REGENERATIVE ENDODONTICS

Miguel Angelo da Cunha Neto,

Antibacterial Efficacy of Triple DDS, MSc,* Jessica de Almeida
^lho, DDS, MSc,†
Coe
Antibiotic Medication With Karem Paula Pinto, DDS, MSc,*
rio Cardenas
Maricel Rosa
Macrogol (3Mix-MP), Cuellar, DDS, MSc,†
Maria Cristina Marcucci, PhD,‡
Traditional Triple Antibiotic Emmanuel Joa ~o Nogueira Leal
Silva, DDS, PhD,*
Paste, Calcium Hydroxide, and Flaviana Bombarda de Andrade,
DDS, PhD,† and Luciana Moura
Ethanol Extract of Propolis: An Sassone, DDS, PhD*

Intratubular Dentin Ex Vivo

Confocal Laser Scanning SIGNIFICANCE
Microscopic Study The use of antibiotic
medications in extremely high
concentrations was not more
effective for intratubular
disinfection than clindamycin-
ABSTRACT modified triple antibiotic paste
Introduction: This study aimed to assess the effectiveness of antibacterial activity of at the concentration
medications used in regenerative endodontic treatment. Methods: Sixty-seven dentin recommended by the
cylinders of single-rooted teeth were contaminated with a culture of Enterococcus faecalis American Association of
(ATCC 29212; American Type Culture Collection, Manassas, VA) for 5 days. Samples were Endodontists. Also, calcium
divided into 1 control group and the following experimental groups according to the medi- hydroxide and propolis
cation applied: traditional triple antibiotic paste (TAP), clindamycin-modified TAP (mTAP), showed great antibacterial
triple antibiotic medication with macrogol (3Mix-MP), clindamycin-modified 3Mix-MP efficacy.
(m3Mix-MP), calcium hydroxide (CH), and ethanol extract of propolis (EEP). After 14 days, the
medications were removed, and the samples were submitted to confocal laser scanning
microscopic analysis to quantify the percentage of viable bacteria. The distribution of data was
confirmed by the Shapiro-Wilk test. The Kruskal-Wallis and Dunn tests were used for inter-
From the From the *PROCLIN
group comparisons, and the Wilcoxon test was used for comparison between superficial and
Department, School of Dentistry, Rio de
deep antibacterial efficacy for the same medication. The level of significance was set at Janeiro State University, Rio de Janeiro,
P , .05. Results: 3Mix-MP and m3Mix-MP presented significantly higher antibacterial effi- Rio de Janeiro, Brazil; †Dentistry,
Endodontics, and Dental Materials
cacy compared with the other tested medications (P , .05), except for mTAP. mTAP was
Department, Bauru School of Dentistry,
more effective than TAP (P , .05). The antibacterial efficacy of EEP and CH did not differ ~o Paulo, Bauru, Sa
University of Sa ~o Paulo,
significantly from TAP and mTAP (P . .05). All medications showed effective antibacterial Brazil; and ‡Biosciences and Oral
Diagnosis Department, Institute of
action compared with the control group (P , .05). Conclusions: 3Mix-MP and m3Mix-MP, ~o Paulo State
Science and Technology, Sa
which present extremely high concentrations of antibiotics (1500 mg/mL), were not more University, Sa~o Jose
 dos Campos, Sa ~o
effective than mTAP at the concentration recommended by the American Association of Paulo, Brazil
Endodontists (5 mg/mL). Moreover, CH and EEP were as effective as TAP and Address requests for reprints to Dr
mTAP. (J Endod 2021;47:1609–1616.) Luciana Moura Sassone, School of
Dentistry, Rio de Janeiro State University,
Boulevard 28 de Setembro, 157–Pavilha ~o
KEY WORDS: Mario Franco Barroso, 2º Andar Vila
Isabel, Rio de Janeiro, RJ, Brazil.
3Mix paste; confocal laser scanning microscopy; endodontics; regenerative endodontic E-mail address: lucianasassone@gmail.
treatment; triple antibiotic paste com
0099-2399/$ - see front matter
Copyright © 2021 American Association
The occurrence of traumatic injury or infection in immature permanent teeth can lead to pulp necrosis and of Endodontists.
the interruption of root development, resulting in short roots, a wide apex, and thin and fragile dental https://doi.org/10.1016/
walls.1 In these situations, a widely recommended treatment has been revascularization, a regenerative j.joen.2021.07.014

