Three-dimensionally printed biological machines powered by skeletal muscle (original) (raw)
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Nature Protocols, 2017
Biological materials have the ability to sense, process, and respond to a range of dynamic environmental signals in real time. This capability allows biological systems to demonstrate complex behaviors such as self-assembly, self-organization, self-healing, self-replication, and constant adaptation of composition and functionality to best suit their environment. Recent advances in manufacturing technologies, such as 3D printing, combined with progress in the field of biomaterials, have synergistically produced robust approaches for manufacturing complex 3D structures from biological materials 1-3. This has driven fundamental advances in the fields of tissue engineering and regenerative medicine by providing a method of reverse-engineering native tissues and organs 4-6. Thus far, the use of these technologies has primarily been limited to replicating biological structures found in nature while largely neglecting applications in forward-engineered biological systems capable of non-natural functional behaviors. Bio-integrated robots, or bio-bots, built using a combination of biological and synthetic materials have the potential to develop enhanced functional attributes as compared with robots made with traditional synthetic materials alone 7,8. The dynamically adaptive nature of biological materials makes them ideal candidates for serving as the building blocks of 'smart' responsive machines and systems for a variety of applications. Nearly all machines require actuators-modules that convert energy into motion-to produce a measurable output in response to varied input stimuli 9. Skeletal muscle is a natural actuator capable of generating larger forces from more compact structures than those of actuators made from synthetic materials, and it is designed to be modular and adaptive to changing environmental loads 10-12. As locomotion is a powerful and intuitive demonstration of force production, we have developed muscle-powered bio-bots that can walk on 2D surfaces in response to external electrical or optical signals 13,14. In this protocol, we describe a repeatable and customizable approach to 3D printing of injection molds for engineered muscle and mechanical bio-bot skeletons. We then describe how to seed and differentiate muscle actuators within these molds and mechanically couple them to printed skeletons to accomplish functional output behaviors when stimulated with external signals. The convergence of the two disciplines of tissue engineering and 3D printing thus enables the iterative design and rapid fabrication of adaptive forward-engineered biological machines whose functionality can be tuned to suit a variety of applications in health, security, and the environment. Development of the protocol The first demonstrations of bio-integrated machines, composed of synthetic skeletons coupled to biological actuators, used the autonomous contraction of engineered cardiac muscle as a source of power 15-19. The continuous beating of cardiac muscle did not provide 'on-off ' control over such machines, motivating the development of bio-integrated machines powered by skeletal muscle. Recent advances in approaches to culturing skeletal muscle cells in vitro have provided a baseline methodology for engineering 3D skeletal muscle tissue constructs 20-22. Although these studies detailed robust techniques for engineering microscale tissues with applicability in high-throughput drug screening and studies of muscle development, they required substantial modifications to suit applications that required force production at the millimeter to centimeter scale. In this protocol, we present a modular and stepwise approach to designing, fabricating, and controlling skeletal-muscle-powered locomotive biological machines at the millimeter to centimeter scale (Fig. 1). We show that the enabling technology of stereolithographic 3D printing can be used to iteratively design and custom-manufacture soft robotic devices for a variety of purposes. These 3D-printed devices, when coupled to tissue-engineered skeletal muscle actuators, can drive locomotion across 2D surfaces and are designed to suit a variety of applications. The design of the 3D-printed skeleton that we use was inspired by the architecture of the musculoskeletal system in vivo. In the
Advanced Materials Technologies, 2018
