Dental Pulp Cells

Basic ResearchBiology
Stimulatory Effects of Low-concentration Reactive Oxygen

Species on Calcification Ability of Human Dental Pulp Cells
Satoshi Matsui, DDS, PhD, Chitaka Takahashi, DDS, Yasuhisa Tsujimoto, DDS, PhD, and
Kiyoshi Matsushima, DDS, PhD
Abstract
The present study was conducted to investigate the
effects of reactive oxygen species (ROS) on the calcification ability of human dental pulp (HDP) cells. HDP
cells were treated with 100 mol/L hydrogen peroxide
(H2O2) for 5 or 10 minutes (5-min ROS group and
10-min ROS group) to investigate the mechanism of
transmission to cells. Untreated cells were used as
controls. Generation of free radicals was quantified by
the electron spin resonance spin-trapping method and
found to be increased by treatment with ROS. Formation of calcified nodules was also investigated by von
Kossa staining and alizarin red S staining. Twenty-eight
days after exposure, calcified nodules were present in
cell cultures that had been treated with ROS for 5 or 10
minutes. Expression of mRNAs for osteopontin (OPN)
and osteocalcin (OCN) was significantly greater in 10min ROS group 6 and 9 days, respectively, after exposure than in controls. Production of OPN and OCN by
10-min ROS group was also greater 12 and 18 days,
respectively, after exposure than in controls. These
results suggested that calcification of HDP cells was
stimulated by H2O2 and by the ROS it generated. (J
Endod 2009;35:6772)
Key Words
Human dental pulp, mineralization, osteocalcin, osteopontin, reactive oxygen species (ROS)
ulp capping is defined as the treatment of exposed vital pulp by sealing the pulpal
wound with a dental material to facilitate the formation of reparative dentin. A
final goal of the application of capping materials is to induce the dentinogenic
potential of pulp cells. Calcium hydroxide and calcium oxide are widely used for
pulp capping (1, 2).
On the other hand, we reported previously that the calcification ability of human
dental pulp (HDP) cells is stimulated by Ga-Al-As laser irradiation, and that reactive
oxygen species (ROS) and hydroxyl radical (OH) generation by laser irradiation is a
trigger for the promotion of HDP cell calcification (3).
One well-known process of free radical production in the body is the transition
from superoxide anion to hydrogen peroxide (H2O2) and thereby to OH (4, 5). Arai
et al. (6) reported that reduced mineralization by MC3T3-E1 cells after H2O2 (400
mmol/L) exposure is the result of up-regulation of the antioxidant system and altered
osteogenic gene expression. Excessive production of ROS is considered to result in the
formation of substances causing various diseases, but recent attention has focused on
the fact that production of lesser quantities can have bactericidal and other biodefensive
roles and can enhance cell proliferative activity and information signaling (3, 79).
Nishida et al. (10) have reported that OH produced from H2O2 plays a role in cell
signaling by the activation of cell membrane G-protein. Lee et al. (11) have also reported that H2O2 exposure significantly increases alkaline phosphatase (ALP) activity
and the formation of calcified nodules in odontoblast-like cells.
Accordingly, our research has focused on H2O2 as one mechanism for the transmission of ROS to cells, and we have been performing in vitro investigations involving
treatment of cultured HDP cells with H2O2. Here we investigated the effect of ROS on
expression of the mRNAs encoding osteopontin (OPN) and osteocalcin (OCN) and of
these proteins themselves, all of which are differentiation markers of accelerated calcified nodule formation.
Materials and Methods

From the Department of Endodontics, Nihon University
School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
Address requests for reprints to Satoshi Matsui, Department of Endodontics, Nihon University School of Dentistry at
Matsudo, 870-1, Sakaecho-Nishi-2, Matsudo, Chiba 271-8587,
Japan. E-mail address: matsui.satoshi@nihon-u.ac.jp.
0099-2399/$0 - see front matter
2008 Published by Elsevier Inc. on behalf of the American Association of Endodontists.
doi:10.1016/j.joen.2008.08.034
JOE Volume 35, Number 1, January 2009
Cell Culture and H2O2 Treatment

