Basic Research—Technology

Antimicrobial Activity of Triantibiotic Paste,

2% Chlorhexidine Gel, and Calcium Hydroxide
on an Intraoral-infected Dentin Biofilm Model
Ronald Ordinola-Zapata, DDS, MSc,* Clovis M. Bramante, DDS, PhD,*
Paloma Gagliardi Minotti, DDS, MSc,* Bruno Cavalini Cavenago, DDS, MSc,*
Roberto Brand~ao Garcia, DDS, PhD,* Norberti Bernardineli, DDS, PhD,*
David E. Jaramillo, DDS,† and Marco A. Hungaro Duarte, DDS, PhD*

Abstract
Introduction: The purpose of this study was to eval-
uate the antimicrobial activity of calcium hydroxide,
2% chlorhexidine gel, and triantibiotic paste (ie,
metronidazole, minocycline, and ciprofloxacin) by
using an intraorally infected dentin biofilm model.
Methods: Forty bovine dentin specimens were in-
I nfection of the pulp canal space of immature teeth is considered a challenging and
fected intraorally using a removable orthodontic device
in order to induce the biofilm colonization of the
dentin. Then, the samples were treated with the medi-
cations for 7 days. Saline solution was used as the
control. Two evaluations were performed: immediately
after the elimination of the medication and after incu-
bation in brain-heart infusion medium for 24 hours. The
Live/Dead technique (Invitrogen, Eugene, OR) and
a confocal microscope were used to obtain the
percentage of live cells. Nonparametric statistical tests
were performed to show differences in the percentage
of live cells among the groups (P < .05). Results:
Calcium hydroxide and 2% chlorhexidine gel did not
show statistical differences in the immediate evalua-
tion. However, after application of the brain-heart infu-
sion medium for 24 hours, 2% gel chlorhexidine
showed a statistically lesser percentage of live cells
in comparison with calcium hydroxide. The triantibiotic
paste significantly showed a lower percentage of live
cells in comparison with the 2% chlorhexidine gel
and calcium hydroxide groups in the immediate and
secondary (after 24 hours) evaluations. Conclusions:
The triantibiotic paste was most effective at killing
the bacteria in the biofilms on the intraorally infected
dentin model in comparison with 2% chlorhexidine
gel and calcium hydroxide. (J Endod 2013;39:115–118)
Key Words
Antimicrobials, bacteria, biofilms, dentin, root canal dressings
complex problem. The thin dentinal walls of these teeth limit the mechanical instru-
mentation with traditional techniques, and the disinfection process is only dependent
on the antimicrobial properties of the irrigant solution and the intracanal medications
(1). Classically, calcium hydroxide has been used to treat necrotic immature teeth in
a procedure called apexification (2). This procedure aims to create an environment
that favors the placement of the root filling materials by the induction of an apical calci-
fied barrier. However, this therapy does not favor the formation of dentin deposition on
the radicular walls of immature teeth, which can lead to an increased risk of radicular
fracture (3).
Several case reports have shown that dentin deposition in root canal walls and
continued root development of immature necrotic teeth can be possible by a treat-
ment-denominated revascularization (1, 4). This treatment includes root canal
disinfection and placement of a matrix for cell ingrowth and a coronal seal to avoid
recontamination (5, 6). In these cases, the aim of the intracanal medication is to
only promote disinfection. In order to support revascularization, sodium
hypochlorite disinfection and the use of a triantibiotic paste have been
recommended based on clinical results and biologic biocompatibility (5–7).
The use of calcium hydroxide is commonly used for the apexification technique or
the standard 2-visit treatment of necrotic mature teeth, but it is not indicated for revas-
cularization procedures, except in isolated clinical reports (8). In order to evaluate the
antimicrobial activity of triantibiotic paste against biofilms, the evaluation of medica-
tions such as calcium hydroxide that are not commonly used for revascularization
procedures is also necessary for comparative purposes.
Another common antimicrobial substance used in endodontic therapy includes
2% chlorhexidine. This antimicrobial substance in its gel form has been proposed as
an intracanal dressing (9–12). It presents low toxicity and effective antimicrobial
activity especially when compared with calcium hydroxide (11, 13). Despite
previous reports addressing the antimicrobial activity of triantibiotic paste (14) or
2% chlorhexidine gel (12), its effect against microbial biofilms in comparison with
commonly used antimicrobial dressings such as calcium hydroxide has not been

From the *Department of Endodontics, Bauru Dental School of Bauru, University of S~ao Paulo, Brazil; and †Department of Endodontics, Loma Linda School of
Dentistry, Loma Linda, California.
