Yamasaki Ken-ichi, Hayashi Hiroyuki, Nishiyama Keiko, Kobayashi Hiroyuki, Uto Sadahito, Kondo Hideo, Hashimoto Shigehiro, Fujisato Toshia
Graduate School of Engineering, Osaka Institute of Technology, Osaka, Japan.
J Artif Organs. 2009;12(2):131-7. doi: 10.1007/s10047-009-0457-4. Epub 2009 Jun 18.
The contractility of tissue-engineered muscle on the application of electrical signals is required for the development of bio-actuators and for muscle tissue regeneration. Investigations have already reported on the contraction of myotubes differentiated from myoblasts and the construction of tissue-engineered skeletal muscle using electrical pulses. However, the relationship between myotube contraction and electrical pulses has not been quantitatively evaluated. We quantitatively investigated the effect of electrical pulse frequency on the excitability of myotubes and developed bio-actuators made of tissue-engineered skeletal muscle. C2C12 cells were seeded on a collagen-coated dish and in collagen gel and were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and antibiotics. When the cells reached confluence or after 2 days in culture, the medium was shifted to DMEM containing 7% horse serum to allow them to differentiate to C2C12 myotubes. We electrically stimulated the myotubes and tissue-engineered skeletal muscle, and contractions were observed under a microscope. The myotubes contracted synchronously with electrical pulses between 0.5 and 5 Hz and unfused tetanus was generated at 10 Hz. The contractile performance of tissue-engineered skeletal muscle made of collagen gel and C2C12 was similar to that of the myotubes. Both the rheobase and chronaxie of the myotubes were lowest when the electric field was applied parallel to the myotube axis, and the values were 8.33 +/- 2.78 mA and 1.19 +/- 0.38 ms, respectively. The motion of C2C12 myotube contraction depended on the pulse frequency and showed anisotropy in the electric field. These results suggest that a tissue-engineered bio-actuator may be controlled using electrical signals.
生物致动器的开发以及肌肉组织再生需要组织工程化肌肉在施加电信号时具有收缩性。已有研究报道了成肌细胞分化而来的肌管的收缩以及使用电脉冲构建组织工程化骨骼肌。然而，肌管收缩与电脉冲之间的关系尚未得到定量评估。我们定量研究了电脉冲频率对肌管兴奋性的影响，并开发了由组织工程化骨骼肌制成的生物致动器。将C2C12细胞接种在胶原包被的培养皿和胶原凝胶中，在含有10%胎牛血清和抗生素的杜氏改良 Eagle 培养基（DMEM）中培养。当细胞达到汇合状态或培养2天后，将培养基换成含有7%马血清的DMEM，使其分化为C2C12肌管。我们对肌管和组织工程化骨骼肌进行电刺激，并在显微镜下观察收缩情况。肌管在0.5至5Hz的电脉冲作用下同步收缩，在10Hz时产生非融合强直收缩。由胶原凝胶和C2C12制成的组织工程化骨骼肌的收缩性能与肌管相似。当电场平行于肌管轴施加时，肌管的基强度和时值最低，分别为8.33±2.78mA和1.19±0.38ms。C2C12肌管收缩的运动取决于脉冲频率，并在电场中表现出各向异性。这些结果表明，可以使用电信号来控制组织工程化生物致动器。
