3D myotube guidance on hierarchically organized anisotropic and conductive fibers for skeletal muscle tissue engineering.

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.