Acta histochemica 112 (2010) 567575

Comparision of biocompatibility and cytotoxicity of

two new root canal sealers
Nimet Gencoglu
a
, Goksel Sener
b
, Gulden Z. Omurtag
c
, Ayfer Tozan
c
,
Bahar Uslu
d
, Serap Arbak
e
, Dilek Helvacioglu
a,
a
Department of Endodontics, Faculty of Dentistry, Marmara University, Buyuk C- iftlik sok. No: 6, Nis-antas-,
Istanbul, Turkey
b
Department of Pharmacology, Faculty of Pharmacy, Marmara University, Haydarpasa, Istanbul, Turkey
c
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Marmara University, Haydarpasa, Istanbul, Turkey
d
Department of Histology and Embryology, Faculty of Medicine, Marmara University, Haydarpasa, Istanbul, Turkey
e
Acibadem University, Department of Histology and Embryology, Gulsuyu Maltepe, Istanbul 34848, Turkey
Received 4 April 2013; received in revised form 9 June 2013; accepted 11 June 2013
KEYWORDS
Biocompatibility;
Cytotoxicity;
EndoREZ;
GuttaFlow;
Kerr pulp canal sea-
ler;
Root canal;
Rats
Summary
The aim of this study was to investigate the remote organ toxicity and connective
tissue reaction of two new root canal sealers (GuttaFlow
s
and EndoREZ
s
) and
to compare them with zinc oxide eugenol sealer using biochemical and histopatho-
logical parameters. A total of 60 white albino Wistar rats were used in the study.
0.1 ml of GuttaFlow
s
, EndoREZ
s
or Kerr Pulp Canal Sealer
s
were administered
subcutaneously into the mid-dorsal thoracic region of rats (15 in each group).
Control rats were given saline only. Rats were decapitated after 24 h, on day 7 and on
day 30 of the experiment and tissue samples from lung, liver, kidney and skin were
removed for the determination of malondialdehyde (MDA) and glutathione (GSH)
levels. In parallel, tissues were also examined histologically. Serum aspartate
aminotransferase (AST), alanine aminotransferase (ALT) levels, and creatinine and
blood urea nitrogen (BUN), concentrations (BUN) were measured to assess liver and
kidney functions, respectively. Tumor necrosis factor (TNF) and lactate dehydro-
genase (LDH) were also assayed in serum samples. No statistical differences were
found among the control and EndoREZ
s
, GuttaFlow
s
and Kerr Pulp Canal sealers
regarding tissue MDA, GSH levels or serum parameters (p40.05) at all time points
examined. Both of the new root canal sealers showed good compatibility and
acceptable tissue toxicity.
& 2013 Elsevier GmbH. All rights reserved.
www.elsevier.de/acthis
0065-1281/$ - see front matter & 2013 Elsevier GmbH. All rights reserved.
doi:10.1016/j.acthis.2013.06.005

Corresponding author. Fax: +90 212 246 52 47.

E-mail address: dhelvacioglu@gmail.com (D. Helvacioglu).
Introduction
The main objective of successful endodontic
treatment is proper cleaning and shaping of the
root canal, as well as total obturation of the canal
space with an inert, dimensionally stable and
biologically compatible material. Currently, the
most frequent obturation methods employ a solid
core such as gutta-percha cemented in the root
canal with a sealer. Since these materials will be in
direct contact with periapical tissues for a pro-
longed period of time, their biocompatibility is of
primary importance (Nguyen, 1984). Previous stu-
dies have shown that almost all sealers have some
degree of toxicity in contact with living tissue
(Browne and Friend, 1968; Friend and Browne,
1968; Olsson and Wennberg, 1985; rstavik and
Mjor, 1988; Yes-ilsoy et al., 1988).
Zinc oxide eugenol sealers, which have been used
for many years, release potentially cytotoxic
concentrations of eugenol (Hume, 1986; Schmalz
et al., 2000). On the other hand, although resin-
based sealers are popular, their cytotoxicity and
mutagenic effects are debatable (Kleinsasser et al.,
2004; Schweikl et al., 1998). Under clinical condi-
tions, the polymerization processes of resin mate-
rials are usually incomplete and almost all the
components can be detected in extracts of poly-
merized materials. Previous studies have shown
that extractable monomers and/or other organic
and inorganic ingredients may induce local and
systemic adverse effects (Spahl et al., 1998).
