Modular Skeletal Muscle Biohybrid Robots: Creation & Multi-Unit Assembly of Functional Muscle Building Blocks

The emerging field of biohybrid robotics aims to create the next generation of soft and sustainable robots by using engineered biological muscle tissues, integrated with soft materials, as actuators. Alongside robotics, such biohybrid muscles will also find biomedical applications in prostheses and drug development and screening. Current skeletal muscle tissue-based robots are powered by bulk-grown 3D configurations of muscle tissue (<i>e.g</i>., blocks or ring-like tissues). The size of these constructs is restricted to the sub-cm scale due to biofabrication constraints and limited nutrient perfusion. Further, lack of internal topography within bulk constructs results in poor control of myofiber formation and thus low contraction force. Therefore, to generate larger biohybrid constructs capable of complex motion while avoiding the issues of bulk muscle tissue manufacture, we have developed a bottom-up fabrication approach based on the assembly of muscle building blocks into designs that exploit the characteristics of the single actuators to form a multi-unit configuration. We created the muscle building blocks by integrating thin layers of muscle-cell laden hydrogels onto hydrogel-based exoskeletons characterized by material properties and microtopographic patterns that enhance muscle tissue development (<i>i.e.</i>, microgrooves). Our thin cell-laden hydrogel layers allowed nutrient diffusion throughout the layer, and we incorporated microtopography with a layer-by-layer approach. <br/><br/>We used a novel linear volumetric printing technique, named xolography, to form the hydrogel exoskeletons (&lt; 1 mm x 5 mm x 8 mm). Adapting xolography for aqueous hydrogel printing allowed us to take advantage of the quick speed, high resolution, and achievable geometric complexity of the technique. We printed exoskeletons with different mechanical properties by combining polyethylene glycol diacrylates with gelatin methacryloyl, e.g. 15% PEGDA700 and 5% GelMA (P700/GelMA, Young's Modulus 140 kPa) and 15% PEGDA575 and 5% GelMA (P575/GelMA, Young's Modulus 270 kPa) . The exoskeletons featured microgrooves of 100 – 300 μm in width to guide formation of uniaxially oriented myofibers. To stably integrate the exoskeletal and the cellular hydrogels, we developed a Matrigel, collagen, and gelatin methacrylate-based cellular hydrogel that, when seeded with myoblasts, allowed for both contractile myofiber formation and effective cross-linking with the exoskeletal hydrogel, as shown via confocal and brightfield imaging and histological analysis.<br/><br/>Directionality analysis of confocal microscopy images of samples stained to visualize myofibers (F-Actin) showed uniaxial alignment in the channel direction with a standard deviation of 10°-20°. The structural deformation of these constructs caused by the contraction via electrical stimulation of their muscle tissue component depended on the exoskeleton composition. Constructs based on P700/GelMA contracted 4% in the direction of myofiber contraction, while constructs based on P575/GelMA contracted &lt;1%. Finally, to form modular multi-unit biohybrid robots with predictable motion abilities, we assembled the muscle building blocks by cross-linking the residual unreacted acrylate groups in the PEGDA/GelMA exoskeletons under UV irradiation.<br/><br/>In conclusion, we developed a method that builds biohybrid robots by the assembly of muscle building blocks with tunable mechanical properties and actuation performance. We have reduced the complexity and increased the predictability of biohybrid muscle fabrication, thus unlocking the potential of engineered muscle tissue as an actuating technology.