Flexible Fabrication of Shape‐Controlled Collagen Building Blocks for Self‐Assembly of 3D Microtissues

Creating artificial tissue‐like structures that possess the functionality, specificity, and architecture of native tissues remains a big challenge. A new and straightforward strategy for generating shape‐controlled collagen building blocks with a well‐defined architecture is presented, which can be used for self‐assembly of complex 3D microtissues. Collagen blocks with tunable geometries are controllably produced and released via a membrane‐templated microdevice. The formation of functional microtissues by embedding tissue‐specific cells into collagen blocks with expression of specific proteins is described. The spontaneous self‐assembly of cell‐laden collagen blocks into organized tissue constructs with predetermined configurations is demonstrated, which are largely driven by the synergistic effects of cell–cell and cell–matrix interactions. This new strategy would open up new avenues for the study of tissue/organ morphogenesis, and tissue engineering applications.     

Cell Encapsulation in Soft,Anisometric Poly(ethylene) Glycol Microgels Using a Novel Radical‐Free Microfluidic System

Complex 3D artificial tissue constructs are extensively investigated for tissue regeneration. Frequently, materials and cells are delivered separately without benefitting from the synergistic effect of combined administration. Cell delivery inside a material construct provides the cells with a supportive environment by presenting biochemical, mechanical, and structural signals to direct cell behavior. Conversely, the cell/material interaction is poorly understood at the micron scale and new systems are required to investigate the effect of micron‐scale features on cell functionality. Consequently, cells are encapsulated in microgels to avoid diffusion limitations of nutrients and waste and facilitate analysis techniques of single or collective cells. However, up to now, the production of soft cell‐loaded microgels by microfluidics is limited to spherical microgels. Here, a novel method is presented to produce monodisperse, anisometric poly(ethylene) glycol microgels to study cells inside an anisometric architecture. These microgels can potentially direct cell growth and can be injected as rod‐shaped mini‐tissues that further assemble into organized macroscopic and macroporous structures post‐injection. Their aspect ratios are adjusted with flow parameters, while mechanical and biochemical properties are altered by modifying the precursors. Encapsulated primary fibroblasts are viable and spread and migrate across the 3D microgel structure.     

Genetically Programmable Self‐Regenerating Bacterial Hydrogels

A notable challenge for the design of engineered living materials (ELMs) is programming a cellular system to assimilate resources from its surroundings and convert them into macroscopic materials with specific functions. Here, an ELM that uses Escherichia coli as its cellular chassis and engineered curli nanofibers as its extracellular matrix component is demonstrated. Cell‐laden hydrogels are created by concentrating curli‐producing cultures. The rheological properties of the living hydrogels are modulated by genetically encoded factors and processing steps. The hydrogels have the ability to grow and self‐renew when placed under conditions that facilitate cell growth. Genetic programming enables the gels to be customized to interact with different tissues of the gastrointestinal tract selectively. This work lays a foundation for the application of ELMs with therapeutic functions and extended residence times in the gut.     

Compartmentalized Jet Polymerization as a High‐Resolution Process to Continuously Produce Anisometric Microgel Rods with Adjustable Size and Stiffness

In the past decade, anisometric rod‐shaped microgels have attracted growing interest in the materials‐design and tissue‐engineering communities. Rod‐shaped microgels exhibit outstanding potential as versatile building blocks for 3D hydrogels, where they introduce macroscopic anisometry, porosity, or functionality for structural guidance in biomaterials. Various fabrication methods have been established to produce such shape‐controlled elements. However, continuous high‐throughput production of rod‐shaped microgels with simultaneous control over stiffness, size, and aspect ratio still presents a major challenge. A novel microfluidic setup is presented for the continuous production of rod‐shaped microgels from microfluidic plug flow and jets. This system overcomes the current limitations of established production methods for rod‐shaped microgels. Here, an on‐chip gelation setup enables fabrication of soft microgel rods with high aspect ratios, tunable stiffness, and diameters significantly smaller than the channel diameter. This is realized by exposing jets of a microgel precursor to a high intensity light source, operated at specific pulse sequences and frequencies to induce ultra‐fast photopolymerization, while a change in flow rates or pulse duration enables variation of the aspect ratio. The microgels can assemble into 3D structures and function as support for cell culture and tissue engineering.     

