Biomaterials 23 (2002) 77–83

The biocompatibility evaluation of epoxy resin-based root canal

sealers in vitro
Tsui-Hsien Huanga, Jaw-Ji Yanga, Huei Lib, Chia-Tze Kaoc,*
a
Dental Department, Chung Shan Medical and Dental College, No.110, Section 1, Chien Kuo N Road, Taichung 402, Taiwan
b
Institute of Toxicology, Chung Shan Medical and Dental College, No.110, Section 1, Chien Kuo N Road, Taichung 402, Taiwan
c
Institute of Stomatology, Chung Shan Medical and Dental College, 620 Shr Jeng Road, Taichung 402, Taiwan
Received 23 October 2000; accepted 19 February 2001

Abstract

The cytotoxic and mutagenic effects of epoxy resin-based root canal sealer AH26 and AH-Plus were determined in vitro. Root
canal sealers were eluted for 24 h in dimethyl sulfoxide (DMSO) and diluted in culture medium. Cytotoxic effects were assessed using
the MTT [tetrazolium dye, 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide, C18H16N5SBr] assay for mitochondrial
enzyme activity and also the cell viability. Genotoxicity assays were assessed using the alkaline single cell gel electrophoresis assay
(comet assay) for DNA damage measurement. Result indicated that both the AH26 and AH-Plus sealers exhibited a dose-dependent
increase in astrocyte toxic effects. Additionally, dose-dependent astrocyte DNA damage was also noted for both sealers. Therefore,
these epoxy resin-based sealers, AH26 and AH-Plus demonstrated both cytotoxicity and genotoxicity in vitro. # 2001 Elsevier
Science Ltd. All rights reserved.

Keywords: Biocompatibility of epoxy resin sealer; MTT assay; Alkaline single cell gel electrophoresis

1. Introduction biocompatiblity and antimicrobial activity of a specific

Clearly, one of the principal requirements of an
endodontic root canal sealer should be that it is
noncytotoxic and immunologically compatible with
peripheral tissue [1]. Sealer-elutable substances or the
degradation or corrosion products from a root canal
sealer may gain access to periodontal tissue through
numerous pathways [2,3]. Root canal sealers and their
diffusible components, therefore, need to be critically
evaluated for their cytocompatibility and genotoxicity
prior to their general clinical use.
Numerous root canal sealers are available, based on
various formulae and containing a variety of different
and partly mutagenic components, such as epoxy resin
sealers, e.g., AH26 and AH Plus, Ca(OH)2-based
materials such as Sealapex and Apeixt, and ZnO-
eugenol cements, e.g., N2 and endomethasone [4]. The
*Corresponding author. Tel.: +886-4-2255-3715; fax: +886-4-2255-
3713.
E-mail address: ctk@mercury.csmc.edu.tw (C.-T. Kao).
root canal sealer remains one of the principal considera-
tions for selecting an appropriate sealer for a dental
restoration [4,5]. From the literature, it would appear
that the side effects of the use of various root canal
sealers have been previously studied to some extent
[6–8]. It has been previously demonstrated that sealer
material based upon zinc oxide-eugenol was moderately
to severely toxic in implantation studies, the results of
such research revealing severe inflammation being
observed following short setting times when the sealers
were implanted subcutaneously into rabbits [7]. Spang-
berg et al. [8] noted that the AH 26 release of
formaldehyde following component mixing will reach
a maximum rate two days post-mixing. Formaldehyde
release from curing endodontic material has been
recognized for many years [8], formaldehyde being
reputed to act as a disinfectant [9]. The disinfective
agent in AH26 is methenamin [10], which is hydrolyzed
to ammonia and formaldehyde [11]. The efficacy of
long-term disinfection of canal by formaldehyde re-
leased from a root canal sealer has previously been
shown to be low [8].