This study was approved by the ethics committee of Nihon University School of
Dentistry at Matsudo (approval number EC 03-025). HDP cells were obtained from
unerupted teeth extracted from young patients in the course of orthodontic treatment.
Patients gave informed consent before providing the samples. After the dental pulp had
been extracted under sterile conditions, it was washed twice with Hanks balanced salt
solution (pH 7.4). Then the pulpal tissue was minced, placed on a 35-mm tissue culture
dish, and covered with a sterilized glass coverslip (12).
The culture medium used was -minimal essential medium (MEM; Gibco, Grand
Island, NY) containing 10% fetal calf serum and antibiotics consisting of 100 g/mL
penicillin G (Meiji Seika, Ltd, Tokyo, Japan), 100 g/mL gentamicin sulfate (Meiji
Seika), and 100 g/mL fungisone (Gibco). Culture was maintained in an atmosphere of
5% CO2, 90% N2, and 5% O2 at 37C.
When cell growth from the explant had reached confluence, the cells were detached with 0.05% trypsin (580 BAEE units/mg; Gibco) in phosphate-buffered saline
(PBS) and subcultured in culture dishes. Cells observed at confluence by phase-contrast microscopy had not formed the small mats typical of epithelial cells.
For the experiment, HDP cells from 3 6 passages were plated at 1 105 cells (in
1.5 mL medium) per dish.
Effects of ROS on the Calcification Ability of HDP Cells
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67
group) or 10 (10-min ROS group) minutes. They were then washed
with fresh -MEM to remove the H2O2. To observe osteoblastic differentiation, the cells were placed in -MEM supplemented with 10% fetal
calf serum, antibiotics, 50 g/mL ascorbic acid (Wako, Osaka, Japan),
and 10 mmol/L sodium -glycerol-phosphate (Wako). The culture medium was changed every 3 days throughout the 28-day experimental
period (3, 13).
Measurement of Free Radical Generation

The amount of free radicals generated during treatment with H2O2
was measured by the electron spin resonance (ESR) spin-trapping
method (7). Nine hundred microliters of 900 mol/L H2O2 medium or
untreated medium plus 100 L of 890 mmol/L 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; Dojin Chemicals, Kumamoto, Japan; as a spintrapping agent for free radicals) was added. OH was released into the
treated culture medium, and the ESR spectrum was recorded after 5 and
10 minutes of treatment with H2O2. The ESR spectrum was recorded
with a JES FA200 electron spin resonance spectrometer (JEOL, Tokyo,
Japan). The conditions for ESR were as follows: microwave power 8
mW, magnetic field 335.0 5 mT, sweep time 2 minutes, field modulation amplitude 0.1 mT at a modulation frequency of 100 kHz, and time
constant 0.03 seconds (3).
Figure 1. ESR signal of DMPO-OH from HDP cells with H2O2. ESR spectrum of
control group (A), 5-min ROS group (B), 10-min ROS group (C), only medium
(D). Culture medium including DMPO was also treated with H2O2, and the ESR
spectrum was recorded.
Cells were recounted with a Coulter Counter ZM (Coulter Electronic Ltd, Luton, England).
After 24 hours, the cells were treated with -MEM containing H2O2
(final concentration, 100 mol/L) and cultured for 5 (5-min ROS
Examination of Calcified Nodule Formation

For examination of calcified nodule formation, HDP cells were
cultured for 28 days in vitro and stained by the von Kossa technique
(3, 6).
Calcified nodules in the HDP cells were first washed for 30 days in
vitro with PBS and then fixed in 10% buffered formalin for 1 hour. They
were then washed again and stained with alizarin red S (13, 14). The
calcium in the nodules was dissolved with 0.5 N HCl (1.0 mL) for 12
hours, and the amount of calcium was then measured by using Calcium
C test Wako (Wako).
Figure 2. von Kossa staining and number calcified nodules formation by ROS. Calcified nodules were visualized by von Kossa staining on the 28th day. The von Kossa
stained on HDP cells was, in order, 10-min ROS group 5-min ROS group control group. Significant difference between 10-min ROS group and control group
(*P .01) and was significantly greater in the 5-min ROS group than in the control group (*P .01).
68
Matsui et al.

