Ritu Raman

Biohybrid robots combining compliant synthetic support structures with biological actuators could enable future applications ranging from precision microsurgery to unmanned exploration. Machines actuated by living skeletal muscles are capable of adaptive behaviors, such as sensing and responding to environmental stimuli in real-time, offering functional advantages over non-biological actuators. However, typical skeletal muscle-powered biohybrid robots depend on 3D tissues which require large cell volumes and offer limited control of muscle fiber alignment, thus reducing efficiency of force generation and transduction. Here, we present a locomotive biohybrid robot powered by 2D monolayers, or thin films, of precisely aligned skeletal muscle fibers on a micropatterned hydrogel skeleton. We demonstrate how varying skeleton design parameters, ranging from material stiffness to microscale topology, impacts muscle fiber alignment and resultant actuation strains, generating forces 10X higher than previous 2D skeletal muscle actuators, improving untethered actuation longevity by ~4500X from < 10 minutes to > 30 days, and increasing efficiency of muscle force output (force per unit volume of muscle) by 20X as compared to 3D muscles. Utilizing our optimized design for skeletal muscle thin films, we create a multi-limbed robot composed of independent muscle-powered fins capable of on/off control and frequency-dependent speed control. With these control inputs, we achieve steered multidirectional locomotion at speeds up to 4 body lengths per minute in straight movement and 1200 degrees per minute in rotational movement, highlighting potential for such actuators to be transformed into long-lasting functional soft robots.

Diseases or injuries that affect neuromuscular tissues severely impact human health, motivating the development of in vitro models of the motor control system. Current platforms either have limited reproducibility or require complex fabrication protocols that preclude high-resolution imaging and scalable functional analysis. We have developed a one-step method for fabricating an all-hydrogel limb-on-a-chip that addresses key challenges of current systems by enabling reproducible and scalable manufacturing of neuromuscular tissues compartmentalized into "spinal cord" and "limb" chambers. Co-cultures of motor neurons and skeletal muscles within this platform enable longitudinal tracking of neuromuscular junction formation and visualization of changes in muscle contraction in response to motor neuron stimulation. We demonstrate tissue-wide recordings of muscle force and single-cell-resolution measurements of muscle calcium activity. Our accessible method for fabricating reproducible in vitro neuromuscular models that are compatible with high-resolution imaging and functional readouts provides a powerful tool for investigating the neuromuscular interface in health and disease.

and intravasated TCs are challenging to isolate. To address these challenges, we developed IntravChip, a continuously perfused microfluidic platform containing a vascularized primary tumor microenvironment (TME) enabling the observation of TC intravasation, and a downstream chamber to collect intravasated TCs. The IntravChip can support a high TC concentration in the TME while maintaining complete vascular perfusion, which we found was necessary to collect intravasated cells. Using MDA-MB-231 breast TCs, we identified an optimal initial TC seeding density that, by day 9, yields a densely populated TME and 100-440 collected intravasated TCs. We validated the IntravChip across several TC types, showing that MDA-MB-231 and MV3 TCs have the highest intravasation rates while MCF-7 TCs have low intravasation efficiency. We also show that the IntravChip is compatible with super-resolution nano-imaging. Our devices enabled high-quality STORM imaging, which revealed that H3K9me3 nanodomains are significantly differentially distributed in intravasated MDA-MB-231 tumor cells compared to those residing in the TME. Finally, the IntravChip was validated as a platform to test the effects of anti-cancer drugs on tumor cells and on the vasculature. We showed that a 5 μM concentration of sorafenib reduced intravasation events by 69% without impacting the morphology of the microvascular networks (MVNs), while a 10 μM concentration led to a significant decrease in vessel diameter. This platform enables quantitative analysis of TC intravasation, collection of intravasated TCs for characterization, and screening of anti-metastatic therapies.

Fostering relationships between the disability and soft robotics communities will spark innovations that could benefit all.

