of the materials did not induce clinically perceptible crown
discoloration (Ho: CIE color difference ΔE<3.7).

ABSTRACT.MATERIAL AND METHODS:
Forty five fully developed, intact, mandibular third molars were sectioned 1 mm
below the cemento-enamel junction. The pulp chambers were chemomechanically
debrided via the cervical access. The specimens were randomly assigned into three
groups Group 1: MTA Fillapex, Group 2: Roth 811, Group 3: Negative control
(unfilled) and immersed in individually marked vials containing distilled water up
to the cervix (37±1o C). The spectral reflectance lines were recorded by utilizing
a UV-VIS spectrophotometer equipped with integration sphere in the visual spectrum
at baseline, 1 week, 1 and 3 months after material placement. Data were
transformed into values of the CIE L*a*b* color system and the corresponding ΔE
values were calculated. Statistical analysis was performed using two-way mixed
ANOVA models, at p=0.05 level of significance. 

ABSTRACT.RESULTS:
A statistically significant increase in a* and b* chromatic parameters of the MTA
Fillapex Group was measured. However, ΔE values did not exceed the human eye
perceptibility threshold (set at ΔE<3.7) during the experimental period
(ΔEt3=2.88). In Roth-811 Group, a statistically significant decrease in L* and a
statistically significant increase in a* and b* chromatic parameters was measured,
during all observation periods. Resultant ΔE values exceeded the human eye
perceptibility threshold after 1 week (ΔEt1=5.65).

ABSTRACT.CONCLUSIONS:
Application of MTA Fillapex in tooth crowns resulted in minimal color alterations,
while Roth 811 induced severe discoloration, in vitro. It could
be suggested that, in terms of aesthetics, the use of MTA Fillapex appears to be
favorable.

BODY.INTRODUCTION:
Poor aesthetic appearance of endodontically treated teeth remains a challenging issue in
clinical dentistry even nowadays. Interestingly, it has been reported that poor
aesthetic appearance of a treated tooth significantly affects the patients' quality of
life10.  A major etiological factor for the occurrence of local intrinsic staining, especially in
the cervical and middle third of the crown, is the presence of root canal filling
materials in contact with the coronal dentin of the pulp chamber16,17. In the long-term, core materials and sealers interact with dentin.
Any change to the optical and chromatic properties of the dentinal structure is likely
to cause an alteration in the outward appearance of the crown caused by its light
transmitting and reflecting properties16,17.  Apart from thorough pulp chamber debridement, a reduction of the coronal aspect of the
root canal filling below the clinical cervix is required for the prevention of
sealer-induced crown discoloration in the anterior aesthetic zone. However, sealer
remnants cannot be always thoroughly removed from the pulp chamber and sometimes are
present due to iatrogenic inadequate manipulations.  Despite the improvement of physicochemical, biomechanical and biological properties of
endodontic sealers, the appearance of coronal discoloration is still evident in daily
practice. Several laboratory studies have shown that some categories of sealers
including ZnOE and epoxy-based sealers are capable of inducing moderate to severe crown
discoloration17,24,25,29. In endodontics, ZnOE sealers are used
in clinical practice for many decades and are regarded as a gold standard in several
laboratory and clinical studies. These materials are regarded to be clinically
satisfactory, providing reasonable seal in the root canal system13. However, after setting reaction, the
formation of a weak porous mass leads to dissolution in contact with tissue
fluids8. Due to gradual
hydrolysis, the release of eugenol leads to long-lasting cytotoxicity and additional
potential for sensitization6.  Recently, Mineral Trioxide Aggregate (MTA) Fillapex® was introduced as a
new generation MTA-based sealer. The main concept for the development of MTA-based
sealers is the exploitation of the physical and biological properties of MTA such as
bioactivity12,
biocompatibility21 and hard
tissue conductivity22.  A developing amount of research data is becoming available upon MTA Fillapex, with
regard to its physical and biological properties2,13,14,23,26. Considering the increasing demands for
aesthetics, biomaterials should be chromatically stable, present optical properties
similar to dental structures and not exert staining effects to hard dental
tissues17. Recent reports showed
that both White and Gray MTA formulations are capable of inducing tooth
discoloration4,5. From that perceptive, it is mandatory for every new
MTA-based material to be tested in terms of aesthetic and color objectives. Currently,
there are no available studies investigating the potential of MTA Fillapex to induce
color alterations to dental tissues.  The aim of this study was to evaluate the chromatic alterations in human tooth crowns
induced by MTA Fillapex and Roth 811 root canal sealer. The null-hypothesis
(Ho) to be tested was that the application of the materials did not induce
clinically perceptible crown discoloration (Ho: CIE color difference
ΔE<3.7)19.

