d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 1205–1210

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Effects of growth factors on dental pulp

cell sensitivity to amalgam toxicity

Smita Cabrera a , Doug Barden a , Megan Wolf b , Doug Lobner b,∗

a Department of Orthodontics, Marquette University Dental School, Milwaukee, WI, United States
b Department of Biomedical Sciences, Marquette University, Milwaukee, WI, United States

a r t i c l e i n f o a b s t r a c t

Article history: Objectives. The objective was to determine the effects of growth factor treatment on dental
Received 8 May 2006 pulp cell sensitivity to toxicity.
Received in revised form Methods. The toxicity of zinc-containing and zinc-free dental amalgam was tested on four
8 October 2006 types of cells; glia, neurons, embryonic stem cells, and dental pulp cells. The effects of six
Accepted 15 November 2006 different growth factors were tested on dental pulp cell sensitivity to amalgam toxicity.
Results. Zinc-containing amalgam was highly toxic to all cell types tested. Zinc-free amalgam
was less toxic, but it was most toxic to dental pulp cells. Exposure of dental pulp cells to the
Keywords: growth factors IGF-I or BMP-7 had no effect on their morphology or rate of cell division, and
Dental pulp did not alter their sensitivity to zinc-free amalgam toxicity. Exposure to EGF, bFGF, or TGF-␤
Amalgam altered the morphology and decreased the rate of cell division, and the cells were no longer
Toxicity sensitive to zinc-free amalgam toxicity. Exposure to BMP-2 also altered the morphology and
Pulp capping decreased the rate of cell division, but the cells remained sensitive to zinc-free amalgam
IGF-I toxicity.
BMP-2 Significance. The results indicate that untreated dental pulp cells are highly sensitive to amal-
BMP-7 gam toxicity, but that sensitivity can be decreased by exposure to certain growth factors.
EGF Therefore, for treatment of conditions in which pulp cells may come in contact with sub-
bFGF stances released from dental materials, such as pulp capping, the use of amalgam should
TGF-␤ be avoided unless the pulp cells are first treated with the proper growth factors.
© 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

1. Introduction stimulate this dentin formation has been widely performed.

Exposure of dental pulp to the oral environment can be the
result of trauma, rapidly progressing caries, or overly aggres-
sive restorative procedures. Such exposure left untreated may
result in either extraction or endodontic therapy. Treatment
options to attempt to save the dental pulp include pulp cap-
ping and pulpotomy procedures. These procedures typically
involve attempts to stimulate the formation of a dentin bridge
over the exposed pulp. The use of calcium hydroxide to
However, the rate of success has been highly variable. When
dental pulp is minimally exposed, a direct pulp cap is placed
and then the restoration, the success rate has been shown to
be only approximately 13% after 10 years [1]. Other studies
have shown higher success rates, particularly in the treat-
ment of traumatically fractured teeth [2], but it is safe to say
that there has been controversy concerning the effectiveness
of pulp capping procedures [3]. Also, the lack of uniform cri-
teria for determining success or failure has led to confusion

