Basic ResearchBiology
Cytotoxic Effects and Antibacterial Efficacy of a 3-Antibiotic
Combination: An In Vitro Study
Sorapong Chuensombat, DDS,* Saengusa Khemaleelakul, DDS, Dip Clin Dent, PhD,*
Siriporn Chattipakorn, DDS, PhD, and Tanida Srisuwan, DDS, Dip Clin Dent, PhD*
Abstract
Introduction: A 3-antibiotic combination (3Mix) is
widely used in endodontics for root canal disinfection,
particularly in pulp revascularization procedures.
However, the cytotoxicity of 3Mix has not been evalu-
ated. The purpose of this study was to determine the
cytotoxicity and antibacterial efficacy of 3Mix and
each single antibiotic component of 3Mix. Methods:
For the cytotoxicity test, human dental pulp cells and
apical papilla cells were exposed to either 3Mix or to
each single antibiotic component of 3Mix using con-
centrations of 0.024, 0.097, 0.39, 1.56, 6.25, and
25.00 mg/mL for 1, 3, 5, and 7 days. Cell viability was
determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide (MTT) assay. For the anti-
bacterial test, 25.00 mg/mL and 0.39 mg/mL 3Mix or
single antibiotic were tested on bacteria isolated from
necrotic teeth by measuring bacterial recovery on blood
agar. Results: The 0.024-mg/mL concentration of all
experimental groups generated the highest dental
pulp cell or apical pulp cell viability at all time periods.
On day 7, 0.39 mg/mL 3Mix produced more than 90%
cell viability; 25.00 mg/mL 3Mix completely eliminated
isolated bacteria, whereas 0.39 mg/mL was unable to
eradicate all bacteria. However, the overall bacterial
reduction was significantly different compared with
the control group (P < .01). Conclusions: All drugs
except metronidazole induced cytotoxicity on cultured
cells. 3Mix generated higher cytotoxicity compared
with a single drug. The cytotoxicity increased in a con-
centration- and time-dependent manner; 0.39 mg/mL
3Mix had less cytotoxicity and was able to significantly
reduce bacteria isolated from necrotic teeth. (J Endod
2013;39:813819)
Key Words
3-antibiotic combination, antibacterial efficacy, apical
papilla cells, cytotoxicity, dental pulp cells
From the Departments of *Restorative Dentistry and Periodontology and Oral Biology and Diagnostic Sciences, Faculty of Dentistry, Chiang Mai University,
Chiang Mai, Thailand.
Address requests for reprints to Dr Sorapong Chuensombat, Department of Restorative Dentistry and Periodontology, Faculty of Dentistry, Chiang Mai University,
Chiang Mai, Thailand. E-mail address: schuensombat@gmail.com
0099-2399/$ - see front matter
Copyright 2013 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2012.11.041
JOE Volume 39, Number 6, June 2013
D ental pulp plays an important role in tooth homeostasis (1). Various types of cells
can be found inside the pulp (eg, odontoblasts, fibroblasts, stem cells, immune
cells, and endothelial cells). During root formation, apical papilla tissue is found.
This tissue has a high collateral circulation and consists of various cells types (eg, fibro-
blasts, immune cells, endothelial cells, and stem cells). These cells play a part in tooth
development and pulpal infection control including the self-repairing process (13).
Microorganisms, especially bacteria, are the main cause of pulpal and periradicular
disease. The invasion of bacteria and their toxins into the pulp is able to induce
pulpal inflammation and lead to pulp death (4, 5). When pulp necrosis occurs
during tooth development, the formation of the root is limited, leaving the tooth with
a thin root structure and wide apical closure, risking root fracture (6).
The treatment for incomplete tooth development is difficult and challenging (7, 8).
Recently, pulp revascularization has become a new treatment modality to manage
immature necrotic tooth using the lesion sterilization and tissue repair (LSTR)
concept (9, 10). In brief, the infection is removed, creating a sterile environment
that promotes tissue regeneration and revitalization (11, 12) and permitting the
tooth to continue the development (6, 13). The cell sources that play a role in this
situation are still unknown. However, remaining vital tissue inside the tooth and
surviving cells around the apical part of the tooth, which have differentiation
potential, are suspected (1417).
A 3-antibiotic combination (3Mix), a mixture of minocycline, ciprofloxacin, and
metronidazole, has been introduced by Hoshino et al (18) along with the LSTR concept.
Various studies reported promising outcomes when 3Mix was used in necrotic teeth
with incomplete root formation (16, 19). However, some recent histologic studies
have shown that regenerated tissue found in the treated tooth was not exactly pulp
tissue (20, 21). Still, the cause for these consequences is unknown.
An interesting issue is the concentration of 3Mix used because the clinical use
of 3Mix has been empirical. Local application of 3Mix using an excessive concentra-
tion might affect the host tissue, causing cell death, which limits tissue regeneration.
Therefore, the purposes of this study were to determine the cytotoxic effects of 3Mix
and each single antibiotic component of 3Mix on cultured human dental pulp cells
(DPCs) and apical papilla cells (APCs) and to evaluate the antibacterial efficacy of
the noncytotoxic concentration of 3Mix and each single antibiotic component of
3Mix against bacteria isolated from necrotic teeth.
Materials and Methods
Primary Cell Culture
DPCs and APCs were extracted from human dental pulp tissue or apical papilla
tissue obtained from nonpathologic mature or immature third molars. All procedures
Cytotoxicity and Antibacterial Efficacy of 3Mix 813
Basic ResearchBiology

