Effect of different electrical stimulation waves on orientation and alignment of adipose derived mesenchymal stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LONG Haiyan ¹ ,  YANG Gang ² , MA Kunlong ³ , XIAO Zhenghua ⁴ , REN Xiaomei ²

1. Center of Engineering-Training, Chengdu Aeronautic Polytechnic, Chengdu Sichuan, 610100, P.R.China;
2. Department of Medical Information and Engineering, School of Electrical Engineering and Information, Sichuan University, Chengdu Sichuan, 610065, P.R.China;
3. Department of Orthopaedics, Yongchuan Hospital, Chongqing Medical University, Yongchuan Chongqing, 402160, P.R.China;
4. Department of Cardiovascular Surgery, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China;

Corresponding author：

YANG Gang, Email: yang_gang@scu.edu.cn

Keywords：

Electrical stimulation; adipose derived mesenchymal stem cells; electrical field intensity; wave form; cell alignment

DOI：

10.7507/1002-1892.201702027

Video：

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ObjectiveTo investigate the effect of different electrical stimulation waves on orientation and alignment of adipose derived mesenchymal stem cells (ADSCs).MethodsADSCs were isolated from 5-week-old Sprague Dawley rats (weight, 100-150 g) and cultivated. The cells at passages 3-5 were inoculated to prepare cell climbing slices, subsequently was exposed to direct-current electrical stimulations (ES) at electric field strengths of 1, 2, 3, 4, 5, and 6 V/cm on a homemade electric field bioreactor (groups A1, A2, A3, A4, A5, and A6); at electric field strength of 6 V/cm, at 50% duty cycle, and at frequency of 1 and 2 Hz (groups B1 and B2) of square wave ES; at electric field strength of 6 V/cm, at pulse width of 2 ms, and at frequency of 1 and 2 Hz (groups C1 and C2) of biphasic pulse wave ES; and no ES was given as a control (group D). The changes of cellular morphology affected by applied ES were evaluated by time-lapse micropho-tography via inverted microscope. The cell alignment was evaluated via average orientation factor (OF). The cytoske-leton of electric field treated ADSCs was characterized by rhodamine-phalloidin staining. The cell survival rates were assessed via cell live/dead staining and intracellular calcium activities were detected by calcium ion fluorescent staining.ResultsThe response of ADSCs to ES was related to the direct-current electric field intensity. The higher the direct-current electric field intensity was, the more cells aligned perpendicular to the direction of electric field. At each time point, there was no obvious cell alignment in groups B1, B2 and C1, C2. The average OF of groups A5 and A6 were significantly higher than that of group D (P<0.05), but no significant difference was found between other groups and group D (P>0.05). The cytoskeleton staining showed that the cells of groups A5 and A6 exhibited a compact fascicular structure of cytoskeleton, and tended to be perpendicular to the direction of the electric field vector. The cellular survival rate of groups A4, A5, and A6 were significantly lower than that of group D (P<0.05), but no significant difference was found between other groups and group D (P>0.05). Calcium fluorescence staining showed that the fluorescence intensity of calcium ions in groups A4, A5, and A6 was slightly higher than that in group D, and no significant difference was found between other groups and group D.ConclusionThe direct-current electric field stimulations with physiological electric field strength (5 V/cm and 6 V/cm) can induce the alignment of ADSCs, but no cell alignment is found under conditions of less than 5 V/cm direct-current electric field, square wave, and biphasic pulse wave stimulation. The cellular viability is negatively correlated with the electric field intensity.

Citation： LONG Haiyan, YANG Gang, MA Kunlong, XIAO Zhenghua, REN Xiaomei. Effect of different electrical stimulation waves on orientation and alignment of adipose derived mesenchymal stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(7): 853-861. doi: 10.7507/1002-1892.201702027 Copy

