Journal of Biomaterials and Nanobiotechnology, 2011, 2, 267-280
doi:10.4236/jbnb.2011.23034 Published Online July 2011 (http://www.SciRP.org/journal/jbnb)
Copyright © 2011 SciRes. JBNB
267
In Vitro Cellular Responses of Human Dental
Primary Cells to Dental Filling Restoratives
Jun Sun1, Yiming Weng2, Fengyu Song1, Dong Xie2*
1Department of Oral Biology, School of Dentistry, Indiana University, Bloomington, USA; 2Department of Biomedical Engineering,
Purdue School of Engineering and Technology, Indiana University-Purdue University Indianapolis, Indianapolis, USA.
Email: *dxie@iupui.edu
Received March 29th, 2011; revised April 20th 2011; accepted May 8th, 2011.
ABSTRACT
In vitro cytotoxicity of six contemporary commercial dental filling restoratives on human dental primary cells, pulp
cells (HPCs) and human gingival fibroblasts (HGFs), were tested using WST-1 assay. Continuous 3T3 mouse fibroblast
cell lines were used for comparison. The results show that conventional glass-ionomer cement (GIC) Fuji II is not cy-
totoxic to all the cells. Resin-modified GIC (RMGIC) Fuji II LC is not cytotoxic to both HPCs and HGFs but cytotoxic
to 3T3 cells. RMGIC Vitremer and resin composite Z100 are very cytotoxic to all the cells. Resin composite P60 is cy-
totoxic but much less cytotoxic than Z100. Polycarboxylate cement Durelon is the most cytotoxic among the six tested
materials. It was found that continuous 3T3 cell lines were more vulnerable to leachable cytotoxic components than
primary HPCs and HGFs. It was also found that the cytotoxcity of the tested materials was dose-dependent.
Keywords: In Vitro Cytotoxicity, Human Pulp Cells, Human Gingival Fibroblasts, 3T3 Mouse Fibroblast Cells, Dental
Cement, Resin Composite
1. Introduction
The biological compatibility of dental materials is of
paramount importance to avoid or limit pulp tissue irrita-
tion and inflammation as well as surrounding gingival
tissue inflammation as well as allergic contact dermatitis
[1-3]. It has been shown that components of dental re-
storatives can be released into the oral cavity [4] and
cause adverse effects such as mucosal irritation, epithe-
lial proliferation, oral lichenoid reaction, hypersensitivity
and anaphylactoid reactions [5]. The released compo-
nents from polymerized resin-based dental materials in-
clude residual or unreacted monomers, initiators, activa-
tors and other additives [3,6,7]. The resin-based dental
materials include resin composites [9,10], dental bonding
agents [11], resin-modified glass-ionomer cements
(RMGICs) [3], conventional GICs (CGICs) [1-3], and
other dental cements [12]. Among all these dental re-
storatives, CGICs are considered to be one of the most
biocompatible restoratives [3,12]. On the other hand,
RMGICs are less biocompatible due to release of unre-
acted monomers 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]. However, 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 ani-
mal usage tests [12,13]. Cell culture studies are fre-
quently used to assess the in vitro cytotoxicity of resin-
based materials, their elutes, or components (such as mo-
nomers or oligomers) [6,16]. So far there have been nu-
merous 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 useful con-
tributions to the area of biocompatibility of dental re-
storatives [5,12,17,18].
This study reports the evaluation of the in vitro cyto-
toxicity of six commercially available filling materials on
two dental primary cells from human pulp and human
In Vitro Cellular Responses of Human Dental Primary Cells to Dental Filling Restoratives
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268
gingival tissues 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
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 composite
resin) and Filtek P60 (light-cured composite resin) were
purchased from 3M ESPE (St. Paul, MN). The detailed
information regarding the materials and their composi-
tions 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, con-
ditioning 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, con-
ditioning 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
volume ratio was 1 cm2/ml, which was set according to
the ISO standards (0.5 - 6.0 cm2/ml) [20]. Five speci-
mens 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.
