Programming Delayed Dissolution Into Sacrificial Bioinks For Dynamic Temporal Control of Architecture within 3D-Bioprinted Constructs

Sacrificial printing allows introduction of architectural cues within engineered tissue constructs. This strategy adopts the use of a 3D-printed sacrificial ink that is embedded within a bulk hydrogel which is subsequently dissolved to leave open-channels. However, current conventional sacrificial inks do not recapitulate the dynamic nature of tissue development, such as the temporal presentation of architectural cues matching cellular requirements during different stages of maturation. To address this limitation, a new class of sacrificial inks is developed that exhibits tailorable and programmable delayed dissolution profiles (1–17 days), by exploiting the unique ability of the ruthenium complex and sodium persulfate initiating system to crosslink native tyrosine groups present in non-chemically modified gelatin. These novel sacrificial inks are also shown to be compatible with a range of biofabrication technologies, including extrusion-based printing, digital-light processing, and volumetric bioprinting. Further embedding these sacrificial templates within cell-laden bulk hydrogels displays precise control over the spatial and temporal introduction of architectural features into cell-laden hydrogel constructs. This approach demonstrates the unique capacity of delaying dissolution of sacrificial inks to modulate cell behavior, improving the deposition of mineralized matrix and capillary-like network formation in osteogenic and vasculogenic culture, respectively.     

3D Microphysiological System-Inspired Scalable Vascularized Tissue Constructs for Regenerative Medicine

Microphysiological systems (MPSs), based on microfabrication technologies and cell culture, can faithfully recapitulate the complex physiology of various tissues. However, 3D tissues formed using MPS have limitations in size and accessibility; their use in regenerative medicine is, therefore, still challenging. Here, an MPS-inspired scale-up vascularized engineered tissue construct that can be used in regenerative medicine is designed. Endothelial cell-laden hydrogels are sandwiched between two through-hole membranes. The microhole array in the through-hole membranes enables the molecular transport across the hydrogel layer, allowing long-term cell culture. Furthermore, the time-controlled delamination of through-hole membranes enables the harvesting of cell-cultured hydrogel constructs without damaging the capillary network. Importantly, when the tissue constructs are implanted in a mouse ischemic model, they protect against necrosis and promoted functional recovery to a greater extent than implanted cells, hydrogels, and simple gel–cell mixtures.     

Induction of Four-Dimensional Spatiotemporal Geometric Transformations in High Cell Density Tissues via Shape-Changing Hydrogels

Developing and healing tissues begin as cellular condensations. Spatiotemporal changes in tissue geometry, transformations in the spatial distribution of the cells, and extracellular matrix are essential for its evolution into a functional tissue. 4D materials, 3D materials capable of geometric changes, may have the potential to recreate the aforementioned biological phenomenon. However, most reported 4D materials are non-degradable and/or not biocompatible, limiting their application in regenerative medicine, and to date, there are no systems controlling the geometry of high density cellular condensations and differentiation. Here, 4D high cell density tissues based on shape-changing hydrogels are described. By sequential photocrosslinking of oxidized and methacrylated alginate (OMA) and methacrylated gelatin (GelMA), bilayered hydrogels presenting controllable geometric changes without any external stimuli are fabricated. Fibroblasts and human adipose-derived stem cells (ASCs) are encapsulated at concentrations up to 1.0 × 10⁸ cells mL^–1 in the 4D constructs, and controllable shape changes are achieved in concert with ASCs differentiated down chondrogenic and osteogenic lineages. Bioprinting of the high density cell-laden OMA and GelMA permits the formation of more complex constructs with defined 4D geometric changes, which may further expand the promise of this approach in regenerative medicine applications.     

