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.     

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.     

Human-Recombinant-Elastin-Based Bioinks for 3D Bioprinting of Vascularized Soft Tissues

Bioprinting has emerged as an advanced method for fabricating complex 3D tissues. Despite the tremendous potential of 3D bioprinting, there are several drawbacks of current bioinks and printing methodologies that limit  the ability to print elastic and highly vascularized tissues. In particular, fabrication of complex biomimetic structure that are entirely based on 3D bioprinting is still challenging primarily due to the lack of suitable bioinks with high printability, biocompatibility, biomimicry, and proper mechanical properties. To address these shortcomings, in this work the use of recombinant human tropoelastin as a highly biocompatible and elastic bioink for 3D printing of complex soft tissues is demonstrated. As proof of the concept, vascularized cardiac constructs are bioprinted and their functions are assessed in vitro and in vivo. The printed constructs demonstrate endothelium barrier function and spontaneous beating of cardiac muscle cells, which are important functions of cardiac tissue in vivo. Furthermore, the printed construct elicits minimal inflammatory responses, and is shown to be efficiently biodegraded in vivo when implanted subcutaneously in rats. Taken together, these results demonstrate the potential of the elastic bioink for printing 3D functional cardiac tissues, which can eventually be used for cardiac tissue replacement.     

Bioprinting of Multiscaled Hepatic Lobules within a Highly Vascularized Construct

Highly vascularized complex liver tissue is generally divided into lobes, lobules, hepatocytes, and sinusoids, which can be viewed under different types of lens from the micro‐ to macro‐scale. To engineer multiscaled heterogeneous tissues, a sophisticated and rapid tissue engineering approach is required, such as advanced 3D bioprinting. In this study, a preset extrusion bioprinting technique, which can create heterogeneous, multicellular, and multimaterial structures simultaneously, is utilized for creating a hepatic lobule (≈1 mm) array. The fabricated hepatic lobules include hepatic cells, endothelial cells, and a lumen. The endothelial cells surround the hepatic cells, the exterior of the lobules, the lumen, and finally, become interconnected with each other. Compared to hepatic cell/endothelial cell mixtures, the fabricated hepatic lobule shows higher albumin secretion, urea production, and albumin, MRP2, and CD31 protein levels, as well as, cytochrome P450 enzyme activity. It is found that each cell type with spatial cell patterning in bioink accelerates cellular organization, which could preserve structural integrity and improve cellular functions. In conclusion, preset extruded hepatic lobules within a highly vascularized construct are successfully constructed, enabling both micro‐ and macro‐scale tissue fabrication, which can support the creation of large 3D tissue constructs for multiscale tissue engineering.     

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.     

Airflow‐Assisted 3D Bioprinting of Human Heterogeneous Microspheroidal Organoids with Microfluidic Nozzle

Hydrogel microspheroids are widely used in tissue engineering, such as injection therapy and 3D cell culture, and among which, heterogeneous microspheroids are drawing much attention as a promising tool to carry multiple cell types in separated phases. However, it is still a big challenge to fabricate heterogeneous microspheroids that can reconstruct built‐up tissues' microarchitecture with excellent resolution and spatial organization in limited sizes. Here, a novel airflow‐assisted 3D bioprinting method is reported, which can print versatile spiral microarchitectures inside the microspheroids, permitting one‐step bioprinting of fascinating hydrogel structures, such as the spherical helix, rose, and saddle. A microfluidic nozzle is developed to improve the capability of intricate cell encapsulation with heterotypic contact. Complex structures, such as a rose, Tai chi pattern, and single cell line can be easily printed in spheroids. The theoretical model during printing is established and process parameters are systematically investigated. As a demonstration, a human multicellular organoid of spirally vascularized ossification is reconstructed with this method, which shows that it is a powerful tool to build mini tissues on microspheroids.     

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.     

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.     

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.     

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.     

High‐Throughput Fabrication and Modular Assembly of 3D Heterogeneous Microscale Tissues

3D hydrogel microstructures that encapsulate cells have been used in broad applications in microscale tissue engineering, personalized drug screening, and regenerative medicine. Recent technological advances in microstructure assembly, such as bioprinting, magnetic assembly, microfluidics, and acoustics, have enabled the construction of designed 3D tissue structures with spatially organized cells in vitro. However, a bottleneck exists that still hampers the application of microtissue structures, due to a lack of techniques that combined high‐throughput fabrication and flexible assembly. Here, a versatile method for fabricating customized microstructures and reorganizing building blocks composed of functional components into a combined single geometric shape is demonstrated. The arbitrary microstructures are dynamically synthesized in a microfluidic device and then transferred to an optically induced electrokinetics chip for manipulation and assembly. Moreover, building blocks containing different cells can be arranged into a desired geometry with specific shape and size, which can be used for microscale tissue engineering.     

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.     

Designer ‘blueprint’ for vascular trees: morphology evolution of vascular tissue constructs

Organ printing is a variant of the biomedical application of rapid prototyping technology or layer-by-layer additive biofabrication of 3D tissue and organ constructs using self-assembled tissue spheroids as building blocks. Bioengineering of perfusable intraorgan branched vascular trees incorporated into 3D tissue constructs is essential for the survival of bioprinted thick 3D tissues and organs. In order to design the optimal ‘blueprint’ for digital bioprinting of intraorgan branched vascular trees, the coefficients of tissue retraction associated with post-printing vascular tissue spheroid fusion and remodelling must be determined and incorporated into the original CAD. Using living tissue spheroids assembled into ring-like and tube-like vascular tissue constructs, the coefficient of tissue retraction has been experimentally evaluated. It has been shown that the internal diameter of ring-like and the height of tubular-like tissue constructs are significantly reduced during tissue spheroid fusion. During the tissue fusion process, the individual tissue spheroids also change their shape from ball-like to a conus-like form. A simple formula for the calculation of the necessary number of tissue spheroids for biofabrication of ring-like structures of desirable diameter has been deduced. These data provide sufficient information to design optimal CAD for bioprinted branched vascular trees of desirable final geometry and size.     