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3.	Wolf CM, Moskowitz IP, Arno S, et al. Somatic events modify hypertrophic cardiomyopathy pathology and link hypertrophy to arrhythmia. Proc Natl Acad Sci U S A, 2005, 102(50): 18123-18128.
4.	Konno M, Hamabe A, Hasegwa S, et al. Adipose-derived mesenchymal stem cells and regenerative medicine. Dev Growth Differ, 2013, 55(3): 309-318.
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6.	Gimble JM, Nuttall ME. Adipose-derived stromal/stem cells (ASC) in regenerative medicine: pharmaceutical applications. Curr Pharm Des, 2011, 17(4): 332-339.
7.	Long H, Yang G, Wang Z. Galvanotactic migration of EA.Hy926 endothelial cells in a novel designed electric field bioreactor. Cell Biochem Biophys, 2011, 61(3): 481-491.
8.	黄文丽, 赵颖异, 杨刚, 等. 外加直流电场对内皮细胞增殖与迁徙的影响. 中国组织工程研究与临床康复, 2010, 14(28): 5167-5171.
9.	李志园, 杨刚, 黄华, 等. 双相脉冲电刺激可促进脂肪干细胞分化为心肌细胞样细胞. 细胞与分子免疫学杂志, 2015, 31(9): 1200-1204.
10.	Yang G, Xiao Z, Ren X, et al. Enzymatically crosslinked gelatin hydrogel promotes the proliferation of adipose tissue-derived stromal cells. Peer J, 2016, 4: e2497.
11.	任小梅, 钱宏, 肖正华, 等. 基于转谷氨酰胺酶交联明胶水凝胶的脂肪干细胞培养研究. 中国修复重建外科杂志, 2016, 30(12): 1532-1537.
12.	Sun S, Titushkin I, Cho M. Regulation of mesenchymal stem cell adhesion and orientation in 3D collagen scaffold by electrical stimulus. Bioelectrochemistry, 2006, 69(2): 133-141.
13.	Yang G, Xiao Z, Ren X, et al. Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels. Sci Rep, 2017, 7: 41781.
14.	Hammerick KE, Longaker MT, Prinz FB. In vitro effects of direct current electric fields on adipose-derived stromal cells. Biochem Biophys Res Commun, 2010, 397(1): 12-17.
15.	Zhao Z, Qin L, Reid B, et al. Directing migration of endothelial progenitor cells with applied DC electric fields. Stem Cell Res, 2012, 8(1): 38-48.
16.	Radisic M, Park H, Shing H, et al. Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proc Natl Acad Sci U S A, 2004, 101(52): 18129-18134.
17.	Radisic M, Fast VG, Sharifov OF, et al. Optical mapping of impulse propagation in engineered cardiac tissue. Tissue Eng Part A, 2009, 15(4): 851-860.
18.	Zorec B, Becker S, Reberšek M, et al. Skin electroporation for transdermal drug delivery: the influence of the order of different square wave electric pulses. Int J Pharm, 2013, 457(1): 214-223.
19.	Krasteva V, Iliev I, Cansell A, et al. Automatic adjustment of biphasic pulse duration in transthoracic defibrillation. J Med Eng Technol, 2000, 24(5): 210-214.
20.	Suárez-Antola R. Contributions to the study of optimal biphasic pulse shapes for functional electric stimulation: an analytical approach using the excitation functional. Conf Proc IEEE Eng Med Biol Soc, 2007, 2007: 2440-2443.
21.	Chiu LL, Iyer RK, King JP, et al. Biphasic electrical field stimulation aids in tissue engineering of multicell-type cardiac organoids. Tissue Eng Part A, 2011, 17(11-12): 1465-1477.
22.	Pietronave S, Zamperone A, Pltolina F, et al. Monophasic and biphasic electrical stimulation induces a precardiac differentiation in progenitor cells isolated from human heart. Stem Cells Dev, 2014, 23(8): 888-898.
23.	Zhu Y, Liu T, Song K, et al. Adipose-derived stem cell: a better stem cell than BMSC. Cell Biochem Funct, 2008, 26(6): 664-675.
24.	Tang L, Yin Y, Zhou H, et al. Proliferative capacity and pluripotent characteristics of porcine adult stem cells derived from adipose tissue and bone marrow. Cell Reprogram, 2012, 14(4): 342-352.
25.	Zhao Z, Walt C, Karystinou A, et al. Directed migration of human bone marrow mesenchymal stem cells in a physiological direct current electric field. Eur Cell Mater, 2011, 22: 344-358.
26.	Zhao H, Steiger A, Nohner M, et al. Specific Intensity Direct Current (DC) Electric Field Improves Neural Stem Cell Migration and Enhances Differentiation towards betaIII-Tubulin+ Neurons. PLoS One, 2015, 10(6): e0129625.