Maintaining uniform cell and hydrogel distribution in extrusion 3D bioprinting is a critical challenge, especially for long-duration or high-throughput prints. We present a magnetically-actuated mixer (MagMix), a compact modular in situ platform that integrates into standard extrusion bioprinters to actively homogenize bioinks in real-time. MagMix utilizes an internal propeller driven by a servo-controlled external magnet, enabling continuous, active, tunable speed mixing without altering bioink formulation. Computational simulations and experimental validation enable optimizing propeller geometry and mixing parameters. We demonstrate the utility of MagMix across commonly used cellular bioinks with different viscosities to demonstrate that active in situ mixing prevents cell sedimentation, improves cell viability, and eliminates nozzle clogging over extended print sessions while preserving 3D print quality and cell differentiation capacity. MagMix can be readily integrated into any extrusion bioprinting workflow and offers a scalable, bioink-agnostic solution to improve reproducibility and functionality in bioprinted constructs for applications in tissue engineering.

Heparan sulfate proteoglycans (HSPGs) have been recognized as key plasma membrane-tethered co-receptors for a broad range of growth factors and cytokines containing cationic heparan-binding domains1,2. However, how HSPGs mechanistically mediate signalling at the cell surface—particularly in the context of cell surface RNA—remain poorly understood. During developmental and disease processes, vascular endothelial growth factor (VEGF-A), a heparan sulfate-binding factor, regulates endothelial cell growth and angiogenesis3. The regulatory paradigm for endothelial cell-mediated selectively of VEGF-A binding and activity has largely been focused on understanding the selective sulfation of the anionic heparan sulfate chains4–8. Here we examine the organizational rules of a new class of anionic cell surface conjugates, glycoRNAs9,10, and cell surface RNA-binding proteins (csRBPs11,12). Leveraging genome-scale knockout screens, we discovered that heparan sulfate biosynthesis and specifically the 6-O-sulfated forms of heparan sulfate chains are critical for the assembly of clusters of glycoRNAs and csRBPs (cell surface ribonucleoproteins (csRNPs)). Mechanistically, we show that these clusters antagonize heparan sulfate-mediated activation of ERK signalling downstream of VEGF-A. We demonstrate that the heparan sulfate-binding domain of VEGF-A165 is responsible for binding RNA, and that disrupting this interaction enhances ERK signalling and impairs vascular development both in vitro and in vivo and is conserved across species. Our study thus uncovers a previously unrecognized regulatory axis by which csRNPs negatively modulate heparan sulfate-mediated signalling in the context of angiogenesis driven by VEGF-A. Heparan sulfate proteoglycans facilitate the assembly of clusters of glycoRNAs and cell surface RNA-binding proteins, which negatively modulate VEGF-A signalling and angiogenesis.

Abstract Exercise promotes human mobility by tuning the function of skeletal muscle, and recent studies highlight exercise's broader impacts on human health via muscle's paracrine and endocrine roles beyond force generation. In vitro models of tissue engineered skeletal muscle enable precise investigation of adaptation to exercise, with emerging approaches for optogenetic muscle stimulation providing a less invasive alternative to traditional techniques for electrical stimulation. In this study, we present a high‐throughput muscle culture and optical exercise protocol for scalable in vitro exercise studies. First, we characterize optical rheobase for 2D muscle monolayers, finding that optical intensities as low as 5 μW mm −2 can trigger functional contraction. We then leverage RNA sequencing to map changes in muscle gene expression in response to various optical exercise regimens, highlighting how changing stimulation parameters impact myogenic and broader physiological and pathological transcriptional responses. Our platform and results establish a practical foundation for high‐throughput in vitro exercise studies of skeletal muscle.