BODY.PREPARATION OF TEETH:
Forty five freshly extracted, fully developed, impacted and semi-impacted mandibular
third molars free of cracks, fractures, caries, abrasions and discoloration due to
systemic intrinsic causes were collected, according to the guidelines of good clinical
practice (Department of Oral and Maxillofacial Surgery, School of Dentistry, Ethical
Committee, Aristotle University of Thessaloniki, Greece).  Soft tissue was removed and the teeth were sectioned in the coronal third of the root
complex, 1 mm below the buccal cemento-enamel junction. Sectioning was performed with
the aid of a low-speed diamond-edge rotary saw microtome (Leica RM2255, Leica, Wetzlar,
Germany). Access cavity preparation was not performed. Pulps were extirpated with a
dental spoon and the internal axial walls of the pulp chambers were chemo-mechanically
debrided with Hedstrom files (No. #60-80) and 10 ml of sodium hypochlorite (2.5% w/w),
through the apical access. Gentle reaming of the internal axial walls was performed
through all directions. Between every file sequence, the pulp chamber was irrigated with
2 ml of sodium hypochlorite (2.5% w/w) (3 file changes x 2 ml=6 ml). A final rinse with
4 ml of sodium hypochlorite (2.5% w/w) was performed at the end of the debridement. The
pulp chambers of the specimens were finally washed with 5 ml sterile saline to remove
sodium hypochlorite remnants.  At the beginning of the experimental period, all crowns were transferred and stored in
individually marked polyethlylene tubes containing distilled water up to the cervix of
the crown in an incubator at 37±1o C. The teeth (N=45) were randomly assigned in one
experimental (n=15), one positive (n=15) and one negative control group (n=15). The
materials to be evaluated were MTA Fillapex (Angelus, Londrina, Brazil) (Group 1) and a
ZnOE based sealer, Roth 811 (Roth's International, Chicago, IL) (Group 2) (Figure 1). The sealers were mixed and prepared
according to the manufacturers' instructions and were placed into the pulp chambers via
the cervical access. A finger plugger was used to coat the internal axial walls with the
sealers. The apical access was sealed with a thin layer of glass-ionomer cement (Ketac
Cem Aplicap, 3M, Espe, Germany) in order to address microleakage and sealers solubility.
Negative controls (Group 3) were only instrumented and remained unfilled.   Figure 1 Study groups 1-3 Group Sample size (n) Materials under study Manufacturer Composition Group 1 15 MTA Fillapex Angelus, Londrina, Brazil resins (salicylate, diluting, natural), bismuth trioxide,
nanoparticulated silica, mineral trioxide aggregate, pigments Group 2 (positive control) 15 Roth 811 Roth's International, Chicago, IL Powder: Zinc oxide, bismouth
bicarbonate, barium sulfate, dehydrated tetraboric sodium Liquid:
Chemically pure eugenol Group 3(negative control) 15 Unfilled     

