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Basic ResearchBiology

Zinc Chloride for Odontogenesis of Dental Pulp Stem Cells via


Metallothionein Up-regulation
Chia-Yung Lin, DDS, PhD,* Hsin-Hua Lin, MA, Mong-Hsun Tsai, PhD,k Shau-Ping Lin, PhD,k
and Min-Huey Chen, DDS, PhD*
Abstract
Introduction: Previous studies have shown that zinc
chloride (ZnCl2) can induce metallthionein (MT) in the
liver and kidney to protect tissues against toxicants
and shows a better corneal wound healing than conventional drugs do. We hypothesized that ZnCl2 can
promote odontogenesis of dental pulp stem cells
(DPSCs) via MT. The purpose of this study was to investigate the effects of ZnCl2 on human DPSCs and the
expression of MT. Methods: DPSCs were isolated by
flow cytometry with selective surface marker CD146
and STRO-1. After they grew into confluence, DPSCs
were induced into odontoblasts with or without ZnCl2
supplemented in the culture medium for 21 days. The
effect of ZnCl2 on DPSCs differentiation was examined
followed by alkaline phosphatase staining/activity and
quantitative real-time polymerase chain reaction analysis. Results: By treating DPSCs with ZnCl2, the duration
of mineralization was shortened and expressions of
differentiation markers into odontoblasts were more
significant than those without ZnCl2 stimulation.
Besides, the MT gene expression was increased with
the increasing expressions of odontoblasts markers
after treated with ZnCl2. Conclusion: This was the first
report that ZnCl2 could promote odontoblastic differentiation of DPSCs through the up-regulation of gene MT.
(J Endod 2011;37:211216)

Key Words
Human dental pulp stem cells, metallothionein, odontoblastic differentiation, odontogenesis, zinc chloride

dontoblasts can generate reparative dentin when teeth are irritated (1). Although
calcium hydroxide is widely used for conservative pulp therapy, its cytotoxicity and
difficult manipulation are still problems (2). Many efforts are made to develop new
materials (35). In addition, mineral trioxide aggregate, bioaggreate (6), and calcium
phosphate cement with its modifications (7) are all under investigations to be better
candidate materials. Dental pulp stem cells (DPSCs) (8) are maintained undifferentiated in the niche provided by the pulp (9, 10). DPSCs can be induced into odontoblasts
in vitro and characterized with expressions of dentinsialophosphoprotine (DSPP) and
dentin matrix acidic phosphoprotein (DMP-1) (11, 12).
Zinc (Zn) is an important regulator involved with various enzymes and proteins
and plays an important role in bone metabolism (13). Low serum Zn and low bone
turnover were reported in small for their gestational age preterm infants (14), and
low Zn intake is associated with low bone mass in adult women (15). Inadequate dietary
intake of Zn causes a decrease in the number of osteoblasts (16), and Zn augments DNA
synthesis in murine osteoblast-like cells in a dose-dependent manner (17). It is also
involved in the stimulation of collagen production in rat calvaria (18) and has an inhibitory effect on bone resorption and a stimulatory effect on bone formation/mineralization (19). Cellular trafficking of Zn is controlled by storage proteins and transporters.
Metallothioneins (MTs) are cellular metal storage proteins induced by essential trace
elements such as Zn and Cu (20). This ubiquitous heavy metal binding and cysteine-rich
protein reacts with free radicals and organic electrophiles and can be induced by zinc
chloride (ZnCl2) treatment (21, 22). MT levels may vary with age and type of tissue and
depend on nutritional and physiological factors. ZnCl2 is used as an additive to
intravenous solutions for total parenteral nutrition. ZnCl2 not only prevents the
kidney and liver from mercury and acetaminophen toxicities but also enhances
corneal wound healing, and these effects were regulated via the MTs (23).
For promoting odontogenesis, novel genes such as hemeoxygenase-1 stimulated
by simvastatin have been identified (24, 25). To our knowledge, no information is
available regarding the effects of MT simulated by Zn on DPSCs. We hypothesized
that ZnCl2 can promote DPSCs differentiation via MT up-regulation.

