Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/
Title
Alkaline extracellular conditions promote the
proliferation and mineralization of a human
cementoblast cell line
Author(s)
Alternative
Muramatsu, T; Kashiwagi, S; Ishizuka, H; Matsuura,
Y; Furusawa, M; Kimura, M; Shibukawa, Y
Journal
International endodontic journal, 52(5): 639-645
URL
http://hdl.handle.net/10130/5124
Right
This is the peer reviewed version of the following
article: Int Endod J. 2019 May;52(5):639-645, which
has been published in final form at
https://doi.org/10.1111/iej.13044. This article may
be used for non-commercial purposes in accordance
with Wiley Terms and Conditions for Use of
Self-Archived Versions.
Original Article:
Alkaline extracellular conditions promote the proliferation and
mineralization of a human cementoblast cell line.
Authors:
T. Muramatsu1*, S. Kashiwagi2, H. Ishizuka1, Y. Matsuura3, M. Furusawa2, M. Kimura4, Y. Shibukawa4, 1: Department of Operative Dentistry, Cariology and Pulp Biology, Tokyo Dental College
2: Department of Endodontics, Tokyo Dental College 3: Oral Health Science Center, Tokyo Dental College 4: Department of Physiology, Tokyo Dental College 2-9-18, Kandamisaki-cho, Chiyoda-ku,
Tokyo, 101-0061, Japan
Corresponding author:
Takashi Muramatsu, DDS, Ph.D. Tokyo Dental College
Department of Operative Dentistry, Cariology and Pulp Biology 2-9-18, Kandamisaki-Cho, Chiyoda-Ku
Tokyo 101-0061 Japan Tel.: +81-3-6380-9125 E-mail: tmuramat@tdc.ac.jp
Abstract
Aim To investigate the proliferation and mineralization of a human cementoblast cell line under alkaline
conditions.
Methodology A human cementoblast cell line was cultured in alkaline media with several pHs (pH 7.6,
8.0 and 8.4) without CO2. Cell numbers, phospho-p44/42 expression, alkaline phosphatase (ALP)
activity, and mineralization were evaluated.
Results Cell numbers increased in a time-dependent manner in the high pH medium groups. Western
blot analysis revealed the up-regulated expression of phospho-p44/42 under alkaline conditions. ALP
activity was also increased at pH 8.0 and 8.4. Alizarin red staining showed increased mineralization in
the high pH medium groups. The incorporation of the transient receptor potential ankyrin subfamily
member 1 (TRPA1) antagonist HC030031 markedly negated the effect on proliferation and
mineralization.
Conclusions Extracellular alkaline conditions promoted the proliferation and mineralization of human
Introduction
Perforation of roots during root canal treatment is a known procedural error, which has been reported to
occur in approximately 2–12% of root filled teeth (Kvinnsland et al. 1989, Farzaneh et al. 2004, Kakani et
al. 2015). If the size of the perforation is small, it may be repaired, but root amputation and tooth
extraction are more likely to be performed when the size of the perforation is large or when bacterial
infection cannot be controlled (Himel et al. 1985, Balla et al. 1991). If the tooth is extracted, the quality of
life (QOL) of the patient is affected (Gerritsen et al. 2010). In general, it is desirable for perforations to be
repaired and covered with cementum as well as normal periodontal tissue. Therefore, the development
of materials to repair root canal perforations is essential.
Mineral trioxide aggregate (MTA) is a powder containing dicalcium silicate, tricalcium silicate,
tricalcium aluminate, gypsum, and tetracalcium aluminoferrite. MTA is used worldwide to repair
perforations (Torabinejad et al. 2018) and has the characteristics of biocompatibility, biostability, and
antibacterial effects. Furthermore, MTA has been reported to induce the formation of hard tissues, such
as bone, dentine and cementum (Keiser et al. 2000). Ideal repair involves sealing the perforated area
with newly formed cementum induced by MTA. The mechanism by which MTA induces the formation of
environment with a high pH of 12.5 (Torabinejad et al. 1995, Camilleri et al. 2005, Mohammadi &
Dummer 2011). When MTA is used at perforated areas, it adheres to periodontal ligament cells, which
become necrotic due to the high pH. Surrounding cementoblasts directly form cementum or
mesenchymal stem cells surrounding the necrotic area differentiate into cementoblasts and newly
formed cementum is induced. However, the mechanisms by which the extracellular alkaline
environment induces hard tissue formation have not yet been elucidated.