BODY.MEASUREMENT OF CROWN CHROMATIC ALTERATIONS:
A double-beam UV-Vis spectrophotometer equipped with integrating sphere was used
(UV-2401PC, Shimadzu Corporation, Kyoto, Japan) and a standardized mounting system was
developed, utilizing a previously validated experimental model17. Standard D65 illumination was chosen (Commission
Internationale de L'Eclairage 1978), as it corresponds approximately to the spectrum of
midday daylight in Western/Northern Europe. The spectrophotometer was linked to a
computer, which recorded the spectral reflectance curves of the crowns, in visual
spectrum (380-780 nm).  The obtained spectral curves in visual spectrum (380-780 nm) were transformed into L*,
a* and b* values of the perceptually uniform CIE L*, a*, b* color space using a
specialized computer software (Color Analysis UV-2401PC). L* values describe lightness,
which range from black (0) to white (100), while a* values represent red (+80a*) to
green (-80a*), and b* values represent yellow (+80b*) to blue (-80b*) color variations.
Total color differences (ΔE) were calculated according to the equation:  ΔE=
[(Li-L0*)2+(ai-a0*)2+(bi-b0*)2]1/2
  The proposed acceptance for color matching adopted in this study was at 3.7 ΔE units
(perceptibility threshold), beyond which the differences are clinically
perceptible19. In dental
science, when ΔE values are less than 1 unit, then color match occurs and any color
differences cannot be identified by independent observers in vitro27. However, color determination in
clinical dentistry may become complicated by adjacent anatomic structures and lighting
conditions. As a result, the proposed acceptance for color matching in dentistry has
been set to 3.7 units (perceptibility threshold), beyond which differences are
clinically visible19.  A standardized mounting system was developed for the customization and the
reproducibility of the crown's position. The cylindrical inner frame (diameter=2 cm,
inner height=0.2 cm) of the black bakelite sample assembly that the spectrophotometer
was equipped with, was filled with black, non-polychromatic, thermo-plasticized
silicone. The lingual surface of each crown was fixed within the silicone mass during
its setting phase in order to construct individualized specimen carriers. The same
individualized, silicone carrier for each specimen was used for measurements in all time
intervals. The specimens were positioned in the in the circular opening of the aperture
mask of the integrating sphere with the aid of an aligning system in order to ensure the
reproducibility of the measured surface.  The dimensions of the polychromatic beam that illuminated the sample were 7x7 mm; thus
the majority of the cervix and the crown surface was exposed and measured. The color
appearance of the buccal surfaces of the crowns was measured in order to simulate their
clinical appearance. Measurements were carried out by the same operator (I.M.) and at
ambient temperature of 23±1o C.  The spectrophotometer was calibrated at each time interval using BaSO4
reference. Measurements were performed prior to the placement of the materials
(baseline: t0) and consecutively 1 week (t1), 1 month
(t2) and 3 months (t3) after sealer placement. All measurements
were repeated twice and averaged. If the total color difference (ΔE) between 2
measurements taken in a row exceeded the threshold of 1 DE unit, new measurements were
obtained.  At the end of the 3rd month, three crowns of the experimental groups were
randomly selected and longitudinally sectioned vertically to the middle of their
mesio-distal dimension, on a bucco-lingual aspect, with the aid of a low-speed
diamond-edge rotary saw microtome (Leica RM2255, Leica, Wetzlar, Germany). Digital
images of the sectioned crown specimens were taken, in day-light conditions (Macro-Lense
100 mm, Canon EOS 1000D, Tokyo, Japan).

BODY.STATISTICAL ANALYSIS:
Sample size was calculated after power analysis based on the results of a pilot study,
according to the equation n=2(z1-α/2 +
zβ)2
σ2 {1+(m -
1)ρ}/mΔ
2 with ρ=0.5, m=4 (repeated measurements), σ2=4,
Δ=2.2, alpha=0.05, power=0.8 (the chosen value of Δ
was greater than the observed one in order to be close to the 3.7 threshold value and
analogously done for the σ
2 )17. Accordingly the
sample size to each group was calculated near to n=12 specimens per
group. In case of drop out during the experimental period, 3 specimens were added in
each group (n=15).  Two-way ANOVA with repeated measurements was used for data analysis of the values of CIE
L*, a*, b* chromatic parameters and the total color differences (ΔE). The significant
effects and interactions of the experimental factors were investigated with pairwise
between-group and within-group comparisons, which were conducted with Bonferroni's
method. The overall analysis was performed with SPSS software (version 16.0, SPSS Inc.,
Chicago, Ill, USA). The level of statistical significance was set at
p<0.05.