Near‐Infrared Light‐Sensitive Polyvinyl Alcohol Hydrogel Photoresist for Spatiotemporal Control of Cell‐Instructive 3D Microenvironments

Advanced hydrogel systems that allow precise control of cells and their 3D microenvironments are needed in tissue engineering, disease modeling, and drug screening. Multiphoton lithography (MPL) allows true 3D microfabrication of complex objects, but its biological application requires a cell‐compatible hydrogel resist that is sufficiently photosensitive, cell‐degradable, and permissive to support 3D cell growth. Here, an extremely photosensitive cell‐responsive hydrogel composed of peptide‐crosslinked polyvinyl alcohol (PVA) is designed to expand the biological applications of MPL. PVA hydrogels are formed rapidly by ultraviolet light within 1 min in the presence of cells, providing fully synthetic matrices that are instructive for cell‐matrix remodeling, multicellular morphogenesis, and protease‐mediated cell invasion. By focusing a multiphoton laser into a cell‐laden PVA hydrogel, cell‐instructive extracellular cues are site‐specifically attached to the PVA matrix. Cell invasion is thus precisely guided in 3D with micrometer‐scale spatial resolution. This robust hydrogel enables, for the first time, ultrafast MPL of cell‐responsive synthetic matrices at writing speeds up to 50 mm s^?1. This approach should enable facile photochemical construction and manipulation of 3D cellular microenvironments with unprecedented flexibility and precision.     

Self‐Assembly of Enzyme‐Like Nanofibrous G‐Molecular Hydrogel for Printed Flexible Electrochemical Sensors

Conducting hydrogels provide great potential for creating designer shape‐morphing architectures for biomedical applications owing to their unique solid–liquid interface and ease of processability. Here, a novel nanofibrous hydrogel with significant enzyme‐like activity that can be used as “ink” to print flexible electrochemical devices is developed. The nanofibrous hydrogel is self‐assembled from guanosine (G) and KB(OH)₄ with simultaneous incorporation of hemin into the G‐quartet scaffold, giving rise to significant enzyme‐like activity. The rapid switching between the sol and gel states responsive to shear stress enables free‐form fabrication of different patterns. Furthermore, the replication of the G‐quartet wires into a conductive matrix by in situ catalytic deposition of polyaniline on nanofibers is demonstrated, which can be directly printed into a flexible electrochemical electrode. By loading glucose oxidase into this novel hydrogel, a flexible glucose biosensor is developed. This study sheds new light on developing artificial enzymes with new functionalities and on fabrication of flexible bioelectronics.     

Volumetric Bioprinting of Complex Living‐Tissue Constructs within Seconds

Biofabrication technologies, including stereolithography and extrusion‐based printing, are revolutionizing the creation of complex engineered tissues. The current paradigm in bioprinting relies on the additive layer‐by‐layer deposition and assembly of repetitive building blocks, typically cell‐laden hydrogel fibers or voxels, single cells, or cellular aggregates. The scalability of these additive manufacturing technologies is limited by their printing velocity, as lengthy biofabrication processes impair cell functionality. Overcoming such limitations, the volumetric bioprinting of clinically relevant sized, anatomically shaped constructs, in a time frame ranging from seconds to tens of seconds is described. An optical‐tomography‐inspired printing approach, based on visible light projection, is developed to generate cell‐laden tissue constructs with high viability (>85%) from gelatin‐based photoresponsive hydrogels. Free‐form architectures, difficult to reproduce with conventional printing, are obtained, including anatomically correct trabecular bone models with embedded angiogenic sprouts and meniscal grafts. The latter undergoes maturation in vitro as the bioprinted chondroprogenitor cells synthesize neo‐fibrocartilage matrix. Moreover, free‐floating structures are generated, as demonstrated by printing functional hydrogel‐based ball‐and‐cage fluidic valves. Volumetric bioprinting permits the creation of geometrically complex, centimeter‐scale constructs at an unprecedented printing velocity, opening new avenues for upscaling the production of hydrogel‐based constructs and for their application in tissue engineering, regenerative medicine, and soft robotics.     