0142-9612/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 0 8 1 - 3
78 T.-H. Huang et al. / Biomaterials 23 (2002) 77–83
There have been case reports of adverse reactions
such as paraesthesia of the inferior alveolar nerve attri-
buted to the formaldehyde released from root canal
sealers [12,13], although any nerve cell reaction to the
formaldehyde released from the use of a root canal
sealer has not been previously reported. Thus, it is of
importance to evaluate the biocompatibility of root
canal sea-lers and neurological cells. In the present study
the astro-cyte was used as the target cell to evaluate the
toxicity.
Genotoxicity, mutagenicity and carcinogenicity are
very important issues associated with the sys-
temic compatibility of root canal sealers [14]. In vitro
test systems can be differentiated into a variety of
different testing procedures, including test systems using
bacterial cells, e.g. the classic Ames test (mutation assay)
and the newly developed umu test (genotoxicity assay),
and the test systems using eukaryotic cells in culture,
such as the hypoxanthine-guanine phosphoribosyl-
transferase test (HGPRT, mutation assay), the chromo-
somal aberration test (mutation assay) and the DNA
synthesis inhibition test (DIT, genotoxicity assay)
[4]. From a review of the literature, it would appear
that the investigation of any genetic effects asso-
ciated with the use of epoxy resin-based root canal
sealer is rare. In a previous study employing the Ames
test for the use of silver-free AH26, in vitro, some degree
of mutagenicity was demonstrated [15], and, further,
AH26 tested with the V79/HGPRT mammalian-cell
assay revealed that this sealer material elicits some
mutagenic effect 24 h subsequent to mixing and admin-
istration [16].
Recently, a new assay for assessing the muta-
genic potential of various compounds has been
developed and is known as the alkaline single-
cell gel electrophoresis assay (comet assay) [17].
This alkaline single-cell gel electrophoresis assay
is both a rapid and sensitive procedure for quan-
titating DNA lesions in mammalian cells, and may be
used to detect specific DNA damage and also DNA
repair [17]. The present study was going to use
this method to evaluate the root canal sealers
genotoxicity.
There is a newly developed epoxy resin sealer
AH-Plus. According to the manufacturer’s instruction
AH Plus will not release formaldehyde. The purpose
of this study was to analyze the biocompatibility of 1st
and 2nd generation epoxy resin sealers, e.g. AH26
and AH Plus sealers, when treated on primary cultured
rat cerebral astrocytes, since only very limited informa-
tion regarding the genotoxicity of resin-based root
canal sealers would appear to be available. The MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] colorimetric test was used as a cytotoxicity
assay and comet assay was used to evaluate the
genotoxicity.
2. Materials and methods
2.1. Sample preparation
Epoxy resin sealers, AH26 and AH-Plus (De Trey
Dentsply, Ballaigues, Switzerland; Table 1) were mixed
according to the manufacturer’s instructions. The mixed
material 100 mg was eluated in 2 ml dimethyl sulfoxide
(DMSO) immediately after mixing at 378C for 24 h [18].
The solution was then centrifuged at 10,000g for 10 min.
The supernatant 1 ml was added to 9 ml of Dulbecco’s
modified Eagle’s medium (DMEM, SIGMA, USA) and
serially diluted until the final concentration of eluate
solution was 0.01, 0.02, 0.04, 0.08, and 0.10 mg/ml for
use in the MTT assay, and the final concentration was
0.01, 0.05, and 0.25 mg/ml for use in the comet assay.
For all culture media, the final concentration of DMSO
in sealer was less than 0.1%. It is reported that DMSO
at a concentration of 1% showed no toxicity to cell
culture [19]. In cytotoxicity test, the pure medium
without cell and sealer was used as control. In
genotoxicity test, the DMSO 0.05% concentration was
used as negative control and 4NQO 0.0003 mg/ml
concentration was used as positive control.
2.2. Rat cerebral astrocyte culture
Astrocytes were prepared as Amruthesh et al. [20]
have previously reported. Briefly, cerebral cortices were
isolated from one- to two-day-old rats, cleaned of white
matter and meninges, minced, and trypsin-digested for
10 min. The tissue was then diluted into feeding medium,
washed, triturated, counted, and placed into 75 cm2
flasks and cultured for 10–14 days at 378C in an air
atmosphere containing 5% CO2. When confluent, the
1
105 cells were plated into 96 culture wells. To
accomplish this, confluent cells were washed with 0.25%
trypsin/0.02% EDTA in saline for exactly 1 min. The
trypsin/EDTA was then aspirated, and the cell mono-
layer, which was still attached to the flask, was covered
with 10 ml Dulbecco’s modified Eagle’s medium
(DMEM) and incubated at 378C, in an air atompho-
sphere of 5% CO2 for approximately 10 min. The lifted
Table 1
The components of the AH26 and AH Plus sealers
Product Composition
AH26 Powder: Bismuth (III) oxide, Methenamine
(silver-free) Liquid: Bisphenol-A-diglycidyl ether
AH Plus Paste A: Epoxy resin, Calcium tungstate, Zirconium
