Comparison of the biocompatibility between 2 endodontic filling

Comparison of the biocompatibility between 2 endodontic
filling materials for primary teeth
CHUNG-WEN CHEN 1
CHIA-TZE KAO 2
TSUI-HSIEN HUANG 1,3
1
Institute of Stomatology, Chung Shan Medical University, Taichung, Taiwan, ROC.
Institute of Oral material science, Chung Shan Medical University, Taichung, Taiwan, ROC.
3
Dental Department, Chung Shan Medical University Hospital Taichung, Taiwan, ROC.
2
The most-popular root canal filling materials for primary teeth are zinc oxide eugenol (ZOE),
iodoform paste, and calcium hydroxide. Root canal filling materials need to be biocompatible with the
periapical tissue. There are few papers which indicate the mechanism of cellular change of root canal
filling materials. The purpose of the present study was to evaluate the cellular changes of a ZOE with
formocresol (FC) and Vitapex-treated human osteosarcoma cell line. The ZOE powder mixed with the
FC liquid and Vitapex alone were eluted in culture medium. The elution solutions were used to treat the
human osteosarcoma cell line. The MTT assay was used to investigate the survival rate of U2OS cells.
Western blot assay was used to compare the c-jun N terminal kinase (JNK) expression. One way ANOVA
was used to compare the survival difference with p < 0.05 accepted as statistically significant. The result
showed that the ZOE sealer was toxic to U2OS cells (p < 0.05). Vitapex was biocompatible with U2OS
cells. JNK kinase expression was seen only in the ZOE with FC group. We concluded that ZOE with FC
is not biocompatible with U2OS cells, and cell death occurred by apoptosis. Clinically, we suggest that
Vitapex is a good choice for a primary tooth root canal filling material. （Chin Dent J, 24(1)：28-34, 2005）
Key words: endodontic filling material for primary teeth, MTT assay, Western blot analysis.
Endondotic therapy for primary teeth has long
been advocated when the criteria for classical
pulpotomy treatment can’t be met1. Resorption of the
filling material is considered one of the requirements
of an ideal root-canal medicament for pulpectomies
of primary teeth2-5. Resorption of the root canal filling
material should occur as the root of the primary tooth
is resorbed during exfoliation, permitting normal
eruption of the succedaneous tooth4. If the material is
expressed beyond the apex, it should be resorbable
and non-toxic to the periapical tissues and the
permanent tooth germ2-5.
The most-popular root canal filling materials for
primary teeth are zinc oxide eugenol (ZOE), iodoform
Received: December 6, 2004
Accepted: February 17, 2005
Reprint requests to: Dr. Tsui-Hsien Huang, Department of Dentistry,
Institute of Stomatology, Chung Shan Medical
University, No. 110, Chien Kuo N. Road, Sec. 1,
Taichung, Taiwan 40201, ROC.
28
paste, and calcium hydroxide6-8. ZOE has been the
material of choice for many years. Although this
agent showed antibacterial effects against pure
cultures of bacteria in several studies9,10, combining it
with formocresol (FC) increased its antibacterial
effect10.
Animal studies using ZOE cement as root canal
filling material have reported chronic inflammatory
reactions and slow resorption of the material11.
Erausquin and Muruzabal1 used ZOE as a root canal
filling material in 141 rats followed for 1 to 90 days.
They noted that ZOE irritated the periapical tissues
and caused necrosis of the bone and cementum. In
addition, they noted that extruded ZOE developed a
fibrous capsule that prevented resorption12. Gould
first reported a 1-visit ZOE pulpectomy study in 1972
in which 39 molars were filled with ZOE. He
concluded that 35 of the 39 molars followed for a
mean time of 16 months had successfully been
treated13.
Root canal filling material for primary teeth has
Chin Dent J 2005‧Vol 24‧No 1
Biocompatibility of filling materials
long been iodoform paste (Kri paste), even though
unfavorable responses of periapical tissue to Kri
paste and increased cytotoxicity were found14,15. A
newer preparation, Vitapex, a mixture of iodoform
and calcium hydroxide (Ca(OH)2), is now available
in North America. A preliminary study suggested that
it too is efficacious for pulpectomies in primary
teeth16. Vitapex demonstrated inhibitory activity
against Streptococcus mutans, Staphylococcus aureus,
and Lactobacillus casei. The main ingredients of
Vitapex are iodoform at 40.4%, calcium hydroxide at
30.3%, and silicone at 22.4%. When extruded into
furcal or apical areas, Vitapex can either diffuse
away17,18 or be resorbed in part by macrophages19,20 in
as short a time as 1 or 2 weeks17. Bone regeneration
has been clinically17,21 and histologically21,22 documented
after using Vitapex. Calcium hydroxide-based
compounds are thought to meet most requirements of
endodontic treatment. Calcium hydroxide-based filling
materials showed mild to moderate tissue-irritating
activities23. Several studies examining toxicity
inflammatory response, demonstrated a generally
mild inflammatory reaction, with an influx of foreign
body giant cells24-26.