the incorporation of biological elements, such as contractile muscle tissue, to develop hybrid bio-robotic devices. This new approach could constitute a change of paradigm by boosting the performance and applicability of soft robotics and by taking advantage of the inherent unique capabilities of biological entities, such as adaptability, self-assembly, self-healing, or responsiveness to external stimuli. [9-13] The integration of living cells into robotic devices has recently been reported with excellent outcomes in terms of controllability, sensing, and response to dynamic environmental stimuli. [14] Both single cells [15-18] and tissues [13,19-21] have already been used as power sources. However, whereas single cells hold potential for the creation of microswimmers, the development of efficient walkers or grippers require the generation of more powerful forces which can only be achieved by engineered muscle tissue. Both cardiac [19,22,23] and skeletal muscle [20,21,24] tissue have yielded good results when used as actuators. Even though cardiac cells possess certain advantages, such as more powerful contractions or the ability to self-contract, skeletal muscle is usually the preferred choice due their controllability and adaptability to 3D shapes. [10] Out of all of the outstanding properties that are attributed to bio-hybrid actuators, which include self-healing, bio-sensing, or adaptability, among others, very little of them have been extensively studied. In particular, Raman et al. analyzed damage and healing of biobots by the addition of fresh myoblasts [25] and their increase in force after applying different stimulation regimes. [24] Although the use of biochemical cues, [9,26,27] topographical features, [28-30] and 3D-printed molds [20,24,31] have proven to be suitable and effective techniques for the fabrication of bio-actuators, the field is still lacking a technique that can increase its versatility, speed, and scalability. Within this regard, 3D bioprinting could emerge as a revolutionary tool to integrate biological tissue into soft robotics. 3D bioprinting of skeletal muscle tissue has already been reported as a viable tool for tissue regeneration, [32-34] but their potential for fabricating complete bio-hybrid actuators in a one-step process has not yet been considered. In the present work, we go beyond the state-of-theart in studying the adaptability and force modulation The integration of biological systems into robotic devices might provide them with capabilities acquired from natural systems and significantly boost their performance. These abilities include real-time bio-sensing, self-organization, adaptability, or self-healing. As many muscle-based bio-hybrid robots and bio-actuators arise in the literature, the question of whether these features can live up to their expectations becomes increasingly substantial. Herein, the force generation and adaptability of skeletalmuscle-based bio-actuators undergoing long-term training protocols are analyzed. The 3D-bioprinting technique is used to fabricate bio-actuators that are functional, responsive, and have highly aligned myotubes. The bio-actuators are 3D-bioprinted together with two artificial posts, allowing to use it as a force measuring platform. In addition, the force output evolution and dynamic gene expression of the bio-actuators are studied to evaluate their degree of adaptability according to training protocols of different frequencies and mechanical stiffness, finding that their force generation could be modulated to different requirements. These results shed some light into the fundamental mechanisms behind the adaptability of muscle-based bio-actuators and highlight the potential of using 3D bioprinting as a rapid and cost-effective tool for the fabrication of custom-designed soft bio-robots.
Electrically Driven Microengineered Bioinspired Soft Robots
Advanced materials (Deerfield Beach, Fla.), 2018
To create life-like movements, living muscle actuator technologies have borrowed inspiration from biomimetic concepts in developing bioinspired robots. Here, the development of a bioinspired soft robotics system, with integrated self-actuating cardiac muscles on a hierarchically structured scaffold with flexible gold microelectrodes is reported. Inspired by the movement of living organisms, a batoid-fish-shaped substrate is designed and reported, which is composed of two micropatterned hydrogel layers. The first layer is a poly(ethylene glycol) hydrogel substrate, which provides a mechanically stable structure for the robot, followed by a layer of gelatin methacryloyl embedded with carbon nanotubes, which serves as a cell culture substrate, to create the actuation component for the soft body robot. In addition, flexible Au microelectrodes are embedded into the biomimetic scaffold, which not only enhance the mechanical integrity of the device, but also increase its electrical conduct...