oxide, Aerosil, Iron oxide
Paste B: Adamantane amine N; N 0 -dibenzyl-
5-oxanonane-diamine-1,9,TCD-diamine calcium
tungstate, Zirconium oxide, Aerosil, Silicone oil
 T.-H. Huang et al. / Biomaterials 23 (2002) 77–83
cells were then transferred to a plastic tube, washed with
DMEM, centrifuged, and counted, and 2
105 cells in
1 ml DMEM containing 10% fetal bovine serum (FBS,
SIGMA, USA) were plated onto collagen-coated
25 mm-diameter silastic membranes, which are 2 mm
thick and form the bottom surface of each well in a six-
well tissue culture flex plate. Utilizing a method
previously reported by Amruthesh et al. [20], the cells
were then characterized for purity. The cells were used
for experiments at a total of four weeks after removal
from the rat.
2.3. MTT assay
A modified MTT assay based upon an original
method described by Guigand et al. [21] was used.
Briefly, a 5 mg/ml MTT solution (SIGMA, USA) was
prepared in 378C warm DMEM immediately prior to
use. A 500 : l aliquot of freshly prepared MTT solution
was added to each well of each plate. The plates were
incubated for 4 h at 378C in an air atmosphere contain-
ing 5% CO2. The medium was then aspirated from the
wells and 500 : l of DMSO was added. The plates were
shaken to maximize MTT formazan dissolution. Spec-
trophotometric absorbance was measured at 570 nm
using an ELISA microplate reader using dimethyl
sulfoxide as the blank. Mitochondrial dehydrogenase
acitivity was calculated as survival %=[(the absorbance
of treatment of samplethe absorbance of medium)/(the
absorbance of DMSOthe absorbance of med-
ium)]
100%.
2.4. Comet assay (alkaline single cell gel electrophoresis
assay)
2.4.1. Slide preparation
Logarithmic growth phase astrocytes in DMEM
medium were treated with root canal sealers after
mixing for 3 h at 378C in an air atmosphere containing
5% CO2 and subsequently renewed medium and
incubated for 24 h prior to sampling. We followed the
technique described by Singh et al. [22] for the
quantitation of low levels of DNA damage in individual
cells. 75 : l of 0.5% agarose (SIGMA, USA) was quickly
layered onto a fully frosted glass slide and covered with
another slide. The slides were placed on ice (5 min) to
allow the agarose to gel, following which the top slide
was removed and 50 : l of agarose containing the
astrocytes (5
104 cells/50 : l) was quickly layered onto
the agarose gel. Finally, 75 : l of 0.5% agarose was
quickly layered on the previous layer. The cells were
lysed immediately by immersion of the slide for 1 h in a
lysis solution (pH=10) containing 2.5 m NaCl, 100 mm
EDTA, 10 mm Trizma [Tris(hydroxymethyl)amino-
methane] (SIGMA,USA), 1% Sarkosyl (SIGMA,
79
USA), 10% DMSO, and 1% Triton X-100 (SIGMA,
USA) maintained at 48C.
2.5. Unwinding and electrophoresis
Following cell lysis, the slides were placed upon a
horizontal gel electrophoresis platform and covered with
an alkaline solution made up of 300 mm NaOH and
1 mm EDTA. The slides were left in the solution for
20 min to allow unwinding of the DNA and expression
of alkali-labile sites. The power supply was set at 15 V
and 250 mA, and the DNA was electrophoresed for
20 min, following which the slides were rinsed gently
three times with 400 mm Trizma buffer (pH=7.5) to
neutralize the excess alkali from the previous solution.
Each slide was stained with 50 : l ethidium bromide
(EthBr) and covered with a coverslip.
2.6. Examination of the cells and statistical analysis
Fifty cells on one slide per treatment group were
examined at 200
magnification using a fluorescence
microscope equipped with an excitation filter of 515–
560 nm and a barrier filter of 590 nm which was
connected through the CCD camera to an image
analysis system (Matrox Inspector, Matrox Electronic
systems, USA). If the cell DNA was damaged, the shape
of the cell was like comet. The entire length of the comet
(length) and the diameter of the head (diameter) were
measured. The ‘‘shape factor’’ was calculated as the
ratio of length to diameter. Migration (m) was calculated
as the difference between length and diameter.
The quantitative results from the MTT tests and
comet assay were analyzed using the one way analysis of
variance (ANOVA) to compare the various means.
3. Results
The relative cytotoxicity of the epoxy resin-based
sealers that was estimated over a 24 h period was
assessed using an MTT assay. A typical dose-response
relationship between eluates of test samples that were
eluted in culture medium and corresponding cell
survival rates is presented in Tables 2 and 3. Resultant
cytotoxic effects of AH26 upon astrocyte cells as
assessed using an MTT assay is indicated in Table 2.
When the exposure concentration of the sealer powder
or liquid or the mixed material was increased, the
astrocyte survival rate (%) dropped ðP50:05Þ.
The cytotoxic effects of AH Plus upon astrocyte cells
as assessed using an MTT assay is depicted in Table 3.
When the concentration of paste A or paste B or mixed
sealer was increased, the astrocyte survival rate (%)
decreased ðP50:05Þ.
80 T.-H. Huang et al. / Biomaterials 23 (2002) 77–83

