Adhesion and proliferation of skeletal muscle cells on single layer poly(lactic acid) ultra-thin films

An increasing interest in bio-hybrid systems and cell-material interactions is evident in the last years. This leads towards the development of new nano-structured devices and the assessment of their biocompatibility. In the present study, the development of free-standing single layer poly(lactic acid) (PLA) ultra-thin films is described, together with the analysis of topography and roughness properties. The biocompatibility of the PLA films has been tested in vitro, by seeding C2C12 skeletal muscle cells, and thus assessing cells shape, density and viability after 24, 48 and 72 h. The results show that free-standing flexible PLA nanofilms represent a good matrix for C2C12 cells adhesion, spreading and proliferation. Early differentiation into myotubes is also allowed. The biocompatibility of the novel ultra-thin films as substrates for cell growth promotes their application in the fields of regenerative medicine, muscle tissue engineering, drug delivery, and-in general-in the field of bio-hybrid devices

Micro- and nanoengineering approaches to control stem cell-biomaterial interactions.

As our population ages, there is a greater need for a suitable supply of engineered tissues to address a range of debilitating ailments. Stem cell based therapies are envisioned to meet this emerging need. Despite significant progress in controlling stem cell differentiation, it is still difficult to engineer human tissue constructs for transplantation. Recent advances in micro- and nanofabrication techniques have enabled the design of more biomimetic biomaterials that may be used to direct the fate of stem cells. These biomaterials could have a significant impact on the next generation of stem cell based therapies. Here, we highlight the recent progress made by micro- and nanoengineering techniques in the biomaterials field in the context of directing stem cell differentiation. Particular attention is given to the effect of surface topography, chemistry, mechanics and micro- and nanopatterns on the differentiation of embryonic, mesenchymal and neural stem cells

Biohybrid robotics: From the nanoscale to the macroscale

Biohybrid robotics is a field in which biological entities are combined with artificial materials in order to obtain improved performance or features that are difficult to mimic with hand-made materials. Three main level of integration can be envisioned depending on the complexity of the biological entity, ranging from the nanoscale to the macroscale. At the nanoscale, enzymes that catalyze biocompatible reactions can be used as power sources for self-propelled nanoparticles of different geometries and compositions, obtaining rather interesting active matter systems that acquire importance in the biomedical field as drug delivery systems. At the microscale, single enzymes are substituted by complete cells, such as bacteria or spermatozoa, whose self-propelling capabilities can be used to transport cargo and can also be used as drug delivery systems, for in vitro fertilization practices or for biofilm removal. Finally, at the macroscale, the combinations of millions of cells forming tissues can be used to power biorobotic devices or bioactuators by using muscle cells. Both cardiac and skeletal muscle tissue have been part of remarkable examples of untethered biorobots that can crawl or swim due to the contractions of the tissue and current developments aim at the integration of several types of tissue to obtain more realistic biomimetic devices, which could lead to the next generation of hybrid robotics. Tethered bioactuators, however, result in excellent candidates for tissue models for drug screening purposes or the study of muscle myopathies due to their three-dimensional architecture

Contractile deficits in engineered cardiac microtissues as a result of MYBPC3 deficiency and mechanical overload.

The integration of in vitro cardiac tissue models, human induced pluripotent stem cells (hiPSCs) and genome-editing tools allows for the enhanced interrogation of physiological phenotypes and recapitulation of disease pathologies. Here, using a cardiac tissue model consisting of filamentous three-dimensional matrices populated with cardiomyocytes derived from healthy wild-type (WT) hiPSCs (WT hiPSC-CMs) or isogenic hiPSCs deficient in the sarcomere protein cardiac myosin-binding protein C (MYBPC3-/- hiPSC-CMs), we show that the WT microtissues adapted to the mechanical environment with increased contraction force commensurate to matrix stiffness, whereas the MYBPC3-/- microtissues exhibited impaired force development kinetics regardless of matrix stiffness and deficient contraction force only when grown on matrices with high fibre stiffness. Under mechanical overload, the MYBPC3-/- microtissues had a higher degree of calcium transient abnormalities, and exhibited an accelerated decay of calcium dynamics as well as calcium desensitization, which accelerated when contracting against stiffer fibres. Our findings suggest that MYBPC3 deficiency and the presence of environmental stresses synergistically lead to contractile deficits in cardiac tissues

Future of smart cardiovascular implants

Cardiovascular disease remains the leading cause of death in Western society. Recent technological advances have opened the opportunity of developing new and innovative smart stent devices that have advanced electrical properties that can improve diagnosis and even treatment of previously intractable conditions, such as central line access failure, atherosclerosis and reporting on vascular grafts for renal dialysis. Here we review the latest advances in the field of cardiovascular medical implants, providing a broad overview of the application of their use in the context of cardiovascular disease rather than an in-depth analysis of the current state of the art. We cover their powering, communication and the challenges faced in their fabrication. We focus specifically on those devices required to maintain vascular access such as ones used to treat arterial disease, a major source of heart attacks and strokes. We look forward to advances in these technologies in the future and their implementation to improve the human condition

Non-linear modeling of active biohybrid materials

Recent advances in engineered muscle tissue attached to a synthetic substrate motivates the development of appropriate constitutive and numerical models. Applications of active materials can be expanded by using robust, non-mammalian muscle cells, such as those of Manduca sexta. In this study, we propose a model to assist in the analysis of biohybrid constructs by generalizing a recently proposed constitutive law for Manduca muscle tissue. The continuum model accounts (i) for the stimulation of muscle fibers by introducing multiple stress-free reference configurations for the active and passive states and (ii) for the hysteretic response by specifying a pseudo-elastic energy function. A simple example representing uniaxial loading-unloading is used to validate and verify the characteristics of the model. Then, based on experimental data of muscular thin films, a more complex case shows the qualitative potential of Manduca muscle tissue in active biohybrid constructs

