Flexible 3D-Printed Piezoelectric and Bioinstructive Platforms for Skeletal Muscle Development and Organization

Volumetric muscle loss (VML) is a devastating injury in which extensive skeletal muscle loss overwhelms endogenous healing. Despite their therapeutic efficacy, acellular constructs fail to provide a comprehensive set of bioactive cues required for muscle development. Notably, very few constructs provide bioelectrical cues necessary for muscle development. To address that need, this study aimed to develop a myogenic platform that provides a comprehensive set of bioactive cues, including bioelectrical cues that can be delivered wirelessly, to promote skeletal muscle development. To achieve this, we engineered a microchanneled, piezoelectric, 3D-printed platform based on a unique interpenetrating hydrogel network of gelatin methacrylate and poly(acrylic acid) that incorporates barium titanate nanoparticles. Rheological studies showed stable gel-like viscoelasticity and shear-thinning behavior of the hydrogels, indicating their suitability for extrusion-3D printing. Physicochemical characterization demonstrated the platform's structural fidelity, precise microchannel definition, pronounced flexibility, and self-adhesiveness. While the platform's stiffness was comparable to that of skeletal muscle, it also generated a surface potential of ∼90 mV when deformed by ultrasound (US), highlighting its piezoelectric nature and ability to harness electromechanically triggered bioelectrical cues. FE-SEM and immunofluorescence analyses revealed that microchannels and US-induced piezoelectricity from the platform can synergistically help form dense, compact, and aligned myotubes. Notably, we demonstrate that the US-induced piezoelectric platforms can promote early-stage myotube development, enhance myogenic progression, and help form mature, elongated, thick myotubes over time. Finally, we observe that the piezoelectric platforms exhibit increased FAK and MAPK activation in adherent cells over time, suggesting that these pathways can contribute to transducing electromechanical cues that promote myotube maturation. Overall, this is the first-of-its-kind comprehensive study to engineer a platform that delivers a comprehensive set of bioactive cues, specifically wirelessly delivered bioelectrical cues, resulting in a spatiotemporally bioinstructive microenvironment for skeletal muscle development. We believe this unique platform could be a promising regenerative therapy for VML.