Human gingival fibroblasts (HGFs) were cultured from
the gingival connective tissue of clinically healthy human
subjects undergoing crown-lengthening surgery, follow-
ing the published protocol [22]. Briefly, the tissues ob-
tained from the clinics were treated consecutively with
sterile PBS, 70% ethanol and PBS. 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).
All the three cells were then cultured at 37˚C in an air
atmosphere containing 5% CO2 and 95% relative humid-
ity, with DMEM containing low glucose, supplemented
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, Carls-
bad, CA) and 2.5 μg/ml amphotericin B fungizone (Lon-
za,Walkersville, MD). Either HPCs or HGFs which grew
out of the explants were sub-cultured and maintained.
Both HPCs and HGFs used for this study were taken
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 [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 incu-
bation at 37˚C overnight, the medium was replaced with
100 μl of the fresh medium containing different concen-
trations of eluate (0%, 10%, 20%, 40%, 60% and 80%).
The cells were then incubated for 72 h before WST test-
ing. 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, In-
dianapolis, IN) and 90 μl of serum minus DMEM into a
In Vitro Cellular Responses of Human Dental Primary Cells to Dental Filling Restoratives
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269
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 vi-
ability (%) = (absorbance of the sample elaute - absorb-
ance of the negative control)/(absorbance of the positive
control - absorbance of the negative control) × 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 and 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 restora-
tives, the most critical concern for biocompatibility is the
cytotoxcity caused by the leachable components such as
unreactive monomers, residual initiators and other resid-
ual additives form the organic resins or cytotoxic metal
ions from the inorganic fillers. In this study, we evalu-
ated six contemporary dental filling restoratives includ-
ing Fuji II, Fuji II LC, Vitremer, P60, Z100 and Durelon.
Their compositions are shown in Table 1 [24-30]. It is
known that chemistry and potentially leachable compo-
nents ultimately determine the cytotoxicity of the filled
restoratives [1-4]. Let’s first describe the chemistry in-
volved in the setting reactions of the six tested materials
as well as discuss the potential leachable species and then
discuss the results from this research.
3.1. Chemistry Involved in the Setting Reactions
of the Tested Materials and Potential
Leachable Species
3.1.1. Durelon
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
cations released from a ZnO/ZnF2 glass and carboxyl
anions pendent on polycarboxylic acid describes the set-
ting reaction mechanism in Durelon [13,31]. During the
setting 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].
3.1.2. Fuji II
Fuji II is a chemically-cured glass-ionomer cement (GIC)
used for dental luting and filling purposes. It is composed
of calcium aluminofluorosilicate glass powder, polycar-
boxylic 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 reaction be-
tween calcium as well as aluminum cations released 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 particles reacts
with the carboxyl groups pendent on polycarboxylate to
form three-dimensional aluminum-carboxylate/calcium-
carboxylate salt-bridges and hardens the cement. The
polymer is hardly leachable. Although not all the glass
particles participate in the setting reaction, the unreacted
glass particles do not easily leach out of the cement be-
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 curedPAA, water Zinc oxide/zinc fluoride powder
Fuji II Conventional GIC Chemically curedPAAIA, water, tartaric acid Ca-AI-F silicate glass powder
Fuji II LC Resin-modified GIC Light-cured TEGDMA, HEMA, PAA, water, CQ, DMAEMASr-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,Ndimethylaminoethyl 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 = urethane dimethacrylate.
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cause they are sintered [32,33].
3.1.3. Fuji II LC
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 limited
conversion in situ [12] and CQ as well as DMAEMA
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].
3.1.4. Vitremer
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), ascor-
bic acid, CQ and diphenyliodonium chloride (DC), where
TA, K2S2O8, ascorbic acid, CQ and DC are used for ad-
justing 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, Vitremer
uses HEMA and polycarboxylic acid with pendent me-
thacrylate groups to form a crosslinked polymer network
via both photo- and redox-initiated polymerizations [6,
29]. Like those in Fuji II and Fuji II LC, the sintered
glass particles usually do not leach out [33]. The poten-
tial leachable components in Vitremer include unreacted
HEMA, CQ, DC, TA, K2S2O8 and ascorbic acid.