The extracellular alkaline pH sensor molecule, the transient receptor potential ankyrin
subfamily member 1 (TRPA1) channel, has recently been investigated and cell responses to alkaline
environments have been extensively examined (Dhaka et al. 2009, Kimura et al. 2016, Kichko et al.
2018). In odontoblasts, high pH-sensing mechanisms are important for activating mineralization induced
by an alkaline environment (Kimura et al. 2016). However, the responses of cementoblasts in
extracellular alkaline environments remain unclear because cementoblast cell lines are rarely
generated.
In the present study, the proliferation and mineralization of a human cementoblast cell line
were investigated under extracellular alkaline conditions, and the role of TRPA1 in human
Materials and Methods
Materials
Powdered NaHCO3-free Eagle’s minimum essential medium (MEM) was purchased from Nissui
Pharmaceutical Co., Ltd. (Tokyo, Japan). Penicillin–streptomycin was obtained from Thermo Fisher
Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Sigma–Aldrich (St. Louis,
MO, USA). The TRPA1 antagonist HC030031 was from Wako Pure Chemical Industries Ltd. (Osaka,
Japan).
Preparation of alkaline pH media
Powdered NaHCO3-free Eagle’s MEM was dissolved in distilled water, sterilized by autoclaving,
supplemented with 10% FBS, and used as the cell medium. Medium samples were adjusted to pH
levels of 7.6, 8.0, and 8.4 by titration with sodium hydroxide solution (Wako Pure Chemical Industries
Ltd.). All media were sterilized by filtration through a 0.2-μm membrane filter (Minisart® plus Syringe
Cell culture
An immortalized cell line from human cementoblasts (HCEM) was used. HCEM were provided by
Professor Takashi Takata (Hiroshima University Graduate School); their characteristics have been
reported previously (Kitagawa et al. 2006). Cells were maintained routinely in MEM supplemented with
10% FBS (Sigma–Aldrich) and 100 U/mL penicillin–streptomycin (Thermo Fisher Scientific) at 37 °C in
a humidified atmosphere of 5% CO2. The culture medium was changed to alkaline pH media and
cultured at 37 °C without CO2.
Cell proliferation
HCEM were seeded at 1 x 104 cells/well on culture plates. At each time point, cells were detached using
0.05% trypsin-EDTA (Thermo Fisher Scientific) and counted using a Coulter Counter (Beckman Coulter,
Fullerton, CA, USA). Results and growth curves were plotted.
Cells were lysed in radio-immunoprecipitation assay buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM
Tris, pH 7.4, containing protease inhibitors and 1 nM sodium orthovanadate) 72 h after changing to high
pH medium. Thirty µg of total lysate proteins were subjected to 7.5% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene
difluoride (PVDF) membranes (BioRad, Melville, NY, USA). Membranes were then incubated with
anti-phospho-p44/42 (1:1000, Cell Signaling Technology, Beverly, MA, USA) and anti-actin antibodies
(dilution 1:1000, Sigma-Aldrich) at 4°C overnight. After washing, the membranes were incubated with
horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:1000, GE Healthcare Ltd., Chalfont St.
Giles, UK) at room temperature for 1 h. Immunoreactive bands were detected with an ECL Western blot
analysis system (GE Healthcare Ltd).
ALP activity
ALP activity was analyzed using Lab AssayTM ALP (Wako Pure Chemical Industries Ltd.) following the
manufacturer’s instructions and absorbance was measured at a wavelength of 450 nm with a microplate
reader. ALP activity (Units/µg protein) is defined as the release of 1 nmol p-nitrophenol per µg total
Alizarin red staining
In order to detect calcium deposits, HCEM were subjected to alizarin red staining. Approximately 1 × 104
HCEM were cultured in MEM at various pHs in 6-well plates for 4 weeks. The cells were washed with
PBS and fixed with 4% paraformaldehyde-0.1 M phosphate buffer (pH 7.4) at room temperature for 30
min. The cells were then stained with Alizarin Red S solution (pH 6.38; Wako Pure Chemical Industries
Ltd.) at room temperature for 5 min.
Incorporation of the TRPA1 antagonist HC030031
The TRPA1 antagonist HC030031 (100 µM, Wako Pure Chemical Industries Ltd.) was incorporated into
the culture medium in order to investigate its effects on proliferation and mineralization.
