From Shape to Function: The Next Step in Bioprinting

In 2013, the “biofabrication window” was introduced to reflect the processing challenge for the fields of biofabrication and bioprinting. At that time, the lack of printable materials that could serve as cell-laden bioinks, as well as the limitations of printing and assembly methods, presented a major constraint. However, recent developments have now resulted in the availability of a plethora of bioinks, new printing approaches, and the technological advancement of established techniques. Nevertheless, it remains largely unknown which materials and technical parameters are essential for the fabrication of intrinsically hierarchical cell–material constructs that truly mimic biologically functional tissue. In order to achieve this, it is urged that the field now shift its focus from materials and technologies toward the biological development of the resulting constructs. Therefore, herein, the recent material and technological advances since the introduction of the biofabrication window are briefly summarized, i.e., approaches how to generate shape, to then focus the discussion on how to acquire the biological function within this context. In particular, a vision of how biological function can evolve from the possibility to determine shape is outlined.     

Microfluidics‐Enabled Multimaterial Maskless Stereolithographic Bioprinting

A stereolithography‐based bioprinting platform for multimaterial fabrication of heterogeneous hydrogel constructs is presented. Dynamic patterning by a digital micromirror device, synchronized by a moving stage and a microfluidic device containing four on/off pneumatic valves, is used to create 3D constructs. The novel microfluidic device is capable of fast switching between different (cell‐loaded) hydrogel bioinks, to achieve layer‐by‐layer multimaterial bioprinting. Compared to conventional stereolithography‐based bioprinters, the system provides the unique advantage of multimaterial fabrication capability at high spatial resolution. To demonstrate the multimaterial capacity of this system, a variety of hydrogel constructs are generated, including those based on poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacryloyl (GelMA). The biocompatibility of this system is validated by introducing cell‐laden GelMA into the microfluidic device and fabricating cellularized constructs. A pattern of a PEGDA frame and three different concentrations of GelMA, loaded with vascular endothelial growth factor, are further assessed for its neovascularization potential in a rat model. The proposed system provides a robust platform for bioprinting of high‐fidelity multimaterial microstructures on demand for applications in tissue engineering, regenerative medicine, and biosensing, which are otherwise not readily achievable at high speed with conventional stereolithographic biofabrication platforms.     

Reduced Graphene Oxide‐GelMA Hybrid Hydrogels as Scaffolds for Cardiac Tissue Engineering

Biomaterials currently used in cardiac tissue engineering have certain limitations, such as lack of electrical conductivity and appropriate mechanical properties, which are two parameters playing a key role in regulating cardiac cell behavior. Here, the myocardial tissue constructs are engineered based on reduced graphene oxide (rGO)‐incorporated gelatin methacryloyl (GelMA) hybrid hydrogels. The incorporation of rGO into the GelMA matrix significantly enhances the electrical conductivity and mechanical properties of the material. Moreover, cells cultured on composite rGO‐GelMA scaffolds exhibit better biological activities such as cell viability, proliferation, and maturation compared to ones cultured on GelMA hydrogels. Cardiomyocytes show stronger contractility and faster spontaneous beating rate on rGO‐GelMA hydrogel sheets compared to those on pristine GelMA hydrogels, as well as GO‐GelMA hydrogel sheets with similar mechanical property and particle concentration. Our strategy of integrating rGO within a biocompatible hydrogel is expected to be broadly applicable for future biomaterial designs to improve tissue engineering outcomes. The engineered cardiac tissue constructs using rGO incorporated hybrid hydrogels can potentially provide high‐fidelity tissue models for drug studies and the investigations of cardiac tissue development and/or disease processes in vitro.     