3.1.5. 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
DMAEMA, where CQ and DMAEMA are used for initi-
ating 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
DMAEMA, TEGDMA is replaced by a mixture of ure-
thane dimethacrylate (UDMA) and bisphenol A polyeth-
ylene 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 initia-
tion the dimethacrylate oligomers in the formulations
lead to formation of the crosslinked polymer networks.
Unlike those in either GICs or zinc polycarboxylate ce-
ment, the glass particles in resin composites are only
used as fillers and do not participate in any chemical re-
actions. They are usually inert to cells or tissues [12,13].
The difference between Z100 and P60 lies in that the
former contains TEGDMA but the latter contains UDMA
and BisEMA. Due to higher MWs of UDMA and Bi-
sEMA, the resin liquid in the P60 formulation is more
viscous. P60 is also claimed to have lower shrinkage as
well as reduced aging and be more hydrophobic as well
as less sensitive to changes in atmospheric moisture. The
potential leachable components in Z100 are TEGDMA,
CQ and DMAEMA whereas those in P60 are UDMA,
BisEMA, CQ and DMAEMA. The unreacted BisGMA
(if any) is hardly leachable due to its higher hydropho-
bicity and MW.
3.2. Results and Discussion on in Vitro
Cytotoxicty Testing
Three types of cells, HPCs, HGFs and 3T3 mouse fibro-
blasts, were used to evaluate the cytotoxicity of the six
materials. HPCs and HGFs, respectively, were isolated
directly from pulp tissues of human teeth and gingival
connective tissues of human subjects whereas 3T3 fibro-
blasts were cultured continuous cell lines. As compared
to 3T3 cells, HPCs and HGFs are more clinically rele-
vant. In this study, the WST-1 assay was used as a tool to
evaluate the cytotoxicity. The WST-1 is a colorimetric
assay based on the cleavage of the water soluble tetra-
zolium salt (WST-1) by mitochondrial dehydrogenases to
a yellow-orange formazan and is claimed to be a more
sensitive assay than MTT [23].
Figure 1(a) shows the HPC viability after the cells
were cultured with the eluates of the six materials at a
concentration of 80%. Fuji II and Fuji II LC showed the
highest cell viability, respectively, after cell exposure to
1-, 3- and 7-day eluates. Durelon showed the lowest vi-
ability. 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 II and Fuji II LC, 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 ±
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271
5.3) > Z100 (6.82 ± 3.7) > Durelon (0.39 ± 0.9), where
Z100 and Durelon as well as Fuji II and Fuji II LC were
not significantly different from each other (p > 0.05); 3)
for the 7-day eluate, Fuji II (101.2 ± 11.3) > Fuji II LC
(93.8 ± 2.3) > P60 (55.5 ± 8.5) > Vitremer (20.5 ± 1.6) >
Z100 (3.65 ± 0.8) > Durelon (3.0 ± 0.7), where Fuji II
and Fuji II LC as well as Z100 and Durelon were not
significantly different from each other (p > 0.05)
Figure 1(b) shows the HGF viability at a concentra-
tion of 80%. Fuji II showed the highest cell viability to
all the three eluates. Durelon showed the lowest viability.
The viability (%) was in the decreasing order: 1) for the
1-day eluate, Fuji II LC (88.5 ± 6.2) > Fuji II (84.0 ± 8.3)
> P60 (61.3 ± 12.8) > Z100 (15.9 ± 0.4) > Vitremer (7.0
± 0.1) > Durelon (1.2 ± 2.2), where Fuji II and Fuji II LC
as well as Vitremer, Z100 and Durelon were not signifi-
cantly different from one another (p > 0.05); 2) for the
3-day eluate, Fuji II LC (88.8 ± 11.7) > Fuji II (88.1 ±
8.1) > P60 (57.9 ± 14.8) > Vitremer (11.3 ± 1.3) > Z100
(7.4 ± 2.6) > Durelon (1.7 ± 0.9), where Fuji II and Fuji
II LC as well as Vitremer, Z100 and Durelon were not
significantly different from one another (p > 0.05); 3) for
the 7-day eluate, Fuji II (83.3 ± 9.6) > Fuji II LC (80.7 ±
5.3) > P60 (57.3 ± 4.0) > Vitremer (12.9 ± 3.1) > Z100
(5.1 ± 3.5) > Durelon (1.8 ± 3.0), where Fuji II and Fuji
II LC as well as Vitremer, Z100 and Durelon were not
significantly different from one another (p > 0.05).