JOE  Volume 47, Number 10, October 2021 Antibacterial Efficacy of Triple Antibiotic Medication 1609
therapy that aims to continue root
development, providing the thickening of
dentin walls, increasing the root length and
apical closure, and decreasing the risk of tooth
fracture.1,2
One of the main principles of
regenerative endodontic therapy is the
disinfection procedure.2 For this purpose, the
American Association of Endodontists (AAE)
and the European Society of Endodontology
recommend the use of calcium hydroxide (CH)
paste or antibiotic pastes composed of 2 or 3
different antibiotics.3,4 The traditional triple
antibiotic paste (TAP) is usually composed of
ciprofloxacin, metronidazole, and minocycline;
diluted in propylene glycol; and has been
shown to be more effective than double
antibiotic pastes (ie, metronidazole and
ciprofloxacin).5,6 A common modification in
TAP formulation is the replacement of
minocycline to clindamycin because of the
discoloration potential of the minocycline. This
clindamycin-modified TAP (mTAP) has also
shown great efficacy in endodontic
infections.7,8
Another antibiotic medication used in
regenerative endodontic therapy is the triple
antibiotic medication with macrogol (3Mix-
MP). It consists of a TAP with the addition of
macrogol, an inert vehicle that improves the
consistency of the medication, which is
indicated to be placed on the orifices of the
root canals.9,10 The inventor of the 3Mix-MP
reports that its “final preparation will be a soft
ball-like structure of 1 mm diameter”.9,10
Therefore, the medication could be easily
handled without touching the coronary dentin
walls, thus avoiding the discoloration caused
by the minocycline. However, a huge amount
of antibiotics must be used to achieve such
consistency. Studies have shown that
commonly used clinical concentrations of
antibiotic medications can exceed more than
200 times the limit stipulated by the AAE and,
therefore, harm the regenerative
therapy.3,11,12
In general, studies have shown
controversial results on which would be the
most effective medication.13 Although most of
the studies report that antibiotic pastes
present higher antibacterial efficacy than
CH,14–16 the CH seems to be less cytotoxic for
regenerative endodontic therapy than
antibiotic pastes.17,18 To overcome this
impasse, alternative intracanal medications
have been proposed, such as the use of
ethanol extract of propolis (EEP). The EEP is a
natural rich substance that shows similar
antibacterial efficacy as antibiotic medications
and great anti-inflammatory and antioxidant
properties, in addition to the stimulation of
stem cells.19
1610 Cunha Neto et al.
It is well-known that 1 of the main
mechanisms of bacterial resistance in
endodontic infections is the ability of
bacterial biofilms to penetrate and colonize
the dentinal tubules.20 To date, there are
no studies assessing the efficacy of
intratubular dentinal disinfection of the
3Mix-MP. Therefore, this study aimed to
compare the intratubular disinfection
efficacy of 3Mix-MP, clindamycin-modified
3Mix-MP (m3Mix-MP), TAP, mTAP, CH,
and EEP using confocal laser scanning
microscopy. The null hypothesis tested
was that there would be no difference in
the disinfection potential among the tested
medications.
MATERIALS AND METHODS
This study was approved by the local ethical
committee of Pedro Ernesto University
Hospital/Rio de Janeiro State University
(UERJ) (Certificate of presentation for ethical
appraisal: 18258819.9.0000.5259/Number:
3.619.523). Sample size calculation was
performed based on previous studies 21,22
using G*Power 3.1 software (Heinrich-Heine-
€sseldorf, Germany). For 6
Universit€at, Du
experimental groups, using an effect size of
0.25, an a error probability of 0.05, and a
power of 0.90, the resultant total sample
size was 60 (ie, 10 for each experimental
group). In addition, 7 teeth were used as
positive controls for intratubular bacterial
growth.
Sample Selection
Sixty-seven human single-rooted teeth were
selected using digital buccolingual and
mesiodistal radiographs out of a total of 90
teeth from the “Human Teeth Biobank” of the
School of Dentistry of UERJ. The teeth
presented fully formed roots and single root
canals, without root resorptions or root canal
calcifications.
Sample Preparation
The teeth were decoronized, and the apical
thirds of the roots were removed using a
double-sided diamond disc
0.10 ! 22 mm (KG Sorensen, Cotia, SP,
Brazil) attached to a low-speed handpiece
under water cooling in order to standardize
dentin cylinders with 8-mm length. The root
canals (ie, the inside diameter of the dentin
cylinders) were prepared using a Gates
Glidden drill size 4 (Dentsply Maillefer,
Ballaigues, Switzerland) to standardize a
diameter of 1.1 mm.23–25
Intratubular Bacterial
Contamination
The dentin cylinders were subjected to
subsequential ultrasonic baths with 1%
sodium hypochlorite (Biodin^amica, Ibipora ~,
PR, Brazil), 17% EDTA (Biodin^amica), and
distilled water for 10 minutes each; covered
with red nail polish (Colorama, Rio de Janeiro,
RJ, Brazil); and sterilized in an autoclave
(Cristofoli, Campo Mour~ao, PR, Brazil) at
121 C for 24 minutes. All subsequent
procedures were performed within a laminar
flow (Grupo Veco, Campinas, SP, Brazil).
The specimens were placed into
microtubes (Eppendorf, Hamburg, Germany)
with sterile brain-heart infusion culture medium
(Difco, Detroit, MI) and subjected to an
ultrasonic bath for 10 minutes for maximum
penetration of the culture broth into the
dentinal tubules.23 A culture of Enterococcus
faecalis obtained from the American Type
Culture Collection (Manassas, VA) (ATCC
29212) was used to contamination. The
bacterial suspension was adjusted to 1
McFarland scale, corresponding to 3 ! 108
colony-forming units per mL, using a
SF325NM spectrophotometer (Bel Photonics
do Brazil Ltda, Osasco, Brazil). Each dentin
cylinder was placed into another 1.5-mL
microtube (Eppendorf), which was filled with
1 mL of the E. faecalis suspension and
incubated at 37 C for 5 days. During this time,
serial periodic changes of the E. faecalis
suspension and culture medium were
performed, in addition to different
centrifugation cycles,26 to reproduce the
penetrability of the E. faecalis into the dentin
tubules following the previously established
protocol of Andrade et al.23
Intracanal Medications
The specimens were randomly assigned to 1
of the following groups, and the intracanal
medications were prepared accordingly:
 TAP:3 metronidazole, ciprofloxacin, and
minocycline (Santa Paula Pharmacy,
Araraquara, SP, Brazil); vehicle: propylene