Thiol–Ene Clickable Gelatin: A Platform Bioink for Multiple 3D Biofabrication Technologies

Bioprinting can be defined as the art of combining materials and cells to fabricate designed, hierarchical 3D hybrid constructs. Suitable materials, so called bioinks, have to comply with challenging rheological processing demands and rapidly form a stable hydrogel postprinting in a cytocompatible manner. Gelatin is often adopted for this purpose, usually modified with (meth‐)acryloyl functionalities for postfabrication curing by free radical photopolymerization, resulting in a hydrogel that is cross‐linked via nondegradable polymer chains of uncontrolled length. The application of allylated gelatin (GelAGE) as a thiol–ene clickable bioink for distinct biofabrication applications is reported. Curing of this system occurs via dimerization and yields a network with flexible properties that offer a wider biofabrication window than (meth‐)acryloyl chemistry, and without additional nondegradable components. An in‐depth analysis of GelAGE synthesis is conducted, and standard UV‐initiation is further compared with a recently described visible‐light‐initiator system for GelAGE hydrogel formation. It is demonstrated that GelAGE may serve as a platform bioink for several biofabrication technologies by fabricating constructs with high shape fidelity via lithography‐based (digital light processing) 3D printing and extrusion‐based 3D bioprinting, the latter supporting long‐term viability postprinting of encapsulated chondrocytes.     

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.     

Adaptable Hydrogel Networks with Reversible Linkages for Tissue Engineering

Adaptable hydrogels have recently emerged as a promising platform for three‐dimensional (3D) cell encapsulation and culture. In conventional, covalently crosslinked hydrogels, degradation is typically required to allow complex cellular functions to occur, leading to bulk material degradation. In contrast, adaptable hydrogels are formed by reversible crosslinks. Through breaking and re‐formation of the reversible linkages, adaptable hydrogels can be locally modified to permit complex cellular functions while maintaining their long‐term integrity. In addition, these adaptable materials can have biomimetic viscoelastic properties that make them well suited for several biotechnology and medical applications. In this review, an overview of adaptable‐hydrogel design considerations and linkage selections is presented, with a focus on various cell‐compatible crosslinking mechanisms that can be exploited to form adaptable hydrogels for tissue engineering.     

3D Bioprinting: from Benches to Translational Applications

Over the last decades, the fabrication of 3D tissues has become commonplace in tissue engineering and regenerative medicine. However, conventional 3D biofabrication techniques such as scaffolding, microengineering, and fiber and cell sheet engineering are limited in their capacity to fabricate complex tissue constructs with the required precision and controllability that is needed to replicate biologically relevant tissues. To this end, 3D bioprinting offers great versatility to fabricate biomimetic, volumetric tissues that are structurally and functionally relevant. It enables precise control of the composition, spatial distribution, and architecture of resulting constructs facilitating the recapitulation of the delicate shapes and structures of targeted organs and tissues. This Review systematically covers the history of bioprinting and the most recent advances in instrumentation and methods. It then focuses on the requirements for bioinks and cells to achieve optimal fabrication of biomimetic constructs. Next, emerging evolutions and future directions of bioprinting are discussed, such as freeform, high‐resolution, multimaterial, and 4D bioprinting. Finally, the translational potential of bioprinting and bioprinted tissues of various categories are presented and the Review is concluded by exemplifying commercially available bioprinting platforms.     

4D Biofabrication Using Shape‐Morphing Hydrogels

Despite the tremendous potential of bioprinting techniques toward the fabrication of highly complex biological structures and the flourishing progress in 3D bioprinting, the most critical challenge of the current approaches is the printing of hollow tubular structures. In this work, an advanced 4D biofabrication approach, based on printing of shape‐morphing biopolymer hydrogels, is developed for the fabrication of hollow self‐folding tubes with unprecedented control over their diameters and architectures at high resolution. The versatility of the approach is demonstrated by employing two different biopolymers (alginate and hyaluronic acid) and mouse bone marrow stromal cells. Harnessing the printing and postprinting parameters allows attaining average internal tube diameters as low as 20 µm, which is not yet achievable by other existing bioprinting/biofabrication approaches and is comparable to the diameters of the smallest blood vessels. The proposed 4D biofabrication process does not pose any negative effect on the viability of the printed cells, and the self‐folded hydrogel‐based tubes support cell survival for at least 7 d without any decrease in cell viability. Consequently, the presented 4D biofabrication strategy allows the production of dynamically reconfigurable architectures with tunable functionality and responsiveness, governed by the selection of suitable materials and cells.     

