Brazilian Dental Journal (2019) 30(6): 536-541

ISSN 0103-6440
http://dx.doi.org/10.1590/0103-6440201902613

A n t i m i c r o b i a l Ac t i v i t y a n d ¹Department of Restorative
Dentistry, Dental Materials and
Endodontics, Bauru Dental
Physicochemical Properties School, USP-Universidade de
São Paulo, Bauru, SP, Brazil
o f A n t i b i o t i c Pa s t e s U s e d I n ²Department of Restorative Dentistry,
UFPR-Universidade Federal do
Regenerative Endodontics Paraná, Curitiba, PR, Brazil

Correspondence: Rafaela Fernandes

Zancan, Al. Octávio Pinheiro Brisola
Rafaela Fernandes Zancan1 , Bruno Cavalini Cavenago2
, Denise Ferracioli no. 9-75, 17012-901 Bauru, SP,
Oda1 , Clovis Monteiro Bramante1 , Flaviana Bombarda de Andrade1 , Brasil. Tel: +55-14-32358344. e-mail:
Marco Antonio Hungaro Duarte1 rafaelazancan@yahoo.com.br

The purpose of this study was to evaluate the pH, solubility and antimicrobial action of
Calcium Hydroxide Paste (CH), Double Antibiotic Paste (metronidazole+ciprofloxacin-DAP),
calcium hydroxide added to DAP (CH/DAP) and Triple Antibiotic Paste (metronidazole +
ciprofloxacin+minocycline-TAP). pH (n=10) were measured by pHmeter. Root canals of
acrylic teeth (n=10) were filled with the above-mentioned intracanal-dressings, immersed
in ultrapure water, and solubility was measured by the difference between the initial and
final volume (7,15 and 30 days) by using micro-computed tomography. Enterococcus
faecalis biofilm was induced on bovine dentin disc surfaces (n=20), and treated with the
pastes for 7 days. Percentage bacterial viability was verified by confocal microscope, with
LIVE/DEAD dye. CH and CH/DAP presented the highest pH values. Regarding solubility, after
7 days, antibiotic groups presented significant volume loss. CH and CH/DAP showed no
statistical difference compared with the Control in antimicrobial action against E. faecalis
biofilm. However, TAP and DAP presented a significant percentage reduction in bacterial
population. Due to high solubility of the pastes, renewing antibiotic dressings every 7 days,
or using the medications for this period in regeneration protocols is recommended. DAP
is indicated for killing E. faecalis in biofilm because it has antimicrobial action similar to
TAP. Adding Calcium Hydroxide to DAP significantly decreased its antimicrobial action. Key Words: calcium hydroxide,
In spite of its the low solubility and high pH values, the CH paste showed a low level of antibiotic pastes, Enterococcus
antimicrobial action against E. faecalis in biofilm.  faecalis, solubility, hydroxyl ions

Introduction its broad spectrum of action against oral microorganisms

Pulp necrosis of immature permanent teeth is a clinical and its ability to disinfect dentin (6,7)., Triple Antibiotic
situation in endodontics associated with traumatic injuries, Paste (TAP), a combination of metronidazole, ciprofloxacin
anatomic anomalies and caries in young patients (1). and minocycline has been widely used. Minocycline can
Depending on the stage of tooth development, the dentin cause tooth discoloration, but the use of only metronidazole
walls may be fragile, making the tooth more susceptible and ciprofloxacin, called Double Antibiotic Paste (DAP),
to fractures (2). The open-apex also increases the chances solved this problem (8). An alternative that has hardly
of extravasion of irrigant solutions and filling materials been studied is the interaction of the antimicrobial action
being over extended, causing chemical irritation, flare ups of DAP with the biological benefits of calcium hydroxide.
and an inappropriate obturation seal (3). Based on the foregoing, the aim of the present study
Clinically treatments have been proposed for artificial was to evaluate the pH, percentage of volumetric loss, the
apical closure (apexification) performed in one visit, with antimicrobial effect of different intracanal dressings used
mineral trioxide aggregate (MTA) that induced an artificial in regenerative procedures, on mono specie (Enterococcus
barrier, and treatment with calcium hydroxide (CH) in faecalis) biofilms. The null hypothesis tested was that the
multiple-visits, which stimulated the formation of a hard pastes would have similar values of pH, volumetric loss
tissue  barrier  (4). However, root development can be and antimicrobial effect.
prevented or restricted by these techniques, leading to the
risk of root canal fracture during masticatory efforts (5). Material and Methods
As an alternative technique, regenerative endodontic Intracanal Dressings
protocols (REPs) are intended to promote continued Four intracanal dressings were evaluated:
root maturation and apical closure. Local application of CH - CH/polyethylene glycol (Calen; SS White Artigos
antibiotics as intracanal dressing has been used to eradicate Dentarios Ltd, Rio de Janeiro, RJ, Brazil); DAP-Double
endodontic pathogens during these procedures. Because of Antibiotic Paste (Metronidazole, Ciprofloxacin)/Saline (500
 Braz Dent J 30(6) 2019