Synthetic biology offers control over cellular and tissue functions. As it moves beyond microbes into humans, synthetic biology enables precise control over gene expression, cell fate, and tissue organization across heart, lung, blood, and sleep systems. By integrating genome engineering, dynamic gene circuits, and high-dimensional biosensors, these advances support scalable, quantitative models of multicellular biology, expanding the need for systems-level models and integration. We highlight emerging systems such as tunable transcriptional regulators, synthetic organizers, and feedback circuits that bridge molecular control with functional outcomes. Furthermore, by combining omics data with artificial intelligence (AI)-guided circuit design, synthetic biology enables high-resolution cellular and tissue-scale models of development, cellular interactions, drug development, gene therapy, and therapeutic response. Key challenges remain-including delivery, transgene stability, and robust spatiotemporal control in physiologically relevant models. This perspective synthesizes field-spanning progress and defines shared priorities for engineering cells and tissues that function reliably across dynamic, multi-organ environments.

Biohybrid robots powered by tissue engineered skeletal muscle have historically relied on architectures in which muscle actuators are placed directly on skeletons, thus limiting the accessible design space for such machines. By contrast, native musculoskeletal architecture relies on tendons to bridge the interface between muscles and skeletons, enabling precise, space-efficient, and energy-efficient force transmission. In this study, a mathematical model of the muscle-tendon-skeleton interface is used to design a biohybrid muscle-tendon unit composed of tissue engineered muscle coupled to adhesive tough hydrogel tendons. It is demonstrated that tuning tendon stiffness and pre-tension optimizes actuator performance, and tuning skeleton stiffness modulates force transmission from muscles to skeletons, with fatigue characteristics measured over > 7000 cycles. Furthermore, an ≈11X improvement in power-to-weight ratio of muscle-tendon units is demonstrated compared to previous demonstrations of robots powered by muscles alone. This work validates a robust approach for designing, manufacturing, and deploying muscle-tendon actuators that promises to enhance the modularity and efficiency of biohybrid robots.

Abstract Engineering organized microvascular networks remains a critical challenge in tissue engineering and regenerative medicine. While biochemical approaches for patterning angiogenesis via growth factor delivery have shown promise, their inability to pattern sustained growth factors with spatiotemporal control limits effectiveness. Here, we demonstrate that dynamically patterned mechanical forces enable precise spatiotemporal control over angiogenic sprouting. We developed a magnetically actuated human vessel-on-a-chip platform that integrates a perfusable endothelialized microchannel within a collagen matrix and allows non-invasive and tunable mechanical stimulation across three spatial dimensions and time (4D). Using an automated 3-axis actuator, we systematically investigated how strain magnitude, frequency, and direction modulate endothelial cell behavior and vessel morphogenesis. Dynamic mechanical stimulation at physiological strain magnitudes (5–15%) enhanced endothelial alignment and barrier function while promoting angiogenesis in a strain-magnitude–dependent manner: lower dynamic strain (5%) maximized sprout initiation, whereas higher dynamic strain (15%) promoted elongation of sprouts. Sequential reorientation of strain direction reprogrammed sprouting trajectories along X, Y, and Z directions, generating complex sprout geometries such as L-shaped branches. RNA sequencing revealed mechanically induced transcriptional profiles distinct from unstimulated controls, characterized by upregulation of genes associated with angiogenesis, mechanotransduction, and extracellular matrix remodeling. Functional perturbation of Piezo1 reduced strain-induced sprouting without altering barrier stabilization, indicating that dynamic mechanical stimulation engages multiple mechanotransduction pathways to regulate angiogenesis. Collectively, these findings establish a strategy for spatiotemporally controlled angiogenesis through 4D force patterning to program vascular morphogenesis while preserving function. This approach provides a foundation for engineering hierarchically organized vascular networks for tissue regeneration. Significance Generation of spatially organized, perfusable microvascular networks is essential for building functional human tissues. Biochemical approaches to pattern angiogenesis rely on diffusive growth factors, which limit control over spatiotemporal sprouting dynamics. Here, we demonstrate that dynamically patterned mechanical forces direct vascular morphogenesis across three spatial dimensions and time (4D). Using a magnetically actuated human vessel-on-a-chip, we show how strain magnitude and orientation govern angiogenic sprouting and reveal transcriptional programs linking mechanical cues to observed functional changes. For the first time, we show that dynamic reorientation of imposed forces can reprogram angiogenic trajectories in real-time. This platform enables programmable mechanical control of angiogenesis and systematic dissection of mechanotransduction pathways, advancing strategies for tissue vascularization, and modeling mechanically regulated vascular diseases.