Figure 1. DPCs treated with 3Mix at different concentrations for 7 days. (A) Control (cells in culture medium) and (BG) 0.024-, 0.097-, 0.39-, 1.56-, 6.25-, and
25.00-mg/mL concentrations (40 magnification). DPCs treated with 0.024, 0.097, 0.39, and 1.56 mg/mL started to form a multilayer when reaching confluence,
whereas DPCs treated with 6.25 and 25.00 mg/mL showed a mixed population of both spindle-shaped cells and polygonal-like cells. 3Mix at a concentration of
25.00 mg/mL produced fewer DPCs when compared with lower concentrations and the control.

were approved by the Human Experimentation Committee of the Faculty
of Dentistry, Chiang Mai University, Chiang Mai, Thailand.
Dental pulp tissues or apical papilla tissues were minced and
digested with a mixture of 3.00 mg/mL collagenase (Gibco/Invitrogen,
Gaithersburg, MD) and 4.00 mg/mL dispase (Sigma-Aldrich, St Louis,
MO) at 37 C for an hour in order to extract DPCs or APCs. The extracted
cells were cultured in the alpha modification of Eagle medium (a-MEM,
Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco/In-
vitrogen), 100.00 U/mL penicillin, 100.00 mg/mL streptomycin (Sigma-
Aldrich), and 100.00 mmol/L L-ascorbic acid (Sigma-Aldrich). The
cells were kept at 37 C in a humidified atmosphere with 95% air and
5% CO2. For the experiment, cells at the third through fifth passages
were harvested and used.
Drug Preparation
A stock solution of 3Mix was prepared by mixing the powder from
minocycline (Qualimed, Samut Prakan, Thailand), ciprofloxacin
(Khandelwal, Mumbai, India), and metronidazole (Piramal Healthcare,
Gujarat, India) at 50.00 mg/mL in distilled water. Then, a 4-time serial
dilution was performed producing 25.00-, 6.25-, 1.56-, 0.39-, 0.097-,
and 0.024-mg/mL concentrations using a-MEM. For the single-drug
solution, minocycline, ciprofloxacin, and metronidazole were sepa-
rately prepared in the same concentrations as previously described.
All samples were prepared at room temperature and sterilized by
a double-filtering technique using 2.00-mm pore size filter paper
(Whatman, Maidstone, England) and 0.2-mm pore size microfilters
(Corning, Oneonta, NY).
Cytotoxicity Test
DPCs or APCs were seeded at 103 cells/well onto 96-well
plates containing 150.00 mL a-MEM. After 24 hours, cells were stim-
ulated with media containing 25.00, 6.25, 1.56, 0.39, 0.097, and
0.024 mg/mL either 3Mix or each single drug. The cells were
monitored for 1, 3, 5, and 7 days under a light microscope
(DP12; Olympus, Melville, NY). An 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide (MTT) assay was performed using