Table 2
Cytotoxicity of AH26 in astrocyte cells evaluated by MTT assaya

Concentration Powder Liquid Mixed

Absorbance Survival % Absorbance Survival % Absorbance Survival %

(M  SD) (M  SD) (M  SD)

Control 0.10  0.02 0.10  0.02 0.10  0.02

DMSO 1.97  0.09 1.97  0.09 1.97  0.09
0.01 mg/ml 1.85  0.09 93.16 2.03  0.05 103.25 1.64  0.03 82.01
0.02 mg/ml 1.88  0.06 94.87 1.88  0.10 94.77 1.72  0.07 86.28
0.04 mg/ml 1.71  0.11 85.75 1.58  0.04 79.24 1.10  0.05 53.30
0.08 mg/ml 0.95  0.12 45.62 0.30  0.01 10.88 0.69  0.02 31.75
0.10 mg/ml 0.79  0.04 36.17 0.25  0.07 7.95 0.25  0.01 7.95

F value 172.9 984.25 1111.22

P50:05 Yes Yes Yes
a
Survival %=[(the absorbance of treatment of sample  the absorbance of medium)/(the absorbance of DMSO  the absorbance of
medium)]
100%. The sample size of each group is five. The absorbance (mean  standard deviation) is analyzed by one way analysis of variance
method. The pure culture medium is taken as control. The DMSO concentration is 0.05% of the medium.

Table 3
Cytotoxicity of AH Plus in astrocyte cells evaluated by MTT assaya

Concentration Paste A Paste B Mixed

Absorbance Survival % Absorbance Survival % Absorbance Survival %

Control 0.10  0.02 0.10  0.02 0.10  0.02

DMSO 1.97  0.09 1.97  0.09 1.97  0.09
0.01 mg/ml 2.02  0.11 102.24 1.92  0.04 96.95 2.04  0.05 103.57
0.02 mg/ml 2.01  0.08 101.97 2.05  0.08 104.10 1.88  0.11 95.03
0.04 mg/ml 1.20  0.10 58.91 2.05  0.05 103.89 0.76  0.10 35.37
0.08 mg/ml 0.64  0.08 28.76 1.90  0.05 53.09 0.35  0.03 13.39
0.10 mg/ml 0.63  0.07 28.44 1.05  0.04 50.74 0.29  0.01 10.13

