J. Biomedical Science and Engineering, 2011, 4, 18-28
doi:10.4236/jbise.2011.41003 Published Online January 2011 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online January 2011 in SciRes. http://www.scirp.org/journal/JBiSE
In vitro responses of human pulp cells and 3T3 mouse
fibroblasts to six contemporary dental restoratives
Jun Sun1, Y im ing Weng2, Fengyu Song1, Dong Xie2
1Department of Oral Biology, School of Dentistry, Indiana University Indianapolis, Indianapolis, USA;
2Department of Biomedical Engineering, Purdue School of Engineering and Technology, Indiana University-Purdue University at
Indianapolis, Indianapolis, USA.
Received 9 November 2010; revised 11 November 2010; accepted 12 November 2010.
ABSTRACT
In vitro responses of human primary pulp cells
(HPCs) and 3T3 mouse fibroblasts to six contempo-
rary commercial dental restoratives were evaluated
using the WST-1 assay. The results show that Fuji II
is not cytotoxic to both cells. Fuji II LC is not cyto-
toxic to HPCs but cytotoxic to 3T3 cells, indicating
that 3T3 cells are more vulnerable to 2-hydroxyethyl
methacrylate (HEMA) than HPCs. Vitremer is very
cytotoxic probably due to having diphenyliodonium
chloride and HEMA in it. Z100 is very cytotoxic
probably due to having triethylene glycol dimethacry-
late (TEGDMA) in it. P60 is cytotoxic but less cyto-
toxic than Z100 probably due to no TEGDMA in it.
Durelon is the most cytotoxic among the six materials
studied probably due to the high cytotoxicity of zinc
ions. Additionally, the cytotoxcity of the tested mate-
rials was found to be dose-dependent.
Keywords: In Vitr o Cytotoxicity; Human Pulp Cells;
3T3 Mouse Fibroblast Cells; Dental Cement;
Glass-Ionomer Cement; Resin Composite
1. INTRODUCTION
It has been shown that components of dental restoratives
can be released into the oral cavity [1-4] and cause ad-
verse effects such as mucosal irritation, epithelial prolif-
eration, oral lichenoid reaction, hypersensitivity and
anaphylactoid reactions [5]. The released components
from polymerized resin-based dental materials include
residual or unreacted monomers, initiators, activators
and other additives [3,6,7]. The resin-based dental mate-
rials include resin composites [8-10], dental bonding
agents [11], resin-modified glass-ionomer cements (RM-
GICs) [3], conventional GICs (CGICs) [1-3], and other
dental cements [12]. Among all these dental restoratives,
CGICs are considered to be one of the most biocompati-
ble restoratives [3,12]. On the other hand, RMGICs are
less biocompatible due to release of unreacted mono-
mers and other components [3]. Dental resin composites,
a current substitute for dental amalgam, are applied in
posterior cavity filling (stress-bearing sites), anterior
teeth repair and core-building up restoration, due to their
high-strength and high-wear-resistant nature [13-15].
Their biocompatibility is somehow still in question and
debate due to release of unreacted monomers, oligomers
and other low molecular weight components.
There are many ways to conduct a biocompatibility or
cytotoxicity test [12,13]. In vitro cytotoxicity tests, a
screening test, are efficient and relatively inexpensive to
conduct although they are not as accurate as in vivo an-
imal usage tests [12,13]. Cell culture studies are fre-
quently used to assess the in vitro cytotoxicity of re-
sin-based materials and their elutes or components such
as monomers, oligomers and additives [6,16]. So far
there have been numerous publications regarding in vitro
cytotoxicity of various dental materials [12,17]. Some of
them have been focused on evaluating the cytotoxicity of
the pure monomers and oligomers [12,17] and the other
on testing the cytotoxicity of the eluates of the materials
[7,8,12,17,18]. All the published results have made dif-
ferent contributions to the area of biocompatibility of
dental restoratives [5,12,17,18].
The objective of this study was to evaluate the in vitro
cytotoxicity of six contemporary commercially available
dental restoratives on human primary cells by testing the
eluates using WST-1 assay. The 3T3 mouse fibroblast
cell lines were evaluated for comparison. The effect of
the eluate concentration on the cytotoxicity was studied
as well.