Figure 2. APCs treated with 3Mix at different concentrations for 7 days. (A) Control (cells in culture medium) and (BG) 0.024-, 0.097-, 0.39-, 1.56-, 6.25-,
and 25.00-mg/mL concentrations (40 magnification). 3Mix affected on APCs similar to DPCs. APCs treated with 0.024 mg/mL 3Mix showed densely cell number, a
descending of cell number of APCs was demonstrated when the cells was exposed to higher concentrations of 3Mix. APCs treated with 6.25 and 25.00 mg/mL
showed a mixed population of both spindle-shaped cells and polygonal-like cells.

814 Chuensombat et al. JOE Volume 39, Number 6, June 2013


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solu-
tion (Sigma-Aldrich) at 5.00 mg/mL in phosphate-buffered saline
solution. The optical density was investigated at l570 nm using an
enzyme-linked immunosorbent assay spectrophotometer (Sunrise;
Tecan, Mannerdrof, Switzerland). Percentages of cell viability were
calculated and grouped according to the criteria of Dalh et al
(22) as follows:
1. <30% cell viability = severe cytotoxicity
2. 30%60% cell viability = moderate cytotoxicity
3. 60%90% cell viability = slight cytotoxicity
4. >90% cell viability = noncytotoxicity
The results were analyzed using the Kruskal-Wallis and Mann-
Whitney U tests.
Bacterial Collection
Bacterial samples were collected from 20 necrotic teeth during
routine endodontic treatment following the protocol of Sassone et al
(23). Briefly, under sterile conditions, an access opening was made
Basic ResearchBiology
using diamond burs with sterile normal saline solution irrigation.
The operating field and pulp chamber were then swabbed with
2.50% NaOCl followed by sterile 5.00% sodium thiosulfate solution.
Sterile normal saline solution was introduced into the canal, and
a no. 15 K-type file was inserted 1.002.00 mm short of the root
apex. After filing the canals, 2 sterile paper points were introduced
into the canal and left for 1 minute. The paper points were transferred
into cryotubes (Corning) containing 500.00 mL reduced transport
fluid. The bacterial samples were immediately transferred to the labo-
ratory, and 10 serial dilutions were performed under an anaerobic
condition.
Bacterial Recovery
Aliquots of 100.00 mL of the diluted bacterial samples were
dropped on and gently spread over the surface of tryptic soy
blood agar (Criterion, Santa Maria, CA) containing either 25.00 or
0.39 mg/mL 3Mix or of each single drug. The plates were incubated
in an anaerobic glove box at 37 C for 7 days. The colonies
were counted and recorded. The data were analyzed using the paired
t test.

Figure 3. DPC and APC viability after being treated with 3Mix at different concentrations and times. (A) DPCs and (B) APCs. For both cell types, 0.024 mg/mL 3Mix
produced the highest cell viability, whereas 25.00 mg/mL 3Mix generated the lowest cell survival at all time periods (P < .001). On day 7, DPC and APC viability was
significantly reduced into less than 90% cell viability when exposed to 25.00, 6.25, and 1.56 mg/mL 3Mix (P < .001). Data are mean  standard deviation.
*A significant difference from the control group (P < .001).

