Nonmulberry Silk Based Ink for Fabricating Mechanically Robust Cardiac Patches and Endothelialized Myocardium‐on‐a‐Chip Application

Bioprinting holds great promise toward engineering functional cardiac tissue constructs for regenerative medicine and as drug test models. However, it is highly limited by the choice of inks that require maintaining a balance between the structure and functional properties associated with the cardiac tissue. In this regard, a novel and mechanically robust biomaterial‐ink based on nonmulberry silk fibroin protein is developed. The silk‐based ink demonstrates suitable mechanical properties required in terms of elasticity and stiffness (≈40 kPa) for developing clinically relevant cardiac tissue constructs. The ink allows the fabrication of stable anisotropic scaffolds using a dual crosslinking method, which are able to support formation of aligned sarcomeres, high expression of gap junction proteins as connexin‐43, and maintain synchronously beating of cardiomyocytes. The printed constructs are found to be nonimmunogenic in vitro and in vivo. Furthermore, delving into an innovative method for fabricating a vascularized myocardial tissue‐on‐a‐chip, the silk‐based ink is used as supporting hydrogel for encapsulating human induced pluripotent stem cell derived cardiac spheroids (hiPSC‐CSs) and creating perfusable vascularized channels via an embedded bioprinting technique. The ability is confirmed of silk‐based supporting hydrogel toward maturation and viability of hiPSC‐CSs and endothelial cells, and for applications in evaluating drug toxicity.     

Void‐Free 3D Bioprinting for In Situ Endothelialization and Microfluidic Perfusion

Two major challenges of 3D bioprinting are the retention of structural fidelity and efficient endothelialization for tissue vascularization. Both of these issues are addressed by introducing a versatile 3D bioprinting strategy, in which a templating bioink is deposited layer‐by‐layer alongside a matrix bioink to establish void‐free multimaterial structures. After crosslinking the matrix phase, the templating phase is sacrificed to create a well‐defined 3D network of interconnected tubular channels. This void‐free 3D printing (VF‐3DP) approach circumvents the traditional concerns of structural collapse, deformation, and oxygen inhibition, moreover, it can be readily used to print materials that are widely considered “unprintable.” By preloading endothelial cells into the templating bioink, the inner surface of the channels can be efficiently cellularized with a confluent endothelial layer. This in situ endothelialization method can be used to produce endothelium with a far greater cell seeding uniformity than can be achieved using the conventional postseeding approach. This VF‐3DP approach can also be extended beyond tissue fabrication and toward customized hydrogel‐based microfluidics and self‐supported perfusable hydrogel constructs.     

Capillary Network‐Like Organization of Endothelial Cells in PEGDA Scaffolds Encoded with Angiogenic Signals via Triple Helical Hybridization

Survival of tissue engineered constructs after implantation depends on proper vascularization. The differentiation of endothelial cells into mature microvasculature requires dynamic interactions between cells, scaffold, and growth factors, which are difficult to recapitulate in artificial systems. Previously, photocrosslinked poly(ethylene glycol) diacrylate (PEGDA) hydrogels displaying collagen mimetic peptides (CMPs), dubbed PEGDA‐CMP, that can be further conjugated with bioactive molecules via CMP‐CMP triple helix hybridization were reported. Here, it is shown that a bifunctional peptide featuring pro‐angiogenic domain mimicking vascular endothelial growth factor (VEGF) and a collagen mimetic domain that can fold into a triple helix conformation can hybridize with CMP side chains of the PEGDA‐CMP hydrogel, which results in presentation of insoluble VEGF‐like signals to endothelial cells. Presentation of VEGF‐like signals on the surface of micropatterned scaffolds in this way transforms cells from a quiescent state to elongated and aligned phenotype suggesting that this system could be used to engineer organized microvasculature. It is also shown that the pro‐angiogenic peptide, when applied topically in combination with modified dextran/PEGDA hydrogels, can enhance neovascularization of burn wounds in mice demonstrating the potential clinical use of CMP‐mediated matrix‐bound bioactive molecules for dermal injuries.     