Supported by the Brazilian Funding Agency FAPESP (grant no. 2010/16002-4).
Address requests for reprints to Dr Ronald Ordinola-Zapata, Faculdade de Odontologia de Bauru–USP, Al Octavio Pinheiro Brisolla, 9-75-CEP 17012-901, Bauru, S~ao
Paulo, Brazil. E-mail address: ronaldordinola@usp.br
0099-2399/$ - see front matter
Copyright ª 2013 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2012.10.004

JOE — Volume 39, Number 1, January 2013 Antimicrobial Activity of Intracanal Medications 115
Basic Research—Technology
previously addressed. Thus, the aim of this study was to evaluate the
antimicrobial activity of calcium hydroxide, 2% chlorhexidine gel,
and triantibiotic paste on the intraorally infected dentin biofilm model.
Material and Methods
The evaluated medications were calcium hydroxide (Biodinamica,
Ibipor~a, Parana, Brazil), 2% chlorhexidine gel (Biodinamica), and tri-
antibiotic paste. The medications were left in contact with the infected
dentin for 7 days. A saline solution was used for 7 days for control
purposes.
Forty sterile bovine dentin sections (2  2  2 mm) were used
(n = 10). The samples were treated with 17% EDTA for 3 minutes to
eliminate the smear layer produced during the sectioning process. To
test the antimicrobial activity against oral bacteria, an in situ model
of intraorally dentin infection was selected. The dentin samples were
fixed into the cavities of a Hawley’s orthodontic device with sticky
wax. The dentin surface in contact with the oral cavity was fixed 1
mm above the surface to favor the accumulation of plaque. The device
was used by 1 volunteer for 72 hours in order to induce the plaque
formation and the subsequent surface infection of dentin. Regular
oral hygiene practices were maintained (Human Committee for Ethical
Research, CEP134/2010). After the intraoral infection process, each
sample was incubated in 2 mL brain-heart infusion (BHI) medium at
37 C for 24 hours in aerobic conditions. Then, 1 mL saline solution
was used to eliminate the culture medium and the nonadherent cells.
For the direct contact test, the infected dentin samples were
immersed in the medications using 12-well tissue culture plates for 7
days at 37 C. The calcium hydroxide paste was prepared by using 1 g
calcium hydroxide powder mixed with 1 mL distilled water. For the
2% chlorhexidine gel medication, 1 mL was used. Metronidazole
(250 mg; Flagyl Aventis Pharma, S~ao Paulo, Brazil), minocycline
(minocycline cloridrate, 100 mg; Ranbaxy, Jacksonville, FL), and cipro-
floxacin (ciprofloxacin cloridrate, 250 mg; Neoquimica, Anapolis,
Goias, Brazil) were used to prepare the triantibiotic medication. Five
hundred milligrams of each antibiotic was homogenized to obtain
a uniform powder. Propylene glycol was added to the antibiotic powder
in an approximate proportion of 7:4 powder/propylene glycol and then
mixed to obtain a paste-like consistency. The saline solution was used
for 7 days for control purposes. Ten dentin blocks were used to test
each medication and the control; 40 blocks were analyzed.
After 7 days, the medications were washed with saline solution for
5 minutes. Five treated dentin blocks were immediately evaluated by
confocal analysis, and 5 blocks were immersed in 2 mL fresh BHI
and incubated at 37 C. These last samples were evaluated after 24 hours
of the elimination of the antimicrobial substances. These samples were
used to test if the residual microflora has the ability to recover from the
antimicrobial stress. After 24 hours, the samples were washed with the
saline solution, stained, and evaluated in a similar fashion to the first
samples.