Real-time Polymerase Chain Reaction
Cultured HDP cells, exposed or not exposed to cultured H2O2,
were homogenized with Trizol reagent (Invitrogen Co, Carlsbad, CA),
after which total RNA was isolated by using a FastPrepFP 120 Instrument
(BIO 101 Inc, Vista, CA). Samples (1 g) of total RNA were then subjected to reverse transcription followed by the addition of oligo-dT
primer and MuLV reverse transcriptase (final volume, 20 L). Then
5-L aliquots from the diluted cDNA (1/100-fold) were used as templates for real-time polymerase chain reaction (PCR). To assess the
effects of H2O2 on the transcription of mRNAs, PCR amplification was
performed with a SYBR Green real-time PCR master mix (Toyobo Co,
Ltd, Osaka, Japan) with the ABI Prism 7700 and Sequence Detection
System software (Applied Biosystems, Foster City, CA) (6). The expression level of each mRNA was observed as the number of cycles by
real-time PCR, with levels normalized to that of the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The PCR
primers for amplification of OPN, OCN, and GAPDH were designed on
the basis of published sequences. The primers were GAPDH (forward)
5=-ATC ACC ATC TTC CAG GAG-3=, (reverse) 5=-ATG GAC TGT GGT CAT
GAG-3=; OPN (forward) 5=-GCT GTA CTA GCG ACA CCC AC-3=, (reverse)
5=-TCA TAA AAC CTG CAA CAG CCA ACT- 3=; OCN (forward) 5=-GAT ACC
TAT CAA TGG CTG GGA GCC-3=, (reverse) 5=-GTC GAC ATA GGC CTC CTG
AAA GCC-3=.
Effects on OPN and OCN Production
OPN released into the culture medium after 6 15 days of incubation was quantified with a Quantkine OPN enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) at a wavelength
of 450 nm in a model 2550 microplate reader. OCN released into the
culture medium after 1224 days of incubation was measured with an
Instant OCN ELISA kit (Bender MedSystems, Burlingame, CA) at 570 nm
in a model 2550 microplate reader.
Statistical Analysis
All values are presented as means standard deviation, and the
significance of differences was determined by Student t test.
Results
Quantification of OH
OH generated in the H2O2 medium was quantified by using an
ESR spin-trapping method with DMPO. ESR spectra typical of the OH
adduct of DMPO (DMPO-OH) were observed after treatment with H2O2
(5 and 10 minutes) (Fig. 1). The hyperfine coupling constant of this
signal was analyzed and found to be AN AH 1.49 mT, which agreed
with the value reported previously (3). The height of the DMPO-OH
signal produced by the H2O2 medium increased with time. H2O2 medium generated a small amount of DMPO-OH, but untreated culture
medium did not generate DMPO-OH (Fig. 1).
Examination of Calcified Nodule Formation
The von Kossa stained on HDP cells was, in order, 10-min ROS
group 5-min ROS group control group. The number of calcified
nodules present in the 35-mm dishes at 28 days was significantly greater
in the 10-min ROS group than in the control (P .01) and in the 5-min
ROS group was significantly greater than in the control group (P .01)
(Fig. 2) (n 15).
After 28 days of culture, calcified nodules present in the 35-mm
dishes were stained with alizarin red S to a markedly greater extent in
the treatment with H2O2 time. The amount of calcium in the 10-min ROS
group was significantly greater than in the control group (P .01) and
in the 5-min ROS group was significantly greater than in the control
group (P .05) (Fig. 3) (n 15).
5 min
Measurement of calcium (mg/dl)
control
10 min
2.5
**
2
1.5
1
0.5
0
control
5 min
10 min
* p<0.01
* *p<0.05
<0.05
(n=15)
Figure 3. Alizarin red S staining and the amount of calcium. Upper photograph shows calcified nodules by alizarin red S staining on the 28th day. The alizarin red S
stained on HDP cells was, in order, 10-min ROS group 5-min ROS group control group. The graph shows the amount of calcium significantly greater in the
10-min ROS group than in the control group (*P .01) and was significantly greater in the 5-min ROS group than in the control group (*P .05).
69
Figure 4. Effects of ROS on mRNA from HDPcells. OPN expression was significantly greater in the 10-min ROS group than in the control group at 6 days (*P .01).
OCN expression was significantly greater in the 10-min ROS group than in the control group at 12 days (*P .01).
Real-time PCR
We used real time-PCR to determine the effects of H2O2 addition on
the expression of mRNAs of OPN and OCN. A significant increase in
mRNA expression in the 10-min ROS group was recognized at 6 days
(OPN) and 9 days (OCN) in comparison with that in the control group
(Fig. 4) (P .01) (n 6).
Effects on OPN and OCN Production
OPN production in the 5-min ROS group, 10-min ROS group, and
control group increased in a time-dependent manner and peaked at 12
days; the production level was significantly greater in the 10-min ROS
group than in the control group (Fig. 4A) (P .05) (n 15). OCN
production in the 10-min ROS group, 5-min ROS group, and control
group increased in a time-dependent manner and peaked at 18 days; the
production level was significantly greater in the 10-min ROS group than
in the control group (Fig. 5) (P .01) (n 18).
Discussion
In the dental clinic, H2O2 is used routinely for tooth bleaching (15)
and root canal irrigation (16). To elucidate the mechanism and promotion of hard-tissue formation after treatment with H2O2, we focused
on ROS generation and its effects on calcified nodule formation by HDP
cells. Although it has been reported that ROS are involved in apoptosis
(17), inflammation (18), DNA damage (19), and aging (6), it has also
become clear that ROS also play roles in cellular signal transduction (3,
20). Nishida et al. (10) reported that activation of mitogen-activated
protein (MAP) kinase was dependent on activation of G-protein when
rat neonatal cardiomyocytes were treated with H2O2, indicating that
70
Matsui et al.
OH directly activates G-protein independently of receptors. In addition, it was reported that MAP kinase, protein kinase B, and extracellular signal-related kinase are pathways to mineralization (21).
Initially, free radicals were analyzed qualitatively and quantitatively
by the ESR spin-trapping method after treatment of the culture medium
with H2O2. ESR is the device that can detect a free radical in real time (3,
16). Generation of OH was detected in the H2O2treated medium but
not in the untreated medium. These findings confirmed that generation
of OH was induced by H2O2.
We next used von Kossa staining to determine whether calcified
nodules were formed by HDP cells after 28 days in culture after H2O2
treatment. The numbers of calcified nodules in the 5-min ROS group
and 10-min ROS group differed significantly from those in the control
group. No significant change in the number of calcified nodules was
observed in the control group at 28 days.
We used alizarin red S staining to observe the effect of ROS on
calcified nodule formation and amount of calcium in HDP cells. It has
been established in vitro that the HDP cell is capable of cell-associated
matrix calcification. The amount of calcium was significantly higher in
the 10-min ROS group than in the control group at 28 days. Von Kossa
stains for phosphorus, and alizarin red S stains for calcium. Our findings suggest that ROS generated as a result of H2O2 treatment promoted
calcium phosphate formation by HDP cells.
It is thought that OPN and OCN play important roles in a series of
processes that lead to mineralization. OPN plays important roles in
osteoblast differentiation and bone formation, growth, and regeneration. It is a phosphorylated glycoprotein expressed by a broad range of
tissues and cells (22), although it was originally characterized as bone