mg of each antibiotic/1 mL); CH/DAP-Double Antibiotic Experiment 4: Antimicrobial Test and Microscopic
Paste (Metronidazole, Ciprofloxacin) /Ca(OH)² ((Merck & Analysis
Co., NJ) /Saline (250 mg of each antibiotic/500 mg Ca(OH)2 Sample Preparation
/1mL); TAP -  Triple Antibiotic Paste (metronidazole, Dentin blocks were obtained from bovine central incisors
ciprofloxacin, minocycline)/Saline (500 mg of each with fully developed roots, by using trephine drills 4.0-mm
antibiotic/1 mL). in diameter, under abundant irrigation. The incisors were
To obtain the antibiotic powders (ciprofloxacin, positioned laterally to provide access to the most flattened
metronidazole and minocycline), their tablets were ground portion of the root. The root was perforated with a trephine
by using a pestle and mortar, and then weighed on a high attached to a counter-angle positioned perpendicular to
precision balance. the teeth passing through the mesial and distal dentin
walls, thus obtaining two dentin blocks measuring 4 mm
Experiment 1 and 2: pH of the Pastes x 1.2 mm (diameter x thickness). These were adjusted by
The pH measurement test was done in two ways. In Test means of polishing with 500 and 800 grit SiC papers on the
1 the pH of the 4 pastes was measured (with a pH meter). pulp surface to smooth it. The smear layer formed during
In Test 2 we tried to simulate the pH of the solubilized dentin specimen preparation, was removed by submerging
root canal pastes in the periapical tissues. Test 1 : 1 mL the specimens in 17% EDTA for 5 min; and after this, they
of the pastes were manipulated as described below and were sterilized by autoclaving at 121° for 20 min.
immersed in 10 mL distilled water. After being agitated
for 5 min the solutions were measured with a pH meter Biofilm Growth
(model 371; Micronal, São Paulo, SP, Brazil). Test 2 and The microbiological procedures and manipulation of the

