Basic Research—Technology
Spectrophotometric Analysis of Crown Discoloration Induced
by Various Antibiotic Pastes Used in Revascularization
Merve Akcay, DDS, PhD,* Hakan Arslan, DDS, PhD,† Bilal Yasa, DDS, PhD,‡ Fevzi Kavrık, DDS,*
and Elif Yasa, DDS, PhD§
Abstract
Introduction: Antibiotic pastes are used for disinfection
in regenerative endodontic procedures. This study evalu-
ated the crown discoloration induced by various antibi-
otic pastes including the mixture of metronidazole and
ciprofloxacin with minocycline, doxycycline, amoxicillin,
or cefaclor. Methods: Seventy extracted bovine incisors
were sectioned to obtain a standardized root length of
10 mm above the facial cementoenamel junction. After
pulp tissue removal, irrigation with sodium hypochlorite
and the placement of temporary filling material and
cotton pellet were performed from the apical aspect.
The specimens were then randomly divided into 7
groups (n = 10 for each group), and each group received
the following antibiotic paste fillings: no filling (control
group), calcium hydroxide, double antibiotic paste
(DAP), triple antibiotic paste (TAP) with minocycline,
TAP with doxycycline, TAP with amoxicillin, and TAP
with cefaclor. Spectrophotometric readings were
obtained on the buccal surfaces of the crown on day 1
to week 3 after filling, and the DE value was calculated.
Data were analyzed with 2-way analysis of variance and
the Tukey post hoc tests (P = .05), and the human
perceptibility threshold was set to 3.7. Results: TAP
with minocycline, doxycycline, and cefaclor induced
more coronal discoloration compared with the control
group (P < .05). The control, calcium hydroxide, and
DAP groups showed no color changes exceeding the
perceptibility threshold at all time points. Conclusions:
The results indicated that all antibiotic pastes, except
DAP, induced crown discoloration. (J Endod
2014;40:845–848)
Key Words
Amoxicillin, calcium hydroxide, cefaclor, discoloration,
doxycycline, minocycline, regenerative, triple antibiotic
paste
From the Departments of *Pediatric Dentistry,
†
Endodontics, and ‡Restorative Dentistry, _Izmir Katip Çelebi
University, _Izmir, Turkey; and Department of Restorative
Dentistry, Sifa University, _Izmir, Turkey.
Address requests for reprints to Dr Hakan Arslan, Depart-
ment of Endodontics, Faculty of Dentistry, _Izmir Katip Çelebi
University, Izmir, 35620, Turkey. E-mail address: hakan.
arslan@ikc.edu.tr
0099-2399/$ - see front matter
Copyright ª 2014 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2013.09.019
JOE — Volume 40, Number 6, June 2014
R evascularization of necrotic pulps has become an alternative conservative treatment
option to the apexification procedure for immature permanent teeth. Revasculari-
zation enables thickening of the root walls by mineralized tissue and continuing phys-
iological root development (1, 2). One of the most important stages of the
revascularization procedure is the disinfection of the root canal system. To effectively
eliminate infection from the root canal system, several investigators have used
antibiotic pastes (2–5).
The traditional triple antibiotic paste (TAP) consisting of ciprofloxacin, metroni-
dazole, and minocycline was developed by Hoshino et al (1). Several reports have
confirmed the good antimicrobial properties of this mixture in infected root canals
(1, 6, 7). Furthermore, Gomes-Filho et al (8) investigated TAP over various experi-
mental periods and found it to be biocompatible. Despite these positive features, several
case reports have shown that minocycline causes visible crown discoloration (9, 10).
Recently, antibiotic alternatives to minocycline have been proposed for use in
combination with ciprofloxacin and metronidazole, including amoxicillin, cefaclor,
and doxycycline (2, 4, 5, 11, 12).