Moreover, resin-based materials may elicit biologi-
cal effects with underlying genetic mechanisms
resulting in mutation in vitro. There are very little
data on the cytotoxic and genotoxic effects of
methacrylate, which is a component of many
sealers, on human cells (Kleinsasser et al., 2004).
Recently EndoREZ
s
, a hydrophilic resin-based
root canal sealer, was developed. It contains zinc
oxide, barium sulphate, resins and pigments within
a matrix of urethane dimethacrylate (UDMA) resin.
Although microleakage of EndoREZ
s
has been
properly investigated, the cytotoxic effects and
biocompatibility of the material are still not clear
(Kardon et al., 2003; Kazemi et al., 2003).
More recently, silicon-based sealers have been
developed and their clinical and biocompatibility
have been found promising. Another new recently
introduced material is GuttaFlow
s
, which includes
both silicon and gutta-percha.
Reports in the literature on cytotoxicity have
been based on in vivo or in vitro tests. However, in
contrast with the in vitro studies, in vivo tests
provide information on acute reactions and long-
term effects of implantation materials. Some
materials may affect distant organs such as kidney,
liver, lung and change biochemical parameters
(Economides et al., 2005; Omurtag et al., 2005).
In some studies, the cytotoxic effects of endodontic
sealers on macrophage activities have been inves-
tigated. Activation of macrophages and cytokines
and the subsequent formation of reactive oxygen
and nitrogen species are of central pathogenic
importance in the development of oxidative tissue
damage and organ dysfunction. Polymorphonuclear
leukocyte (PMN) extravasation can lead to vascular
dysfunction as well as parenchymal cell dysfunction
(Neviere et al., 1999). Besides their direct dama-
ging effects on tissues, these reactive species
appear to trigger the accumulation of leukocytes
in the tissues involved, and thus cause further
injury through activated neutrophils. It has been
shown that activated neutrophils secrete enzymes,
such as myeloperoxidase, elastase, proteases and
liberate oxygen radicals (Reiter et al., 2001). Lipid
peroxidation mediated by oxygen free radicals is
believed to be an important cause of destruction
and damage to cell membranes.
Accordingly, this study was designed to investi-
gate if GuttaFlow
s
, EndoREZ
s
or Kerr Pulp Canal
Sealers have any toxic effects by activating macro-
phages and thus generating reactive species, which
ultimately could lead to oxidative tissue damage in
the lungs, liver, kidneys and skin. Toxicity was
examined using biochemical measurements, such
as tissue malondialdehyde (MDA) and glutathione
(GSH) or serum lactate dehydrogenase (LDH) and
TNF-a analysis, and parallel histopathological ap-
proaches, while the functional impairments were
monitored by tests for hepatic and renal functions.
Materials and methods
Sixty albino Wistar rats (weighing 200250 g)
were used in this study. The study was performed
on 4 groups of animals; Control, Kerr Pulp Canal
Sealer
s
(Kerr, Romulus, MI, USA), GuttaFlow
s
(Roeko, Coltene Whaledent, Langenau, Germany)
and EndoREZ
s
(Ultradent Products Inc, South
Jordan, UT, USA) groups. Each group consisted of
15 rats. Rats were administered a single dose of
0.1 ml dental materials subcutaneously into the
dorsal thoracic mid-region and decapitated after
24 h, 7 days and 30 days of the experiment. All
experimental protocols were approved by the
Marmara University Animal Care and Use Commit-
tee (No: 09.28.2004-49). The authors have no
commercial interest in the products investigated
in this study.
N. Gencoglu et al. 568
Histological analysis
For histological analysis, samples of the tissues
were xed in 10% neutral buffered formalin and
prepared for routine parafn wax embedding. 6 mm
thick section of lung, kidney and liver tissue were
stained with hematoxylin and eosin and skin
sections were stained with Massons trichrome
stain. All the slides were examined under a bright-
eld light microscope (Olympus-BH-2, Olympus
Corporation, Tokyo, Japan). An experienced histol-
ogist who was unaware of the treatment conditions
made the histological assessments.
Assays
Serum levels of blood urea nitrogen, AST, ALT,
creatinine, LDH and TNF-a
After decapitation, trunk blood was collected,
the serum was separated to measure the aspartate
aminotransferase (AST), alanine aminotransferase
(ALT) levels and creatinine and blood urea nitrogen
(BUN) as indicators of liver and kidney functions,
respectively. Lactate dehydrogenase and TNF-a
were also assayed in serum samples for the
evaluation of generalized tissue damage.