Remotely Light‐Powered Soft Fluidic Actuators Based on Plasmonic‐Driven Phase Transitions in Elastic Constraint

Materials capable of actuation through remote stimuli are crucial for untethering soft robotic systems from hardware for powering and control. Fluidic actuation is one of the most applied and versatile actuation strategies in soft robotics. Here, the first macroscale soft fluidic actuator is derived that operates remotely powered and controlled by light through a plasmonically induced phase transition in an elastomeric constraint. A multiphase assembly of a liquid layer of concentrated gold nanoparticles in a silicone or styrene–ethylene–butylene–styrene elastic pocket forms the actuator. Upon laser excitation, the nanoparticles convert light of specific wavelength into heat and initiate a liquid‐to‐gas phase transition. The related pressure increase inflates the elastomers in response to laser wavelength, intensity, direction, and on–off pulses. During laser‐off periods, heating halts and condensation of the gas phase renders the actuation reversible. The versatile multiphase materials actuate—like soft “steam engines”—a variety of soft robotic structures (soft valve, pnue‐net structure, crawling robot, pump) and are capable of operating in different environments (air, water, biological tissue) in a single configuration. Tailored toward the near‐infrared window of biological tissue, the structures actuate also through animal tissue for potential medical soft robotic applications.     

Rapid Production of Cell‐Laden Microspheres Using a Flexible Microfluidic Encapsulation Platform

This study establishes a novel microfluidic platform for rapid encapsulation of cells at high densities in photocrosslinkable microspherical hydrogels including poly(ethylene glycol)‐diacrylate, poly(ethylene glycol)‐fibrinogen, and gelatin methacrylate. Cell‐laden hydrogel microspheres are advantageous for many applications from drug screening to regenerative medicine. Employing microfluidic systems is considered the most efficient method for scale‐up production of uniform microspheres. However, existing platforms have been constrained by traditional microfabrication techniques for device fabrication, restricting microsphere diameter to below 200 µm and making iterative design changes time‐consuming and costly. Using a new molding technique, the microfluidic device employs a modified T‐junction design with readily adjustable channel sizes, enabling production of highly uniform microspheres with cell densities (10–60 million cells mL^?1) and a wide range of diameters (300–1100 µm), which are critical for realizing downstream applications, through rapid photocrosslinking (≈1 s per microsphere). Multiple cell types are encapsulated at rates of up to 1 million cells per min, are evenly distributed throughout the microspheres, and maintain high viability and appropriate cellular activities in long‐term culture. This microfluidic encapsulation platform is a valuable and readily adoptable tool for numerous applications, including supporting injectable cell therapy, bioreactor‐based cell expansion and differentiation, and high throughput tissue sphere‐based drug testing assays.     

Microscale Electro‐Hydrodynamic Cell Printing with High Viability

Cell printing has gained extensive attentions for the controlled fabrication of living cellular constructs in vitro. Various cell printing techniques are now being explored and developed for improved cell viability and printing resolution. Here an electro‐hydrodynamic cell printing strategy is developed with microscale resolution (<100 µm) and high cellular viability (>95%). Unlike the existing electro‐hydrodynamic cell jetting or printing explorations, insulating substrate is used to replace conventional semiconductive substrate as the collecting surface which significantly reduces the electrical current in the electro‐hydrodynamic printing process from milliamperes (>0.5 mA) to microamperes (<10 µA). Additionally, the nozzle‐to‐collector distance is fixed as small as 100 µm for better control over filament deposition. These features ensure high cellular viability and normal postproliferative capability of the electro‐hydrodynamically printed cells. The smallest width of the electro‐hydrodynamically printed hydrogel filament is 82.4 ± 14.3 µm by optimizing process parameters. Multiple hydrogels or multilayer cell‐laden constructs can be flexibly printed under cell‐friendly conditions. The printed cells in multilayer hydrogels kept alive and gradually spread during 7‐days culture in vitro. This exploration offers a novel and promising cell printing strategy which might benefit future biomedical innovations such as microscale tissue engineering, organ‐on‐a‐chip systems, and nanomedicine.     