No known studies have compared the effect of the different antibiotic combina-
tions used for regenerative procedures on crown discoloration. Therefore, this study
used spectrophotometric techniques to assess the coronal discoloration potential of
different antibiotic combinations of ciprofloxacin and metronidazole with minocycline,
amoxicillin, cefaclor, and doxycycline. The null hypothesis of this study was that the
tested antibiotics would not affect the color of the crown.
Materials and Methods
The sample size was calculated considering 80% power and a significance level of
0.05 using data (effect size = 4.5) obtained from the study by Lenherr et al (13).
Although according to the data of this study, 14 teeth were sufficient for the analysis
(n = 2), a worst-case scenario was proposed with a 0.48 effect size. According to
the worst-case scenario, the sample size was calculated as 70 considering 83% power
at a significance level of 0.05. Seventy bovine maxillary incisors were collected and
disinfected by immersion in 0.5% Chloramine-T solution (Merck, Darmstadt,
Germany) for 48 hours. Each specimen was sectioned using a bur to obtain a standard-
ized root length of 10 mm above the facial cementoenamel junction (CEJ). The bucco-
lingual and mesiodistal dimensions at the CEJ and the thickness between the root canal
wall and external root surface were measured using electronic digital calipers and
recorded for each specimen. On the basis of the sum of these 3 measurements, teeth
with similar measurements were distributed equally across the 7 groups. Statistical
analysis by 1-way analysis of variance (ANOVA) confirmed no significant differences
among the groups in terms of their buccolingual dimensions (P = .230), mesiodistal
dimensions (P = .921), and root canal wall thickness (P = .788).
All the procedures, including the removal of pulp tissue, irrigation, placement of
antibiotic pastes, and placement of cotton pellet and temporary filling material, were per-
formed from the apical aspect to avoid disruption of the intact crown and to prevent cor-
onal microleakage (Fig. 1A). The pulp tissue was removed, and the root canal was
irrigated with 10 mL 5.25% sodium hypochlorite to remove any remaining pulp tissue;
10 mL 17% EDTA was added for 2 minutes followed by 10 mL distilled water to simulate
clinical irrigation. After the root canal space was dried using cotton, the pulp chamber
Spectrophotometric Analysis of Crown Discoloration 845
Basic Research—Technology
Figure 1. (A) A schematic presentation of apically performed procedures. Temporary filling material was placed first. A cotton pellet, tested antibiotic paste, and
wax were then placed, respectively. (B) A representative image of the analysis of crown color of the tooth using a standardized circular strip.
was filled with temporary filling material (META Biomed Co Ltd,
Cheongju, Korea) from the pulp chamber ceiling up to 2 mm below
the CEJ. A cotton pellet was placed loosely on the temporary filling ma-
terial up to the facial CEJ (Fig. 1A). The specimens were then filled ac-
cording to the following groups (n = 10):
1. Control group: The tooth specimens in this group were left empty.
2. Calcium hydroxide group: The specimens in this group received cal-
cium hydroxide (Spotdent, Izmir, Turkey) mixed with distilled water.
3. Double antibiotic paste (DAP) group: The specimens in this group
received equal portions of metronidazole (Eczacıbası, Istanbul,
Turkey) and ciprofloxacin (Biofarma, Istanbul, Turkey) mixed
with distilled water (a powder/liquid ratio of 3:1).
4. TAP with minocycline group: The specimens in this group received
equal portions of metronidazole,ciprofloxacin, and minocycline
(Ratiopharm, Ulm, Germany) mixed with distilled water (at a pow-
der/liquid ratio of 3:1).
5. TAP with doxycycline group: The specimens in this group received
equal portions of metronidazole, ciprofloxacin, and doxycycline
(Deva, Istanbul, Turkey) mixed with distilled water as mentioned
previously.
6. TAP with amoxicillin group: The specimens in this group
received amoxicillin (Bilim,Istanbul, Turkey), metronidazole,
and ciprofloxacin.
7. TAP with cefaclor group: The specimens in this group received ce-
faclor (Sanovel, Istanbul, Turkey), metronidazole, and ciprofloxacin
The apical openings were sealed with sticky wax and all samples
were stored at 100% humidity in an incubator at 37
C for 3 weeks.
