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Biohybrid robots, composed of cellular actuators and synthetic scaffolds, have garnered much attention in recent years owing to the advantages provided by their biological components. In recent years, various forms of biohybrid robots have been developed that are capable of life-like movements, such as walking, swimming, and gripping. Specifically, for walking or crawling biorobots, there is a need for complex functionality and versatile and robust fabrication processes. Here, we designed and fabricated multi-actuator biohybrid walkers with multi-directional walking capabilities in response to noninvasive optical stimulation through a scalable modular biofabrication process. Our new fabrication approach provides a constant mechanical strain throughout the cellular differentiation and maturation process. This maximizes the myotube formation and alignment, limits passive bending, and produces higher active forces. These demonstrations of the new fabrication process and bioactuator designs can pave the way for advanced multi-cellular biohybrid robots and enhance our understanding of the emergent behaviors of these multi-cellular engineered living systems. 

Biohybrid centimeter-scale robots developed from optoelectronics and optogenetic muscles can be controlled wirelessly. 

Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, leads to progressive and fatal muscle weakness through yet‐to‐be‐fully deciphered molecular perturbations. Emerging evidence implicates RhoA/Rho‐associated protein kinase (ROCK) signalling in DMD pathology, yet its direct role in DMD muscle function, and related mechanisms, are unknown.

Three‐dimensionally engineered dystrophin‐deficientmdxskeletal muscles andmdxmice were used to test the role of ROCK in DMD muscle functionin vitroandin situ, respectively. The role of ARHGEF3, one of the RhoA guanine nucleotide exchange factors (GEFs), in RhoA/ROCK signalling and DMD pathology was examined by generatingArhgef3knockoutmdxmice. The role of RhoA/ROCK signalling in mediating the function of ARHGEF3 was determined by evaluating the effects of wild‐type or GEF‐inactive ARHGEF3 overexpression with ROCK inhibitor treatment. To gain more mechanistic insights, autophagy flux and the role of autophagy were assessed in various conditions with chloroquine.

Inhibition of ROCK with Y‐27632 improved muscle force production in 3D‐engineeredmdxmuscles (+25% from three independent experiments,P < 0.05) and in mice (+25%,P < 0.001). Unlike suggested by previous studies, this improvement was independent of muscle differentiation or quantity and instead related to increased muscle quality. We found that ARHGEF3 was elevated and responsible for RhoA/ROCK activation inmdxmuscles, and that depleting ARHGEF3 inmdxmice restored muscle quality (up to +36%,P < 0.01) and morphology without affecting regeneration. Conversely, overexpressing ARHGEF3 further compromisedmdxmuscle quality (−13% vs. empty vector control,P < 0.01) in GEF activity‐ and ROCK‐dependent manner. Notably, ARHGEF3/ROCK inhibition exerted the effects by rescuing autophagy which is commonly impaired in dystrophic muscles.

Our findings uncover a new pathological mechanism of muscle weakness in DMD involving the ARHGEF3‐ROCK‐autophagy pathway and the therapeutic potential of targeting ARHGEF3 in DMD.

 

Tissue-on-chip systems represent promising platforms for monitoring and controlling tissue functions in vitro for various purposes in biomedical research. The two-dimensional (2D) layouts of these constructs constrain the types of interactions that can be studied and limit their relevance to three-dimensional (3D) tissues. The development of 3D electronic scaffolds and microphysiological devices with geometries and functions tailored to realistic 3D tissues has the potential to create important possibilities in advanced sensing and control. This study presents classes of compliant 3D frameworks that incorporate microscale strain sensors for high-sensitivity measurements of contractile forces of engineered optogenetic muscle tissue rings, supported by quantitative simulations. Compared with traditional approaches based on optical microscopy, these 3D mechanical frameworks and sensing systems can measure not only motions but also contractile forces with high accuracy and high temporal resolution. Results of active tension force measurements of engineered muscle rings under different stimulation conditions in long-term monitoring settings for over 5 wk and in response to various chemical and drug doses demonstrate the utility of such platforms in sensing and modulation of muscle and other tissues. Possibilities for applications range from drug screening and disease modeling to biohybrid robotic engineering.

 

Integration of conductive electrodes with 3D tissue models can have great potential for applications in bioelectronics, drug screening, and implantable devices. As conventional electrodes cannot be easily integrated on 3D, polymeric, and biocompatible substrates, alternatives are highly desirable. Graphene offers significant advantages over conventional electrodes due to its mechanical flexibility and robustness, biocompatibility, and electrical properties. However, the transfer of chemical vapor deposition graphene onto millimeter scale 3D structures is challenging using conventional wet graphene transfer methods with a rigid poly (methyl methacrylate) (PMMA) supportive layer. Here, a biocompatible 3D graphene transfer method onto 3D printed structure using a soft poly ethylene glycol diacrylate (PEGDA) supportive layer to integrate the graphene layer with a 3D engineered ring of skeletal muscle tissue is reported. The use of softer PEGDA supportive layer, with a 10⁵times lower Young's modulus compared to PMMA, results in conformal integration of the graphene with 3D printed pillars and allows electrical stimulation and actuation of the muscle ring with various applied voltages and frequencies. The graphene integration method can be applied to many 3D tissue models and be used as a platform for electrical interfaces to 3D biological tissue system.

 

The intriguing opportunities enabled by the use of living components in biological machines have spurred the development of a variety of muscle‐powered biohybrid robots in recent years. Among them, several generations of tissue‐engineered biohybrid walkers have been established as reliable platforms to study untethered locomotion. However, despite these advances, such technology is not mature yet, and major challenges remain. Herein, steps are taken to address two of them: the lack of systematic design approaches, common to biohybrid robotics in general, and in the case of biohybrid walkers specifically, the lack of maneuverability. A dual‐ring biobot is presented which is computationally designed and selected to exhibit robust forward motion and rotational steering. This dual‐ring biobot consists of two independent muscle actuators and a four‐legged scaffold asymmetric in the fore/aft direction. The integration of multiple muscles within its body architecture, combined with differential electrical stimulation, allows the robot to maneuver. The dual‐ring robot design is then fabricated and experimentally tested, confirming computational predictions and turning abilities. Overall, a design approach based on modeling, simulation, and fabrication exemplified in this versatile robot represents a route to efficiently engineer complex biological machines with adaptive functionalities.

 

Advancing biologically driven soft robotics and actuators will involve employing different scaffold geometries and cellular constructs to enable a controllable emergence for increased production of force. By using hydrogel scaffolds and muscle tissue, soft biological robotic actuators that are capable of motility have been successfully engineered with varying morphologies. Having the flexibility of altering geometry while ensuring tissue viability can enable advancing functional output from these machines through the implementation of new construction concepts and fabrication approaches. This study reports a forward engineering approach to computationally design the next generation of biological machines via direct numerical simulations. This was subsequently followed by fabrication and characterization of high force producing biological machines. These biological machines show millinewton forces capable of driving locomotion at speeds above 0.5 mm s⁻¹. It is important to note that these results are predicted by computational simulations, ultimately showing excellent agreement of the predictive models and experimental results, further providing the ability to forward design future generations of these biological machines. This study aims to develop the building blocks and modular technologies capable of scaling force and complexity of these devices for applications toward solving real world problems in medicine, environment, and manufacturing.