2. MATERIALS AND METHODS
2.1. Materials
Fuji II (conventional glass-monomer cement) and Fuji II

J. Sun et al. / J. Biomedical Science and Engineering 4 (2011) 18-28
19
LC (light-cured glass-monomer cement) were used as
received from GC America Inc (Alsip, IL). Durelon
(zinc polycarboxylate cement), Vitremer (light-cured
glass-monomer cement), Filtek Z100 (light-cured com-
posite resin) and Filtek P60 (light-cured composite resin)
were purchased from 3M ESPE (St. Paul, MN). The de-
tailed information regarding the materials and their
compositions are described in Table 1.
2.2. Preparation of Specimens
Cylindrical specimens of the materials used in this study
were prepared at room temperature in glass tubing molds
with dimensions of 4 mm in diameter by 2 mm in length
[19]. A two-component (glass powder and liquid) system
for Fuji II, Fuji II LC, Vitremer and Durelon and a single
syringe (paste) system for Z100 and P60 were used and
their specimens were prepared per manufacturers’ in-
structions. For Fuji II and Durelon, specimens were pre-
pared by thoroughly mixing glass powder and polymer
liquid at a ratio of 2.7 and 2.0, respectively, followed by
placing in the mold, conditioning in 100% humidity for
15 min, removing from the mold and immediately steril-
izing with 70% alcohol. For Fuji II LC and Vitremer,
specimens were prepared by thoroughly mixing glass
powder and polymer liquid at a ratio of 3.2 and 2.5, re-
spectively, followed by placing in the mold, exposing to
blue light (EXAKT 520 Blue Light Polymerization Unit,
9W/71, power = 30, WGmbH, Germany) for 2 min,
conditioning in 100% humidity for 15 min, removing
from the mold and immediately sterilizing with 70%
alcohol. For Z100 and P60, specimens were prepared by
placing the premixed paste from the product syringe into
the mold, followed by exposing to blue light for 2 min,
conditioning in 100% humidity for 15 min, removing
from the mold and immediately sterilizing with 70%
alcohol.
2.3. Preparation of Eluates of the Test Materials
Immediately after removing form the molds, the speci-
mens were quickly rinsed with 70% ethanol and sterile
phosphate buffer saline (PBS), followed by immersing in
a 48-well plate containing 300 μl serum minus DMEM
(Dulbecco’s modified Eagle’s medium or DMEM, Hy-
clone Laboratories, Inc. Logan, UT) in a humidified in-
cubator at 37˚C with 5% CO2 and 95% air for 1, 3 and 7
days, for preparation of eluates. The surface area to vo-
lume ratio was 1 cm2/ml, which was set according to the
ISO standards (0.5-6.0 cm2/ml) [20]. Five specimens of
each material for every eluate preparation were prepared
and used for statistical analysis.
2.4. Cell Culture Preparation
Human pulp cells (HPCs) were isolated from the pulp
tissue of healthy young permanent teeth undergoing or-
thodontic treatment, following the published protocol
[21]. Briefly, the extracted teeth were cleaned consecu-
tively with sterile PBS, 70% ethanol and PBS, followed
by cutting to obtain the pulp tissues. The tissues were
then placed in a culture dish and minced to small pieces.
Balb/c 3T3 mouse fibroblast cells were obtained directly
from the American Type Culture Collection (Manassas,
VA ) .
Either HPCs or 3T3 cells were then cultured at 37°C
in an air atmosphere containing 5% CO2 and 95% rela-
tive humidity, with DMEM containing low glucose, sup-
plemented with 10% heat-inactivated fetal bovine serum
(Hyclone Laboratories), 4 mM L-glutamine (Hyclone
Laboratories), 100 U/ml penicillin (Sigma-Aldrich, St.
Louis, MO), 50 μg/ml gentamicin (Invitrogen Life
Technologies, Carlsbad, CA) and 2.5 μg/ml amphotericin
B fungizone (Lonza,Walkersville, MD). The HPCs
which grew out of the pulp explants were sub-cultured
Table 1. Information related to the materials used in this study.