Cell growth, division, differentiation, and death
are now known to be regulated in part by the
mitogen-activated protein kinase (MAPK) pathway27,28.
Cellular signal transduction is a 2-step process: first,
a signaling molecule is sensed by a receptor at a
target cell, and then the receptor is activated. The
c-Jun N-terminal kinase (JNK), one of the MAP
kinases, is a member of an evolutionarily conserved
subfamily of mitogen-activated protein (MAP) kinases.
Selective phosphorylation of c-Jun by JNK is
determined by a specific docking motif in c-Jun, the
delta region, which enables JNK to physically
associate with c-Jun. Signals relayed by JNK through
c-Jun regulate a range of cellular processes including
cell proliferation, tumourigenesis, apoptosis, and
embryonic development29-32.
Root canal filling materials for primary teeth
have slow resorption rates in tissue. Long-term
clinical studies reveal retention rates of material in
28.7% to 73.3% of cases33. Another study found that
the retained material altered the paths of eruption of
succedaneous teeth in 20% of cases34. The
mechanism of the cell reaction after root canal filling
material treatment has seldom been reported. The
purpose of the present study was to evaluate cellular
changes of a ZOE- and Vitapex-treated human
osteosarcoma cell line.
MATERIAL AND METHODS
Materials and sample preparation
The experimental groups containing the 2 root
canal filling materials are listed in Table 1. The zinc
oxide eugenol materials were mixed in the following
rate: ZnO: eugenol: FC of 6 g: 1 ml: 1 ml. The
Vitapex was directly injected into the ring. Samples
tested for cytotoxicity were prepared as follows:
freshly mixed materials were filled in glass rings (2
mm in height, 6 mm in diameter) and allowed to set
for 24 h at 37 ℃ in a humidified chamber. One to 3
test specimens were then eluted in 10 ml of cell
culture medium at 37 ℃ for 24 h in a 5% CO2 air
atmosphere. The concentration of the test material
was diluted by adding different volumes of medium.
The culture medium without adding test material
served as the control group.
MTT assay
The human osteosacorma cell line (U2OS) was
routinely cultivated in McCoy’s medium (Sigma
Chemical, St. Louis, MO, USA) supplemented
with 5% fetal bovine serum (Sigma) at 37 ℃ in an
air atmosphere containing 5% CO2. Single-cell
suspensions of U2OS cells were obtained from
monolayer cell cultures close to confluency after
trypsinization. Cell numbers were determined by
Table 1. Composition of the experimental group
Group
Composition
Vitapex
(Neo Dental Clinical,Tokyo, Japan)
Calcium hydroxide, iodoform, Oily agents.
Zinc oxide
(Pulpdent, Watertown, MA, USA)
Powder: zinc oxide,
Liquid: eugenol and formocresol
Chin Dent J 2005‧Vol 24‧No 1
29
C.W. Chen, C.T. Kao and T.H. Huang.
hemocytometric counting, and 104 cells/well were
seeded into 96-well plates. Cells were then incubated
for 24 h in a humidified atmosphere of air and 5%
CO2 at 37 ℃. Cell cultures were exposed to 2-, 6-,
and 10-μl aliquots of serially diluted eluates. Five
wells were used for each eluate concentration in each
of 3 independent experiments. Exposure of cell
cultures was stopped by discarding the exposure
medium after 24 h. Viable cells in both treated and
untreated cell cultures were stained with the
formazan dye MTT (1 mg/ml) (Sigma) dissolved in
200 μl culture medium as described35-37. After 3 h at
37 ℃ , the MTT solution was discarded, and
formazan crystals were solubilized with 200 µl of
DMSO. Optical densities were measured at 570 nm
in a multiwell spectrophotometer (Hitachi, Tokyo,
Japan). The survival rate was calculated as Survival
% = absorbance of the treated sample / absorbance of
the medium × 100%. Results were compared using
the one-way analysis of variance (ANOVA).
Differences in treatment means were analyzed by the
Student-Newman-Keul’s test and were considered to
be significant at p < 0.05.
material, 4 µl of Vitapex and 4 µl of the ZnO group
were used to detect kinase expression38. U2OS cells
treated for 1 hour were washed once with cold PBS.
Five million cells were lysed in 50 µl of lysis buffer
(1% Triton X-100, 0.5% NP40, 10 mM EGTA, 0.2
mM Na3VO4, 0.2 mM NaF, and 0.2 mM PMSF). Cell
lysates were cleared at 15,000 rpm for 15 minutes at
4 ℃. Twenty-five micrograms of protein from each
sample was boiled for 5 min in 1X SDS gel-loading
buffer (125 mM Tris (pH 6.8), 5% glycerol, 28 mM
SDS, 1% beta-mercaptoethanol, and 0.006% brompophenol blue). Proteins were separated by 12.5%
SDS-PAGE and transferred onto polyvinylidene
difluoride membranes. Membranes were blocked for
1 hour at room temperature in 3% BSA, 5% nonfat
dried milk, 10 mM Tris (pH 7.5), 100 mM NaCl, and
0.1% Tween 20. After 4 washes in TBS-T buffer, the
membrane was incubated with 0.5 µg/ml rabbit JNK
antibody for overnight. After four more washes, the
membrane was overlayed with a second antibody
(rabbit anti-JNK antibody) for 1 hour, then washed
with TBS-T buffer for 20 minutes.
