Nitric oxide modulates the responses of osteoclast formation to static magnetic fields

Nitric oxide (NO) is involved in osteoclast differentiation. Our previous studies showed that static magnetic fields (SMFs) could affect osteoclast differentiation. The inhibitory effects of 16 T of high SMF (HiMF) on osteoclast differentiation was correlated with increased production of NO. We raised the hypothesis that NO mediated the regulatory role of SMFs on osteoclast formation. In this study, 500 nT of hypomagnetic field (HyMF), 0.2 T of moderate SMF (MMF) and 16 T of high SMF (HiMF) were utilized as SMF treatment. Under 16 T, osteoclast formation was markedly decreased with enhanced NO synthase (NOS) activity, thus producing a high level of NO. When treated with NOS inhibitor N-Nitro-L-Arginine Methyl Ester (L-NAME), NO production could be inhibited, and osteoclast formation was restored to control group level in a concentration-dependent manner. However, 500 nT and 0.2 T increased osteoclast formation with decreased NOS activity and NO production. When treated with NOS substrate L-Arginine (L-Arg) or NO donor sodium nitroprusside (SNP), the NO level in the culture medium was obviously elevated, thus inhibiting osteoclast differentiation in a concentration-dependent manner under 500 nT or 0.2 T. Therefore, these findings indicate that NO mediates the regulatory role of SMF on osteoclast formation.     

Regulation of Osteoblast Differentiation and Iron Content in MC3T3-E1 Cells by Static Magnetic Field with Different Intensities

Many studies have indicated that static magnetic fields (SMFs) have positive effects on bone tissue, including bone formation and bone healing process. Evaluating the effects of SMFs on bone cell (especially osteoblast) function and exploring the mechanism, which is critical for understanding the possible risks or benefits from SMFs to the balance of bone remodeling. Iron and magnetic fields have the natural relationship, and iron is an essential element for normal bone metabolism. Iron overload or deficiency can cause severe bone disorders including osteoporosis. However, there are few reports regarding the role of iron in the regulation of bone formation under SMFs. In this study, hypomagnetic field (HyMF) of 500 nT, moderate SMF (MMF) of 0.2 T, and high SMF (HiMF) of 16 T were used to investigate how osteoblast (MC3T3-E1) responses to SMFs and iron metabolism of osteoblast under SMFs. The results showed that SMFs did not pose severe toxic effects on osteoblast growth. During cell proliferation, iron content of osteoblast MC3T3-E1 cells was decreased in HyMF, but was increased in MMF and HiMF after exposure for 48 h. Compared to untreated control (i.e., geomagnetic field, GMF), HyMF and MMF exerted deleterious effects on osteoblast differentiation by simultaneously retarding alkaline phosphatase (ALP) activity, mineralization and calcium deposition. However, when exposed to HiMF of 16 T, the differentiation potential showed the opposite tendency with enhanced mineralization. Iron level was increased in HyMF, constant in MMF and decreased in HiMF during cell differentiation. In addition, the mRNA expression of transferrin receptor 1 (TFR1) was promoted by HyMF but was inhibited by HiMF. At the same time, HiMF of 16 T and MMF of 0.2 T increased the expression of ferroportin 1 (FPN1). In conclusion, these results indicated that osteoblast differentiation can be regulated by altering the strength of the SMF, and iron is possibly involved in this process.

     

Effects of static magnetic fields on bone microstructure and mechanical properties in mice

All the living organisms originate, evolve and live under geomagnetic field (GMF, 20–70 µT). With rapid development in science and technology, exposure to various static magnetic fields (SMFs) from natural and man-made sources remains a public environmental topic in consideration of its probable health risk for humans. Many animal studies related to health effect have demonstrated that SMF could improve bone formation and enhance bone healing. Moreover, most of the studies focused on local SMF generated by rod-type magnet. It was difficult to come to a conclusion that how SMF affected bone metabolism in mice. The present study employed hypomagnetic field (HyMF, 500 nT), and moderate SMF (MMF, 0.2 T) to systematically investigate the effects of SMF with continuous exposure on microstructure and mechanical properties of bone. Our results clearly indicated that 4-week MMF exposure did not affect bone biomechanical properties or bone microarchitecture, while HyMF significantly inhibited the growth of mice and elasticity of bone. Furthermore, mineral elements might mediate the biological effect of SMF.     

