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.     

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.     

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.     

Freeform,Reconfigurable Embedded Printing of All‐Aqueous 3D Architectures

Aqueous microstructures are challenging to create, handle, and preserve since their surfaces tend to shrink into spherical shapes with minimum surface areas. The creation of freeform aqueous architectures will significantly advance the bioprinting of complex tissue‐like constructs, such as arteries, urinary catheters, and tracheae. The generation of complex, freeform, three‐dimensional (3D) all‐liquid architectures using formulated aqueous two‐phase systems (ATPSs) is demonstrated. These all‐liquid microconstructs are formed by printing aqueous bioinks in an immiscible aqueous environment, which functions as a biocompatible support and pregel solution. By exploiting the hydrogen bonding interaction between polymers in ATPS, the printed aqueous‐in‐aqueous reconfigurable 3D architectures can be stabilized for weeks by the noncovalent membrane at the interface. Different cells can be separately combined with compartmentalized bioinks and matrices to obtain tailor‐designed microconstructs with perfusable vascular networks. The freeform, reconfigurable embedded printing of all‐liquid architectures by ATPSs offers unique opportunities and powerful tools since limitless formulations can be designed from among a breadth of natural and synthetic hydrophilic polymers to mimic tissues. This printing approach may be useful to engineer biomimetic, dynamic tissue‐like constructs for potential applications in drug screening, in vitro tissue models, and regenerative medicine.     

A 3D Self‐Shaping Strategy for Nanoresolution Multicomponent Architectures

3D printing or fabrication pursues the essential surface behavior manipulation of droplets or a liquid for rapidly and precisely constructing 3D multimaterial architectures. Further development of 3D fabrication desires a self‐shaping strategy that can heterogeneously integrate functional materials with disparate electrical or optical properties. Here, a 3D liquid self‐shaping strategy is reported for rapidly patterning materials over a series of compositions and accurately achieving micro‐ and nanoscale structures. The predesigned template selectively pins the droplet, and the surface energy minimization drives the self‐shaping processing. The as‐prepared 3D circuits assembled by silver nanoparticles carry a current of 208–448 µA at 0.01 V impressed voltage, while the 3D architectures achieved by two different quantum dots show noninterfering optical properties with feature resolution below 3 µm. This strategy can facilely fabricate micro‐nanogeometric patterns without a modeling program, which will be of great significance for the development of 3D functional devices.     

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.     

Responsive biomaterials for 3D bioprinting: A review

Three-dimensional (3D) bioprinting enables a controlled deposition of cells, biomaterials, and biological compounds (i.e., bioinks) to build complex 3D biological models, biological living systems, and therapeutic products. Developing responsive biomaterials as novel bioinks has been a central focus of research in the field of bioprinting because of their controllable material properties in response to printing-induced external or internal stimuli. In this review, we highlight the most recent advances of responsive biomaterials for 3D bioprinting applications. We review commonly used stimuli-responsive biomaterials and strategies for utilizing multifunctional responsiveness to achieve desirable printability, structural formability, cell viability, and construct bioactivity for 3D bioprinting. We also summarize major bioink formulation strategies currently adopted in 3D bioprinting. We subsequently discuss several promising applications of 3D printing involving responsive biomaterials, such as bioprinting in a supporting bath, 4D bioprinting, and bioprinting new controlled drug delivery systems. Future perspectives on the design and development of novel multifunctional bioinks based on responsive biomaterials and technological innovations are also presented.     

Emerging Biofabrication Strategies for Engineering Complex Tissue Constructs

The demand for organ transplantation and repair, coupled with a shortage of available donors, poses an urgent clinical need for the development of innovative treatment strategies for long‐term repair and regeneration of injured or diseased tissues and organs. Bioengineering organs, by growing patient‐derived cells in biomaterial scaffolds in the presence of pertinent physicochemical signals, provides a promising solution to meet this demand. However, recapitulating the structural and cytoarchitectural complexities of native tissues in vitro remains a significant challenge to be addressed. Through tremendous efforts over the past decade, several innovative biofabrication strategies have been developed to overcome these challenges. This review highlights recent work on emerging three‐dimensional bioprinting and textile techniques, compares the advantages and shortcomings of these approaches, outlines the use of common biomaterials and advanced hybrid scaffolds, and describes several design considerations including the structural, physical, biological, and economical parameters that are crucial for the fabrication of functional, complex, engineered tissues. Finally, the applications of these biofabrication strategies in neural, skin, connective, and muscle tissue engineering are explored.     