∗
Corresponding author at: Department of Biomedical Sciences, Marquette University, 561N. 15th Street, Rm 426, Milwaukee, WI 53233,
United States. Tel.: +1 414 288 6569; fax: +1 414 288 6564.
E-mail address: Doug.Lobner@marquette.edu (D. Lobner).
0109-5641/$ – see front matter © 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.dental.2006.11.003
1206 d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 1205–1210
regarding the effectiveness of vital pulp therapies [4]. A further
complication of treatment involving calcium hydroxide is that
if this therapy fails subsequent endodontic therapy is difficult
due to calcification in the root canal [5]. An alternative to the
use of calcium hydroxide, or other similar materials, to stim-
ulate dentin formation is the use of growth factors. Growth
factors can stimulate odontoblast differentiation and dentin
formation [6]. However, before growth factor treatments can
be fully implemented, further studies need to be conducted
to understand in detail the effects of growth factors on pulp
cells and how they may alter their sensitivity to the potential
toxicity of dental materials.
Another possible situation in which dental material may
come in contact with pulp cells is the potential use of dental
pulp stem, or progenitor, cells for the re-growth of odon-
toblasts. Stem cells from dental pulp have been isolated
and characterized [7,8]. It has been proposed that stem cells
derived from dental pulp could be guided into differentiat-
ing into odontoblasts [9]. However, if such a procedure was
attempted a restoration would still be required. The same
questions regarding which growth factors to use and the sen-
sitivity of the cells to dental material toxicity would apply to
this situation as they did for pulp capping.
The interaction between dental pulp and dentin is com-
plex. Odontoblasts produce predentin composed of collagen
fibers embedded in a matrix of phosphoprotein and gly-
cosaminoglycans. The predentin is later mineralized at its
border with previously mineralized dentin. Infection of the
dental pulp can occur due to exposure of the dental pulp to the
oral environment or through the dentinal tubules [10]. Infec-
tion and inflammation can lead to pulpal necrosis. However,
these processes can also stimulate recruitment of odonto-
blasts to the site of injury where they can mediate dentin
repair.
In order to determine the least cytotoxic material to use
in situations where it may come in contact with pulp cells it
would be useful to know the toxicity of dental materials to
the dental pulp cells, both in their differentiated and undif-
ferentiated states. We have shown previously that amalgam
containing zinc is toxic to fetal murine neuronal cells and this
toxicity is primarily from the zinc [11]. The comparative toxi-
city of amalgam with or without zinc on the dental pulp cells
is unknown.
Growth factors are proteins that act as signaling molecules
between cells by binding to receptors on the target cell surface,
causing cellular proliferation, differentiation or maturation
[12]. The effects of a number of growth factors on dental pulp
cells are tested in this study. The growth factors were chosen
because of their varying effects on dental pulp cells. IGF-I is
produced in response to growth hormone and induces sub-
sequent cellular alterations and is involved in the formation
of hard tissue like bone and teeth. IGF-I also enhances repar-
ative dentinogenesis in comparison to controls [13]. BMP-7
can induce the formation of reparative dentin [14]. EGF is a
mitogenic protein that stimulates proliferation and differen-
tiation of a wide variety of cell types. The receptor for EGF has
been shown to be present in differentiating odontoblasts [15].
BasicFGF has been associated with tooth morphogenesis by
contributing to the mineralization of dentin [16]. Specifically,
bFGF has been shown to have the capability to promote the dif-
ferentiation of dental pulp cells to odontoblasts in culture [17].
Also, bFGF has been shown to play a role in dental pulp devel-
opment and maturation [18]. TGF-␤ has been shown to induce
differentiation of odontoblast-like cells and to stimulate pri-
mary odontoblasts [19] and BMP-2 can induce differentiation
of odontoblasts [20].
The aim of the present study was to determine the in
vitro effects of dental amalgam on mouse glial and neu-
ronal cells, mouse embryonic stem cells, and human dental
pulp cells. The experiments involved determining if zinc-
containing amalgam is more toxic to the dental pulp cells
than zinc-free amalgam and compare the sensitivity to amal-
gam toxicity of dental pulp cells to other cell types. We also
tested the effects of the growth factors, insulin-like growth
factor-I (IGF-I), bone morphogenic peptide-7 (BMP-7), epider-
mal growth factor (EGF), basic fibroblast growth factor (bFGF),
transforming growth factor-␤ (TGF-␤), and bone morphogenic
peptide-2 (BMP-2) on dental pulp cells to determine if they
alter the cells sensitivity to amalgam toxicity.
2. Materials and methods
2.1. Materials
Timed, pregnant, Swiss Webster mice were obtained from
Charles River Laboratories (Wilmington, DE, USA). Serum was
obtained from Life Technologies (Gaithersburg, MD, USA).
Amalgam that contained zinc (Dispersalloy; composition—
silver: 34.8%; tin: 9.0%; copper: 5.9%; zinc: 0.5%; mercury:
49.8%) and amalgam that did not contain zinc (Megalloy EZ;
composition—silver: 32.0%; tin: 16.2%; copper: 8.3%; mercury:
43.5%) were obtained from Dentsply (Milford, DE, USA). BMP-
2,7 were obtained from R&D systems (Minneapolis, MN, USA).
All other chemicals were obtained from Sigma (St. Louis, MO,
USA).
2.2. Subjects and human dental pulp cell cultures
Normal human impacted third molars were collected from
adults at the Marquette University School of Dentistry Surgical
Services Department under a protocol approved by the Insti-
tutional Review Board at Marquette University. Tooth surfaces
were cleaned and cut around the cementum–enamel junction
by using sterilized diamond stones to access the pulp cham-
ber. The pulp tissue was separated from the tooth and digested
in a solution of 3 mg/ml collagenase type I and 4 mg/ml dis-
pase for 1 h at 37 ◦ C [7]. The cells were then plated onto
24-well plates coated with poly-d-lysine and laminin in Eagle’s
medium supplemented with 20% fetal calf serum/100 ␮M
l-ascorbic acid 2-phosphate/2 mM l-glutamine/100 units/ml
penicillin/100 ␮g/ml streptomycin, and then incubated at 37 ◦ C
with 5% CO2 . Experiments were performed on cultures 7–9
days in vitro.
2.3. Mouse cell cultures (glial, pure neuronal, and
embryonic stem cell)
Cortical cell cultures were prepared from fetal (15–16 days’
gestation) mice as previously described [21]. Mice were han-
 d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 1205–1210 1207