The role of the calmodulin‐dependent pathway in static magnetic field‐induced mechanotransduction

While the effects of static magnetic fields (SMFs) on osteoblastic differentiation are well demonstrated, the mechanotransduction pathways of SMFs are still unclear. The aim of this study was to explore the role of calmodulin in the biophysical effects of SMFs on osteoblastic cells. MG63 cells were exposed to a 0.4 T SMF. The expression of phosphodiesterase RNA in the cytoplasm was tested using real‐time polymerase chain reaction. The differentiation of the cells was assessed by detecting changes in alkaline phosphatase activity. The role of calmodulin antagonist W‐7 was used to evaluate alterations in osteoblastic proliferation and differentiation after the SMF simulations. Our results showed that SMF exposure increased alkaline phosphatase activity and phosphodiesterase 1C gene expression in MG63 cells. Addition of W‐7 significantly inhibited the SMF‐induced cellular response. We suggest that one possible mechanism by which SMFs affects osteoblastic maturation is through a calmodulin‐dependent mechanotransduction pathway. Bioelectromagnetics 31:255–261, 2010. © 2009 Wiley‐Liss, Inc.     

Effects of strong static magnetic fields used in magnetic resonance imaging on insulin-secreting cells

The magnetic flux density of MRI for clinical diagnosis has been steadily increasing. However, there remains very little biological data regarding the effect of strong static magnetic fields (SMFs) on human health. To evaluate the effects of strong SMFs on biological systems, we cultured insulin-secreting cells under exposure to sham and SMF conditions (3-10 T of magnetic flux density, and 0-41.7 T/m of magnetic field gradient) for 0.5 or 1 h, and analyzed insulin secretion, mRNA expression, glucose-stimulated insulin secretion, insulin content, cell proliferation and cell number. Exposure to SMF with a high magnetic field gradient for 1 h significantly increased insulin secretion and insulin 1 mRNA expression. Exposure to SMF with a high magnetic flux density for 0.5 h significantly enhanced responsiveness to glucose stimulation. Exposure to SMF did not affect the insulin content, cell proliferation or cell number. Our results suggested that MRI systems with a higher magnetic flux density might not cause cell proliferative or functional damages on insulin-secreting cells, and that SMF with a high magnetic field gradient might be used clinically after thorough in vivo investigations are conducted.     

An upward 9.4 T static magnetic field inhibits DNA synthesis and increases ROS-P53 to suppress lung cancer growth

Studies have shown that 9.4 Tesla (9.4 T) high-field magnetic resonance imaging (MRI) has obvious advantages in improving image resolution and capacity, but their safety issues need to be further validated before their clinical approval. Meanwhile, emerging experimental evidences show that moderate to high intensity Static Magnetic Fields (SMFs) have some anti-cancer effects.We examined the effects of two opposite SMF directions on lung cancer bearing mice and found when the lung cancer cell-bearing mice were treated with 9.4 T SMFs for 88 h in total, the upward 9.4 T SMF significantly inhibited A549 tumor growth (tumor growth inhibition=41%), but not the downward 9.4 T SMF. In vitro cellular analysis shows that 9.4 T upward SMF treatment for 24 h not only inhibited A549 DNA synthesis, but also significantly increased ROS and P53 levels, and arrested G2 cell cycle. Moreover, the 9.4 T SMF-treatments for 88 h had no severe impairment to the key organs or blood cell count of the mice.Our findings demonstrated the safety of 9.4 T SMF long-term exposure for their future applications in MRI, and revealed the anti-cancer potential of the upward direction 9.4 T SMF.     

Magnetic field direction differentially impacts the growth of different cell types