Tooth Color Assessment
Color measurements were recorded immediately after tooth prep-
aration, on day 1, and at weeks 1, 2, and 3 after placement of the medi-
cation. The color of each specimen was assessed by the CIE Lab system,
which was defined according to the International Commission on
Illumination in 1967 and referred to as CIE Lab (Commission Interna-
846 Akcay et al.
tionale de L’Eclairage, 1978) in L*a*b* mode by using a spectropho-
tometer (Spectro Shade Micro; MHT, Milan, Italy) on the buccal
surfaces of the crown (Fig. 1B). A standardized circular strip with a
diameter of 6 mm was bonded to the buccal surface of the crown 2
mm above the CEJ to ensure that color measurement was performed
on the same region at every turn with a vertical angle. Before the
measurement, the spectrophotometer was calibrated according to the
manufacturer’s instructions. The color measurements were performed
3 times at each time point on a white background, and the mean of the 3
measurements was calculated.
According to the manufacturer’s recommendation, the spectro-
photometer was calibrated on the white calibration tile. L* represents
the value of lightness or darkness, a* represents the measurement along
the red-green axis, and b* represents the measurement along the
yellow-blue axis. The total color differences or distances between 2
colors (DE) were calculated using the following formula:
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
DE ¼ DL2 þ Da2 þ Db2 .
Statistical Analysis
The control and test groups were compared for differences in the
baseline mean L*, a*, and b* scores by using 1-way ANOVA. The groups
were compared for differences in mean DE at different time intervals using
2-way ANOVA. The human perceptibility threshold was set to 3.7 units to
determine which differences were clinically visible (14–16).
Results
The treatment groups did not have significantly different baseline
L* (P = .307), a* (P = .719), or b* (P = .697). The control, calcium
hydroxide, and DAP groups did not induce color changes exceeding the
perceptibility threshold at any time interval (Fig. 2A–G). The TAP with
minocycline group induced more coronal discoloration than the other
groups at all time points tested. TAP with minocycline, doxycycline, and
cefaclor induced severe color changes exceeding the perceptibility
JOE — Volume 40, Number 6, June 2014
 Basic Research—Technology

Figure 2. The color of teeth after 3 weeks according to the groups: (A) control group, (B) calcium hydroxide, (C) DAP, (D) TAP with minocycline, (E) with
doxycycline, (F) with amoxicillin, and (G) with cefaclor.

threshold from day 1 of evaluation. TAP with amoxicillin induced severe (metronidazole, ciprofloxacin, and minocycline) and found that mino-
color changes from week 1 of evaluation (Fig. 3). cycline caused tooth discoloration. The results of the aforementioned
The discoloration in the teeth increased with time. There were sta- studies confirm our results. The mechanisms by which TAP with mino-
tistically significant differences in the induced coronal discoloration be- cycline impacts on coronal tooth discoloration may relate to the binding
tween the initial color and the color observed on day 1 (P < .001), week of minocycline to the calcium ions via chelation (18).
1 (P = .001), week 2 (P < .001), and week 3 (P < .001). Limited data are available in the literature concerning the tooth
discoloration induced by antibiotic pastes, except for TAP with minocy-
cline, which has been studied previously. Interestingly, TAP with
Discussion
Despite the increase in the number of regenerative endodontic cases
reported in the literature (2–5, 9, 11), there is no standardized protocol
for regenerative procedures. Disinfection of the root canal is a common
procedure, and antibiotic pastes are used to achieve this objective.
Previous studies have shown that antibiotic pastes, including
minocycline, resulted in tooth discoloration (3, 9, 10). Thus, our study
aimed to assess the coronal discoloration potential of different antibiotic
combinations.