Tissue Scaffolds: Robotic Hydrogel Extrusions for Advanced Design Applications
WAD?24, 2024
Tissue engineering refers to the attempt to create functional human tissues from cells in a laboratory. Its goals include repairing or replacing failed tissues and organs through 3D organ printing, as well as acting as a replacement for animal-based experiments. Tissue engineering relies on three important factors: identifying the right cells to do the job, the right scaffold to support the cell growth, and the right bioreactor to influence the development of the cells. This thesis takes part in developing the scaffolds, with the purpose of creating self-supportive hydrogel structures through novel fabrication techniques developed from existing cutting-edge references in biomedical engineering, aiming at robotic extrusion printing tissue scaffolds for mammal organs and large-scale applications. Hydrogels made from 4% w/v sodium alginate solution with three times the mass of methylcellulose are used for printing, and CaCl2 solution is used for crosslinking if needed. Three robotic extrusion printing techniques are tested and compared, and a customized syringe pump end effector is derived, which allows for printing both paste and liquid alginate-based hydrogels. The rehydration property of paste alginate-based hydrogels is studied to print a 1:1 scale knee joint, and the self-supportive potential is challenged when printing a 1:1 heart. A marine landscape of size 400 x 350 x 90 mm is created to be demonstrated as a large-scale application.
Tissue engineered in-vitro models are an essential tool in biomedical research. Tissue geometry is a key determinant of function, but controlling geometry of micro-scale tissues remains a challenge. We developed a new double molding approach that allows precise replication of high-resolution stereolithographic prints into poly(dimethylsiloxane), facilitating rapid design iterations and highly parallelized sample production. Hydrogels are used as an intermediary mold, and gel mechanical properties including crosslink density predict replication fidelity. We leveraged this approach to study the effects of geometry on the electrophysiology of miniaturized heart muscles engineered from human induced pluripotent stem cells (iPSC). Geometries predicted to increase tissue prestress globally affected cardiomyocyte structure and tissue electrophysiology. Strikingly, pharmacologic studies revealed a prestress threshold is required for sodium channel function. Analysis of RNA and protein level...
A novel 3D bioprinted flexible and biocompatible hydrogel bioelectronic platform
Biosensors & bioelectronics, 2017
Bioelectronics platforms are gaining widespread attention as they provide a template to study the interactions between biological species and electronics. Decoding the effect of the electrical signals on the cells and tissues holds the promise for treating the malignant tissue growth, regenerating organs and engineering new-age medical devices. This work is a step forward in this direction, where bio- and electronic materials co-exist on one platform without any need for post processing. We fabricate a freestanding and flexible hydrogel based platform using 3D bioprinting. The fabrication process is simple, easy and provides a flexible route to print materials with preferred shapes, size and spatial orientation. Through the design of interdigitated electrodes and heating coil, the platform can be tailored to print various circuits for different functionalities. The biocompatibility of the printed platform is tested using C2C12 murine myoblasts cell line. Furthermore, normal human de...
Indirect 3D and 4D Printing of Soft Robotic Microstructures
Advanced Materials Technologies
today among small-scale roboticists is to substitute hard components with softer elements, including parts made from elastomers, hydrogels, macromolecular systems, and biomolecules. This paradigm shift in materials is a logical development from the locomotion and actuation point of view. Soft materials allow for more sophisticated movements such as deformation, shrinkage/swelling, and changes in morphology. [19,20] Additionally, soft materials display mechanical properties that are much closer to those of biological structures such as tissues, and they usually exhibit enhanced biocompatibility characteristics. Soft matter can also be programmed to biodegrade by the body's chemistry, for example, by enzymes or pH. [9,10] Consequently, the production of micro-and nanorobotic platforms from soft building blocks will advance the field of small-scale robotics (in terms of material constituents) toward medical applications. An impediment to the miniaturization of small soft components lies in the available manufacturing methods and their related limitations, which are mainly a result of the physicochemical nature of the soft material. In order to fabricate microand nanodevices with superior capabilities, the production of components with any conceivable shape is essential. To date, 3D printing (3DP) techniques have offered a wealth of opportunities for creating 3D structures with virtually unlimited shapes or materials. 