A multifunctional bioreactor was fabricated in this study to investigate the facilitation efficiency of electrical and mechanical stimulations on myogenic differentiation. This bioreactor consisted of a highly stretchable conductive membrane prepared by depositing polypyrrole (PPy) on a flexible polydimethylsiloxane (PDMS) film. The tensile deformation of the PPy/PDMS membrane can be tuned by adjusting the channel depth. In addition, PPy/PDMS maintained its electrical conductivity under continuous cyclic stretching in the strain range of 6.5%–13% for 24 h. This device can be used to individually or simultaneously perform cyclic stretching and electrical stimulation. The results of single stimulation showed that either cyclic stretching or electrical stimulation upregulated myogenic gene expression and promoted myotube formation, where electrical stimulation improved better than cyclic stretching. However, only cyclic stretching can align C2C12 cells perpendicular to the stretching direction, and electrical stimulation did not affect cell morphology. Myosin heavy chain (MHC) immunostaining demonstrated that oriented cells under cyclic stretching resulted in parallel myotubes. The combination of these two stimuli exhibited synergetic effects on both myogenic gene regulation and myotube formation, and the incorporated electrical field did not affect the orientation effect of the cyclic stretching. These results suggested that these two treatments likely influenced cells through different pathways. Overall, the simultaneous application of cyclic stretching and electrical stimulation preserved both stimuli's advantages, so myo-differentiation can be highly improved to obtain abundant parallel myotubes, suggesting that our developed multifunctional bioreactor should benefit muscle tissue engineering applications.

中文翻译：

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开发多功能生物反应器评估循环拉伸和电刺激对肌肉分化的促进作用

本研究制造了一个多功能生物反应器，以研究电和机械刺激对生肌分化的促进效率。该生物反应器由高度可拉伸的导电膜组成，该膜是通过在柔性聚二甲基硅氧烷（PDMS）薄膜上沉积聚吡咯（PPy）而制备的。PPy/PDMS膜的拉伸变形可以通过调节通道深度来调节。此外，PPy/PDMS在6.5%~13%的应变范围内连续循环拉伸24小时后仍保持其导电性。该装置可用于单独或同时进行循环拉伸和电刺激。单次刺激的结果表明，循环拉伸或电刺激均可上调生肌基因表达并促进肌管形成，其中电刺激的改善效果优于循环拉伸。然而，只有循环拉伸才能使C2C12细胞垂直于拉伸方向排列，并且电刺激不影响细胞形态。肌球蛋白重链 (MHC) 免疫染色表明，循环拉伸下的定向细胞会产生平行的肌管。这两种刺激的组合对生肌基因调节和肌管形成表现出协同效应，并且合并的电场不影响循环拉伸的定向效应。这些结果表明这两种治疗可能通过不同的途径影响细胞。总体而言，同时应用循环拉伸和电刺激保留了两种刺激的优点，因此可以高度改善肌分化以获得丰富的平行肌管，这表明我们开发的多功能生物反应器应有利于肌肉组织工程应用。