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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\u2010culture with motor neurons. We accomplished this by creating micro\u2010nano biomaterial interfaces that presented nanoclusters of integrin\u2010binding ligands to promote mechanotransduction on the surface of aligned electrospun microfibers. Controlling surface topography and nanoscale ligand clustering led to 1.5\u2010 to 2.5\u2010fold increases in myoblast proliferation, myotube formation, elongation, and alignment, resulting in spontaneous twitching and enhanced myotube\u2010neuron 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\u2010resolution platform for advancing skeletal muscle tissue engineering, studying muscle development and neuromuscular diseases, and shaping therapeutic screening strategies.<\/jats:p>","DOI":"10.1002\/smll.75566","type":"journal-article","created":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T04:30:51Z","timestamp":1788496251000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering In Vitro Models of Skeletal Muscle With Neuromuscular Junctions Using Hierarchical Micro\u2010Nano Biomaterials: Cooperative Effect of Adhesion Ligand Nanoclustering and Surface Anisotropy"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0025-1171","authenticated-orcid":false,"given":"Shirin","family":"Nour","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering The University of Melbourne  Parkville Victoria Australia"},{"name":"Graeme Clark Institute The University of Melbourne  Parkville Victoria Australia"},{"name":"Polymer Science Group Department of Chemical Engineering The University of Melbourne  Parkville Victoria Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2509-4619","authenticated-orcid":false,"given":"Kristy","family":"Swiderski","sequence":"additional","affiliation":[{"name":"Centre for Muscle Research Department of Anatomy and Physiology The University of Melbourne  Parkville Victoria Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6740-4195","authenticated-orcid":false,"given":"Sahar","family":"Salehi","sequence":"additional","affiliation":[{"name":"Department of Biomaterials Faculty of Engineering Science University of Bayreuth  Bayreuth Germany"},{"name":"Institute of Food Science and Biotechnology University of Hohenheim  Stuttgart Germany"},{"name":"Fraunhofer Institute for Manufacturing Engineering and Automation  Stuttgart Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9220-9810","authenticated-orcid":false,"given":"Gordon S.","family":"Lynch","sequence":"additional","affiliation":[{"name":"Centre for Muscle Research Department of Anatomy and Physiology The University of Melbourne  Parkville Victoria Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6925-885X","authenticated-orcid":false,"given":"Andrea J.","family":"O'Connor","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering The University of Melbourne  Parkville Victoria Australia"},{"name":"Graeme Clark Institute The University of Melbourne  Parkville Victoria Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2771-9675","authenticated-orcid":false,"given":"Greg 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