BUN, AST, ALT and creatinine concentrations and
LDH levels were determined spectrophotometri-
cally using an automated analyzer (Olympus AU
600, Diamond Diagnostic, Holliston, MA, USA)
(Martinek, 1972). TNF-a was evaluated by an
RIA-IRMA (radioimmunoassay-immunoradiometric
assay) method. All samples were assayed in
duplicates using a commercial kit (Biosource
Europe S.A., Nivelles, Belgium). The activity of
radioactive assays was measured by a gamma
counter (LKB Wallac 1270 Rack Gamma Counter,
Turku, Finland). TNF-a in the serum samples was
expressed as ng/ml.
Tissue levels of MDA, GSH and collagen
In order to evaluate the presence of tissue injury
in the skin and distant organs, tissue samples from
the lung, liver, kidney and skin samples were stored
at 80 1C for the determination of levels of
malondialdehyde (an end-product of lipid perox-
idation), glutathione (a key antioxidant), glu-
tathione levels and collagen content (a marker of
brosis).
Tissue samples were homogenized with ice-cold
150mM KCl for the determination of malondialde-
hyde and glutathione levels. The MDA levels were
assayed for products of lipid peroxidation by
monitoring thiobarbituric acid reactive substance.
Lipid peroxidation was expressed in terms of MDA
equivalents using an extinction coefcient of
1.5610
5
M
1
cm
1
and results are expressed as nmol
MDA/g tissue. GSH measurements were performed
using a modication of the Ellman procedure
(Beutler, 1975). Briey, after centrifugation at
3000rev/min for 10min, 0.5ml of supernatant was
added to 2ml of 0.3mol/l Na
2
HPO
4
2H
2
O solution. A
0.2ml solution of dithiobisnitrobenzoate (0.4mg/ml
1% sodium citrate) was added and the absorbance at
412nm was measured immediately after mixing. GSH
levels were calculated using an extinction coefcient
of 13600M
1
cm
1
. Results are expressed in mmol
GSH/g tissue.
Tissue collagen was measured as a free radical-
induced marker of brosis. Fresh tissue samples
were cut with a razor blade, immediately xed in
10% formalin in 0.1 M phosphate buffer (pH, 7.2)
and processed for embedding in parafn wax.
Sections, approximately 15 mm thick, were ob-
tained. Evaluation of collagen content was investi-
gated according to the method of Lopez de Leon
and Rojkind (1985), which is based on selective
binding of the dyes Sirius red and fast green FCF to
collagen and non-collagenous components, respec-
tively. Both dyes were eluted simultaneously by
using 0.1 N NaOHmethanol (1:1 v/v). Finally, the
absorbances at 540 and 605 nm were used to
determine the amount of collagen and protein,
respectively.
Statistics
Statistical analysis was carried out using Graph-
Pad Prism 3.0 (GraphPad Software, San Diego, CA,
USA). All data were expressed as mean7SEM.
Groups of data were compared with an analysis of
variance (ANOVA) followed by Tukeys multiple
comparison tests. Values of po0.05 were regarded
as signicant.
Results
Histological analysis
Liver
In all groups, the liver tissue sections were
investigated to determine signs of hepatic degen-
eration, sinusoidal dilatation, vasocongestion and
increased activity of Kupffer cells. Among the three
root canal sealers, EndoREZ
s
was the most dama-
ging sealer on the liver tissue. Especially on day 7
and day 30, hepatocyte degeneration, sinusoidal
dilalation and vasocongestion were prominent. No
vasocongestion was noticed on day 1 and day 7. The
Comparision of biocompatibility and cytotoxicity of two new root canal sealers 569
activities of Kupffer cells were generally normal at
all times. There was no inammatory tissue
reaction in liver. We conclude that all of the three
sealers caused only a minor degree of liver injury
(Figure 1).
Kidney
In all groups, the kidney tissue sections were
investigated to determine signs of tubular degen-
eration, vasocongestion, glomerular degeneration
and leukocyte inltration. Of the three sealers,
GuttaFlow
s
proved to be the least toxic with
regard to cellular injury. Vasocongestion, glomeru-
lar degeneration and leukocyte inltration were
nearly absent in all kidney tissue sections for all
days with GuttaFlow
s
. In Kerr Pulp Canal Sealer
and EndoREZ
s
groups, a slight tissue injury in terms
of tubular cellular degeneration was revealed
(Figure 2).