Photosynthetic Cyanobacteria can Clearly Induce Efficient Muscle Tissue Regeneration of Bioprinted Cell-Constructs

Tissue engineering strategies using cell-laden constructs have shown promising results in the treatment of various types of damaged tissues. However, inadequate oxygen delivery to the macroscale 3D cell-constructs for regenerating skeletal muscle tissue has remained a multiplex issue owing to the pivotal factors including cell metabolism and several regulatory intercellular pathways that eventually influence various cellular activities and determines cell phenotype. To overcome this issue, a photosynthetic cyanobacterium (Synechococcus elongatus) is employed in a methacrylated gelatin bioink. Furthermore, to effectively induce cell alignment in the bioink, in situ electric field stimulation is used in a bioprinting system to fabricate cell-laden scaffolds for regenerating skeletal muscle tissue. Owing to the synergistic effects of the bioactive microenvironment that rescues cells from hypoxic conditions and activations of voltage-gated ion channels, highly aligned, multi-nucleated myofibers are obtained as well as significant upregulation (7–10-fold) of myogenic-related genes compared with conventionally prepared cell-constructs. In addition, in vivo studies using a mouse volumetric muscle loss model demonstrate considerable restoration of muscle functionality and regeneration.     

Direct 3D Printed Biomimetic Scaffolds Based on Hydrogel Microparticles for Cell Spheroid Growth

Biocompatible hydrogel inks with shear‐thinning, appropriate yield strength, and fast self‐healing are desired for 3D bioprinting. However, the lack of ideal 3D bioprinting inks with outstanding printability and high structural fidelity, as well as cell‐compatibility, has hindered the progress of extrusion‐based 3D bioprinting for tissue engineering. In this study, novel self‐healable pre‐cross‐linked hydrogel microparticles (pcHμPs) of chitosan methacrylate (CHMA) and polyvinyl alcohol (PVA) hybrid hydrogels are developed and used as bioinks for extrusion‐based 3D printing of scaffolds with high fidelity and biocompatibility. The pcHμPs display excellent shear thinning when injected through a syringe and subsequently self‐heal into gels as shear forces are removed. Numerical simulations indicate that the pcHμPs experience a plug flow in the nozzle with minimal disturbance, which favors a steady and continuous printing. Moreover, the pcHμPs show a self‐supportive yield strength (540 Pa), which is critical for the fidelity of printed constructs. A series of biomimetic constructs with very high aspect ratio and delicate fine structures are directly printed by using the pcHμP ink. The 3D printed scaffolds support the growth of bone‐marrow‐derived mesenchymal stem cells and formation of cell spheroids, which are most important for tissue engineering.     

A Stimuli‐Responsive Nanocomposite for 3D Anisotropic Cell‐Guidance and Magnetic Soft Robotics

Stimuli‐responsive materials have the potential to enable the generation of new bioinspired devices with unique physicochemical properties and cell‐instructive ability. Enhancing biocompatibility while simplifying the production methodologies, as well as enabling the creation of complex constructs, i.e., via 3D (bio)printing technologies, remains key challenge in the field. Here, a novel method is presented to biofabricate cellularized anisotropic hybrid hydrogel through a mild and biocompatible process driven by multiple external stimuli: magnetic field, temperature, and light. A low‐intensity magnetic field is used to align mosaic iron oxide nanoparticles (IOPs) into filaments with tunable size within a gelatin methacryloyl matrix. Cells seeded on top or embedded within the hydrogel align to the same axes of the IOPs filaments. Furthermore, in 3D, C2C12 skeletal myoblasts differentiate toward myotubes even in the absence of differentiation media. 3D printing of the nanocomposite hydrogel is achieved and creation of complex heterogeneous structures that respond to magnetic field is demonstrated. By combining the advanced, stimuli‐responsive hydrogel with the architectural control provided by bioprinting technologies, 3D constructs can also be created that, although inspired by nature, express functionalities beyond those of native tissue, which have important application in soft robotics, bioactuators, and bionic devices.     