Summary Diseases or injuries that impact neuromuscular tissues have a severe negative impact on human health, mobility, and quality-of-life, motivating the development of tissue engineered in vitro models of the motor control system. Current neuromuscular organoids and organ-on-a-chip platforms either rely on stochastic self-assembly that limits reproducibility or require complex microfabrication processes that preclude high-resolution imaging and scalable functional analysis. We have developed a Limb-on-a-Chip platform that addresses key challenges of current model systems by enabling reproducible and scalable manufacturing of neuromuscular tissues compartmentalized into “spinal cord” and “limb” chambers, while promoting biochemical crosstalk between cell types. Our fabrication method leverages 3D printed molds to perform 1-step micropatterning of an all-hydrogel chip containing precise features to guide muscle fiber alignment and motor neuron axonal outgrowth. We demonstrate the ability to co-culture motor neurons and skeletal muscles within this hydrogel platform, enabling tissue-wide readouts of muscle force as well as single cell-resolution measurements of muscle fiber calcium activity. Our accessible method for fabricating reproducible in vitro neuromuscular models that are compatible with high-resolution imaging and functional readouts provides a powerful new tool for investigating the neuromuscular interface in health and disease.

Abstract Biohybrid robots powered by tissue engineered skeletal muscle have historically relied on architectures in which muscle actuators are placed directly on skeletons, thus limiting the accessible design space for such machines. By contrast, native musculoskeletal architecture relies on tendons to bridge the interface between muscles and skeletons, enabling precise, space-efficient, and energy-efficient force transmission. In this study, we use a mathematical model of the muscle-tendon-skeleton interface to design a biohybrid muscle-tendon unit composed of tissue engineered muscle coupled to adhesive tough hydrogel tendons. We show how tuning tendon stiffness and pre-tension modulates actuator performance, measure fatigue characteristics of our actuators over &gt;7000 cycles, and tune skeleton stiffness to increase force transmission muscles to skeletons by ∼29X. Furthermore, we demonstrate an ∼11X improvement in power-to-weight ratio of muscle-tendon units as compared to previous demonstrations of robots powered by muscles alone. This work validates a robust approach for designing, manufacturing, and deploying muscle-tendon actuators that promises to enhance the modularity and efficiency of biohybrid robots.

Abstract Real-time monitoring and in situ data analysis are increasingly vital for enhancing precision, reproducibility, and defect detection in embedded bioprinting. As interest grows in improving the capabilities of existing bioprinting systems, accessible strategies for integrating real-time sensing and analysis are becoming essential to ensure consistent quality and to optimize printing parameters. Here, we present a modular, low-cost, and printer-agnostic platform that combines a compact sensing architecture with an effective image analysis pipeline to enable in situ process monitoring, defect detection, and print quality assessment. the platform integrates a digital microscope aligned on-axis with the extrusion printhead to capture high-resolution images during fabrication. We applied and compared two segmentation methods, thresholding and the Segment Anything Model (SAM) on in situ and ex situ datasets acquired via confocal fluorescence imaging, finding SAM to yield stronger correlations (R = 0.85–0.86) between in situ and ex situ measurements. Additionally, we demonstrated that 2D in situ images provide reliable approximations of 3D filament geometries, supporting their use for real-time morphological assessment. the system also revealed pressure-related effects on the diameters, and a critical velocity threshold for printing stability, highlighting its value for process optimization. together, these findings establish the approach as a low-cost, scalable and adaptable solution that can be readily implemented across embedded bioprinting workflows, offering a practical path toward greater reproducibility and automation.