Material Type Setting Mode Liquid Composition1 (by weight) Glass Composition (by volume)
Durelon Polycarboxylate cementChemically cured PAA, water Zinc oxide (most)/zinc
fluoride powder
Fuji II Conventional GIC Chemically cured PAAIA, water, tartaric acid Ca-AI-F silicate glass powder
Fuji II LC Resin-modified GIC Light-cured TEGDMA, HEMA, PAA, water, CQ, DMAEMA Sr-AI-F silicate glass powder
Vitremer Resin-modified GIC Light-cured HEMA, PAA-g-IEM, water, tartaric acid, CQ, DC,
K2S2O8, ascorbic acid Al-F silicate glass powder
P60 Composite resin Light-cured BisGMA, UDMA, BisEMA, CQ, DMAEMA 61% ZrO2-SiO2 filler
Z100 Composite resin Light-cured BisGMA, TEGDMA, CQ, DMAEMA 66% ZrO2-SiO2 filler
1PAA = poly(acrylic acid), PAAIA = poly(acrylic acid-co-itaconic acid), TEGDMA = triethylene glycol dimethacrylate, HEMA = 2-hydroxyethyl methacrylate,
CQ = camphorquinone, DMAEMA = N,N-dimethylaminoethyl methacrylate, PAA-g-IEM = poly(acrylic acid) grafted with 2-isocyanatoethyl methacrylate, DC =
diphenyliodonium chloride, BisGMA = Bisphenol A glycidyl dimethacrylate, BisEMA = Bisphenol A polyethylene glycol diether dimethacrylate, and UDMA =
rethane dimethacrylate. u

J. Sun et al. / J. Biomedical Science and Engineering 4 (2011) 18-28
20
and maintained. The HPCs used for this study were tak-
en between passage 3 and 8.
2.5. Evaluation of Cytotoxicity Using WST-1
Assay
The water soluble tetrazolium salt-1 (WST-1) test was
performed as described elsewhere [22,23]. Briefly, the
cells were plated in a 96-well plate at 2 × 103 cells per
well in 100 μl of DMEM supplemented with 10% FBS,
100 U/ml penicillin and 100 μg/ml streptomycin. After
incubation at 37˚C overnight, the medium was replaced
with 100 μl of the fresh medium containing different
concentrations of eluate (0%, 10%, 20%, 40%, 60% and
80%). The cells were then incubated for 72 h before the
WST testing. The positive control was serum minus
DMEM with untreated cells and the negative control was
serum minus DMEM without cells. The WST-1 test was
conducted by adding 10 µl of WST-1 reagent (Roche
Diagnostics, Indianapolis, IN) and 90 μl of serum minus
DMEM into a well and then incubating the plate at 37˚C
for 2 h. The absorbance of the solution was measured at
450 nm using a microplate reader (Molecular devices,
Sunnyvale, CA). Cell viability (%) was obtained by the
equation: cell viability (%) = (absorbance of the sample
elaute-absorbance of the negative control)/(absorbance
of the positive control-absorbance of the negative con-
trol) × 100%. Cell morphology was obtained at 100X
magnification using Nikon eclipse TS100 microscope
(Nikon Corp., Japan).
2.6. Statistical Analysis
One-way analysis of variance (ANOVA) with the post
hoc Tukey-Kramer multiple range test was used to de-
termine significant differences of in vitro cytotoxicity
among the materials. A level of α = 0.05 was used for
statistical significance.
3. RESULTS
Figure 1(a) shows the HPC viability after the cells were
cultured with the eluates of the six materials tested at a
concentration of 80%. Fuji II and Fuji II LC showed the
highest cell viability, respectively, after cell exposure to
1-day and 3-day eluates. Durelon showed the lowest
viability. The viability (%) was in the decreasing order:
(1) for the 1-day eluate, Fuji II (100.3 ± 6.3) > Fuji LC
(88.0 ± 11) > P60 (54.2 ± 7.2) > Vitremer (37.9 ± 3.8) >
Z100 (12.4 ± 2.7) > Durelon (0.19 ± 0.3), where Z100
and Durelon, Fuji LC and Fuji II, and Vitremer and P60
were not significantly different from each other (p >
0.05); (2) for the 3-day eluate, Fuji LC (105.9 ± 10.3) >
Fuji II (98.8 ± 7.8) > P60 (50.7 ± 3.6) > Vitremer (27.1 ±
5.3) > Z100 (6.82 ± 3.7) > Durelon (0.39 ± 0.9), where
Z100 and Durelon as well as Fuji LC and Fuji II were
not significantly different from each other (p > 0.05).
Figure 1(b) shows the 3T3 fibroblast viability after
the cells were cultured with the eluates of the six materi-
als tested at a concentration of 80%. Fuji II showed the
highest cell viability but Z100 showed the lowest viabil-
ity. The viability (%) was in the decreasing order: 1) for
the 1-day eluate, Fuji II (99.4 ± 2.3) > P60 (64.9 ± 8.4) >
Fuji LC (53.6 ± 2.9) > Durelon (2.63 ± 2.6) > Vitremer
(0.78 ± 0.7) > Z100 (0.47 ± 1.9), where Vitremer, Z100
and Durelon were not significantly different from one
another (p > 0.05); 2) for the 3-day eluate (Figure 2(b)),
Fuji II (100.8 ± 5.5) > P60 (70.4 ± 6.1) > Fuji LC (68.0 ±
3.3) > Vitremer (4.89 ± 0.3) > Durelon (3.15 ± 0.4) >
Z100 (0.07 ± 2.3), where Vitremer, Z100 and Durelon as
well as Fuji LC and P60 were not significantly different
from each other (p > 0.05).