F value 301.04 304.67 688.41

P50:05 Yes Yes Yes
a
Survival %=[(the absorbance of treatment of sample  the absorbance of medium)/(the absorbance of DMSO  the absorbance of
medium)]
100%. The sample size of each group is five. The absorbance (mean  standard deviation) is analyzed by a one way analysis of variance
method. The pure culture medium is as control. The DMSO concentration is 0.05% of the medium.

Table 4
The inhibition dose (ID 50) of the AH26 and AH Plus sealers

AH26 AH plus

Powder Liquid Mixed Paste A Paste B Mixed

Concentration (mg/ml) 0.08 0.06 0.05 0.05 0.11 0.04

Cellular inhibition dose 50 data are based upon a 50%
reduction in mitochondrial dehydrogenase activity. The
inhibition dose (ID 50) of the AH26 and AH Plus sealers
is referred to in Table 4. In AH26, the lowest ID 50 was
the mixed group (0.05 mg/ml). The highest ID 50 was the
powder group (0.08 mg/ml). In AH Plus, the lowest ID
50 was the mixed group (0.04 mg/ml). The highest ID50
was the paste B group (0.11 mg/ml) (Table 4).
In genotoxicity test, a negative control (DMSO) and a
positive control (4NQO) were included in each experi-
ment and their results are summarized in Table 5.
Treatment with AH26 led to a noted dose-dependent
increase in all measures of DNA damage.
In addition, the AH-Plus resin was also able to induce
DNA damage after treatment. The DNA migration of
AH Plus at this concentration (0.25 mg/ml) is higher
than that for the concentration of AH26 ðP50:05Þ,
suggesting that the DNA damage elicited by AH Plus is
more substantial than that for AH26 when applied at
the same dose.
 T.-H. Huang et al. / Biomaterials 23 (2002) 77–83 81

Table 5
The comet assay of the AH26 and AH Plus sealers

Condition N Migration (M  SD) Shape factor (M  SD)

(lengthdiameter) (length/diameter)

DMSO 50 44.16  1.47 F ¼ 5:20 1.00  0 F ¼ 89:23

(Negative control)
4NQO 50 57.89  5.84 P ¼ 0:025a 2.75  0.19 P ¼ 0a
(Positive control)
AH26 (mg/ml)
0.01 50 58.26  4.69 F ¼ 19:91 2.62  0.17 F ¼ 14:90
0.05 50 67.74  2.32 P ¼ 0a 3.63  0.14 P ¼ 0a
0.25 50 89.13  3.21 3.54  0.12
AH Plus (mg/ml)
0.01 50 80.20  4.90 F ¼ 21:42 3.89  0.27 F ¼ 11:24
0.05 50 79.04  3.23 P ¼ 0a 3.73  0.14 P ¼ 0a
0.25 50 110.83  3.33 4.97  0.17

F value 29.2 48.44

a a
P50:05
a
It represents that the comparison is statistically significant difference at P50:05. The entire length of the comet (including the head) is defined as
its length and the diameter of the head is defined as diameter. Shape factor was calculated as the ratio of length to diameter. Migration (m) was
calculated as the difference between length and diameter. The negative control: DMSO concentration is 0.05% of the medium. The positive control:
4NQO concentration is 0.0003 mg/ml.