Magnetic resonance imaging (MRI) machines have horizontal or upright static magnetic field (SMF) of 0.1–3 T (Tesla) at sites of patients and operators, but the biological effects of these SMFs still remain elusive. We examined 12 different cell lines, including 5 human solid tumor cell lines, 2 human leukemia cell lines and 4 human non-cancer cell lines, as well as the Chinese hamster ovary cell line. Permanent magnets were used to provide 0.2–1 T SMFs with different magnetic field directions. We found that an upward magnetic field of 0.2–1 T could effectively reduce the cell numbers of all human solid tumor cell lines we tested, but a downward magnetic field mostly had no statistically significant effect. However, the leukemia cells in suspension, which do not have shape-induced anisotropy, were inhibited by both upward and downward magnetic fields. In contrast, the cell numbers of most non-cancer cells were not affected by magnetic fields of all directions. Moreover, the upward magnetic field inhibited GIST-T1 tumor growth in nude mice by 19.3% (p < 0.05) while the downward magnetic field did not produce significant effect. In conclusion, although still lack of mechanistical insights, our results show that different magnetic field directions produce divergent effects on cancer cell numbers as well as tumor growth in mice. This not only verified the safety of SMF exposure related to current MRI machines but also revealed the possible antitumor potential of magnetic field with an upward direction.     

静磁场对小鼠黑色素瘤细胞B16生长和氧化应激的影响

目的:利用小鼠黑色素瘤细胞B16,研究静磁场对肿瘤细胞生长和氧化应激的影响,探讨氧化应激介导静磁场影响肿瘤细胞生长的机制,为磁场在肿瘤疾病的治疗中的应用提供理论依据。方法:采用MTT法测定磁场对B16细胞活力的影响;利用流式细胞仪测定静磁场暴露对B16细胞周期分布的影响;利用生物化学方法测定磁场暴露对细胞氧化防御系统相关蛋白酶活性的影响。结果:24 h内50 m T-200 m T静磁场暴露可以抑制B16生长,但超过24 h的磁场暴露可以促进B16生长;100 m T和200 m T静磁场暴露对B16的细胞周期分布没有影响;B16暴露于100 m T和200 m T静磁场48 h,GST活性和GSH/GSSG水平表现为先上升后下降,SOD活性和T-AOC水平先下降后上升,CAT活性没有受到影响。结论:50 m T-200 m T静磁场可以抑制小鼠黑色素瘤细胞B16的生长,诱导肿瘤细胞产生氧化应激。     

Application of Weak Sinusoidal Magnetic Field on Flavobacterium Species in the Treatment of Paper Mill Effluent

This paper presents the results of a preliminary study on the effects of sinusoidal magnetic fields on the growth and degradation potential of Flavobacterium species in paper mill effluents. The paper presents a brief account of the experimental set-up, protocol and the essential parameters employed. The study was carried out using a pure colony of Flavobacterium species and subjecting them to Sinusoidal Magnetic Fields (SMF) at different frequencies, intensities and duration of exposure in order to obtain the “frequency window” of optimum response. The organisms were subjected to 1 Hz (100 nT, 1500 nT and 4000 nT) for 5 hours per day for 3, 6, 9, 12, 15, 18 and 20 days and to 10 Hz (100 nT, 1500 nT and 4000 nT) for 5 hours per days for 3, 6, 9, 12, 15, 18 and 20 days. The organism has been primarily analyzed for its efficacy in the treatment of paper mill effluent by using a sinusoidal magnetic field. The growth kinetics of the bacterium with the application of sinusoidal magnetic fields was studied. As judged from the physico-chemical properties of the effluent, Flavobacterium species was found to have a four fold increase with respect to growth when exposed to SMF of 10 Hz, 100 nT for 30 hrs (5 hours per day for 6 days). The BOD, COD, lignin, phenol and protein content were found reduced in the effluent using SMF treated cells.

Pre-treatment of Flavobacterium species with Sinusoidal Magnetic Fields (SMF) appears to result in more effective degradation of the paper mill effluents.     

Exposure to a MRI‐type high‐strength static magnetic field stimulates megakaryocytic/erythroid hematopoiesis in CD34+ cells from human placental and umbilical cord blood

The biological response after exposure to a high‐strength static magnetic field (SMF) has recently been widely discussed from the perspective of possible health benefits as well as potential adverse effects. To clarify this issue, CD34⁺ cells from human placental and umbilical cord blood were exposed under conditions of high‐strength SMF in vitro. The high‐strength SMF exposure system was comprised of a magnetic field generator with a helium‐free superconducting magnet with built‐in CO₂ incubator. Freshly prepared CD34⁺ cells were exposed to a 5 tesla (T) SMF with the strongest magnetic field gradient (41.7 T/m) or a 10 T SMF without magnetic field gradient for 4 or 16 h. In the harvested cells after exposure to 10 T SMF for 16 h, a significant increase of hematopoietic progenitors in the total burst‐forming unit erythroid‐ and megakaryocytic progenitor cells‐derived colony formation was observed, thus producing 1.72‐ and 1.77‐fold higher than the control, respectively. Furthermore, early hematopoiesis‐related and cell cycle‐related genes were found to be significantly up‐regulated by exposure to SMF. These results suggest that the 10 T SMF exposure may change gene expressions and result in the specific enhancement of megakaryocytic/erythroid progenitor (MEP) differentiation from pluripotent hematopoietic stem cells and/or the proliferation of bipotent MEP. Bioelectromagnetics 30:280–285, 2009. © 2009 Wiley‐Liss, Inc.     