RecommendationsB iological systems are capable of dexterous and adaptable behaviors across length scales.Replicating these behaviors in human-made machines thus requires drawing inspiration from nature.In recent years, roboticists have identified compliance as a key design feature of biological sensors and actuators, enabling closed-loop control of complex behaviors such as locomotion, feeding, and manipulation that are central to life.Integrating compliance into the functional components of autonomous machines has inspired and accelerated the growth of "soft robotics" as a discipline.In this special issue on Soft Robotics, we highlight emerging frontiers in compliant sensing and actuation, novel materials and manufacturing techniques for fabricating soft bioinspired and biohybrid systems, and real-world applications of compliant machines.The featured reviews outline an exciting vision for the future of soft robotics that promises to advance the safety, reliability, and sustainability of autonomous machines.Despite rapid progress in soft materials, sensors, and actuators, achieving seamless sensorimotor integration in soft robots remains a challenge.A comprehensive review from Xiaodong Chen and colleagues explores this emerging topic by examining the foundations of sensorimotor functions. 1The authors first outline the current state-of-the-art in soft sensing mechanisms (pressure, strain, temperature, optical, chemical, acoustic, and electromagnetic) and actuation mechanisms (fluidic, electroactive, magnetic, optical, thermal, chemical) and then highlight efforts to combine these into sensorimotor control architectures, drawing inspiration from biological systems.In particular, this review considers how artificial intelligence (AI) integrated with soft robotics can enable adaptive and responsive control in dynamic environments, enabling high-level functional behaviors such as decision making and autonomous learning.Adaptive and responsive control requires improvements in stretchable electronics, motivating a review by Michael Dickey and colleagues on methods to manufacture flexible conductors via sintering of liquid metal particles. 2The review surveys the benefits and limitations of ten sintering methods (mechanical, thermal, laser, sonication, electrochemical, Ag flake bridges, chemical, evaporation-induced, field-based alignment, and freezing-activated) for forming soft, stretchable, and conductive materials for functional use in soft robotics.The authors also highlight key technical challenges, including the development of practical manufacturing and processing methods, that need to be addressed to enable scalable fabrication of high-performance soft electronics for real-world applications.

Abstract Exercise has long been considered an essential part of human health and longevity. Recent physiological studies have expanded muscle’s role beyond simply acting as an actuator, revealing muscle’s exercise-mediated paracrine and endocrine relationships with other organ systems. In vitro engineered skeletal muscle models can address physiological questions about exercise adaptation with the precision of cell biology. Optogenetic tools have enabled a noninvasive approach to stimulating muscle contraction that avoids the potential off-target effects of electrical stimulation techniques. In this article we propose high-throughput culture and optical exercise protocols to generate statistically robust cellular exercise response datasets. We characterize optical rheobase for 2D muscle tissue morphology, finding that optical intensities as low as 5 μW mm -2 can trigger contraction. We then analyze bulk RNA sequencing data collected from high throughput, acute exercise protocols and find a rich display of transcriptional behavior that is consistent with experimental observations. The spontaneous contractility of our tissue constructs introduced oxygen diffusion challenges when maintained in a 24 well plate, and our analysis shows divergent myogenic and pathological transcriptional consequences of hypoxia. We believe our techniques provide a practical foundation for conducting future high-precision in vitro exercise studies of skeletal muscle. Translational impact High-fidelity, engineered skeletal muscle has potential to elucidate exercise-mediated response pathways at the cell and tissue level. We leverage optogenetic techniques to develop a high-throughput assay that optically stimulates 2D muscle monolayers, avoiding potential cell damage from electrical stimulation. Our culture and exercise protocol generates statistically robust RNA sequencing datasets which reveal myogenic and pathological responses to exercise and in vitro culture conditions, informing practical next steps to cultivate stronger, more physiologically relevant muscle models.

Organized cell architecture and dynamic forces are key for (re)creating native-like tissue function (e.g., contractile soft tissues). However, few studies have explored the combined effects of material-guided 3D cell organization with mechanical stimulation. Herein we underscore the importance of converging material-driven guidance of cell organization with stimulus-responsive actuation for multiscale biomaterial design, outlining strategies to engineer such biomaterials. Given the state-of-the-art biomaterials for multiscale spatiotemporally controlled organization and actuation, we propose a synergistic approach ('meta-adaptive biomaterials') that unlocks complexity in engineered biomaterials, harnessing adaptive feedback pathways arising from cell-material interactions. These can be designed similarly to cell-extracellular matrix (ECM) interactions to reinforce user-specified behaviors and yield functionalities that resemble or surpass native tissues, expanding possibilities in tissue engineering, in vitro models, and biohybrid robotics.

