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236]

Original Article

Comparative evaluation of cytotoxic effects of MTAD and

sodium hypochlorite using lactate dehydrogenase and trypan
blue assays: An in vitro study
Anant Singh, Pooja Kakkar, A. B. Pant1
Department of Conservative Dentistry and Endodontics, Sardar Patel Post Graduate Institute of Dental and Medical Sciences,
1
Sr. Scientist, In Vitro Toxicology, Indian Institute of Toxicology Research-CSIR, Lucknow, Uttar Pradesh, India

Abstract Aim: This study aims to examine the cytotoxic effects of BioPure MTAD and three clinical concentrations of
sodium hypochlorite (NaOCl) solutions (1%, 3%, and 5%) on cultured human periodontal ligament (PDL) cells.
Materials and Methods: Human PDL cells were grown on cell culture plates and were placed in contact with
various concentrations of  tested irrigants. Cytotoxicity of both BioPure MTAD and different concentrations
of NaOCl was assessed for immediate short‑term intervals (1, 5, 10, and 15 min) and midterm intervals as
well (24, 48, 72, and 96 h). Different parameters of toxicity such as lactate dehydrogenase assay and  trypan
blue dye exclusion assay were studied along with morphological changes at all the different time points.
The mean zones of inhibition were calculated for each group and were statistically analyzed.
Results: BioPure MTAD was least toxic of all test irrigants in short‑term and midterm time intervals. 5%
NaOCl showed the maximum cytotoxicity at short‑term time intervals. All cells died within the first 24 h
for all three NaOCl concentrations.
Conclusions: BioPure MTAD showed minimal toxicity and the three concentrations (1%, 3%, and 5%) of NaOCl
showed detrimental effects on the PDL cells and its toxicity was dose dependent.

Keywords: BioPure MTAD, cytotoxicity, lactate dehydrogenase assay, periodontal ligament cells, trypan
blue dye exclusion assay

Address for correspondence: Dr. Anant Singh, Department of Conservative Dentistry and Endodontics, Sardar Patel Post Graduate Institute of Dental and
Medical Sciences, Lucknow ‑ 226 025, Uttar Pradesh, India.
E‑mail: dranantsingh83@gmail.com

INTRODUCTION have when deciding an endodontic irrigation solution for

During the microbial control phase of endodontic
treatment, irrigating solutions assist in the dissolution of
the pulp tissue and antimicrobial activity inside all aspects
of the root canal system.[1] However, endodontic irrigants
may inadvertently come in contact with periradicular tissue.
Tissue toxicity is therefore an important consideration to
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root canal therapy.[2] Minimal toxicity to the surrounding
tissues is required for irrigants because they can be
accidentally extruded beyond the apical foramen as a result
of poor operator technique and/or anatomical variations
at the root apex resulting in periapical damage.[3]
This is an open access journal, and articles are distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to
remix, tweak, and build upon the work non-commercially, as long as appropriate credit
is given and the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com

How to cite this article: Singh A, Kakkar P, Pant AB. Comparative

DOI: evaluation of cytotoxic effects of MTAD and sodium hypochlorite using
10.4103/sej.sej_75_17 lactate dehydrogenase and trypan blue assays: An in vitro study. Saudi
Endod J 2018;8:189-95.