RESULTS
Western blot analysis
The survival rate of the Vitapex group showed
From the MTT test of the root canal filling
Vitapex treated U2OS cell's survival rate (%).
Survival rate (%)
120
100
80
60
40
20
0
0
2
6
10
Concentration (µl)
Figure 1. Survival rate (%) of Vitapex-treated U2OS cell.
30
Chin Dent J 2005‧Vol 24‧No 1
Biocompatibility of filling materials
no statistical difference (p > 0.05) with control group
(Figure 1). The survival rate of the ZOE with FC
group showed a dose dependent decrease (p < 0.05)
(Figure 2).
The morphology of U2OS cell treated with the
filling materials is shown in Figure 3. Cell
membranes in the ZOE group were not intact and
contained vesicles. The morphology of the Vitapex
group showed that cells were the same as those of the
control group.
JNK expression was shown in ZOE-treated
U2OS cells. The control group and Vitapex group
exhibited no JNK band expression according to the
Western blot analysis (Figure 4).
might cause damage to periapical tissue. This result is
similar to that of Erausquin and Muruzaba’s study
which demonstrated ZOE to be highly toxic to
periapical tissues in rats, causing necrosis of the hard
tissues it contacted39. A previous report showed that
long-term clinical studies revealed retention rates of
the material in 28.7% to 73.3% of cases34,35. It was
also shown that the retained material altered the paths
of eruption of succedaneous teeth34,35. From the
present results, the biocompatibility of the root canal
filling material may be the main factor affecting
alteration of the eruption path.
Traditionally, zinc oxide eugenol has been the
most-commonly used root canal filling material for
primary teeth. It has been used in combination with
silver nitrate, formocresol, or iodoform, all of which
have fixative properties. Eugenol (4-allyl-2methoxyphenol, a natural substance derived from the
oil of cloves) was previously found to be genotoxic in
several in vitro systems including the Ames test40.
Furthermore, a zinc oxide-eugenol- based sealer was
found to be cytotoxic, and this action was ascribed to
the eugenol component41.
Since the traditional ZOE with FC is toxic to
U2OS cells, cellular changes after U2OS cell contact
with ZOE were discovered. JNK was expressed in the
ZOE group but not in the control or Vitapx groups
(Figure 4). This means that in ZOE-treated U2OS cell,
DISCUSSION
Because of the anatomy of primary roots and
furcal areas, it is difficult to avoid extrusion of sealer
beyond the root canals in all pulpectomy cases.
Vitapex contains calcium hydroxide and iodoform. In
1991, iodoform paste was advocated as a pulpectomy
filler in primary teeth due to its resorbability and
disinfectant properties33. Result of the present study
show that the Vitapex is more biocompatibile with
U2OS cells than is the ZOE group. So, the use of
ZnO+eugenol+FC as endodontic filling material
120
Survival rate (%)
100
80
*
60
40
20
*
*
6
10
0
0
2
Concentration (µl)
Figure 2. Survival rate (%) of U2OS cells treated with ZOE with FC cement.
*: Statistically significant difference at p < 0.05.
Chin Dent J 2005‧Vol 24‧No 1
31
C.W. Chen, C.T. Kao and T.H. Huang.
Figure 3. Morphology of the U2OS cells treated with test materials. A. Control group. B. ZOE with FC group. C. Vitapex group.
(magnification, 10X)
Control
Vitapex
ZOE+FC
JNK
B-actin
Figure 4. JNK expression by Western blot analysis. The JNK band
was expressed in the ZOE with FC group but not in control on Vitapex
groups.
32
its toxic mechanism may be through the JNK
pathway. Signals relayed by JNK through c-Jun
regulate a range of cellular processes including cell
proliferation,
tumourigenesis,
apoptosis,
and
embryonic development29-32. The toxicity of ZOE
might cause cell apoptosis rather than necrosis. This
finding has not been shown in any previous study.
Although the success rates of ZOE root fillings
range from 68.7% to 86.1%, overfilling with ZOE
resulted in a much lower-success rate (41%)42.
Vitapex, used as a root canal filling material for
pulpectomy treatment for primary teeth is resorbed
extraradicularly and intraradicularly without apparent
ill effects, and has been proven to be clinically and
radiographically successful43. From the present study,
we suggest that Vitapex can be a good choice of an
endodontic filling material for primary teeth.
Chin Dent J 2005‧Vol 24‧No 1
Biocompatibility of filling materials
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