Figure 1(c) shows the 3T3 fibroblast viability at a
concentration of 80%. Fuji II showed the highest cell
viability to the three eluates. Z100 showed the lowest
viability. The viability (%) was in the decreasing order: 1)
for the 1-day eluate (Figure 2(a)), Fuji II (99.4 ± 2.3) >
P60 (64.9 ± 8.4) > Fuji II 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 signifi-
cantly 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 II LC (68.0 ± 3.3) > Vitremer (4.89 ±
0.3) > Durelon (3.15 ± 0.4) > Z100 (0.07 ± 2.3), where
Fuji II LC and P60 as well as Vitremer, Z100 and Dure-
lon were not significantly different from each other (p >
0.05); 3) for the 7-day eluate, Fuji II (94.4) > P60 (45.7 ±
4.8) > Fuji II LC (43.1 ± 4.6) > Durelon (2.4 ± 1.7) >
Z100 (0.5 ± 0.023) > Vitremer (0.18 ± 2.1), where Fuji II
LC and P60 as well as Vitremer, Z100 and Durelon were
not significantly different from each other (p > 0.05).
From Figures 1(a)-(c), Fuji II was the most biocom-
patible restorative but Durelon was nearly the most cyto-
toxic one. The results are interpreted below with the help
of the compositions and potential leachable species dis-
cussed in the previous section. For Fuji II, this cement
simply consists of a sintered calcium aluminofluorosili-
cate glass powder, polycarboxylic acid, TA and water.
(a)
(b)
(c)
Figure 1. Cell viability comparison after cultured with the
eluates from different cements for 72 h. (a) HPC viability;
(b) HGF viability; (c) 3T3 fibroblast viability. Eluates were
obtained from the 1-, 3- and 7-day incubation at a concen-
tration of 80%.
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272
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 pos-
sibly cause any significant cytotoxicity [33]. Meanwhile,
there may be some leachable polycarboxylic acid and TA
[34]; however, these acidic species would not possibly
cause any significant cytotoxicity as well because the
tested specimens were immersed in DMEM where these
acidic species could be buffered. That is why Fuji II
showed no cytotoxicity at all to all the cells. For Fuji II
LC, this cement showed nearly no cytotoxicity to HPCs
and HGFs but was cytotoxic to 3T3 cells. Except for the
similar components 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 considered to be the least toxic [7] and
they are only used in 1% - 2% [13,29]. It was found that
both HEMA and TEGDMA were cytotoxic and TEG-
DMA was even more cytotoxic than HEMA [6,7]. How-
ever, since Fuji II LC only contains a very small amount
of TEGDMA and most of it was used to crosslink with
HEMA, Fuji II LC showed a better biocompatibility than
the other tested materials except for Fuji II. Furthermore,
by comparing the cell viability results among HPCs,
HGFs and 3T3 cells, we found that 3T3 cells seemed
more sensitive to HEMA and TEGDMA (if any) than
HPCs and HGFs. In other words, 3T3 cells responded
more sensitively than either HPCs or HGFs. For Vi-
tremer, 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 polycarbox-
ylic acid with pendent methacrylate groups, K2S2O8,
ascorbic acid and DC. DC has been reported to be the
most cytotoxic component which is 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 addition, similar to the
results for Fuji II LC, HPCs and HGFs showed higher
viability values than 3T3 cells, indicating that 3T3 cells
are more vulnerable to both HEMA and DC. For Z100,
this resin composite contains ZrO2-SiO2 fillers, BisGMA
(approximately 50% by mole), TEGDMA (approximately
50%), CQ (approximately 1%) and DMAEMA (approxi-
mately 2%). Except for CQ and DMAEMA, both Bis-
GMA 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 hydrophobic 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. Further-
more, HPCs and HGFs seemed less vulnerable to Z100
than 3T3 cells. The similar result was demonstrated
elsewhere [18]. For P60, this resin composite was found
not to be as cytotoxic as Z100. Except for the 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 Bi-
sEMA (MW = 540) have higher MWs and are more hy-
drophobic than TEGDMA (MW = 286), their mobility
and aqueous solubility, respectively, should be slower
and lower than TEGDMA. Although BisGMA, UDMA
and BisEMA have been found to be more cytotoxic in
the DMSO/water mixture than TEGDMA [7,12], TEG-
DMA is more leachable in aqueous solution or culture
medium than the other two due to its lower MW and
higher hydrophilicity. That is probably why P60 showed
considerably 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.