© 2018 Saudi Endodontic Journal | Published by Wolters Kluwer ‑ Medknow 189

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Singh, et al.: Cytotoxicity of MTAD and NaOCl using cell membrane integrity assays
Sodium hypochlorite (NaOCl) as an endodontic irrigant
is considered as the gold standard in chemomechanical
cleansing of the root canal. [4] Previous literature is
replete with evidence of its antimicrobial activity and
tissue‑dissolving capacity.[5‑7] In endodontic practice, several
concentrations of NaOCl ranging from 0.5% to 5.25% are
routinely used for irrigation. It is also well known that the
antimicrobial effectiveness and tissue dissolution properties
of NaOCl are strongly influenced by the concentration of
the solution.[8,9]
It has been reported that the cytotoxic effects of NaOCl
were observed at lower concentrations.[10] Furthermore,
effective concentrations of this solution are known to
exhibit cytotoxicity to vital tissues resulting in hemolysis,
inhibition of neutrophil migration, and damage to
endothelial and fibroblast cells.[11] Clinically, this could
manifest as pain, ulceration, and delayed wound healing
in the periradicular tissues.[12]
An ideal endodontic irrigant should, therefore, be nontoxic
and nonirritating to the periodontal tissues.
In 2003, a new endodontic irrigant was introduced by
Torabinejad and Johnson, which was later marketed
under the trade name BioPure MTAD (Dentsply, Tulsa
Dental, Tulsa, OK, USA).[13] BioPure MTAD is an aqueous
mix of a broad‑spectrum antibiotic (3% doxycycline),
demineralizing agent (4.25% citric acid), and detergent
(0.5% polysorbate 80).[13] A cytotoxic study conducted
by Zhang et  al. comparing MTAD to commonly
used irrigants and medications using  methyl thiazol
tetrazolium (MTT)‑tetrazolium assay concluded that it
could fulfill the ideal requirement of minimal toxicity while
retaining its antibiotic and demineralizing potential.[14]
In the past, colorimetric and luminescence‑based assays
have been employed for in vitro cytotoxic evaluation of
endodontic irrigants based on one parameter: the viable cell
metabolic activity.[14] However, the cytotoxicity of MTAD
and different clinical concentrations of NaOCl (1%, 3%,
and 5%) on the parameter of cell membrane integrity has
not yet been analyzed. Hence, the aim of this study was to
determine the cytotoxic potential of MTAD and NaOCl
in its various clinical concentrations of 1%, 3%, and 5%
on cultured human PDL cells using two cell membrane
integrity assays: lactate dehydrogenase (LDH) assay and
trypan blue assay.
MATERIALS AND METHODS
The materials tested in this study were BioPure
MTAD (Dentsply Tulsa Dental, Tulsa, OK) and three
190 Saudi Endodontic Journal | Volume 8 | Issue 3 | September-December 2018
concentrations (1%, 3%, and 5%) of NaOCl (Hyposol,
Jammu and Kashmir, India). Diluted NaOCl solutions
were prepared by adding distilled water (Minitube, New
Delhi, India) to 5% NaOCl. Test solutions were divided
into respective groups for evaluation (Group I: 1% NaOCl,
Group II: 3% NaOCl, Group III: 5%, Group IV: BioPure
MTAD, and Control group: distilled water).
Cell culture
All patients who volunteered were briefed about the nature
of the study and informed consent was taken for the
experimental procedures. Approval for the study protocol
was obtained from the institutional review board. Healthy
PDL tissue was taken from freshly extracted human
premolar teeth extracted atraumatically for orthodontic
reasons from adult male/female patients. All teeth used
for the study were free from any evidence of caries, root
resorption, periapical pathology, or root canal therapy.
The PDL tissue collected was immediately stored in
Hanks’ balanced salt solution containing an antibiotic
(penicillin/streptomycin) and antifungal (amphotericin B)
agent. The specimens were then stored at 4°C for 6 h.
The primary cultures of human PDL fibroblasts were
obtained by following the methods of Pant et al. with desired
modifications.[15] The teeth were washed in Dulbecco’s
minimum essential medium (MEM) (Gibco BRL, Grand
Island, NY, USA) containing antibiotic‑antimycotic, and
then, adherent soft tissues were removed from the crown and
the coronal one‑third of the root and discarded. The crown
and coronal one‑third of the root were then placed in 5.25%
NaOCl for 2 min to reduce the bacterial contamination,
as well as to kill any remaining gingival epithelial cells.
The middle 1/3rd of the root was then scraped to obtain
PDL tissue specimen. The tissue specimens were placed in
sterile Petri dish containing a thin layer of MEM containing
10% fetal bovine serum (Medox biotech, Chennai,
Tamil Nadu, India). The PDL tissue was disaggregated
using 0.2% collagenase (Roche, Mumbai, India) and 0.125%
trypsin (Sigma‑Aldrich, MI, USA) for 30 min at 37°C, and the
cells were collected by centrifugation at 1000 rpm for 5 min.
The pellet of packed cells was then resuspended in the 6‑well
culture plates (Corning Glass Works, Corning NY, USA) in
complete MEM and incubated at 37°C with an atmosphere
of 95% air and 5% CO2 for the attachment. Growth was
permitted to continue until the cell attained the confluent
monolayer, at which time they were trypsinized (trypsin
0.05%–ethylenediaminetetraacetic acid 0.53 mM) and
passaged into T‑25 culture flasks (Nunc, Denmark) to
expand the cell population (first cell passage). The cells of
third and fourth passages were trypsinized and pooled for
experimentation (to control the cell variability). Cell number
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Singh, et al.: Cytotoxicity of MTAD and NaOCl using cell membrane integrity assays

