Effect of low frequency low intensity electromagnetic fields on maturation and mineralization of rat skull osteoblasts <i>in vitro</i>

doi:10.3785/j.issn.1008-9292.2017.12.03

J Zhejiang Univ (Med Sci)

2017, Vol. 46

Issue (6): 585-592 DOI: 10.3785/j.issn.1008-9292.2017.12.03

Effect of low frequency low intensity electromagnetic fields on maturation and mineralization of rat skull osteoblasts in vitro

ZHU Baoying(

),ZHOU Jian,GAO Yuhai,SHI Wengui,WEI Zhenlong,LI Wenyuan,WANG Yuanyuan,CHEN Keming*(

)

Institute of Orthopedic Research, Lanzhou General Hospital of PLA, Lanzhou 730050, China

Download:

HTML( 6 )

PDF(1118KB)
Export: BibTeX | EndNote (RIS)

Abstract

Objective: To compare the effects of 50 Hz 1.8 mT sinusoidal magnetic field (SEMF) and 50 Hz 0.6 mT pulsed electromagnetic field(PEMF) on the maturation and mineralization of rat calvaria osteoblasts. Methods: Primary cultured rat calvarial osteoblasts were divided into 3 groups:blank control group, SEMF group and PEMF group. The rats in SEMT and PEMT groups were treated with 50 Hz 1.8 mT SEMF or 50 Hz 0.6 mT PEMF for 90 min/d, respectively. Western blotting and Real-time RT-PCR were used to detect the protein and mRNA expressions of Collagen-1, bone morphogenetic protein 2 (BMP-2), osterix (OSX) and Runt-associated transcription factor 2(Runx-2). The alkaline phosphatase(ALP) activity was detected by ALP test kits at d6 and d9 after treatment, and by ALP staining using azo coupling at d10 after treatment. The formation of calcium nodules was observed by alizarin red staining. Results: Compared with blank control group, the protein and mRNA expressions of Collagen-1, BMP-2, OSX and Runx-2 in SEMT and PEMT groups were significantly increased (P < 0.01 or P < 0.05); while the mRNA expressions of Collagen-1 and BMP-2 in PEMF group were significantly higher than those in SEMF group. After 6 days treatment, the activity of ALP in PEMF group was significantly higher than that in blank control group (P < 0.05), while such difference was not observed in SEMF group (P>0.05); after 9 days treatment, the activities of ALP in both PEMF and SEMP groups were significantly higher than that in blank control group (all P < 0.05), but the difference between PEMF and SEMF groups was not significant (P>0.05). After 10 days treatment, ALP staining was increased in both PEMF and SEMF groups compared with that in blank control group (all P < 0.01), and the stained area was bigger in PEMF group than that in SEMF group (P < 0.05). After 12 days treatment, calcium nodules were increased in PEMF and SEMF groups compared with that in blank control group (all P < 0.01), and more calcium nodules were observed in PEMF group than SEMF group (P < 0.05). Conclusion: Both 50 Hz 1.8 mT that in SEMF and 50 Hz 0.6 mT PEMF can promote the maturation and mineralization of osteoblasts, and the effect of PEMF is more marked.

Key words： Osteoblasts/cytology Skull/cytology Electromagnetic fields Cell differentiation

Received: 21 September 2017 Published: 25 December 2017

CLC:	Q683
	R68

Corresponding Authors: CHEN Keming E-mail: zby19930118@126.com;chenkm@lut.cn

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	ZHU Baoying
	ZHOU Jian
	GAO Yuhai
	SHI Wengui
	WEI Zhenlong
	LI Wenyuan
	WANG Yuanyuan
	CHEN Keming

Cite this article:

ZHU Baoying,ZHOU Jian,GAO Yuhai,SHI Wengui,WEI Zhenlong,LI Wenyuan,WANG Yuanyuan,CHEN Keming. Effect of low frequency low intensity electromagnetic fields on maturation and mineralization of rat skull osteoblasts in vitro. J Zhejiang Univ (Med Sci), 2017, 46(6): 585-592.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2017.12.03 OR http://www.zjujournals.com/med/Y2017/V46/I6/585