Except for polycarboxylic acid and water, Durelon uses
zinc oxide and zinc fluoride as a reactive glass in its sys-
tem. Zinc cations have been 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 much cytotoxcity from the cement due to
buffering of the culture medium. However, leachable
zinc cations especially from zinc fluoride could possibly
cause significant cytotoxicity to surrounding cells or tis-
sues. Apparently the zinc-con- taining cement Durelon
showed the highest cytotoxcity to all the cells among all
the six materials.
Figures 2(a) and (b) show the HPC viability vs. eluate
concentration at the 1-day and 7-day extractions, respec-
tively. For the 1-day eluate, Fuji II showed no cytotoxic-
ity at all; Fuji II LC showed nearly no cytotoxicty;
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 7-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 10%, 20%, 10% and 40%, with the vi-
ability values of 59%, 83%, 65% and 1.3%.
Figures 3(a) and (b) show the HGF viability vs. eluate
concentration at the 1-day and 7-day extractions, respec-
tively. For the 1 day eluate, Fuji II and Fuji II LC showed
nearly no cytotoxicity; Vitremer, P60, Z100 and Durelon
started to show the cytotoxicty, respectively, at a concen-
tration of 20%, 60%, 40% and 40%, with the viability
values of 62, 80, 63 and 36%. For the 7-day eluate, Fuji
II showed no cytotoxicity; Fuji II LC showed slight cy-
In Vitro Cellular Responses of Human Dental Primary Cells to Dental Filling Restoratives
Copyright © 2011 SciRes. JBNB
273
(a)
(b)
Figure 2. HPC viability (%) vs. cement eluate concentration.
(a) eluates obtained from the 1-day incubation; (b) eluates
obtained from the 7-day incubation. The cells were incu-
bated with the medium containing different concentrations
of the eluates at 37˚C for 72 h before WST-1 testing.
totoxicty at a concentration of 80 with the viability of
80%; Vitremer, P60, Z100 and Durelon started to show
the cytotoxicty, respectively, at a concentration of 10%,
40%, 10% and 40%, with the viability values of 52%,
70%, 83% and 0.9%.
Figures 4(a) and (b) show the 3T3 fibroblast viability
vs. eluate concentration at the 1-day and 7-day extrac-
tions, respectively. For the 1 day eluate, Fuji II showed
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 viability values of 82%, 70%, 77%, 62% and 36%.
For the 7-day eluate, Fuji II showed no cytotoxicity; Fuji
II LC, Vitremer, P60, Z100 and Durelon started to show
the cytotoxicty, respectively, at a concentration of 20%,
10%, 20%, 10% and 10%, with the viability values of
(a)
(b)
Figure 3. HGF viability (%) vs. cement eluate concentration.
(a) eluates obtained from the 1-day incubation; (b) eluates
obtained from the 7-day incubation. The cells were incu-
bated with the medium containing different concentrations
of the eluates at 37˚C for 72 h before WST-1 testing.
70%, 60%, 79%, 35% and 59%.