Skeletal muscle's elegant protein-based architecture powers motion throughout the animal kingdom, with its constituent actomyosin complexes driving intra- and extra-cellular motion. Classical motors and recently developed soft actuators cannot match the packing density and contractility of individual muscle fibers that scale to power the motion of ants and elephants alike. Accordingly, the interdisciplinary fields of robotics and tissue engineering have combined efforts to build living muscle actuators that can power a new class of robots to be more energy-efficient, dexterous, and safe than existing motor-powered and hydraulic paradigms. Doing so ethically and at scale─creating meter-scale tissue constructs from sustainable muscle progenitor cell lines─has inspired innovations in biomaterials and tissue culture methodology. We weave discussions of muscle cell biology, materials chemistry, tissue engineering, and biohybrid design to review the state of the art in soft actuator biofabrication. Looking forward, we outline a vision for meter-scale biohybrid robotic systems and tie discussions of recent progress to long-term research goals.

Neuromuscular Tissue Engineering Exercise has systemic impacts on our bodies. In article 2403712, Ritu Raman and co-workers demonstrate that exercise accelerates the growth of motor neurons through both mechanical and biochemical signaling. This cover image shows a sphere of motor neurons growing outward, encouraged by both mechanical forces (left) and biochemical factors (right). Art by Ella Marushchenko.

Mechanomedicine The emergence of magneto-mechanical actuation systems in biomedical research and mechanomedicine, ranging from single particles to 3D scaffolds, enable dynamic and remote modulation of cellular processes. By mechanically simulating physiological and pathological processes, these systems offer a new path for invitro research, that may be translated to in vivo innovation for therapeutic discoveries. More details can be found in article number 2400638 by Daniel Garcia-Gonzalez and co-workers.

micro-topographical patterning), an easily accessible and cost-effective one-step method to pattern microtopography of various sizes and configurations on the surface of hydrogels using reusable 3D printed stamps. We demonstrate that STAMP enables precisely controlling the alignment of mouse and human skeletal muscle fibers without negatively impacting their maturation or function. To showcase the versatility of our technique, we designed a planar soft robot inspired by the iris, which leverages spatially segregated regions of concentric and radial muscle fibers to control pupil dilation. Optogenetic skeletal muscle fibers grown on a STAMPed iris substrates formed a multi-oriented actuator, and selective light stimulation of the radial and concentric fibers was used to control the function of the iris, including pupil constriction. Computational modeling of the biohybrid robot as an active bilayer matched experimental outcomes, showcasing the robustness of our STAMP method for designing, fabricating, and testing planar biohybrid robots capable of complex multi-DOF motion.

In the perspective of this article, the emergence of materials and systems for magneto‐mechanical actuation in the field of mechanobiology is presented, and their potential to promote and advance biomedical research is discussed. These materials, ranging from single particles to compliant 2D substrates to 3D scaffolds, enable mechanical modulation of cells in a remote, dynamic, and reversible fashion. These features represent a major advance enabling researchers to reproduce time‐evolving physiological and pathological processes in vitro and transmit mechanical forces and deformations to activate cellular responses or promote directed cell migration. As smart in vitro platforms, magneto‐responsive systems may accelerate the discovery of mechanically mediated cellular mechanisms as therapeutic targets. In addition, the low magnetic susceptibility of biological tissues may facilitate the translation of in vitro approaches to in vivo settings, opening new routes for biomedical applications.