for experimentation was determined using an Electronic to exposure of different concentrations (1%, 3%, and
Coulter Counter (Model Zf, Coulter Electronics, Hialeah, 5%) of NaOCl and MTAD for the time interval of 1, 5,
Florida, USA). The numbers of viable cells in each batch 10, and 15 min and 24 h. The effect of the irrigants on
were measured by trypan blue dye exclusion (TBDE) cell morphology and cell proliferation was documented
test (Sigma‑Aldrich, MI, USA) before each experiment and under ×100 magnification.
batches showed that cell viability >95% was used for the
experiment. Statistical analysis
All assays were done in triplicate. The mean and standard
Cytotoxicity assays deviations of zones of inhibition were calculated for each
Lactate dehydrogenase release assay group. The data were analyzed using two‑way analysis of
The LDH assay is a quantitative analysis designed to variance  (ANOVA). When a significant difference was
measure the intactness of the cell membrane on damaged found, Tukey’s honest significant difference test was used
cells by the amount of cytoplasmic LDH released into to determine the significance among the means. P < 0.05
a commercially available medium kit (LDH Assay Kit, was considered statistically significant.
Sigma‑Aldrich, MI, USA), following the exposure of
various concentrations of test irrigants at different time RESULTS
intervals. After exposure to experimental irrigants, the
The results of these assays are expressed in percentage cell
plates were incubated as per the experimental schedule in
viability. The cell growth and proliferation values of the
CO2 incubator and centrifuged at 250 x g for 4 min. Then,
negative and positive control groups obtained from each
supernatant of each well was transferred to a fresh flat
experiment were within the historical data range of the
bottom 96‑well culture microtiter plates and proceeded
laboratory. The untreated batches of cells were designated
further for enzymatic analysis as per the instructions given
as positive controls and run simultaneously under exact
in the kit. The untreated sets that served as positive controls
conditions. To make the data comparable among the test
were also run under identical conditions
materials, which induced different and a wide range of
Trypan blue dye exclusion assay cytotoxic responses, the experiments were carried out at
The TBDE assay test was conducted to study the cell immediate short‑term intervals (1, 5, 10, and 15 min) and
viability by assessing the loss of membrane integrity at midterm (24, 48, 72, and 96 h) intervals for BioPure
following the protocols of Siddiqui et  al. with desired MTAD alone. In the pilot study we conducted, all cells were
modifications.[16] The cells (1 × 106) were seeded in  T‑25 cm2 found dead within 24 h for the different concentrations of
culture flasks and allowed to grow for 24 h in 5% CO2– NaOCl used; hence, the time period for NaOCl was limited
95% atmosphere at 37°C under high humid conditions. to short‑term intervals only [Figure 1].
Then, the medium was replaced with serum‑free medium
The time‑related inhibition of cell proliferation by BioPure
supplemented with different dilutions of experimental
MTAD and three concentrations of NaOCl (1%, 3%,
irrigant to test for various time intervals. The treated cells
and 5%) is discussed in our results. It is evident from
were then allowed to incubate for intervals of 24–96 h.
the data that there was a minimum percentage of LDH
Immediately after the completion of respective incubations,
release activity at short‑term time intervals for BioPure
cell suspensions were aspirated and centrifuged at 600 rpm
MTAD compared to all NaOCl groups for the same time
for 5 min and washed with sterile phosphate‑buffered
intervals [Figure 2].
saline (pH 7.4) twice, before stain with trypan blue dye. The
cell suspension was then mixed with trypan blue dye (0.4%
solution) at a ratio of 1:5 vol/vol (dye: cell suspension) and
placed in a hemocytometer (Sigma‑Aldrich, MI, USA). The
live (unstained transparent) and dead (blue stained) cells
were counted under ×100 magnification in a phase‑contrast
inverted microscope (Leica, Wetzler, Germany). The
untreated sets were also run simultaneously under the
identical conditions and served as control.