Statistical analysis
The significance of differences between groups was assessed using a two-way analysis of variance
Results
Cell growth
In order to clarify whether alkaline conditions contribute to cell growth, cell numbers were measured
after changing to alkaline media without CO2. The numbers of alkaline-stimulated HCEM were
measured on days 0, 1, 3, and 7. All cell groups grew in a time-dependent manner and the number of
cells was higher in the alkaline-stimulated group (pH 8.0 and 8.4) than in pH 7.6 group, with significant
differences being observed on days 3 and 7 (P<0.01) (Fig. 1a). Cell numbers in the HC030031 treated
groups also increased in a time-dependent manner, and were lower than those in the non-HC030031
group, but no significant differences were observed among the pH 7.6, 8.0, and 8.4 groups (P<0.01) (Fig.
1b).
Expression of phospho-p44/42
Western blot analyses were performed in order to investigate the expression of phospho-p44/42, which
is also known as mitogen-activated protein kinase (MAPK), a marker of cell proliferation, after changing
(as a loading control) in all samples. The expression of phospho-p44/42 was stronger in the pH 8.0 and
pH 8.4 medium than in the pH 7.6 medium at 72 h. The expression of phospho-p44/42 was significantly
weaker in the HC030031-treated groups than in the non-HC030031-treated groups (Fig. 2).
ALP activity
ALP activity was measured on day 7 and was stronger in the pH 8.0 and 8.4 medium than in the pH 7.6
medium (Fig. 3a). ALP activity was lower in the HC030031-treated group than in non-HC030031-treated
group, with significant differences being observed on day 7.
Alizarin red staining
The effect of TRPA1 channel activity on mineralization was examined using alizarin red staining. Alizarin
red staining revealed increased mineralization in the high pH groups, whereas staining was markedly
weaker in the HC030031-treated groups (Fig. 3b). Alizarin red staining revealed mineralization in both
groups (with or without HC030031). There was no difference between the pH 7.6 group with or without
the TRPA1 antagonist. Red staining, which indicates mineralization by alizarin red staining, was
HC030031. Darker red staining was seen in the pH 8.4 group and was reduced in the pH 8.4 group
treated with the TRPA1 antagonist HC030031.
Discussion
The present study investigated the responses of a human cementoblast cell line to extracellular alkaline
conditions.
Previous studies examined cell responses to extracellular alkaline environments in various
types of cells, such as odontoblasts (Hirose et al. 2016, Kimura et al. 2016). Difficulties are associated
with controlling high pH conditions in a CO2 incubator because an alkaline pH in the culture medium is
changed to a neutral pH in the presence of CO2. Kimura et al. (2016) performed cell culture using a high
pH medium at 37 °C without CO2 for 14 h per day for 14 or 28 days. However, the experimental
conditions of previous studies needed to be improved because they were not similar to in vivo or in situ
conditions. In the present study, high pH conditions were maintained using NaHCO3-free culture
medium in an incubator without CO2. The preliminary results revealed that changes in pH were less
(NaHCO3-free culture medium without CO2) used in the present study are suitable for investigating cell
responses to changes in pH.
It remains unclear whether alkaline conditions induce proliferation. Fliefel et al. (2016)
reported greater proliferation rates of human mesenchymal stem cells cultured in alkaline medium (pH
8.0) than in normal pH medium. Hirose et al. (2016) reported a higher proliferation of human dental pulp
cells cultured in pH 7.9 medium with NaHCO3 and in pH 9.5 medium without NaHCO3. However, those
studies used culture conditions in a CO2 incubator, and thus the pH became neutral. In the present study,
cell proliferation was evaluated under culture conditions without CO2, and demonstrated that
proliferation was enhanced with alkaline media, such as pH 8.0 and 8.4, which is consistent with
previous findings. Furthermore, it has been reported previously that a high extracellular pH induces the
activation of TRPA1 (Kimura et al. 2016), and the activation of TRPA1 by phenytoin has been shown to
increase the proliferation of human gingival fibroblasts (Lopez-Gonzalez et al. 2017). Therefore, it was
hypothesized that an alkaline pH stimulates channels and activates extracellular-regulated kinase
(ERK; phospho-p44/42) in HCEM. The present results revealed that the expression of the proliferation
marker phospho-p44/42 was stronger at pH 8.0 and pH 8.4, and was inhibited by HC030031, a TRPA1
channels and increases the expression of phospho-p44/42.
