Void‐Free 3D Bioprinting for In Situ Endothelialization and Microfluidic Perfusion

Two major challenges of 3D bioprinting are the retention of structural fidelity and efficient endothelialization for tissue vascularization. Both of these issues are addressed by introducing a versatile 3D bioprinting strategy, in which a templating bioink is deposited layer‐by‐layer alongside a matrix bioink to establish void‐free multimaterial structures. After crosslinking the matrix phase, the templating phase is sacrificed to create a well‐defined 3D network of interconnected tubular channels. This void‐free 3D printing (VF‐3DP) approach circumvents the traditional concerns of structural collapse, deformation, and oxygen inhibition, moreover, it can be readily used to print materials that are widely considered “unprintable.” By preloading endothelial cells into the templating bioink, the inner surface of the channels can be efficiently cellularized with a confluent endothelial layer. This in situ endothelialization method can be used to produce endothelium with a far greater cell seeding uniformity than can be achieved using the conventional postseeding approach. This VF‐3DP approach can also be extended beyond tissue fabrication and toward customized hydrogel‐based microfluidics and self‐supported perfusable hydrogel constructs.     

Marine-Derived Hydrogels for Biomedical Applications

Marine organisms provide novel and broad sources for the preparations and applications of biomaterials. Since the urgent requirement of bio-hydrogels to mimic tissue extracellular matrix (ECM), the natural biomacromolecule hydrogels derived from marine sources have received increasing attention. Benefiting from their outstanding bioactivity and biocompatibility, many attempts have been made to reconstruct ECM components by applying marine-derived natural hydrogels. Moreover, marine hydrogels have been successfully applied in biomedicine by means of microfluidics, electrospray, and bioprinting. In this review, the classification and characteristics of marine-derived hydrogels are summarized. In particular, their role in the development of biomaterials is also introduced. Then, the recent advances in bio-fabrication strategies for various hydrogel materials are focused upon. Besides, the influences of hydrogel types on their functions in biomedical applications are discussed in depth. Finally, critical reflections on the limitations and future development of marine-derived hydrogels are presented.     

3D Bioprinting using UNIversal Orthogonal Network (UNION) Bioinks

Three-dimensional (3D) bioprinting is a promising technology to produce tissue-like structures, but a lack of diversity in bioinks is a major limitation. Ideally each cell type would be printed in its own customizable bioink. To fulfill this need for a universally applicable bioink strategy, a versatile bioorthogonal bioink crosslinking mechanism that is cell compatible and works with a range of polymers is developed. This family of materials is termed UNIversal, Orthogonal Network (UNION) bioinks. As demonstration of UNION bioink versatility, gelatin, hyaluronic acid (HA), recombinant elastin-like protein (ELP), and polyethylene glycol (PEG) are each used as backbone polymers to create inks with storage moduli spanning from 200 to 10 000 Pa. Because UNION bioinks are crosslinked by a common chemistry, multiple materials can be printed together to form a unified, cohesive structure. This approach is compatible with any support bath that enables diffusion of UNION crosslinkers. Both matrix-adherent human corneal mesenchymal stromal cells and non-matrix-adherent human induced pluripotent stem cell-derived neural progenitor spheroids are printed with UNION bioinks. The cells retained high viability and expressed characteristic phenotypic markers after printing. Thus, UNION bioinks are a versatile strategy to expand the toolkit of customizable materials available for 3D bioprinting.     

3D Printing of Multifunctional Hydrogels

3D printing technology has been widely explored for the rapid design and fabrication of hydrogels, as required by complicated soft structures and devices. Here, a new 3D printing method is presented based on the rheology modifier of Carbomer for direct ink writing of various functional hydrogels. Carbomer is shown to be highly efficient in providing ideal rheological behaviors for multifunctional hydrogel inks, including double network hydrogels, magnetic hydrogels, temperature‐sensitive hydrogels, and biogels, with a low dosage (at least 0.5% w/v) recorded. Besides the excellent printing performance, mechanical behaviors, and biocompatibility, the 3D printed multifunctional hydrogels enable various soft devices, including loadable webs, soft robots, 4D printed leaves, and hydrogel Petri dishes. Moreover, with its unprecedented capability, the Carbomer‐based 3D printing method opens new avenues for bioprinting manufacturing and integrated hydrogel devices.     