低频率低强度电磁场对体外培养大鼠颅骨成骨细胞成熟和矿化的影响

目的: 比较研究50 Hz 1.8 mT正弦电磁场（SEMF）和50 Hz 0.6 mT脉冲电磁场（PEMF）对大鼠颅骨成骨细胞成熟和矿化的影响。方法: 原代培养大鼠颅骨成骨细胞，传代后随机分为空白对照组、SEMF组和PEMF组。SEMF组和PEMF组每天接受电磁场处理一次，每次90 min。电磁场处理2 d时，采用蛋白质印迹法和实时定量RT-PCR检测Ⅰ型胶原（Collagen-1）、骨形态发生蛋白2（BMP-2）、Osterix（OSX）和Runt相关转录因子2（Runx-2）的蛋白和基因表达；电磁场处理第6天和第9天时，采用碱性磷酸酶（ALP）试剂盒检测ALP活性；电磁场处理第10天时采用偶氮偶合染色检测ALP活性；电磁场处理第12天时，以茜素红染色观察钙化结节形成情况并作定量分析。结果: 与空白对照组比较，SEMF组和PEMF组的Collagen-1、BMP-2、OSX、Runx-2蛋白和基因表达水平均显著升高（P < 0.01或P < 0.05），且Collagen-1、BMP-2在PEMF组中的mRNA表达量显著高于SEMF组（均P < 0.05）。电磁场处理第6天时，PEMF组ALP活性显著高于空白对照组（P < 0.05），SEMF组与空白对照组比较差异无统计学意义（P>0.05）；第9天时，PEMF组和SEMF组ALP活性均显著高于空白对照组（均P < 0.01），而PEMF组和SEMF组之间差异无统计学意义（P>0.05）。PEMF组和SEMF组偶氮偶合染色面积均大于空白对照组（均P < 0.01），且PEMF组染色面积大于SEMF组（P < 0.05）。茜素红染色结果显示，PEMF组和SEMF组钙化结节染色面积显著大于空白对照组（均P < 0.01），且PEMF组钙化结节染色面积大于SEMF组（P < 0.01）。结论: 50 Hz 1.8 mT SEMF和50 Hz 0.6 mT PEMF均能促进大鼠颅骨成骨细胞成熟和矿化，其中PEMF效果更为明显。

关键词： 成骨细胞/细胞学, 颅骨/细胞学, 电磁场, 细胞分化

Fig 1 Magnetic field generator and electromagnetic oscillographs

Tab 1 Primer sequences for real-time RT-PCR

Fig 2 Electropherogram of Collagen-1, BMP-2, OSX and Runx-2 expression in each group

Fig 3 Protein expressions of Collagen-1, BMP-2, OSX and Runx-2 in each group

Fig 4 mRNA expressions of Collagen-1, BMP-2, OSX and Runx-2 in each group

Fig 5 Activities of alkaline phosphatase (ALP) in rat calvarial osteoblasts at different time after electromagnetic field treatment

Fig 6 Immunohistochemical results of ALP in rat calvarial osteoblasts after 10 days of electromagnetic field treatment

Fig 7 Alizarin red staining showed calcium nodule in rat calvarial osteoblasts after 12 days of electromagnetic field treatment


[1]	SHEN W W , ZHAO J H . Pulsed electromagnetic fields stimulation affects BMD and local factor production of rats with disuse osteoporosis[J]. Bioelectromagnetics, 2010, 31 (2): 113- 119

[2]	HUANG L Q , HE H C , HE C Q et al. Clinical update of pulsed electromagnetic fields on osteoporosis[J]. Chin Med J(Engl), 2008, 121 (20): 2095- 2099

[3]	CHAO P H , LU H H , HUNG C T et al. Effects of applied DC electric field on ligament fibroblast migration and wound healing[J]. Connect Tissue Res, 2007, 48 (4): 188- 197 doi: 10.1080/03008200701424451

[4]	DEL S C , GHIONE S , LUSCHI P et al. Pain perception and electromagnetic fields[J]. Neurosci Biobehav Rev, 2007, 31 (4): 619- 642 doi: 10.1016/j.neubiorev.2007.01.003

[5]	程国政, 王鸣刚, 陈克明 et al. 不同干预时间正弦电磁场对大鼠骨髓基质干细胞成骨性分化的影响[J]. 现代生物医学进展, 2010, 10 (23): 4469- 4473 CHENG Guozheng , WANG Mingang , CHEN Keming et al. Effects of sinusoidal electromagnetic fields on osteoblastic differentiation of rats bone marrow mesenchymal stem cells at different treatment time[J]. Progress in Modern Biomedicine, 2010, 10 (23): 4469- 4473

