Controlling Mechanical Properties of Cell‐Laden Hydrogels by Covalent Incorporation of Graphene Oxide

Graphene‐based materials are useful reinforcing agents to modify the mechanical properties of hydrogels. Here, an approach is presented to covalently incorporate graphene oxide (GO) into hydrogels via radical copolymerization to enhance the dispersion and conjugation of GO sheets within the hydrogels. GO is chemically modified to present surface‐grafted methacrylate groups (MeGO). In comparison to GO, higher concentrations of MeGO can be stably dispersed in a pre‐gel solution containing methacrylated gelatin (GelMA) without aggregation or significant increase in viscosity. In addition, the resulting MeGO‐GelMA hydrogels demonstrate a significant increase in fracture strength with increasing MeGO concentration. Interestingly, the rigidity of the hydrogels is not significantly affected by the covalently incorporated GO. Therefore, this approach can be used to enhance the structural integrity and resistance to fracture of the hydrogels without inadvertently affecting their rigidity, which is known to affect the behavior of encapsulated cells. The biocompatibility of MeGO‐GelMA hydrogels is confirmed by measuring the viability and proliferation of the encapsulated fibroblasts. Overall, this study highlights the advantage of covalently incorporating GO into a hydrogel system, and improves the quality of cell‐laden hydrogels.     

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.     

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.     

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.     

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.     

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.     

3D Printable Gelatin Methacryloyl (GelMA)-Dextran Aqueous Two-Phase System with Tunable Pores Structure and Size Enables Physiological Behavior of Embedded Cells In Vitro

The restricted porosity of most hydrogels established for in vitro 3D tissue engineering applications limits embedded cells with regard to their physiological spreading, proliferation, and migration behavior. To overcome these confines, porous hydrogels derived from aqueous two-phase systems (ATPS) are an interesting alternative. However, while developing hydrogels with trapped pores is widespread, the design of bicontinuous hydrogels is still challenging. Herein, an ATPS consisting of photo-crosslinkable gelatin methacryloyl (GelMA) and dextran is presented. The phase behavior, monophasic or biphasic, is tuned via the pH and dextran concentration. This, in turn, allows the formation of hydrogels with three distinct microstructures: homogenous nonporous, regular disconnected-pores, and bicontinuous with interconnected-pores. The pore size of the latter two hydrogels can be tuned from ≈4 to 100 µm. Cytocompatibility of the generated ATPS hydrogels is confirmed by testing the viability of stromal and tumor cells. Their distribution and growth pattern are cell-type specific but are also strongly defined by the microstructure of the hydrogel. Finally, it is demonstrated that the unique porous structure is sustained when processing the bicontinuous system by inkjet and microextrusion techniques. The proposed ATPS hydrogels hold great potential for 3D tissue engineering applications due to their unique tunable interconnected porosity.     

Interface‐Directed Self‐Assembly of Cell‐Laden Microgels

Cell‐laden hydrogels show great promise for creating engineered tissues. However, a major shortcoming with these systems has been the inability to fabricate structures with controlled micrometer‐scale features on a biologically relevant length scale. In this Full Paper, a rapid method is demonstrated for creating centimeter‐scale, cell‐laden hydrogels through the assembly of shape‐controlled microgels or a liquid–air interface. Cell‐laden microgels of specific shapes are randomly placed on the surface of a high‐density, hydrophobic solution, induced to aggregate and then crosslinked into macroscale tissue‐like structures. The resulting assemblies are cell‐laden hydrogel sheets consisting of tightly packed, ordered microgel units. In addition, a hierarchical approach creates complex multigel building blocks, which are then assembled into tissues with precise spatial control over the cell distribution. The results demonstrate that forces at an air–liquid interface can be used to self‐assemble spatially controllable, cocultured tissue‐like structures.     

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.     

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.     

Gravity Drawing of Micro‐ and Nanofibers for Additive Manufacturing of Well‐Organized 3D‐Nanostructured Scaffolds

This work introduces a gravity fiber drawing (GFD) method of making single filament nanofibers from polymer solutions and precise alignment of the fibers in 3D scaffolds. This method is advantageous for nanofiber 3D alignment in contrast to other known methods. GFD provides a technology for the fabrication of freestanding filament nanofibers of well‐controlled diameter, draw ratio, and 3D organization with controllable spacing and angular orientation between nanofibers. The GFD method is capable of fabricating complex 3D scaffolds combining fibers with different diameters, chemical compositions, mechanical properties, angular orientations, and multilayer structures in the same construct. The scaffold porosity can be as high as 99% to secure transport of nutrients and space for cell infiltration and differentiation in tissue engineering and 3D cell culture applications.