Interdisciplinary Nanoscience Center, Aarhus University, DK-8000 Aarhus C, Denmark.
Department of Engineering, Aarhus University, DK-8000 Aarhus C, Denmark.
Mater Sci Eng C Mater Biol Appl. 2020 Nov;116:111070. doi: 10.1016/j.msec.2020.111070. Epub 2020 May 11.
Tissue engineering represents a promising approach for the functional restoration of large volumetric skeletal muscle loss. However, design and fabrication of 3D hierarchically organized scaffolds that closely mimic the natural micro-/nanostructures of skeletal muscle extracellular matrix (ECM) is still an ongoing challenge. Here, we constructed a hierarchically organized, anisotropic and conductive scaffold with microscale melt electrowriting (MEW) grooves parallel aligned on the top of unidirectionally oriented nanofibrous mesh to guide myoblast cell alignment, elongation and differentiation into myotubes. A 7-days study of H9c2 myoblast cells cultured on the scaffolds indicated that the combination of nanoscale and microscale anisotropic surface topography enhanced myogenesis. Specifically, the parallel patterned scaffold effectively enhanced both the elongation and maturation of myotubes, as indicated by the increased myotube length (>600 μm), higher heavy myosin chain (MHC) surface coverage and maturation index on the parallel patterned scaffold compared to their pure (2.4-fold MHC surface coverage, 1.6-fold maturation index) and perpendicular patterned counterparts (1.7-fold MHC surface coverage, 1.5-fold maturation index). Furthermore, a gold nanolayer coating on the aligned nanofibrous mesh surface provided electroactive cues to enhance the formation of myotubes. With the increase of Au coating thickness, improved myoblasts alignment (74.6%, 80.5%, 85.6%, 94.9% for Au 0, 10, 40, 70 nm respectively) and higher MHC-positive expression (57.5% for Au 0 nm, 90.3%, 92.5%, 95.0% for Au10, 40, 70 nm respectively) were observed. Finally, the formation and morphology of myotubes were also found dependent on the spacing between microgrooves (100, 200, 300 μm), where myotubes formed on 200 μm spacing scaffold showed the highest alignment (within 10° along nanofibers) and elongation (>850 μm). The hierarchically organized, anisotropic and conductive fibers hold great potential for regeneration of skeletal muscle tissue.
组织工程代表了一种有前途的方法，可以实现大体积骨骼肌缺失的功能恢复。然而，设计和制造模仿骨骼肌细胞外基质（ECM）的天然微观/纳米结构的三维层次结构支架仍然是一个持续的挑战。在这里，我们构建了一种具有微观结构的各向异性和导电性支架，其微尺度熔融电纺（MEW）凹槽平行排列在单向定向纳米纤维网格的顶部，以引导成肌细胞的排列、伸长和分化为肌管。在支架上培养 H9c2 成肌细胞 7 天的研究表明，纳米级和微级各向异性表面形貌的结合增强了成肌作用。具体来说，平行图案化支架有效地增强了肌管的伸长和成熟，这表现为肌管长度增加（>600μm），以及与纯（2.4 倍肌球蛋白重链（MHC）表面覆盖率，1.6 倍成熟指数）和垂直图案化支架相比，平行图案化支架上的 MHC 表面覆盖率更高（2.4 倍 MHC 表面覆盖率，1.6 倍成熟指数）和成熟指数（1.7 倍 MHC 表面覆盖率，1.5 倍成熟指数）。此外，在定向纳米纤维网格表面上涂覆金纳米层提供了电活性线索，以增强肌管的形成。随着金涂层厚度的增加，观察到成肌细胞的排列更加整齐（Au0、10、40、70nm 分别为 74.6%、80.5%、85.6%、94.9%）和 MHC 阳性表达更高（Au0nm 为 57.5%，Au10、40、70nm 分别为 90.3%、92.5%、95.0%）。最后，肌管的形成和形态也发现依赖于微槽之间的间距（100、200、300μm），其中在 200μm 间距支架上形成的肌管具有最高的排列（沿纳米纤维<10°）和伸长（>850μm）。这种具有层次结构的各向异性和导电性纤维在骨骼肌组织再生方面具有巨大的潜力。
