In Situ Expansion, Differentiation, and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid.

RATIONALE: One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes-a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. OBJECTIVE: To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. METHODS AND RESULTS: We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell-laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. CONCLUSIONS: This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting.

A Bionic Testbed for Cardiac Ablation Tools.

Bionic-engineered tissues have been proposed for testing the performance of cardiovascular medical devices and predicting clinical outcomes ex vivo. Progress has been made in the development of compliant electronics that are capable of monitoring treatment parameters and being coupled to engineered tissues; however, the scale of most engineered tissues is too small to accommodate the size of clinical-grade medical devices. Here, we show substantial progress toward bionic tissues for evaluating cardiac ablation tools by generating a centimeter-scale human cardiac disk and coupling it to a hydrogel-based soft-pressure sensor. The cardiac tissue with contiguous electromechanical function was made possible by our recently established method to 3D bioprint human pluripotent stem cells in an extracellular matrix-based bioink that allows for in situ cell expansion prior to cardiac differentiation. The pressure sensor described here utilized electrical impedance tomography to enable the real-time spatiotemporal mapping of pressure distribution. A cryoablation tip catheter was applied to the composite bionic tissues with varied pressure. We found a close correlation between the cell response to ablation and the applied pressure. Under some conditions, cardiomyocytes could survive in the ablated region with more rounded morphology compared to the unablated controls, and connectivity was disrupted. This is the first known functional characterization of living human cardiomyocytes following an ablation procedure that suggests several mechanisms by which arrhythmia might redevelop following an ablation. Thus, bionic-engineered testbeds of this type can be indicators of tissue health and function and provide unique insight into human cell responses to ablative interventions.

3D Printed Neural Regeneration Devices.

Neural regeneration devices interface with the nervous system and can provide flexibility in material choice, implantation without the need for additional surgeries, and the ability to serve as guides augmented with physical, biological (e.g., cellular), and biochemical functionalities. Given the complexity and challenges associated with neural regeneration, a 3D printing approach to the design and manufacturing of neural devices could provide next-generation opportunities for advanced neural regeneration via the production of anatomically accurate geometries, spatial distributions of cellular components, and incorporation of therapeutic biomolecules. A 3D printing-based approach offers compatibility with 3D scanning, computer modeling, choice of input material, and increasing control over hierarchical integration. Therefore, a 3D printed implantable platform could ultimately be used to prepare novel biomimetic scaffolds and model complex tissue architectures for clinical implants in order to treat neurological diseases and injuries. Further, the flexibility and specificity offered by 3D printed in vitro platforms have the potential to be a significant foundational breakthrough with broad research implications in cell signaling and drug screening for personalized healthcare. This progress report examines recent advances in 3D printing strategies for neural regeneration as well as insight into how these approaches can be improved in future studies.

3D Printed Stem-Cell Derived Neural Progenitors Generate Spinal Cord Scaffolds.

A bioengineered spinal cord is fabricated via extrusion-based multi-material 3D bioprinting, in which clusters of induced pluripotent stem cell (iPSC)-derived spinal neuronal progenitor cells (sNPCs) and oligodendrocyte progenitor cells (OPCs) are placed in precise positions within 3D printed biocompatible scaffolds during assembly. The location of a cluster of cells, of a single type or multiple types, is controlled using a point-dispensing printing method with a 200 µm center-to-center spacing within 150 µm wide channels. The bioprinted sNPCs differentiate and extend axons throughout microscale scaffold channels, and the activity of these neuronal networks is confirmed by physiological spontaneous calcium flux studies. Successful bioprinting of OPCs in combination with sNPCs demonstrates a multicellular neural tissue engineering approach, where the ability to direct the patterning and combination of transplanted neuronal and glial cells can be beneficial in rebuilding functional axonal connections across areas of central nervous system (CNS) tissue damage. This platform can be used to prepare novel biomimetic, hydrogel-based scaffolds modeling complex CNS tissue architecture in vitro and harnessed to develop new clinical approaches to treat neurological diseases, including spinal cord injury.

The Role of Nanoparticle Design in Determining Analytical Performance of Lateral Flow Immunoassays.