Formation and optogenetic control of engineered 3D skeletal muscle bioactuators

Densely arrayed skeletal myotubes are activated individually and as a group using precise optical stimulation with high spatiotemporal resolution. Skeletal muscle myoblasts are genetically encoded to express a light-activated cation channel, Channelrhodopsin-2, which allows for spatiotemporal coordination of a multitude of skeletal myotubes that contract in response to pulsed blue light. Furthermore, ensembles of mature, functional 3D muscle microtissues have been formed from the optogenetically encoded myoblasts using a high-throughput device. The device, called “skeletal muscle on a chip”, not only provides the myoblasts with controlled stress and constraints necessary for muscle alignment, fusion and maturation, but also facilitates the measurement of forces and characterization of the muscle tissue. We measured the specific static and dynamic stresses generated by the microtissues and characterized the morphology and alignment of the myotubes within the constructs. The device allows testing of the effect of a wide range of parameters (cell source, matrix composition, microtissue geometry, auxotonic load, growth factors and exercise) on the maturation, structure and function of the engineered muscle tissues in a combinatorial manner. Our studies integrate tools from optogenetics and microelectromechanical systems (MEMS) technology with skeletal muscle tissue engineering to open up opportunities to generate soft robots actuated by a multitude of spatiotemporally coordinated 3D skeletal muscle microtissues.National Science Foundation (U.S.) (Science and Technology Center—Emergent Behaviors of Integrated Cellular Systems (EBICS) grant No. CBET-0939511)National Institutes of Health (U.S.) (EB00262)National Science Foundation (U.S.) (GM74048)National Science Foundation (U.S.) (HL90747)National Institute for Biomedical Imaging and Bioengineering (U.S.) (RESBIO, Integrapted Technologies for Polymeric Biomaterial)University of Pennsylvania. Center for Engineering Cells and RegenerationSingapore-MIT Alliance for Research and Technolog

Design and Implementation of a Passive Neurostimulator with Wireless Resonance-Coupled Power Delivery and Demonstration on Frog Sciatic Nerve and Gastrocnemius Muscle

The thesis presented has four goals: to perform a comprehensive literature review on current neurostimulator technology; to outline the current issues with the state-of-the-art; to provide a neurostimulator design that solves these issues, and to characterize the design and demonstrate its neurostimulation features. The literature review describes the physiology of a neuron, and then proceeds to outline neural interfaces and neurostimulators. The neurostimulator design process is then outlined and current requirements in the field are described. The novel neurostimulator circuit that implements a solution that has wireless capability, passive control, and small size is outlined and characterized. The circuit is demonstrated to operate wirelessly with a resonance-coupled multi-channel implementation, and is shown powering LEDs. The circuit was then fabricated in a miniature implementation which utilized a 10 x 20 x 3 mm&179 antenna, and occupied a volume approximating 1 cm&179. This miniature circuit is used to stimulate frog sciatic nerve and gastrocnemius muscle in vitro. These demonstrations and characterization show the device is capable of neurostimulation, can operate wirelessly, is controlled passively, and can be implemented in a small size, thus solving the aforementioned neurostimulator requirements. Further work in this area is focused on developing an extensive characterization of the device and the wireless power delivery system, optimizing the circuit design, and performing in vivo experiments with restoration of motor control in injured animals. This device shows promise to provide a comprehensive solution to many application-specific problems in neurostimulation, and be a modular addition to larger neural interface systems

Stable, covalent attachment of laminin to microposts improves the contractility of mouse neonatal cardiomyocytes.

The mechanical output of contracting cardiomyocytes, the muscle cells of the heart, relates to healthy and disease states of the heart. Culturing cardiomyocytes on arrays of elastomeric microposts can enable inexpensive and high-throughput studies of heart disease at the single-cell level. However, cardiomyocytes weakly adhere to these microposts, which limits the possibility of using biomechanical assays of single cardiomyocytes to study heart disease. We hypothesized that a stable covalent attachment of laminin to the surface of microposts improves cardiomyocyte contractility. We cultured cells on polydimethylsiloxane microposts with laminin covalently bonded with the organosilanes 3-glycidoxypropyltrimethoxysilane and 3-aminopropyltriethoxysilane with glutaraldehyde. We measured displacement of microposts induced by the contractility of mouse neonatal cardiomyocytes, which attach better than mature cardiomyocytes to substrates. We observed time-dependent changes in contractile parameters such as micropost deformation, contractility rates, contraction and relaxation speeds, and the times of contractions. These parameters were affected by the density of laminin on microposts and by the stability of laminin binding to micropost surfaces. Organosilane-mediated binding resulted in higher laminin surface density and laminin binding stability. 3-glycidoxypropyltrimethoxysilane provided the highest laminin density but did not provide stable protein binding with time. Higher surface protein binding stability and strength were observed with 3-aminopropyltriethoxysilane with glutaraldehyde. In cultured cardiomyocytes, contractility rate, contraction speeds, and contraction time increased with higher laminin stability. Given these variations in contractile function, we conclude that binding of laminin to microposts via 3-aminopropyltriethoxysilane with glutaraldehyde improves contractility observed by an increase in beating rate and contraction speed as it occurs during the postnatal maturation of cardiomyocytes. This approach is promising for future studies to mimic in vivo tissue environments