Properties of regenerative endodontic pastes

3: Based on the Duarte et al. (9) methodology, 40 acrylic pastes were conducted under aseptic conditions in a laminar
teeth (n=10), with an artificial foramen standardized to a flow chamber (VecoFlow Ltda, Campinas, SP, Brazil). For
diameter of 800 µm were filled with the antibiotic pastes, Enterococcus faecalis (E. faecalis) biofilm, 15 µL standard
and the crown access was sealed. Afterwards, the teeth strain (ATCC 4083) was put into 3 mL sterile BHI (Oxoid,
were individually immersed in a plastic flask containing Basingstoke, UK) for growth overnight at 37° in air. After
10 mL of ultrapure water. After time intervals of 7, 15 this, bacterial density was adjusted at 109cells/mL for E.
and 30 days, the teeth were moved to a new plastic flask faecalis (ATCC 4083) in a spectrophotometer (UV-VISIBLI,
with an equal volume of new ultrapure water. The pH in Shimadzu, Japan) at an optical density (OD) of 1 at 600
the solutions was analyzed with a pH meter (Model 371; nm according to the 0.5 MacFarland standard.
Micronal, São Paulo, SP, Brazil).
Dentin Surface Infection
Experiment 3: Micro-CT Volumetric Solubility After density adjustment, the dentin surfaces were
The canals of 40 artificial maxillary central incisors infected. For E. faecalis biofilm a dentin block + 100µL
fabricated of resin acrylic, with a foramen standardized of E. faecalis + 900 µL of BHI was put into each well of
to a diameter of 800 µm, were filled immediately after a 24-well multiwell plate. For biofilm growth, all plates
mixing the experimental pastes (n=10) Subsequently, the were incubated aerobically at 37 °C for 21 days. The BHI
samples were scanned using an X -ray desktop microfocus was refreshed every 2 days.
CT scanner (SkyScan 1174v2; SkyScan, Kontich, Belgium).
The acrylic teeth were stored, individually immersed in Antimicrobial Test for E. faecalis Biofilm
plastic flasks containing 10 mL of deionized water in a warm After the incubation period, the infected samples
room at 37° C. After time intervals of 7, 15 and 30 days, were washed with 1 mL of distilled water to remove
the acrylic teeth were removed from the flasks and scanned loosely adherent planktonic bacteria. Then they were
again. The image capture parameters used were the same randomly divided into four groups (n=20) according to
for all stages: voxel size 19.70 µm; 0.5º rotation step and the experimental pastes + 1 control group that received no
360º rotation. Each scan consisted of 373.tif images with treatment. The contact test was performed by immersing
1024 x 1304 pixels. Digital data were further elaborated dentin samples for 7 days in the experimental intracanal
by reconstruction software (NReconvl.6.4.8, SkyScan) and dressings and incubating them at 37°C.
the volume (mm³) of the pastes was obtained by means of
CTan software (CTan v1.11.10.0, SkyScan). The solubility of Microbiological Analysis
each specimen was considered the difference between the Biofilm viability was analyzed by using the SYTO 9/
initial and final volume scanning, showing the proportion propidium iodide technique (Live/Dead, Baclight, Invitrogen,
of the dissolved material. Eugene, OR). SYTO-9 is a green fluorescent nucleic acid
537
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stain that acts as a dye, generally labeling both live and Results
dead microorganisms; and propidium iodide is a red Table 1 presents the OH- and Ca2+ ion release results
fluorescent nucleic acid stain that only penetrates the cells provided by the experimental pastes in all time intervals
with damaged membranes, signaling dead microorganisms. analyzed. The CH and DAP/CH presented the highest values,
After the established time of contact with the pastes, with no statistically significant differences (p<0.05)
the blocks were washed with phosphate-buffered saline between the two. DAP and TAP showed acid pH values.
(PBS) and stained with 15 µL of the dyes for 15 min pH measured in direct contact with the pastes showed
in a dark environment. Then they were washed again, higher pH values for CH- 12.51, DAP/CH-12.36, and lower
and directly observed using an inverted confocal laser for DAP-5.49 and TAP-4.32.
scanning microscope (Leica TCS-SPE; Leica Biosystems CMS, Table 2 presents the percentage of solubility of the
Mannheim, Germany). Four confocal “stacks” of random pastes in 7, 15 and 30 days. At all periods there was
areas were obtained for each sample using a 40x oil lens. high solubility with no statistically significant differences
In total, there were 5 samples per group, therefore, 20 (p>0.05) between DAP/CH, DAP and TAP. For CH, lower
stacks for each medication. For quantification purposes, values were shown (p<0.05). For all groups in 30 days the
bioImage_L software (www.bioImageL.com) was used to volume of solubilized pastes was higher than 7, with no
calculate the total biovolume and the percentage of red statistical difference intragroup for CH (p>0.05).
(dead cells) found after the antimicrobial treatment. Table 3 presents the percentages of live cells in the
biofilms after contact with experimental pastes. The
Statistical Analysis weakest antimicrobial activity occurred in Group CH
The data from all the analyses were subjected to the followed by DAP/CH, showing no statistical differences
Shapiro-Wilks test to verify normality, which was shown to compared with the control group (p>0.05). Groups DAP
be absent. Then, for the global and individual comparisons, and TAP showed the best antimicrobial activity (p<0.05).
the Kruskal-Wallis and Dunn tests, respectively, were used. Figure 1 presents micro-CT 3D reconstructions of the
R. F. Zancan et al.