3D Patterning within Hydrogels for the Recreation of Functional Biological Environments

Biological structures are inherently complex in nature. Structural hierarchy, chemical anisotropy, and compositional heterogeneity are ubiquitous in biological systems and play a key role in the functionality of living systems. For decades, methods such as soft lithography have enabled recreation of such arrangements through precise spatial control of molecular patterns in 2D. With technological advances and increasing understanding of molecular and structural biology, there has been an increasing interest in recreating such spatial organizations in 3D. In this review, a comprehensive summary of the latest technologies being used to create 3D patterns of functional molecules within hydrogels for tissue engineering applications is presented. The review is divided into five groups of technologies defined according to the main driving force used to fabricate the patterns including light, precise chemical design, microfluidics, 3D printing, and non-contact forces (i.e. electric, magnetic, or acoustic fields and self-assembly).     

Photon Upconversion Hydrogels for 3D Optogenetics

The ability to optically induce biological responses in 3D has been dwarfed by the physical limitations of visible light penetration to trigger photochemical processes. However, many biological systems are relatively transparent to low-energy light, which does not provide sufficient energy to induce photochemistry in 3D. To overcome this challenge, hydrogels that are capable of converting red or near-IR (NIR) light into blue light within the cell-laden 3D scaffolds are developed. The upconverted light can then excite optically active proteins in cells to trigger a photochemical response. The hydrogels operate by triplet–triplet annihilation upconversion. As proof-of-principle, it is found that the hydrogels trigger an optogenetic response by red/NIR irradiation of HeLa cells that have been engineered to express the blue-light sensitive protein Cry2olig. While it is remarkable to photoinduce the clustering of Cry2olig with blanket NIR irradiation in 3D, it is also demonstrated how the hydrogels trigger clustering within a single cell with great specificity and spatiotemporal control. In principle, these hydrogels may allow for photochemical control of cell function within 3D scaffolds, which can lead to a wealth of fundamental studies and biochemical applications.     

DNA Hydrogels as Functional Materials and Their Biomedical Applications

With the remarkable development of DNA nanotechnology, interest in DNA molecules has expanded beyond its biological role to building blocks in materials science. As a unique branch of DNA-based materials, DNA hydrogels have exhibited many fascinating characteristics, including broad biocompatibility, precise programmability, convenient modification, and tunable mechanical properties, which make DNA hydrogels ideal biomaterials. Moreover, by combining with functional nucleic acids, such as aptamers, i-motif nanostructures, CpG oligodeoxynucleotides, and DNAzymes, DNA hydrogels can be further tailored to provide additional target recognition, therapeutic potential, and catalytic activities, allowing them to play important roles in biosensing and medical applications. This review, aims to provide readers with an up-to-date overview of the important developments of biomedical DNA hydrogels. First, it introduces different synthetic strategies of hydrogels that utilize DNA as building materials and functional units within the hydrogel networks and discuss their advantages in biomedical applications. Subsequently, new approaches and applications of biomedical DNA hydrogels in the recent years are highlighted, such as therapeutic systems, cell culture platforms, tissue engineering materials, and biosensors. Finally, future perspectives and remaining challenges of DNA hydrogels in biomedicine are presented.     

Intra-Operative Bioprinting of Hard,Soft, and Hard/Soft Composite Tissues for Craniomaxillofacial Reconstruction

Reconstruction of complex craniomaxillofacial (CMF) defects is challenging due to the highly organized layering of multiple tissue types. Such compartmentalization necessitates the precise and effective use of cells and other biologics to recapitulate the native tissue anatomy. In this study, intra-operative bioprinting (IOB) of different CMF tissues, including bone, skin, and composite (hard/soft) tissues, is demonstrated directly on rats in a surgical setting. A novel extrudable osteogenic hard tissue ink is introduced, which induced substantial bone regeneration, with ≈80% bone coverage area of calvarial defects in 6 weeks. Using droplet-based bioprinting, the soft tissue ink accelerated the reconstruction of full-thickness skin defects and facilitated up to 60% wound closure in 6 days. Most importantly, the use of a hybrid IOB approach is unveiled to reconstitute hard/soft composite tissues in a stratified arrangement with controlled spatial bioink deposition conforming the shape of a new composite defect model, which resulted in ≈80% skin wound closure in 10 days and 50% bone coverage area at Week 6. The presented approach will be absolutely unique in the clinical realm of CMF defects and will have a significant impact on translating bioprinting technologies into the clinic in the future.     