Integration of Self‐Assembled Microvascular Networks with Microfabricated PEG‐Based Hydrogels

Despite tremendous efforts, tissue engineered constructs are restricted to thin, simple tissues sustained only by diffusion. The most significant barrier in tissue engineering is insufficient vascularization to deliver nutrients and metabolites during development in vitro and to facilitate rapid vascular integration in vivo. Tissue engineered constructs can be greatly improved by developing perfusable microvascular networks in vitro in order to provide transport that mimics native vascular organization and function. Here a microfluidic hydrogel is integrated with a self‐assembling pro‐vasculogenic co‐culture in a strategy to perfuse microvascular networks in vitro. This approach allows for control over microvascular network self‐assembly and employs an anastomotic interface for integration of self‐assembled microvascular networks with fabricated microchannels. As a result, transport within the system shifts from simple diffusion to vessel supported convective transport and extra‐vessel diffusion, thus improving overall mass transport properties. This work impacts the development of perfusable prevascularized tissues in vitro and ultimately tissue engineering applications in vivo.     

High‐Throughput Scaffold System for Studying the Effect of Local Geometry and Topology on the Development and Orientation of Sprouting Blood Vessels

Live tissues require vascular networks for cell nourishing. Mimicking the complex structure of native vascular networks in vitro requires understanding the governing factors of early tubulogenesis. Current vascularization protocols allow for spontaneous formation of vascular networks; however, there is still a need to provide control over the defined network structure. Moreover, there is little understanding on sprouting decision and migration, especially within 3D environments. Here, tessellated polymer scaffolds with various compartment geometries and a novel two‐step seeding protocol are used to study vessel sprouting decisions. Endothelial cells first organize into hollow vessels tracing the shape contour with high fidelity. Subsequent sprouts emerge in specific directions, responding to compartment geometry. Time‐lapse imaging is used to track vessel migration, evidencing that sprouts frequently emerge from the side centers, mainly migrating toward opposing corners, where the density of support cells (SCs) is the highest, providing the highest levels of angiogenic factors. SCs distribution is quantified by smooth muscle actin expression, confirming the cells preference for curved compartment surfaces and corners. Displacements within the hydrogel correlate with SCs distribution during the initial tubulogenesis phase. This work provides new insight regarding vessel sprouting decisions that should be considered when designing scaffolds for vascularized engineered tissues.     

Microfabricated Porous Silk Scaffolds for Vascularizing Engineered Tissues

There is critical clinical demand for tissue‐engineered (TE), 3D constructs for tissue repair and organ replacements. Current efforts toward this goal are prone to necrosis at the core of larger constructs because of limited oxygen and nutrient diffusion. Therefore, critically sized 3D TE constructs demand an immediate vascular system for sustained tissue function upon implantation. To address this challenge the goal of this project was to develop a strategy to incorporate microchannels into a porous silk TE scaffold that could be fabricated reproducibly using microfabrication and soft lithography. Silk is a suitable biopolymer material for this application because it is mechanically robust, biocompatible, slowly degrades in vivo, and has been used in a variety of TE constructs. Here, the fabrication of a silk‐based TE scaffold that contains an embedded network of porous microchannels is reported. Enclosed porous microchannels support endothelial lumen formation, a critical step toward development of the vascular niche, while the porous scaffold surrounding the microchannels supports tissue formation, demonstrated using human mesenchymal stem cells. This approach for fabricating vascularized TE constructs is advantageous compared to previous systems, which lack porosity and biodegradability or degrade too rapidly to sustain tissue structure and function. The broader impact of this research will enable the systemic study and development of complex, critically‐sized engineered tissues, from regenerative medicine to in vitro tissue models of disease states.     

Two‐Phase Emulgels for Direct Ink Writing of Skin‐Bearing Architectures

Direct ink writing (DIW) provides programmable and customizable platforms to engineer hierarchically organized constructs. However, one‐step, facile synthesis of such architectures via DIW has been challenging. This study introduces inks based on two‐phase emulgels for direct printing and in situ formation of protecting layers enveloping multicomponent cores, mimicking skin‐bearing biological systems. The emulgel consists of a Pickering emulsion with an organic, internal phase containing poly(lactic acid) stabilized by chitin/cellulose nanofibers and a continuous, cross‐linkable hydrogel containing cellulose nanofibers and any of the given solid particles. The shear during ink extrusion through nozzles of low surface energy facilitates the generation of the enveloped structures via fast and spontaneous phase separation of the emulgel. The skin‐bearing architectures enable control of mass transport as a novel configuration for cargo release. As a demonstration, a hydrophilic molecule is loaded in the hydrogel, which is released through the core and skin, enabling regulation of diffusion and permeation phenomena. This 3D‐printed functional material allows independent control of strength owing to the hierarchical construction. The new method of fabrication is proposed as a simple way to achieve protection, regulation, and sensation, taking the example of the functions of skins and cuticles, which are ubiquitous in nature.     