The significance level was established at 5%. superimposed intracanal dressings at the root canal thirds
and cross-sections in all groups.
Figure 2 presents confocal
Table 1. Medians (Med), minimum and maximum (Min-Max) values for pH of the pastes in different laser scanning microscopy
studied periods
images of biofilms treated with
3 days 7 days 15 days 30 days the four intracanal dressing
CH 9.52 (8.6-12.3)Aa 8.1 (7.6 - 9.07)Aab 7.9 (7.4-8.15)ABb 8.9 (7.9-11.4)Aa materials and control.
DAP 5.3 (4.8-7.2)BCa 6.1 (5.2-6.6)Bab 6.2 (5.9 -6.6)BCab 5.5 (4.3-6.7)BCb
DAP/CH 9.2 (8.2-10.3)ABa 8.85 (7.8-9.76)Aa 8.0 (7.3-8.64)Aab 7.1 (6.5 - 8.0)ABb Discussion
TAP 5.46 (4.8-7.34)Ca 6.17 (5.29-6.54)Ba 6.23 (5.78-6.54)Ca 5.39 (4.4-6.6)Ca The null hypothesis tested
*Kruskal-Wallis with Dunn’s post-hoc p-value <0.05; Different capital letters in columns indicate was rejected because the pastes
statistically significant intergroup differences in the same time period. presented differences in pH,

Table 2. Median (Med), minimum and maximum (Min - Max) values of intracanal dressing volume, initial and after 7, 15 and 30 days of immersion
in 10 mL of deionized water and the percentage of lost volume (solubility) comparing 7, 15 and 30 days volume with the initial

Initial 7 days 15 days 30 days

Med (Min-Max) Med (Min-Max) Med (Min-Max) Med (Min-Max)
Groups
% of lost V % of lost V % of lost V
Volume Volume Volume Volume
(7d-I) (15d - I) (30d - I)
9.7 8.3 2.35 8.05 5.23 8.05 16,13
CH
(8.34-11.96 )A (5.5-11.6)AB (0-44.95)b (5.2-11.6)B (1.25-47.72)b (5.2 - 11.3)B (3,41-47.4)b
9.8 6.17 41.0 5.2 52.3 3.3 65.8
DAP
(8.21- 11.3)A (3.9 - 8.0)AB (22.35-58.0)a (2.2-7.12)BC (32.25-75.84)a (2.2-5.1)C (51.8-75.8)a
9.7 5.6 41.71 4.1 57.9 3.4 63.5
DAP/CH
(7.4 - 11.5)A (0.05 - 8.9)AB (17.11 -99.41)a (0.05-7.71)BC (31.13-99.41)a (0.04-6.16)C (44.99-99.4)a
10.5 6.3 41.70 6.0 42.31 3.5 63.89
TAP
(8.5-12.12)A (4.5-9.3)AB (20.5-57.09)a (0.5-8.4) B (24.3-93.38)a (0.5-6.9)B (39.8– 93.3)a

*Friedman p-value <0.05; Different capital letters in rows indicate statistically significant intragroup differences; *Kruskal-Wallis with Dunn’s
post-hoc p-value <0.05; Different lowercase letters in columns indicate statistically significant intergroup differences in the same time period.
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solubility and antimicrobial action. proliferation, and differentiation, and has been shown to
As the dentin walls of infected immature teeth are be beneficial for regenerative endodontic therapies.(14). It
fragile, the revitalization approach is the treatment that is also preferred for the survival of stem cells from apical
allows root development to continue, in terms of both papilla when compared with antibiotic pastes used in high
length and thickness (5). In this method, hemorrhage is concentrations (15,16). However, it may be ineffective for
induced in the cleaned and disinfected canal space and killing E. faecalis in biofilm, a species commonly used for
the blood clot formed serves as a fibrin scaffold, allowing microbiological tests in endodontics (Table 3). The presence
the formation of new living tissue. For effective antisepsis of microorganisms and their antigens has a profound
in root canals, care must be taken to perform the copious effect on the final phenotype of stem cells transferred
irrigation without extruding the irrigant; and also when to root canals during REPs (17). Thus, the presence of
performing instrumentation, because this is capable of residual bacteria that lead to periapical and intracanal
weakening the dentin walls. For microbial control, it is inflammation has been a major risk to the success of this
necessary to place an intracanal medication with crown therapy (18). Further antimicrobial tests are necessary with
restoration to prevent recontamination (10). other bacterial species found in immature teeth to evaluate
Intracanal applications of Triple Antibiotic Paste (TAP) the antimicrobial action of CH in REPs.
have been used as intracanal medication endodontics Therefore, the purpose of using CH associated with
in regeneration procedures due to the broad spectrum DAP was to associate their biologic and antimicrobial
antimicrobial activity by the combination of metronidazole, action, respectively, as suggested by Maniglia-Ferreira (19).
ciprofloxacin and minocycline. This has been beneficial The pH measurements in direct contact with these pastes
because of the polymicrobial nature of root canal showed an acid pH, which may have had a demineralizing