Lung
In all groups, lung tissue sections were evaluated
for signs of edema, intra-alveolar hemorrhage,
leukocytic inltration, vasocongestion and dis-
rupted alveolar architecture. Among those
criteria, intra-alveolar hemorrhage was not noticed
in any of the experimental groups. All of the agents
presented a slight degree of lung tissue damage
including edema, leukocytic inltration, vasocon-
gestion and disrupted alveolar architecture
(Figure 3).
Skin and connective tissue
Skin tissue was investigated and scored to
determine the degree of tissue degeneration at
the levels of epidermis, dermis, hair follicles and
subcutaneous tissue. Very few signs of epidermal
degeneration were seen in the experimental
groups. The total of other scoring criteria was
nearly equal in all groups revealing only slight skin
tissue damage (Figure 4).
Assays
Serum AST and ALT levels were determined as a
measure of hepatic function, while BUN and serum
creatinine levels were used for the assessment of
renal function. The experimental root canal sealers
did not change the hepatic and renal functions
(Table 1). Serum lactate dehydrogenase, which was
analyzed to evaluate generalized tissue injury, was
found not to be different from that of the control
group. TNF-a levels increased signicantly
(po0.05) in all groups.
There were no differences in MDA levels deter-
mined in the lung, liver, kidney and skin tissues
between the control and the experimental groups
(Table 2). Similarly, GSH levels in all the studied
tissues were not affected by the root canal sealers
(Table 3).
Figure 1. Micrographs of liver from (a) EndoREZ
s
group showing vacuolated hepatocytes ( ) and sinusoidal dilatation
( ), (b) Kerr Pulp Canal Sealer and (c) GuttaFlow
s
groups on day 30 after sealer treatment showing a slight degree of
hepatocytic degeneration ( ). (d) Control group. Stained hematoxylin and eosin. Bars: 10 mm.
N. Gencoglu et al. 570
As an indicator of the level of brotic activity in
tissues, the collagen content was analyzed and
when compared with the control, no differences
were seen within experimental groups (Table 4).
Discussion
Many different studies have evaluated cytotoxi-
city of root canal sealers using in vitro cell culture
Figure 3. Micrographs of lung showing a low degree of interalveolar fusion (-), vasocongestion ( ) and slight edema
in (a) EndoREZ
s
, (b) Kerr Pulp Canal Sealer and (c) Gutta treated groups on day 30 after sealer treatment. (d) Control
group. Stained hematoxylin and eosin. Bars: 10 mm.
Figure 2. Micrographs of kidney showing slightly damaged tubular cells (-) in (a) EndoREZ
s
, (b) Kerr Pulp Canal Sealer
and (c) GuttaFlow
s
groups on day 30 after sealer treatment indicating a low degree of kidney parenchymal damage. (d)
Control group. Stained hematoxylin and eosin. Bars: 10 mm.
Comparision of biocompatibility and cytotoxicity of two new root canal sealers 571
tests, or in vivo tests such as implantation into
connective tissue, osseous implantation and peri-
radicular tissue response. In vivo tests are based on
clinical or histological appraisal of tissue response
and probably correlate better than the in vitro test
(Langeland, 1978).
In the present study, GuttaFlow
s
, EndoREZ
s
and
Kerr Pulp Canal sealers showed slight cytotoxicity
in some organs such as liver, kidney and lung. These
phenomena occurred while the subcutaneously
injected sealer was in direct contact with the
blood circulation in the connective tissue of the
test animals. Apparently some of the sealer
ingredients (such as UDMA, eugenol or silicon)
passed into the blood stream and were carried into
tissues and affected distant organs (Langeland,
1978). In the present study, tissue toxicity was
found acceptable at the end of 30 days for all
groups. Blood serum parameters were not found
statistically different from that of the control
group. Moreover, the root canal sealers were
subcutaneously injected in order to avoid the
trauma due to the surgical incision for the
implantation.