JOE Volume 39, Number 6, June 2013 Cytotoxicity and Antibacterial Efficacy of 3Mix 815
Basic ResearchBiology
Results
Morphology of DPCs and APCs
Monitoring DPCs and APCs under a light microscope showed that
DPCs or APCs in the control group were a mixed population containing
both spindle-shaped cells and polygonal-like cells. In the experimental
groups that contained antibiotics, a reduced cell number was shown
only for DPCs or APCs stimulated by 25.00 mg/mL 3Mix. Polygonal
and round-shaped cells were observed in a monolayer (Figs. 1 and 2).
Cytotoxicity of 3Mix and Each Single Antibiotic
The 0.024-mg/mL concentration of 3Mix produced the highest cell
viability, whereas 25.00 mg/mL 3Mix generated the lowest cell survival at
all time periods (P < .001). On day 7, DPC and APC survival was reduced
to less than 90% cell viability when stimulated by 25.00, 6.25, and 1.56
mg/mL 3Mix (Fig. 3). All experiments on day 1 produced the highest cell
viability, whereas day 7 generated the lowest cell viability when 3Mix,
minocycline, and ciprofloxacin were tested (P < .001, Fig. 4). When
comparing all the drugs, 3Mix had greater cytotoxicity than the other
single antibiotics. Minocycline and ciprofloxacin at 25.00, 6.25, and
1.56 mg/mL generated less than 90% cell viability, whereas metronida-
zole at all concentrations had little effect on cell viability (Fig. 5).
Antibacterial Efcacy of Antibiotics
3Mix at 25.00 mg/mL completely eradicated all isolated
bacteria with a significant reduction of bacterial recovery (P < .01).
Figure 4. DPC and APC viability after being treated with 3Mix at 25.00, 6.25, and 1.56 mg/mL for 1, 3, 5, and 7 days. (A) DPCs and (B) APCs. 3Mix generated the
highest percentage of cell viability on day 1, whereas the lowest percentage of cell viability was observed on day 7. DPC and APC viability was significantly reduced
when the exposure time increased (P < .001). Data are mean  standard deviation. A significant difference from *day 1, **day 3, and ***day 5.
816 Chuensombat et al.
In the presence of 0.39 mg/mL 3Mix or a single drug at the concentration
of 25.00 mg/mL, the bacterial number was reduced, but bacteria were
not completely eliminated. However, bacterial recovery also significantly
decreased compared with the control group (P < .01, Fig. 6).
Discussion
This study showed that all drugs except metronidazole were cyto-
toxic to cultured human DPCs and APCs in a dose- and time-dependent
manner. When comparing all drugs at the same concentration, 3Mix
had greater cytotoxicity than the other single drugs; 25.00 mg/mL
3Mix produced moderate cytotoxicity with the highest bacteria reduc-
tion, whereas 0.39 mg/mL 3Mix had a less cytotoxic effect on cultured
cells but showed less bacterial elimination. However, this concentration
was able to significantly reduce the number of bacteria when compared
with the control group.
A 3-antibiotic combination has been used in treating immature
teeth with necrotic pulp using the LSTR concept (10, 18). Many
studies have reported the successful outcomes by which immature
teeth showed continued root formation (13, 24). However, histologic
studies regarding the regenerated tissue inside the necrotic human
tooth are lacking. Recent histologic studies (20, 21) of dogs teeth
treated with the LSTR technique revealed that the newly generated
tissue was not pulp tissue. Periodontal tissue, including bone, was
generally observed inside the treated tooth. Therefore, the question
should be raised whether this treatment option is valid and how to
prevent these consequences.
JOE Volume 39, Number 6, June 2013
 Basic ResearchBiology

Figure 5. Cell viability after being treated with antibiotics at different concentrations for 7 days. (A) DPCs and (B) APCs. For both cell types, 3Mix produced more
cytotoxicity compared with each single drug at all concentrations. Minocycline and ciprofloxacin at 25.00, 6.25, and 1.56 mg/mL generated less than 90% cell
viability, whereas metronidazole at all concentrations had a small effect on cell viability. Data are mean  standard deviation. *A significant difference from
the control group (P < .001).