Previous studies demonstrated that TRP channels exist in odontoblasts (Okumura et al. 2005,
Son et al. 2009, Tsumura et al. 2012, 2013, Sato et al. 2013, Shibukawa et al. 2015). The activation of
TRPA1 by an alkaline stimulation was recently reported to result in earlier mineralization in an
odontoblast cell line (Kimura et al. 2016). However, the involvement of TRPA1 in the mineralization of
cementoblasts currently remains unknown. A few cementoblast cell lines have been generated and their
characteristics and responses to stimuli have been elucidated (Kitagawa et al. 2006, Matsunaga et al.
2016). In the present study, the results of alizarin red staining showed that mineralization increased in
the high pH medium compared to the pH 7.6 medium, and was inhibited by the TRPA1 antagonist
HC030031. The results indicate that TRPA1 is involved in the mineralization of cementoblasts as well as
odontoblasts as shown in a previous study (Kimura et al. 2016). Further studies, such as the
measurement of intracellular and extracellular calcium ions after application of the TRPA1 antagonist,
will be necessary to clarify the involvement of TRPA1 in the mineralization of cementoblasts.
ALP is one marker of differentiation and mineralization in osteogenic cells. The behavior of
osteoblasts in the proliferating phase differs from that in the differentiating phase. A time-dependent
proliferation of osteoblasts was found to be predominant following differentiation in the early stages
(Katagiri et al. 1990). On the other hand, pH was shown to affect the proliferation and mineralization of
mesenchymal stem cells without influencing osteogenic differentiation (Fliefel et al. 2016). In the present
study, ALP activity increased in the high pH medium, and decreased in the HC030031-treated groups,
although HCEM was used instead of osteoblastic cells. Furthermore, the results of alizarin red staining
revealed an increase of mineralization by HCEM in the high pH medium and a reduction of
mineralization following treatment with the TRPA1 antagonist HC030031. These results suggest that
alkaline conditions promote differentiation and mineralization via the activation of TRPA1 in HCEM.
Conclusions
Extracellular alkaline conditions promote the proliferation and mineralization of human
cementoblasts in vitro via TRPA1.
We thank Professor Takashi Takata and Dr. Masae Kitagawa, Hiroshima University Graduate School,
for providing HCEM. This study was supported in part by a grant from the Multidisciplinary Research
Center for Jaw Disease (MRCJD) from Tokyo Dental College.
Conflicts of Interest
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Figure legends
Figure 1: Cell growth
(a) Cell number in high pH medium. Cell numbers increased in a time-dependent manner in all 3 pH
groups. The number of cells in the high pH groups (pH 8.0 and 8.4) was significantly higher than in the
normal pH group (pH 7.6) on days 3 and 7 (P<0.01).
(b) Cell numbers in the HC030031-treated groups. Cell numbers in the HC030031-treated groups
increased in a time-dependent manner as well in the groups without HC030031. The number of cells in
the HC030031-treated groups was lower than in the non-HC030031-treated groups on days 3 and 7. No
significant differences were observed among the pH 7.6, 8.0 and 8.4 groups.
*
statistical significance P < 0.01; (-): without HC030031, (+): with HC030031Figure 2: Expression of phospho-p44/42
Western blot analysis showing that the expression of phospho-p44/42 was stronger in the high pH
groups than in the pH 7.6 group at 72 h. The expression of phospho-p44/42 was significantly weaker in
Figure 3: ALP activity and alizarin red staining
(a) ALP activity in cells cultured in normal and high pH media.
ALP activity was measured on day 7, and was stronger in the high pH medium groups (pH 8.0 and 8.4)
than in the pH 7.6 medium group. ALP activity was weaker in the HC030031-treated groups than in the
HC030031-untreated groups, with significant differences observed on day 7.
*
statistical significance (P < 0.01)(b) Alizarin red staining
Alizarin red staining revealed mineralization in both groups (treated with or without HC030031). There
was no difference between the pH 7.6 group treated with or without the TRPA1 antagonist. Stronger red
staining, indicating mineralization by alizarin red staining, was seen in the pH 8.0 group compared to the
pH 8.0 group treated with the TRPA1 antagonist. Darker red staining was seen in the pH 8.4 medium