(a)
(b)
Figure 1. Cell viability comparison after cultured with the
eluates from different cements for 72 h; (a) HPC viability
comparison; (b) 3T3 fibroblast viability comparison. Eluates
were obtained from the 1-day and 3-day incubation at a con-
centration of 80%.

J. Sun et al. / J. Biomedical Science and Engineering 4 (2011) 18-28
21
Figures 2(a) and (b) show the HPC viability vs. elu-
ate concentration at the 1-day and 3-day extractions,
respectively. For the 1-day eluate, Fuji II showed no
cytotoxicity at all; Fuji II LC showed nearly no cyto-
toxicty; Vitremer, P60, Z100 and Durelon started to
show the cytotoxicty, respectively, at a concentration of
20, 80, 40 and 40%, with the viability values of 80, 54,
66 and 4.8%. For the 3-day eluate, both Fuji II and Fuji
II LC showed no cytotoxicity; Vitremer, P60, Z100 and
Durelon started to show the cytotoxicty, respectively, at
a concentration of 20, 40, 10 and 40%, with the viability
values of 70, 68, 85 and 0.5%.
Figures 3(a) and (b) show the 3T3 fibroblast viability
vs. eluate concentration at the 1-day and 3-day extrac-
tions, respectively. For the 1 day eluate, Fuji II showed
(a)
(b)
Figure 2. HPC viability (%) vs. cement eluate concentration:
(a) eluates obtained from the 1-day incubation; (b) eluates
obtained from the 3-day incubation. The cells were incubated
with the medium containing different concentrations of the
eluates at 37˚C for 72 h before WST-1 testing.
(a)
(b)
Figure 3. 3T3 fibroblast viability (%) vs. cement eluate con-
centration: (a) eluates obtained from the 1-day incubation; (b)
eluates obtained from the 3-day incubation. The cells were
incubated with the medium containing different concentrations
of the eluates at 37°C for 72 h before WST-1 testing.
no cytotoxicity; Fuji II LC, Vitremer, P60, Z100 and
Durelon started to show the cytotoxicty, respectively, at
a concentration of 60, 20, 60, 10 and 20%, with the vi-
ability values of 82, 70, 77, 62 and 36%. For the 3-day
eluate, Fuji II showed no cytotoxicity at all. Fuji II LC,
Vitremer, P60, Z100 and Durelon started to show the
cytotoxicty, respectively, at a concentration of 20, 10, 60,
10 and 10%, with the viability values of 87, 79, 74, 37
and 85%.
Figure 4 is a set of optical photomicrographs de-
scribing the HPC morphology after contact with the cor-
responding 3-day eluate. Figures 4(a-g) represent the
HPC morphology after cultured with blank, Fuji II, Fuji
II LC, Vitremer, P60, Z100 and Durelon, respectively. In
Figures 4(a) (control) and (b) (Fuji II), numerous healthy

J. Sun et al. / J. Biomedical Science and Engineering 4 (2011) 18-28
22
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 4. HPC morphology and density (100X magnification):
(a) HPC; (b) Fuji II; (c) Fuji II LC; (d) Vitremer; (e) P60; (f)
Z100; (g) Durelon. Cell morphology photomicrograph was
obtained after the cells incubated with the 3-day eluates for
72 h.
cells with an elongated and spindle shape (typical HPC
morphology) are observed. In Figure 4(c) (Fuji II LC)
and e (P60), some small black round spots (dead or un-
healthy cells) are observed although there still exist
many elongated and spindle shaped cells. In Figures 4(d)
(Vitremer), (f) (Z100) and (g) (Durelon), large black spots
(condensed irregular nuclei of the dead cells) are clearly
seen and the intact cells disappeared. Furthermore, the

J. Sun et al. / J. Biomedical Science and Engineering 4 (2011) 18-28
23
lysed cell pieces are found in Figure 4(g)
Figure 5 is a set of optical photomicrographs de-
scribing the 3T3 cell morphology after contact with the
corresponding 3-day eluate. In Figures 5(a) (control)
and (b) (Fuji II), numerous cells with a multipolar shape
(typical 3T3 cell morphology) are observed. In Figure
5(c) (Fuji II LC) and e (P60), black spots (dead cells)
and deformed 3T3 cells are clearly seen, although the
multipolar-shaped cells are still noticed. In Figure 5(d)
(Vitremer) and f (Z100), nearly no multipolar-shaped
cells are observed except for the round dead cells. In
Figure 5(g), the cells are found to be significantly lysed
and different sizes of black spots or lysed pieces are no-
ticed everywhere.
