Department of Biomedical Engineering, Ankara University Faculty of Engineering, Ankara, Turkey. Ankara University Biotechnology Institute, Ankara, Turkey.
Biomed Mater. 2020 Jul 31;15(5):055017. doi: 10.1088/1748-605X/ab95e2.
Determination of a stem cell source with sufficient myogenic differentiation capacity that can be easily obtained in large quantities is of great importance in skeletal muscle regeneration therapies. Adipose-derived stem cells (ASCs) are readily available, can be isolated from fat tissue with high yield and possess myogenic differentiation capacity. Consequently, ASCs have high applicability in muscle regenerative therapies. However, a key challenge is their low differentiation efficiency. In this study, we have explored the potential of mimicking the natural microenvironment of the skeletal muscle tissue to enhance ASC myogenesis by inducing 3D cellular alignment and using dynamic biomimetic culture. ASCs were entrapped and 3D aligned in parallel within fibrin-based microfibers and subjected to uniaxial cyclic stretch. 3D cell alignment was shown to be necessary for achieving and maintaining the stiffness of the construct mimicking the natural tissue (12 ± 1 kPa), where acellular aligned fibers and cell-laden random fibers had stiffness values of 4 ± 1 and 5 ± 2 kPa, respectively, at the end of 21 d. The synergistic effect of 3D cell alignment and biomimetic dynamic culture was evaluated on cell proliferation, viability and the expression of muscle-specific markers (immunofluorescent staining for MyoD1, myogenin, desmin and myosin heavy chain). It was shown that the myogenic markers were only expressed on the aligned-dynamic culture samples on day 21 of dynamic culture. These results demonstrate that 3D skeletal muscle grafts can be developed using ASCs by mimicking the structural and physiological muscle microenvironment.
确定具有足够的成肌分化能力且易于大量获得的干细胞来源，对于骨骼肌再生治疗至关重要。脂肪来源干细胞（ASCs）易于获得，可从脂肪组织中高产量分离，并且具有成肌分化能力。因此，ASCs 在肌肉再生治疗中有很高的适用性。然而，一个关键的挑战是它们的低分化效率。在本研究中，我们探索了通过模拟骨骼肌组织的自然微环境来增强 ASC 成肌分化的潜力，方法是诱导 3D 细胞排列并用动态仿生培养。ASCs 被包埋并在纤维蛋白基微纤维中平行排列，并进行单轴循环拉伸。结果表明，3D 细胞排列对于实现和维持模仿自然组织的结构（12±1kPa）是必要的，其中无细胞排列纤维和细胞填充的随机纤维在 21 天结束时的刚度值分别为 4±1kPa 和 5±2kPa。3D 细胞排列和仿生动态培养的协同作用对细胞增殖、活力和肌肉特异性标志物（MyoD1、myogenin、结蛋白和肌球蛋白重链的免疫荧光染色）的表达进行了评估。结果表明，只有在动态培养的第 21 天，排列动态培养样品上才表达肌生成标志物。这些结果表明，可以通过模拟结构和生理肌肉微环境来使用 ASC 开发 3D 骨骼肌移植物。