glycol (Santa Paula Pharmacy); 1: 1: 1 ratio
of antibiotics; and concentration: 5 mg/mL
 mTAP:3 metronidazole, ciprofloxacin, and
clindamycin (Santa Paula Pharmacy);
vehicle: propylene glycol (Santa Paula
Pharmacy); 1: 1: 1 ratio of antibiotics; and
concentration: 5 mg/mL
 3Mix-MP:8,9 metronidazole, ciprofloxacin,
and minocycline (Santa Paula Pharmacy);
vehicles: macrogol (Solbase; Meiji, Tokyo,
Japan) and propylene glycol (Santa Paula
Pharmacy); 1: 1: 1 ratio of antibiotics; 1:1
ratio of vehicles; and concentration:
1500 mg/mL
JOE  Volume 47, Number 10, October 2021
  m3Mix-MP:8,9 metronidazole,
ciprofloxacin, and clindamycin (Santa Paula
Pharmacy); vehicles: propylene glycol
(Santa Paula Pharmacy) and macrogol
(Solbase); 1:1:1 ratio of antibiotics; 1:1 ratio
of vehicles; and concentration: 1500 mg/
mL
 CH paste:27,28 CH (Biodina ^mica), vehicle:
propylene glycol (Santa Paula Pharmacy),
and concentration: 3 mg/mL
 EEP:27,28 solid green propolis extracted
from the state of Parana  (Brazil), diluted in
pure ethanol (10%), and typified as Brazilian
Green Propolis (Marcucci et al29) using
high-performance liquid chromatography;
vehicle: propylene glycol (Santa Paula
Pharmacy); and concentration: 0.1 mg/mL
 Positive control (PC) group: contaminated
dentin cylinders without medication
The antibiotics, calcium hydroxide, and
macrogol were weighted using a digital
analytical balance (Ohaus Adventurer AR2140;
OHAUS, Parsippany, NJ), whereas the EEP
and propylene glycol were measured using
micropipettes. All medications were
manipulated at the time of application.
Before applying the medications, the
specimens were removed from the microtubes
and fixed on a sterilized stainless table. The
TAP, mTAP, and EEP were inserted into the
dentin cylinders using a hypodermic syringe
(100 IU) with a 30-G 5/16-inch needle
(8 ! 0.30 mm) (Solidor, Osasco, SP, Brazil)
until completely filled; a ball-like structure
(Supplemental Fig. S1 is available online at
www.jendodon.com) of a 1-mm diameter of
3Mix-MP and m3Mix-MP was inserted into the
dentin cylinders using a vertical condenser
(Golgran Ltda, S~ao Caetano do Sul, SP, Brazil),
and the CH was inserted into the cylinders with
a K-file size 40 (Dentsply Maillefer) until
completely filled. After insertion of the
FIGURE 1 – The percentage of viable bacteria of each group in the superficial, deep, and total areas of the specimens. Different capital letters indicate a statistically significant
difference between medications in the same area. Letters in bold show a statistically significant difference between the superficial and deep area for the same medication. Bars indicate
the median. Vertical dashes above the bars indicate the standard deviation.
JOE  Volume 47, Number 10, October 2021
medications, a temporary sealer (Coltosol;
Coltene, Rio de Janeiro, RJ, Brazil) was
applied at the ends of the dentin cylinders. The
specimens were stored in Petri dishes coated
with gauze soaked in distilled water and
incubated in a bacteriological oven for 14 days
at 37 C.26
Confocal Laser Scanning
Microscopic Analysis
After 14 days, the temporary sealer was
removed with a Hollemback 3S Carver
(SSWhite Duflex, Juiz de Fora, MG, Brazil), and
the medication was removed by irrigation with
5 mL sterilized distilled water. The specimens
were sectioned longitudinally using an Isomet
cutting machine (Isomet, Buehler, Lake Bluff,
IL) with a diamond disk cooled with sterilized
saline solution, and 1 of the halves was
randomly selected for analysis. The smear
layer produced was removed using 2.5 mL
17% EDTA (Biodynamics, Ibipora ~, PR, Brazil)
for 3 minutes followed by irrigation with
sterilized saline solution, and the specimens
were dried using absorbent paper.
The specimens were stained with 20 mL
of the LIVE/DEAD BacLight Bacterial Viability
Kit (Molecular Probes, Eugene, OR) for
15 minutes in a dark environment. This kit
contains the SYTO 9 dye, which marks viable
bacteria with a green pigment, and propidium
iodide dye, which marks dead bacteria with a
red pigment, thus allowing viable bacteria to be
easily identified. The specimens were
examined with an inverted Leica TCS-SPE
laser scanning microscope (Leica
Microsystems GmbH, Mannheim, Baden-
Wu €rttemberg, Germany) using a 40!
magnification lens in immersion oil by an expert
in confocal laser scanning microscopic
analysis blinded to the experiment. Images of
the specimen’s surface were obtained using
Antibacterial Efficacy of Triple Antibiotic Medication
23 sections with 1-mm depth and a 1024 !
1024 format.23,24,30 The respective excitation
and emission wavelengths were 480/500 nm
for SYTO 9 and 490/635 nm for propidium
iodide dye. Eight sequential images were
obtained from each sample, 4 near the main
root canal (superficial area) and 4 furthest from
the main canal (deep area) 23,24,30
(Supplemental Fig. S2 is available online at
www.jendodon.com), with the superficial area
ranging from 0- to 275-mm depth from the root
canal wall and the deep area ranging from 275-
to 550-mm depth. The first image obtained
from each sample was from the area 1A
followed by 1B; then images of the areas 2A
and 2B; and then 3A, 3B, 4A, and 4B
sequentially (Supplemental Fig. S2 is available
online at www.jendodon.com). For quantitative
analysis, all captured images were fragmented
into many layers and converted into TIFF
format by the Leica Application Suite
Advanced Fluorescence software (Leica
Microsystems GmbH). These images were
exported to the BioImageL v2.1 software
(www.bioImageL.com; Faculty of Odontology,
Malmo € University, Malmo
€, Sweden) to quantify
the percentage of viable bacteria (stained in
green) and nonviable bacteria (stained in
red).23 The image layers were used to
reconstruct the sections of the contaminated
dentin for evaluation of the amount and
distribution of the contamination. Each layer
was evaluated individually using the “surface
and volume distribution” function, and bacterial
viability data were obtained.23
Statistical Analysis
Statistical analysis was performed using
Prisma 8.3 software (GraphPad Software Inc,
La Jolla, CA). The results were calculated using
the values of percentage of viable cells from the
8 areas from each sample, totaling 80 values
1611
 per experimental group. The Shapiro-Wilk
normality test showed the absence of a normal
distribution. The Kruskal-Wallis test followed
by the Dunn test were used for comparisons
between medications, and the Wilcoxon test
was used for comparison between superficial
and deep areas for the same medication. The
level of significance was established at
P , .05.