dled in accordance with a protocol approved by the Marquette

University institutional animal care committee. Dissociated
3. Results
cortical cells were plated on 24-well plates coated with poly-d-
We first determined the sensitivity of various cell types to
lysine and laminin in Eagle’s Minimal Essential Medium (MEM,
zinc-containing and zinc-free amalgam toxicity. Cultures of
Earle’s salts, supplied glutamine-free) supplemented with 5%
glial cells, pure neuronal cells, embryonic stem cells, and
heat-inactivated horse serum, 5% fetal bovine serum, 2 mM
dental pulp cells were exposed to zinc-containing amalgam
glutamine, and glucose (total, 21 mM). Cultures were main-
(Fig. 1A). The zinc-containing amalgam caused near com-
tained in humidified 5% CO2 incubators at 37 ◦ C. Near-pure
plete cell death in all of the cell types tested and there was
neuronal cultures were obtained by adding cytosine arabi-
no significant difference in the percentage of cell survival
noside (10 ␮M) to the cultures 48 h after plating to inhibit glial
between the four cell types. We next determined the effects
replication. Less than 1% of cells in these cultures stain for glial
of zinc-free amalgam. Cultures of glial cells, pure neuronal
fibrillary acidic protein [22]. Glial cultures were prepared using
cells, embryonic stem cells, and dental pulp cells were tested
the same method except cortical cells were dissected from 1-
with zinc-free amalgam (Fig. 1B). Comparison of Fig. 1A and B
to 3-day-old mice and cytosine arabinoside was not added. ES-
shows that zinc-free dental amalgam was less toxic than zinc-
D3 embryonic mouse stem cells were purchased from ATCC
containing amalgam in each of the cell cultures. Dental pulp
(Manassas, VA, USA). All experiments were performed on cul-
cells were the most sensitive to zinc-free amalgam toxicity of
tures 7–14 days in vitro. This time in culture allows for the
the four types of cells tested, with them being significantly
various cell types to divide until becoming confluent.
more sensitive than glia and embryonic stem cell cultures.
Cultures of dental pulp cells were treated with the six dif-
2.4. Preparation of amalgam rods and exposure to
ferent growth factors IGF-I, BMP-7, EGF, bFGF, TGF-␤, or BMP-2.
cultures
Fig. 2 shows the appearance of normal cultured pulp cells (A)
and those cells following 24 h treatment with zinc-free amal-
A messing gun was used to make standard-sized rods of
gam (B). There were clear morphological differences in the
amalgam (0.010 ± 0.002 g each). The freshly prepared amalgam
appearance of the cultures treated with EGF, bFGF, TGF-␤, or
(with or without zinc) was placed into the wells containing cul-
tured cells for 24 h. The area of the cells directly impacted by
the amalgam was only slightly more than 1% of the total area
covered by the cells (amalgam area approximately: 2.5 mm2 ;
culture area: 201 mm2 ). The amalgam was placed in the cul-
tures and we observed no evidence that it moved during the
duration of the experiment.