1. Li Y, Huang G, Zhang X, et al. Engineering cell alignment in vitro. Biotechnol Adv, 2014, 32(2): 347-365.
2. Sepp R, Severs NJ, Gourdie RG. Altered patterns of cardiac intercellular junction distribution in hypertrophic cardiomyopathy. Heart, 1996, 76(5): 412-417.
3. Wolf CM, Moskowitz IP, Arno S, et al. Somatic events modify hypertrophic cardiomyopathy pathology and link hypertrophy to arrhythmia. Proc Natl Acad Sci U S A, 2005, 102(50): 18123-18128.
4. Konno M, Hamabe A, Hasegwa S, et al. Adipose-derived mesenchymal stem cells and regenerative medicine. Dev Growth Differ, 2013, 55(3): 309-318.
5. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res, 2007, 100(9): 1249-1260.
6. Gimble JM, Nuttall ME. Adipose-derived stromal/stem cells (ASC) in regenerative medicine: pharmaceutical applications. Curr Pharm Des, 2011, 17(4): 332-339.
7. Long H, Yang G, Wang Z. Galvanotactic migration of EA.Hy926 endothelial cells in a novel designed electric field bioreactor. Cell Biochem Biophys, 2011, 61(3): 481-491.
8. 黄文丽, 赵颖异, 杨刚, 等. 外加直流电场对内皮细胞增殖与迁徙的影响. 中国组织工程研究与临床康复, 2010, 14(28): 5167-5171.
9. 李志园, 杨刚, 黄华, 等. 双相脉冲电刺激可促进脂肪干细胞分化为心肌细胞样细胞. 细胞与分子免疫学杂志, 2015, 31(9): 1200-1204.
10. Yang G, Xiao Z, Ren X, et al. Enzymatically crosslinked gelatin hydrogel promotes the proliferation of adipose tissue-derived stromal cells. Peer J, 2016, 4: e2497.
11. 任小梅, 钱宏, 肖正华, 等. 基于转谷氨酰胺酶交联明胶水凝胶的脂肪干细胞培养研究. 中国修复重建外科杂志, 2016, 30(12): 1532-1537.
12. Sun S, Titushkin I, Cho M. Regulation of mesenchymal stem cell adhesion and orientation in 3D collagen scaffold by electrical stimulus. Bioelectrochemistry, 2006, 69(2): 133-141.
13. Yang G, Xiao Z, Ren X, et al. Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels. Sci Rep, 2017, 7: 41781.
14. Hammerick KE, Longaker MT, Prinz FB. In vitro effects of direct current electric fields on adipose-derived stromal cells. Biochem Biophys Res Commun, 2010, 397(1): 12-17.
15. Zhao Z, Qin L, Reid B, et al. Directing migration of endothelial progenitor cells with applied DC electric fields. Stem Cell Res, 2012, 8(1): 38-48.
16. Radisic M, Park H, Shing H, et al. Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proc Natl Acad Sci U S A, 2004, 101(52): 18129-18134.
17. Radisic M, Fast VG, Sharifov OF, et al. Optical mapping of impulse propagation in engineered cardiac tissue. Tissue Eng Part A, 2009, 15(4): 851-860.
18. Zorec B, Becker S, Reberšek M, et al. Skin electroporation for transdermal drug delivery: the influence of the order of different square wave electric pulses. Int J Pharm, 2013, 457(1): 214-223.
19. Krasteva V, Iliev I, Cansell A, et al. Automatic adjustment of biphasic pulse duration in transthoracic defibrillation. J Med Eng Technol, 2000, 24(5): 210-214.
20. Suárez-Antola R. Contributions to the study of optimal biphasic pulse shapes for functional electric stimulation: an analytical approach using the excitation functional. Conf Proc IEEE Eng Med Biol Soc, 2007, 2007: 2440-2443.
21. Chiu LL, Iyer RK, King JP, et al. Biphasic electrical field stimulation aids in tissue engineering of multicell-type cardiac organoids. Tissue Eng Part A, 2011, 17(11-12): 1465-1477.
22. Pietronave S, Zamperone A, Pltolina F, et al. Monophasic and biphasic electrical stimulation induces a precardiac differentiation in progenitor cells isolated from human heart. Stem Cells Dev, 2014, 23(8): 888-898.
23. Zhu Y, Liu T, Song K, et al. Adipose-derived stem cell: a better stem cell than BMSC. Cell Biochem Funct, 2008, 26(6): 664-675.
24. Tang L, Yin Y, Zhou H, et al. Proliferative capacity and pluripotent characteristics of porcine adult stem cells derived from adipose tissue and bone marrow. Cell Reprogram, 2012, 14(4): 342-352.
25. Zhao Z, Walt C, Karystinou A, et al. Directed migration of human bone marrow mesenchymal stem cells in a physiological direct current electric field. Eur Cell Mater, 2011, 22: 344-358.
26. Zhao H, Steiger A, Nohner M, et al. Specific Intensity Direct Current (DC) Electric Field Improves Neural Stem Cell Migration and Enhances Differentiation towards betaIII-Tubulin+ Neurons. PLoS One, 2015, 10(6): e0129625.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of different electrical stimulation waves on orientation and alignment of adipose derived mesenchymal stem cells

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