RESULTS
The percentages of viable bacteria after
contact with the medications for 14 days
assessed in the superficial, deep, and total
areas of the dentinal tubules are presented in
Figure 1. The 3Mix-MP and m3Mix-MP
showed significantly higher antibacterial
efficacy compared with the other tested
medications (P , .05), except for the m-TAP,
in both evaluated areas. The mTAP was more
effective than the TAP in the deep and total
areas (P , .05). The EEP and CH presented
similar antibacterial efficacy to the TAP and
mTAP in the superficial, deep, and total areas
(P . .05). In the superficial and deep areas, the
antibacterial efficacy of the TAP did not differ
significantly from the PC (P . .05). Considering
the total area, all medications were
demonstrated to have an effective antibacterial
action compared with the PC (P , .05).
Comparing superficial and deep antibacterial
efficacy of each medication, statistical
difference was observed only in the m3Mix-MP
and mTAP groups, with less viable bacteria in
the deep areas (P , .05). Representative
confocal laser scanning microscopic images of
the groups are shown in Figures 2A–G and
3A–G, Supplemental Figure S3 (available
online at www.jendodon.com), and
Supplemental Figure S4 (available online at
www.jendodon.com).

DISCUSSION
This study aimed to evaluate the effects of
different medications indicated for regenerative
endodontic therapy (TAP, mTAP, 3Mix-MP,
m3Mix-MP, CH, and EEP) on intratubular
disinfection of root canals. The null hypothesis
was rejected because there were differences in
the antibacterial efficacy among the tested
medications.
The antibacterial efficacy was assessed
using E. faecalis biofilm, as performed by
previous similar in vitro studies.26,31 E. faecalis
is a gram-positive facultative anaerobic
bacterium that is tolerant to high pH levels and
presents several genes associated with drug
resistance to a wide range of antibiotic
FIGURE 2 – Representative confocal laser scanning microscopic images from the superficial areas of dentinal tubules medications.32 In addition, like most of the
(near the root canal). (A ) 3Mix-MP, (B ) m3Mix-MP, (C ) TAP, (D ) mTAP, (E ) CH, (F ) EEP, and (G ) PC. Green, viable bacteria related to endodontic infections, E.
cells; red, dead cells. The pictures represent areas of 275 ! 275 mm. The scale bar represents 20 mm.

