Muscle-fiber-inspired nanofibrillar microbundles induce myogenic differentiation in human adipose-derived stem cells

Skeletal muscle function relies on uniaxially organized myofibers, whose aligned extracellular matrix provides instructive topographical cues that regulate myogenic behavior. Here, we introduce a melt electrofibrillation strategy, melt electrowriting (MEW) of poly (ε-caprolactone)/poly (vinyl acetate) blends, followed by selective polyvinyl acetate removal, to fabricate highly aligned nanofibrillar microbundle scaffolds that present collagen-like nanotopography. We first verified biocompatibility and alignment guidance using primary human skeletal muscle cells compared with 2D tissue culture polystyrene controls. We then assessed the myogenic response of human adipose-derived stem cells (hASCs) on nanofibrillar scaffolds relative to conventional MEW-printed microfibers and 2D controls. Nanofibrils supported sustained viability over 35 days and promoted pronounced cell alignment, aligned collagen type-I deposition, and enhanced myogenic differentiation. hASCs on the scaffolds formed myosin-positive, multinucleated myotube-like structures by days 28-35 and exhibited increased MYOG and MYF6 expression by qPCR. Bulk RNA sequencing (day 21) further showed that nanofibrils induce a distinct transcriptional state enriched for muscle development/differentiation and contraction-associated programs, accompanied by activation of mechanosensitive signaling and adhesion-cytoskeleton remodeling pathways (PI3K-Akt, MAPK, Wnt, Hippo, and TGF-β). Titin immunostaining revealed early sarcomere assembly, indicating progression toward maturation. Collectively, nanofibrillar scaffolds establish a programmable, muscle-mimetic fibrillar niche that strengthens topography-driven myogenesis of hASCs and supports prolonged culture, providing a versatile platform for in vitro muscle tissue engineering.