Materials and Methods


From the Graduate Institute of *Clinical Dentistry and Oral
Biology, School of Dentistry, National Taiwan University, Taipei,
Taiwan ROC; National Taiwan University Hospital, National
Taiwan University, Taipei, Taiwan; Institute of Biotechnology,
College of Bio-Resources and Agriculture, National Taiwan
University, Taipei, Taiwan ROC; and kAgricultural Biotechnology Research Center, Academia Sinica, Taiwan ROC.
Address requests for reprints to Dr. Shau-Ping Lin, Institute
of Biotechnology, College of Bio-Resources and Agriculture,
National Taiwan University, Taipei, Taiwan ROC., and Dr. MinHuey Chen, Graduate Institute of Clinical Dentistry, School
of Dentistry, National Taiwan University, Taipei, Taiwan ROC.
E-mail address: shaupinglin@ntu.edu.tw; minhueychen@ntu.
edu.tw.
0099-2399/$ - see front matter
Copyright 2011 American Association of Endodontists.
doi:10.1016/j.joen.2010.11.009

JOE Volume 37, Number 2, February 2011

Cell Culture
Isolation of DPSCs. All experimental procedures were performed according to
protocols approved by the Review Committee of College of Medicine, National Taiwan
University. Dental pulp cells were isolated from normal human third molars. Eight third
molars were collected from two healthy adults (18-year-old woman and a 25-year-old
man) in National Taiwan University Hospital. Teeth surfaces were cleaned with phosphate buffered saline (PBS) and cut by sterilized diamond burs. The pulp tissue was
gently separated by forceps and digested in a solution of 3 mg/mL collagenase type I
and 4 mg/mL dispase for 1 hour at 37 C. Single-cell suspensions were obtained by
passing through a 70-mm strainer (Pharmingen; BD, San Jose, CA) and then cultured
in 6-cm dishes (Costar, Cambridge, MA) with Dulbeccos Modified Eagle Media (low
glucose) (Gibco Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10%
fetal bovine serum (FBS) (HyClone, Logan, UT)/100 U/mL penicillin + 100 mg/mL
streptomycin (Biological Technologies, Beit Haemek, Israel)/2 mmol/L L-glutamine
(Gibco, Grand Island, NY).

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TABLE 1. Oligonucleotide Primer Sequences Used in the Reverse-Transcription PCR To Identify Dental Pulp Stem Cells
Gene name
DSPP
DMP1
OCN
GAPDH

Sequence

Tm

Size

F:5GCC ATT CCA GTT CCT CAA GC 3


R:5 CAT GCA CCA GGA CAC CAC TT 3
F:5 GAT CAG CAT CCT GCT CAT GTT C 3
R:5 GAG CCA AAT GAC CTT CCA TT 3
F:5 ATG AGA GCC CTC AGA CTC CTC 3
R:5 CGG GCC GTA GAA GCG CCG ATA 3
F:5 GTC TTC ACC ACC ATG GAG AAG GCT 3
R:5 CAT GCC AGT GAG CTT CCC GTT 3

59 C

144bp

59 C

109bp

After 1 week, dental pulp cells were observed to grow at approximately 90% confluence; then, cells were subcultured in 10-cm dishes
for another 2 weeks. Approximately 107 cells could be derived from
each tooth. All cells from eight teeth were mixed together to acquire
an average situation for the selection of DPSCs. Adherent cells were
detached by 0.25% trypsin-EDTA, centrifuged for 5 minutes at 500 g,
and resuspended in PBS. Aliquots containing 107 cells were incubated
with mouse antihuman CD146 conjugated with phycoerythrin (Pharmingen) (maximum absorption at 495 nm) and mouse antihuman
STRO-1 (Invitogen, Carlsbad, CA) for 30 minutes at 4 C; cells were
washed with PBS with 2% FBS followed by staining with 1 mg FITC conjugate goat antimouse immunoglobulin G for 30 minutes at 4 C. All cells
were then passed through a 70-mm strainer and collected by Becton
Dickinson FACS Aria fluorescence activated cell-sorting system..

