Organ-Level Functional 3D Tissue Constructs with Complex Compartments and their Preclinical Applications

There is an increasing interest in organ-level 3D tissue constructs, owing to their mirroring of in vivo-like features. This has resulted in a wide range of preclinical applications to obtain cell- or tissue-specific responses. Additionally, the development and improvement of sophisticated technologies, such as organoid generation, microfluidics, hydrogel engineering, and 3D printing, have enhanced 3D tissue constructs to become more elaborate. In particular, recent studies have focused on including complex compartments, i.e., vascular and innervation structured 3D tissue constructs, which mimic the nature of the human body in that all tissues/organs are interconnected and physiological phenomena are mediated through vascular and neural systems. Here, the strategies are categorized according to the number of dimensions (0D, 1D, 2D, and 3D) of the starting materials for scaling up, and novel approaches to introduce increased complexity in 3D tissue constructs are highlighted. Recent advances in preclinical applications are also investigated to gain insight into the future direction of 3D tissue construct research. Overcoming the challenges in improving organ-level functional 3D tissue constructs both in vitro and in vivo will ultimately become a life-saving tool in the biomedical field.     

2D to 3D Manipulation and Assembly of Microstructures Using Optothermally Generated Surface Bubble Microrobots

Hydrogel microstructures that encapsulate cells can be assembled into tissues and have broad applications in biology and medicine. However, 3D posture control for a single arbitrary microstructure remains a challenge. A novel 3D manipulation and assembly technique based on optothermally generated bubble robots is proposed. The generation, rate of growth, and motion of a microbubble robot can be controlled by modulating the power of a laser focused on the interface between the substrate and a fluid. In addition to 2D operations, bubble robots are able to perform 3D manipulations. The 3D properties of hydrogel microstructures are adjusted arbitrarily, and convex and concave structures with different heights are designed. Furthermore, annular micromodules are assembled into 3D constructs, including tubular and concentric constructs. A variety of hydrogel microstructures of different sizes and shapes are operated and assembled in both 2D and 3D conformations by bubble robots. The manipulation and assembly methods are simple, rapid, versatile, and can be used for fabricating tissue constructs.     

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.     

Associative Liquid-In-Liquid 3D Printing Techniques for Freeform Fabrication of Soft Matter

Shaping soft materials into prescribed 3D complex designs has been challenging yet feasible using various 3D printing technologies. For a broader range of soft matters to be printable, liquid-in-liquid 3D printing techniques have emerged in which an ink phase is printed into 3D constructs within a bath. Most of the attention in this field has been focused on using a support bath with favorable rheology (i.e., shear-thinning behavior) which limits the selection of materials, impeding the broad application of such techniques. However, a growing body of work has begun to leverage the interaction or association of the two involved phases (specifically at the liquid–liquid interface) to fabricate complex constructs from a myriad of soft materials with practical structural, mechanical, optical, magnetic, and communicative properties. This review article has provided an overview of the studies on such associative liquid-in-liquid 3D printing techniques along with their fundamentals, underlying mechanisms, various characterization techniques used for ensuring the structural stability, and practical properties of prints. Also, the future paths with the potential applications are discussed.     

3D Microstructures of Liquid Crystal Networks with Programmed Voxelated Director Fields

The shape-shifting behavior of liquid crystal networks (LCNs) and elastomers (LCEs) is a result of an interplay between their initial geometrical shape and their molecular alignment. For years, reliance on either one-step in situ or two-step film processing techniques has limited the shape-change transformations from 2D to 3D geometries. The combination of various fabrication techniques, alignment methods, and chemical formulations developed in recent years has introduced new opportunities to achieve 3D-to-3D shape-transformations in large scales, albeit the precise control of local molecular alignment in microscale 3D constructs remains a challenge. Here, the voxel-by-voxel encoding of nematic alignment in 3D microstructures of LCNs produced by two-photon polymerization using high-resolution topographical features is demonstrated. 3D LCN microstructures (suspended films, coils, and rings) with designable 2D and 3D director fields with a resolution of 5 µm are achieved. Different shape transformations of LCN microstructures with the same geometry but dissimilar molecular alignments upon actuation are elicited. This strategy offers higher freedom in the shape-change programming of 3D LCN microstructures and expands their applicability in emerging technologies, such as small-scale soft robots and devices and responsive surfaces.     