4. DISCUSSION
Biocompatibility of dental restoratives is very important
in dentistry [1-3]. Non-biocompatible or cytotoxic mate-
rials can cause short-term or long-term tissue inflamma-
tion or cell death [1-5]. Regarding dental cavity filling
restoratives, the most critical concern for biocompatibil-
ity is the cytotoxcity caused by the leachable compo-
nents such as unreactive monomers, initiators and other
additives form the organic resins or cytotoxic metal ions
from the inorganic fillers. These leachable components
can penetrate into pulp chamber through dental tubules
and cause pulp inflammation [1,5]. In this study, we
evaluated six contemporary commercial dental filling
restoratives including Fuji II, Fuji II LC, Vitremer, P60,
Z100 and Durelon. Their compositions are shown in
Tab le 1 [24-30]. It is known that chemistry and poten-
tially leachable components ultimately determine the
cytotoxicity of the filled restoratives [1-4].
4.1. Chemistry Involved in the Setting Reactions
of the Tested Materials and Potential
Leachable Species
4.1.1. Durel on
Durelon is a chemically-cured dental luting cement. It is
composed of zinc oxide, zinc fluoride, polycarboxylic
acid and water. An acid-base reaction between zinc ca-
tions released from a ZnO/ZnF2 glass and carboxyl ani-
ons pendent on polycarboxylic acid describes the setting
reaction mechanism in Durelon [13,31]. During the set-
ting reaction, with the help of water the surface of the
ZnO/ZnF2 glass particles reacts with the carboxyl groups
pendent from polycarboxylate to form zinc carboxylate
salt-bridges and hardens the cement. It is known that not
all the glass particles participate in the setting reaction
[13]. Therefore, the unreacted zinc cations can leach out
of the cement. The polycarboxylic acid is hardly leach-
able due to its high molecular weight (MW) [3,13].
(a)
(b)
(c)
(d)

J. Sun et al. / J. Biomedical Science and Engineering 4 (2011) 18-28
24
(e)
(f)
(g)
Figure 5. 3T3 fibroblast morphology and density (100X mag-
nification): (a) HPC; (b) Fuji II; (c) Fuji II LC; (d) Vitremer; (e)
P60; (f) Z100; (g) Durelon. Cell morphology photomicrograph
was obtained after the cells incubated with the 3-day eluates
for 72 h.
4.1.2. Fuji II
Fuji II is a chemically-cured glass-ionomer cement (GIC)
used for dental luting and filling purposes. It is com-
posed of calcium aluminofluorosilicate glass powder,
polycarboxylic acid (a copolymer of acrylic acid and
itaconic acid), tartaric acid (TA) and water, where TA is
used for extending the working time. An acid-base reac-
tion between calcium as well as aluminum cations re-
leased from a reactive sintered glass and carboxyl anions
pendent on polyacid describes the setting mechanism in
conventional GIC [32]. During the setting reaction, with
the help of water the surface of the sintered glass parti-
cles reacts with the carboxyl groups pendent on poly-
carboxylate to form three-dimensional aluminum-car-
boxylate/calcium-carboxylate salt-bridges and hardens
the cement. The polymer is hardly leachable. Although
not all the glass particles participate in the setting reac-
tion, the unreacted glass particles do not easily leach out
of the cement because they are sintered [32,33].
4.1.3. Fuj i II L C
Fuji II LC is a light-cured resin-modified GIC (RMGIC)
mainly used for cavity filling and core-building purposes.