4. Discussion recognized that during the setting process, AH26 may

The MTT biological testing results of root canal
sealers revealed a dose-dependent toxicity for AH26 and
AH Plus, such results being in agreement with the
observations of other workers applying AH26 to other
cell culture systems [14,23]. The mixed group of AH26
sealer appears to be capable of inducing a greater degree
of toxicity to astrocytes than is the case for either the
pure powder or liquid form of AH26 ðP50:05Þ.
Various in vitro and in vivo studies have shown that
freshly prepared and cured specimens of the epoxy resin-
based root canal sealer AH26 may induce strong
cytotoxic effects [7,15,16,21]. These experimental ob-
servations have been confirmed by some clinical case
reports [23–25]. It has been reported that the formalde-
hyde emanating from the curing sealer may be the main
causative factor for the high cytotoxicity of AH26
during, particularly, the early setting period [8]. The
liquid component of the AH26 is prepared using
bisphenol-A-diglycidyl ether (Table 1). From our work,
it is apparent that the survival rate of astrocytes treated
with AH26 liquid (0.08 mg/ml) is lower than that for
either powder or mixed formats ðP50:05Þ (Table 2).
From the work of others, in vitro mammalian cell (V79/
hprt) mutation assay, it has been demonstrated that
diglycidyl ether is the causative factor of toxicity [16],
thus, clearly, it is dangerous if the mixing sealer is
incomplete. There is a suggestion that the presence of
uncured (liquid) sealer in the oral cavity may be a cause
for health concern [16]. Thus, any remnant liquid sealer
post-mixing may be more toxic than cured sealer. It is
release formaldehyde [8], and that formaldehyde in the
oral cavity is a toxic agent [26].
From our experiments, it is apparent that the cured
AH26 sealer is toxic to astrocytes in a dose-dependent
manner. The strongest cell inhibition elicited by the
sealer mix occurs at a concentration of 0.10 mg/ml, at
which concentration, both the liquid and mixed groups
exhibit the same degree of toxicity, and, by contrast, the
powder seems to be somewhat more compatible with
astrocyte survival.
The AH Plus product is a relatively new epoxy resin-
based sealer, its manufacturer emphasizing that the
cured resin will not release formaldehyde and is thus
more biocompatible than AH26. From our experiments,
the AH plus-induced astrocyte toxicity appears to be
dose dependent, both pastes (independently) and the
resin mixture groups demonstrating cytotoxicity, such
results contrasting the results of Leyhausen et al. [13].
These authors suggest that minor or no cytotoxic effects
were observed for this new epoxy resin-based sealer [13].
Schweikl et al. found that DMSO eluated of the mixed
material, paste A and paste B clearly reduced the
viability of V79 cells and was mutagenic in a dose-
dependent manner in V79 cells [18].
From the chemical composition of the AH Plus sealer
(Table 1), it seems likely that numerous substances may
be released from the material into the adjacent tissue as
a result of its use in the oral cavity, and thus, there
clearly exists a cogent need to undertake further
investigation into the potential toxicity of the use of
eluting material. The degradation of root canal sealers
82 T.-H. Huang et al. / Biomaterials 23 (2002) 77–83
within the oral cavity, with time, may lead to component
leakage into the adjacent tissue through the dentinal
tubules or apex, such that the surrounding tissue
of the tooth, bone or nerve, may be subsequently
affected by resin-released compounds. The use of the
astrocyte as target cell in this study is based upon its
nerve origin.
The other purpose of this study was to determine
genotoxic effects due to dimethyl sulfoxide extracts of
the AH26 and AH-Plus sealers, acknowledging that
detectable chemical cytotoxic effects may not reflect as
analogous gentoxic effects. There is only scant informa-