Cell morphology a b
Primary human PDL fibroblast cells were seeded in 12‑well Figure 1: (a) Untreated periodontal ligament cells (b) All the periodontal
culture plates, incubated to confluency and subjected ligament cells dead after 24 h of exposure to sodium hypochlorite

Saudi Endodontic Journal | Volume 8 | Issue 3 | September-December 2018 191

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Singh, et al.: Cytotoxicity of MTAD and NaOCl using cell membrane integrity assays

As expected, there was a strong correlation between the
cytotoxicity of NaOCl and its concentration [Figure 3]. For
1% NaOCl, the LDH release activity at 1 and 15 min was
between 100% and 150%. Whereas, 5% NaOCl showed
a significant percentage release of 125%–225% at the
same time interval. Five percentage NaOCl could induce
the maximum percentage of LDH release even at 15 min
of exposure. The results showed that the cytotoxicity
of BioPure MTAD in comparison to NaOCl groups of
various concentrations at different time intervals was the
least toxic.
A similar trend in the percentage cell viability was observed
in both exposures using TBDE assay. In BioPure MTAD
group, the percentage of cell viability was maximum at
all the short‑term time intervals [Figure 4 and Table 1].
Overall, the response of NaOCl was dose‑time dependent.
1%, 3%, and 5% NaOCl remained toxic even after 5 min
and onward exposure periods [Figure 5 and Table 2].
For midterm evaluation, MTAD showed statistically
significant (P < 0.001) increases in the LDH release even
at 24 h, but this increase reached near to control levels by
96 h [Figure 6 and Table 3].
As evident from Figure 5, NaOCl exposure resulted
in concentration and time‑dependent alterations in
morphology as well as cell number. When the cells were
exposed for 1 min to NaOCl, slight differences in the cell
morphology were noted. In sharp contrast, for 5, 10, and
15 min of NaOCl exposure, the cells were seen to be less
healthy with disruption of plasma membrane and release
of cytosolic contents [Figure 5]. When cells were exposed
for 24 h to NaOCl, all the cells were found to be dead with
no viable cells [Figure 1].
In case of MTAD, there was no significant difference noted
in the cell morphology at 1, 5, 10, and 15 min [Figure 6].

Figure 3: Dose‑related inhibition of periodontal ligament cell

Figure 2: Comparison of % cell viability as an indicator of lactate proliferation by BioPure MTAD compared with 1%, 3%, and 5%
dehydrogenase release activity of BioPure MTAD compared with 1%, sodium hypochlorite using lactate dehydrogenase assay at 1, 5, 10,
3%, and 5% sodium hypochlorite at 1, 5, 10, and 15 min time intervals and 15 min time intervals

Table 1: Comparison of sodium hypochlorite solutions (1%, 3%, and 5%) and BioPure MTAD at different time intervals using
trypan blue dye exclusion assay
Time interval 1% NaOCl (n=3) 3% NaOCl (n=3) 5% NaOCl (n=3) MTAD (n=3) ANOVA
Mean SD Mean SD Mean SD Mean SD F P
1 min 68.0 1.2 58.9 2.3 37.0 1.4 95.6 1.5 634.538 <0.001
5 min 36.0 1.5 22.3 1.3 14.0 0.9 88.9 2.6 1222.705 <0.001
10 min 24.9 1.6 17.1 1.2 10.1 2.1 85.2 2.0 1138.671 <0.001
15 min 15.0 0.8 13.0 1.4 7.0 1.1 81.7 2.5 1490.916 <0.001
ANOVA: Analysis of variance, SD: Standard deviation, NaOCl: Sodium hypochlorite

Table 2: Comparison of NaOCl (1%, 3%, and 5%) and BioPure MTAD at different time intervals using lactate dehydrogenase assay
Time interval 1% NaOCl (n=3) 3% NaOCl (n=3) 5% NaOCl (n=3) MTAD (n=3) ANOVA
Mean SD Mean SD Mean SD Mean SD F P
1 min 101.2 1.3 109.0 2.3 125.0 1.7 5.1 1.7 2760.039 <0.001
5 min 120.5 2.0 150.1 1.4 169.8 1.5 7.2 1.3 6556.343 <0.001
10 min 135.9 1.9 165.2 1.8 205.0 2.5 11.2 1.5 5530.798 <0.001
15 min 155.7 2.3 201.4 4.0 230.3 2.1 15.1 2.9 3231.505 <0.001
ANOVA: Analysis of variance, SD: Standard deviation, NaOCl: Sodium hypochlorite