This is the first study to investigate coronal color alterations induced
by various antibiotic pastes. Within the limitations of this experimental
study, in acceptance of the null hypothesis, calcium hydroxide, DAP,
and TAP with amoxicillin did not induce clinically perceptible crown
discoloration. In rejection of the null hypothesis, TAP with minocycline,
doxycycline, or cefaclor induced crown discoloration exceeding the
perceptibility threshold 1 day after paste placement. TAP with amoxicillin
induced severe color changes from week 1 of evaluation.
The highest level of coronal discoloration occurred in the teeth
filled with TAP with minocycline at all time intervals. The degree of
discoloration increased with time and exceeded the perceptibility
threshold from day 1 of the evaluation. Nosrat et al (17) used a TAP
with minocycline for 4 weeks in immature central incisors. They noted
moderate discoloration after 6 years. Lenherr et al (13) used a bovine
tooth model to investigate the discoloration potential of TAP with min- Figure 3. The color changes at different time periods in the groups. Note that
ocycline and other endodontic materials. They showed that TAP with TAP with minocycline, doxycycline, amoxicillin, and cefaclor induced color
minocycline induced discoloration. Kim et al (9) tested 3 antibiotics changes exceeding the perceptibility threshold.

JOE — Volume 40, Number 6, June 2014 Spectrophotometric Analysis of Crown Discoloration 847
Basic Research—Technology
doxycycline, amoxicillin, or cefaclor also resulted in clinically visible
crown discoloration. However, DAP and calcium hydroxide did not
cause visible discoloration at any of the studied time points. However,
there are no previous study data to compare the results of TAP with
doxycycline, amoxicillin, or cefaclor and DAP. Lenherr et al (13) re-
ported that the calcium hydroxide dressings did not show any discolor-
ation or differ from the negative controls at any time point. This result is
in agreement with the results obtained in the present study.
A previous study used a cuboid enamel-dentine block to evaluate
tooth discoloration (13). However, this block does not imitate the
whole tooth. In the present study, all the procedures were performed
from the apical aspect to avoid disruption of the intact crown and to pre-
vent coronal microleakage. Temporary filling material and cotton pellet
were placed before the antibiotics were applied. Moreover, the color
analysis was conducted on the same region in each specimen using stan-
dardized circular strips. Despite its advantage, the present model has
limitations in fully standardizing tooth thickness. To eliminate this
limitation, the specimens were equally distributed across the groups
according to their buccolingual dimensions (P = .230), mesiodistal
dimensions (P = .921), and thickness (P = .788).
In the present study, bovine incisors were used to assess discolor-
ation by various antibiotic pastes. Schilke et al (19) reported that the
number of dentinal tubules in bovine coronal dentine did not signifi-
cantly differ from the dentine of human teeth. In the present study,
we assessed coronal discoloration in bovine incisors; therefore, the re-
sults of the aforementioned study suggest that bovine coronal dentine
may be suitable for the assessment of discoloration (13). Another
reason for the use of bovine teeth was that the pulp was removed
from the apical aspect; therefore, this was more predictable in the
bovine model.
Previous studies of root canal treatment and revascularization
treatment have studied the effects of antibiotic pastes left in the root
canal for various time periods (4, 20). Some studies reported that
the application of antibiotic pastes for short periods (24–48 hours)
is sufficient for the effective disinfection of the infected root dentin
(1, 6). In regenerative endodontic procedures, the pastes are left in
the canal for up to 3 weeks (5, 21). In a recent study, TAP with
minocycline and DAP were found to have detrimental effects on the
survival of stem cells with less than 20% viability (22). In another study,
antibiotic pastes were found to degrade superficial collagen and demin-
eralize radicular dentin (23). Thus, the application of antibiotics should
be limited if possible. Within the limitations of this study, all antibiotic
pastes, except DAP, induced crown discoloration. DAP did not induce
clinically visible coronal discoloration up to 3 weeks after placement.
Future studies should be conducted to compare the microbial efficiency
of various antibiotic pastes.
Acknowledgments
We would like to thank to Dr B€ €
ulent Ozkan for his statistical
recommendations.
The authors deny any conflicts of interest related to this study.
848 Akcay et al.
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