3D-printed drug testing platform based on a 3D model of aged human skeletal muscle (original) (raw)
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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.
Three-dimensionally printed biological machines powered by skeletal muscle
Proceedings of the National Academy of Sciences of the United States of America, 2014
Combining biological components, such as cells and tissues, with soft robotics can enable the fabrication of biological machines with the ability to sense, process signals, and produce force. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. Whereas cardiac cell-driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel "bio-bots" with an asymmetric physical design and powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot. Geometric design and material properties of the hydrogel bio-bots were optimized using stereolithographic 3D printing, and the effect of collagen I and fibrin extracell...
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.
3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect?
Frontiers in Bioengineering and Biotechnology
Skeletal muscle is a fundamental tissue of the human body with great plasticity and adaptation to diseases and injuries. Recreating this tissue in vitro helps not only to deepen its functionality, but also to simulate pathophysiological processes. In this review we discuss the generation of human skeletal muscle three-dimensional (3D) models obtained through tissue engineering approaches. First, we present an overview of the most severe myopathies and the two key players involved: the variety of cells composing skeletal muscle tissue and the different components of its extracellular matrix. Then, we discuss the peculiar characteristics among diverse in vitro models with a specific focus on cell sources, scaffold composition and formulations, and fabrication techniques. To conclude, we highlight the efficacy of 3D models in mimicking patient-specific myopathies, deepening muscle disease mechanisms or investigating possible therapeutic effects.
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
Biochemistry and Molecular Biology Education, 2015
Interdisciplinary exploration is vital to education in the 21st century. This manuscript outlines an innovative laboratory-based teaching method that combines elements of biochemistry/molecular biology, kinesiology/health science, computer science, and manufacturing engineering to give students the ability to better conceptualize complex biological systems. Here, we utilize technology available at most universities to print three-dimensional (3D) scale models of actual human muscle cells (myofibers) out of bioplastic materials. The same methodological approach could be applied to nearly any cell type or molecular structure. This advancement is significant because historically, two-dimensional (2D) myocellular images have proven insufficient for detailed analysis of organelle organization and morphology. 3D imaging fills this void by providing accurate and quantifiable myofiber structural data. Manipulating tangible 3D models combats 2D limitation and gives students new perspectives and alternative learning experiences that may assist their understanding. This approach also exposes learners to 1) human muscle cell extraction and isolation, 2) targeted fluorescence labeling, 3) confocal microscopy, 4) image processing (via open-source software), and 5) 3D printing bioplastic scale-models (×500 larger than the actual cells). Creating these physical models may further student's interest in the invisible world of molecular and cellular biology. Furthermore, this interdisciplinary laboratory project gives instructors of all biological disciplines a new teaching tool to foster integrative thinking. © 2015 by The International Union of Biochemistry and Molecular Biology, 2015.
Mechanical stimulation improves tissue-engineered human skeletal muscle
AJP: Cell Physiology, 2002
Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This...
Engineered Muscle Tissues for Disease Modeling and Drug Screening Applications
Current Pharmaceutical Design, 2017
Animal models have been the main resources for drug discovery and prediction of drugs' pharmacokinetic responses in the body. However, noticeable drawbacks associated with animal models include high cost, low reproducibility, low physiological similarity to humans, and ethical problems. Engineered tissue models have recently emerged as an alternative or substitute for animal models in drug discovery and testing and disease modeling. In this review, we focus on skeletal muscle and cardiac muscle tissues by first describing their characterization and physiology. Major fabrication technologies (i.e., electrospinning, bioprinting, dielectrophoresis, textile technology, and microfluidics) to make functional muscle tissues are then described. Finally, currently used muscle tissue models in drug screening are reviewed and discussed.
Proceedings of the National Academy of Sciences, 2021
Significance Tissue-on-chip systems offer important capabilities in engineering of living tissues for diverse biomedical applications in disease model studies, drug screening, and regenerative medicine. Conventional approaches use two-dimensional layouts that cannot suppport interfaces to geometrically complex three-dimensional (3D) tissue constructs in a deterministic fashion. Here, we present concepts in engineering systems that allow intimate contact with and stable mechanical coupling to 3D tissues for high-precision measurements of tissue contractility, as demonstrated in 3D, millimeter-scale engineered skeletal muscle tissues. These compliant, 3D frameworks instrumented with advanced sensors and other functional electronics, may significantly enhance the capabilities of tissue-on-chip systems.
Generation of 3D skeletal muscle-like fibrin constructs via the application of mechanical stimuli
2017
Skeletal muscle tissue engineering demonstrates a promising tool for the creation of mature muscle tissue constructs in vitro and in vivo. The aim of engineering mature muscle-like constructs ranges from substituting lost tissue after traumata, cancer ablation to conduct research on in vitro muscle disease models. Therefore the interplay of three key factors is crucial, which constitute of the right biomaterial, myogenic cells moreover mechanical stimulation. In our 3D studies we were using fibrin based hydrogels with C2C12 mouse myoblasts incorporated, as fibrin constitutes a biomaterial suitable for skeletal muscle tissue engineering. The novel bioreactor system (MagneTissue) was used to apply mechanical stimuli to our ring shaped fibrin constructs. We were able to engineer skeletal muscle-like tissue constructs via parallel alignment of fibrin fibers and cells due to the applied strain causing them to differentate into myofibers. Static strain resulted in a positive effect on myo...