Hydrogel Bioink Reinforcement for Additive Manufacturing: A Focused Review of Emerging Strategies

Bioprinting is an emerging approach for fabricating cell-laden 3D scaffolds via robotic deposition of cells and biomaterials into custom shapes and patterns to replicate complex tissue architectures. Bioprinting uses hydrogel solutions called bioinks as both cell carriers and structural components, requiring bioinks to be highly printable while providing a robust and cell-friendly microenvironment. Unfortunately, conventional hydrogel bioinks have not been able to meet these requirements and are mechanically weak due to their heterogeneously crosslinked networks and lack of energy dissipation mechanisms. Advanced bioink designs using various methods of dissipating mechanical energy are aimed at developing next-generation cellularized 3D scaffolds to mimic anatomical size, tissue architecture, and tissue-specific functions. These next-generation bioinks need to have high print fidelity and should provide a biocompatible microenvironment along with improved mechanical properties. To design these advanced bioink formulations, it is important to understand the structure–property–function relationships of hydrogel networks. By specifically leveraging biophysical and biochemical characteristics of hydrogel networks, high performance bioinks can be designed to control and direct cell functions. In this review article, current and emerging approaches in hydrogel design and bioink reinforcement techniques are critically evaluated. This bottom-up perspective provides a materials-centric approach to bioink design for 3D bioprinting.     

Emerging Trends in Information‐Driven Engineering of Complex Biological Systems

Synthetic biological systems are used for a myriad of applications, including tissue engineered constructs for in vivo use and microengineered devices for in vitro testing. Recent advances in engineering complex biological systems have been fueled by opportunities arising from the combination of bioinspired materials with biological and computational tools. Driven by the availability of large datasets in the “omics” era of biology, the design of the next generation of tissue equivalents will have to integrate information from single‐cell behavior to whole organ architecture. Herein, recent trends in combining multiscale processes to enable the design of the next generation of biomaterials are discussed. Any successful microprocessing pipeline must be able to integrate hierarchical sets of information to capture key aspects of functional tissue equivalents. Micro‐ and biofabrication techniques that facilitate hierarchical control as well as emerging polymer candidates used in these technologies are also reviewed.     

Design,physical prototyping and initial characterisation of ‘lockyballs’

Directed tissue self-assembly or bottom-up modular approach in tissue biofabrication is an attractive and potentially superior alternative to a classic top-down solid scaffold-based approach in tissue engineering. For example, rapidly emerging organ printing technology using self-assembling tissue spheroids as building blocks is enabling computer-aided robotic bioprinting of three-dimensional (3D) tissue constructs. However, achieving proper material properties while maintaining desirable geometry and shape of 3D bioprinted tissue engineered constructs using directed tissue self-assembly, is still a challenge. Proponents of directed tissue self-assembly see the solution of this problem in developing methods of accelerated tissue maturation and/or using sacrificial temporal supporting of removable hydrogels. In the meantime, there is a growing consensus that a third strategy based on the integration of a directed tissue self-assembly approach with a conventional solid scaffold-based approach could be a potential optimal solution. We hypothesise that tissue spheroids with ‘velcro®-like’ interlockable solid microscaffolds or simply ‘lockyballs’ could enable the rapid in vivo biofabrication of 3D tissue constructs at desirable material properties and high initial cell density. Recently, biocompatible and biodegradable photo-sensitive biomaterials could be fabricated at nanoscale resolution using two-photon polymerisation (2PP), a development rendering this technique with high potential to fabricate ‘velcro®-like’ interlockable microscaffolds. Here we report design studies, physical prototyping using 2PP and initial functional characterisation of interlockable solid microscaffolds or so-called ‘lockyballs’. 2PP was used as a novel enabling platform technology for rapid bottom-up modular tissue biofabrication of interlockable constructs. The principle of lockable tissue spheroids fabricated using the described lockyballs as solid microscaffolds is characterised by attractive new functionalities such as lockability and tunable material properties of the engineered constructs. It is reasonable to predict that these building blocks create the basis for a development of a clinical in vivo rapid biofabrication approach and form part of recent promising emerging bioprinting technologies.     