1612 Cunha Neto et al. JOE  Volume 47, Number 10, October 2021
 faecalis can penetrate and colonize dentinal
tubules, which is an important mechanism of
bacterial resistance.19 For this reason,
confocal laser scanning microscopy was used,
which is an adequate technique to assess
microbial reduction inside dentinal tubules in
depth.23,26 Confocal lasers can deeply
penetrate in a substrate, up to 23 mm in soft
tissue and around 15 mm in the tubular dentin
(a great dentin mass when considering single-
rooted teeth), which interestingly means that
evaluation using a confocal microscope allows
3-dimensional analysis, making it possible to
visualize the architecture and spatial
distribution of the biofilm inside the dentinal
tubules in different planes of the dentin mass.30
The present results showed that all
tested medications were effective in the total
area of the dentinal tubules compared with the
PC (P , .05). 3Mix-MP and m3Mix-MP were
significantly more effective than the other
tested medications, except for mTAP. This can
be expected because of the high
concentration of 3Mix-MP and m3Mix-MP. To
date, the ideal concentration of antibiotic
medications for adequate disinfection of the
root canal system in regenerative endodontic
therapy is still controversial; some studies
reported a wide range varying from 0.01–
1000 mg/mL.11,19,33 At this point, it is
important to clarify the difference between
proportion and concentration. Both TAP
indicated by the AAE 3 and 3-Mix-MP
suggested by Hoshino and Takushige 9,10
have to be formulated with an equal proportion
of each antibiotic in a ratio of 1:1:1. On the
other hand, concentration is related to the ratio
between the antibiotics and vehicles used.
Although the recommendation of the AAE is a
concentration from 1 mg/mL to a maximum of
5 mg/mL for TAPs,3 Hoshino 10 indicates that
the preparation of 3Mix-MP should start with a
ratio antibiotic/vehicle of 7:1, and more
antibiotic powder must be added until
achieving a solid consistency of a soft rounded
ball-like structure of 1-mm diameter.9,10 In the
present study, the final concentration of 3Mix-
MP and m3Mix-MP was 1500 mg/mL.
This is the first study reporting the
concentration of 3Mix-MP manipulated as
recommended by Hoshino;10 however, it
seems to be consistent with previous studies
that reported that a concentration of 1000 mg/
mL is needed to create a thick antibiotic
paste.11,12 Although very low amounts of
3Mix-MP and m3Mix-MP are used compared
with the other antibiotic medications that
completely fill the root canals, the high
concentration could harm the regenerative
therapy. Different in vitro studies showed that
FIGURE 3 – Representative confocal laser scanning microscopic images from the deep areas of dentinal tubules high concentrations of antibiotic combinations
(furthest from the root canal). (A ) 3Mix-MP, (B ) m3Mix-MP, (C ) TAP, (D ) mTAP, (E ) CH, (F ) EEP, and (G ) PC. Green, can cause changes in the dentinal structure,
viable cells; red, dead cells. The pictures represent areas of 275 ! 275 mm. The scale bar represents 20 mm.