The results from Figures 2-4 clearly indicate that the
cytotoxicity of the tested materials was dose- dependent,
as reported elsewhere [36]. In the case of HPCs (Figure
2), Fuji II showed no cytotoxicity at all the eluate con-
centrations with both 1-day and 7-day extractions. Fuji II
LC showed nearly no cytotoxicity at all the eluate con-
centrations. Vitremer started to show the cytotoxicity
with the cell viability of 80% at 20% and 59% at 10%
and ended up with 38% and 21% at 80% for the 1-day
and 7-day eluates, respectively, suggesting that at the
eluate concentration of 20% the leachable species 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 83% at 20% and 55% at 80% for the
7-day eluate, suggesting that the leachable species in P60
In Vitro Cellular Responses of Human Dental Primary Cells to Dental Filling Restoratives
Copyright © 2011 SciRes. JBNB
274
(a)
(b)
Figure 4. 3T3 fibroblast viability (%) vs. cement eluate con-
centration: (a) eluates obtained from the 1-day incubation;
(b) eluates obtained from the 7-day incubation. The cells
were incubated with the medium containing different con-
centrations of the eluates at 37˚C for 72 h before WST-1
testing.
release very slowly. Z100 started to show the cytotoxicity
with the viability of 66% at 40% and 65% at 10% and
ended with 12% and 3.6% at 80%, suggesting that a large
quantity of TEGDMA in Z100 lead to a higher cytotox-
icity. Durelon started to show the cytotoxicity with the
viability of 4.8% and 1.3% at 40% and ended up with
0.2% and 3.0% at 80%. The results indicate that HPCs
can tolerate the eluate concentration below 40% in
Durelon. However, once reaching 40%, the cells almost
completely died, suggesting that above a certain concen-
tration 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].
Regarding HGFs (Figure 3), similar to HPCs, both
Fuji II and Fuji II LC showed nearly no cytotoxicity at all
the eluate concentrations. Vitremer started to show the
cytotoxicity with the cell viability of 62% at 20% and
52% at 10% and ended up with 7% and 13% at 80% for
the 1-day and 7-day eluates, respectively, suggesting that
at the eluate concentration of 10% or 20% the leachable
species in Vitremer started to kill the cells. P60 started to
show the cytotoxcity with the cell viability of 80% at
60% and 70% at 40% and end up with 61% and 57% at
80%, suggesting that the leachable species such as Bi-
sEMA and UGDMA in P60 release very slowly. Unlike
P60, Z100 started to show the cytotoxicity with the vi-
ability of 63% at 40% and 83% at 10% and ended with
16% and 5.1% at 80%, suggesting that a large quantity of
TEGDMA leaching speeds up the cytotoxicity of Z100.
Durelon started to show the cytotoxicity with the viabil-
ity of 36% and 0.9% at 40% and ended up with 1.2% and
1.8% at 80%, indicating that HGFs can tolerate the eluate
concentration of Durelon below 40%. The results for
HFGs showed the same trend as those for HPCs.
Considering 3T3 cells (Figure 4), 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 70% at 20% and ended with 54% and
43% at 80%, for the 1-day and 3-day eluates, respec-
tively. The results suggest that Fuji II LC is selectively
cytotoxic to 3T3 cells or continuous cell lines but not to
human primary cells (see Figures 2 and 3). The results
were consistent with those published elsewhere [18,19].
Vitremer started to show the cytotoxicity with the cell
viability of 70% at 20% and 60% at 10% and ended with
0.8% and 0.2% at 80%. By comparing with HPCs and
HGFs, 3T3 is more vulnerable to Vitremer. P60 started to
show the cytotoxicity with the cell viability of 77% at
60% and 79% at 20% and ended with 65% and 46% at
80%. Z100 started to show the cytotoxicity with the vi-
ability of 62% and 35% at 10% and ended with 0.5% and
0.5% at 80%. Durelon started to show the cytotoxicity
with the viability of 36% at 20% and 59% at 10% and
ended up with 2.6% and 2.5% at 80%, indicating that
3T3 cells can tolerate the eluate concentration of Durelon
below 10% or 20%. Comparing with HPCs and HGFs,
3T3 showed lower tolerance to Durelon.