The analysis of biofilm viability was performed by using the
SYTO 9/propidium iodide technique (Live/Dead, Bacligth, Invitro-
gen, Eugene, OR) (15, 16); SYTO 9 is a green-fluorescent stain
that labels both live and dead microorganisms. Propidium iodide
is a red-fluorescent nucleic acid stain and only penetrates the cells
with damaged membranes (dead microbes). A confocal laser scan-
ning microscope (Leica TCS-SPE; Leica Biosystems CMS, Mannheim,
Germany) was used to perform the analysis. The respective absorp-
tion and emission wavelengths were 494/518 nm for SYTO 9 and
536/617 nm for propidium iodide. Four confocal ‘‘stacks’’ from
random areas were obtained from each sample using a 40 oil
lens, a 1-mm step size, and a format of 512  512 pixels. At least
116 Ordinola-Zapata et al.
5 mm of the scanning included the subsurface level of the dentin.
In total, 20 stacks (4 operative fields  5 samples) were obtained
for each medication immediately after the removal of the medica-
tion, and 20 stacks were obtained after 24 hours of incubation
with BHI. For quantification purposes, bioImage_L software
(www.bioImageL.com) was used (17) to calculate the percentage
of the volume of live cells found after the antimicrobial treatment.
Statistical analysis of the percentage of live cells in the samples
evaluated after 7 days of contact or after an additional 24 hours of re-
incubation was performed using the nonparametric Kruskal-Wallis and
Dunn tests (P < .05) by the absence of a normal distribution confirmed
in the preliminary analysis. The Mann-Whitney U test was used to
compare the effect of an immediate and a 24-hour evaluation. Prisma
5.0 software (GraphPad Software Inc, La Jolla, CA) was used as the
analytic tool.
Results
The median and range of the percentage of live cells in the evalu-
ated groups are shown in Table 1. Overall, calcium hydroxide showed
the weakest antimicrobial activity, and the triantibiotic mix showed the
highest with or without 24 hours of additional reincubation in BHI.
Calcium hydroxide and 2% chlorhexidine gel did not show statis-
tical differences (P > .05) in the immediate evaluation; however, after
the application of BHI for 24 hours, 2% chlorhexidine gel showed
a statistically (P < .05) lesser percentage of live cells in comparison
with calcium hydroxide. This effect was observed because the calcium
hydroxide–treated dentin significantly increased the percentage of live
cells from 63.71% to 83.88% after the additional BHI treatment (Mann-
Whitney U test, P < .05). The additional BHI treatment did not show any
effect on 2% chlorhexidine gel or in the triantibiotic paste–treated
dentin (Mann-Whitney U test, P > .05). The triantibiotic paste showed
the lowest percentage of live cells in comparison with the 2% chlorhex-
idine gel and the calcium hydroxide groups in the immediate and
secondary (after 24 hours) evaluations (P < .05). Representative
pictures of the evaluated samples are shown in Figure 1A–D.
Discussion
Disinfection of immature teeth can be considered a challenge that
needs specific disinfection procedures when compared with conven-
tional endodontic treatment. In this context, the use of triantibiotic paste
has been previously reported for the treatment of necrotic teeth with
open apexes (4, 6). For methodologic purposes, this study was
designed to test the isolated effect of 3 root canal medications used
for disinfection purposes in endodontic therapy. However, during
clinical situations intracanal dressings are only one of the steps to
treat endodontic infections. The antimicrobial control stage also
includes the use of irrigant solutions and mechanical instrumentation
to treat the previously infected dentinal biofilm.
Two percent chlorhexidine gel has been proposed as an intracanal
dressing (10) because of its effective antimicrobial activity (11, 18).
TABLE 1. Median and Range Values of the Percentage of Live Cells after
Contact with the Experimental Medicaments for 1 Week Evaluated Immediately
(Green) and after an Additional 24 Hours of Incubation in BHI (Green 24 h)
Green (%) Green 24 h (%)
Saline (control) 93.36 (45.87–98.98) a
82.69 (35.49–97.74)a
CaOH2 + DW 63.71 (9.20–99.23)ab 83.88 (55.46–96.51)a
2% CHX 28.33 (0.00–78.32)b 18.70 (5.53–74.69)b
TRIMIX 1.37 (0.00–13.18)c 1.57 (0.00–25.68)c
CHX, chlorhexidine; DW, distilled water; TRIMIX, triantibiotic paste.
Different superscript letters in each column represent statistical significance (P < .05).