Out‐of‐Plane 3D‐Printed Microfibers Improve the Shear Properties of Hydrogel Composites

One challenge in biofabrication is to fabricate a matrix that is soft enough to elicit optimal cell behavior while possessing the strength required to withstand the mechanical load that the matrix is subjected to once implanted in the body. Here, melt electrowriting (MEW) is used to direct‐write poly(ε‐caprolactone) fibers “out‐of‐plane” by design. These out‐of‐plane fibers are specifically intended to stabilize an existing structure and subsequently improve the shear modulus of hydrogel–fiber composites. The stabilizing fibers (diameter = 13.3 ± 0.3 µm) are sinusoidally direct‐written over an existing MEW wall‐like structure (330 µm height). The printed constructs are embedded in different hydrogels (5, 10, and 15 wt% polyacrylamide; 65% poly(2‐hydroxyethyl methacrylate) (pHEMA)) and a frequency sweep test (0.05–500 rad s^?1, 0.01% strain, n = 5) is performed to measure the complex shear modulus. For the rheological measurements, stabilizing fibers are deposited with a radial‐architecture prior to embedding to correspond to the direction of the stabilizing fibers with the loading of the rheometer. Stabilizing fibers increase the complex shear modulus irrespective of the percentage of gel or crosslinking density. The capacity of MEW to produce well‐defined out‐of‐plane fibers and the ability to increase the shear properties of fiber‐reinforced hydrogel composites are highlighted.     

Writing with Fluid: Structuring Hydrogels with Micrometer Precision by AFM in Combination with Nanofluidics

Hydrogels have many applications in biomedical surface modification and tissue engineering. However, the structuring of hydrogels after their formation represents still a major challenge, in particular due to their softness. Here, a novel approach is presented that is based on the combination of atomic force microscopy (AFM) and nanofluidics, also referred to as FluidFM technology. Its applicability is demonstrated for supramolecular hydrogel films that are prepared from low‐molecular weight hydrogelators, such as derivates of 1,3,5‐benzene tricarboxamides (BTAs). BTA films can be dissolved selectively by ejecting alkaline solution through the aperture of a hollow AFM‐cantilever connected to a nanofluidic controller. The AFM‐based force control is essential in preventing mechanical destruction of the hydrogels. The resulting “chemical writing” process is studied in detail and the influence of various parameters, such as applied pressure and time, is validated. It is demonstrated that the achievable structuring precision is primarily limited by diffusion and the aperture dimensions. Recently, various additive techniques have been presented to pattern hydrogels. The here‐presented subtractive approach can not only be applied to structure hydrogels from the large class of reversibly formed gels with superior resolution but would also allow for the selective loading of the hydrogels with active substances or nanoparticles.     

25th Anniversary Article: Rational Design and Applications of Hydrogels in Regenerative Medicine

Hydrogels are hydrophilic polymer‐based materials with high water content and physical characteristics that resemble the native extracellular matrix. Because of their remarkable properties, hydrogel systems are used for a wide range of biomedical applications, such as three‐dimensional (3D) matrices for tissue engineering, drug‐delivery vehicles, composite biomaterials, and as injectable fillers in minimally invasive surgeries. In addition, the rational design of hydrogels with controlled physical and biological properties can be used to modulate cellular functionality and tissue morphogenesis. Here, the development of advanced hydrogels with tunable physiochemical properties is highlighted, with particular emphasis on elastomeric, light‐sensitive, composite, and shape‐memory hydrogels. Emerging technologies developed over the past decade to control hydrogel architecture are also discussed and a number of potential applications and challenges in the utilization of hydrogels in regenerative medicine are reviewed. It is anticipated that the continued development of sophisticated hydrogels will result in clinical applications that will improve patient care and quality of life.     

Molding Micropatterns of Elasticity on PEG‐Based Hydrogels to Control Cell Adhesion and Migration

We present an innovative and simple, soft UV lithographic method “FIll‐Molding In Capillaries” (FIMIC) that combines soft lithography with capillary force driven filling of micro‐channels to create smooth hydrogel substrates with a 2D micro‐pattern on the surface. The lithographic procedure involves the molding of a polymer; in our case a bulk PEG‐based hydrogel, via UV‐curing from a microfabricated silicon master. The grooves of the created regular line pattern are consequently filled with a second hydrogel by capillary action. As a result, a smooth surface is obtained with a well‐defined pattern design of the two different polymers on its surface. The FIMIC method is very versatile; the only prerequisite is that the second material is liquid before curing in order to enable the filling process. In this specific case we present the proof of principle of this method by applying two hydrogels which differ in their crosslinking density and therefore in their elasticity. Preliminary cell culture studies on the fabricated elasticity patterned hydrogels indicate the preferred adhesion of the cells to the stiffer regions of the substrates, which implies that the novel substrates are a very useful platform for systematic cell migration studies, e.g. more fundamental investigation of the concept of “durotaxis”.     