192 Saudi Endodontic Journal | Volume 8 | Issue 3 | September-December 2018

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Singh, et al.: Cytotoxicity of MTAD and NaOCl using cell membrane integrity assays

Table 3: Comparison of cytotoxicity parameters at different time intervals of BioPure MTAD at midterm (24, 48, 72, and 96 h)
time interval
Variable 24 h 48 h 72 h 96 h ANOVA
Mean SD Mean SD Mean SD Mean SD F P
Percentage TBDE cell viability 43.7 2.1 57.5 1.7 64.5 2.2 71.8 1.4 122.418 <0.001
Percentage LDH release 126.1 3.1 114.7 3.6 110.3 1.8 105.3 2.4 29.959 <0.001
TBDE: Trypan blue dye exclusion, LDH: Lactate dehydrogenase, SD: Standard deviation, ANOVA: Analysis of variance

Figure 4: Dose‑related inhibition of periodontal ligament cell

proliferation by BioPure MTAD compared with 1%, 3%, and 5% sodium
hypochlorite using trypan blue dye exclusion assay at 1, 5, 10, and
15 min time intervals

At 24 h, the cell number and proliferative capacity were

significantly affected when compared to unexposed control
groups with the increase in exposure periods. Cell rupture
and damage with release of cytoplasmic contents into the
cell medium can be observed [Figure 6].
DISCUSSION
One of the main goals of root canal therapy is to
completely disinfect root canals. The use of irrigants is
to essentially facilitate intracanal cleansing by washing out
debris, disinfecting, and dissolving any tissue tags that may
remain within the complex canal anatomy.[1]
The physical, chemical, and biologic properties are
important factors to consider when deciding which
irrigating solution to be used. The toxic effects of irrigating
solutions used in endodontics are of particular interest
because their untoward extrusion into the periapical area
may be detrimental to healing capacity of the periradicular
tissues.[16]
It is a well‑known fact that the worldwide use of NaOCl
irrigation is because of its inherent antibacterial and
pulp‑dissolving capacity.[5‑7] Both these properties are,
however, strongly influenced by their concentration.[8,9]
Saudi Endodontic Journal | Volume 8 | Issue 3 | September-December 2018 193
Figure 5: Decreased cell viability and altered morphology of exposed
periodontal ligament fibroblast cells exposed to the indicated
concentrations of sodium hypochlorite and MTAD for 1, 5, 10, and 15 min
Therefore, biocompatibility plays a pivotal role in the
choice of an endodontic irrigant during root canal
preparation.
Studying the cytotoxic properties of an endodontic irrigant
on PDL cells at various concentrations is on the way
to determine the choice of irrigant. The primary PDL
cell cultures have shown to retain their metabolic state
when compared to their original tissue and therefore are
commonly used for cytotoxicity evaluations. Due to this
nearly unchanged metabolic state, in vitro studies closely
approximate the in vivo situation. For these reasons, these
cell cultures have been commonly used for cytotoxicity
evaluation.[17,18]
Several modern assays are available to study the viability and
proliferative capacity of cells in culture.[19] The advantage of
using microtiter plates (96‑well format) is that the analysis
of samples is quick and simultaneous.   One parameter
to evaluate cell death is to measure the cell membrane
integrity.[20] In this study, two assays were used to evaluate
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Singh, et al.: Cytotoxicity of MTAD and NaOCl using cell membrane integrity assays
Figure 6: Altered morphology of exposed periodontal ligament cells
to BioPure MTAD. The primary periodontal ligament cells were
seeded in 96-well culture plates and exposed to MTAD for 24, 48,
72, and 96 h
the viability and cytotoxicity of PDL cells by the intactness
of their cell membrane.
