Engineering In Vitro Models of Skeletal Muscle With Neuromuscular Junctions Using Hierarchical Micro‐Nano Biomaterials: Cooperative Effect of Adhesion Ligand Nanoclustering and Surface Anisotropy

The development of high-fidelity in vitro contractile skeletal muscle containing neuromuscular junctions (NMJs) is an unmet challenge, largely because current systems cannot accurately reproduce hierarchical micro- to nano-scale biointerfacial cues of the native extracellular matrix. This limitation impairs tissue regeneration and limits the clinical relevance of existing models for drug screening and studies of disease pathophysiology. Herein, we present hierarchical, anisotropic biomaterials that induced early maturation of myotubes and NMJ development during co-culture with motor neurons. We accomplished this by creating micro-nano biomaterial interfaces that presented nanoclusters of integrin-binding ligands to promote mechanotransduction on the surface of aligned electrospun microfibers. Controlling surface topography and nanoscale ligand clustering led to 1.5- to 2.5-fold increases in myoblast proliferation, myotube formation, elongation, and alignment, resulting in spontaneous twitching and enhanced myotube-neuron connections, including increased acetylcholine receptor clustering, neurite branching, and myotube contraction compared to control surfaces without the requirement for exogenous neurotrophic factors or electrical stimulation. These findings highlight the importance of tailoring the distribution and presentation of adhesive ligands for in vitro development of NMJs and synaptic organization. Our approach offers a scalable, high-resolution platform for advancing skeletal muscle tissue engineering, studying muscle development and neuromuscular diseases, and shaping therapeutic screening strategies.