Monolithic Dual‐Material 3D Printing of Ionic Skins with Long‐Term Performance Stability

Artificial “ionic skin” is of great interest for mimicking the functionality of human skin, such as subtle pressure sensing. However, the development of ionic skin is hindered by the strict requirements of device integration and the need for devices with satisfactory performance. Here, a dual‐material printing strategy for ionic skin fabrication to eliminate signal drift and performance degradation during long‐term use is proposed, while endowing the ionic skins with high sensitivity by 3D printing of ionic hydrogel electrodes with microstructures. The ionic skins are fabricated by alternative digital light processing 3D printing of two photocurable precursors: hydrogel and water‐dilutable polyurethane acrylate (WPUA), in which the ionically conductive hydrogel layers serve as soft, transparent electrodes and the electrically insulated WPUA as flexible, transparent dielectric layers. This novel dual‐material printing strategy enables strong chemical bonding between the hydrogel and the WPUA, endowing the device with designed characteristics. The resulting device has high sensitivity, minimal hysteresis, a response time in the millisecond range, and excellent repetition durability for pressure sensing. The results demonstrate the potential of the dual‐material 3D printing strategy as a pathway to realize highly stable and high‐performance ionic skin fabrication to monitor human physiological signals and human–machine interactions.     

Switchable Supercapacitors with Transistor‐Like Gating Characteristics (G‐Cap)

A novel three‐electrode electrolyte supercapacitor (electric double‐layer capacitor [EDLC]) architecture in which a symmetrical interdigital “working” two‐electrode micro‐supercapacitor array (W‐Cap) is paired with a third “gate” electrode that reversibly depletes/injects electrolyte ions into the system controlling the “working” capacity effectively is described. All three electrodes are based on precursor‐derived nanoporous carbons with well‐defined specific surface area (735 m² g^?1). The interdigitated architecture of the W‐Cap is precisely manufactured using 3D printing. The W‐Cap operating with a proton conducting PVA/H₂SO₄‐hydrogel electrolyte and high capacitance (6.9 mF cm^?2) can be repeatedly switched “on” and “off”. By applying a low DC bias potential (?0.5 V) at the gate electrode, the AC electroadsorption in the coupled interdigital nanoporous carbon electrodes of the W‐Cap is effectively suppressed leading to a stark capacity drop by two orders of magnitude from an “on” to an “off” state. The switchable micro‐supercapacitor is the first of its kind. This general concept is suitable for implementing a broad range of nanoporous materials and advanced electrolytes expanding its functions and applications in future. The integration of intelligent functions into EDLC devices has extensive implications for diverse areas such as capacitive energy management, microelectronics, iontronics, and neuromodulation.     

A Stimuli‐Responsive Nanocomposite for 3D Anisotropic Cell‐Guidance and Magnetic Soft Robotics

Stimuli‐responsive materials have the potential to enable the generation of new bioinspired devices with unique physicochemical properties and cell‐instructive ability. Enhancing biocompatibility while simplifying the production methodologies, as well as enabling the creation of complex constructs, i.e., via 3D (bio)printing technologies, remains key challenge in the field. Here, a novel method is presented to biofabricate cellularized anisotropic hybrid hydrogel through a mild and biocompatible process driven by multiple external stimuli: magnetic field, temperature, and light. A low‐intensity magnetic field is used to align mosaic iron oxide nanoparticles (IOPs) into filaments with tunable size within a gelatin methacryloyl matrix. Cells seeded on top or embedded within the hydrogel align to the same axes of the IOPs filaments. Furthermore, in 3D, C2C12 skeletal myoblasts differentiate toward myotubes even in the absence of differentiation media. 3D printing of the nanocomposite hydrogel is achieved and creation of complex heterogeneous structures that respond to magnetic field is demonstrated. By combining the advanced, stimuli‐responsive hydrogel with the architectural control provided by bioprinting technologies, 3D constructs can also be created that, although inspired by nature, express functionalities beyond those of native tissue, which have important application in soft robotics, bioactuators, and bionic devices.     