The LDH is a stable cytoplasmic enzyme present in all cells.
It is rapidly released into the cell culture supernatant on
damage of the plasma membrane. The LDH release assay
is a simple, accurate, and reproducible assay that determines
the integrity of the cell membrane by measuring the
cytoplasmic enzyme activity released by damaged/necrotic
cells.[21]
TBDE assay is a diazo dye used to color dead tissues or
cells a distinctive blue color under a microscope. Live cells
are excluded from staining.[22]
The results of the present study have shown minimal
cytotoxicity of BioPure MTAD against PDL cells when
compared to the conventional gold standard of irrigation,
NaOCl. The cytotoxicity of dental materials has been
evaluated on the basis of changes in their cell number,
intracellular metabolism, and morphology.
In the case of BioPure MTAD, maximum reduction in
cell viability was recorded in the first 5 min of exposure,
but these values were significantly less when compared
to 1% concentration of NaOCl used for the same time
period. Yasuda et  al. have stated that BioPure MTAD
has minimal cytotoxicity against MC3T3‑E1 and PDL
cells compared with conventional irrigants.[23] Zhang
et al. compared BioPure MTAD to the most commonly
used intracanal irrigants using MTT‑tetrazolium method
on L929 fibroblasts and concluded that BioPure MTAD
showed the least cytotoxic effects.[14]
Regarding the cytotoxicity of various concentrations of
NaOCl, Chang et  al., with the help of PI fluorescence
assay, reported that the toxic effects of NaOCl increased
with concentration. PDL cells were destroyed at
3 and 24 h time interval when 5.25% NaOCl was
diluted.[10]
194 Saudi Endodontic Journal | Volume 8 | Issue 3 | September-December 2018
Our results concur with those obtained by Silva et  al.[24]
and Spangberg et  al.[25] The results of their studies also
concluded that 5% NaOCl showed the maximum
cytotoxicity at short‑time intervals [24] and various
concentrations of NaOCl are dose dependent.[25] Similar
findings were observed by Essner et al. using CellTiter‑Glo
luminescent cell viability assay to determine the viability of
the pulp cells. They found that as NaOCl concentration
increased from 0.04% to 0.33%, the pulpal cells viability
decreased correspondingly.[26]
For midterm evaluation, BioPure MTAD showed a
significant increase in cell viability. All cells were dead
within 24 h when exposed to all the three concentrations
of NaOCl.
CONCLUSIONS
The present study showed that in both cytotoxic assays,
BioPure MTAD had a lower toxicity against PDL compared
to NaOCl. Overall response of NaOCl was concentration
dependent with 1%, 3%, and 5% NaOCl remained toxic
even after 5 min and onward exposure periods. Further
studies will be needed to determine the long‑term toxic
effects of BioPure MTAD on cells in vivo.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
REFERENCES
1. Zehnder M. Root canal irrigants. J Endod 2006;32:389‑98.
2. Mehdipour O, Kleier DJ, Averbach RE. Anatomy of sodium
hypochlorite accidents. Compend Contin Educ Dent 2007;28:544‑6,
548, 550.
3. Spencer HR, Ike V, Brennan PA. Review: The use of sodium
hypochlorite in endodontics  –  Potential complications and their
management. Br Dent J 2007;202:555‑9.
4. Ruddle CJ. Endodontic disinfection: Tsunami irrigation. Saudi Endod
J 2015;5:1‑12.
5. Mittal R, Singla MG, Garg A, Gupta S, Dahiya V. Comparative
evaluation of the antimicrobial efficacy of MTAD, oxytetracycline,
sodium hypochlorite and chlorhexidine against Enterococcus faecalis: An
ex‑vivo study. Saudi Endod J 2012;2:70‑4.
6. Siqueira JF Jr., Batista MM, Fraga RC, de Uzeda M. Antibacterial effects
of endodontic irrigants on black‑pigmented gram‑negative anaerobes
and facultative bacteria. J Endod 1998;24:414‑6.
7. Beltz RE, Torabinejad M, Pouresmail M. Quantitative analysis of the
