Biocompatibility of Various Formula Root Filling Materials for Primary Teeth

Biocompatibility of Various Formula Root Filling Materials for
Primary Teeth
Tsui-Hsien Huang,1 Shinn-Jyh Ding,2 Chia-Tze Kao2
1 Dental Department, Chung Shan Medical University Hospital, Taichung, Taiwan
2 Institute of Oral Material Science, Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan
Received 29 November 2005; revised 7 February 2006; accepted 7 April 2006
Published online 21 July 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.30621
Abstract:
The aim of this study was to compare the effects of different materials used in
primary root canal fillings on the cell viability of human osteosarcoma cell lines. The
experimental group contained six different types of root canal filling materials, including zinc
oxide (ZnO)
eugenol
formocresol (FC), Ca(OH)
FC, Ca(OH)
Iodoform, Ca(OH)
2
2
2
Iodoform
camphorated parachlorophenol (CPC), Ca(OH)
CPC, and Vitapex. Cell
2
viability tests were performed using tetrazolium bromide colorimetric (MTT) assay on human
osteosacorma cell lines (U2OS). The results were analyzed using one-way analysis of variance
(ANOVA) and Student–Newman–Keul’s test with p < 0.05 showed statistical differences. The
ZnO
eugenol
FC group and Ca(OH)
FC group showed the lowest survival rates (p <
2
0.05). The Ca(OH)
Iodoform
CPC group and Ca(OH)
CPC group showed signifi-
2
2
cantly lower survival rates at concentrations above 6
L/mL (p < 0.05). The Ca(OH)2
Iodoform group and Vitapex group showed the highest survival rates (p < 0.05). We
concluded that the use of calcium hydroxide with iodoform as a root filling base material is a
better option than other medications. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl
Biomater 80B: 486 – 490, 2007
Keywords:
primary teeth; zinc oxide eugenol; formocresol; iodoform; biocompatibility
INTRODUCTION
menthol) or VitapexTM paste (a mixture of iodoform 40.4%,
calcium hydroxide 30.3%, and silicone 22.4%).6,7 Recently, a
Endodontic therapy for primary teeth has long been advo-
biocompatible material–mineral trioxide aggregate (MTA)
cated when the criteria for classic pulpectomy treatment
has been applied as root filling material.8
cannot be met.1 Resorption of the filling material is consid-
The use of ZnOE or ZnO to fill root canals of primary
ered one of the requirements of an ideal root canal medica-
teeth was first described by Sweet in 1930.9 For many years
ment following pulpectomy of a primary tooth.2,3 Resorption
ZnOE or ZnO was the material of choice. Although this agent
of the root canal filling material should occur as the root of
showed antibacterial effects against pure cultures of bacteria
the primary tooth is resorbed during exfoliation, permitting
in other studies,10,11 combining it with formocresol increased
normal eruption of the succedaneous tooth.4 If the material is
the antibacterial effects.11 Kri paste was found to have long
expressed beyond the apex, it should be resorbable and non-
lasting bactericidal potential. The excess of paste extruded
toxic to the periapical tissues and the permanent tooth germ.
into periapical granulomatous tissue, which is removed rap-
Jerrell and Ronk presented a case of arrested tooth formation
idly from the apical region and replaced by healthy connec-
in a mandibular second premolar after zinc oxide– eugenol/
tive tissue.12 MTA has demonstrated cemental repair, forma-
formocresol paste was extruded from the apex of the primary
tion of bone, and regeneration of the periodontal ligament
second molar pulpectomy.5
when used as a root canal sealer.8
The traditional root canal filling materials for primary
Tchaou et al. used 10 types of filling materials to evaluate
teeth are calcium hydroxide (Ca(OH) ), zinc oxide (ZnO),
2
the antibacterial ability.13 Their results showed strong anti-
zinc oxide eugenol (ZnOE) with or without mixed with
bacterial effects for calcium hydroxide (Ca(OH) ) mixed with
2
formocresol (FC), iodoform paste such as Kri 1 paste (a
camphorated parachlorophenol (CPC), ZnO mixed with CPC,
mixture of iodoform and pararchlorophenol, camphor, and
and ZnO mixed with FC. The minimum antibacterial effects
were for Vitapex and Ca(OH) added with water.13 The null
2
hypotheses are that (a) the composition of root filling mate-
Correspondence to: C.-T. Kao (e-mail: ctk@csmu.edu.tw)
rials does not affect biocompatibility as measured by cell
© 2006 Wiley Periodicals, Inc.