tion regarding the mutagenicity of these root canal
sealers. Schweikl et al. investigated the mutagenicity of
AH26 in the V79/HGPRT mammalian cell assay [16].
They found that this material induces mutagenic effects
24 h after mixing which significantly decrease within
1 week. Stea et al. (Ames test) [27] and Heil et al. (umu,
DIT) [28] found mutagenic substances even in the set
material. The alkaline single cell gel electrophoresis
assay (comet assay) is a sensitive method to investigate
DNA breakage in individual cells as a consequence of
their in vitro or in vivo exposure to genotoxic
compounds [29]. In our experimental series, the muta-
genic effects of AH26 and AH Plus demonstrated
genotoxicity to astrocytes (Table 5). Resin-based sealers’
mutagenic potencies were noted to occur in a dose-
dependent manner; following exposure of astrocytes to
these two compounds, an increased migration factor was
noted.
Observed DNA damage at a sealer concentration of
0.25 mg/ml of AH26 and AH Plus in culture medium
revealed a more pronounced migration for the AH Plus
group than for its analog, suggesting that the AH Plus
sealer elicits more substantial DNA damage than is the
case for the AH26 group. The effect of dying or dead
cells upon the assay-derived data may be to influence the
estimate of the positive response of resin-released
chemicals, since dying or dead cells may increase DNA
migration in this assay [17]. In our experiments, the AH
Plus (ID 50=0.04 mg/ml) is more toxic than AH26
(ID 50=0.05 mg/ml) (Table 4); the migration of the AH
Plus moiety is larger than that of its analog in the assay.
There is a reduced possibility that dead cells participated
in the positive responses of chemicals. In shape factor
evaluation, the AH26 and AH-Plus sealers exert their
influence dose-dependently, such results being similar to
the results of the migration factor assessment.
The study by Ersev et al. [15] indicated that mixed,
silver-free AH26 elicited mutagenicity in eukaryotic and
prokaryotic cells; they speculate that the mutagenic
effect of AH26 may arise from the liquid component
bisphenol-A-diglycidyl ether and also formaldehyde.
Their experimental results were similar to those from
our experiments that AH26 can elicit astrocyte DNA
damage.
Our experimental series indicated that the epoxy
resin-based sealers AH26 or AH Plus are not truly
biocompatible [30,31]. From this work, we have
demonstrated a direct dose-dependent in vitro relation-
ship between the concentration of administered sealer
and cytotoxic and mutagenic effects. In conclusion, in
this study the epoxy resin-based sealers, AH26 and AH-
Plus demonstrated both cytotoxicity and genotoxicity
in vitro.
Acknowledgements
The authors acknowledge the Chung Shan Medical
and Dental College (CSMC-88-OM-B-033) for support-
ing this project.
References
[1] Willershausen B, Marroquin BB, Schulze R. Cytotoxicity of root
canal filling materials to three different human cell lines. J
Endodon 2000;26:703–7.
[2] DeDenus QD. Frequency, location and direction of the lateral
secondary and accessory canals. J Endodon 1975;1:361–6.
[3] Dongari A, Lambrianids T. Periodontally derived pulpal lesion.
Endodon. Dent Traumatol 1988;4:49–52.
[4] Geusten W, Leyhausen. Biological aspects of root canal filling
materials}histocompatibility, cytotoxicity and mutagenicity.
Clin Oral Invest 1997;1:5–11.
[5] Broisman H, Van Houte J, Gron P, Karkow AA. Antimicrobial
effects of N2 in vitro. Oral Surg Oral Med Oral Pathol
1978;45:116–22.
[6] Cox ST, Hembree JH, McKnight JP. The bactericidal potential of
various endodontic materials for primary teeth. Oral Surg Oral
Med Oral Pathol 1978;45:947–54.
[7] Mittal M, Chandra S. Comparative tissue toxicity evaluation of
four endodontic sealers. J Endodon 1995;21:622–4.
[8] Spangberg LSW, Barbosa SV, Lavigne GD. AH 26 release
formaldehyde. J Endodon 1993;19:596–8.
