Emergence of functional neuromuscular junctions in an engineered, multicellular spinal cord-muscle bioactuator

Three-dimensional (3D) biomimetic systems hold great promise for the study of biological systems in vitro as well as for the development and testing of pharmaceuticals. Here, we test the hypothesis that an intact segment of lumbar rat spinal cord will form functional neuromuscular junctions (NMJs) with engineered, 3D muscle tissue, mimicking the partial development of the peripheral nervous system (PNS). Muscle tissues are grown on a 3D-printed polyethylene glycol (PEG) skeleton where deflection of the backbone due to muscle contraction causes the displacement of the pillar-like "feet." We show that spinal cord explants extend a robust and complex arbor of motor neurons and glia in vitro. We then engineered a "spinobot" by innervating the muscle tissue with an intact segment of lumbar spinal cord that houses the hindlimb locomotor central pattern generator (CPG). Within 7 days of the spinal cord being introduced to the muscle tissue, functional neuromuscular junctions (NMJs) are formed, resulting in the development of an early PNS in vitro. The newly innervated muscles exhibit spontaneous contractions as measured by the displacement of pillars on the PEG skeleton. Upon chemical excitation, the spinal cord-muscle system initiated muscular twitches with a consistent frequency pattern. These sequences of contraction/relaxation suggest the action of a spinal CPG. Chemical inhibition with a blocker of neuronal glutamate receptors effectively blocked contractions. Overall, these data demonstrate that a rat spinal cord is capable of forming functional neuromuscular junctions ex vivo with an engineered muscle tissue at an ontogenetically similar timescale.