Effect of Magnetic Fields on Osteoblasts and Fibroblasts in vitro

 

R. Fukuzawa¹, S. Ozawa¹, K. Kubo², Y. Sugita², W. Yoshida², H. Maeda² and Y. Tanaka¹

 

¹Department of Removable Prosthodontics, School of Dentistry, Aichi-Gakuin University

²Department of Oral Pathology, School of Dentistry, Aichi-Gakuin University

Introduction

Effects of magnetic fields on bone biology has been interested, however, those specificity and mechanism of the magnetic fields on bone have not been revealed yet^1-2). The purpose of this study is to compare osteoblasts which are responsible to bone formation with fibroblasts which do not make bone, and explore a mechanism of accelerated bone formation by the magnetic field exposure.

 

Materials and Methods

In this study MC3T3-E1 cells that are established osteoblast cell strain form mouse calvaria and L929 that is standard fibroblast cell strain were used. Those cells were inoculated onto 12 well plate by 1 x 10⁴ cells per well and incubated in 37゜C, 5%CO₂ atmosphere. A time varying electro-magnetic power unit generates maximum 1T magnetic field in the culture area of 120mm x 120mm (Fig.1). Frequency of the magnetic field can also be changed from 0 to 1Hz. This unit equipped cell culture chamber fitted to magnetic field exposure area. The chamber can carry a culture plate inside the slot. A strength and frequency of extremely low magnetic fields (ELMF) was set to 0.4T and 0.17 Hz respectively, and the ELMF was exposed to semi-confluent culture plates for 6 hours.

Cell proliferation was assessed by using a colorimetric proliferation assay (WST-8 Cell counting kit , Dojindo, Kumamoto, Japan) at day 1, 3, 7, and day 10 after ELMF exposure. We determined the hormazan content in the samples by measuring the absorbance at 450 nm. Alkaline phosphatase (ALP) activities of MC3T3-E1 cells were measured at day 3, 7,and day 10 after ELMF exposure to evaluate osteoblast differentiation. ALP activities were standardized by total protein content that were measured by the Bradford method. Statistical calculation was performed by student t-test at significant level of 5%.

 

Results

Proliferation of MC3T3-E1 cells were promoted at day 3 after ELMF exposure, although day 1, day 7 and day 10 cultures did not show significant differences between the control and the exposed groups. On the proliferation of L929 cells, the exposed culture did not show any differences at any time points compared with the control cultures. ALP activity in MC3T3-E1 cell significantly increased at day 7 and day10 as compared with the controls(Fig.2-4).

Discussion

This study revealed that ELMF stimulated proliferation of MC3T3-E1 osteoblast like cells in early stage, and then promoted osteoblastic differentiation at later stage. Whereas ELMF did not have significant effects on proliferation of L929 fibroblastic cells.

Soda et al. reported that collagen synthesis stimulated by ELMF could be mediated by p38 MARK pathway and suppressed the collagen synthesis by PI3K pathway³⁾. Moreover, Nakano suggested that ELMF induced differentiation of osteoblasts, but not act like Tri-iodothyronine (T3); a regulator of osteoblastic differentiation⁴⁾. Since various studies related to the biological effect of magnetic field have been done, mechanism of bone formation and the magnetic field stimulation have not been elucidated yet. Further study is needed to understand ELMF effects on osteoblastic cell by molecular biological methods.

 Conclusion

ELMF of 0.4T and 0.17Hz stimulated mouse osteoblast-like cell proliferation at early stage, and differentiation to mature osteoblasts, whereas fibroblasts did not show significant differences in proliferation by the exposure. These results suggested that osteoblasts have specific response on the magnetic fields.

 

References

 

1.                Y. Imaizumi, S. Ozawa , T. Shigemori, et al.: Effect of extremely low frequency strong magnetic field on proliferation of osteoblastic cells, J J Mag Dent, 15-2, 2006.

2.                K. Shoumura: Effects of pulsed electromagnetic stimulation for proliferation and calcification of osteoblast like MC3T3-E1 cell. J Jpn Orthod Soc 56(4) 211-223, 1997.

3.                A. Soda, T. Ikehara, Y. Kinouchi, et al.: Effect of exposure to an extremely low frequency-electromagnetic field on the cellular collagen with respect to signaling pathways in osteoblast-like cells. The Journal of Medical Investigation, 55, 2008. 

4.                Y. Nakano, K. Hosokawa, H. Yamaguchi et al.: Effects of ELF magnetic fields on physiological functions in cultured osteoblastic cells. IEICE Technical Report , 101(182), 19-24, 2001.