Rapid, simple, and cost-effective diagnostics are needed to improve healthcare at the point of care (POC). However, the most widely used POC diagnostic, the lateral flow immunoassay (LFA), is ∼1000-times less sensitive and has a smaller analytical range than laboratory tests, requiring a confirmatory test to establish truly negative results. Here, a rational and systematic strategy is used to design the LFA contrast label (i.e., gold nanoparticles) to improve the analytical sensitivity, analytical detection range, and antigen quantification of LFAs. Specifically, we discovered that the size (30, 60, or 100 nm) of the gold nanoparticles is a main contributor to the LFA analytical performance through both the degree of receptor interaction and the ultimate visual or thermal contrast signals. Using the optimal LFA design, we demonstrated the ability to improve the analytical sensitivity by 256-fold and expand the analytical detection range from 3 log10 to 6 log10 for diagnosing patients with inflammatory conditions by measuring C-reactive protein. This work demonstrates that, with appropriate design of the contrast label, a simple and commonly used diagnostic technology can compete with more expensive state-of-the-art laboratory tests.

3D Printed Programmable Release Capsules.

The development of methods for achieving precise spatiotemporal control over chemical and biomolecular gradients could enable significant advances in areas such as synthetic tissue engineering, biotic-abiotic interfaces, and bionanotechnology. Living organisms guide tissue development through highly orchestrated gradients of biomolecules that direct cell growth, migration, and differentiation. While numerous methods have been developed to manipulate and implement biomolecular gradients, integrating gradients into multiplexed, three-dimensional (3D) matrices remains a critical challenge. Here we present a method to 3D print stimuli-responsive core/shell capsules for programmable release of multiplexed gradients within hydrogel matrices. These capsules are composed of an aqueous core, which can be formulated to maintain the activity of payload biomolecules, and a poly(lactic-co-glycolic) acid (PLGA, an FDA approved polymer) shell. Importantly, the shell can be loaded with plasmonic gold nanorods (AuNRs), which permits selective rupturing of the capsule when irradiated with a laser wavelength specifically determined by the lengths of the nanorods. This precise control over space, time, and selectivity allows for the ability to pattern 2D and 3D multiplexed arrays of enzyme-loaded capsules along with tunable laser-triggered rupture and release of active enzymes into a hydrogel ambient. The advantages of this 3D printing-based method include (1) highly monodisperse capsules, (2) efficient encapsulation of biomolecular payloads, (3) precise spatial patterning of capsule arrays, (4) "on the fly" programmable reconfiguration of gradients, and (5) versatility for incorporation in hierarchical architectures. Indeed, 3D printing of programmable release capsules may represent a powerful new tool to enable spatiotemporal control over biomolecular gradients.

3D Printed Anatomical Nerve Regeneration Pathways.

An imaging-coupled 3D printing methodology for the design, optimization, and fabrication of a customized nerve repair technology for complex injuries is presented. The custom scaffolds are deterministically fabricated via a microextrusion printing principle which enables the simultaneous incorporation of anatomical geometries, biomimetic physical cues, and spatially controlled biochemical gradients in a one-pot 3D manufacturing approach.

3D printed quantum dot light-emitting diodes.

Developing the ability to 3D print various classes of materials possessing distinct properties could enable the freeform generation of active electronics in unique functional, interwoven architectures. Achieving seamless integration of diverse materials with 3D printing is a significant challenge that requires overcoming discrepancies in material properties in addition to ensuring that all the materials are compatible with the 3D printing process. To date, 3D printing has been limited to specific plastics, passive conductors, and a few biological materials. Here, we show that diverse classes of materials can be 3D printed and fully integrated into device components with active properties. Specifically, we demonstrate the seamless interweaving of five different materials, including (1) emissive semiconducting inorganic nanoparticles, (2) an elastomeric matrix, (3) organic polymers as charge transport layers, (4) solid and liquid metal leads, and (5) a UV-adhesive transparent substrate layer. As a proof of concept for demonstrating the integrated functionality of these materials, we 3D printed quantum dot-based light-emitting diodes (QD-LEDs) that exhibit pure and tunable color emission properties. By further incorporating the 3D scanning of surface topologies, we demonstrate the ability to conformally print devices onto curvilinear surfaces, such as contact lenses. Finally, we show that novel architectures that are not easily accessed using standard microfabrication techniques can be constructed, by 3D printing a 2 × 2 × 2 cube of encapsulated LEDs, in which every component of the cube and electronics are 3D printed. Overall, these results suggest that 3D printing is more versatile than has been demonstrated to date and is capable of integrating many distinct classes of materials.