[6]	IVANCSITS S , PILGER A , DIEM E et al. Cell type-specific genotoxic effects of intermittent extremely low-frequency electromagnetic fields[J]. Mutat Res, 2005, 583 (2): 184- 188 doi: 10.1016/j.mrgentox.2005.03.011

[7]	BERG H . Problems of weak electromagnetic field effects in cell biology[J]. Bioelectrochem Bioenerg, 1999, 48 (2): 355- 360 doi: 10.1016/S0302-4598(99)00012-4

[8]	周建, 葛宝丰, 陈克明 et al. 正弦交变电磁场促进体外培养成骨细胞成熟矿化的时间效应[J]. 生物医学工程学杂志, 2011, 28 (6): 1085- 1088 ZHOU Jian , GE Baofeng , CHEN Keming et al. Time effect of sinusoidal electromagnetic field on enhancing the maturation and mineralization of osteoblasts in vitro[J]. Journal of Biomedical Engineering, 2011, 28 (6): 1085- 1088

[9]	ZHOU J , MING L G , GE B F et al. Effects of 50 Hz sinusoidal electromagnetic fields of different intensities on proliferation, differentiation and mineralization potentials of rat osteoblasts[J]. Bone, 2011, 49 (4): 753- 761 doi: 10.1016/j.bone.2011.06.026

[10]	YAN J L , ZHOU J , MA H P et al. Pulsed electromagnetic fields promote osteoblast mineralization and maturation needing the existence of primary cilia[J]. Mol Cell Endocrinol, 2015, 404 132- 140 doi: 10.1016/j.mce.2015.01.031

[11]	陈克明, 葛宝丰, 刘兴炎 et al. 大鼠骨髓基质干细胞体外定向诱导成骨[J]. 兰州大学学报:自然科学版, 2003, 39 (12): 70- 72 CHEN Keming , GE Baofeng , LIU Xingyan et al. Directed differentiation of rat bone marrow stromal cells into osteoblast in vitro[J]. Journal of Lanzhou University:Natural Sciences, 2003, 39 (12): 70- 72

[12]	宋涛, 霍小林, 吴石增 . 生物电磁特性及其应用[M]. 北京: 北京工业大学出版社, 2008: 1- 12 SONG Tao , HUO Xiaolin , WU Shizeng . Electromagnetic properties and its application[M]. Beijing: Beijing University of Technology Press, 2008: 1- 12

[13]	孙文均, 姜槐 . 电磁场生物效应的细胞膜及蛋白激酶信号转导机制研究[J]. 国外医学生物医学工程分册, 2004, 27 (5): 260- 263 SUN Wenjun , JIANG Huai . Effects of electromagnetic field on biological membrane and protein kinase signal transduction mechanism[J]. Biomedical Engineering Foreign Medical Sciences, 2004, 27 (5): 260- 263

[14]	ZHOU J , MA X N , GAO Y H et al. Sinusoidal electromagnetic fields promote bone formation and inhibit bone resorption in rat femoral tissues in vitro[J]. Electromagn Biol Med, 2016, 35 (1): 75- 83 doi: 10.3109/15368378.2014.971958

[15]	LOHMANN C H , SCHWARTZ Z , LIU Y et al. Pulsed electromagnetic field stimulation of MG63 osteoblast-like cells affects differentiation and local factor production[J]. J Orthop Res, 2000, 18 (4): 637- 646 doi: 10.1002/(ISSN)1554-527X

[16]	LOHMANN C H , SCHWARTZ Z , LIU Y et al. Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells[J]. J Orthop Res, 2003, 21 (2): 326- 334 doi: 10.1016/S0736-0266(02)00137-7

[17]	PESSINA G P , ALDINUCCI C , PALMI M et al. Pulsed electromagnetic fields affect the intracellular calcium concentrations in human astrocytoma cells[J]. Bioelectromagnetics, 2001, 22 (7): 503- 510 doi: 10.1002/(ISSN)1521-186X

[18]	BAUD'HUIN M , LAMOUREUX F , DUPLOMB L et al. RANKL, RANK, osteoprotegerin:key partners of osteoimmunology and vascular diseases[J]. Cell Mol Life Sci, 2007, 64 (18): 2334- 2350 doi: 10.1007/s00018-007-7104-0

