骨骼肌是由肌母細胞(Myoblasts)相互融合分化成多核之肌小管(Myotube)，最後再成熟成為肌纖維(Myofiber)，以便進行收縮功能。因此，由肌母細胞分化成為成熟肌小管的過程，在骨骼肌組織工程中扮演相當重要的角色。在本研究中，我們利用具有順向排列與導電性的複合奈米纖維作為細胞支架(Cell scaffold)以刺激肌母細胞分化，接著搭配電脈衝刺激進一步使肌小管成熟並促進其功能性收縮單位肌節(Sacromere)之形成。實驗中，將含有導電性高分子Polyaniline (PANi)之Poly‐ε‐caprolactone (PCL)溶液，以靜電紡織技術結合磁場輔助收集器，製備出具有高度順向性與導電性之奈米纖維。Aligned‐PCL/PANi奈米纖維之直徑大小為307.6 ± 39.6 nm，且擁有高度順向之特性(夾角小於10°的纖維絲占90%)；細胞相容性測試結果顯示，C2C12肌母細胞可正常貼附並順向生長於奈米纖維上，且摻雜PANi後的奈米纖維相較於純PCL奈米纖維更能促進肌母細胞的增生；在細胞分化5天後，Aligned‐PCL/PANi相較於Random‐PCL/PANi可明顯提升其肌小管數目(38.9 ± 2.0 vs. 30.3 ± 2.4)、長度(198.3 ± 22.2 μm vs. 147.2 ± 30.7 μm)、融合指數(36.7 ± 1.3% vs. 27.9 ± 3.2%)和成熟指數(53.2 ± 2.2% vs. 44.2 ± 3.0%)之分化程度。最後探討Aligned‐PCL/PANi搭配電脈衝刺激對促進肌小管分化之程度與肌節形成之影響。結果表示，經電脈衝刺激後，可以大幅度的提升肌小管分化之程度，在分化9天後其肌小管數目(76.3 ± 2.1vs. 65.7 ± 3.3)、長度(425.5 ± 54.2 μm vs. 369.4 ± 30.2 μm)和融合指數(86.0 ± 2.2% vs. 80.3 ± 1.6%)均較未經電脈衝刺激者優異；在肌節形成之比例上，經第七天開始電脈衝刺激並且連續刺激24小時後，能夠獲得約47%的肌節橫紋比例，證實電脈衝刺激，可以有效的幫助肌小管發展成功能性的收縮單位。以上結果顯示，本研究成功開發出具有順向排列和導電性的奈米纖維，將其搭配電脈衝刺激後，可以有效促進肌小管分化與肌節形成，將可應用於骨骼肌組織修復與再生。

Skeletal muscle tissue is compared of bundles of highly oriented and densely packed muscle fibers, each with multinucleated myotubes derived from myoblasts. Therefore, the process of myotube formation from myoblasts plays an important role for skeletal muscle tissue engineering. The goal of this study was to examine the differentiation of skeletal myoblasts on aligned and electrically conductive nanofibrous scaffolds, and then investigate the effect of electrical pulse stimulation (EPS) combined with this scaffolds on myotubes maturation and functional sarcomere assembly. The aligned and electrically conductive nanofibers composed of poly-ε-caprolactone (PCL) and polyaniline (PANi) were fabricated by a magnetic-field-assisted electrospinning (MFAES) method. The diameter of Aligned-PCL/PANi nanofibers was 307.6 ± 39.6 nm with highly oriented structure (> 90% fibers within ± 10° of the preferred direction). The cell viability results indicated that Aligned-PCL/PANi nanofibers induced myoblasts alignment and significantly enhanced cell proliferation compared to Aligned-PCL nanofibers. After differentiation for 5 days, the myotube number and length, fusion index and degree of maturation of Aligned-PCL/PANi were greater (38.9 ± 2.0, 198.3 ± 22.2 μm, 36.7 ± 1.3% and 53.2 ± 2.2%) than that of Random-PCL/PANi (30.3 ± 2.4, 147.2 ± 30.7 μm, 27.9 ± 3.2% and 44.2 ± 3.0%). Finally, we investigated the effect of EPS on myotube maturation and sarcomere assembly on the Aligned-PCL/PANi. We found EPS could enhance the myotube formation and maturation. The myotube number and length, fusion index and maturation index of myocytes on Aligned-PCL/PANi with EPS at day 9 for 12 h were greater (76.3 ± 2.1, 425.5 ± 54.2 μm and 86.0 ± 2.2%) than that without EPS (65.7 ± 3.3, 369.4 ± 30.2 μm and 80.3 ± 1.6%). Additionally, when EPS was applied at day 7 for 24 h, the percentage of striated myotubes can reach to 47%. These results indicate that aligned and electrically conductive nanofibers can modulate the induction of myoblasts into mature and striated myotubes when EPS was applied, suggesting that the combined strategy have great potential for skeletal muscle tissue engineering.

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