JOE — Volume 39, Number 1, January 2013

Figure 1. Confocal laser scanning microscopy of biofilms treated with (A) saline solution, (B) calcium hydroxide, (C) 2% chlorhexidine gel, and (D) triantibiotic
paste. Live cells are seen in green, and dead cells are seen in red. Each picture represents an area of 275  275 mm. Bars represents 20 mm.
However, a previous clinical study showed a limited ability of this
medication to kill bacteria (19). In the present work, contrary to the
results found in the triantibiotic paste, the variability of live cells found
in 2% chlorhexidine and calcium hydroxide was higher. This probably
could be explained by the effect of neutralizing substances (20, 21) that
can be found in the biofilms as dead cells or an exopolymeric matrix.
However, the antimicrobial effect of 2% chlorhexidine gel was superior
to calcium hydroxide, which does not show a significant effect against
the biofilm.
According to previous studies, an alkaline microbial effect is not
effective in killing bacteria in the form of biofilms (22–25). The
findings of the current research are consistent with the cited
studies. The reason for this is that the amount of hydroxyl ions
reached after 1 week is probably not suitably high enough to
promote antimicrobial activity. These results suggest that previous
disorganization of biofilm by sodium hypochlorite treatment could be
a necessary condition to enhance the effect of intracanal dressings.
Triantibiotic paste includes metronidazole and minocycline. These
antibiotics have been used in periodontics in order to suppress subgin-
gival microbiota as an adjunctive therapy (26, 27). Previous studies
have shown the antimicrobial activity of these medications against
oral bacteria, and its ability to sterilize infected dentin has been
confirmed (14, 28, 29). Our findings showed a good ability of the
triantibiotic paste to kill bacteria inside the biofilms in comparison
with calcium hydroxide and 2% chlorhexidine gel.
The antimicrobial medications evaluated in this study could not
kill 100% of the bacteria inside the biofilm. Thus, it was important to
JOE — Volume 39, Number 1, January 2013
Basic Research—Technology
test whether the residual bacteria could recolonize the chemically
treated biofilms or not. In order to reach this hypothesis, an additional
evaluation of the stressed biofilm after 24 hours of incubation in a rich-
medium was performed (BHI). The results showed that the triantibiotic
paste– and 2% chlorhexidine gel–treated dentin do not significantly
increase the number of live bacteria in comparison with calcium
hydroxide. It is known that minocycline (a tetracycline derivate) present
in a triantibiotic paste formula and chlorhexidine presents substantivity
effects (18, 30, 31). This could explain the difficulty of residual live
bacteria to repopulate in the triantibiotic- and chlorhexidine-treated
biofilms. In contrast, calcium hydroxide–treated dentin significantly
increased the percentage of live cells from 63.71% to 83.88% after
an additional BHI treatment. This result shows the following:
1. The alkaline effect can be neutralized.
2. The biofilm can increase the proportion of live cells if new nutrients
are available.
The inactivation of medications is essential for culture-based
methods. In culture plate methodology, the antimicrobial activity is
measured by the ability of surviving bacteria to produce clones. This
procedure is evaluated after the detachment of the surface-associated
biofilm, suspension in a transport media, and inoculation in agar plates.
If residual antimicrobial compounds are introduced during these
procedures, the conditions could not be permissive for the growth of
new cells or clones. On the other hand, an advantage of confocal meth-
odology is that it can evaluate in situ by direct observation the viability of
microorganisms on the infected dentin by assessing the membrane
Antimicrobial Activity of Intracanal Medications 117
Basic Research—Technology
permeability without disturbing the attached cells. This evaluation
procedure in which the cell viability is measured directly on the infected
surface is similar to clinical conditions in which no inactivation of anti-
microbial substances is performed during the endodontic treatment.
Although the triantibiotic paste showed good antimicrobial
activity, it presented undesirable effects such as dentin staining (32).
In addition, an adequate antibiotic concentration that avoids toxicity
to host stem cells has not been completely addressed yet (33). Thus,
there is the necessity to search for other antimicrobial or antibiotic
medications with similar useful properties to the tested triantibiotic
paste but without having its associated deleterious side effects.
Conclusion
The triantibiotic paste was most effective at killing bacteria in the
biofilms on the intraorally infected dentin model in comparison with 2%
chlorhexidine gel and calcium hydroxide paste.
Acknowledgments
The authors deny any conflicts of interest related to this study.
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