Versatile Functionalization of the Micropatterned Hydrogel of Hyperbranched Poly(ether amine) Based on “Thiol‐yne” Chemistry

The functionalization of a hydrogel with target molecules is one of the key steps in its various applications. Here, a versatile approach is demonstrated to functionalize a micropatterned hydrogel, which is formed by “thiol‐yne” photo‐click reaction between the yne‐ended hyperbranched poly(ether amine) (hPEA‐yne) and thiol‐containing polyhedral oligomeric silsesquioxane (PEG‐POSS‐SH). By controlling the molar ratio between hPEA‐yne and PEG‐POSS‐SH, patterned hydrogels containing thiol or yne groups are obtained. A series of thiol‐based click chemistry such as “thiol‐epoxy”, “thiol‐halogen”, “thiol‐ene”, and “thiol‐isocyanate” are used to functionalize the thiol‐containing hydrogel (Gel‐1), while the yne‐containing hydrogel (Gel‐2) is functionalized through a typical copper‐catalysed alkyne‐azide reaction (CuAAC). FTIR, UV‐vis spectra and confocal laser scanning microscopy (CLSM) are used to trace these click reactions. Due to the selective adsorption to the hydrophilic dyes, the obtained patterned hydrogel of hPEA modified with fluorescence dye is further demonstrated in application for the recognition of guest molecules.     

Direct 3D Printed Biomimetic Scaffolds Based on Hydrogel Microparticles for Cell Spheroid Growth

Biocompatible hydrogel inks with shear‐thinning, appropriate yield strength, and fast self‐healing are desired for 3D bioprinting. However, the lack of ideal 3D bioprinting inks with outstanding printability and high structural fidelity, as well as cell‐compatibility, has hindered the progress of extrusion‐based 3D bioprinting for tissue engineering. In this study, novel self‐healable pre‐cross‐linked hydrogel microparticles (pcHμPs) of chitosan methacrylate (CHMA) and polyvinyl alcohol (PVA) hybrid hydrogels are developed and used as bioinks for extrusion‐based 3D printing of scaffolds with high fidelity and biocompatibility. The pcHμPs display excellent shear thinning when injected through a syringe and subsequently self‐heal into gels as shear forces are removed. Numerical simulations indicate that the pcHμPs experience a plug flow in the nozzle with minimal disturbance, which favors a steady and continuous printing. Moreover, the pcHμPs show a self‐supportive yield strength (540 Pa), which is critical for the fidelity of printed constructs. A series of biomimetic constructs with very high aspect ratio and delicate fine structures are directly printed by using the pcHμP ink. The 3D printed scaffolds support the growth of bone‐marrow‐derived mesenchymal stem cells and formation of cell spheroids, which are most important for tissue engineering.     

Programming Delayed Dissolution Into Sacrificial Bioinks For Dynamic Temporal Control of Architecture within 3D-Bioprinted Constructs

Sacrificial printing allows introduction of architectural cues within engineered tissue constructs. This strategy adopts the use of a 3D-printed sacrificial ink that is embedded within a bulk hydrogel which is subsequently dissolved to leave open-channels. However, current conventional sacrificial inks do not recapitulate the dynamic nature of tissue development, such as the temporal presentation of architectural cues matching cellular requirements during different stages of maturation. To address this limitation, a new class of sacrificial inks is developed that exhibits tailorable and programmable delayed dissolution profiles (1–17 days), by exploiting the unique ability of the ruthenium complex and sodium persulfate initiating system to crosslink native tyrosine groups present in non-chemically modified gelatin. These novel sacrificial inks are also shown to be compatible with a range of biofabrication technologies, including extrusion-based printing, digital-light processing, and volumetric bioprinting. Further embedding these sacrificial templates within cell-laden bulk hydrogels displays precise control over the spatial and temporal introduction of architectural features into cell-laden hydrogel constructs. This approach demonstrates the unique capacity of delaying dissolution of sacrificial inks to modulate cell behavior, improving the deposition of mineralized matrix and capillary-like network formation in osteogenic and vasculogenic culture, respectively.     