Properties of regenerative endodontic pastes

infections, (8) since TAP has been shown to be capable of effect on dentin (2). DAP/CH, showed the highest OH- ion
eradicating anaerobic, gram-positive and gram-negative release favoring an alternative biological approach, with
microorganisms (6). The Triple Antibiotic Paste has caused no statistical difference when compared with the alkaline
color change in teeth due to the minocycline in its CH paste. However, instead of any increase in antimicrobial
formulation, which is incorporated into the tooth matrix activity, the association of CH to the DAP had the same
and binds to the calcium ions via chelation, causing the behavior as that of CH alone in the antibacterial action
discoloration (11). Thus, the use of metronidazole and against E. faecalis, without differing from the Control
ciprofloxacin only - called a Double Antibiotic Paste (DAP) (Fig. 2). The alkaline environment provided by CH could
was tested. Hoshino clarified the antibacterial effect of a probably influence the permeability of the antibiotic into
mixture of antibiotics (TAP) in 1996 (6) but the efficacy of the bacterial cells. Thus, this combination is unnecessary
each agent alone needed to be studied, to investigate the and should be avoided. Maniglia-Ferreira (19) found
real necessity of the mixture. Some studies have shown that CH did not alter the antimicrobial effects of DAP,
no difference between antimicrobial activity of DAP and but these conflicting results may be due to the different
TAP, which significantly reduced the viability of cells in a methodologies; in the present study the antibacterial action
biofilm (12,13) according to the results of these studies. was tested in a biofilm model, and the above-mentioned
CH is known to amplify release of the growth factor study used the radial diffusion method in agar that involved
(TGF-β1), which modulates pulpal  stem cell  migration, planktonic cells.
The solubility of pastes was measured based on a novel
methodology using microcomputed tomography to produce
Table 3. Median and minimum and maximum values of the percentage their volumetric losses in the different time intervals (20).
of live cells of E. faecalis biofilm after contact with the experimental
Acrylic teeth with standardized root canals and foraminal
medicaments for a week
diameter of 600 µm were used to simulate immature
Group % live cells teeth. CH paste had the lowest solubility values. However,
Control 75.59 (42.9-96.54)A approximately 40% of the antibiotic pastes were solubilized
CH 44.64 (4.08-92.03)A
in 7 days and 65% in 30 days (Fig. 1). The favorable
outcomes of the treatment depend on effective antisepsis
DAP 0.05 (0.0-31.56)BC
of the root canal. In the antimicrobial test, the DAP and
DAP/CH 59.98 (11.46-80.33)AB TAP in the period of contact analyzed (7 days) showed the
TAP 0.06 (0.001- 1.23)C highest level of antimicrobial activity, demonstrating that
1 week was sufficient to promote effective antibacterial
Kruskal-Wallis with Dunn’s post-hoc p-value < 0.05; On % of live cells,
different capital letters in columns indicate statistically significant action. High solubility leads to a higher ionic dissociation
intergroup differences in the same biofilm. of the molecules of intracanal pastes, which can favor
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antimicrobial properties such as OH- ion release from solubility test of antibiotic pastes may lead to an empty
the Calcium Hydroxide paste (20). Antibiotics induce root canal, blocking their direct contact with bacteria and
bacterial cell death  by physical or chemical interaction consequently their antimicrobial action.
between a drug molecule and its bacterial-specific target. Furthermore, when the volume of water in the
Nevertheless, the high percentage of volume loss found in mixture of pastes was increased, higher solubility was
observed (21). At present, there are no defined protocols
for endodontic regeneration. Recommendations of
the American Association Endodontics (AAE), namely
“Considerations for regeneration procedures” based on
clinical evidences and recent articles, have indicated
that the placement of an intracanal dressing with CH or
TAP in the patient first appointment, with the treatment
time required varying between 1-4 weeks, depended
on patient’s signs and symptoms (22). The proportion
of powder/liquid in regenerative procedures varies in
concentration of up to a million-fold (0.001-1000 mg/
mL) (23). Due to the high solubility, in cases of low
medication concentration, the root canal is probably
empty for the greater part of time, in periods longer than
7 days. Thus, in regeneration protocols it is important
to consider renewing antibiotic pastes every 7 days,
or using the medication for this period, and thereby
Figure 1. Representative micro-CT 3D reconstructions of the initial induce clot formation. The DAP is indicated because it
(green) and final (red) images of the superimposed intracanal dressings
R. F. Zancan et al.

in root canals and cross-sections at the coronal (c), middle (m), and has a percentage of bacterial reduction similar to that
apical (a) thirds. G1: CH, G2: DAP, G3:DAP/CH, G4:TAP. of TAP at 7 days.

