Exercise-Mediated Biochemical Secretion Guides Maturation and Repair of Neuromuscular Actuators

<b>Introduction: </b>Stimuli-responsive actuators that match the flexibility and adaptability of skeletal muscle are of significant interest in powering the next-generation of responsive machines. However, abiotic actuators have yet to match muscle’s energy efficiency or its ability to dynamically adapt to gain-of-function cues, such as exercise, and loss-of-function cues, such as damage. We have engineered bioactuators, powered by skeletal muscle and controlled by motor neurons, and deployed them as functional actuators in soft robots. Deeper understanding of the stimuli-responsive nature of these bioactuators requires studying how damage that disrupts intercellular signaling between motor neurons and skeletal muscle can impact mobility.<br/>Measuring and modulating intercellular signaling requires tools for longitudinal tracking of biochemicals exchanged between cells within tissues. We leveraged a custom-built micro-invasive interstitial fluid sampling tool to measure biochemical signaling in space and time within our bioactuators. We used this tool to identify muscle-secreted cytokines that are upregulated in response to exercise, driving hierarchical tissue assembly. Moreover, we show that exercise-mediated biochemical secretion enhances repair after injury and accelerates functional integration of bioactuators implanted in animals. We anticipate that this will advance understanding of how to design bioactuators that are robustly responsive to injury when deployed in real-world applications.<br/><b>Methods: </b>We use our established protocols to engineer neuromuscular bioactuators using mouse and human skeletal muscle and motor neuron lines. Briefly, bioactuators are formed by embedding optogenetic skeletal muscle myoblasts and motor neurons within separate layers of a fibrin/Matrigel hydrogel and growing them around a flexible skeleton. Neuromuscular junctions are formed within 3 days and muscle contraction can be controlled via light stimulation of the muscle or upstream chemical stimulation of the neurons. We have developed a tool, composed of a nanofluidic pump coupled to a micro-scale probe, that enables minimally-invasive sampling of interstitial fluid from tissues. We use mass spectrometry to quantify secretion of hundreds of peptides and proteins in parallel.<br/><b>Results & Discussion: </b>Our bioactuator and sampling tool enabled investigating the impact of exercise-mediated biochemical secretion on tissue maturation and response to injury. Sampling interstitial fluid in engineered tissues showed significant proteomic differences between control and exercised groups. Specifically, light-stimulated exercise of optogenetic muscle upregulates the secretion of cytokines that enhance nerve growth including axon guidance pathways mediated by netrin and nicotinic acetylcholine receptor signaling pathways. When bioactuators were implanted in a volumetric muscle loss model <i>in vivo</i>, targeted exercise upregulated secretion of cytokines that promoted innervation and angiogenesis, enhanced functional recovery after injury. These results indicate: 1) our sampling tool can be used to monitor intercellular signaling in stimuli-responsive living materials; 2) exercise-mediated cytokine secretion can be used to guide assembly of such materials; 3) stimuli-responsive living materials can modulate intercellular signaling <i>in vivo</i> and enhance integration of implanted tissues.<br/><b>Conclusion: </b>There is a critical need to develop bioactuators for functional applications, and to monitor and modulate intercellular communication within such stimuli-responsive living materials. We have developed a bioactuator and sampling tool that enables monitoring intercellular signaling, and established how exercise-mediated biochemical secretion guides tissue maturation. This platform is being used to advance mechanistic understanding of neuromuscular tissues and develop stimuli-responsive bioactuators for engineered living machines.