Emerging in vivo evidence suggests that repeated muscle contraction, or exercise, impacts peripheral nerves. However, the difficulty of isolating the muscle-specific impact on motor neurons in vivo, as well as the inability to decouple the biochemical and mechanical impacts of muscle contraction in this setting, motivates investigating this phenomenon in vitro. This study demonstrates that tuning the mechanical properties of fibrin enables longitudinal culture of highly contractile skeletal muscle monolayers, enabling functional characterization of and long-term secretome harvesting from exercised tissues. Motor neurons stimulated with exercised muscle-secreted factors significantly upregulate neurite outgrowth and migration, with an effect size dependent on muscle contraction intensity. Actuating magnetic microparticles embedded within fibrin hydrogels enable dynamically stretching motor neurons and non-invasively mimicking the mechanical effects of muscle contraction. Interestingly, axonogenesis is similarly upregulated in both mechanically and biochemically stimulated motor neurons, but RNA sequencing reveals different transcriptomic signatures between groups, with biochemical stimulation having a greater impact on cell signaling related to axonogenesis and synapse maturation. This study leverages actuating extracellular matrices to robustly validate a previously hypothesized role for muscle contraction in regulating motor neuron growth and maturation from the bottom-up through both mechanical and biochemical signaling.

Innovations in control mechanisms for muscle-powered robots are advancing the sophistication of biohybrid machines.

Abstract Engineering skeletal muscle tissue with precisely defined alignment is of significant importance for applications ranging from drug screening to biohybrid robotics. Aligning 2D contractile muscle monolayers, which are compatible with high-content imaging and can be deployed in planar soft robots, typically require micropatterned cues. However, current protocols for integrating microscale topographical features in extracellular matrix hydrogels require expensive microfabrication equipment and multi-step procedures involving error-prone manual handling steps. To address this challenge, we present STAMP (Simple Templating of Actuators via Micro-topographical Patterning), an easily accessible and cost-effective one-step method to pattern microtopography of various sizes and configurations on the surface of hydrogels using reusable 3D printed stamps. We demonstrate that STAMP enables precisely controlling the alignment of mouse and human skeletal muscle fibers, and thus their force-generating axes, without impacting their maturation or function. To showcase the versatility of our technique, we designed a planar soft robot inspired by the iris, which leverages spatially segregated regions of concentric and radial muscle fibers to control pupil dilation. Optogenetic skeletal muscle fibers grown on a STAMPed iris substrates formed a multi-oriented actuator, and selective light stimulation of the radial and concentric fibers was used to control the function of the iris, including pupil constriction. Computational modeling of the biohybrid robot as an active bilayer matched experimental outcomes, showcase the robustness of our method of designing, fabricating, and testing planar biohybrid robots capable of complex multi-degree-of-freedom motion.

The impact of tissue engineering has extended beyond a traditional focus in medicine to the rapidly growing realm of biohybrid robotics. Leveraging living actuators as functional components in machines has been a central focus of this field, generating a range of compelling demonstrations of robots capable of muscle-powered swimming, walking, pumping, gripping, and even computation. In this review, we highlight key advances in fabricating tissue-scale cardiac and skeletal muscle actuators for a range of functional applications. We discuss areas for future growth including scalable manufacturing, integrated feedback control, and predictive modeling and also propose methods for ensuring inclusive and bioethics-focused pedagogy in this emerging discipline. We hope this review motivates the next generation of biomedical engineers to advance rational design and practical use of living machines for applications ranging from telesurgery to manufacturing to on- and off-world exploration.

Muscle Actuators with Flexures Biohybrid robots require robust, reproducible, and predictable muscle actuators. Ritu Raman and co-workers have designed compliant mechanisms, termed “flexures”, that enhance the performance and decode the contractile dynamics of biological actuators with unprecedented accuracy and precision (see article number 2300834). The authors’ platform enables user-defined control of muscle contractile dynamics and fatigue behavior, enabling real-world applications of muscle as an actuator for adaptive machines. [Cover art by Ella Marushchenko.]