3DP has also strongly impacted the field of robotics, by enabling the fabrication of soft robotic engines with sophisticated 3D continuous movements. Although substantial research has been devoted to the production of 3D printed small-scale robotic tools, these are typically made of nonresponsive, stiff materials [21-23] or, in the case of soft materials, their geometrical features are either rudimentary, or too large (from ≈200 µm to a few mm) to be used as biomedical micro or nanorobots. [24-26] Here, we propose a method that uses the capabilities of direct laser writing (DLW) [27] to produce complex 3D sacrificial templates for molding polymers that cannot be directly 3D printed at the microscale by any other technique. One of the first examples of using sacrificial templates obtained by DLW showed the fabrication of gold helices as photonic metamaterials as broadband circular polarizer. [28] Electrodeposition in DLW templates has also been utilized for the fabrication of
Acta Biomaterialia, 2015
The generation of functional biomimetic skeletal muscle constructs is still one of the fundamental challenges in skeletal muscle tissue engineering. With the notion that structure strongly dictates functional capabilities, a myriad of cell types, scaffold materials and stimulation strategies have been combined. To further optimize muscle engineered constructs, we have developed a novel bioreactor system (MagneTissue) for rapid engineering of skeletal muscle-like constructs with the aim to resemble native muscle in terms of structure, gene expression profile and maturity. Myoblasts embedded in fibrin, a natural hydrogel that serves as extracellular matrix, are subjected to mechanical stimulation via magnetic force transmission. We identify static mechanical strain as a trigger for cellular alignment concomitant with the orientation of the scaffold into highly organized fibrin fibrils. This ultimately yields myotubes with a more mature phenotype in terms of sarcomeric patterning, diameter and length. On the molecular level, a faster progression of the myogenic gene expression program is evident as myogenic determination markers MyoD and Myogenin as well as the Ca 2+ dependent contractile structural marker TnnT1 are significantly upregulated when strain is applied. The major advantage of the MagneTissue bioreactor system is that the generated tension is not exclusively relying on the strain generated by the cells themselves in response to scaffold anchoring but its ability to subject the constructs to individually adjustable strain protocols. In future work, this will allow applying mechanical stimulation with different strain regimes in the maturation process of tissue engineered constructs and elucidating the role of mechanotransduction in myogenesis.
Muscular thin films for building actuators and powering devices
Science, 2007
We demonstrate the assembly of biohybrid materials from engineered tissues and synthetic polymer thin films. The constructs were built by culturing neonatal rat ventricular cardiomyocytes on polydimethylsiloxane thin films micropatterned with extracellular matrix proteins to promote spatially ordered, two-dimensional myogenesis. The constructs, termed muscular thin films, adopted functional, three-dimensional conformations when released from a thermally sensitive polymer substrate and were designed to perform biomimetic tasks by varying tissue architecture, thin-film shape, and electrical-pacing protocol. These centimeter-scale constructs perform functions as diverse as gripping, pumping, walking, and swimming with fine spatial and temporal control and generating specific forces as high as 4 millinewtons per square millimeter. M uscle cells are microscale linear actuators driven by the activation of actinmyosin motors, coordinated in space and time through excitation-contraction (EC) coupling (1, 2). Structure-function relations are conserved over several orders of spatial magnitude, from the sarcomere to the muscle bundle, by virtue of a hierarchical architecture. These architectures are achieved by morphogenesis programs that are responsible for coupling a broad range of processes, from sarcomeregenesis to the integration of the biochemical and electrical networks that support muscle function (1). Muscle actuation occurs over a wide range of frequencies (0 to~100 Hz), spatial dimensions (5 mm to ≥1 m), and force regimes (~5 mN to ≥1 kN) (3, 4). Artificial muscles can match certain temporal, spatial, or force regimes typical of biological muscle (5, 6), but they cannot fully replicate all of these capabilities, nor can they use the same high-density energy sources. Thus, engineered muscle remains an attractive method for building actuators and powering devices from the micro to macro scales.