A biomimetic three-dimensional woven composite scaffold for functional tissue engineering of cartilage

Tissue engineering seeks to repair or regenerate tissues through combinations of implanted cells, biomaterial scaffolds and biologically active molecules. The rapid restoration of tissue biomechanical function remains an important challenge, emphasizing the need to replicate structural and mechanical properties using novel scaffold designs. Here we present a microscale 3D weaving technique to generate anisotropic 3D woven structures as the basis for novel composite scaffolds that are consolidated with a chondrocyte-hydrogel mixture into cartilage tissue constructs. Composite scaffolds show mechanical properties of the same order of magnitude as values for native articular cartilage, as measured by compressive, tensile and shear testing. Moreover, our findings showed that porous composite scaffolds could be engineered with initial properties that reproduce the anisotropy, viscoelasticity and tension-compression nonlinearity of native articular cartilage. Such scaffolds uniquely combine the potential for load-bearing immediately after implantation in vivo with biological support for cell-based tissue regeneration without requiring cultivation in vitro.     

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.     

A Novel Top‐Down Synthesis of Ultrathin 2D Boron Nanosheets for Multimodal Imaging‐Guided Cancer Therapy

Single atom nonmetal 2D nanomaterials have shown considerable potential in cancer nanomedicines, owing to their intriguing properties and biocompatibility. Herein, ultrathin boron nanosheets (B NSs) are prepared through a novel top‐down approach by coupling thermal oxidation etching and liquid exfoliation technologies, with controlled nanoscale thickness. Based on the PEGylated B NSs, a new photonic drug delivery platform is developed, which exhibits multiple promising features for cancer therapy and imaging, including: i) efficient NIR‐light‐to‐heat conversion with a high photothermal conversion efficiency of 42.5%, ii) high drug‐loading capacity and triggered drug release by NIR light and moderate acidic pH, iii) strong accumulation at tumor sites, iv) multimodal imaging properties (photoacoustic, photothermal, and fluorescence imaging), and v) complete tumor ablation and excellent biocompatibility. As far as it is known, this is the first report on the top‐down fabrication of ultrathin 2D B NSs by the combined thermal oxidation etching and liquid exfoliation, as well as their application as a multimodal imaging‐guided drug delivery platform. The newly prepared B NSs are also expected to provide a robust and useful 2D nanoplatform for various biomedical applications.     

Crosslinking Strategies for 3D Bioprinting of Polymeric Hydrogels

Three‐dimensional (3D) bioprinting has recently advanced as an important tool to produce viable constructs that can be used for regenerative purposes or as tissue models. To develop biomimetic and sustainable 3D constructs, several important processing aspects need to be considered, among which crosslinking is most important for achieving desirable biomechanical stability of printed structures, which is reflected in subsequent behavior and use of these constructs. In this work, crosslinking methods used in 3D bioprinting studies are reviewed, parameters that affect bioink chemistry are discussed, and the potential toward improving crosslinking outcomes and construct performance is highlighted. Furthermore, current challenges and future prospects are discussed. Due to the direct connection between crosslinking methods and properties of 3D bioprinted structures, this Review can provide a basis for developing necessary modifications to the design and manufacturing process of advanced tissue‐like constructs in future.     

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.     

Nanoscale Assembly of 2D Materials for Energy and Environmental Applications

Rational design of 2D materials is crucial for the realization of their profound implications in energy and environmental fields. The past decade has witnessed significant developments in 2D material research, yet a number of critical challenges remain for real-world applications. Nanoscale assembly, precise control over the orientational and positional ordering, and complex interfaces among 2D layers are essential for the continued progress of 2D materials, especially for energy storage and conversion and environmental remediation. Herein, recent progress, the status, future prospects, and challenges associated with nanoscopic assembly of 2D materials are highlighted, specifically targeting energy and environmental applications. Geometric dimensional diversity of 2D material assembly is focused on, based on novel assembly mechanisms, including 1D fibers from the colloidal liquid crystalline phase, 2D films by interfacial tension (Marangoni effect), and 3D nanoarchitecture assembly by electrochemical processes. Relevant critical advantages of 2D material assembly are highlighted for application fields, including secondary batteries, supercapacitors, catalysts, gas sensors, desalination, and water decontamination.     

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.     

Similarity among tandem mass spectra from proteomic experiments: detection,significance, and utility

Liquid chromatography paired with tandem mass spectrometry is a standard technique for identifying peptides from complex protein mixtures. Most fragment ion spectra acquired by this technique are unique, but some are repeated. Similarities among the spectra from 1D and 2D liquid chromatography experiments were calculated by the dot product algorithm. Similar spectra were grouped, and the degree of duplication was calculated for each sample. In 1D liquid chromatography data from 1D gel bands, 18% of the fragment ion spectra were duplicates. A six-cycle 2D liquid chromatographic separation of more than 200 proteins produced 28% duplicate spectra. A rat hippocampal homogenate analyzed by a 12-cycle 2D liquid chromatographic separation contained 25% duplicate spectra. Removal of these duplicate spectra, however, resulted in fewer peptides being successfully identified by SEQUEST. We propose a modification for peptide identification algorithms that would improve their performance and accuracy by explicitly recognizing and making use of spectral similarity.     