3D Printing of Poly-ε-Caprolactone (PCL) Auxetic Implants with Advanced Performance for Large Volume Soft Tissue Engineering

A successful 3D printing of a novel 3D architected auxetic for large-volume soft tissue engineering is reported. The 3D auxetic design is analyzed through finite element (FE) simulation and created by selective laser sintering (SLS) of Poly-ε-caprolactone (PCL) for further in-depth mechanical and biological analysis. High initial flexibility and nonlinear stress–strain response to the uniaxial compression are achieved despite the use of PCL, which is one of the biomaterials that is clinically approved but has the disadvantage of having relatively stiff and linear mechanical properties. The high mass transport properties of the 3D auxetic are also demonstrated by not only high cell viability but also cell functionality within a cell-laden hydrogel in large sizes of the auxetic. The outstanding mechanical and biological performance of the 3D auxetic is a consequence of the synergistic effect of the novel architected auxetic design combined with the inherent printing characteristic of SLS. The current study demonstrates great potential of SLS-based printing of 3D auxetics toward the development of clinically viable 3D implants for the reconstruction of large-volume soft tissues.     

Functional Human Vascular Network Generated in Photocrosslinkable Gelatin Methacrylate Hydrogels

The generation of functional, 3D vascular networks is a fundamental prerequisite for the development of many future tissue engineering-based therapies. Current approaches in vascular network bioengineering are largely carried out using natural hydrogels as embedding scaffolds. However, most natural hydrogels present a poor mechanical stability and a suboptimal durability, which are critical limitations that hamper their widespread applicability. The search for improved hydrogels has become a priority in tissue engineering research. Here, the suitability of a photopolymerizable gelatin methacrylate (GelMA) hydrogel to support human progenitor cell-based formation of vascular networks is demonstrated. Using GelMA as the embedding scaffold, it is shown that 3D constructs containing human blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs) generate extensive capillary-like networks in vitro. These vascular structures contain distinct lumens that are formed by the fusion of ECFC intracellular vacuoles in a process of vascular morphogenesis. The process of vascular network formation is dependent on the presence of MSCs, which differentiate into perivascular cells occupying abluminal positions within the network. Importantly, it is shown that implantation of cell-laden GelMA hydrogels into immunodeficient mice results in a rapid formation of functional anastomoses between the bioengineered human vascular network and the mouse vasculature. Furthermore, it is shown that the degree of methacrylation of the GelMA can be used to modulate the cellular behavior and the extent of vascular network formation both in vitro and in vivo. These data suggest that GelMA hydrogels can be used for biomedical applications that require the formation of microvascular networks, including the development of complex engineered tissues.     

3D Printed Template-Assisted Casting of Biocompatible Polyvinyl Alcohol-Based Soft Microswimmers with Tunable Stability

The past decade has seen an upsurge in the development of small-scale magnetic robots for various biomedical applications. However, many of the reported designs comprise components with biocompatibility concerns. Strategies for fabricating biocompatible and degradable microrobots are required. In this study, polyvinyl alcohol (PVA)-based magnetic hydrogel microrobots with different morphologies and tunable stability are developed by combining a 3D printed template-assisted casting with a salting-out process. 3D sacrificial micromolds are prepared via direct laser writing to shape PVA-magnetic nanoparticle composite hydrogel microrobots with high architectural complexity. By adjusting the PVA composition and salting-out parameters, the hydrogel dissolubility can be customized. Due to their high mobility, tunable stability, and high biocompatibility, these PVA-based magnetic microrobots are suitable platforms for targeted drug and cell delivery.     

Chinese‐Noodle‐Inspired Muscle Myofiber Fabrication

Much effort has been made to engineer artificial fiber‐shaped cellular constructs that can be potentially used as muscle fibers or blood vessels. However, existing microfiber‐based approaches for culturing cells are still limited to 2D systems, compatible with a restricted number of polymers (e.g., alginate) and always lacking in situ mechanical stimulation. Here, a simple, facile, and high‐throughput technique is reported to fabricate 3D cell‐laden hydrogel microfibers (named hydrogel noodles), inspired by the fabrication approach for Chinese Hele noodle. A magnetically actuated and noncontact method to apply tensile stretch on hydrogel noodles has also been developed. With this method, it is found that cellular strain‐threshold and saturation behaviors in hydrogel noodles differ substantially from their 2D analogs, including proliferation, spreading, and alignment. Moreover, it is shown that these cell‐laden microfibers can induce muscle myofiber formation by tensile stretching alone. This easily adaptable platform holds great potential for the creation of functional tissue constructs and probing mechanobiology in three dimensions.     