Functional Nanomaterials on 2D Surfaces and in 3D Nanocomposite Hydrogels for Biomedical Applications

An emerging approach to improve the physicobiochemical properties and the multifunctionality of biomaterials is to incorporate functional nanomaterials (NMs) onto 2D surfaces and into 3D hydrogel networks. This approach is starting to generate promising advanced functional materials such as self‐assembled monolayers (SAMs) and nanocomposite (NC) hydrogels of NMs with remarkable properties and tailored functionalities that are beneficial for a variety of biomedical applications, including tissue engineering, drug delivery, and developing biosensors. A wide range of NMs, such as carbon‐, metal‐, and silica‐based NMs, can be integrated into 2D and 3D biomaterial formulations due to their unique characteristics, such as magnetic properties, electrical properties, stimuli responsiveness, hydrophobicity/hydrophilicity, and chemical composition. The highly ordered nano‐ or microscale assemblies of NMs on surfaces alter the original properties of the NMs and add enhanced and/or synergetic and novel features to the final SAMs of the NM constructs. Furthermore, the incorporation of NMs into polymeric hydrogel networks reinforces the (soft) polymer matrix such that the formed NC hydrogels show extraordinary mechanical properties with superior biological properties.     

3D Printing of Strong and Tough Double Network Granular Hydrogels

Many soft natural tissues display a fascinating set of mechanical properties that remains unmatched by manmade counterparts. These unprecedented mechanical properties are achieved through an intricate interplay between the structure and locally varying the composition of these natural tissues. This level of control cannot be achieved in soft synthetic materials. To address this shortcoming, a novel 3D printing approach to fabricate strong and tough soft materials is introduced, namely double network granular hydrogels (DNGHs) made from compartmentalized reagents. This is achieved with an ink composed of microgels that are swollen in a monomer-containing solution; after the ink is additive manufactured, these monomers are converted into a percolating network, resulting in a DNGH. These DNGHs are sufficiently stiff to repetitively support tensile loads up to 1.3 MPa. Moreover, they are more than an order of magnitude tougher than each of the pure polymeric networks they are made from. It is demonstrated that this ink enables printing macroscopic, strong, and tough objects, which can optionally be rendered responsive, with high shape fidelity. The modular and robust fabrication of DNGHs opens up new possibilities to design adaptive, strong, and tough hydrogels that have the potential to advance, for example, soft robotic applications.     

Universal Peptide Hydrogel for Scalable Physiological Formation and Bioprinting of 3D Spheroids from Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) are used for drug discoveries, disease modeling and show great potential for human organ regeneration. 3D culture methods have been demonstrated to be an advanced approach compared to the traditional monolayer (2D) method. Here, a self-healing universal peptide hydrogel is reported for manufacturing physiologically formed hiPSC spheroids. With 100 000 hiPSCs encapsulated in 500 µL hydrogel, ≈50 000 spheroids mL⁻¹ (diameter 20–50 µm) are generated in 5 d. The spheroids in the universal peptide hydrogel are viable (85–96%) and show superior pluripotency and differentiation potential based on multiple biomarkers. Cell performance is influenced by the degradability of the hydrogel but not by gel strength. Without postprinting crosslinking aided by UV or visible lights or chemicals, various patterns are easily extruded from a simple star to a kidney-like organ shape using the universal peptide hydrogel bioink showing acceptable printability. A 20.0 × 20.0 × 0.75 mm³ sheet is finally printed with the universal peptide hydrogel bioink encapsulating hiPSCs and cultured for multiple days, and the hiPSC spheroids are physiologically formed and well maintained.     

Encapsulation of Human Natural and Induced Regulatory T‐Cells in IL‐2 and CCL1 Supplemented Alginate‐GelMA Hydrogel for 3D Bioprinting

Regulatory T‐cells (Tregs) are important modulators of the immune system through their intrinsic suppressive functions. Systemic adoptive transfer of ex vivo expanded Tregs has been extensively investigated for allogeneic transplantation. Due to the time‐consuming and costly expansion protocols of Tregs, more targeted approaches could be beneficial. The encapsulation of human natural and induced Tregs for localized immunosuppression is described for the first time. Tregs encapsulated in alginate‐gelatin methacryloyl hydrogel remain viable, phenotypically stable, functional, and confined in the structure. Supplementation of the hydrogel with the Treg‐specific bioactive factors interleukin‐2 and chemokine ligand 1 improves Treg viability, suppressive phenotype, and function, and attracts to the structure CCR8⁺ T‐cells enriched with anti‐inflammatory subpopulations, including Tregs, from human peripheral blood. Furthermore, these findings are applicable to 3D bioprinting. Co‐axial printing of murine pancreatic islets with human natural and induced Tregs protects the islets from xenoresponse upon co‐culture with human peripheral blood mononuclear cells. This establishes the co‐encapsulation of Tregs by co‐axial 3D bioprinting as a valid option for providing local immune protection to allogeneic cellular transplants such as pancreatic islets.     