One interesting issue is whether excessive concentrations of
3Mix are used in the treatment. Hoshino et al (10) reported that
25.00 mg/mL 3Mix was able to eradicate all isolated bacteria.
However, the exact concentration used in the clinic, especially in
the pulp revascularization technique, has never been measured. It
seems that excessive concentrations are currently being used, which
may damage the remaining cells and impair tissue healing and regen-
eration. It is possible that the remaining vital pulp tissue and perira-
dicular area, both containing stem cells (1417), are damaged by
the high concentration of 3Mix. Therefore, an expected
differentiation pattern (eg, odontoblast differentiation and dentin
formation) might not occur.
In this study, DPCs or APCs were exposed to different concentra-
tions of 3Mix. DPCs were selected for this study because these cells may
survive in the infected tooth. Many published case reports revealed the
observation of vital tissue in the tooth diagnosed as necrotic (1417).
Moreover, APCs were also tested because stem cells may still remain in
the apical region, which may have some roles in pulp regeneration.
From our study, we discovered that 25.00 mg/mL 3Mix produced
moderate cytotoxicity. However, in a pilot study, concentrations
higher than 25.00 mg/mL produced even greater toxicity, leading to
total cell death (data not shown). Therefore, these results ensure that
the concentration of 3Mix in general clinical usage is too strong,
causing cytotoxicity on the remaining vital tissues. On the other hand,
3Mix at 0.39 mg/mL had a less cytotoxic effect on DPC and APC
viability. This concentration is suggested to be the best candidate for
pulpal regeneration along the concentrations of drugs tested in this
experiment.
Interestingly, recently published data concerning the cytotoxicity
of 3Mix reported that 0.010.10 mg/mL did not have an effect on
cultured stem cells from apical papilla (25). This result was different
from our study, which showed that 0.39 mg/mL 3Mix was a safe
dose. Also, some possible reasons might be the different source of anti-
biotics used in the experiment, the different techniques for 3Mix solu-
tion preparation, and the different method for cell viability
measurement that may affect the results. It is important for clinicians
to be aware that diverse sources of antibiotics have varying levels of
toxicity; these antibiotics also have varying levels of effectiveness, which
is an additional concern. More information and research into this area
are needed.