Flexible piezoelectric PMN-PT nanowire-based nanocomposite and device.

Piezoelectric nanocomposites represent a unique class of materials that synergize the advantageous features of polymers and piezoelectric nanostructures and have attracted extensive attention for the applications of energy harvesting and self-powered sensing recently. Currently, most of the piezoelectric nanocomposites were synthesized using piezoelectric nanostructures with relatively low piezoelectric constants, resulting in lower output currents and lower output voltages. Here, we report a synthesis of piezoelectric (1 - x)Pb(Mg1/3Nb2/3)O3-xPbTiO3 (PMN-PT) nanowire-based nanocomposite with significantly improved performances for energy harvesting and self-powered sensing. With the high piezoelectric constant (d33) and the unique hierarchical structure of the PMN-PT nanowires, the PMN-PT nanowire-based nanocomposite demonstrated an output voltage up to 7.8 V and an output current up to 2.29 µA (current density of 4.58 µA/cm(2)); this output voltage is more than double that of other reported piezoelectric nanocomposites, and the output current is at least 6 times greater. The PMN-PT nanowire-based nanocomposite also showed a linear relationship of output voltage versus strain with a high sensitivity. The enhanced performance and the flexibility of the PMN-PT nanowire-based nanocomposite make it a promising building block for energy harvesting and self-powered sensing applications.

3D printed bionic ears.

The ability to three-dimensionally interweave biological tissue with functional electronics could enable the creation of bionic organs possessing enhanced functionalities over their human counterparts. Conventional electronic devices are inherently two-dimensional, preventing seamless multidimensional integration with synthetic biology, as the processes and materials are very different. Here, we present a novel strategy for overcoming these difficulties via additive manufacturing of biological cells with structural and nanoparticle derived electronic elements. As a proof of concept, we generated a bionic ear via 3D printing of a cell-seeded hydrogel matrix in the anatomic geometry of a human ear, along with an intertwined conducting polymer consisting of infused silver nanoparticles. This allowed for in vitro culturing of cartilage tissue around an inductive coil antenna in the ear, which subsequently enables readout of inductively-coupled signals from cochlea-shaped electrodes. The printed ear exhibits enhanced auditory sensing for radio frequency reception, and complementary left and right ears can listen to stereo audio music. Overall, our approach suggests a means to intricately merge biologic and nanoelectronic functionalities via 3D printing.

Biotemplated synthesis of PZT nanowires.

Piezoelectric nanowires are an important class of smart materials for next-generation applications including energy harvesting, robotic actuation, and bioMEMS. Lead zirconate titanate (PZT), in particular, has attracted significant attention, owing to its superior electromechanical conversion performance. Yet, the ability to synthesize crystalline PZT nanowires with well-controlled properties remains a challenge. Applications of common nanosynthesis methods to PZT are hampered by issues such as slow kinetics, lack of suitable catalysts, and harsh reaction conditions. Here we report a versatile biomimetic method, in which biotemplates are used to define PZT nanostructures, allowing for rational control over composition and crystallinity. Specifically, stoichiometric PZT nanowires were synthesized using both polysaccharide (alginate) and bacteriophage templates. The wires possessed measured piezoelectric constants of up to 132 pm/V after poling, among the highest reported for PZT nanomaterials. Further, integrated devices can generate up to 0.820 µW/cm(2) of power. These results suggest that biotemplated piezoelectric nanowires are attractive candidates for stimuli-responsive nanosensors, adaptive nanoactuators, and nanoscale energy harvesters.

Biomimetic peptide nanosensors.