Osteoporosis regulation by salubrinal through eIF2α mediated differentiation of osteoclast and osteoblast

Nuclear factor-κB (NF-κB) ligand (RANKL) was shown to induce osteoclast differentiation by increasing the expression of c-Fos, NFATc1 and TRAP. Salubrinal treatment to bone marrow macrophage (BMM) cells, however, significantly blocked NFATc1 expression and osteoclast differentiation by RANKL. Overexpression of NFATc1 further confirmed that NFATc1 is a key factor affected by salubrinal in osteoclast differentiation by RANKL. Unexpectedly, NFATc1 and c-Fos mRNA expressions were not affected by salubrinal, implicating that NFATc1 expression is regulated at a translational stage. In support of this, salubrinal increased the phosphorylation of a translation factor eIF2α, decreasing the global protein synthesis including NFATc1. In contrast, a phosphorylation mutant plasmid pLenti-eIF2α-S51A restored RANKL-induced NFATc1 expression and osteoclast differentiation even in the presence of salubrinal. Furthermore, knockdown of ATF4 significantly reduced salubrinal-induced osteoblast differentiation as evidenced by decreased calcium accumulation and lowered expressions of the osteoblast differentiation markers, alkaline phosphatase and RANKL in MC3T3-E1 osteoblast cells. Salubrinal treatment to co-cultured BMM and MC3T3-E1 cells also showed reduction of osteoclast differentiation. Finally, salubrinal efficiently blocked osteoporosis in mice model treated with RANKL as evidenced by elevated bone mineral density (BMD) and other osteoporosis factors. Collectively, our data indicate that salubrinal could affect the differentiation of both osteoblast and osteoclast, and be developed as an excellent anti-osteoporosis drug. In addition, modulation of ATF4 and NFATc1 expressions through eIF2α phosphorylation could be a valuable target for the treatment of osteoporosis.     

Stimulation of Bone Formation in Cortical Bone of Mice Treated with a Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-binding Peptide That Possesses Osteoclastogenesis Inhibitory Activity

To date, parathyroid hormone is the only clinically available bone anabolic drug. The major difficulty in the development of such drugs is the lack of clarification of the mechanisms regulating osteoblast differentiation and bone formation. Here, we report a peptide (W9) known to abrogate osteoclast differentiation in vivo via blocking receptor activator of nuclear factor-κB ligand (RANKL)-RANK signaling that we surprisingly found exhibits a bone anabolic effect in vivo. Subcutaneous administration of W9 three times/day for 5 days significantly augmented bone mineral density in mouse cortical bone. Histomorphometric analysis showed a decrease in osteoclastogenesis in the distal femoral metaphysis and a significant increase in bone formation in the femoral diaphysis. Our findings suggest that W9 exerts bone anabolic activity. To clarify the mechanisms involved in this activity, we investigated the effects of W9 on osteoblast differentiation/mineralization in MC3T3-E1 (E1) cells. W9 markedly increased alkaline phosphatase (a marker enzyme of osteoblasts) activity and mineralization as shown by alizarin red staining. Gene expression of several osteogenesis-related factors was increased in W9-treated E1 cells. Addition of W9 activated p38 MAPK and Smad1/5/8 in E1 cells, and W9 showed osteogenesis stimulatory activity synergistically with BMP-2 in vitro and ectopic bone formation. Knockdown of RANKL expression in E1 cells reduced the effect of W9. Furthermore, W9 showed a weak effect on RANKL-deficient osteoblasts in alkaline phosphatase assay. Taken together, our findings suggest that this peptide may be useful for the treatment of bone diseases, and W9 achieves its bone anabolic activity through RANKL on osteoblasts accompanied by production of several autocrine factors.     