Effective Light Directed Assembly of Building Blocks with Microscale Control

Light‐directed forces have been widely used to pattern micro/nanoscale objects with precise control, forming functional assemblies. However, a substantial laser intensity is required to generate sufficient optical gradient forces to move a small object in a certain direction, causing limited throughput for applications. A high‐throughput light‐directed assembly is demonstrated as a printing technology by introducing gold nanorods to induce thermal convection flows that move microparticles (diameter = 40 µm to several hundreds of micrometers) to specific light‐guided locations, forming desired patterns. With the advantage of effective light‐directed assembly, the microfluidic‐fabricated monodispersed biocompatible microparticles are used as building blocks to construct a structured assembly (≈10 cm scale) in ≈2 min. The control with microscale precision is approached by changing the size of the laser light spot. After crosslinking assembly of building blocks, a novel soft material with wanted pattern is approached. To demonstrate its application, the mesenchymal stem‐cell‐seeded hydrogel microparticles are prepared as functional building blocks to construct scaffold‐free tissues with desired structures. This light‐directed fabrication method can be applied to integrate different building units, enabling the bottom‐up formation of materials with precise control over their internal structure for bioprinting, tissue engineering, and advanced manufacturing.     

Capillary Origami Inspired Fabrication of Complex 3D Hydrogel Constructs

Hydrogels have found broad applications in various engineering and biomedical fields, where the shape and size of hydrogels can profoundly influence their functions. Although numerous methods have been developed to tailor 3D hydrogel structures, it is still challenging to fabricate complex 3D hydrogel constructs. Inspired by the capillary origami phenomenon where surface tension of a droplet on an elastic membrane can induce spontaneous folding of the membrane into 3D structures along with droplet evaporation, a facile strategy is established for the fabrication of complex 3D hydrogel constructs with programmable shapes and sizes by crosslinking hydrogels during the folding process. A mathematical model is further proposed to predict the temporal structure evolution of the folded 3D hydrogel constructs. Using this model, precise control is achieved over the 3D shapes (e.g., pyramid, pentahedron, and cube) and sizes (ranging from hundreds of micrometers to millimeters) through tuning membrane shape, dimensionless parameter of the process (elastocapillary number C_e), and evaporation time. This work would be favorable to multiple areas, such as flexible electronics, tissue regeneration, and drug delivery.     

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.     

Fabrication of hierarchical polycaprolactone/gel scaffolds via combined 3D bioprinting and electrospinning for tissue engineering

It is a severe challenge to construct 3D scaffolds which hold controllable pore structure and similar morphology of the natural extracellular matrix(ECM).In this study,a compound technology is proposed by combining the 3D bioprinting and electrospinning process to fabricate 3D scaffolds,which are composed by orthogonal array gel microfibers in a grid-like arrangement and intercalated by a nonwoven structure with randomly distributed polycaprolactone(PCL) nanofibers.Human adiposederived stem cells(hASCs) are seeded on the hierarchical scaffold and cultured 21 d for in vitro study.The results of cells culturing show that the microfibers structure with controlled pores can allow the easy entrance of cells and the efficient diffusion of nutrients,and the nanofiber webs layered in the scaffold can significantly improve initial cell attachment and proliferation.The present work demonstrates that the hierarchical PCL/gel scaffolds consisting of controllable 3D architecture with interconnected pores and biomimetic nanofiber structures resembling the ECM can be designed and fabricated by the combination of 3D bioprinting and electrospinning to improve biological performance in tissue engineering applications.     

The Next Frontier of 3D Bioprinting: Bioactive Materials Functionalized by Bacteria

3D bioprinting has become a flexible technical means used in many fields. Currently, research on 3D bioprinting is mainly focused on the use of mammalian cells to print organ and tissue models, which has greatly promoted progress in the fields of tissue engineering, regenerative medicine, and pharmaceuticals. In recent years, bacterial bioprinting has gradually become a rapidly developing research fields, with a wide range of potential applications in basic research, biomedicine, bioremediation, and other field. Here, this works reviews new research on bacterial bioprinting, and discuss its future research direction.     

Universal Nanocarrier Ink Platform for Biomaterials Additive Manufacturing

Ink engineering is a fundamental area of research within additive manufacturing (AM) that designs next‐generation biomaterials tailored for additive processes. During the design of new inks, specific requirements must be considered, such as flowability, postfabrication stability, biointegration, and controlled release of therapeutic molecules. To date, many (bio)inks have been developed; however, few are sufficiently versatile to address a broad range of applications. In this work, a universal nanocarrier ink platform is presented that provides tailored rheology for extrusion‐based AM and facilitates the formulation of biofunctional inks. The universal nanocarrier ink (UNI) leverages reversible polymer–nanoparticle interactions to form a transient physical network with shear‐thinning and self‐healing properties engineered for direct ink writing (DIW). The unique advantage of the material is that a range of functional secondary polymers can be combined with the UNI to enable stabilization of printed constructs via secondary cross‐linking as well as customized biofunctionality for tissue engineering and drug delivery applications. Specific UNI formulations are used for bioprinting of living tissue constructs and DIW of controlled release devices. The robust and versatile nature of the UNI platform enables rapid formulation of a broad range of functional inks for AM of advanced biomaterials.