2.5D Actuating Substrates Enable Decoupling the Mechanical and Biochemical Effects of Muscle Exercise on Motor Neurons

Emerging in vivo evidence suggests that exercise impacts peripheral nerves, but the difficulty of isolating and studying the muscle-specific impact on motor neurons in vivo , as well as the inability to decouple the biochemical and mechanical impacts of exercise in this setting, motivate investigating this phenomenon in vitro . In this study, we show that tuning the mechanical properties of fibrin hydrogels can generate stable 2.5D motor neuron and contractile skeletal muscle cultures that enable long-term efficient secretome harvesting from exercised tissues. Motor neurons stimulated with muscle-secreted cytokines significantly upregulate neurite outgrowth and migration, with an effect size dependent on exercise intensity. Actuating magnetic microparticles embedded within 2.5D substrates enabled us to dynamically stretch motor neurons and non-invasively mimic the mechanical effects of exercise, revealing that dynamic stretch has an equally significant impact on axonogenesis. RNA sequencing revealed different transcriptomic signatures between groups, with biochemical stimulation having a significantly greater impact on cell signaling related to axon growth and development, neuron projection guidance, and neuron-muscle synapse maturation. Our study thus leverages 2.5D actuating substrates to robustly validate a hypothesized role for muscle exercise in regulating motor neuron growth and maturation through both mechanical and biochemical signaling.