25th Anniversary Article: Engineering Hydrogels for Biofabrication

With advances in tissue engineering, the possibility of regenerating injured tissue or failing organs has become a realistic prospect for the first time in medical history. Tissue engineering – the combination of bioactive materials with cells to generate engineered constructs that functionally replace lost and/or damaged tissue – is a major strategy to achieve this goal. One facet of tissue engineering is biofabrication, where three‐dimensional tissue‐like structures composed of biomaterials and cells in a single manufacturing procedure are generated. Cell‐laden hydrogels are commonly used in biofabrication and are termed “bioinks”. Hydrogels are particularly attractive for biofabrication as they recapitulate several features of the natural extracellular matrix and allow cell encapsulation in a highly hydrated mechanically supportive three‐dimensional environment. Additionally, they allow for efficient and homogeneous cell seeding, can provide biologically‐relevant chemical and physical signals, and can be formed in various shapes and biomechanical characteristics. However, despite the progress made in modifying hydrogels for enhanced bioactivation, cell survival and tissue formation, little attention has so far been paid to optimize hydrogels for the physico‐chemical demands of the biofabrication process. The resulting lack of hydrogel bioinks have been identified as one major hurdle for a more rapid progress of the field. In this review we summarize and focus on the deposition process, the parameters and demands of hydrogels in biofabrication, with special attention to robotic dispensing as an approach that generates constructs of clinically relevant dimensions. We aim to highlight this current lack of effectual hydrogels within biofabrication and initiate new ideas and developments in the design and tailoring of hydrogels. The successful development of a “printable” hydrogel that supports cell adhesion, migration, and differentiation will significantly advance this exciting and promising approach for tissue engineering.     

Centering Single Cells in Microgels via Delayed Crosslinking Supports Long‐Term 3D Culture by Preventing Cell Escape

Single‐cell‐laden microgels support physiological 3D culture conditions while enabling straightforward handling and high‐resolution readouts of individual cells. However, their widespread adoption for long‐term cultures is limited by cell escape. In this work, it is demonstrated that cell escape is predisposed to off‐center encapsulated cells. High‐speed microscopy reveals that cells are positioned at the microgel precursor droplets' oil/water interface within milliseconds after droplet formation. In conventional microencapsulation strategies, the droplets are typically gelled immediately after emulsification, which traps cells in this off‐center position. By delaying crosslinking, driving cells toward the centers of microgels is succeeded. The centering of cells in enzymatically crosslinked microgels prevents their escape during at least 28 d. It thereby uniquely enables the long‐term culture of individual cells within <5‐µm‐thick 3D uniform hydrogel coatings. Single cell analysis of mesenchymal stem cells in enzymatically crosslinked microgels reveals unprecedented high cell viability (>90%), maintained metabolic activity (>70%), and multilineage differentiation capacity (>60%) over a period of 28 d. The facile nature of this microfluidic cell‐centering method enables its straightforward integration into many microencapsulation strategies and significantly enhances control, reproducibility, and reliability of 3D single cell cultures.     