Tissue engineering can provide in vitro models for drug testing, disease modeling, and perhaps someday, tissue/organ replacements. For building 3D heart tissue, the alignment of cardiac cells or cardiomyocytes (CMs) is important in generating a synchronously contracting tissue. To that end, researchers have generated several fabrication methods for building heart tissue, but direct comparisons of pros and cons using the same cell source is lacking. Here, we derived cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) and compare the assembly of these cells using three fabrication methods: cardiospheres, muscle rings, and muscle strips. All three protocols successfully generated compacted tissue comprised of hiPSC-derived CMs stable for at least 2 weeks. The percentage of aligned cells was greatest in the muscle strip (55%) and the muscle ring (50%) compared with the relatively unaligned cardiospheres (35%). The iPSC-derived CMs within the muscle strip also exhibited the greatest elongation, with elongation factor at 2.0 compared with 1.5 for the muscle ring and 1.2 for the cardiospheres. This is the first direct comparison of various fabrication techniques using the same cell source.

Leveraging living muscle as an efficient and adaptive actuator for soft robots has been of increasing interest over the past decade, with a focus on proof‐of‐concept demonstrations of function. Reproducible design and scalable manufacturing of biohybrid machines requires methods to increase the stroke output of strain‐limited muscle actuators and enable accurate and precise quality control and performance monitoring. Compliant mechanical elements, termed flexures, are designed to enhance muscle contractile stroke to ≈5× previously reported values and decode contraction dynamics with high spatiotemporal resolution. Combining rigid and flexible elements within a linear elastic flexure enables us to outperform the sensitivity of gold standard elastomeric beam‐based measurements of muscle contraction at both low‐ and high‐frequency stimulations. Flexures are leveraged to make quantitative comparisons of force, work, and power outputs in muscle actuators, driving us to discover a new observation of frequency‐dependent fatigue in muscle, and also develop a novel method for tuning muscle contractile dynamics in a frequency‐independent manner. By enhancing the contractile stroke of muscle actuators and precisely tuning contractile dynamics and endurance with unprecedented precision, this study sets the stage for leveraging flexures to improve robust, reproducible, and predictive design and manufacturing of next‐generation biohybrid robots.

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.

The hierarchical design and adaptive functionalities of biological tissues are driven by dynamic biochemical, electrical, and mechanical signaling between cells and their extracellular matrices. While existing tools enable monitoring and controlling biochemical and electrical signaling in multicellular systems, there is a significant need for techniques that enable mapping and modulating intercellular mechanical signaling. We have developed a magnetically actuated extracellular matrix that serves as a mechanically active substrate for cells and can program morphological and functional anisotropy in tissues such as skeletal muscle. This method improves the ease and efficiency of programming muscle force directionality and synchronicity for applications ranging from medicine to robotics. Additionally, we present an open-source computational framework enabling quantitative analyses of muscle contractility. Our actuating matrices and accompanying tools are broadly applicable across cell types and hydrogel chemistries, and they can drive fundamental studies in mechanobiology as well as translational applications of engineered tissues in medicine and machines.

Damage that affects large volumes of skeletal muscle tissue can severely impact health, mobility, and quality-of-life. Efforts to restore muscle function by implanting tissue engineered muscle grafts at the site of damage have demonstrated limited restoration of force production. Various forms of mechanical and biochemical stimulation have been shown to have a potentially beneficial impact on graft maturation, vascularization, and innervation. However, these approaches yield unpredictable and incomplete recovery of functional mobility. Here we show that targeted actuation of implanted grafts, via non-invasive transcutaneous light stimulation of optogenetic engineered muscle, restores motor function to levels similar to healthy mice 2 weeks post-injury. Furthermore, we conduct phosphoproteomic analysis of actuated engineered muscle in vivo and in vitro to show that repeated muscle contraction alters signaling pathways that play key roles in skeletal muscle contractility, adaptation to injury, neurite growth, neuromuscular synapse formation, angiogenesis, and cytoskeletal remodeling. Our study uncovers changes in phosphorylation of several proteins previously unreported in the context of muscle contraction, revealing promising mechanisms for leveraging actuated muscle grafts to restore mobility after volumetric muscle loss.