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.     

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.     

2D Intrinsic Ferromagnetic MnP Single Crystals

2D intrinsic ferromagnetic materials are highly anticipated in spintronic devices due to their coveted 2D limited magnetism. However, 2D non‐layered intrinsic ferromagnets have received sporadic attention, which is largely attributed to the fact that their synthesis is still a great challenge. Significantly, manganese phosphide (MnP) is a promising non‐layered intrinsic ferromagnet with excellent properties. Herein, high‐quality 2D MnP single crystals formed over liquid metal tin (Sn) is demonstrated through a facile chemical vapor deposition technique. The introduction of liquid metal Sn provides a fertile ground for the growth of 2D MnP single crystals. Interestingly, 2D MnP single crystals maintain their intrinsic ferromagnetism and exhibit a Curie temperature above room temperature. The research enriches the diversity of 2D intrinsic ferromagnetic materials, opening up opportunities for further exploration of their unique properties and rich applications.     

Geometrical approach for automatic detection of liquid surfaces in 3D computed tomography baggage imagery

This study presents a novel method for liquid detection within three-dimensional (3D) computed tomography (CT) baggage inspection imagery. Liquid detection within airport security is currently of significant interest due to security threats associated with liquid explosives. In this paper, we propose a robust technique based on the automatic identification of universal geometric properties of liquids within 3D space. The proposed approach is based on two stages of geometric fitting. First, we identify the 3D plane which fits to the horizontally oriented surface of the liquid recognising the universal self-levelling property of liquids in any given container. Second, we conduct two-dimensional shape analysis to highlight the shape of the liquid surface at a given level within the container using a least squares elliptical fitting approach. The proposed approach relies on the fact that occurrences of such perfectly aligned horizontal planes within a 3D CT security baggage scan are generally unlikely. Occurrences of such instance are thus indicative of liquid presence. Our results, over an extended set of complex test examples, confirm a liquid detection rate of 85–98% with a moderate processing time. Furthermore, as this proposed approach is based purely on the geometric properties of liquids and robust geometrical shape detection, this methodology is intrinsic to the 3D nature of the resulting CT data and not dependent on any exemplar training imagery.     

Development of three-dimensional tissue engineered bone-oral mucosal composite models

Tissue engineering of bone and oral mucosa have been extensively studied independently. The aim of this study was to develop and investigate a novel combination of bone and oral mucosa in a single 3D in vitro composite tissue mimicking the natural structure of alveolar bone with an overlying oral mucosa. Rat osteosarcoma (ROS) cells were seeded into a hydroxyapatite/tri-calcium phosphate scaffold and bone constructs were cultured in a spinner bioreactor for 3 months. An engineered oral mucosa was fabricated by air/liquid interface culture of immortalized OKF6/TERET-2 oral keratinocytes on collagen gel-embedded fibroblasts. EOM was incorporated into the engineered bone using a tissue adhesive and further cultured prior to qualitative and quantitative assessments. Presto Blue assay revealed that ROS cells remained vital throughout the experiment. The histological and scanning electron microscope examinations showed that the cells proliferated and densely populated the scaffold construct. Micro computed tomography (micro-CT) scanning revealed an increase in closed porosity and a decrease in open and total porosity at the end of the culture period. Histological examination of bone-oral mucosa model showed a relatively differentiated parakeratinized epithelium, evenly distributed fibroblasts in the connective tissue layer and widely spread ROS cells within the bone scaffold. The feasibility of fabricating a novel bone-oral mucosa model using cell lines is demonstrated. Generating human ‘normal’ cell-based models with further characterization is required to optimize the model for in vitro and in vivo applications.     

3D Printing by Multiphase Silicone/Water Capillary Inks

3D printing of polymers is accomplished easily with thermoplastics as the extruded hot melt solidifies rapidly during the printing process. Printing with liquid polymer precursors is more challenging due to their longer curing times. One curable liquid polymer of specific interest is polydimethylsiloxane (PDMS). This study demonstrates a new efficient technique for 3D printing with PDMS by using a capillary suspension ink containing PDMS in the form of both precured microbeads and uncured liquid precursor, dispersed in water as continuous medium. The PDMS microbeads are held together in thixotropic granular paste by capillary attraction induced by the liquid precursor. These capillary suspensions possess high storage moduli and yield stresses that are needed for direct ink writing. They could be 3D printed and cured both in air and under water. The resulting PDMS structures are remarkably elastic, flexible, and extensible. As the ink is made of porous, biocompatible silicone that can be printed directly inside aqueous medium, it can be used in 3D printed biomedical products, or in applications such as direct printing of bioscaffolds on live tissue. This study demonstrates a number of examples using the high softness, elasticity, and resilience of these 3D printed structures.     

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.