Cold‐Responsive Nanocapsules Enable the Sole‐Cryoprotectant‐Trehalose Cryopreservation of β Cell–Laden Hydrogels for Diabetes Treatment

Islet transplantation has been one promising strategy in diabetes treatment, which can maintain patient's insulin level long‐term and avoid periodical insulin injections. However, donor shortage and temporal mismatch between donors and recipients has limited its widespread use. Therefore, searching for islet substitutes and developing efficient cryopreservation technology (providing potential islet bank for transplantation on demand) is in great need. Herein, a novel cryopreservation method is developed for islet β cells by combining microfluidic encapsulation and cold‐responsive nanocapsules (CR‐NCs). The cryopreserved cell‐laden hydrogels (calcium alginate hydrogel, CAH) can be transplanted for diabetes treatment. During the freezing process, trehalose is released inside β cells through the CR‐NCs and serves as the sole cryoprotectant (CPA). Additionally, CAH helps cells to survive the freeze–thaw process and provide cells with a natural immune barrier in vivo. Different from traditional cryopreservation methods, this method combining the CR‐NCs and hydrogel encapsulation replaces the toxic CPAs with natural trehalose. Great preservation results are obtained and transplantation experiments of diabetic rats further prove the excellent glucose regulation ability of such β cell–laden hydrogels post cryopreservation. This novel cryopreservation method helps to establish a reliable and ready‐to‐use bank of biological samples for transplantation therapy and other biomedical applications.     

Injectable Granular Hydrogels with Multifunctional Properties for Biomedical Applications

Injectable hydrogels are useful for numerous biomedical applications, such as to introduce therapeutics into tissues or for 3D printing. To expand the complexity of available injectable hydrogels, shear‐thinning and self‐healing granular hydrogels are developed from microgels that interact via guest–host chemistry. The microgel properties (e.g., degradation, molecule release) are tailored through their crosslinking chemistry, including degradation in response to proteases. When microgels of varied formulations are mixed, complex release and degradation behaviors are observed, including after injection to permit cellular invasion.     

Vapor‐Sensitive Materials from Polysaccharide Fibers with Self‐Assembling Twisted Microstructures

Polysaccharides play a variety of roles in nature, including molecular recognition and water retention. The microscale structures of polysaccharides are seldom utilized in vitro because of the difficulties in regulating self‐assembled structures. Herein, it is demonstrated that a cyanobacterial polysaccharide, sacran, can hierarchically self‐assemble as twisted fibers from nanoscale to microscale with diameters of ≈1 µm and lengths >800 µm that are remarkably larger than polysaccharides previously reported. Unlike other rigid fibrillar polysaccharides, the sacran fiber is capable of flexibly transforming into two‐dimensional (2D) snaking and three‐dimensional (3D) twisted structures at an evaporative air–water interface. Furthermore, a vapor‐sensitive film with a millisecond‐scale response time is developed from the crosslinked polymer due to the spring‐like behavior of twisted structures. This study increases understanding of the functions of fibers in nature and establishes a novel approach to the design of environmentally adaptive materials for soft sensors and actuators.     

Buoyancy‐Driven Gradients for Biomaterial Fabrication and Tissue Engineering

The controlled fabrication of gradient materials is becoming increasingly important as the next generation of tissue engineering seeks to produce inhomogeneous constructs with physiological complexity. Current strategies for fabricating gradient materials can require highly specialized materials or equipment and cannot be generally applied to the wide range of systems used for tissue engineering. Here, the fundamental physical principle of buoyancy is exploited as a generalized approach for generating materials bearing well‐defined compositional, mechanical, or biochemical gradients. Gradient formation is demonstrated across a range of different materials (e.g., polymers and hydrogels) and cargos (e.g., liposomes, nanoparticles, extracellular vesicles, macromolecules, and small molecules). As well as providing versatility, this buoyancy‐driven gradient approach also offers speed (<1 min) and simplicity (a single injection) using standard laboratory apparatus. Moreover, this technique is readily applied to a major target in complex tissue engineering: the osteochondral interface. A bone morphogenetic protein 2 gradient, presented across a gelatin methacryloyl hydrogel laden with human mesenchymal stem cells, is used to locally stimulate osteogenesis and mineralization in order to produce integrated osteochondral tissue constructs. The versatility and accessibility of this fabrication platform should ensure widespread applicability and provide opportunities to generate other gradient materials or interfacial tissues.     