Figure 2. Confocal laser scanning microscopy of biofilms treated with CH (G1), DAP (G2), DAP/CH (G3), TAP (G4) and control (G5). Live cells are
seen in green, and dead cells are seen in red. Each picture represents an area of 275 x 275 µm.
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Evaluation of residual antibacterial effect of human radicular dentin

Resumo treated with triple and double antibiotic pastes. J Endod 2015;41:1081-
A proposição deste estudo foi avaliar o pH, solubilidade e ação 1084.
antimicrobiana da pasta de Hidróxido de Cálcio (HC), Pasta Diantibiótica  9. Duarte MA, Midena RZ, Zeferino MA, Vivan RR, Weckwerth PH,
(metronidazol+ciprofloxacina-DAP), Hidróxido de Cálcio adicionado a Dos Santos F, et al. Evaluation of pH and calcium ion release of
DAP (HC/DAP) e pasta Triantibiótica (metronidazol + ciprofloxacina + calcium hydroxide pastes containing different substances. J Endod
miociclina - TAP). a medição do pH e liberação de íons cálcio (n=10) foram 2009;35:1274-1277.
avaliadas através de peagômetro e espectofotômetro de absorção atômica. 10. Yassen GH, Sabrah AH, Eckert GJ, Platt JA. Effect of different
Canais de dentes de acrílico (n=10) foram preenchidos com as medicações endodontic regeneration protocols on wettability, roughness, and
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aferida pela diferença entre o volume inicial e final (7,15 e 30 dias) 11. Tanase S, Tsuchiya H, Yao J, Ohmoto S, Takagi N, Yoshida S. Reversed-
usando micro tomografia computadorizada. O biofilme de Enterococcus phase ion-pair chromatographic analysis of tetracycline antibiotics.
faecalis foi induzido em blocos de dentina bovina (n=20) e tratados Application to discolored teeth. J Chromatogr B Biomed Sci Appl
com as pastas em questão por 7 dias. A porcentagem de bactérias vivas 1998;706:279-285
foi verificada usando o microscópio confocal com corante LIVE/DEAD. 12. Sabrah AH, Yassen GH, Gregory RL. Effectiveness of antibiotic
HC e HC/DAP apresentaram os maiores valores de pH e liberação de medicaments against biofilm formation of Enterococcus faecalis and
íons cálcio. A respeito da solubilidade, depois de 7 dias, os grupos com Porphyromonas gingivalis. J Endod 2013;39:1385-1389.
pastas antibióticas apresentaram perda de volume significante. HC e HC/ 13. A Algarni A, H Yassen G, L Gregory R. Inhibitory effect of gels loaded
DAP não tiveram diferenças estatísticas com o grupo controle na ação with a low concentration of antibiotics against biofilm formation
antimicrobiana contra biofilme. No entanto, a TAP e a DAP mostraram by Enterococcus faecalis and Porphyromonas gingivalis. J Oral Sci
porcentagem significante de redução bacteriana. Conclusões: Devido 2015;57:213-218.
à alta solubilidade das pastas, renovar as medicações antibióticas a 14. Galler KM, Buchalla W, Hiller KA, Federlin M, Eidt A, Schiefersteiner M,
cada 7 dias ou usa-las por esse período em protocolos regenerativos é et al. Influence of root canal disinfectants on growth factor release
recomendado. DAP é indicada para matar E. faecalis na forma de biofilme from dentin. J Endod 2015;41:363-368.
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significantemente sua ação antimicrobiana. Apesar da baixa solubilidade intracanal medicaments on survival of stem cells of the apical papilla.
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large periradicular lesions. J Investig Clin Dent 2012;3:72-76. Received January 30, 2019
 8. Sabrah AH, Yassen GH, Spolnik KJ, Hara AT, Platt JA, Gregory RL. Accepted April 8, 2019

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