Tissue Engineered Motor Units Featuring Spinal Motor Neurons Innervating Myofiber Bundles

Introduction: Tissue engineering strategies are being pursued to treat severe neuromuscular injuries by mimicking aspects of native myofascicular architecture and providing topographical guidance to regenerating motor units. A motor unit is the functional unit of muscle contraction and consists of a motor neuron and the population of skeletal muscle fibers innervated by the neuron's axon terminals. Previously developed pre-innervated tissue engineered muscle on nanofiber sheets showed that the presence of motor neurons facilitated myofiber maturation in vitro and promoted a pro-regenerative microenvironment following implant in a rat model of neuromuscular injury1. While promising, the planar nature of this pre-innervated muscle did not reflect the three-dimensional (3D) bundled architecture of myofibers in vivo. In response, we have developed Tissue Engineered Motor Units (TEMUs) comprised of centimeter-scale aligned bundles of myofibers encased within a 3D collagen extracellular matrix (ECM) and innervated by axons projecting from a discrete population(s) of spinal motor neurons.

Materials and

Methods: Our system facilitates the self-assembly of myoblasts into aligned 3D bundles of myofibers measuring millimeters in diameter and spanning centimeters in length. Using this platform, we investigated the combined effects of channel topography and innervation on myocyte maturation and contraction. We dispensed a homogenous solution comprising ECM + C2C12 mouse myoblasts into a 3D-printed channel scaffolding and placed spinal motor neuron aggregates on either end of the channel. After variable time – 7 or 21 days in culture – myofiber bundle thickness, fusion index, axon ingrowth, and neuromuscular junction formation were assessed through immunocytochemistry and confocal microscopy. Likewise, histological data was acquired in scaled up and bulked iterations.

Results, Conclusions, and Discussions:

Results: Skeletal myocytes were successfully co-cultured with motor neurons in a columnar architecture. Cross-sectional histological analysis showed that TEMUs exhibited fascicle-like organization, exhibiting myofiber bundles and axons projecting along and within the ECM (Fig. 1). The engineered microtissue was formed at variable lengths (1, 2, 4, 8cm) with uniform myocyte cell density, neuron cell bodies at the extremes, and axons projecting along the length. Individual microtissue units were bundled to increase the cumulative depth of a volumetric repair strategy (Fig. 2). Ongoing studies are using the TEMU platform to further establish the critical role of motor axon innervation on the development, maturation, functionality, and regenerative potential of engineered 3D myofiber bundles.

Conclusion: To overcome the inherent limitations of engineering complex multicellular muscle tissue, there is a critical need for an approach that can simultaneously (1) recapitulate the fascicular architecture and cellular niche of skeletal muscle, (2) provide axonal inputs from multiple neuronal populations to recreate motor units, and (3) fabricate scalable tissue appropriate for preclinical testing. TEMUs comprised of centimeter-scale aligned bundles of myocytes with axonal innervation from discrete motor neuron populations may provide such a platform. The innervated skeletal muscle in the TEMU contracts when induced with glutamate and can be scaled up in size via length and width. TEMUs may aid in addressing key challenges in tissue engineering muscle and may serve as both an in vitro test platform as well as an implantable composite tissue to facilitate muscle replacement after severe trauma.

Acknowledgements (Optional): 3D printed object courtesy of the University of Pennsylvania Libraries' Holman Biotech Commons.