Autonomous Ultrafast Self‐Healing Hydrogels by pH‐Responsive Functional Nanofiber Gelators as Cell Matrices

The synthesis of hybrid hydrogels by pH‐controlled structural transition with exceptional rheological properties as cellular matrix is reported. “Depsi” peptide sequences are grafted onto a polypeptide backbone that undergo a pH‐induced intramolecular O–N–acyl migration at physiological conditions affording peptide nanofibers (PNFs) as supramolecular gelators. The polypeptide–PNF hydrogels are mechanically remarkably robust. They reveal exciting thixotropic behavior with immediate in situ recovery after exposure to various high strains over long periods and self‐repair of defects by instantaneous reassembly. High cytocompatibility, convenient functionalization by coassembly, and controlled enzymatic degradation but stability in 2D and 3D cell culture as demonstrated by the encapsulation of primary human umbilical vein endothelial cells and neuronal cells open many attractive opportunities for 3D tissue engineering and other biomedical applications.     

Microfluidic Templating of Spatially Inhomogeneous Protein Microgels

3D scaffolds in the form of hydrogels and microgels have allowed for more native cell‐culture systems to be developed relative to flat substrates. Native biological tissues are, however, usually spatially inhomogeneous and anisotropic, but regulating the spatial density of hydrogels at the microscale to mimic this inhomogeneity has been challenging to achieve. Moreover, the development of biocompatible synthesis approaches for protein‐based microgels remains challenging, and typical gelation conditions include UV light, extreme pH, extreme temperature, or organic solvents, factors which can compromise the viability of cells. This study addresses these challenges by demonstrating an approach to fabricate protein microgels with controllable radial density through microfluidic mixing and physical and enzymatic crosslinking of gelatin precursor molecules. Microgels with a higher density in their cores and microgels with a higher density in their shells are demonstrated. The microgels have robust stability at 37 °C and different dissolution rates through enzymolysis, which can be further used for gradient scaffolds for 3D cell culture, enabling controlled degradability, and the release of biomolecules. The design principles of the microgels could also be exploited to generate other soft materials for applications ranging from novel protein‐only micro reactors to soft robots.     

Ultrastrong Anchoring Yet Barrier‐Free Adsorption of Composite Microgels at Liquid Interfaces

Microgel particles display an interesting duality with properties attributed typically both to polymeric and colloidal systems. When adsorbed at a liquid‐liquid interface, this duality becomes particularly apparent as the various phenomena at play are governed by different aspects of these soft and responsive particles. The introduction of a solid, fluorescently labeled, polystyrene core into the microgels allows direct and accurate visualization without the necessity of potential perturbing sample preparation techniques. By combining in‐situ imaging and tensiometry, we determine that composite microgels at a wide variety of oil‐water interfaces anchor strongly, with adsorption energies of approximately 10⁶ k_BT, typical for particle adsorption, yet accumulate at the interface spontaneously without any energy barrier, which is more typical for polymers. The high adsorption energies allow the particle to spontaneously form very dense crystalline packings at the liquid interface in which the microgels are significantly compressed with respect to their swollen state in bulk solutions. Finally, we demonstrate the unique nature of these particles by producing highly stable and monodisperse microgel‐stabilized droplets using microfluidics.     

Ultrastretchable and Wireless Bioelectronics Based on All‐Hydrogel Microfluidics

Hydrogel bioelectronics that can interface biological tissues and flexible electronics is at the core of the growing field of healthcare monitoring, smart drug systems, and wearable and implantable devices. Here, a simple strategy is demonstrated to prototype all‐hydrogel bioelectronics with embedded arbitrary conductive networks using tough hydrogels and liquid metal. Due to their excellent stretchability, the resultant all‐hydrogel bioelectronics exhibits stable electrochemical properties at large tensile stretch and various modes of deformation. The potential of fabricated all‐hydrogel bioelectronics is demonstrated as wearable strain sensors, cardiac patches, and near‐field communication (NFC) devices for monitoring various physiological conditions wirelessly. The presented simple platform paves the way of implantable hydrogel electronics for Internet‐of‐Things and tissue–machine interfacing applications.