1 Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology , Daejeon, Korea.
2 Micro/Nano-Scale Manufacturing R&BD Group, Korea Institute of Industrial , Cheonan, Korea.
Tissue Eng Part A. 2018 May;24(9-10):752-760. doi: 10.1089/ten.TEA.2017.0055. Epub 2017 Nov 17.
Engineered muscular substitutes can restore the impaired muscle functions when integrated properly into the host tissue. To generate functional muscles with sufficient contractility at the site of transplant, the in vitro construction of fully differentiated muscle fibers would be desired. Many previous reports have identified either topographical alignment or electrical stimulation as an effective tool to promote myogenic differentiation. However, optimization of spatial and temporal arrangement of these two physical cues for better differentiation and maturation of skeletal muscles has not been investigated. In this article, we introduce a novel cell culture system that allows simultaneous application of these two independent directional cues at both orthogonal and parallel arrangements. We then show that the parallel arrangement of the aligned topography and the electric field synergistically facilitates better differentiation and maturation of C2C12, generating myotubes with more fused nuclei. Addition of the electric stimulation at the late stage of myogenic differentiation is found to further improve cell fusion to form multinucleate myotubes through a phosphatidylinositol-3-OH-kinase-dependent pathway. As such, we successfully demonstrated that the combined stimulation of topographical and electrical cues could effectively enhance both myogenic differentiation and maturation in a temporal and orientation-dependent manner, providing the basis for therapeutic strategies for regenerative tissue engineering.
工程化的肌肉替代物可以在适当整合到宿主组织中时恢复受损的肌肉功能。为了在移植部位生成具有足够收缩力的功能性肌肉，需要在体外构建完全分化的肌纤维。许多先前的报告已经确定了拓扑排列或电刺激作为促进成肌分化的有效工具。然而，对于这两种物理线索的空间和时间排列的优化，以更好地分化和成熟骨骼肌肉，尚未进行研究。在本文中，我们介绍了一种新的细胞培养系统，该系统允许同时在正交和平行排列上应用这两种独立的定向线索。然后我们表明，对齐的拓扑结构和平行电场的平行排列协同促进 C2C12 的更好分化和成熟，生成具有更多融合核的肌管。在成肌分化的后期施加电刺激被发现通过磷脂酰肌醇-3-羟激酶依赖性途径进一步促进细胞融合形成多核肌管。因此，我们成功地证明了拓扑和电线索的联合刺激可以有效地增强成肌分化和成熟，具有时间和方向依赖性，为再生组织工程的治疗策略提供了基础。