Multicolor Printing Using Electric‐Field‐Responsive and Photocurable Photonic Crystals

Efficient and large scale printing of photonic crystal patterns with multicolor, multigrayscale, and fine resolution is highly desired due to its application in smart prints, sensors, and photonic devices. Here, an electric‐field‐assisted multicolor printing is reported based on electrically responsive and photocurable colloidal photonic crystal, which is prepared by supersaturation‐induced self‐assembly of SiO₂ particles in the mixture of propylene carbonate (PC) and trimethylolpropane ethoxylate triacrylate (ETPTA). This colloidal crystal suspension, named as E‐ink, has tunable structural color, controllable grayscale, and instantly fixable characteristics at the same time because the SiO₂/ETPTA‐PC photonic crystal has metastable and reversible assembly as well as polymerizable features. Lithographical printing with photomask and maskless pixel printing techniques are developed respectively to efficiently prepare multicolor and high‐resolution photonic patterns using a single‐component E‐ink.     

Glycocalyx‐Like Hydrogel Coatings for Small Diameter Vascular Grafts

Novel biological vascular conduits, such as decellularized tissue engineered vascular grafts (TEVGs) are hindered by high thrombogenicity. To mimic the antithrombogenic surface of native vessels with a continuous glycosaminoglycan layer that is present on endothelial cells (ECs), a hyaluronic acid (HA) modified surface is established, to effectively shield blood platelets from collagen‐triggered activation. Using the amine groups present on 4 mm diameter decellularized TEVGs, a continuous HA hydrogel coating is built via a bifunctional thiol‐reactive cross‐linker, thereby avoiding nonspecific collagen matrix cross‐linking. The HA hydrogel layer recreates a luminal wall, “hiding” exposed collagen from the bloodstream. In vitro blood tests show that adhered platelets, fibrinogen absorption, and fibrin formation on HA‐coated decellularized TEVGs are significantly lower than on uncoated decellularized TEVGs. The HA surface also inhibits macrophage adhesion in vitro. HA‐coated decellularized syngeneic rat aortae (≈1.5 mm diameter), and TEVGs in rat and canine models, respectively, are protected from aggressive thrombus formation, and preserve normal blood flow. Re‐endothelialization is also observed. HA‐coated TEVGs may be an off‐the‐shelf small‐diameter vascular graft with dual benefits: antithrombogenic protection and promotion of endothelium.     

Full Color Light Responsive Diarylethene Inks for Reusable Paper

“Digitalization” represents one approach to shift society's dependence on paper‐based communication. However, thus far, this tactic has not had a significant impact on global paper consumption, which has risen over the past few decades. The escalating demand of paper making and consumption has resulted in an intensified negative effect on the environment. Because of this, the development of rewritable paper or erasable ink appears to be an ideal approach to alleviate the increasing demand for paper. In the investigation described herein, novel light‐stimulated (UV–vis), reversible color switching, photochromic diarylethene (DE) derivatives are designed, which serve as cyan, magenta, and yellow colored ink materials for full color ink‐jet printing. The structures of the DE derivatives are unique in that they contain hydrophilic ethylene glycol chains that enable them to be compatible with aqueous based, ink‐jet printing systems. The results of these studies demonstrate that the new DE derivatives can be used in a printing system based on the “write–erase–write” concept that utilizes the same paper multiple times. The approach appears to be ideal for reducing the negative environmental consequences of paper production and consumption.     

Plasmonic Color Filters as Dual‐State Nanopixels for High‐Density Microimage Encoding

Plasmonic color filtering has provided a range of new techniques for “printing” images at resolutions beyond the diffraction‐limit, significantly improving upon what can be achieved using traditional, dye‐based filtering methods. Here, a new approach to high‐density data encoding is demonstrated using full color, dual‐state plasmonic nanopixels, doubling the amount of information that can be stored in a unit‐area. This technique is used to encode two data sets into a single set of pixels for the first time, generating vivid, near‐full sRGB (standard Red Green Blue color space)color images and codes with polarization‐switchable information states. Using a standard optical microscope, the smallest “unit” that can be read relates to 2 × 2 nanopixels (370 nm × 370 nm). As a result, dual‐state nanopixels may prove significant for long‐term, high‐resolution optical image encoding, and counterfeit‐prevention measures.     

