Long Haiyan, Yang Gang, Ma Kunlong, Xiao Zhenghua, Ren Xiaomei
Center of Engineering-Training, Chengdu Aeronautic Polytechnic, Chengdu Sichuan, 610100, P.R.China.
Department of Medical Information and Engineering, School of Electrical Engineering and Information, Sichuan University, Chengdu Sichuan, 610065,
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2017 Jul 15;31(7):853-861. doi: 10.7507/1002-1892.201702027.
To investigate the effect of different electrical stimulation waves on orientation and alignment of adipose derived mesenchymal stem cells (ADSCs).
ADSCs 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 ( ). 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.
The 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 groups A5 and A6 were significantly higher than that of group D ( <0.05), but no significant difference was found between other groups and group D ( >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 ( <0.05), but no significant difference was found between other groups and group D ( >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.
The 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.
探讨不同电刺激波对脂肪来源间充质干细胞（ADSCs）定向排列的影响。
从5周龄的Sprague Dawley大鼠（体重100 - 150 g）中分离并培养ADSCs。将第3 - 5代细胞接种以制备细胞爬片，随后在自制的电场生物反应器上分别给予电场强度为1、2、3、4、5和6 V/cm的直流电刺激（ES）（A1、A2、A3、A4、A5和A6组）；在电场强度为6 V/cm、占空比为50%、频率为1和2 Hz的方波ES刺激下（B1和B2组）；在电场强度为6 V/cm、脉冲宽度为2 ms、频率为1和2 Hz的双相脉冲波ES刺激下（C1和C2组）；不给予ES作为对照（D组）。通过倒置显微镜的延时显微摄影评估施加ES后细胞形态的变化。通过平均取向因子（ ）评估细胞排列。用罗丹明 - 鬼笔环肽染色对经电场处理的ADSCs的细胞骨架进行表征。通过细胞活/死染色评估细胞存活率，并用钙离子荧光染色检测细胞内钙活性。
ADSCs对ES的反应与直流电场强度有关。直流电场强度越高，垂直于电场方向排列的细胞越多。在每个时间点，B1、B2组和C1、C2组均无明显的细胞排列。A5和A6组的平均 显著高于D组（ <0.05），但其他组与D组之间无显著差异（ >0.05）。细胞骨架染色显示，A5和A6组的细胞呈现紧密的束状细胞骨架结构，并倾向于垂直于电场矢量方向。A4、A5和A6组的细胞存活率显著低于D组（ <0.05），但其他组与D组之间无显著差异（ >0.05）。钙荧光染色显示，A4、A5和A6组的钙离子荧光强度略高于D组，其他组与D组之间无显著差异。
生理电场强度（5 V/cm和6 V/cm）的直流电场刺激可诱导ADSCs排列，但在低于5 V/cm的直流电场、方波和双相脉冲波刺激条件下未发现细胞排列。细胞活力与电场强度呈负相关。