2.5. Cell viability assessment

Cell injury was quantified by the measurement of the reduc-

tion of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium
bromide (MTT) to produce a dark blue formazan product
[23,24]. MTT was added to each well 24 h after the begin-
ning of the insult to the cells. After a 30 min incubation,
the media was removed, and cells dissolved in DMSO. The
formation of formazan was measured as the amount of reac-
tion product by absorbance change using a VersaMax tunable
microplate reader (Molecular Devices, Sunnyvale, CA). Levels
of formazan formation from cultures exposed to 10 ␮M of the
calcium ionophore A23187 (100% cell death) were subtracted
from insult formazan levels, and results were normalized to a
sham wash.

2.6. Phase contrast microscopy imaging

Cell cultures were viewed using a phase contrast microscope.

Digital images were taken with a Diagnostic Instruments Spot
Camera at 400X.
2.7. Statistical analysis
Statistical calculations were performed using one-way ANOVA
followed by the Bonferroni t-test. P-values < 0.05 were consid-
ered to indicate significant differences.
Fig. 1 – The toxicity of zinc-containing amalgam is not
significantly different between glia (Glia), pure neuronal
cultures (PNC), embryonic stem cells (Stem) and dental pulp
cultures (Pulp) (A). Zinc-free amalgam is significantly more
toxic to dental pulp cells than to glia or embryonic stem
cells (B). Amalgam was present for 24 h. Bars show %
survival (mean ± S.E.M., n = 15–24) quantified by inhibition
of MTT reduction.
1208 d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 1205–1210
Fig. 2 – The growth factors EGF, bFGF, TGF-␤, and BMP-2, but not IGF-I or BMP-7 alter dental pulp cell morphology. Phase
contrast microscopy image of dental pulp cells. (A) Blank; (B) blank exposed to zinc-free amalgam for 24 h; (C) IGF-I; (D)
BMP-7; (E) EGF; (F) bFGF; (G) TGF-␤; (H) BMP-2. Growth factors (100 ng/ml) were added at plating. Scale bar = 50 ␮m.
Fig. 3 – Effect of growth factor treatment on zinc-free
amalgam toxicity to dental pulp cells. Empty bars are
without zinc-free amalgam. Hatched bars are with zinc-free
amalgam. BK: Blank; Zn Am: zinc-free amalgam. Zinc-free
amalgam was present for 24 h. Growth factors were added
at plating. Bars show % neuronal survival (mean ± S.E.M.,
n = 8–12) quantified by inhibition of MTT reduction.
* Significant difference (P < 0.05).
BMP-2, but not those treated with IGF-I or BMP-7 (Fig. 2C–H).
The sensitivity of these growth factor treated cultures to zinc-
free amalgam was then tested (Fig. 3). Cultures treated with
IGF-I or BMP-7 had similar levels of control MTT metabolism
as untreated cultures and had similar sensitivity to zinc-free
amalgam toxicity. Cultures treated with EGF, bFGF, or TGF-␤
had diminished MTT metabolism, but were no longer sensi-
tive to zinc-free amalgam toxicity. Cultures treated with BMP-2
had diminished MTT metabolism, but still remained sensitive
to zinc-free amalgam toxicity.
4. Discussion
The high failure rate of pulp capping was originally attributed
to toxicity of dental materials [3,25]. However, recent think-
ing has focused on the role of bacterial infection. While
infection is undoubtedly a major factor in many pulp cap-
ping failures, the evidence against a role of toxicity of dental
materials is fairly weak. The primary evidence comes from
animal studies. For example, in rats it was found that when
pulp capping was performed under germ free conditions it
resulted in minimal pulpal inflammation and good dentin
bridge formation [26]. However, the rats were only observed
for a maximum of 42 days. Furthermore, the fact that bacte-