Injectable and Tunable Gelatin Hydrogels Enhance Stem Cell Retention and Improve Cutaneous Wound Healing

Stem cells have shown substantial promise for various diseases in preclinical and clinical trials. However, low cell engraftment rates significantly limit the clinical translation of stem cell therapeutics. Numerous injectable hydrogels have been developed to enhance cell retention. Yet, the design of an ideal material with tunable properties that can mimic different tissue niches and regulate stem cell behaviors remains an unfulfilled promise. Here, an injectable poly(ethylene glycol) (PEG)–gelatin hydrogel is designed with highly tunable properties, from a multifunctional PEG‐based hyperbranched polymer and a commercially available thiolated gelatin. Spontaneous gelation occurs within about 2 min under the physiological condition. Murine adipose‐derived stem cells (ASCs) can be easily encapsulated into the hydrogel, which supports ASC growth and maintains their stemness. The hydrogel mechanical properties, biodegradability, and cellular responses can be finely controlled by changing hydrogel formulation and cell seeding densities. An animal study shows that the in situ formed hydrogel significantly improves cell retention, enhances angiogenesis, and accelerates wound closure using a murine wound healing model. These data suggest that injectable PEG–gelatin hydrogel can be used for regulating stem cell behaviors in 3D culture, delivering cells for wound healing and other tissue regeneration applications.     

Magnetic Helical Hydrogel Motor for Directing T Cell Chemotaxis

Micro/nanomotors are attracting booming research enthusiasm with their revolutionary potential in biomedicine, sensing, and nanoengineering. Among the motors proposed, magnetic micro/nanomotors are of great interest with their high controllability and field biocompatibility. Yet the fabrication of magnetic actuated especially helical motors requires expensive and complicated instruments, 3D printing or glancing angle deposition, etc. Here, a soft and biocompatible helical poly(vinyl alcohol) (PVA) hydrogel motor via a versatile set-up is engineered. The obtained helical hydrogel motor offers high capacity for chemokine CXCL12 and superparamagnetic iron oxide (Fe₃O₄) nanoparticles, which can then allow magnetic manipulation. With a low strength rotating magnetic field, the system is able to perform 3D precision navigation, necessary to steer the robotic system to a model diseased area. The chemokine cues from the hydrogel motor, acting as the synthetic leader cell, then directs immune T cell chemotactic migration. In a previously reported cell manipulating motor system, towing or pushing a single/two cell was demonstrated, with limited efficiency. This motor platform represents a novel approach for directing endogenous cell chemotaxis and organizing immune response.     

A Self-Sustaining Hydrogels with Autonomous Supply of Nutrients and Bioactive Domains for 3D Cell Culture

Photo-crosslinkable platelet lysate (PL)-based hydrogels have been proven to support human-derived cell cultures owing to their high content of bioactive molecules, such as cytokines and growth factors. As a unique self-maintained and biocompatible 3D scaffold, the recently reported self-feeding hydrogels with enzyme-empowered degradation capacity have shown high biological performance in vitro and in vivo. To take advantage of all features of both PL and self-feeding hydrogels, here UV responsive laminaran-methacrylate (LamMA) and PL-methacrylate (PLMA) derivatives plus glucoamylase (GA), which significantly improve the overall features of a 3D system, is coupled. This self-sustaining hybrid hydrogel emerges as a unique scaffold due to the sustained delivery of glucose produced via enzymatic degradation of laminaran while granting the release of growth factors through the presence of PL. This biomaterial is applied to fabricate high-throughput freestanding microgels with controlled geometric shapes. Furthermore, this multicomponent hybrid hydrogel is successfully implemented as the first reported glucose supplier bioink to manufacture intricate and precisely defined cell-laden structures using a support matrix. Finally, such hydrogels are utilized as a proof of concept to serve as 3D in vitro cancer models, with the aim of recapitulating the tumor microenvironment.     