BODY.RESULTS:
Tables 1 and 2 present the mean L*, a*, b* and ΔE values of all groups, in all time
intervals respectively. With regard to L*, a* and b* parameters, MTA Fillapex caused a
statistically significant increase in a* (Δa*to-t3=1.043,
p<0.001) and b* (Δb* to-t3=2.292, p<0.001) values. L* values
remained stable. However, resultant ΔE values indicate that MTA Fillapex induced overall
color changes below the human perceptibility threshold, ranging between 2.12-2.88 units.
Roth 811 induced severe color changes exceeding the perceptibility threshold from the
1st week of evaluation (ΔE=5.65). A statistically significant increase in
a* (Δa*to-t3=-3,335, p<0.001) and b* (Δb*
to-t3=-4,014, p<0.001) values was observed, while L* values
statistically decreased (ΔL*to-t3=4,926, p<0.001). In Group 3,
L*, a*, b* values remained stable in all time intervals.   Table 1 CIE L*, a*, b* mean standard deviation values of Groups 1-3, in all experimental
periods     baseline 1 st 
week 1 st 
month 3 rd 
month   (CIE) L* parameter           Groups n L 0 L 1 L 2 L 3 MTA Fillapex 15 84.95(3.14) 85.36(2.54) 84.35(3.10) 84.91(2.61) Roth 811 15 84.21(1.98) 81.89(1.18) 79.38(1.82) 79.28(1.71) Negative control 15 84.55(2.22) 84.77(2.26) 84.48(2.34) 84.55(2.17)                             baseline 1 st  week 1 st  month 3 rd  month   (CIE) a*
parameter           Groups n a 0 a 1 a 2 a 3 MTA Fillapex 15 0.15(0.68) 0.57(0.67) 0.77(0.68) 1.19(0.65) Roth 811 15 0.01(0.79) 2.03(0.82) 2.71(1.00) 3.34(1.05) Negative control 15 0.42(0.74) 0.39(0.66) 0.38(0.73) 0.41(0.73)                             baseline 1 st
 week 1 st 
month 3 rd 
month   (CIE) b* parameter           Groups n b 0 b 1 b 2 b 3 MTA Fillapex 15 21.60(2.82) 23.36(2.21) 22.64(2.74) 23.89(2.35) Roth 811 15 21.26(2.33) 25.67(2.36) 24.76(2.72) 25.28(2.89) Negative control 15 20.62(3.11) 20.33(2.89) 20.49(2.94) 20.57(3.07) Within-group comparisons: a statistically significant differences when compared
to t 0 , b when compared to t 1 , c when compared to
t 2 , d when compared to t 3 Between-group comparisons: a statistically significant differences when
compared to Fillapex, B when compared to Roth 811   Table 2 Mean standard deviation ΔE values of Groups 1-3 in all experimental periods     1 st 
week 1 st 
month 3 rd 
month Groups n (ΔΕ 1 ) (ΔΕ 2 ) (ΔΕ 3 ) MTA Fillapex 15 2.13(1.18) 2.12(0.72) 2.88(1.49) Roth 811 15 5.65(2.34) 6.70(2.68) 7.37(3.02) Negative control 15 0.51(0.37) 0.61(0.30) 0.51(0.20) Within-group comparisons: a statistically significant differences when compared
to ΔΕ1, b when compared to ΔΕ2, c when compared to
ΔΕ3 Between-group comparisons: A statistically significant differences when
compared to MTA Fillapex, B when compared to Roth 811   The macroscopic examination of the sectioned specimens of Group 1 (MTA Fillapex) showed
stability of sealer color within its mass and absence of dentinal staining (Figure 2). On the contrary, in Group 2 (Roth 811) the
set sealer displayed a granular, grayish appearance. Dark orange dentinal staining
accompanied by varying leaching within tubules was evident, in areas where sealer
remnants were in contact with the axial walls of the pulp chamber (Figure 3).  Figure 2Appearance of Group 1 (MTA Fillapex) after 3 months. Color stability of MTA
Fillapex sealer within its mass and absence of dentinal staining  Figure 3Appearance of Group 2 (Roth 811) after 3 months. Granular, grayish appearance of
Roth 811 sealer and dark orange dentinal staining accompanied by varying leaching
within tubules