Figure 5 is a set of optical photomicrographs describ-
ing the HPC morphology after contact with the corre-
sponding 7-day eluate. Figures 5(a)-(g) represent the
HPC morphology after cultured with blank, Fuji II, Fuji
II LC, Vitremer, P60, Z100 and Durelon, respectively. In
Figure 5(a) (control) and (b) (Fuji II), numerous healthy
cells with an elongated and spindle shape (typical HPC
morphology) are observed. In Figure 5(c) (Fuji II LC)
and (e) (P60), some small black round spots (dead or
unhealthy cells) are observed although there still exist
many elongated and spindle shaped cells. In Figure 5(d)
In Vitro Cellular Responses of Human Dental Primary Cells to Dental Filling Restoratives
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(a) (b)
(c) (d)
(e) (f)
(g)
Figure 5. 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 7-day eluates for 72 h.
In Vitro Cellular Responses of Human Dental Primary Cells to Dental Filling Restoratives
Copyright © 2011 SciRes. JBNB
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(a) (b)
(c) (d)
(e) (f)
(g)
Figure 6. HGF 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 7-day eluates for 72 h.
In Vitro Cellular Responses of Human Dental Primary Cells to Dental Filling Restoratives
Copyright © 2011 SciRes. JBNB
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(a) (b)
(c) (d)
(e) (f)
(g)
Figure 7. 3T3 fibroblast 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 7-day eluates for 72 h.
In Vitro Cellular Responses of Human Dental Primary Cells to Dental Filling Restoratives
Copyright © 2011 SciRes. JBNB
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(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
lysed cell pieces are found in Figure 5(g).
Figure 6 is a set of optical photomicrographs describ-
ing the HGF morphology after contact with the corre-
sponding 7-day eluate. The results are very similar to
those shown in Figure 5, except that the healthy HGFs
look more elongated than healthy HPCs. Furthermore,
more lysed cell pieces are observed in Figure 6(g).
Figure 7 is a set of optical photomicrographs describ-
ing the 3T3 cell morphology after contact with the cor-
responding 3-day eluate. In Figures 7(a) (control) and (b)
(Fuji II), numerous cells with a multipolar shape (typical
3T3 cell morphology) are observed. In Figures 7(c) (Fuji
II LC) and (e) (P60), black spots (dead cells) and de-
formed 3T3 cells are clearly seen, although the multipo-
lar-shaped cells are still noticed. In Figures 7(d)
(Vitremer) and (f) (Z100), nearly no multipolar-shaped
cells are observed except for the round dead cells. In Fig-
ure 7(g), the cells are found to be significantly lysed and
different sizes of black spots or lysed pieces are noticed
everywhere.
From the above photomicrographs, it is clear that the
results for cell morphology matched the results shown
for the cell viability and well explained the cell viability
values described in Figure 1.
4. Conclusions
In vitro cytotoxicity of six commercial dental filling re-
storatives on human pulp cells (HPCs) and human gingi-
val fibroblasts (HGFs) were tested using WST-1 assay.
Continuous 3T3 mouse fibroblast cell lines were used for
comparison. The results show that conventional glass-
ionomer cement (GIC) Fuji II is not cytotoxic to all the
cells. Resin-modified GIC (RMGIC) Fuji II LC is not
cytotoxic to both HPCs and HGFs but cytotoxic to 3T3
cells. RMGIC Vitremer and resin composite Z100 are
very cytotoxic to all the cells. Resin composite P60 is
cytotoxic but much less cytotoxic than Z100. Dental ce-
ment Durelon is the most cytotoxic among the six tested
materials. It was found that continuous 3T3 cell lines
were more vulnerable to leachable cytotoxic components
than human primary HPCs and HGFs. It was also found
that the cytotoxcity of the tested materials was dose-de-
pendent. This study reports the in vitro responses of hu-
man primary pulp and gingival cells as well as continu-
ous 3T3 mouse fibroblasts to six commercial dental fill-
ing restoratives. The in vivo tissue responses in animals
or human to these materials might be different. Therefore,
it would be necessary if some in vivo animal study could
be done with these restoratives before making a final
conclusion, i.e., whether these materials are in vivo cy-
totoxic or in other words whether the tissues are tolerable
to the leachable components in vivo.
5. Acknowledgements
This work was partially sponsored by NIH challenge
grant (RC1) DE020614.
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