viability; (b) composition does not affect cell morphology;
486

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VARIOUS FORMULA ROOT FILLING MATERIALS FOR PRIMARY TEETH
487
TABLE 1. Composition of Experimental Root Filling Materials
wells were used for each eluate concentration in each of three
independent experiments. Exposure of cell cultures was
Material
Composition
stopped by discarding the exposure medium after 24 h. Via-
Vitapex (Ca(OH)
Iodoform
Silicone oil)
0.5 g
2
ble cells in both treated and untreated cell cultures were
ZnO
Eugenol
FC
6 g:1 mL:1 mL
stained with formazan dye MTT (1 mg/mL) (Sigma) dis-
Ca(OH)
FC
6 g: 1 mL
2
solved in 200 L culture medium. After 3 h at 37°C, the MTT
Ca(OH)
Iodoform
deionize water
6 g: 0.6 g: 2 mL
2
solution was discarded and formazan crystals were solubi-
Ca(OH)
Iodoform
CPC
6 g: 0.6 g: 2 mL
2
lized with 200
L of DMSO. Optical densities were mea-
Ca(OH)
CPC
6 g: 2 mL
2
sured at 570 nm in a multi-well spectrophotometer (Hitachi,
The dilute solution used is 1 mL McCoy’s medium.
Tokyo, Japan). The survival rate was calculated as Survival
Immersion time is 24 h.
%
absorbance of the treated sample/absorbance of the
medium
100%. Results were compared using the one-way
analysis of variance (ANOVA). Differences in treatment
and (c) the concentration of eluate does not affect cell via-
means were analyzed using the Student–Newman–Keul’s test
bility. The aim of this study was to compare the effects of
and were considered to be significant at p
0.05.
different materials used in primary root canal fillings on the
cell viability of human osteosarcoma cell lines.
Assessment of Morphology Observation
After U2OS being treated with 6
L/mL concentrations of
MATERIALS AND METHODS
root filling materials 24 h, each well was observed by phase
contrast microscopy with an inverted optical microscope
(Leica DM IRB inverted microscope), for a total magnifica-
Materials and Sample Preparation
tion of 10 . Observation of the U2OS focused on assessing
The experimental group comprised of six formulas of root
malformation, degeneration, sloughing, or lysis of the cul-
canal filling materials. They are listed in Table I. The differ-
tures.
ent root filling material formulas were mixed in the following
proportions: ZnO
eugenol
FC
6 g: 1 mL: 1 mL;
Ca(OH)
FC
6 g: 1 mL; Ca(OH)
Iodoform
2
2
RESULTS
deionized water
6 g: 0.6 g: 2 mL; Ca(OH)
Iodoform
2
CPC
6 g: 0.6 g: 2 mL; and Ca(OH)
CPC
6 g: 2 mL.
2
Figure 1 shows U2OS cell morphology after being treated
Vitapex (Neo Dental Chemical Co. Tokyo, Japan) was a
with 6
L/mL concentrations of root filling materials under
nonmixing-type material and was examined as supplied by
10
magnification during microscopic examination. In Fig-
the manufacturer. Samples were prepared as follows: freshly
ure 1(A), the Vitapex group shows good growth of U2OS
mixed materials were filled in glass rings (2 mm in height, 6
cells with intact cell morphology. In Figure 1(B), the ZnO
mm in diameter) and allowed to set for 24 h at 37°C in a
Eugenol
FC group shows most of the U2OS cells are dead,
humidified chamber. In the experimental group, five samples
a decrease in numbers, and cell lysis or membrane destruc-
of each root filling material were then eluted in 10 mL of cell
tion. In Figure 1(C), the Ca(OH)
FC group shows some of
culture medium at 37°C, 5% CO air atmosphere for 24 h.
2
2
the U2OS cells are dead, and the numbers have decreased. In
The concentration of the test materials were diluted by adding
addition, the morphology of the U2OS cells have changed to
culture medium to final concentrations as 2, 6, and 10
L/
irregular shaped. In Figure 1(D), the Ca(OH)
Iodoform
mL. The culture medium without adding experimental mate-
2
group, the U2OS cells show good growth and no morphology
rial served as the control group.
changes. In Figure 1(E), the Ca(OH)
Iodoform
CPC
2
group shows that the U2OS cell number has decreased and no
Cell Viability Test by MTT (3-[4,5-dimethylthiazol-2-yl]-
changes in morphology. In Figure 1(F), the Ca(OH)
CPC
2,5-diphenyl tetrazolium bromide colorimetric) Assay
2
group shows that the U2OS cells have a minor decrease in
The assay was the same as mentioned in our previous study.14
numbers, but no morphology changes. Figure 1(G) is the
Briefly, the human osteosarcorma cell line (U2OS) was rou-
control group cell morphology.
tinely cultivated in McCoy’s medium (Sigma Chemical, St.
