Application of Cyclic Strain for Accelerated Skeletal Myogenic Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stromal Cells with Cell Alignment
Application of Cyclic Strain for Accelerated Skeletal Myogenic Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stromal Cells with Cell Alignment
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DOI：
10.1089/ten.tea.2012.0164
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发表时间：
2013-03-01
影响因子：
4.1
通讯作者：
Yatani, Hirofumi
中科院分区：
文献类型：
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作者：
Egusa, Hiroshi;Kobayashi, Munemasa;Yatani, Hirofumi
仿生骨骼肌细胞结构的制备继续对功能性组织工程提出挑战。骨髓间充质基质细胞（BMSCs）模拟天然组织结构的骨骼肌形成提供了巨大的治疗前景，但仍然特别困难。本研究的目的是研究应用循环应变加速具有排列结构的骨髓间充质干细胞分化的可能性。将小鼠骨髓间充质干细胞（mBMSCs）镀在涂有纤维连接蛋白的硅胶片上，当细胞融合度达到80%-90%时，进行10%的单轴循环应变。在频率为0.17 Hz（10次/分钟）的循环应变下培养的细胞在48小时内从完全随机的方向转变为排列整齐的形态，具有与应变载体平行的组织良好的肌动蛋白应力纤维。循环菌株限制了排列的mBMSCs在生长培养基中的运动和增殖，导致细胞群体中的细胞接触紧密。当mBMSCs在生肌培养基中进行循环应变时，逆转录-聚合酶链反应分析显示骨骼肌生成标记基因（肌生成因子5 [Myf5]、肌生成素和肌生成调节因子4 [MRF4]）上调，但平滑肌标记基因（心肌素和a-平滑肌肌动蛋白）未上调。此外，免疫细胞化学显示，mBMSCs在5天内沿施加张力的方向融合形成多核肌球蛋白和肌原蛋白阳性的肌管。这些结果表明，我们将循环菌株应用于在成肌培养基中培养的细胞的简单方法大大加速了具有排列结构的骨髓间充质干细胞的骨骼肌分化，并强调了细胞排列对于创造生理相关环境来研究骨髓间充质干细胞的成肌和工程骨骼肌的重要性。
The fabrication of biomimetic skeletal myocyte constructs continues to present a challenge to functional tissue engineering. The skeletal myogenesis of bone marrow-derived mesenchymal stromal cells (BMSCs) to mimic the native tissue architecture offers great therapeutic promise, but remains particularly difficult. The aim of this study was to examine the possibility of accelerating the skeletal myogenic differentiation of BMSCs with an aligned structure by applying cyclic strain. Mouse BMSCs (mBMSCs) were plated on silicone sheets that were coated with fibronectin and subjected to cyclic 10% uniaxial strain when they reached 80%-90% cell confluency. Cells cultured in a growth medium that were subjected to cyclic strain at a frequency of 0.17 Hz (10 times/min) demonstrated a shift of alignment within 48 h from a completely random orientation to a well-aligned morphology with well-organized actin stress fibers that were parallel to the strain vector. The cyclic strain restricted the motility and proliferation of the aligned mBMSCs in the growth medium, which resulted in tight cellular contact in the cell population. When mBMSCs were subjected to cyclic strain in a myogenic medium, reverse transcription-polymerase chain reaction analysis demonstrated the upregulation of skeletal myogenic marker genes (myogenic factor 5 [Myf5], myogenin, and myogenic regulatory factor 4 [MRF4]), but not smooth muscle marker genes (myocardin and a-smooth muscle actin). In addition, immunocytochemistry showed that the mBMSCs fused to form multinucleated myosin-and myogenin-positive myotubes in the direction of the applied tension within 5 days. These results demonstrate that our simple method of applying of cyclic strain to cells cultured in a myogenic medium greatly accelerates the skeletal myogenic differentiation of mBMSCs with an aligned structure, and they highlight the importance of cellular alignment for creating physiologically relevant environments to study the myogenesis of BMSCs and engineer skeletal muscle.