The development of a miniaturized sensing platform tailored for sensitive and selective detection of a variety of biochemical analytes could offer transformative fundamental and technological opportunities. Due to their high surface-to-volume ratios, nanoscale materials are extremely sensitive sensors. Likewise, peptides represent robust substrates for selective recognition due to the potential for broad chemical diversity within their relatively compact size. Here we explore the possibilities of linking peptides to nanosensors for the selective detection of biochemical targets. Such systems raise a number of interesting fundamental challenges: What are the peptide sequences, and how can rational design be used to derive selective binders? What nanomaterials should be used, and what are some strategies for assembling hybrid nanosensors? What role does molecular modeling play in elucidating response mechanisms? What is the resulting performance of these sensors, in terms of sensitivity, selectivity, and response time? What are some potential applications? This Account will highlight our early attempts to address these research challenges. Specifically, we use natural peptide sequences or sequences identified from phage display as capture elements. The sensors are based on a variety of nanomaterials including nanowires, graphene, and carbon nanotubes. We couple peptides to the nanomaterial surfaces via traditional surface functionalization methods or self-assembly. Molecular modeling provides detailed insights into the hybrid nanostructure, as well as the sensor detection mechanisms. The peptide nanosensors can distinguish chemically camouflaged mixtures of vapors and detect chemical warfare agents with sensitivities as low as parts-per-billion levels. Finally, we anticipate future uses of this technology in biomedicine: for example, devices based on these sensors could detect disease from the molecular components in human breath. Overall, these results provide a novel platform for the development of highly sensitive and selective "nanoelectronic noses".

Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides.

The development of a robust and portable biosensor for the detection of pathogenic bacteria could impact areas ranging from water-quality monitoring to testing of pharmaceutical products for bacterial contamination. Of particular interest are detectors that combine the natural specificity of biological recognition with sensitive, label-free sensors providing electronic readout. Evolution has tailored antimicrobial peptides to exhibit broad-spectrum activity against pathogenic bacteria, while retaining a high degree of robustness. Here, we report selective and sensitive detection of infectious agents via electronic detection based on antimicrobial peptide-functionalized microcapacitive electrode arrays. The semiselective antimicrobial peptide magainin I--which occurs naturally on the skin of African clawed frogs--was immobilized on gold microelectrodes via a C-terminal cysteine residue. Significantly, exposing the sensor to various concentrations of pathogenic Escherichia coli revealed detection limits of approximately 1 bacterium/µL, a clinically useful detection range. The peptide-microcapacitive hybrid device was further able to demonstrate both Gram-selective detection as well as interbacterial strain differentiation, while maintaining recognition capabilities toward pathogenic strains of E. coli and Salmonella. Finally, we report a simulated "water-sampling" chip, consisting of a microfluidic flow cell integrated onto the hybrid sensor, which demonstrates real-time on-chip monitoring of the interaction of E. coli cells with the antimicrobial peptides. The combination of robust, evolutionarily tailored peptides with electronic read-out monitoring electrodes may open exciting avenues in both fundamental studies of the interactions of bacteria with antimicrobial peptides, as well as the practical use of these devices as portable pathogen detectors.

3D printed microfluidics: advances in strategies, integration, and applications.

The ability to construct multiplexed micro-systems for fluid regulation could substantially impact multiple fields, including chemistry, biology, biomedicine, tissue engineering, and soft robotics, among others. 3D printing is gaining traction as a compelling approach to fabricating microfluidic devices by providing unique capabilities, such as 1) rapid design iteration and prototyping, 2) the potential for automated manufacturing and alignment, 3) the incorporation of numerous classes of materials within a single platform, and 4) the integration of 3D microstructures with prefabricated devices, sensing arrays, and nonplanar substrates. However, to widely deploy 3D printed microfluidics at research and commercial scales, critical issues related to printing factors, device integration strategies, and incorporation of multiple functionalities require further development and optimization. In this review, we summarize important figures of merit of 3D printed microfluidics and inspect recent progress in the field, including ink properties, structural resolutions, and hierarchical levels of integration with functional platforms. Particularly, we highlight advances in microfluidic devices printed with thermosetting elastomers, printing methodologies with enhanced degrees of automation and resolution, and the direct printing of microfluidics on various 3D surfaces. The substantial progress in the performance and multifunctionality of 3D printed microfluidics suggests a rapidly approaching era in which these versatile devices could be untethered from microfabrication facilities and created on demand by users in arbitrary settings with minimal prior training.