Gold Nanocomposite Bioink for Printing 3D Cardiac Constructs

Bioprinting is the most convenient microfabrication method to create biomimetic three‐dimensional (3D) cardiac tissue constructs, that can be used to regenerate damaged tissue and provide platforms for drug screening. However, existing bioinks, which are usually composed of polymeric biomaterials, are poorly conductive and delay efficient electrical coupling between adjacent cardiac cells. To solve this problem, a gold nanorod (GNR)‐incorporated gelatin methacryloyl (GelMA)‐based bioink is developed for printing 3D functional cardiac tissue constructs. The GNR concentration is adjusted to create a proper microenvironment for the spreading and organization of cardiac cells. At optimized concentrations of GNR, the nanocomposite bioink has a low viscosity, similar to pristine inks, which allows for the easy integration of cells at high densities. As a result, rapid deposition of cell‐laden fibers at a high resolution is possible, while reducing shear stress on the encapsulated cells. In the printed GNR constructs, cardiac cells show improved cell adhesion and organization when compared to the constructs without GNRs. Furthermore, the incorporated GNRs bridge the electrically resistant pore walls of polymers, improve the cell‐to‐cell coupling, and promote synchronized contraction of the bioprinted constructs. Given its advantageous properties, this gold nanocomposite bioink may find wide application in cardiac tissue engineering.     

Formation Theory and Printability of Photocurable Hydrogel for 3D Bioprinting

Bio-ink has gradually transited from ionic-crosslinking to photocrosslinking due to photocurable bio-hydrogel having good formability and biocompatibility. It is very important to understand and quantify the crosslinking process of photocurable hydrogels, otherwise, bioprinting cannot be standardized and scalable. However, there are few studies on hydrogel formation process and its photocrosslinking behavior which cannot be accurately predicted. Herein, the photoinitiated radical polymerized bio-hydrogels are taken as an example to establish the formation theory. Three typical crosslinking reactions are first distinguished. It is further proposed that not all double-bonds consumed during crosslinking contributeequally to polymerization. Then the concept of effective double-bond conversion (EDBC) is elicited. Deriving from EDBC, several important formation indices are defined. According to theory, it is predicted that slow crosslinking can improve the crosslinking degree. Furthermore, based on the slow crosslinking effect, a new strategy of projection-based 3D printing (PBP) is proposed, which significantly improved printing quality and efficiency. Overall, this work will fill the gap in hydrogel's formation theory, making it possible to accurately quantify the formation process.     

Biomimetic Scaffolds for Tissue Engineering

Biomimetic scaffolds mimic important features of the extracellular matrix (ECM) architecture and can be finely controlled at the nano‐ or microscale for tissue engineering. Rational design of biomimetic scaffolds is based on consideration of the ECM as a natural scaffold; the ECM provides cells with a variety of physical, chemical, and biological cues that affect cell growth and function. There are a number of approaches available to create 3D biomimetic scaffolds with control over their physical and mechanical properties, cell adhesion, and the temporal and spatial release of growth factors. Here, an overview of some biological features of the natural ECM is presented and a variety of original engineering methods that are currently used to produce synthetic polymer‐based scaffolds in pre‐fabricated form before implantation, to modify their surfaces with biochemical ligands, to incorporate growth factors, and to control their nano‐ and microscale geometry to create biomimetic scaffolds are discussed. Finally, in contrast to pre‐fabricated scaffolds composed of synthetic polymers, injectable biomimetic scaffolds based on either genetically engineered‐ or chemically synthesized‐peptides of which sequences are derived from the natural ECM are discussed. The presence of defined peptide sequences can trigger in situ hydrogelation via molecular self‐assembly and chemical crosslinking. A basic understanding of the entire spectrum of biomimetic scaffolds provides insight into how they can potentially be used in diverse tissue engineering, regenerative medicine, and drug delivery applications.     