solubilizing action of MTAD, sodium hypochlorite, and EDTA on
bovine pulp and dentin. J Endod 2003;29:334‑7.
8. Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC,
de Souza‑Filho FJ, et al. In vitro evaluation of the antimicrobial activity
of chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral
Pathol Oral Radiol Endod 2004;97:79‑84.
9. Spanó JC, Barbin EL, Santos TC, Guimarães LF, Pécora JD. Solvent
[Downloaded free from http://www.saudiendodj.com on Thursday, August 16, 2018, IP: 10.32.67.236]
Singh, et al.: Cytotoxicity of MTAD and NaOCl using cell membrane integrity assays
action of sodium hypochlorite on bovine pulp and physico‑chemical
properties of resulting liquid. Braz Dent J 2001;12:154‑7.
10. Chang YC, Huang FM, Tai KW, Chou MY. The effect of sodium
hypochlorite and chlorhexidine on cultured human periodontal
ligament cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2001;92:446‑50.
11. Shetty KP, Satish SV, Kilaru K, Ponangi KC, Venumuddala VR,
Ratnakar P. Comparative evaluation of the cytotoxicity of 5.25%
sodium hypochlorite, 2% chlorhexidine and mixture of a tetracycline
isomer, an acid and a detergent on human red blood corpuscles: An
in vitro study. Saudi Endod J 2014;4:1‑6.
12. Gatot A, Arbelle J, Leiberman A, Yanai‑Inbar I. Effects of sodium
hypochlorite on soft tissues after its inadvertent injection beyond the
root apex. J Endod 1991;17:573‑4.
13. Torabinejad M, Khademi AA, Babagoli J, Cho Y, Johnson WB,
Bozhilov K, et al. A new solution for the removal of the smear layer.
J Endod 2003;29:170‑5.
14. Zhang W, Torabinejad M, Li Y. Evaluation of cytotoxicity of MTAD
using the MTT‑tetrazolium method. J Endod 2003;29:654‑7.
15. Pant V, Dixit J, Agrawal AK, Seth PK, Pant AB. Behaviour of human
periodontal ligament cells on CO2 laser irradiated dentinal root
surfaces: An in vitro study. J Periodontal Res 2004;39:373‑9.
16. Siddiqui MA, Singh G, Kashyap MP, Khanna VK, Yadav S,
Chandra D, et al. Influence of cytotoxic doses of 4‑hydroxynonenal
on selected neurotransmitter receptors in PC‑12 cells. Toxicol In Vitro
2008;22:1681‑8.
17. Barnhart BD, Chuang A, Lucca JJ, Roberts S, Liewehr F, Joyce AP,
Saudi Endodontic Journal | Volume 8 | Issue 3 | September-December 2018 195
et al. An in vitro evaluation of the cytotoxicity of various endodontic
irrigants on human gingival fibroblasts. J Endod 2005;31:613‑5.
18. Al‑Shaher A, Wallace J, Agarwal S, Bretz W, Baugh D. Effect of propolis
on human fibroblasts from the pulp and periodontal ligament. J Endod
2004;30:359‑61.
19. Cook JA, Mitchell JB. Viability measurements in mammalian cell
systems. Anal Biochem 1989;179:1‑7.
20. Weyermann J, Lochmann D, Zimmer A. A practical note on the use
of cytotoxicity assays. Int J Pharm 2005;288:369‑76.
21. Korzeniewski C, Callewaert DM. An enzyme‑release assay for natural
cytotoxicity. J Immunol Methods 1983;64:313‑20.
22. DeRenzis FA, Schechtman A. Staining by neutral red and trypan blue
in sequence for assaying vital and nonvital cultured cells. Stain Technol
1973;48:135‑6.
23. Yasuda Y, Tatematsu Y, Fujii S, Maeda H, Akamine A, Torabinejad M,
et al. Effect of MTAD on the differentiation of osteoblast‑like cells.
J Endod 2010;36:260‑3.
24. Silva FT, Barcelos R, Petrópolis DB, Azevedo BR, Primo LG,
Silva‑Filho FC. Cytotoxicity of different concentrations of
sodium hypochlorite on human osteoblasts. Rev Gaúcha Odontol
2009;57:317‑21.
25. Spangberg L, Engström B, Langeland K. Biologic effects of dental
materials 3. Toxicity and antimicrobial effect of endodontic antiseptics
in vitro. Oral Surg Oral Med Oral Pathol 1973;36:856‑71.
26. Essner MD, Javed A, Eleazer PD. Effect of sodium hypochlorite on
human pulp cells: An in vitro study. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod 2011;112:662‑6.