Highly Stretchable,Elastic, and Ionic Conductive Hydrogel for Artificial Soft Electronics

High conductivity, large mechanical strength, and elongation are important parameters for soft electronic applications. However, it is difficult to find a material with balanced electronic and mechanical performance. Here, a simple method is developed to introduce ion‐rich pores into strong hydrogel matrix and fabricate a novel ionic conductive hydrogel with a high level of electronic and mechanical properties. The proposed ionic conductive hydrogel is achieved by physically cross‐linking the tough biocompatible polyvinyl alcohol (PVA) gel as the matrix and embedding hydroxypropyl cellulose (HPC) biopolymer fibers inside matrix followed by salt solution soaking. The wrinkle and dense structure induced by salting in PVA matrix provides large stress (1.3 MPa) and strain (975%). The well‐distributed porous structure as well as ion migration–facilitated ion‐rich environment generated by embedded HPC fibers dramatically enhances ionic conductivity (up to 3.4 S m^?1, at f = 1 MHz). The conductive hybrid hydrogel can work as an artificial nerve in a 3D printed robotic hand, allowing passing of stable and tunable electrical signals and full recovery under robotic hand finger movements. This natural rubber‐like ionic conductive hydrogel has a promising application in artificial flexible electronics.     

Creating Physicochemical Gradients in Modular Microporous Annealed Particle Hydrogels via a Microfluidic Method

Microporous annealed particle (MAP) hydrogels are an attractive platform for engineering biomaterials with controlled heterogeneity. Here, a microfluidic method is introduced to create physicochemical gradients within poly(ethylene glycol) based MAP hydrogels. By combining microfluidic mixing and droplet generator modules, microgels with varying properties are produced by adjusting the relative flow rates between two precursor solutions and collected layer‐by‐layer in a syringe. Subsequently, the microgels are injected out of the syringe and then annealed with thiol‐ene click chemistry. Fluorescence intensity measurements of constructs annealed in vitro and after mock implantation into a tissue defect show that a continuous gradient profile is achieved and maintained after injection, indicating utility for in situ hydrogel formation. The effects of physicochemical property gradients on human mesenchymal stem cells (hMSCs) are also studied. Microgel stiffness is studied first, and the hMSCs exhibit increased spreading and proliferation as stiffness increased along the gradient. Microgel degradability is also studied, revealing a critical degradability threshold above which the hMSCs spread robustly and below which they are isolated and exhibit reduced spreading. This method of generating spatial gradients in MAP hydrogels can be further used to gain new insights into cell–material interactions, which can be leveraged for tissue engineering applications.     

Injectable Biomimetic Hydrogel Guided Functional Bone Regeneration by Adapting Material Degradation to Tissue Healing

The treatment of irregular bone defects remains a clinical challenge since the current biomaterials (e.g., calcium phosphate bone cement (CPC)) mainly act as inert substitutes, which are incapable of transforming into a regenerated host bone (termed functional bone regeneration). Ideally, the implant degradation rate should adapt to that of bone regeneration, therefore providing sufficient physicochemical support and giving space for bone growth. This study aims to develop an injectable biomaterial with bone regeneration-adapted degradability, to reconstruct a biomimetic bone-like structure that can timely transform into new bone, facilitating functional bone regeneration. To achieve this goal, a hybrid (LP-CPC@gelatin, LC) hydrogel is synthesized via one-step incorporation of laponite (LP) and CPC into gelatin hydrogel, and the LC gel degradation rate is controlled by adjusting the LP/CPC ratio to match the bone regeneration rate. Such an LC hydrogel shows good osteoinduction, osteoconduction, and angiogenesis effects, with complete implant-to-new bone transformation capacity. This 2D nanoclay-based bionic hydrogel can induce ectopic bone regeneration and promote ligament graft osseointegration in vivo by inducing functional bone regeneration. Therefore, this study provides an advanced strategy for functional bone regeneration and an injectable biomimetic biomaterial for functional skeletal muscle repair in a minimally invasive therapy.     

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.     

