| Co-Investigator(Kenkyū-buntansha) |
KANNO Masamoto Hiroshima University, Graduate School of Biomedical Sciences, Professor, 大学院・医歯薬学総合研究科, 教授 (40161393)
KATAOKA Katsuko Hiroshima University, Graduate School of Biomedical Sciences, Professor, 大学院・医歯薬学総合研究科, 教授 (30034002)
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| Research Abstract |
Physical environment including gravity are believed to play an important role in muscle and bone atrophy and / or regeneration in space flight. We developed here new methods for studying the effects of magnetic force and multidirectional gravity vector environment on cell differentiation.
I. Effect of magnetic force
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 cell 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). We did not notice the difference in cell numbers among the four experimental groups throughout the culture. The cells in group MP-MF became larger and were elongated along the axis of the magnetic poles. Appearance of alkaline phospha
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tase (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 phenomena. 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 MAPK between group MP-MF and group C. Phosphorylation of p38^<MAPK> (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 magnetic field offers anew approach to osteoblast differentiation.
II. Effect of multidirectional gravity vector
A 3D-clinostat which is a three-dimensional (3D)-clinostat is a device for multi-direction G force generation. By controlled rotation of two axes, a 3D-clinostat cancels the cumulative gravity vector at the center of the device and produces an environment with an average of 10^<-3>G over time. We cultured a human osteoblast cell line in a 3D-clinostat (group CL), examined differentiation of the cells including morphology, histological detection of calcification and MAPK cascades and compared to those in a normal 1G environment (group C). Cell numbers were not different between the two groups thought the culture. In the group C, AlPase activity was detected on day 7 of culture, bone nodules were formed on day 121 and calcium deposits were seen on day 20. In the group CL, the cells looked larger and bulged. AlPase activity was detected on day 10 of culture. However, neither bone nodules nor calcification was found up to day 21. The expression levels of Cbfal, and osteocalcin increased in group C but decreased in group CL. Phosphorylation of p38^<MAPK> (p38) was repressed in culture in group CL, while total p38 as well as total and phosphorylated forms of ERK 1/2 and SAPK / JNK were not changed in group CL. 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 strongly inhibited. On the other hand, they were inhibited only partially by a MAPKK inhibitor, U-0126. Based on these results, it is concluded that 1) osteoblast differentiation is inhibited in culture in a 3D-clinostat, and 2) this inhibition is mainly due to the suppression of p38 phosphorylation. Less
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