Studies reported in the literature have shown that
most dental materials pose signicant cytotoxic
Figure 4. Micrographs of skin of (a) EndoREZ
s
, (b) Kerr Pulp Canal Sealer and (c) GuttaFlow
s
groups on day 30 after
sealer treatment showing a thinning of epithelium (-). (d) Control group. Massons trichrome stain. Bars: 10 mm.
Table 1. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine,
lactate dehydrogenase (LDH) and TNF-a levels in all groups (n 5 per group).
ALT (mg/dl) AST (mg/dl) BUN (mg/dl) Creatinine (mg/dl) LDH (U/L) TNF-a (pg/ml)
Control 78.078.6 172716.5 36.074.5 0.5670.02 19907165 4.070.2
GR
24 h
76.078.5 185714.2 41.074.6 0.570.04 18907361 8.670.3

GR
7d
69.0712.3 201720.1 39.073.5 0.670.02 24217420 6.971.1

GR
30d
68.079.9 176721.5 36.072.5 0.670.02 21107165 6.970.5

ER
24 h
72.0710.2 196714.2 38.073.6 0.570.06 19347125 8.771.9

ER
7d
70.0712.3 198719.5 41.273.8 0.570.01 24317156 7.571.9

ER
30d
74.078.1 168724.3 42.074.0 0.670.08 19907189 8.171.2

KR
24 h
66710.2 16879.8 34.077.5 0.570.03 22107230 6.070.4

KR
7d
61.079.8 211712.3 35.073.2 0.670.01 25007320 5.070.9
KR
30d
72.0712.6 198712.8 38.072.2 0.670.01 26807256 8.270.2

GR: GuttaFlow, ER: EndoREZ, KR: Kerr Pulp Canal Sealer.

po0.05: compared with control group.

N. Gencoglu et al. 572
risks, particularly in the freshly mixed condition, in
a progressively decreasing time-related manner
(rstavik and Mjor, 1988). Kardon et al. (2003),
however, found in their study that EndoREZ
s
did
not consistently set even after 7 days. In the
present study, EndoREZ
s
was found the most
damaging on liver tissue at 24 h, but this harmful
effect was alleviated with time. In the literature,
there are only limited studies describing the
cytotoxic effects of EndoREZ
s
sealer.
Becce and Pameijer (2003) investigated the
biocompatibility of EndoREZ
s
by in vitro cell
culture using L929 cell lines and found a mild
irritation with 60% rat survival. The result of in
vitro cytotoxicity tests may not highly correlate
with in vivo data. However, a limited number of in
vivo studies reported in the literature indicate the
cytotoxicity of EndoREZ
s
sealer. Louw et al. (2001)
obturated teeth of primates with gutta-percha and
EndoREZ
s
or AH Plus (De Trey Dentsply, Konstanz,
Germany) with either short, ush or extruded root
canal llings and found inammatory reaction in 90
days only in overextended root llings. Although
limited studies were found in literature about
cytotoxicty of EndoREZ
s
sealer, several studies
have been performed on urethane dimethacrylate,
which is a component of EndoREZ
s
.
Ratanasathien et al. (1995) found bisphenol-A
diglycidyl dimethacrylate (Bis-GMA) and urethane
dimethacrylate to be more toxic than triethylene-
glycol dimethacrylate (TEGDMA) and hydroxyethyl
methacrylate (HEMA) in a period of 24 and 72 h
after exposing mouse broblasts and performing an
MTT assay, which evaluates the mitochondrial
enzyme activity. Kleinsasser et al. (2004) investi-
gated the cytotoxicty of HEMA, TEGDMA, UDMA,
Bis-GMA using a single cell microgel electrophoresis
(comet) assay and found a relevant cytotoxicity
effect for all materials, but vitality levels were only
at a critical level for Bis-GMA and TEGDMA.
Regarding this information, it seems that extrac-
table monomers or other ingredients in resin-based
EndoREZ
s
sealer may produce some cytotoxic
effects at the outset, but these effects subside
with time.
Recently introduced GuttaFlow
s
sealer includes
the silicon-based sealer Roekoseal (RSA, Roeko,
Coltene/Whaledent Inc, Langenau, Germany) com-
bined with gutta-percha. Polydimethyl siloxane has
been used widely in prosthodontics because of its
low dimensional change (about 0.60.15%) and low
water absorption. Silicon soft liners have been
proposed for use in patients with irritation of the
denture-bearing mucosa. Vulcanized or room tem-
perature silicon is popular as maxillofacial materi-
als for correcting facial defects because of its ease
of processing and good physical properties. Also
silicon-low temperature isotropic carbon (LTI) alloy
Table 3. Tissue glutathione (GSH; mmol/g tissue) levels
in all groups (n 5).