Rapid Magnetically Directed Assembly of Pre-Patterned Capillary-Scale Microvessels

Capillary scale vascularization is critical to the survival of engineered 3D tissues and remains an outstanding challenge for the field of tissue engineering. Current methods to generate micro-scale vasculatures such as 3D printing, two photon hydrogel ablation, angiogenesis, and vasculogenic assembly face challenges in rapidly creating organized, highly vascularized tissues at capillary length-scales. Within metabolically demanding tissues, native capillary beds are highly organized and densely packed to achieve adequate delivery of nutrients and oxygen and efficient waste removal. Here, two existing techniques are adopted to fabricate lattices composed of sacrificial microfibers that can be efficiently and uniformly seeded with endothelial cells (ECs) by magnetizing both lattices and ECs. Ferromagnetic microparticles are incorporated into microfibers produced by solution electrowriting and fiber electropulling. By loading ECs with superparamagnetic iron oxide nanoparticles, the cells could be seeded onto magnetized microfiber lattices. Following encapsulation in a hydrogel, the capillary templating lattice is selectively degraded by a bacterial lipase that does not impact mammalian cell viability or function. This study introduces a novel approach to rapidly producing organized capillary networks within metabolically demanding engineered tissue constructs which should have broad utility in the fields of tissue engineering and regenerative medicine.     

Novel Biocompatible Polysaccharide‐Based Self‐Healing Hydrogel

A novel biocompatible polysaccharide‐based self‐healing hydrogel, CEC‐l‐OSA‐l‐ADH hydrogel (“l” means “linked‐by”), is developed by exploiting the dynamic reaction of N‐carboxyethyl chitosan (CEC) and adipic acid dihydrazide (ADH) with oxidized sodium alginate (OSA). The self‐healing ability, as demonstrated by rheological recovery, macroscopic observation, and beam‐shaped strain compression measurement, is attributed to the coexistence of dynamic imine and acylhydrazone bonds in the hydrogel networks. The CEC‐l‐OSA‐l‐ADH hydrogel shows excellent self‐healing ability under physiological conditions with a high healing efficiency (up to 95%) without need for any external stimuli. In addition, the CEC‐l‐OSA‐l‐ADH hydrogel exhibits good cytocompatibility and cell release as demonstrated by three‐dimensional cell encapsulation. With these superior properties, the developed hydrogel holds great potential for applications in various biomedical fields, e.g., as cell or drug delivery carriers.     

Stimuli‐Responsive Donor–Acceptor and DNA‐Crosslinked Hydrogels: Application as Shape‐Memory and Self‐Healing Materials

Carboxymethyl cellulose (CMC) chains are functionalized with self‐complementary nucleic acid tethers and electron donor or electron acceptor functionalities. The polymer chains crosslinked by the self‐complementary duplex nucleic acids and the donor–acceptor complexes as bridging units, yield a stiff stimuli‐responsive hydrogel. Upon the oxidation of the electron donor units, the donor–acceptor bridging units are separated, leading to a hydrogel of lower stiffness. By the cyclic oxidation and reduction of the donor units, the hydrogel is reversibly transformed across low and high stiffness states. The controlled stiffness properties of the hydrogel are used to develop shape‐memory hydrogels. In addition, CMC hydrogels crosslinked by donor–acceptor complexes and K⁺‐stabilized G‐quadruplexes reveal stimuli‐responsive properties that exhibit dually triggered stiffness functions. While the hydrogel bridged by the two crosslinking motifs reveals high stiffness, the redox‐stimulated separation of the donor–acceptor complexes or the crown‐ether‐stimulated separation of the G‐quadruplex bridges yields two alternative hydrogels exhibiting low stiffness states. The control over the stiffness properties of the dually triggered hydrogel is used to develop shape‐memory hydrogels, where the donor–acceptor units or G‐quadruplex bridges act as “memories”, and to develop triggered self‐healing process of the hydrogel.