Myotube orientation using strong static magnetic fields

In this experiment, we evaluated the effects of strong static magnetic fields (SMF) on the orientation of myotubes formed from a mouse-derived myoblast cell line, C2C12. Myogenic differentiation of C2C12 cells was conducted under exposure to SMF at a magnetic flux density of 0-10 T and a magnetic gradient of 0-41.7 T/m. Exposure to SMF at 10 T led to significant formation of oriented myotubes. Under the high magnetic field gradient and a high value of the product of the magnetic flux density and magnetic field gradient, myotube orientation increased as the myogenic differentiation period increased. At the 3 T exposure position, where there was a moderate magnetic flux density and moderate magnetic field gradient, myotube orientation was not observed. We demonstrated that SMF induced the formation of oriented myotubes depending on the magnetic flux density, and that a high magnetic field gradient and a high value of the product of the magnetic flux density and magnetic field gradient induced the formation of oriented myotubes 6 days after myogenic differentiation. We did not detect any effect of the static magnetic fields on myogenic differentiation or cell number. To the best of our knowledge, this is the first report to demonstrate that myotubes orient to each other under a SMF without affecting the cell number and myogenic differentiation.     

Effects of homogeneous and inhomogeneous static magnetic fields combined with gamma radiation on DNA and DNA repair

The aim of this study was to reveal whether static magnetic fields (SMFs) influence the repair of radiation‐damaged DNA on leukocytes or has any effect on DNA. After 4 Gy of ⁶⁰Co‐γ irradiation, some of the samples were exposed to inhomogeneous SMFs with a lateral magnetic flux density gradient of 47.7, 1.2, or 0.3 T/m by 10 mm lateral periodicity, while other samples were exposed to homogeneous SMF of 159.2 ± 13.4 mT magnetic flux density for a time period of 0.5 min, 1, 2, 4, 6, 18, 20, or 24 h. Another set of samples was exposed to the aforementioned SMFs before gamma irradiation. The following three groups were examined: (i) exposed to SMF only, (ii) exposed to SMF following irradiation by ⁶⁰Co‐γ, and (iii) exposed to SMF before ⁶⁰Co‐γ irradiation. The analysis of the DNA damage was made by single‐cell gel electrophoresis technique (comet assay). Statistically significant differences were found at 1 h (iSMF), 4 h (hSMF), and 18 h (hSMF) if samples were exposed to only SMF, compared to control. When the SMF exposure followed the ⁶⁰Co‐γ irradiation, statistically significant differences were found at 1 h (iSMF) and 4 h (hSMF). If exposure to SMF preceded ⁶⁰Co‐γ irradiation, no statistically significant difference was found compared to 4 Gy gamma‐irradiated group. Bioelectromagnetics 31:488–494, 2010. © 2010 Wiley‐Liss, Inc.     

The Influence of a gradient static magnetic field on an unstirred Belousov-Zhabotinsky reaction

It is believed that static magnetic fields (SMF) cannot affect the pattern formation of the Belousov-Zhabotinsky (BZ) reaction, which has been frequently studied as a simplified experimental model of a nonequilibrium open system, because SMF produces no induced current and the magnetic force of SMF far below 1 T is too low to expect the effects on electrons in the BZ reaction. In the present study, we examined whether the velocity of chemical waves in the unstirred BZ reaction can be affected by a moderate-intensity SMF exposure depending on the spatial magnetic gradient. The SMF was generated by a parallel pair of attracting rectangular NdFeB magnets positioned opposite each other. The respective maximum values of magnetic flux density (B(max)), magnetic flux gradient (G(max)), and the magnetic force product of the magnetic flux density its gradient (a magnetic force parameter) were 206 mT, 37 mT/mm, and 3,000 mT(2)/mm. The ferroin-catalyzed BZ medium was exposed to the SMF for up to 16 min at 25 degrees C. The experiments demonstrated that the wave velocity was significantly accelerated primarily by the magnetic gradient. The propagation of the fastest wave front indicated a sigmoid increase along the peak magnetic gradient line, but not along the peak magnetic force product line. The underlying mechanisms of the SMF effects on the anomalous wave propagation could be attributed primarily to the increased concentration gradient of the paramagnetic iron ion complexes at the chemical wave fronts induced by the magnetic gradient.