Solution Mask Liquid Lithography (SMaLL) for One‐Step,Multimaterial 3D Printing

A novel methodology for printing 3D objects with spatially resolved mechanical and chemical properties is reported. Photochromic molecules are used to control polymerization through coherent bleaching fronts, providing large depths of cure and rapid build rates without the need for moving parts. The coupling of these photoswitches with resin mixtures containing orthogonal photo‐crosslinking systems allows simultaneous and selective curing of multiple networks, providing access to 3D objects with chemically and mechanically distinct domains. The power of this approach is showcased through the one‐step fabrication of bioinspired soft joints and mechanically reinforced “brick‐and‐mortar” structures.     

Multiphoton Direct Laser Writing and 3D Imaging of Polymeric Freestanding Architectures for Cell Colonization

The realization of 3D architectures for the study of cell growth, proliferation, and differentiation is a task of fundamental importance for both technological and biological communities involved in the development of biomimetic cell culture environments. Here we report the fabrication of 3D freestanding scaffolds, realized by multiphoton direct laser writing and seeded with neuroblastoma cells, and their multitechnique characterization using advanced 3D fluorescence imaging approaches. The high accuracy of the fabrication process (≈200 nm) allows a much finer control of the micro‐ and nanoscale features compared to other 3D printing technologies based on fused deposition modeling, inkjet printing, selective laser sintering, or polyjet technology. Scanning electron microscopy (SEM) provides detailed insights about the morphology of both cells and cellular interconnections around the 3D architecture. On the other hand, the nature of the seeding in the inner core of the 3D scaffold, inaccessible by conventional SEM imaging, is unveiled by light sheet fluorescence microscopy and multiphoton confocal imaging highlighting an optimal cell colonization both around and within the 3D scaffold as well as the formation of long neuritic extensions. The results open appealing scenarios for the use of the developed 3D fabrication/3D imaging protocols in several neuroscientific contexts.     

Recent Advances in Enabling Technologies in 3D Printing for Precision Medicine

Advances in areas such as data analytics, genomics, and imaging have revealed individual patient complexities and exposed the inherent limitations of generic therapies for patient treatment. These observations have also fueled the development of precision medicine approaches, where therapies are tailored for the individual rather than the broad patient population. 3D printing is a field that intersects with precision medicine through the design of precision implants with patient-directed shapes, structures, and materials or for the development of patient-specific in vitro models that can be used for screening precision therapeutics. Toward their success, advances in 3D printing and biofabrication technologies are needed with enhanced resolution, complexity, reproducibility, and speed and that encompass a broad range of cells and materials. The overall goal of this progress report is to highlight recent advances in 3D printing technologies that are helping to enable advances important in precision medicine.     

A Review on the 3D Printing of Functional Structures for Medical Phantoms and Regenerated Tissue and Organ Applications

Medical models, or “phantoms,” have been widely used for medical training and for doctor-patient interactions. They are increasingly used for surgical planning, medical computational models, algorithm verification and validation, and medical devices development. Such new applications demand high-fidelity, patient-specific, tissue-mimicking medical phantoms that can not only closely emulate the geometric structures of human organs, but also possess the properties and functions of the organ structure. With the rapid advancement of three-dimensional (3D) printing and 3D bioprinting technologies, many researchers have explored the use of these additive manufacturing techniques to fabricate functional medical phantoms for various applications. This paper reviews the applications of these 3D printing and 3D bioprinting technologies for the fabrication of functional medical phantoms and bio-structures. This review specifically discusses the state of the art along with new developments and trends in 3D printed functional medical phantoms (i.e., tissue-mimicking medical phantoms, radiologically relevant medical phantoms, and physiological medical phantoms) and 3D bio-printed structures (i.e., hybrid scaffolding materials, convertible scaffolds, and integrated sensors) for regenerated tissues and organs.     

Recent Advances in Engineering Bioinks for 3D Bioprinting

The 3D bioprinting can controllably deposit bioink containing cells and fabricate complex bionic tissue structures in a fast and scalable way, which is expected to completely change the scenario of clinical organ transplantation. Bioprinting holds broad application prospect in tissue engineering, life sciences, and clinical medicine. In the process of 3D bioprinting, bioink, as the carrier of cells and bioactive substances, influences cell activity and accuracy of organ structure after printing. To better understand and design bioink, in this review, the concept, development, and basic composition of bioink are introduced, while focusing on the advantages and disadvantages of various biomaterials, and the use of common cells and biomolecules that constitute bioink. In addition, the properties and applications of various stimuli-responsive smart materials for 4D bioprinting are mentioned. The challenges and development trends of bioink are also summarized.     