JOE  Volume 47, Number 10, October 2021 Antibacterial Efficacy of Triple Antibiotic Medication 1613
modifying or inactivating growth factors, and
can lead to deleterious effects on the survival of
pulp stem cells.11,12 Moreover, the present
results demonstrate that this huge
concentration of antibiotics did not result in a
greater effect on bacterial death compared
with the use of mTAP in a concentration of
5 mg/mL, which proves that higher
concentrations of antibiotic medications are
not necessary for adequate intratubular
disinfection. In addition, the use of mTAP
within the concentration recommended by the
AAE is detrimental for the survival of stem cells
from the apical papilla.3,12 The antibiotic
medication has to be efficient in the elimination
of the endodontic biofilm without being
cytotoxic to the precursor cells of the new
tissue and without stimulating an inflammatory
response that could hinder the regenerative
process. Higher concentrations of antibiotic
medication could damage the regenerative
therapy because the cytotoxicity of the
medication is directly related to its
concentration.12 It was shown that the use of
TAP and mTAP in concentrations above 6 mg/
mL can lead to the death of more than 50% of
stem cells from the apical papilla.
mTAP was shown to be as effective as
3Mix-MP and m3Mix-MP (P . .05) in
superficial, deep, and total areas, and more
effective than TAP in the deep and total areas
(P , .05). Also, a statistical difference in the
reduction of E. faecalis was observed only in
the clindamycin-modified groups (m3Mix-MP
and mTAP) (P , .05) when comparing the
superficial and deep areas. Clindamycin-
modified antibiotic medications have been
successfully used in regenerative endodontic
therapy and have been shown to be as
effective as triple antibiotic medications
containing minocycline.8,34,35 A recent in vitro
study reported that clindamycin was less
cytotoxic to dental pulp stem cells and
enhanced their angiogenic potential compared
with minocycline.7 Moreover, clindamycin has
a great potential to deeply penetrate dentinal
REFERENCES
1. Bose R, Nummikoski P, Hargreaves KA. Retrospective evaluation of radiographic outcomes in
immature teeth with necrotic root canal systems treated with regenerative endodontic
procedures. J Endod 2014;35:1343–9.
2. Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis:
new treatment protocol? J Endod 2004;30:196–200.
1614 Cunha Neto et al.
tubules,36 which can explain the best results of
the present study with this drug.
CH and EEP presented similar
antibacterial efficacy to TAP and mTAP in the
total area (P . .05). CH is 1 of the medications
recommended by the AAE and the European
Society of Endodontology for regenerative
endodontic therapy and presents high
antibacterial efficacy because of its alkaline pH
and the ability to degrade
lipopolysaccharides.3,4,37 Also, it was reported
that CH is less cytotoxic and induces less
inflammatory response than TAP.17 Moreover,
CH induces proliferation and great attachment
of apical papilla stem cells, which is 1 of the
main goals of regenerative endodontic
therapy.18 Previous studies comparing the
antibacterial efficacy between CH and TAP
show controversial results. Although several
studies report a higher intracanal antibacterial
efficacy for TAP,14–16 studies assessing
intratubular decontamination have shown no
significant differences and endorse the present
results.38
Propolis is a natural compound that
presents great antibacterial, antifungal, and
anti-inflammatory properties. Previous in vitro
studies also showed similar antibacterial
efficacy of EEP compared with CH and TAP
and a similar intratubular penetrability
compared with CH.27,28,39,40 In addition,
in vivo studies have shown that the use of EEP
in regenerative endodontic therapy results in
less inflammatory reaction and increased
bioactivity, with major tissue deposition on
dentin walls and stimulation of growth factors
and stem cells compared with TAP and
CH.4,41
In the present study, the antibacterial
efficacy of 3Mix-MP prepared as
recommended by Hoshino was assessed (ie,
the antibiotic medication has to present a final
solid moldable consistency to achieve a soft
rounded ball-like structure of 1-mm
diameter).9,10 The main limitation is that,
following this recommendation, 3Mix-MP was
prepared based on its visual consistency,
which defined the amount of antibiotic powder
to be used and, therefore, its final
concentration. Moreover, in the present study,
the medication was placed inside the root
canals because dentin cylinders were used;
therefore, the method described by Hoshino of
placing the medication on the orifices of the
root canals could not be replicated. Because
this is the first study assessing the intratubular
antibacterial efficacy of 3Mix-MP, further
studies are needed to evaluate its final
concentration, its cytotoxicity to the stem cells
of the apical papilla, and the intratubular
antibacterial efficacy of 3Mix-MP using
multispecies biofilm, taking into consideration
the polymicrobial nature of endodontic
infections.
Based on the present results, 3Mix-
MP and m3Mix-MP at 1500 mg/mL were
not more effective than mTAP at the
concentration recommended by the AAE.
Among the antibiotic medications assessed
in the present study, mTAP was shown to
be the most recommended because of its
high efficacy, concentration, and the
benefit of avoiding tooth discoloration.
Moreover, CH and EEP presented similar
antibacterial efficacy as TAP and mTAP
and are also good options as intracanal
medication.
ACKNOWLEDGMENTS
Supported by the Coordenaç~ao de
Aperfeiçoamento de Pessoal de Nível
Superior–Brasil (finance code 001).
The authors deny any conflicts of
interest related to this study.
SUPPLEMENTARY MATERIAL
Supplementary material associated with this
article can be found in the online version at
www.jendodon.com (https://doi.org/10.1016/
j.joen.2021.07.014).
JOE  Volume 47, Number 10, October 2021
 3. American Association of Endodontists. Clinical considerations for regenerative endodontic
procedures revised 4/1/2018. IOP Publishing American Association of Endodontists (AAE). 2018.
Available at: https://www.aae.org/specialty/wp-content/uploads/sites/2/2018/04/Consideratio
nsForRegEndo_AsOfApril2018.pdf. Accessed March 12, 2021.
4. Galler KM, Krastl G, Simon S, et al. European Society of Endodontology position statement:
Revitalization procedures. Int Endod J 2016;49:717–23.