Figure 5. Effect of ROS on OPN and OCN production. OPN production was significantly greater in the 10-min ROS group than in the control group at 12 days (*P
.05). OCN production was significantly greater in the 10-min ROS group than in the control group at 18 days (*P .01).
matrix protein (23). OPN is synthesized in response to the release of

calcitropic hormones. The observation of OCN in the odontoblastic cell
and in the odontoblastic process, as well as in the dentin matrix, but
not in predentin, supports the view that OCN is synthesized within the
odontoblasts, transported through the odontoblastic processes, and
deposited in the mineralizing dentin (3, 12, 13, 24, 25). However,
a more likely explanation is that OCN is secreted by young odontoblasts but is not contained within the matrix because of the absence
of mineral. Such a concept would imply that the presence or absence
of OCN is not the limiting factor in dentin mineralization, but that the
deposition of OCN in dentin follows calcification, as observed in
bone. It is generally believed that OCN production occurs after cell
differentiation (25).
We investigated expression of the mRNAs of OPN and OCN at 6
and 12 days. We also investigated the production of OPN protein
from 6 15 days and that of OCN from 1218 days. The greatest
increases in expression of OPN and OCN mRNAs and the proteins
they encoded were observed in the 10-min ROS group. These findings indicate that the presence of ROS generated by treatment with
H2O2 led HDP cells to differentiate into cells with the ability to form
hard tissue. Gericke et al. (22) reported that OPN complexes with
OCN to form hydroxyapatite crystals. Moreover, Yao et al. (26) have
reported that the mRNAs of collagen, ALP, and OCN are expressed
before the formation of mineralized nodules, but that OCN is not
produced during the initial formation of bony tissue. Their results
support our data.
In conclusion, ROS are by-products that are generally harmful to
aerobic life and are a primary cause of aging and numerous diseases.
These data, however, suggest that ROS can induce in vitro cell differentiation, and that they play a more complex role in cell physiology than
simply causing oxidative damage. Our findings indicate that HDP cells
differentiated into cells with the ability to exhibit calcification, and that
this was mediated by the generation of ROS from low concentrations of
H2O2. Our findings indicate that the ROS generated by low-concentraJOE Volume 35, Number 1, January 2009
tion H2O2 promote calcification in HDP cells. The ROS appear to activate
cell signaling and increase OPN and OCN production, thereby promoting the calcification ability of HDP cells.
Therefore, we speculate that irrigation of pulp exposure with lowconcentration H2O2 is effective for dentinogenesis.
Acknowledgments
This work was supported in part by a Start-up Grant-in-Aid for
Young Scientists from the Japan Society for the Promotion of Science (No. 19890226, 2007, 2008) and by a Nihon University Individual Research Grant (No. 07-097, 2007).
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Basic ResearchBiology

Stimulatory Effects of Low-concentration Reactive Oxygen

Materials and Methods

JOE Volume 35, Number 1, January 2009

Cell Culture and H2O2 Treatment

Effects of ROS on the Calcification Ability of HDP Cells

Measurement of Free Radical Generation

Examination of Calcified Nodule Formation

JOE Volume 35, Number 1, January 2009

Measurement of calcium (mg/dl)

JOE Volume 35, Number 1, January 2009

Effects of ROS on the Calcification Ability of HDP Cells

JOE Volume 35, Number 1, January 2009

matrix protein (23). OPN is synthesized in response to the release of

Effects of ROS on the Calcification Ability of HDP Cells

JOE Volume 35, Number 1, January 2009

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