JOE Volume 39, Number 6, June 2013 Cytotoxicity and Antibacterial Efficacy of 3Mix 817
Basic ResearchBiology
Figure 6. The antibacterial efficacy of 3Mix and each single antibiotic against bacteria isolated form necrotic teeth. 3Mix at 25.00 mg/mL could completely erad-
icate all isolated bacteria with a significant reduction of bacterial recovery (P < .01). In the presence of 0.39 mg/mL 3Mix or a single drug at the concentration of
25.00 mg/mL, the bacteria number was reduced but not completely eliminated. Data are mean  standard deviation. *A significant difference from the control
group (P < .01).
The cytotoxicity of 3Mix was investigated regarding its combination
because 3 antibiotics were mixed. Therefore, each drug was also tested
at the same concentration as 3Mix using an MTT assay. The result indi-
cated that all drugs except metronidazole generated cytotoxicity. The
cytotoxic effect was dose and time dependent. 3Mix had greater cytotox-
icity than other single drugs. The greater cytotoxicity of 3Mix might
result from a combination of the toxicities of each drug. However,
the study was conducted in an in vitro environment, which may not
represent the real clinical situation. No evidence has been published
regarding the depth of 3Mix penetration in immature tooth whether it
reaches the apical and surrounding tissue. Therefore, the clinical impli-
cation of these results should be further tested.
One reason for its cytotoxicity might be the low pH (pH = 4.04.6)
of 3Mix. Minocycline hydrochloride and ciprofloxacin hydrochloride
are generally used for 3Mix preparation. The release of hydrogen
ions from HCl groups resulted in an acidic condition that was an unfa-
vorable condition for culturing cells (26). In addition, a low pH allowed
more agents to remain soluble and available for uptake into the cells;
thus, the retained drug was able to develop more cytotoxicity (27).
Conversely, in this study, metronidazole did not inversely affect DPCs
and APCs even at the 25.00-mg/mL concentration. It is possible that
metronidazole solution has a neutral pH (pH = 6.84); thus, cytotoxicity
did not occur. In contrast, this result differed from that of a previous
study by Ferreira et al (28), who reported that only 5.00 mg/mL metro-
nidazole resulted in 74% human gingival fibroblast cell line viability.
However, many studies indicated that there is a variation in the degree
of drug-induced cytotoxicity in different cell types (29, 30). Therefore,
the sensitivity of DPCs or APCs and the gingival fibroblast cell line
on drugs might be different. From this point of view, the cytotoxic
effect of 3Mix from its acidic condition should be a concern. Other
antibiotics that have a neutral pH should be selected as a substitute
for minocycline and ciprofloxacin. However, those alternative
antibiotics should intensely offer a potent antibacterial efficacy.
The antibacterial efficacy of antibiotics used in vital pulp treatment,
especially the LSTR concept, is important because bacteria remnant
might affect the treatment outcome. Interestingly, 0.39 mg/mL 3Mix,
which had a less cytotoxic effect on cultured cells, significantly reduced
isolated bacteria compared with the control group. However, this effec-
tive concentration differed from Hoshino et al (10), who reported much
higher concentrations (100200 mg/mL) with antibacterial effects. The
reasons might be the differences in the source of antibiotics used and the
bacteria isolation techniques. When single antibiotics were tested at the
818 Chuensombat et al.
same concentrations as 3Mix, it was found that even a 25.00-mg/mL
concentration of a single drug alone was unable to eradicate all isolated
bacteria. This might be because each drug is not active against all bacte-
rial types. Minocycline and ciprofloxacin, which are broad-spectrum
antibiotics, have an efficient action against some gram-positive and
gram-negative bacteria (31, 32), whereas metronidazole is able to kill
only anaerobic bacteria (33). Thus, the combination of 3 drugs provides
a broader antibacterial spectrum than a single drug alone. However,
balancing between the cytotoxic effect and antibacterial efficacy is impor-
tant and still needs further study.
However, there is concern whether the types of bacteria presented
in necrotic immature teeth are the same as in necrotic mature teeth. In
this study, only bacteria isolated from necrotic mature teeth were
applied using a protocol adapted from Sassone et al (23). The dentin
was scratched by means of a K-file, assuming that bacteria presented
in dentin might be dispersed and collected after absorbing with a paper
point. After incubation, collected bacteria samples were grossly clarified
using gram staining. Main types of bacteria observed were similar to
previously published data representing bacteria found in necrotic tooth
(34). However, these bacterial samples may or may not completely
represent the bacteria observed in immature necrotic teeth. Further
studies are suggested because current evidence is lacking.
The use of 3Mix has been popular for a decade. It offers many bene-
fits in treating patients with infected teeth. However, the results from this
study should stimulate a concern with 3Mix usage, especially in pulp
revascularization, because using high doses of 3Mix produced cytotox-
icity. The reduction of the 3Mix concentration into an appropriate dose
may improve the outcome of pulp revascularization treatment because
it causes less cytotoxicity and still has a potent antibacterial efficacy.
Conclusions
3Mix, minocycline, and ciprofloxacin were cytotoxic to cultured
DPCs and APCs. 3Mix induced a higher toxicity than minocycline, cipro-
floxacin, and metronidazole. The cytotoxicity of each antibiotic except
metronidazole increased in a concentration- and time-dependent
manner. The 0.39-mg/mL concentration of 3Mix had no cytotoxicity
to DPCs and APCs, yet it was able to significantly reduce bacteria isolated
from necrotic teeth.
Acknowledgments
The authors deny any conflicts of interest related to this study.
JOE Volume 39, Number 6, June 2013

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Cytotoxicity and Antibacterial Efficacy of 3Mix 819