The U2OS cell survival rates after being treated with
Louis, MO) supplemented with 5% fetal bovine serum
different concentrations of root filling materials are shown in
(Sigma) at 37°C in an air atmosphere containing 5% CO .
Figure 2.
2
Single-cell suspensions of U2OS cells were obtained from
The survival rate of the Vitapex group showed statistical
monolayer cell cultures close to confluency after trypsiniza-
difference with that of the control group ( p
0.00
0.05).
tion. Cell numbers were determined by hemocytometric
The survival rate at concentrations above 6
L/mL showed
counting, and 104 cells/well were seeded into 96-well plates.
no differences [Figure 2(A)].
Cells were then incubated for 24 h in a humidified atmo-
The survival rate of the ZnO
Eugenol
FC group
sphere of air and 5% CO at 37°C. Cell cultures were exposed
showed a dose-dependent decrease as the concentration in-
2
to 2-, 6-, and 10- L aliquots of serially diluted eluates. Five
creased ( p
0.00
0.05). At concentrations above 6 L/mL
Journal of Biomedical Materials Research Part B: Applied Biomaterials
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488
HUANG, DING, AND KAO
DISCUSSION
The concepts of endodontic treatment of primary teeth and
permanent teeth are different. First, the root canal morphol-
ogy of a primary tooth is different from a permanent tooth.
With many lateral canals and accessory canals in the primary
teeth, an infected primary tooth root canal is difficult to clean
using mechanical instruments. Second, the root canal filling
materials need to easily flow into the narrow and tortuous root
canals of the primary tooth. Third, the filling material ex-
truded to the periapical tissue of the primary tooth must be
biocompatible.
To increase antibacterial effects, antibacterial medicines
need to be added to the root canal filling material. In addition
to the antibacterial effects, the root canal filling material of
primary teeth requires more cell biocompatibility. Materials
that have been used routinely in root canal therapy of primary
teeth include zinc oxide eugenol (ZnOE), Vitapex or other
calcium hydroxide-based products with different additives.
Figure 1. The U2OS cell survival rate after treated with 6
L/mL
concentrations of root filling materials. (A) Control (B) ZnO
Eugenol
FC (C) Ca(OH)
FC (D) Ca(OH)
Iodoform (E) Ca(OH)
2
2
2
Iodoform
CPC (F) Ca(OH)
CPC (G) Vitapex.
2
the survival rate of the ZnO
Eugenol
FC group was
severely decreased [Figure 2(B)].
The survival rate of the Ca(OH)
FC group showed a
2
dose-dependent decrease as the concentration increased
( p
0.00
0.05). At concentrations above 6
L/mL of
Ca(OH) l
FC, the survival rate was severely decreased
2
[Figure 2(C)].
The survival rate of the Ca(OH)
Iodoform group was
2
higher than that of the control group at 2
L/mL concentra-
tions ( p
0.02
0.05) [Figure 2(D)].
The survival rate of the Ca(OH)
Iodoform
CPC
2
group showed a dose dependent decrease as the concentration
increased (F
1148.12, p
0.00). At concentrations above
6
L/mL, the survival rate of the Ca(OH)
Iodoform
2
CMCP group was severely decreased [Figure 2(E)].
The survival rate of the Ca(OH)
CPC group showed a
2
dose-dependent decrease as the concentration increased ( p
Figure 2. The U2OS cell survival rate after treated with different
0.00
0.05). At concentrations above 6
L/mL, the survival
concentrations of root filling materials. (A) Vitapex (B) ZnO
Eugenol
rate of the Ca(OH)
CPC group was severely decreased
2
FC. (C) Ca(OH)
FC (D) Ca(OH)
Iodoform (E) Ca(OH)
2
2
2
[Figure 2(F)].
Iodoform
CPC (F) Ca(OH)
CPC. Control group concentration is
2
The comparison of same concentration survival rate in
0
L/mL. Experimental group concentrations are 2, 6, and 10
L/mL.
One-way ANOVA was used to test for significant differences. The
different groups was shown in Figure 3. The Ca(OH)2
Student–Newman–Keul (SNK) multiple comparison of means proce-
Iodoform group showed highest survival rate in all experi-
dure at p
0.05 was used to show differences. The SNK ranking with
mental groups. ( p
0.05)
the same letters do not significantly differ at p
0.05.