[19]	林佳声, 赵承斌 . BMP/bFGF对关节软骨损伤修复的作用[J]. 中国伤残医学, 2009, 17 (2): 131- 133 LIN Jiasheng , ZHAO Chengbin . BMP/bFGF on articular cartilage injury repair[J]. Chinese Journal of Trauma and Disability Medicine, 2009, 17 (2): 131- 133

[1]	WEI Zhenlong,SHI Wengui,CHEN Keming,ZHOU Jian,WANG Minggang. Icaritin promotes maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signal pathway[J]. J Zhejiang Univ (Med Sci), 2017, 46(6): 571-577.

[2]	FANG Qingqing, LI Zhizhong, ZHOU Jian, SHI Wengui, YAN Juanli, CHEN Keming. Genic and non-genic regulation of low frequence pulsed electromagnetic fields on osteoblasts differentiation[J]. J Zhejiang Univ (Med Sci), 2016, 45(6): 568-574.

[3]	ZHOU Yanfeng, GAO Yuhai, ZHEN Ping, CHEN Keming. Effects of 1.8 mT sinusoidal alternating electromagnetic fields of different frequencies on bone biomechanics of young rats[J]. J Zhejiang Univ (Med Sci), 2016, 45(6): 561-567.

[4]	CHEN Xiao, ZOU Xiaohui, YU Guangyan, FU Xin, CAO Tong, XIAO Yin, OUYANG Hongwei. Expert consensus on induction of human embryonic stem cells into tenocytes[J]. J Zhejiang Univ (Med Sci), 2016, 45(2): 105-111.

[5]	CUI Guang-ying, DIAO Hong-yan. Research advances of anti-tumor immune response induced by pulse electric field ablation[J]. J Zhejiang Univ (Med Sci), 2015, 44(6): 672-677.

[6]	LIAO Wei-chao, HE Ying, WANG Bin-sheng, HUANG He. Everolimus combined with all-trans retinoid acid reverses drug resistance in acute promyelocytic leukemia NB4-R1 cells[J]. J Zhejiang Univ (Med Sci), 2015, 44(5): 525-531.

[7]	YANG Min-li, YE Zhao-ming. Extremely low frequency electromagnetic field induces apoptosis of osteosarcoma cells via oxidative stress[J]. J Zhejiang Univ (Med Sci), 2015, 44(3): 323-328.

[8]	. Expression of Junctophilin 1 during cardiogenesis of mouse embryonic stem cells and rat embryos[J]. J Zhejiang Univ (Med Sci), 2012, 41(4): 359-365.

[9]	. Screening of differentially expressed genes during adipocyte differentiation by suppression subtractive hybridization technique[J]. J Zhejiang Univ (Med Sci), 2012, 41(3): 267-273.

[10]	. Screening of differentially expressed microRNAs in bone marrow-derived mesenchymal stem cells during osteoporosis and their function in stem cell differentiation[J]. J Zhejiang Univ (Med Sci), 2012, 41(1): 75-80.

[11]	. Effects of mycophenolic acid on human bone marrow-derived mesenchymal stem cells in vitro[J]. J Zhejiang Univ (Med Sci), 2011, 40(5): 467-474.

[12]	. Repetitive transcranial magnetic stimulation combined with antidepressant medication in treatment of first-episode patients with major depression[J]. J Zhejiang Univ (Med Sci), 2011, 40(3): 286-290.

[13]	. Effects of agonist and antagonist of cysteinyl leukotriene receptors on differentiation of rat glioma C6 cells[J]. J Zhejiang Univ (Med Sci), 2011, 40(2): 139-144.

[14]	. Traditional Chinese medicine Drynaria fortunei J.Smith naringin promotes proliferation and differentiation of human periodontal ligament cells [J]. J Zhejiang Univ (Med Sci), 2010, 39(1): 79-83.

[15]	Zhou Xiaohong, Zhu Weijian, Lou Jiangen. DEPENDENCE OF CARDIAC EXCITATION ON THE DIRECTION OF ELECTRICAL POTENTIAL GRADIENT FIELD[J]. J Zhejiang Univ (Med Sci), 1992, 21(5): 193-196.

Viewed

Full text

Abstract

Cited

Shared

Discussed