[9] Tronstad L, Yang ZP, Trope M, Barnett F, Hammond B.
Controlled release of medicaments in endodontic therapy.
Endodon Dent Trauma 1985;1:130–4.
[10] Schroder A. Mitteilungen uber die abschludichitigkeit von
wurzelfullmaterialien und erster Hinweis auf ein neuartiges
Wurzelfullmaterial. Schweizerische Monatsschrift fur Zahnheilk-
unde 1954;64:921–31.
[11] Koch MJ. Formaldehyde release from root canal sealers:
influence of method. Int Endodon 1999;32:10–6.
[12] Erisen R, Yucel T, Kucukay S. Endomethasone root canal filling
material in the mandibular canal: a case report. Oral Surg Oral
Med Oral Pathol 1989;63:343–5.
[13] Gumru OZ, Yalcin S. Surgical treatment of paresthesia following
over extension of root canal filling material: a case report. J Nihon
University school of Dentistry 1991;33:49–53.
[14] Leyhausen G, Heil J, Reifferscheid G, Waldmann P, Geurtsen W.
Genotoxicity and cytotoxicity of the epoxy resin based root canal
sealer AH plus. J Endodon 1999;25:109–13.
[15] Ersev H, Schmalz G, Bayirli G, Schweikl H. Cytotoxicity and
mutagenic potencies of various root canal filling materials
 T.-H. Huang et al. / Biomaterials 23 (2002) 77–83
in eukaryotic and prokaryotic cells in vitro. J Endodon 1999;25:
359–63.
[16] Schweikl H, Schmalz G, Stimmelmayer H, Bey B. Mutagenicity of
AH26 in an in vitro mammalian cell mutation assay. J Endodon
1995;21:407–10.
[17] Miyamae Y, Zaizen K, Ohara K, Mine Y, Sasaki YF. Detection
of DNA lesions induced by chemical mutagens by the single cell
gel electrophoresis (comet) assay. 1. Relationship between the
onset of DNA damage and the characteristics of mutagens. Mutat
Res 1998;415:229–35.
[18] Schweikl H, Schmalz G. The induction of micronuclei in V79
cells by the root canal filling material AH plus. Biomat 2000;21:
939–44.
[19] Huveneers-Oorsprong MBM, Hoogenboom LAP, Kuiper HA.
The use of the MTT test for determining the cytotoxicity
of veterinary drugs in pig hepatocytes. Toxicol Vitro 1997;11:
385–92.
[20] Amruthesh SC, Boerschel MF, McKinney JS, Willoughby KA,
Ellis EF. Metabolism of arachidonic acid to epoxyeicosatrienoic
acids, hydroxyeicosatetraenoic acids and prostaglandins in
cultured rat hippocampal astrocytes. J Neurochem 1993;61:150–9.
[21] Guigand M, Pellen-Mussi P, Le Goff A, Vulcain JM, Bonnaure-
Mallet M. Evaluation of the cytocompatibility of three endodon-
tic material. J Endodon 1999;25:419–23.
[22] Singh NP, McCoy MT, Tice RR. A simple technique for
quantitation of low levels of DNA damage in individual cells.
Exp Cell Res 1988;175:184–91.
83
[23] Spangberg L, Langeland K. Biological effects of dental materials.
1. Toxicity of root canal filling materials on HeLa cells in vitro.
J Oral Surg 1973;35:402–14.
[24] Tagger M, Tagger E. Periapical reactions to calcium hydroxide
containing sealers and AH26 in monkeys. Endo Dent Traumatol
1989;5:139–46.
[25] Briseno BM, Willershausen B. Root canal sealer cytotoxicity on
human gingival fibroblasts. 2. Silicon and resin based sealers.
J Endodon 1991;11:537–40.
[26] Lweis BB, Chestner SB. Formaldehyde in dentistry: a review
of mutagenic and carcinogenic potential. JADA 1981;103:
429–34.
[27] Stea S, Savarino L, Ciabetti G, Cenni E, Stea St, Trotta F,
Morozzi G, Pizzoferrato A. Mutagenic potential of root canal
sealers: evaluation through Ames testing. J Biomed Mater Res
1994;28:319–28.
[28] Heil J, Reifferscheid G, Waldmann P, Leyhausen G, Geurtsen W.
Genotoxicity of dental materials. Mutat Res 1996;368:181–94.
[29] Kuchenmeister F, Schmezer P, Engelhardt G. Genotoxic bifunc-
tional aldehydes produce specific images in the comet assay.
Mutat Res 1998;419:68–78.
[30] Huang TH, Kao CT, Huang MF, Liao PH, Chou MY, Lee H.
Cytotoxicity of AH26 and AH Plus root canal sealers on oral
cancer cell line OC2. Chin Dent J 1999;18:101–8.
[31] Huang TH, Lii CK, Chou MY, Kao CT. Lactate dehydrogenase
leakage of hepatocytes with AH26 and AH Plus sealer treatment.
J Endodon 2000;26:509–12.