An extremely low magnetic field exposure in dental pulp stem cells
M. Hata, M. Omi, R. Fukuzawa, N. Kojima, Y. Nakamura, S. Ozawa, and Y. Tanaka
Departments of Removable Prosthodontics, School of Dentistry, Aichi-Gakuin University

Introduction
Dental pulp stem cell (DPSCs) which are a sort of mesenchymal stem cells locates in the dental
pulp cavity. DPSCs can be obtained from wasted tissue and have differentiation capability. SoDPSCs are suitable for transplantation therapy, which need to ensure a large amount of transplanted
cell. The stimulation to cells before transplantation is thought to be a crucial point. We havepreviously studied mechanical stress increased the proliferation activity of DPSCs.

Objective
Aim of this study is to evaluate the response of an extremely low magnetic field (ELMF) inDPSCs.

Materials and Methods
Isolation and characterization of DPSCs
DPSCs were isolated and cultured from 6-week-old male Sprague-Dawley rats as previously
described (Fig.1). Identification of DPSCs was analyzed by a fluorescence activated cell sorter(FACS). Cells were differentiated adipocytes or osteoblasts using differentiation medium.

Fig.1 Dental pulp stem cells

ELF magnetic exposure condition
The electro-magnetic power unit produce maximum 1T magnetic field in the culture area of 120
mm x 120 mm. This units is equipped with cell culture chamber fitted to the magnetic field exposurearea. A control chamber is also prepared in order to compare another plate under the same condition.
The strength and frequency of the magnetic fields was set to 0.4 T and 0.17 Hz respectively for 6hours.

Proliferation analysis of DPSCs
The proliferation ability of DPSCs was investigated by MTT assay according to the manufacturer’sprocedure. After magnetic field exposure for 6 h, cells were seeded in a 96-well plate and cultured
for an additional 6 h. Then, the MTT Reagent was added to each well and the plates were incubatedfor 3 h. Formazan crystals were dissolved and the absorbance was measured at 570nm using a
multilabel luminescence counter.

Osteogenic differentiation of DPSCs
The osteogenic differentiation were conducted as described above with or without magnetic field
exposure once a three day for 2 weeks. The osteogenic differentiation was detected by the staining
with ALP and osteocalcin.Total cell count was calculated by the nuclei staining with
4¢-6-Diamidino-2-phenylindole (DAPI). We randomly counted the number of theosteocalcin-positive cells and the cell nuclei stained by DAPI at 10 different areas and averaged thecounting number in each chamber.

Results
Proliferation analysis of DPSCs
The effect of magnetic field exposure on the proliferation of DPSCs was evaluated by MTT
assay. MTT assay revealed that this stimulation increased the proliferation activity of DPSCs by
1.5-fold compared with control (p<0.05)(Fig.2).

ratio

p<0.05Fig.2 Effect of ELMF on the proliferation of DPSCs.

Osteogenic differentiation of DPSCs
To examine the effect of magnetic field exposure in DPSCs, an extremely low magnetic field
was inducted to DPSCs once a three days for 2 weeks. The osteogenic differentiation was evaluatedby ALP and the immunohistological staining of osteocalcin. ALP-staining cells were observed by
ELMF (Fig.3A:control, B:ELMF). The rate of osteocalcin-positive cells to whole cells weresignificantly increased by ELMF compared with control cells (ELMF exposed cells;8.84%±1.92%,control cells; 0.77%±0.3%, p<0.05) (Fig.3C).

A

B

C%

p<0.05

Fig.3 Effect of ELMF on the osteogenic differentation of DPSCs

Discussions
DPSCs are expected to be a promising source of regenerative medicine, since collection of cellis easy to access. We have revealed magnetic field promote proliferation and osteogenic
differentiation on DPSCs. Fukuzawa et al. indicated that osteoblasts (MC3T3-E1) were stimulated
3days after ELMF exposure and increased 7 and 10 days after the exposure on ALP activities.
Calcified nodule formation was also promoted as compared to the no exposed control cells on day
21.We showed that ALP-staining cells and osteocalcin-positive cells were observed by ELMF
exposure once a three days for two weeks, though differential rate is low percent. Further study isrequired to clarify this issue in effect of magnetic field on DPSCs.

Conclusions
An extremely low magnetic field exposure may be thought to promote application of DPSCs forbone regeneration.

References
1. Gronthos, S., Mankani, M., Brahim, J., Robey, P.G., Shi,S., Postnatal human dental pulp stemcells (DPSCs) in vitro and in vivo., Proc Natl Acad Sci U S A 97, 13625, 2000.
2. Fukuzawa R, Ozawa S, Anuudari E, Komori A, Yoshiyama M, Kubo K, Sugita Y, Yoshida W,
Maeda H, Tanaka Y, Different Responses of Osteoblasts and Fibroblasts to a Changing MagneticField, J. Hard Tissue Biol 22[2] (2013) p183-188
3. Hata M, Naruse K, Ozawa S, Kobayashi Y, Nakamura N, Kojima N, Omi M, Katanosaka Y,Nishikawa T, Naruse K, Tanaka Y, Matsubara T. Mechanical stretch increases the proliferation
while inhibiting the osteogenic differentiation in dental pulp stem cells. Tissue Eng Part A. 2013
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