Lung Liver Kidney Skin
Control 2.170.2 2.670.4 2.370.3 2.570.2
GR
24 h
1.970.3 1.870.8 2.070.4 2.070.4
GR
7d
2.070.4 1.970.3 2.370.2 1.870.4
GR
30d
2.470.2 2.370.5 2.270.2 1.970.1
ER
24 h
2.170.3 2.470.1 1.970.1 2.170.1
ER
7d
2.570.4 2.070.2 2.170.2 1.970.1
ER
30d
2.570.1 1.770.1 2.270.2 2.370.2
KR
24 h
2.470.01 2.070.2 1.870.2 2.370.2
KR
7d
2.370.2 2.370.2 2.270.2 2.270.3
KR
30d
1.970.5 2.270.3 2.170.1 2.370.1
GR: GuttaFlow, ER: EndoREZ, KR: Kerr Pulp Canal Sealer. Values
are represented as mean7SEM. The differences are not
signicant.
Table 2. Tissue malondialdehyde (MDA; nmol/g tissue)
levels in all groups (n 5 per group).
Groups Lung Liver Kidney Skin
Control 38.171.2 43.772.7 39.472.3 42.372.6
GR
24 h
41.372.1 47.773.6 44.774.5 40.674.3
GR
7d
40.372.0 44.774.3 43.272.4 47.674.2
GR
30d
37.071.8 48.575.0 35.676.7 46.274.1
ER
24 h
37.973.6 47.775.6 38.072.6 41.273.7
ER
7d
40.471.6 47.773.2 43.175.6 46.075.1
ER
30d
38.972.1 50.273.9 41.275.5 44.573.4
KR
24 h
43. 172.3 42.572.6 41.273.4 42.972.3
KR
7d
39.373.1 47.075.4 42.674.4 47.674.5
KR
30d
41.972.5 44.273.9 39.57 6.5 45.575.3
GR: GuttaFlow, ER: EndoREZ, KR: Kerr Pulp Canal Sealer. Values
are represented as mean7SEM. The differences are not
signicant.
Table 4. Tissue collagen content (mg/mg protein) of all
groups (n 5).
Lung Liver Kidney Skin
Control 19.570.4 19.670.97 9.570.3 27.071.1
GR
24 h
17.870.8 21.470.6 9.170.3 25.870.5
GR
7d
18.670.2 18.670.2 9.370.1 25.570.5
GR
30d
20.670.2 21.670.4 9.670.1 25.870.2
ER
24 h
18.570.6 19.970.4 8.870.3 23.270.9
ER
7d
19.170.3 20.370.7 9.270.2 29.270.3
ER
30d
19.770.2 21.170.5 9.770.3 24.770.3
KR
24 h
17.970.4 20.670.4 8.770.2 25.970.7
KR
7d
20.870.6 21.770.7 9.470.2 28.770.5
KR
30d
20.970.2 20.370.4 10.570.2 27.970.4
GR: GuttaFlow, ER: EndoREZ, KR: Kerr Pulp Canal Sealer. Values
are represented as mean7SEM. The differences are not
signicant.
Comparision of biocompatibility and cytotoxicity of two new root canal sealers 573
implants, which are usually placed on a metal
graphite substrate in the form of either a subper-
iosteal implant or an endosteal blade, are found to
be very biocompatible (Philiphs, 1981). However,
there are still not many reported studies on the
cytotoxicity of GuttaFlow
s
. Gerosa et al. (2003)
compared the cytotoxicity of GuttaFlow with Pulp
Canal Sealer and resin-based Acroseal (Septodont,
Saint-Maur-des-Fosses, Cedex, France) and found
GuttaFlow
s
less cytotoxic than Pulp Canal Sealer
and Acroseal. Miletic et al. (2005) found no
cytotoxic effects of the silicon-based sealer Roe-
koseal
s
on HeLa cells and mouse skin broblasts
(L929). Gencoglu et al. (2003) investigated the
connective tissue response of RSA (Roekoseal
s
) and
found new granulation tissue with brous tissue
adjacent to RSA on day 30 after treatment. In the
present study, GuttaFlow
s
showed slightly cyto-
toxic effects in all the investigated organs within a
period of 24 h and this effect decreased in time and
was found acceptable. However, a lesser degree of
tissue damage was found in kidney tissue of the
GuttaFlow
s
-applied group compared to the other
groups.