It is composed of strontium aluminofluorosilicate glass
powder, 2-hydroxylethyl methacrylate (HEMA), triethyl-
ene glycol dimethacrylate (TEGDMA), poly(acrylic acid),
water, camphorquinone (CQ) and dimethylaminoethyl
methacrylate (DMAEMA), where CQ and DMAEMA
are used for initiating the photo polymerization. Except
for the acid-base reaction similar to that in Fuji II,
HEMA and TEGDMA can copolymerize to form a
crosslinked network via covalent bond formation upon
blue light initiation. This polymer network is somehow
stronger than that formed by salt-bridges in Fuji II, espe-
cially in toughness and tensile strength [13,29]. However,
unreacted HEMA as well as TEGDMA due to the lim-
ited conversion in situ [12] and CQ as well as DMA-
EMA may possibly leach out of the cement. The sintered
glass particles, like the glass in Fuji II, usually do not
easily leach out from Fuji II LC [33].
4.1.4. Vi tre mer
Vitremer is a tri-cured RMGIC mainly used for dental
luting. It contains aluminofluorosilicate glass powder,
HEMA, poly(carboxylic acid) with pendent methacrylate
groups, TA, water, potassium persulfate (K2S2O8), as-
corbic acid, CQ and diphenyliodonium chloride (DC),
where TA, K2S2O8, ascorbic acid, CQ and DC are used
for adjusting the working time and initiating the redox as
well as photo polymerizations. Except for the acid-base
and photo polymerization reactions shown in Fuji II LC,
there also exists a redox polymerization initiated by a
pair of redox initiators K2S2O8 and ascorbic acid. The
photo-activator used in Vitremer is also different from
that used in Fuji II LC, i.e., Vitremer uses DC but Fuji II
LC uses DMAEMA instead [6,29]. Furthermore, Vitre-
mer uses HEMA and polycarboxylic acid with pendent
methacrylate groups to form a crosslinked polymer net-
work via both photo- and redox-initiated polymeriza-
tions [6,29]. Like those in Fuji II and Fuji II LC, the sin-
tered glass particles usually do not leach out [33]. The

J. Sun et al. / J. Biomedical Science and Engineering 4 (2011) 18-28
25
potential leachable components in Vitremer include un-
reacted HEMA, CQ, DC, TA, K2S2O8 and ascorbic acid.
4.1.5. Z100 and P60
Z100 is a light-cured resin composite for cavity filling. It
contains ZrO2-SiO2 fillers, bisphenol A diglycidyl ether
dimethacrylate (BisGMA), TEGDMA, CQ and DMA-
EMA, where CQ and DMAEMA are used for initiating
the photo polymerization. P60 is also a light-cured resin
composite but it is an improved version of Z100. In P60,
except for ZrO2-SiO2 fillers, BisGMA, CQ and DMA-
EMA, TEGDMA is replaced by a mixture of urethane
dimethacrylate (UDMA) and bisphenol A polyethylene
glycol diether dimethacrylate (BisEMA). It is claimed to
have lower shrinkage as well as reduced aging and be
more hydrophobic as well as less sensitive to changes in
atmospheric moisture. Upon the photo initiation the di-
methacrylate oligomers in the formulations lead to for-
mation of the crosslinked polymer networks. Unlike
those in either GICs or zinc polycarboxylate cement, the
glass particles in resin composites are only used as fillers
and do not participate in any chemical reactions. They
are usually inert to cells or tissues [12,13]. The differ-
ence between Z100 and P60 lies in that the former con-
tains TEGDMA but the latter contains UDMA and Bi-
sEMA. Due to higher MWs of UDMA and BisEMA, the
resin liquid in the P60 formulation is more viscous. P60
is also claimed to have lower shrinkage as well as re-
duced aging and be more hydrophobic as well as less
sensitive to changes in atmospheric moisture. The poten-
tial leachable components in Z100 are TEGDMA, CQ
and DMAEMA whereas those in P60 are UDMA, Bi-
sEMA, CQ and DMAEMA. The unreacted BisGMA (if
any) is hardly leachable due to its higher hydrophobicity
and MW.
4.2. In Vitro Cytotoxicty
Two types of cells, HPC and 3T3 fibroblast, were used
to evaluate the cytotoxicity of the six materials. HPCs
were isolated directly from pulp tissues of human teeth
whereas 3T3 mouse fibroblasts were cultured cell lines.
As compared to 3T3 cells, HPCs are more clinically
relevant. In this study the WST-1 assay was used as a
tool to evaluate the cytotoxicity. The WST-1 is a colori-
metric assay based on the cleavage of the water soluble
tetrazolium salt (WST-1) by mitochondrial dehydro-
genases to a yellow-orange formazan and is claimed to
be a more sensitive assay than MTT [23].