Complex 3D‐Printed Microchannels within Cell‐Degradable Hydrogels

3D‐printing is emerging as a technology to introduce microchannels into hydrogels, for the perfusion of engineered constructs. Although numerous techniques have been developed, new techniques are still needed to obtain the complex geometries of blood vessels and with materials that permit desired cellular responses. Here, a printing process where a shear‐thinning and self‐healing hydrogel “ink” is injected directly into a “support” hydrogel with similar properties is reported. The support hydrogel is further engineered to undergo stabilization through a thiol‐ene reaction, permitting (i) the washing of the ink to produce microchannels and (ii) tunable properties depending on the crosslinker design. When adhesive peptides are included in the support hydrogel, endothelial cells form confluent monolayers within the channels, across a range of printed configurations (e.g., straight, stenosis, spiral). When protease‐degradable crosslinkers are used for the support hydrogel and gradients of angiogenic factors are introduced, endothelial cells sprout into the support hydrogel in the direction of the gradient. This printing approach is used to investigate the influence of channel curvature on angiogenic sprouting and increased sprouting is observed at curved locations. Ultimately, this technique can be used for a range of biomedical applications, from engineering vascularized tissue constructs to modeling in vitro cultures.     

3D Printing of Bioinspired Alginate-Albumin Based Instant Gel Ink with Electroconductivity and Its Expansion to Direct Four-Axis Printing of Hollow Porous Tubular Constructs without Supporting Materials

A successful 3D printable hydrogel ink needs not only biofunctionalities but also minimal fabrication steps such as multiple crosslinking sites, high printability, cytocompatibility, high shape fidelity, stability, shear thinning, robust properties, and less time-consuming processing steps, by maximizing known material chemistries and functionalities. This work reports a novel bioinspired conjugate with polysaccharide (alginate)–tannic acid (TA)–protein (bovine serum albumin) to fabricate proteoglycan-like gels, which are 3D printable with multilayers, shear-thinning, elastic, electroconductive (with carbon nanotubes), controlled crosslinking/degradation through multiple crosslinking mechanisms (TA, Ca²⁺ ions, and NaIO₄ oxidation), and interactions with cytocompatible hydrogel system. The synthesis process is simple, and gelation (within 2 h) is ensured without any chemical crosslinking agents (at room temperature). While cell-adhesive albumin largely improves cytocompatibility, carbon nanotubes in the gel give electrical conductivity in the different four-axis 3D printed structures, including large hollow tubular constructs. This work demonstrates promising results of electroconductive proteoglycan-like gel ink to address the challenges in 3D/four-axis ink printing such as synthesis, printability, shape fidelity, electroconductivity, controlled fabrication and degradation, cytocompatibility, and multiple crosslinking abilities to maintain the dimensions of the diversely printed constructs.     

A Facile Strategy for the Fabrication of Cell-Laden Porous Alginate Hydrogels based on Two-Phase Aqueous Emulsions

Porous alginate (Alg) hydrogels possess many advantages as cell carriers. However, current pore generation methods require either complex or harsh fabrication processes, toxic components, or extra purification steps, limiting the feasibility and affecting the cellular survival and function. In this study, a simple and cell-friendly approach to generate highly porous cell-laden Alg hydrogels based on two-phase aqueous emulsions is reported. The pre-gel solutions, which contain two immiscible aqueous phases of Alg and caseinate (Cas), are cross-linked by calcium ions. The porous structure of the hydrogel construct is formed by subsequently removing the Cas phase from the ion-cross-linked Alg hydrogel. Those porous Alg hydrogels possess heterogeneous pores ≈100 µm and interconnected paths. Human white adipose progenitors (WAPs) encapsulated in these hydrogels self-organize into spheroids and show enhanced viability, proliferation, and adipogenic differentiation, compared to non-porous constructs. As a proof of concept, this porous Alg hydrogel platform is employed to prepare core-shell spheres for coculture of WAPs and colon cancer cells, with WAP clusters distributed around cancer cell aggregates, to investigate cellular crosstalk. This efficacious approach is believed to provide a robust and versatile platform for engineering porous-structured Alg hydrogels for applications as cell carriers and in disease modeling.