Physical stress by magnetic force accelerates differentiation of human osteoblasts

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Abstract

We examined the effect of magnetic force on differentiation of cultured human osteoblasts. Magnetic microparticles (MPs) were introduced into the cytoplasm of a human osteoblast cell line and the cells were cultured in a magnetic field (MF) in group MP–MF. Three groups of controls were used: cells without MPs were cultured out of MF (group C), cells without MPs were cultured in MF (group MF), and cells with MPs were cultured out of MF (group MP). The cells in group MP–MF became larger and were elongated along the axis of the magnetic poles. Appearance of alkaline phosphatase (AlPase) activity, formation of bone nodules, and calcium deposition were accelerated depending on the intensity of the magnetic field. It takes longer culture in the other three groups to exhibit these changes. Core-binding factor A1 (Cbfa1: transcription factor for osteoblast differentiation) and osteocalcin (a bone-matrix protein involved in controlling osteogenesis) were expressed earlier or stronger in group MP-MF than the other groups. Then we compared phosphorylation of mitogen-activated protein kinase (MAPK) between group MP–MF and group C. Phosphorylation of p38MAPK (p38) was increased in group MP–MF, while total p38 as well as total and phosphorylated forms of MAPK/ERK 1/2 and SAPK/JNK were not changed between the two groups. When a p38 inhibitor, SB 203580, was added to the culture medium in group C, AlPase activity, formation of bone nodules, and calcium deposits were completely inhibited. On the other hand, they were inhibited only partially by a MAPK/ERK 1/2 inhibitor, U-0126. Based on these results, it is concluded that (1) osteoblast differentiation is accelerated by a magnetic force, (2) this acceleration is mainly attributed to the activation of p38 phosphorylation, and (3) the stimulus induced by a magnetic field offers a new approach to osteoblast differentiation.

Section snippets

Materials and methods

Cell culture. A normal human osteoblast (NHOst) cell system was purchased from BioWhittaker (Walkersville, MD). The cells were seeded in 60-mm culture dishes (Nalge Nunc International, Roskilde, Denmark). The cell concentration was 2 × 106 cells/ml and maintained in an osteoblast basal medium (OBM, CC-3208: BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS: CC-4102), 200 nM ascorbic acid (CC-4398), and 200 nM gentamicin/amphotericin-B (CC-4381).

An osteoblast

Uptake of trypan blue and BrdU

There was no difference in the percentages of viable cells (trypan blue-negative cells) or in the percentages of BrdU-uptake cells among group C, group MP, group MF, and group MP–MF 0.01, 0.03, 0.05 T (Fig. 1).

Morphology of cultured osteoblasts in the magnetic force

Differentiation of human osteoblasts in group C was compared with that in group MP, group MF, and group MP–MF (0.01, 0.03, and 0.05 T). In the group C the osteoblasts were thin spindle-shaped. The cells are oriented randomly on day 1 (Fig. 2A). They began to form colonies by day 3. The

Discussion

We originated a physical stimulation method, in which magnetic microparticles were introduced into cultured cells and the culture was placed in a magnetic field [5]. This physical stimulation accelerated differentiation of myoblasts [5], as well as osteoblasts shown in the present study in which the appearance of AlPase activity, formation of bone nodules, and calcification were seen earlier. In the present study we also found that this osteoblast differentiation acceleration is mainly

Acknowledgements

We are grateful to Mr. Toshiaki Mouri of Himeji Denshi Co. for providing technical information. We also thank Mrs. N.J. Johnson for comments on the manuscript. This study was supported by the “Ground Research Announcement for Space Utilization” promoted by the National Space Development Agency of Japan and the Japan Space Forum (to L.Y.), “Space Utilization Research” from ISAS (to L.Y.), and by Grants-in-Aid for scientific research (C), No. 10838021 and No. 12832032 (to L.Y.), from the Ministry

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Abbreviations: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinases; SAPK/JNK, stress-activated protein kinase/jun N-terminal kinase; MAPKK, mitogen-activated protein kinase kinase; AlPase, alkaline phosphatase; Cbfa 1, core-binding factor A1.

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