rial infection plays an important role in the success or failure
of pulp capping does not exclude the possibility that toxicity
of dental materials is also a factor that should be consid-
ered.
These present studies confirm our previous study that
the presence of zinc in dental amalgam greatly increases its
toxicity and that the increased toxicity is due to release of
zinc [11]. All four cell types tested, glial, neuronal, embry-
onic stem cells, and dental pulp cells were highly sensitive to
zinc-containing amalgam toxicity. Zinc is known to be cyto-
toxic, particularly to neurons [27,28]. Release of zinc from
synaptic vesicles has been shown to contribute to neuronal
death induced by global ischemia in rats [29]. However, zinc
levels in the brain are tightly regulated and zinc has been
used for the treatment of Wilson’s disease for many years
without adverse effects [30]. Therefore, the release of zinc
from amalgam is not of concern regarding systemic toxi-
city. However, if released zinc comes in contact with pulp
cells, cytotoxicity is of concern. Another interesting result
was that dental pulp cells exhibited the greatest sensitivity
to zinc-free amalgam toxicity. This suggests that any type
of amalgam should be avoided in conditions in which sub-
stances released from it may come in contact with dental
pulp cells. Exactly under what conditions substances leached
from dental amalgam will come in contact with dental pulp
is not certain. Typically in pulp capping, a material such as
calcium hydroxide is applied to the dental pulp before the
restoration is applied. However, calcium hydroxide has been
shown to be mechanically weak and soluble over time [25]
 d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 1205–1210
allowing for possible leakage from the restoration to the dental
pulp.
When dental pulp cells were treated with EGF, bFGF, TGF-
␤, or BMP-2 there was a marked difference in the appearance
of the cells. Interestingly, the level of MTT metabolism was
decreased to approximately half of the controls. Since MTT
metabolism is a measure of cell number [31], it is clear that
these growth factors decrease cell number. Since the growth
factors were added at the time of plating, the most likely
explanation for the results is that these growth factors induce
differentiation of the cells, and also inhibit their replica-
tion. Further analysis will be required to determine exactly
what types of cells are present following each growth fac-
tor treatment. Treatment of cultures with IGF-I or BMP-7
caused no morphological changes to the cells or change in
cell number. The cultures treated with IGF-I or BMP-7 also
responded to zinc-free amalgam toxicity with the same sen-
sitivity as untreated cultures. The most interesting result in
the current study is that cultures treated with EGF, bFGF, or
TGF-␤ were no longer sensitive to zinc-free amalgam tox-
icity, while those treated with BMP-2, which also induced
morphological changes and decreased cell number, remained
sensitive to zinc-free amalgam toxicity. Further analysis will
be required to determine why this difference in sensitivity
occurred, but the results suggest that BMP-2 may have unfa-
vorable effects.
5. Conclusions
The use of zinc-containing dental amalgam should be avoided
under conditions when released zinc may come in contact
with dental pulp cells. Zinc-free amalgam is also of concern.
The use of specific growth factors should be considered as a
method to differentiate dental pulp cells into a state in which
they are less sensitive to toxicity.
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