機械和電訊號對肌肉生成有正向作用。在我們先前研究中成功地以聚二甲基矽氧烷(PDMS)薄膜作為彈性基底將導電高分子聚吡咯(PPy)製備於其上得到高拉伸性導電薄膜。可用於組裝生物反應器對細胞進行拉伸及電刺激。然而，如何將刺激過後的細胞完整取下來應用於後續肌肉修復是一大挑戰。因此本研究將製備同時具有導電性及溫敏性的薄膜，並以小鼠骨骼成肌細胞C2C12作為研究對象，研究所開發薄膜是否合適對貼附的細胞進行拉伸跟電刺激，並探討降溫細胞脫附後細胞外基質及肌管排列是否得以保留。在電刺激的部份我們發現此薄膜上的細胞在1 V/cm的電場刺激下有最好效果。在細胞排列實驗中9%循環拉伸促使C2C12沿著與拉伸方向垂直的方向進行高度排列。而複合刺激實驗中其中細胞排列取決於拉伸方向，可得到高度分化且排列的肌管。在西方點墨的實驗中，證實利用降溫得到的細胞片相較於傳統胰蛋白酶消化細胞方法仍保有細胞外基質 。qPCR結果證實拉伸刺激或電刺激皆能夠增加分化標誌基因的表達，肌球重鏈蛋白(MHC)免疫染色實驗指出兩種刺激都會有效地增加肌管的數量及提升分化率，且拉伸刺激有助於控制肌管的排列。與單一刺激相比，複合刺激不僅保有維持肌管排列的效果，還能使分化標誌基因更加上調控或維持高水準，並在降溫脫附後仍保留這些排列的肌管。這些結果證實本研究所開發的可助於收集拉伸及電刺激過後的細胞片，以作為組織工程及再生醫學上的應用。;In our previous study, our lab has successfully deposited conductive polypyrrole (PPy) onto elastic polydimethylsiloxane (PDMS) substrate to obtain a highly stretchable conductive film. It can be used to construct a bioreactor to cyclicaly stretch and electrically stimulate surface cells. However, how to completely harvest these stimulated muscle tissue to repair damage muscle is a challenge. Therefore, in this study, we intend to develop a polymer film owns both conductivity and thermo-sensitivity. Therefore, cells after stimulation can be completely harvested as cell sheets through breducing temperature. Mouse skeletal myoblast, C2C12 cells, were applied to examine our hypothesis. In electrical stimulation, C2C12 cells demonstrated the best myo-differentiation under the electric field of 1 V/cm. Regarding cyclic stretching, the strain equal to or higher than 9% can highly align C2C12 perpendicular to the stretching direction. The Western blotting experiments demonstrated that the cell sheets harvested by cooling reserved more extracellular matrix (ECM) than cells collected by the traditional trypsin digestion method. The qPCR results revealed that both cyclic stretching and electrical treatment increased the expression of differentiation myogenic marker genes. Immunostaining of myosin heavy chain protein (MHC) indicated that both mechanical and electrical stimuli effectively increased the number of myotubes and the differentiation ratio, and the myotubes can be aligned by the cyclic stretching. Compared the sole stimulation, dual stimulation not only aligned myotubes, but also highly increased the up-regulation myogenic differentiation markers. Stimulated cell sheets can be harvested by cooling and the alignment of myotubes was still maintained. These results suggested that our developed polymeric film can be applied to harvest intact cell sheets after cyclic stretching and electrical stimulation, which increased the feasibility bioreactor for the application of tissue engineering and regenerative medicine.