Visible Light-Induced 3D Bioprinting Technologies and Corresponding Bioink Materials for Tissue Engineering: A Review

Three-dimensional (3D) bioprinting based on traditional 3D printing is an emerging technology that is used to precisely assemble biocompatible materials and cells or bioactive factors into advanced tissue engineering solutions. Similar technology, particularly photo-cured bioprinting strategies, plays an important role in the field of tissue engineering research. The successful implementation of 3D bioprinting is based on the properties of photopolymerized materials. Photocrosslinkable hydrogel is an attractive biomaterial that is polymerized rapidly and enables process control in space and time. Photopolymerization is frequently initiated by ultraviolet (UV) or visible light. However, UV light may cause cell damage and thereby, affect cell viability. Thus, visible light is considered to be more biocompatible than UV light for bioprinting. In this review, we provide an overview of photo curing-based bioprinting technologies, and describe a visible light crosslinkable bioink, including its crosslinking mechanisms, types of visible light initiator, and biomedical applications. We also discuss existing challenges and prospects of visible light-induced 3D bioprinting devices and hydrogels in biomedical areas.     

Electro‐Assisted Bioprinting of Low‐Concentration GelMA Microdroplets

Low‐concentration gelatin methacryloyl (GelMA) has excellent biocompatibility to cell‐laden structures. However, it is still a big challenge to stably fabricate organoids (even microdroplets) using this material due to its extremely low viscosity. Here, a promising electro‐assisted bioprinting method is developed, which can print low‐concentration pure GelMA microdroplets with low cost, low cell damage, and high efficiency. With the help of electrostatic attraction, uniform GelMA microdroplets measuring about 100 μm are rapidly printed. Due to the application of lower external forces to separate the droplets, cell damage during printing is negligible, which often happens in piezoelectric or thermal inkjet bioprinting. Different printing states and effects of printing parameters (voltages, gas pressure, nozzle size, etc.) on microdroplet diameter are also investigated. The fundamental properties of low‐concentration GelMA microspheres are subsequently studied. The results show that the printed microspheres with 5% w/v GelMA can provide a suitable microenvironment for laden bone marrow stem cells. Finally, it is demonstrated that the printed microdroplets can be used in building microspheroidal organoids, in drug controlled release, and in 3D bioprinting as biobricks. This method shows great potential use in cell therapy, drug delivery, and organoid building.     

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.     

3D Printing of Ultralight Biomimetic Hierarchical Graphene Materials with Exceptional Stiffness and Resilience

Biological materials with hierarchical architectures (e.g., a macroscopic hollow structure and a microscopic cellular structure) offer unique inspiration for designing and manufacturing advanced biomimetic materials with outstanding mechanical performance and low density. Most conventional biomimetic materials only benefit from bioinspired architecture at a single length scale (e.g., microscopic material structure), which largely limits the mechanical performance of the resulting materials. There exists great potential to maxime the mechanical performance of biomimetic materials by leveraging a bioinspired hierarchical structure. An ink‐based three‐dimensional (3D) printing strategy to manufacture an ultralight biomimetic hierarchical graphene material (BHGMs) with exceptionally high stiffness and resilience is demonstrated. By simultaneously engineering 3D‐printed macroscopic hollow structures and constructing an ice‐crystal‐induced cellular microstructure, BHGMs can achieve ultrahigh elasticity and stability at compressive strains up to 95%. Multiscale finite element analyses indicate that the hierarchical structures of BHGMs effectively reduce the macroscopic strain and transform the microscopic compressive deformation into the rotation and bending of the interconnected graphene flakes. This 3D printing strategy demonstrates the great potential that exists for the assembly of other functional materials into hierarchical cellular structures for various applications where high stiffness and resilience at low density are simultaneously required.     

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.     

Recent Progress in Biomimetic Additive Manufacturing Technology: From Materials to Functional Structures

Nature has developed high‐performance materials and structures over millions of years of evolution and provides valuable sources of inspiration for the design of next‐generation structural materials, given the variety of excellent mechanical, hydrodynamic, optical, and electrical properties. Biomimicry, by learning from nature's concepts and design principles, is driving a paradigm shift in modern materials science and technology. However, the complicated structural architectures in nature far exceed the capability of traditional design and fabrication technologies, which hinders the progress of biomimetic study and its usage in engineering systems. Additive manufacturing (three‐dimensional (3D) printing) has created new opportunities for manipulating and mimicking the intrinsically multiscale, multimaterial, and multifunctional structures in nature. Here, an overview of recent developments in 3D printing of biomimetic reinforced mechanics, shape changing, and hydrodynamic structures, as well as optical and electrical devices is provided. The inspirations are from various creatures such as nacre, lobster claw, pine cone, flowers, octopus, butterfly wing, fly eye, etc., and various 3D‐printing technologies are discussed. Future opportunities for the development of biomimetic 3D‐printing technology to fabricate next‐generation functional materials and structures in mechanical, electrical, optical, and biomedical engineering are also outlined.