From Figures 1(a) and (b), Fuji II was the most bio-
compatible but Durelon was the most cytotoxic. The
results are interpreted below with the help of the compo-
sitions discussed in the previous section. For Fuji II, this
cement simply consists of a sintered calcium alu-
minofluorosilicate glass powder, polycarboxylic acid, TA
and water. During the initial setting, the Al3+ and Ca2+
react with –COO– groups. Although there may be a very
small amount of Al3+ and Ca2+ ions leached, it would not
cause any significant cytotoxicity [33]. Meanwhile, there
may be some leachable polycarboxylic acid and TA [34];
However, these acidic species would not cause any sig-
nificant cytotoxicity as well because the tested speci-
mens were immersed in DMEM where these acidic spe-
cies could be buffered. That is why Fuji II showed no
cytotoxicity at all to both cells. For Fuji II LC, this ce-
ment showed nearly no cytotoxicity to HPCs but was
cytotoxic to 3T3 cells. Except for the similar compo-
nents shown in Fuji II, Fuji II LC also contains HEMA
(30-60% by weight), TEGDMA (1-5%), CQ and
DMAEMA. Among them, CQ and DMAEMA are con-
sidered to be the least toxic [7] and only 1-2% of them
are included in the formulation [13,29]. It was found that
both HEMA and TEGDMA were cytotoxic and TEG-
DMA was even more cytotoxic than HEMA [6,7].
However, there was only a very small amount of TEG-
DMA in Fuji II LC and most of it was crosslinked with
HEMA. That may be why Fuji II LC showed a better
biocompatibility than the other tested materials except
for Fuji II. Furthermore, by comparing the cell viability
results between HPCs and 3T3 cells, we found that 3T3
cells were more sensitive to HEMA and TEGDMA (if
any) than HPCs. In other words, 3T3 cells responded
more sensitively than HPCs. For Vitremer, we found that
this cement was the most cytotoxic restorative among
the three GICs tested. Except for the components shown
in Fuji II and HEMA as well as CQ shown in Fuji II LC,
Vitremer also contains polycarboxylic acid with pendent
methacrylate groups, K2S2O8, ascorbic acid and DC. DC
was found to be the most cytotoxic component to be
responsible for the cell death in Vitremer and Vitrobond
[29]. Both DC and HEMA are the main possible reason
to cause very low cell viability values [6,7,29]. In addi-
tion, similar to the results for Fuji II LC, HPCs showed
higher viability values than 3T3 cells, indicating that
3T3 cells are more vulnerable to both DC and HEMA.
For Z100, this resin composite contains ZrO2-SiO2 fillers,
BisGMA (approximately 50% by mole), TEGDMA (ap-
proximately 50%), CQ and DMAEMA. Except for CQ
and DMAEMA, both BisGMA and TEGDMA were
found to be very cytotoxic and BisGMA was even more
cytotoxic than TEGDMA when testing in a DMSO/water
mixture [7]. However, since BisGMA is more hydro-
phobic than TEGDMA [7,12] and its MW (MW = 512)
is also higher than that of TEGDMA (MW = 286), the
leaching probability of BisGMA in aqueous solution is
much lower than that for TEGDMA. A substantial
amount of TEGDMA in Z100 should be responsible for
its high cytotoxicity. Furthermore, HPCs seemed less

J. Sun et al. / J. Biomedical Science and Engineering 4 (2011) 18-28
26
vulnerable to Z100 than 3T3 cells. The similar result was
also demonstrated elsewhere [18]. For P60, this resin
composite was not as cytotoxic as Z100. Except for the
same fillers, BisGMA, CQ and DMAEMA present in
Z100, P60 does not contain TEGDMA but contains
UDMA and BisEMA instead [27]. Because both UDMA
(MW = 470) and BisEMA (MW = 540) have higher
MWs and are more hydrophobic than TEGDMA (MW =
286), their mobility and aqueous solubility, respectively,
should be slower and lower than TEGDMA. Although
BisGMA, UDMA and BisEMA were found to be more
cytotoxic in the DMSO/water mixture than TEGDMA
[7,12], TEGDMA is more leachable in aqueous solution
or culture medium than both due to its lower MW and
higher hydrophilicity. That is why P60 showed consid-
erably lower cytotoxicity than Z100. For Durelon, this is
a dental luting cement and its setting chemistry is very
similar to most conventional GICs such as Fuji II. Ex-
cept for polycarboxylic acid and water, Durelon uses
zinc oxide and zinc fluoride as a reactive glass in its
system. Zinc cations are found to be very cytotoxic in
vitro and considered to be a dangerous cations to cells,
unless combining with other cations such as Fe++ or Ca++
[35]. As mentioned earlier, polycarboxylic acid would
not lead to cytotoxcity due to buffering of the culture
medium. However, leachable zinc cations especially
from zinc fluoride can cause significant cytotoxicity to
surrounding cells or tissues. Apparently the zinc-con-
taining cement Durelon showed the highest cytotoxcity
to both cells.