Characterization of DPSCs
For characterization, odontoblastic differentiation was performed
by using odontoinduction medium (OM) containing Dulbeccos Modified Eagle Media (low glucose), 10% FBS, 100 U/mL penicillin, 100 mg/
mL streptomycin, 0.1 mmol/L ascorbic acid, 100 mmol/L b-glycerophosphate (Fluka, Buchs, Switzerland), and 108 mmol/L dexamethasone (Sigma-Aldrich, St. Louis, MO). When cells grew into 90%
confluence, the original medium was replaced by OM and cultured
for the following 21 days. The differentiation potentials were identified
by reverse-transcription polymerase chain reaction (RT-PCR) for
expressions of DSPP, DMP-1, and OCN. The oligonucleotide RT-PCR
primers were listed in Table 1. DPSCs in the third passage were used
for subsequent experiments.
Preparation of Induction Medium With ZnCl2
To evaluate effects of ZnCl2, 500 mL OM was added with 0.0034 g,
0.0068 g, and 0.0136 g ZnCl2 (Sigma-Aldrich), respectively, at concentrations of 50 mmol/L, 100 mmol/L, and 200 mmol/L. Those without
ZnCl2 were used as controls.
MTT Assay
For each experiment, DPSCs were seeded at a density of 1  104/
mL in 24-well plates. Experimental groups were those cultured in OM
with 50 mmol/L, 100 mmol/L, and 200 mmol/L ZnCl2; those in OM
without ZnCl2 served as controls. After 1, 4, and 7-day of cells cultured
in OM with different concentrations of ZnCl2, media were removed and
100 mL of MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) solution (2 mg/mL in PBS) was added into
each well and following procedures were performed as described
previously (26).
Alkaline Phosphatase Stain
DPSCs were cultured in OM with different concentrations of ZnCl2
(0, 50, 100, and 200 mmol/L) in six 12-well plates with the density of 1
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Lin et al.

60 C

297bp

58 C

392bp

 104 cells/mL (n = 6). On days 7, 10, and 14, each well of cells with
a different concentration of ZnCl2 were fixed by 4% paraformaldehyde.
After this, the alkaline phosphatase (ALP) stain kit (BioPioneer, San
Diego, CA) was applied for alkaline phosphatase staining by manufacturers instructions.

ALP Activity
DPSCs were cultured in OM with different concentrations of ZnCl2
as described. ALP activity was expressed as micromoles of reaction
product (p-nitrophenol) per 30 minutes from milligram of cellular
protein as described (27).
Quantitative RT-PCR
DPSCs cultured in OM with 100 mmol/L ZnCl2 were used for quantitative RT-PCR (qRT-PCR) analysis at 0, 7, and 14 days. Those in OM
without ZnCl2 were used as controls. Reactions were prepared with
the TaqMan (Applied Biosystems by Life Technologies, Foster City,
CA) method according to the manufacturers instructions. The product
was aggregated on the bottom of the well by a 2,000-rpm centrifugal
force for 1 minute, and the plate was put in ABI Prism 7900HT (Applied
Biosystems by Life Technologies) for PCR reaction and data collection.
The GAPDH (Hs99999905_ml), DSPP (Hs00171962_ml), OCN
(Hs00758162_ml), and MT (Hs00744661_sH) were standardized
by the TaqMan gene expression assays and quantified by Sequence
Detector Software 2.21 (Applied Biosystems by Life Technologies).
Statistical Analysis
One-way analysis of variance was used to estimate the difference
that resulted from the addition of ZnCl2, and a Scheffe test was used
for further analysis. Statistical significance was defined as p < 0.05.

Results
DPSCs
After a 1-week culture of isolated pulp cells, colony formation was
found. About 1.22% STRO-1+ CD146+ cells from isolated cells were
sorted as DPSCs. Expressions of DSPP, DMP-1, and OCN after induction
were also confirmed by RT-PCR (Fig. 1A and B).
MTT Assay
MTT assay indicates the activity of vital cells. Figure 1C showed the
ratio of MTT conversion of cells in OM with 50, 100, and 200 mmol/L
ZnCl2 relative to that of cells in OM without ZnCl2 (controls) after incubation for 1, 4, and 7 days. At all data points, the ratio of MTT conversion
of DPSCs in ZnCl2 showed no significant difference with that of controls
(p > 0.05). According to this result, ZnCl2 had no toxicity to DPSCs.
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Basic ResearchBiology

Figure 1. (A) The isolation of DPSCs was performed by the Fluorescence Activated Cell Sorting System. About 1.22% STRO-1+ CD146+ cells from the mixture
isolated cells of eight teeth were sorted as DPSCs. (B) By the reverse-transcription PCR, the DPSCs were examined to show the DSPP, DMP-1, and OCN expression.
(C) The MTT of DPSCs was cultured in the odontogenic induction medium with different concentration of ZnCl2 for 1, 7, and 14 days. No significant differences were
observed between DPSCs within or without ZnCl2. (D) The ALP staining of these DPSCs showed that with 100 mmol/L ZnCl2 the ALP staining was more evident in
fastening the mineralization at 10 days than that with other concentrations of ZnCl2. (E) The ALP activities of DPSCs cultured in the odontogenic induction medium
with different concentrations of ZnCl2 after 7, 10, and 14 days showed that the ALP activity of DPSCs cultured in 100 mmol/L ZnCl2 at 10 day was the highest (*p <
0.05; **p < 0.01).

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Figure 1. (continued).