5. Devaraj S, Jagannathan N, Neelakantan P. Antibiofilm efficacy of photoactivated curcumin, triple

and double antibiotic paste, 2% chlorhexidine and calcium hydroxide against Enterococcus
faecalis in vitro. Sci Rep 2016;6:24797.

6. Tiwari UO, Begum A, Jain J, et al. Comparative evaluation of antimicrobial efficacy of triple
antibiotic paste and modified double antibiotic paste using different vehicles against Candida
albicans. Int J Adv Res 2020;8:528–33.

7. Dubey N, Xu J, Zhang Z, et al. Comparative evaluation of the cytotoxic and angiogenic effects of
minocycline and clindamycin: an in vitro study. J Endod 2019;45:882–9.

8. Karczewsk A, Feitosa SA, Hamer EI, et al. Clindamycin-modified triple antibiotic nanofibers: a
stain-free antimicrobial intracanal drug delivery system. J Endod 2018;44:155–62.
9. Hoshino E, Takushige T. LSTR 3MIX-MP method-better and efficient clinical procedures of lesion
sterilization and tissue repair (LSTR) therapy. Dent Rev 1998;666:57–106.

10. Hoshino E. Information on LSTR 3Mix-MP therapy: lesion sterilization and tissue repair (LSTR)
therapy – LSTR 3Mix-MP therapy. 2007. Available at: http://www.lstr.jp/e/information.html.
Accessed March 10, 2021.

11. Althumairy RI, Teixeira FB, Diogenes A. Effect of dentin conditioning with intracanal medicaments
on survival of stem cells of apical papilla. J Endod 2014;40:521–5.
12. Ruparel NB, Teixeira FB, Ferraz CC, Diogenes A. Direct effect of intracanal medicaments on
survival of stem cells of the apical papilla. J Endod 2012;38:1372–5.
13. €nchow EA, Ferreira Bordini EA, et al. Antimicrobial therapeutics in regenerative
Ribeiro JS, Mu
endodontics: a scoping review. J Endod 2020;46:S115–27.

14. Abbaszadegan A, Dadolahi S, Gholami A, et al. Antimicrobial and cytotoxic activity of

Cinnamomum zeylanicum, calcium hydroxide, and triple antibiotic paste as root canal dressing
materials. J Contemp Dent Pract 2016;17:105–13.

15. Moradi Eslami L, Vatanpour M, Aminzadeh N, et al. The comparison of intracanal medicaments,
diode laser and photodynamic therapy on removing the biofilm of Enterococcus faecalis and
Candida albicans in the root canal system (ex-vivo study). Photodiagnosis Photodyn Ther
2019;26:157–61.

16. Mozayeni MA, Haeri A, Dianat O, Jafari AR. Antimicrobial effects of four intracanal medicaments
on Enterococcus faecalis: an in vitro study. Iran Endod J 2014;9:195–8.

17. Yadlapati M, Souza LC, Dorn S, et al. Deleterious effect of triple antibiotic paste on human
periodontal ligament fibroblasts. Int Endod J 2014;47:769–75.
18. Kitikuson P, Srisuwan T. Attachment ability of human apical papilla cells to root dentin surfaces
treated with either 3Mix or calcium hydroxide. J Endod 2016;42:89–94.

19. El-Tayeb MM, Abu-Seida AM, El Ashry SH, et al. Evaluation of antibacterial activity of propolis on
regenerative potential of necrotic immature permanent teeth in dogs. BMC Oral Health
2019;19:174.

20. Siqueira JF Jr, De Uzeda M, Fonseca ME. A scanning electron microscopic evaluation of in vitro
dentinal tubules penetration by selected anaerobic bacteria. J Endod 1996;22:308–10.