Tissue Engineering: Scaffold‐Mediated 2D Cellular Orientations for Construction of Three Dimensionally Engineered Tissues Composed of Oriented Cells and Extracellular Matrices (Adv. Funct. Mater. 7/2009)

Various hydrogels, such as poly(γ‐glutamic acid) (γ‐PGA), gelatin (GT), alginic acid (Alg), and agarose (Aga), with 3D interconnected and oriented fibrous pores (OP gels) are prepared for 3D polymeric cellular scaffolds by using silica fiber cloth (SC) as template. After the preparation of these hydrogels with the SC templates, the latter are subsequently removed by washing with hydrofluoric acid solution. Scanning electron microscopy (SEM) clearly shows OP structures in the hydrogels. These various types of OP gels are successfully prepared in this way, independently of the crosslinking mechanism, such as chemical (γ‐PGA or GT), coordinate‐bonded (Alg), or hydrogen‐bonded (Aga) crosslinks. SEM, confocal laser scanning microscopy, and histological evaluations clearly demonstrate that mouse L929 fibroblast cells adhere to and extend along these OP structures on/in γ‐PGA hydrogels during 3D cell culture. The L929 cells that adhere on/in the oriented hydrogel are viable and proliferative. Furthermore, 3D engineered tissues, composed of the oriented cells and extracellular matrices (ECM) produced by the cells, are constructed in vitro by subsequent decomposition of the hydrogel with cysteine after 14 days of cell culture. This novel technology to fabricate 3D‐engineered tissues, consisting of oriented cells and ECM, will be useful for tissue engineering.     

Silk Hydrogels as Soft Substrates for Neural Tissue Engineering

There is great need for soft biomaterials that match the stiffness of human tissues for tissue engineering and regeneration. Hydrogels are frequently employed for extracellular matrix functionalization and to provide appropriate mechanical cues. It is challenging, however, to achieve structural integrity and retain bioactive molecules in hydrogels for complex tissue formation that may take months to develop. This work aims to investigate mechanical and biochemical characteristics of silk hydrogels for soft tissue engineering, specifically for the nervous system. The stiffness of 1 to 8% silk hydrogels, measured by atomic force microscopy, is 4 to 33 kPa. The structural integrity of silk gels is maintained throughout embryonic chick dorsal root ganglion (cDRG) explant culture over 4 days whereas fibrin and collagen gels decrease in mass over time. Neurite extension of cDRGs cultured on 2 and 4% silk hydrogels exhibit greater growth than softer or stiffer gels. Silk hydrogels release <5% of neurotrophin‐3 (NT‐3) over 2 weeks and 11‐day old gels show maintenance of growth factor bioactivity. Finally, fibronectin‐ and NT‐3‐functionalized silk gels elicit increased axonal bundling suggesting their use in bridging nerve injuries. These results support silk hydrogels as soft and sustainable biomaterials for neural tissue engineering.     

Stimuli-Responsive Nanocomposite Hydrogels for Biomedical Applications

The complex tissue-specific physiology that is orchestrated from the nano- to the macroscale, in conjugation with the dynamic biophysical/biochemical stimuli underlying biological processes, has inspired the design of sophisticated hydrogels and nanoparticle systems exhibiting stimuli-responsive features. Recently, hydrogels and nanoparticles have been combined in advanced nanocomposite hybrid platforms expanding their range of biomedical applications. The ease and flexibility of attaining modular nanocomposite hydrogel constructs by selecting different classes of nanomaterials/hydrogels, or tuning nanoparticle-hydrogel physicochemical interactions widely expands the range of attainable properties to levels beyond those of traditional platforms. This review showcases the intrinsic ability of hybrid constructs to react to external or internal/physiological stimuli in the scope of developing sophisticated and intelligent systems with application-oriented features. Moreover, nanoparticle-hydrogel platforms are overviewed in the context of encoding stimuli-responsive cascades that recapitulate signaling interplays present in native biosystems. Collectively, recent breakthroughs in the design of stimuli-responsive nanocomposite hydrogels improve their potential for operating as advanced systems in different biomedical applications that benefit from tailored single or multi-responsiveness.     

Hydrogel Scaffolds of Amphiphilic and Acidic β‐Sheet Peptides

Amphiphilic and acidic β‐sheet‐forming peptides (AAβPs) having the sequence Pro‐Y‐(Z‐Y)₅‐Pro, Y = Glu or Asp and Z = Phe or Leu may assemble into hydrogel structures at near neutral pH values, several units higher than the intrinsic pKa of their acidic amino acid side chains. The bottom‐to‐top design strategy enables the rationally supported association between the peptides' amino acids composition and bulk pH hydrogelation. Hydrogen bonds between the acidic amino acids side chains in the β‐sheet structure are found to contribute substantially to the stabilization of AAβPs hydrogels. The negatively charged peptides are also found to form gels at lower concentration in presence of calcium ions. Bone forming cells may be cultured on two‐dimensional films of AAβPs hydrogels that form at physiological pH values as well as within three dimensional hydrogel matrices. These acidic‐rich peptides hydrogels may become advantageous in applications related to engineering of mineralized tissues providing controllable, multifunctional calcified scaffolds to affect both the biological activity and the inorganic mineralization.