Enhanced piezoelectricity and stretchability in energy harvesting devices fabricated from buckled PZT ribbons.

The development of a method for integrating highly efficient energy conversion materials onto soft, biocompatible substrates could yield breakthroughs in implantable or wearable energy harvesting systems. Of particular interest are devices which can conform to irregular, curved surfaces, and operate in vital environments that may involve both flexing and stretching modes. Previous studies have shown significant advances in the integration of highly efficient piezoelectric nanocrystals on flexible and bendable substrates. Yet, such inorganic nanomaterials are mechanically incompatible with the extreme elasticity of elastomeric substrates. Here, we present a novel strategy for overcoming these limitations, by generating wavy piezoelectric ribbons on silicone rubber. Our results show that the amplitudes in the waves accommodate order-of-magnitude increases in maximum tensile strain without fracture. Further, local probing of the buckled ribbons reveals an enhancement in the piezoelectric effect of up to 70%, thus representing the highest reported piezoelectric response on a stretchable medium. These results allow for the integration of energy conversion devices which operate in stretching mode via reversible deformations in the wavy/buckled ribbons.

3D Printed Skin-Interfaced UV-Visible Hybrid Photodetectors.

Photodetectors that are intimately interfaced with human skin and measure real-time optical irradiance are appealing in the medical profiling of photosensitive diseases. Developing compliant devices for this purpose requires the fabrication of photodetectors with ultraviolet (UV)-enhanced broadband photoresponse and high mechanical flexibility, to ensure precise irradiance measurements across the spectral band critical to dermatological health when directly applied onto curved skin surfaces. Here, a fully 3D printed flexible UV-visible photodetector array is reported that incorporates a hybrid organic-inorganic material system and is integrated with a custom-built portable console to continuously monitor broadband irradiance in-situ. The active materials are formulated by doping polymeric photoactive materials with zinc oxide nanoparticles in order to improve the UV photoresponse and trigger a photomultiplication (PM) effect. The ability of a stand-alone skin-interfaced light intensity monitoring system to detect natural irradiance within the wavelength range of 310-650 nm for nearly 24 h is demonstrated.

3D-printed flexible organic light-emitting diode displays.

The ability to fully 3D-print active electronic and optoelectronic devices will enable unique device form factors via strategies untethered from conventional microfabrication facilities. Currently, the performance of 3D-printed optoelectronics can suffer from nonuniformities in the solution-deposited active layers and unstable polymer-metal junctions. Here, we demonstrate a multimodal printing methodology that results in fully 3D-printed flexible organic light-emitting diode displays. The electrodes, interconnects, insulation, and encapsulation are all extrusion-printed, while the active layers are spray-printed. Spray printing leads to improved layer uniformity via suppression of directional mass transport in the printed droplets. By exploiting the viscoelastic oxide surface of the printed cathode droplets, a mechanical reconfiguration process is achieved to increase the contact area of the polymer-metal junctions. The uniform cathode array is intimately interfaced with the top interconnects. This hybrid approach creates a fully 3D-printed flexible 8 × 8 display with all pixels turning on successfully.

Preferential binding of peptides to graphene edges and planes.

Peptides identified from combinatorial peptide libraries have been shown to bind to a variety of abiotic surfaces. Biotic-abiotic interactions can be exploited to create hybrid materials with interesting electronic, optical, or catalytic properties. Here we show that peptides identified from a combinatorial phage display peptide library assemble preferentially to the edge or planar surface of graphene and can affect the electronic properties of graphene. Molecular dynamics simulations and experiments provide insight into the mechanism of peptide binding to the graphene edge.

Nanotechnology: high-speed integrated nanowire circuits.

Macroelectronic circuits made on substrates of glass or plastic could one day make computing devices ubiquitous owing to their light weight, flexibility and low cost. But these substrates deform at high temperatures so, until now, only semiconductors such as organics and amorphous silicon could be used, leading to poor performance. Here we present the use of low-temperature processes to integrate high-performance multi-nanowire transistors into logical inverters and fast ring oscillators on glass substrates. As well as potentially enabling powerful electronics to permeate all aspects of modern life, this advance could find application in devices such as low-cost radio-frequency tags and fully integrated high-refresh-rate displays.