Speaker
Description
Recreating the highly aligned and hierarchical structure of native extracellular matrix (ECM) remains a pivotal challenge in musculoskeletal tissue engineering, particularly for skeletal muscle, where anisotropic architecture is critical for function. In this study, we present a novel strategy that integrates melt electrofibrillation and 3D cell spheroid bioassembly to fabricate structurally organized muscle-like tissue constructs. Using a blend of polycaprolactone (PCL) and polyvinyl acetate (PVAc), we generated precisely printed box-shaped scaffolds via melt electrowriting, followed by selective removal of PVAc to yield collagen-mimicking nanofibrillar bundles. These fibrillated scaffolds provided aligned nano-topographical cues, essential for guiding myoblast alignment and subsequent tissue maturation. Murine C2C12 myoblasts were assembled into spheroids using honeycomb-inspired microwell arrays and seeded onto the fibrillated scaffolds. Optimal spheroid size and number per scaffold compartment were established to ensure homogeneous scaffold coverage. Within five days of culture, spheroids disassembled and cells migrated along the aligned fibrils, showing strong infiltration and colonization of the scaffold architecture. Prolonged cultivation (up to 21 days) under both growth and myogenic differentiation conditions resulted in high cellular viability, significant proliferation (as evidenced by DNA content), and enhanced myotube formation. Immunostaining and gene expression analyses confirmed myogenic differentiation, with aligned myotubes expressing myosin heavy chain and elevated levels of myogenic regulatory factors MyoD1 and myogenin, particularly under differentiation conditions. SEM and FIB-SEM imaging further corroborated the formation of tissue-like structures, with cells forming dense, longitudinal bundles throughout the scaffold matrix. Notably, the combination of spheroid-based seeding and aligned nanofibrillar scaffolds led to significantly improved myotube length and width compared to conventional single-cell seeding methods. This work demonstrates the feasibility of using synthetic, collagen-mimicking nanofibrillar scaffolds in conjunction with bioassembled myoblast spheroids to recapitulate key features of skeletal muscle tissue. The melt electrofibrillation approach not only provides precise control over scaffold geometry and fiber alignment but also offers scalability, reproducibility, and mechanical robustness. Altogether, our results underscore the potential of this system as a versatile platform for bottom-up muscle tissue engineering and biofabrication of other anisotropic tissues. Future investigations may expand this model to human muscle cells and further explore its translational relevance for regenerative medicine applications.
32028905047