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.     

Amino Acids and Peptide‐Based Supramolecular Hydrogels for Three‐Dimensional Cell Culture

Supramolecular hydrogels assembled from amino acids and peptide‐derived hydrogelators have shown great potential as biomimetic three‐dimensional (3D) extracellular matrices because of their merits over conventional polymeric hydrogels, such as non‐covalent or physical interactions, controllable self‐assembly, and biocompatibility. These merits enable hydrogels to be made not only by using external stimuli, but also under physiological conditions by rationally designing gelator structures, as well as in situ encapsulation of cells into hydrogels for 3D culture. This review will assess current progress in the preparation of amino acids and peptide‐based hydrogels under various kinds of external stimuli, and in situ encapsulation of cells into the hydrogels, with a focus on understanding the associations between their structures, properties, and functions during cell culture, and the remaining challenges in this field. The amino acids and peptide‐based hydrogelators with rationally designed structures have promising applications in the fields of regenerative medicine, tissue engineering, and pre‐clinical evaluation.     

Rapid biofabrication of tubular tissue constructs by centrifugal casting in a decellularized natural scaffold with laser-machined micropores

Centrifugal casting allows rapid biofabrication of tubular tissue constructs by suspending living cells in an in situ cross-linkable hydrogel. We hypothesize that introduction of laser-machined micropores into a decellularized natural scaffold will facilitate cell seeding by centrifugal casting and increase hydrogel retention, without compromising the biomechanical properties of the scaffold. Micropores with diameters of 50, 100, and 200 mum were machined at different linear densities in decellularized small intestine submucosa (SIS) planar sheets and tubular SIS scaffolds using an argon laser. The ultimate stress and ultimate strain values for SIS sheets with laser-machined micropores with diameter 50 mum and distance between holes as low as 714 mum were not significantly different from unmachined control SIS specimens. Centrifugal casting of GFP-labeled cells suspended in an in situ cross-linkable hyaluronan-based hydrogel resulted in scaffold recellularization with a high density of viable cells inside the laser-machined micropores. Perfusion tests demonstrated the retention of the cells encapsulated within the HA hydrogel in the microholes. Thus, an SIS scaffold with appropriately sized microholes can be loaded with hydrogel encapsulated cells by centrifugal casting to give a mechanically robust construct that retains the cell-seeded hydrogel, permitting rapid biofabrication of tubular tissue construct in a "bioreactor-free" fashion.     

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.     

3D Photo-Fabrication for Tissue Engineering and Drug Delivery

The most promising strategies in tissue engineering involve the integration of a triad of biomaterials, living cells, and biologically active molecules to engineer synthetic environments that closely mimic the healing milieu present in human tissues, and that stimulate tissue repair and regeneration. To be clinically effective, these environments must replicate, as closely as possible, the main characteristics of the native extracellular matrix(ECM) on a cellular and subcellular scale. Photo-fabrication techniques have already been used to generate 3D environments with precise architectures and heterogeneous composition, through a multi-layer procedure involving the selective photocrosslinking reaction of a light-sensitive prepolymer. Cells and therapeutic molecules can be included in the initial hydrogel precursor solution, and processed into 3D constructs. Recently, photofabrication has also been explored to dynamically modulate hydrogel features in real time, providing enhanced control of cell fate and delivery of bioactive compounds. This paper focuses on the use of 3D photo-fabrication techniques to produce advanced constructs for tissue regeneration and drug delivery applications. State-of-the-art photo-fabrication techniques are described, with emphasis on the operating principles and biofabrication strategies to create spatially controlled patterns of cells and bioactive factors. Considering its fast processing, spatiotemporal control, high resolution, and accuracy, photo-fabrication is assuming a critical role in the design of sophisticated 3D constructs. This technology is capable of providing appropriate environments for tissue regeneration, and regulating the spatiotemporal delivery of therapeutics.