BODY.DISCUSSION:
MTA Fillapex is a newly developed MTA-based root canal sealer. According to the
manufacturer, its composition after mixture is basically MTA particles incorporated in a
matrix of salicylate resin, natural resin, bismuth and silica. Other proposed MTA-based
sealers consist of MTA with other additives, including HEMA, TEGDMA resins (lc-MTA) and
water-soluble polymers (ProRoot-Endo sealer)7,11. Recent data reported
that MTA Fillapex presents acceptable physical properties, antibacterial properties and
intracanal resistance to dislodgement similar to AH Plus2,23. The sealer
presents increased cytotoxicity during the setting reaction which decreases over
time26. After setting, the
sealer is capable of retaining alkaline pH levels due to calcium release, and presents
bioactivity stimulating hydroxyapatite crystal nucleation in human osteoblast-like cell
cultures26 and mineralization in
connective tissue14.  The rational for organizing this research study was the increasing publication of case
reports, in which the discoloring effect of MTA has been reported. Both Gray and White
MTA repair materials were capable of inducing coronal discoloration, when applied in
cases of root and furcal perforation, pulpotomy and pulp revascularization
procedures4,5. In addition, two recent laboratory studies confirmed the
previous clinical observations with the aid of instrumental colorimetry1 and visual spectrophotometry18. It has been proposed that metal oxides,
such as iron and manganese, could be responsible for the discoloring effects of Gray
MTA18. Moreover, recent data
have shown that the discoloring effects of White MTA have been attributed to its
progressive mass darkening due the presence of reduced black crystals of bismuth atoms
under light conditions28.  This laboratory study is the first to report tooth color alterations induced by MTA
Fillapex, utilizing the methodology of visual spectrophotometry. Within the limitations
of this experimental study, in acceptance of the null hypothesis, MTA Fillapex did not
induce clinically perceptible crown discoloration. In rejection of the null hypothesis,
Roth 811 induced fast and severe discoloration and exceeded the perceptibility threshold
1 week after sealer placement.  The results of this study indicated that the tested sealer had minimum potential to
induce color alterations in human teeth in vitro, since the measured
color alterations did not exceed the threshold of perceptibility set (ΔE<3.7). The
descriptive analysis of the CIE L*, a*, b* chromatic parameters showed a statistically
significant color change towards red and yellow after three months, while lightness was
not affected. The macroscopic findings confirmed that the sealer did not present color
changes within its mass, maintaining its yellowish color throughout the experimental
period. Dentinal staining was not evident as well. The increase of CIE a* and b* values
of the crowns from the first week of investigation may be attributed to alterations in
specimens' optical properties due to the physical presence of the sealer in the internal
dentinal surface.  Roth 811 sealer induced fast and severe discoloration, which was clinically perceptible
1 week following sealer placement. The chromogenic potential of ZnOE sealers has been
attributed to the unstable chemical bond between ZnO and eugenol. Even after the end of
the setting reaction, eugenol release leads to self-oxidation and becomes darker with
time. The results of this study are in complete accordance with results of previous
studies17,25,29.  Several methods have been proposed for the evaluation or measurement of sealer-induced
discoloration, including visual technique and computer analysis of digital
photos24,25,29. Inherent
objectivity and standardization difficulties may be improved by the use of a
spectrophotometer15. This
methodology has been reported as accurate and reliable in dentistry for quantitative
tooth color measurements17,20. A major advantage of visual
spectrophotometry is that tooth color measurement is based on the measurement of
spectral reflectance, which describes the total reflection of a sample in visual
spectrum17.  Random errors in this study were minimized by strict control of the environmental
factors along with multiple measurements and averaging. One of the main limitations of
instrumental color measurement, however, is posed by systematic errors that can be
attributed to variations in instrument geometrical design, metamerism and calibration
techniques27.  The comparison of absolute tristimulus values found in that study with other studies is
discouraged. Evaluation and comparison of total color differences (expressed by ΔE),
however, is well documented and considered as safe since differential measurement is
highly reproducible between instruments9. The results of this study are comparable to those of a previous
study which was conducted utilizing the same methodology and under the same experimental
conditions. After 3 months, non-perceptible total color differences (ΔE) of MTA Fillapex
were similar to the ΔE values of Guttaflow (Roeko, Coltene, Whaledent Ltd., Germany) and
Epiphany SE (Pentron Clinical Technologies, Wallingford, CT), indicating that they
likewise exert minimal chromogenic effects17.  The results of this study do not directly represent the in vivo tooth
discoloration potential of root canal sealers in good clinical practice. In this
laboratory study, the investigation of the discoloration potential of root canal sealers
was based on the generation of a "worst case scenario" by leaving a significant amount
of sealer in direct contact with the axial dentinal walls and several anatomical
features of the pulp chamber17.
However, the knowledge of the magnitude of the sealers' chromogenic potential indicates
that thorough cleaning measures are essential to prevent discoloration
postoperatively.  Regardless of the minimal staining effects of new generation root canal sealers
including MTA Fillapex, the clinician should always ensure thorough removal of sealer
remnants. Apart from basic properties, such as biocompatibility and good sealing
ability, it could be suggested that the chromogenic potential of sealers may also play
an important role in selecting proper root canal filling materials.

BODY.CONCLUSION:
Within the limitations of this experimental study, MTA Fillapex did not induce
clinically perceptible crown discoloration. Roth 811 induced fast and severe
discoloration and exceeded the perceptibility threshold 1 week after sealer placement.
Although the incorporated MTA has proved chromogenic potential, MTA Fillapex posed
minimal risk for potential staining effects. It could be suggested that, in terms of
aesthetics, the use of MTA Fillapex appears to be favorable.

Question: Compared to ZnOE-based sealer (Roth-811) what was the result of MTA Fillapex sealer on Chromatic alterations?
Evidence: vely. With regard to L*, a* and b* parameters, MTA Fillapex caused a
statistically significant increase in a* (Δa*to-t3=1.043,
p<0.001) and b* (Δb* to-t3=2.292, p<0.001) values. L* values
remained stable. However, resultant ΔE values indicate that MTA Fillapex induced overall
color changes below the human perceptibility threshold, ranging between 2.12-2.88 units.
Roth 811 induced severe color changes exceeding the perceptibility threshold from the
1st week of evaluation (ΔE=5.65). 
Answer: significantly decreased