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Figure 2. (A) By the quantitative RT PCR, DPSCs cultured in the odontogenic


induction medium with or without 100 mmol/L ZnCl2 at days 0, 7, and 14 were
analyzed. MT was seldom shown in the control group (without ZnCl2 in the
odontogenic induction medium), but it could be induced by ZnCl2 stimulation.
(B and C) Although the similar tendency with that in the control group, the
expressions of DSPP and OCN were also higher by ZnCl2 stimulation at days
7 and 14.
Figure 1. (continued).

ALP Stain
When DPSCs were cultured in OM with 100 mmol/L ZnCl2, the most
evident expression of ALP was appeared at day 10, which was earlier than
those of controls. However, there were no differences between media
with either 50 or 200 mmol/L ZnCl2 and controls (Fig. 1D).
ALP Activity
When DPSCs were cultured in OM with 50 mmol/L ZnCl2 and 100
mmol/L ZnCl2, the ALP activities were significantly higher than those of
cells in the medium without ZnCl2 at day 10 ( p < 0.05, p < 0.01)
(Fig. 1E).
Gene Expressions After Stimulation
Results showed that in OM without ZnCl2, the gene of MT in DPSCs
was expressed less than those in OM with 100 mmol/L ZnCl2, where the
MT expression was evident from day 7 to day 14 (Fig. 2A). The expressions of both DSPP and OCN in experimental groups treated with ZnCl2
were significantly higher than those of controls (Fig. 2B). The gene
expression of DSPP was the highest at day 10, and the expression of
OCN was the highest at day 7 in the experimental group.
JOE Volume 37, Number 2, February 2011

Discussion
This is the first study to show the promotion effect of ZnCl2 in
DPSCs odontoblastic differentiation. In our work, dental pulp was
treated by the enzyme-digestion technique (10), which preserved
more complete cells (28), and DPSCs were sorted by surface markers
(8). Expressions of differentiation markers in these DPSCs were also
identified by RT-PCR.
Although natural functions of MT remain elusive, studies have indicated that MT is involved in the homeostatic regulation of essential
metals and detoxification of nonessential metals (23). MT, induced
by ZnCl2 and regulated by platelet-derived growth factor-BB, which
has the ability to increase the periodontal ligament cells divisions
(29), also could protect bone marrow stem cells from the inhibition
of osteogenesis (30). Therefore, we hypothesized that ZnCl2 may help
to fasten damaged dental pulp healing.
Traditionally, OM is made for DPSCs to differentiate toward odontoblasts phenotypes (31, 32). In our results, by adding ZnCl2 in OM,
both ALP expression and activities were increased compared with
those of controls without ZnCl2. The ALP expression indicates that
ZnCl2 could enforce the mineralization of DPSCs, and with a 100mmol/L supplement, this effect appeared earlier (Fig. 1D). The mineral
deposits at day 14 in the experimental groups with ZnCl2 stimulation
were obviously much more than those in control groups. Besides, in
groups with 50 and 100 mmol/L ZnCl2, 10 days after the induction,
the ALP activities in both groups were significantly higher than that of

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other experimental groups ( p < 0.05) (Fig. 1E), but the relative value
of ALP activity (amount of ALP/amount of total protein) on day 10 was
lower than that of day 7; we assumed that on day 10 the total protein
increase was more than the ALP increase because some undifferentiated
DPSCs were still proliferated. According to the previous statements, 100
mmol/L ZnCl2 was chosen for qRT-PCR.
qRT-PCR revealed that ZnCl2 would induce the expression of MT,
which was seldom shown in the control group. Expressions of DSPP and
OCN were also greater than those in the control group. The decreased
OCN expression on 10 day was noted, which may result from variances
of primary cells we used but it still regained on day 14; this was consistent with a previous study that showed that OCN was a differentiation
marker (33). The expression of MT increased with increasing intracellular storage of Zn concentrations, and Zn exposure has been shown to
augment MT expression in a dose-dependent fashion in various cells
(34). In our study, the tendency for MT expression was also similar
with this dose-dependent fashion. On the other hand, when cells started
to differentiate and mineralize through the pronounced MT upregulation, the intracellular Zn storage was smaller, which may result
in the decreased MT expression at days 10 and 14. This might explain
why the expression of OCN increased from day 0 to day 7 and decreased
at day 10.
We conclude that by the Zn stimulation, it is associated with the
expression of MT as mineralization and differentiation proceeds. This
novel study suggested that ZnCl2 is nontoxic and able to promote DPSCs
differentiation through MT up-regulation.

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