Although zinc oxide eugenol (ZOE) containing
sealers are the most widely used sealers in
endodontics, non-specic biocompatibility tests
showed eugenol elicited a pronounced tissue
irritation (Langeland, 1978). Araki et al. (1993)
demonstrated that the increased cytotoxicity of
ZOE sealer was caused by its eugenol content.
However, Hume (1986) indicated that variations in
the powder/liquid ratio of the ZOE mixture had
only slight effects on eugenol availability. He
demonstrated that when ZOE was in contact with
the soft tissue, a release of eugenol in sufcent
concentrations (10
3
M) to cause local cell death
was likely to have occurred. If the contact area of
ZOE at the root apex is small, then the area of cell
death is relatively conned and the rate of eugenol
release declines sufciently within 12 weeks to
allow healing. A local effect at the root apex that
might be therapeutic would be sensory nerve
inhibition or death of local nerve endings (Hume,
1986).
Kolokouris et al. (1998) also demonstrated that
eugenol liberation from ZOE containing compounds
was initially high immediately after mixing, but
decreased over time with a progressive decrease in
connective tissue reaction. In the present study,
Kerr Pulp Canal Sealer presented a slight cytotoxi-
city compared to other two sealers, but this effect
subsided and was found acceptable in living organs
at the end of 30 days. The effects of sealers
components on living organs have also been
investigated by other authors. Kolokouris et al.
(1998) investigated the effects of ZOE sealer on Zn,
Ca and Cu concentrations in some living organs of
rats. They found that subcutaneous injection of a
ZOE sealer (Roth 811) changed the concentrations
of Zn, Ca and Cu in some organs on days 4 and 5
after treatment. Economides et al. (2005) found
Calciobiotic root canal sealer (CRCS, Coltene/
Whaledent Inc., Cuyahoga Falls, OH, USA) and Roth
811 (Roth International, Chicago, IL, USA) to induce
redistribution of zinc, whereas AH26 (De Trey
Dentsply, Konstanz, Germany) induced changes in
calcium content in some organs of rats. These
results indicate that ZOE sealers might release
considerable amounts of those compounds, which
were then deposited in vital organs.
It appears that all sealers may cause some
irritation reactions in organs distant from the sites
of injection or application. However, after nal
setting this reaction decreases. This phenomenon
was also conrmed in the present study. Although
these results were conducted in experimental
animals, they are signicant as such experimental
studies cannot easily be conducted in humans.
Nevertheless further clinical studies are needed to
conrm these results and to evaluate their rele-
vance to the outcome of the treatment.
Acknowledgements
We would like to honor the memory of Dr. Nursal
Gedik, our dear friend, who passed away in 2007.
Without her contribution, it would not be possible
to accomplish the current study.
This research was presented in part at the 12th
Biennial Congress of the European Society of
Endodontology (September, 2005), in Dublin, Ire-
land.
References
Araki K, Suda H, Spangberg LSW. Reduced cytotoxicity
of a root canal sealer through eugenol substitution.
J Endod 1993;19:5547.
Becce C, Pameijer CH. Biocompatibility of a new
endodontic sealer. J Dent Res 2003;82:321.
Beutler E. Glutathione in red blood cell metabolism. In:
A manual of biochemical methods. New York: Grune
and Stratton; 1975. p. 1124.
Browne RM, Friend LA. An investigation into the irritant
properties of some root lling materials. Arch Oral Biol
1968;13:135569.
Economides N, Kotsaki-Kovatsi VP, Poulopoulos A,
Kolokuris I, Rozos G, Shore R. Experimental study of
the biocompatibility of four root canal sealers and
N. Gencoglu et al. 574
their inuence on the zinc and calcium content of
several tissues. J Endod 2005;21:1227.
Friend LA, Browne RM. Tissue reaction to endodontic
lling materials. Br Dent J 1968;125:2918.
Gencoglu N, Turkmen T, Ahiskali R. A new silicon-based
root canal sealer (Roekoseal-Automix). J Oral Rehabil
2003;30:15.