21. Zancan RF, Vivan RR, Milanda Lopes MR, et al. Antimicrobial activity and physicochemical
properties of calcium hydroxide pastes used as intracanal medication. J Endod 2016;42:1822–8.
22. Porciuncula MA, Cunha-Neto MA, Pinto KP, et al. Antibacterial efficacy and discolouration
potential of antibiotic pastes with macrogol for regenerative endodontic therapy. Aust Endod J
2020. https://doi.org/10.1111/aej.12438 [Epub ahead of print].
23. Andrade FB, Arias MP, Maliza AG, et al. A new improved protocol for in vitro intratubular dentinal
bacterial contamination for antimicrobial endodontic tests: standardization and validation by
confocal laser scanning microscopy. J Appl Oral Sci 2015;23:591–8.
24. Oda DF, Duarte MA, Andrade FB, et al. Antimicrobial action of photodynamic therapy in root
canals using LED curing light, curcumin and carbopol gel. Int Endod J 2019;52:1010–9.

JOE  Volume 47, Number 10, October 2021 Antibacterial Efficacy of Triple Antibiotic Medication 1615
25. Haapasalo M, Orstavik D. In vitro infection and disinfection of dentinal tubules. J Dent Res
1987;66:1375–9.

26. Ma J, Wang Z, Shen Y, Haapasalo M. A new noninvasive model to study the effectiveness of
dentin disinfection by using confocal laser scanning microscopy. J Endod 2011;37:1380–5.
27. Ferreira FB, Torres SA, Rosa OP, et al. Antmicrobial effect of propolis and other substances
against selected endodontic pathogens. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2007;104:709–16.
28. Pereira TC, da Silva Munhoz Vasconcelos LR, Graeff MS, et al. Intratubular decontamination
ability and physicochemical properties of calcium hydroxide pastes. Clin Oral Investig
2019;23:1253–62.
29. Marcucci MC, Sawaya AC, Custodio AR, et al. HPLC and ESI-MS typification: new approaches
for natural therapy with Brazilian propolis. In: Orsolic N, Basic I, editors. Scientific Evidence of the
Use of Propolis in Ethnomedicine. Kerala, India: Transworld Research Network; 2008. p. 33–54.
30. Pereira TC, Dijkstra RJ, Petridis X, et al. Chemical and mechanical influence of root canal irrigation
on biofilm removal from lateral morphological features of simulated root canals, dentine discs and
dentinal tubules. Int Endod J 2021;54:112–29.
31. Latham J, Fong H, Jewett A, et al. Disinfection efficacy of current regenerative endodontic
protocols in simulated necrotic immature permanent teeth. J Endod 2016;42:1218–25.

32. Seneviratne CJ, Suriyanarayanan T, Swarup S, et al. Transcriptomics analysis reveals putative
genes involved in biofilm formation and biofilm-associated drug resistance of Enterococcus
faecalis. J Endod 2017;43:949–55.

33. Diogenes AR, Ruparel NB, Teixeira FB, Hargreaves KM. Translational science in disinfection for
regenerative endodontics. J Endod 2014;40:52–7.
34. Montero-Miralles P, Martín-Gonza lez J, Alonso-Ezpeleta O, et al. Effectiveness and clinical
implications of the use of topical antibiotics in regenerative endodontic procedures: a review. Int
Endod J 2018;51:981–8.
35. Lin J, Zeng Q, Wei X, et al. Regenerative endodontics versus apexification in immature
permanent teeth with apical periodontitis: a prospective randomized controlled study. J Endod
2017;43:1821–7.
36. Lin S, Levin L, Peled M, et al. Reduction of viable bacteria in dentinal tubules treated with
clindamycin or tetracycline. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:751–6.

37. Safavi KE, Nichols FC. Effect of calcium hydroxide on bacterial lipopolysaccharide. J Endod
1993;19:76–8.

38. Pereira TC, Vasconcelos LR, Graeff MS, et al. Intratubular disinfection with tri-antibiotic and
calcium hydroxide pastes. Acta Odontol Scand 2017;75:87–93.
39. € u
Kayaoglu G, Om €rlu
€ H, Akca G, et al. Antibacterial activity of propolis versus conventional
endodontic disinfectants against Enterococcus faecalis in infected dentinal tubules. J Endod
2011;37:376–81.
40. Madhubala MM, Srinivasan N, Ahamed S. Comparative evaluation of propolis and triantibiotic
mixture as an intracanal medicament against Enterococcus faecalis. J Endod 2011;37:1287–9.

41. Ahangari Z, Naseri M, Jalili M, et al. Effect of propolis on dentin regeneration and the potential role
of dental pulp stem cell in guinea pigs. Cell J 2012;13:223–8.

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