Journal of Biomedical Materials Research Part B: Applied Biomaterials
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VARIOUS FORMULA ROOT FILLING MATERIALS FOR PRIMARY TEETH
489
calcium hydroxide combined with iodoform resulted in a
high survival rate of the U2OS cells. Similar results of
high survival rates of the U2OS cells were found in the
Vitapex group (Figure 3). From this point of view, it may
be assumed that the medication plays an important role in
the biocompatibility of the root canal filling material. FC is
known for its severe cytotoxicity.16 CPC can inhibit the
human periodontal ligament cells viability and prolifera-
tion in a dose-dependent manner.19 The findings are in
agreement with our present results that calcium hydroxide
and CPC mixing materials resulted in low survival rate of
the U2OS cells (Figure 3). It was reported that iodoform-
based tooth filling pastes caused considerable tissue necro-
sis and had a higher cytotoxicity than ZnOE.20 This is
contrary to our present results, when U2OS cells were
treated with calcium hydroxide and iodoform, the U2OS
Figure 3. The U2OS cell survival rate after treatment with different
concentrations of root filling materials. One-way ANOVA was used to
cells showed the highest survival rate (Figure 3). In our
test for significant differences. The Student–Newman–Keul (SNK)
present study, the Vitapex group showed the U2OS cells
multiple comparison of means procedure at p
0.05 was used to
had a similar high survival rate (Figure 3). From above
show differences. The SNK ranking with the same letters do not
findings, the results of the U2OS cells treated with calcium
significantly differ at p
0.05.
hydroxide and iodoform with CPC mixing should be in-
teresting. The present results showed that the U2OS cell
These additives are to inhibit various species of bacteria and
survival rate was high under low concentrations (2
L/mL)
to increase the success rate of primary tooth pulpectomy. In
of calcium hydroxide and iodoform with CPC and the
the study by Tchaou et al., they showed that the most effec-
survival rate was low under high concentrations (above 6
tive antibacterial materials were ZnOE and Ca(OH) -based
L/mL) of calcium hydroxide and iodoform with CPC
2
materials with additions such as FC, CPC, and chlorhexidine
[Figure 2(E)].
dihydrochloride (CHX).13 Materials with no or minimal an-
One factor that caused failure of endodontic treatment of
tibacterial effects included Vitapex, Ca(OH)
H O and
primary teeth was the slow rate of resorption of ZnOE. When
2
2
Vaseline.13 From our biocompatibility study results, root
ZnOE filling material is forced beyond the apex, there is a
canal filling materials with strong antibacterial additions
risk of deflection of erupting succedaneous teeth because of
showed definitive cytotoxicity to U2OS cells [Figures
its hardness.21 Because of the shortcomings, the use of an
2(B,C,E, and F)]. Contrary to the antibacterial effects, Vi-
iodoform base or calcium hydroxide containing material as
tapex and Ca(OH) with iodoform paste showed good bio-
substitute is more popular. Replacement of the ZnOE and FC
2
compatibility but had low antibacterial effects (Figure 3).
with Ca(OH) and FC for the U2OS cell toxicity test found
2
Zinc oxide eugenol has been the material of choice for
that the Ca(OH) and FC treated U2OS cells showed a low
2
many years. Although this agent showed antibacterial effects
survival rate even at low concentrations [Figure 2(C)]. Thus,
against pure cultures of bacterial in several studies,10,11,15
the addition of strong antibacterial medicine does not meet
combining it with formocresol increased its antibacterial ef-
the basic requirements of the root canal treatment. When
fects.13 Formorcresol has been demonstrated to be toxic to
primary root filling materials have strong antibacterial prop-
pulp fibroblast and eugenol has been shown to be toxic to
erties, the cytotoxicity is strong too.
human submandibular gland carcinoma (HSG) cells.16,17
Thus, it can be predicted that ZnOE with FC as the root canal
CONCLUSION
material should be have the strongest cytotoxic effects (Fig-
The results of our study reject the hypothesis that the com-
ure 3). Our present results support this finding. The success
position of root filling materials does affect biocompatibility
rates of zinc oxide and eugenol, and Vitapex for root canal
as measured by cell viability. The different compositions do
treatment of necrotic primary teeth were 78.5% and 100%,
affect cell morphology. The concentration of elutes does
respectively.18 The root filling material with strong antibac-
affect cell viability. During pulpectomy therapy of primary
terial effects did not match with the high success rate of
teeth, balance between antibacterial ability and cytocompat-
primary endodontic treatment.
ibility needs to be reached. The use of calcium hydroxide
Calcium hydroxide did not always inhibit bacterial
with iodoform as a root filling base material is a better option
growth.13 When applying calcium hydroxide during the
than other medications.
endodontic treatment of primary teeth, mixing it with other
antibacterial medicine is needed for the antibacterial ef-
fects. But in our present results, ZnOE or calcium hydrox-
ACKNOWLEDGEMENT
ide base materials containing FC or CPC exerted low
This study was supported by the grant of National Science
biocompatibility [Figures 2(B,C,E,F) and 3]. However,
Council of Taiwan (NSC-94-2314-B040-017).
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490
HUANG, DING, AND KAO
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