Modular Tissue-Engineered Motor Units Featuring Spinal Motor Neurons Innervating Self-Assembled Myofiber Bundles

A motor unit is the functional unit of muscle contraction, consisting of a population of skeletal muscle fibers innervated by axon terminals from a motor neuron. Tissue engineering strategies are being pursued to treat neuromuscular injuries by mimicking aspects of native myofascicular architecture; however, the critical role of innervation in myofiber development is often overlooked. Our group previously developed a pre-innervated tissue-engineered muscle on nanofiber sheets, demonstrating that innervation facilitated myofiber maturation and function in vitro. The current study builds on this framework to biofabricate pre-innervated three-dimensional (3D) bundles of individual myofibers that more closely replicate in vivo architecture. Specifically, we established a methodology to generate centimeter-scale Tissue Engineered Motor Units (TEMUs) comprising aligned myofiber bundles within a collagenous hydrogel and innervated by axons projecting from discrete population(s) of spinal motor neurons. A custom-built polydimethylsiloxane micro-scale channel system facilitated the alignment and self-assembly of myoblasts. The presence of aggregated motor neurons and axonal integration significantly enhanced myofiber maturation and contractility compared to non-innervated controls. We also evaluated the effects of media constituents on myofiber maturation, as assessed by myocyte fusion and sarcomere formation. Importantly, this TEMU biofabrication protocol is fully scalable, generating modular myofiber bundles at least 8 cm in length that can be aligned in parallel to achieve large-scale myofiber macro-bundles. TEMUs address key challenges in muscle tissue engineering by providing a 3D biofidelic platform to study the role of innervation in muscle development and function in vitro, as well as an implantable composite tissue to facilitate muscle replacement after severe trauma.