The results from Figures 2 and 3 clearly indicate that
the cytotoxicity of the tested materials was dose-de-
pendent, as reported elsewhere [36]. In the case of HPCs
(Figure 2), Fuji II showed no cytotoxicity at all the elu-
ate concentrations to both 1-day and 3-day extractions.
Fuji II LC showed nearly no cytotoxicity at all the eluate
concentrations. Vitremer started to show the cytotoxicity
with the cell viability of 80% and 70% at 20% and ended
up with 38% and 27% at 80% for the 1-day and 3-day
eluates, respectively, suggesting that at a concentration
of 20% the components in Vitremer started to kill the
cells. P60 started to show the cytotoxcity with the cell
viability of 54% at 80% for the 1-day eluate and 68% at
40% and 51% at 80% for the 3-day eluate, suggesting
that the components in P60 release very slowly. Z100
started to show the cytotoxicity with the viability of 66%
at 40% and 85% at 10% and ended up with 12% and
6.8% at 80%, suggesting that a large quantity of TEG-
DMA in Z100 lead to higher cytotoxicity. Durelon
started to show the cytotoxicity with the viability of
4.8% and 0.5% at 40% and ended up with 0.2% and
0.4% at 80%. The results indicate that HPCs can tolerate
the eluate concentration below 40% in Durelon. How-
ever, once reaching 40%, the cells almost completely
died, suggesting that above a certain concentration
threadshold the zinc ions are deadly species to cells,
unless it can be buffered or combined with other cations
such as Fe++ or Ca++ [35].
Considering 3T3 cells (Figure 3), only Fuji II showed
no cytotoxicity at all the eluate concentrations. Fuji II
LC started to show the cytotoxicity with the cell viability
of 82% at 60% and 86% at 20% and ended up with 54%
and 68% at 80%, for the 1-day and 3-day eluates, re-
spectively. The results suggest that Fuji II LC is selec-
tively cytotoxic to 3T3 cell lines but not to human pri-
mary cells. The results were consistent with those pub-
lished elsewhere [18,19]. Vitremer started to show the
cytotoxicity with the cell viability of 70% at 20% and
80% at 10% and ended up with 4.9% and 0.8% at 80%.
By comparing with HPCs, 3T3 is more vulnerable to
Vitremer. P60 started to show the cytotoxicity with the
cell viability of 77% and 74 % at 60% and ended up with
65% and 70% at 80%. Z100 started to show the cytotox-
icity with the viability of 62% and 37% at 10% and
ended up with 0.5% and 0.1% at 80%. Durelon started to
show the cytotoxicity with the viability of 36% at 20%
and 80% at 10% and ended up with 2.6% and 3.2% at
80%, indicating that 3T3 cells can tolerate the eluate
concentration of Durelon below 10 or 20%. Comparing
with HPCs, 3T3 showed lower tolerance to Durelon.
From the photomicrographs shown in Figures 4 and 5,
it is clear that the results for cell morphology matched
those shown for the cell viability and well explained the
cell viability values described in Figure 1.
5. CONCLUSIONS
In vitro responses of human primary pulp cells (HPCs)
and 3T3 mouse fibroblasts to six contemporary dental
restoratives on HPCs and 3T3 mouse fibroblasts were
tested. Fuji II is not cytotoxic to both cells. Fuji II LC is
not cytotoxic to HPCs but cytotoxic to 3T3 cells. Vitre-
mer is very cytotoxic probably due to having DC and
HEMA in it. Z100 is very cytotoxic probably due to
having a large quantity of TEGDMA in it. P60 is cyto-
toxic but less cytotoxic than Z100 probably due to no
TEGDMA in it. Durelon is the most cytotoxic among the
tested materials probably due to the high cytotoxicity of
zinc ions. It was found that 3T3 cell lines were more
vulnerable to leachable cytotoxic components than pri-
mary HPCs. It was also found that the cytotoxcity of the
tested materials was dose-dependent.
6. ACKNOWLEDGEMENTS
This work was partially sponsored by NIH challenge grant (RC1)

J. Sun et al. / J. Biomedical Science and Engineering 4 (2011) 18-28
27
DE020614.
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