Gerosa R, Pongione G, Testarelli L, Gallottini L, Gambar-
ini G. Cytotoxicity of a new experimental endodontic
sealer: a comparative study. Int End J 2003;36:953.
Hume WR. The pharmacologic and toxicological proper-
ties of zinc oxide-eugenol. J Am Dent Assoc 1986;113:
78991.
Kardon BR, Kuttler S, Hardigan P, Dorn SO. An in vitro
evaluation of sealing ability of a new root canal
obturation system. J Endod 2003;29:65861.
Kazemi RB, Safavi KE, Pameijer CH. Sealing properties of
a new injectable root canal lling material. J Dent Res
2003;82:309.
Kleinsasser NH, Wallner BC, Harreus UA, et al. Genotoxi-
city and cytotoxicity of dental materials in human
lymphocytes as assessed by single cell microgel
electrophoresis (comet) assay. J Dent 2004;32:22934.
Kolokouris I, Kotsaki-Kovatsi VP, Economides N, Poulo-
poulos A, Ruzos G, Vlemmas I. Inuence of zinc oxide
and eugenol sealer on concentration of zinc, calcium
and copper in rat tissues. Endod Dent Traumatol
1998;14:2103.
Langeland K. Correlation of screening tests to usage
tests. J Endod 1978;4:3003.
Lopez De Leon A, Rojkind M. A simple micromethod for
collagen and total protein determination in formalin-
xed parafn-embedded sections. J Histochem Cyto-
chem 1985;33:73743.
Louw NP, Pameijer CH, Norval G. Histopathological
evaluation of root canal sealer in subhuman primates.
J Dent Res 2001;80:654.
Martinek RG. A rapid ultraviolent spectrophotometric
lactic dehydrogenase assay. Clin Chem Acta 1972;40:
919.
Miletic I, Devcic N, Anic I, Borcic J, Karlovic Z, Osmak M.
The cytotoxicity of Roekoseal and AH Plus compared
during different setting periods. J Endod 2005;31:
3079.
Neviere RR, Ceepinskas G, Madorin W, et al. LPS
pretreatment ameliorates peritonitis-induced myocar-
dial inammation and dysfunction: role of myocytes.
Am J Physiol Heart Circ Physiol 1999;277:H88592.
Nguyen NT. Obturation of the root canal system. In:
Cohens S, Burns RC, editors. Pathways of the pulp. 3rd
ed. St.Louis: CV Mosby Co; 1984. p. 20599.
Olsson B, Wennberg A. Early tissue reaction to endodontic
lling materials. Endod Dent Traumatol
1985;1:13841.
Omurtag GZ, Guranliog lu FD, Sehirli O, et al. Protective
effect of aqueous garlic extract against naphthalene-
induced oxidative stress in mice. J Pharm Pharmacol
2005;57:62330.
rstavik D, Mjor IA. Histopathology and X-ray micro-
analyses of the subcutaneous tissue response to
endodontic sealers. J Endod 1988;14:1323.
Philiphs RW. Skinners science of dental materials, 8th ed.
Philadelphia: W.B. Saunders; 1981. p. 137.
Ratanasathien S, Wataha JC, Hanks CT, Dennison JB.
Cytotoxic interactive effects of dentin bonding com-
ponents on mouse broblasts. J Dent Res 1995;74:
16026.
Reiter RJ, Acuna-Castroviejo D, Tan DX, Burkhart S. Free
radical-mediated molecular damage. Ann NY Acad Sci
2001;939:20015.
Schmalz G, Hoffmann M, Weis K, Schweikl H. Inuence of
albumin and collagen on the cell mortality evoked by
zinc oxideeugenol in vitro. J Endod 2000;26:2847.
Schweikl H, Schmalz G, Federlin M. Mutagenicity of the
root canal sealers AH Plus in the Ames test. Clin Oral
Invest 1998;2:1259.
Spahl W, Budzikiewicz H, Geurtsen W. Determination of
leachable components from four commercial dental
composites by gas and liquid chromatography/mass
spectrometry. J Dent 1998;26:13745.
Yes-ilsoy C, Koren LZ, Morse DR, Kobayashi C. A compara-
tive tissue toxicity evaluation of established and
newer root canal sealers. Oral Surg Oral Med Oral
Pathol 1988;65:45967.
Comparision of biocompatibility and cytotoxicity of two new root canal sealers 575