To reduce costs and delays related to developing new and effective drugs, there is a critical need for improved human liver tissue models. Here we describe an approach for 3D bioprinting functional human liver tissue models, in which we fabricate disc-shaped structures (discoids) 200 μm in thickness and 1-3 mm in diameter from mixtures of cells and collagen-1, embedded in a highly permeable support medium made from packed polyethylene glycol (PEG) microgels. We demonstrate that the method is precise, accurate, and scalable; up to 100 tissues/h can be manufactured with a variability and error in diameter of about 4 %. Histologic and immunohistochemical evaluation of printed discs reveal self-organization, cell cohesion, and key liver marker expression. Over the course of three weeks in culture, the tissues stably synthesize albumin and urea at high levels, outperforming spheroid tissue models. We find the tissues express >100 genes associated with molecular absorption, distribution, metabolism, and excretion (ADME) at levels within the range of human liver. The liver tissue models exhibit enzymatic formation of metabolites after exposure to multiple test compounds. Together, these results demonstrate the promise of 3D printed discoids for pharmacological and toxicological applications.

With the rapid development of flexible electronics in wearable devices, healthcare devices, and the Internet of Things (IoT), liquid metals (LMs)-based hydrogels have emerged as cutting-edge functional materials due to their high electrical conductivity, tunable mechanical properties, excellent biocompatibility, and unique self-healing properties. Through various physical or chemical methods, LMs can be integrated to form multifunctional LMs-based hydrogels, thus broadening the potential application fields. In this Review, the recent advancement in LMs-based hydrogels for flexible electronics is comprehensively and systematically reviewed from three aspects of synthesis methods, properties, and applications. For the first time, the existing innovative synthesis methods of LMs-based hydrogels are classified and summarized, including patterned LMs on/inside hydrogel substrates, LMs as conductive fillers in polymeric hydrogels, LMs as initiators in hydrogels, and LMs as cross-linkers with organic/inorganic materials. The synthesis mechanism is also stated in detail to highlight the multiple roles of LMs in adjusting the hydrogel properties. The versatile applications of LMs-based hydrogels in flexible electronics, including flexible sensors, wireless communications, electromagnetic interference (EMI) shielding, soft robot actuators, energy storage and conversion, etc., are separately described. Finally, the current challenges and future prospects of LMs-based hydrogels are proposed.

Embedded ink writing (EIW) is an emerging 3D printing technique that fabricates complex 3D structures from various biomaterial inks but is limited to a printing speed of ∼10 mm s-1 due to suboptimal rheological properties of particulate-dominated yield-stress fluids when used as liquid baths. In this work, a particle-hydrogel interactive system to design advanced baths with enhanced yield stress and extended thixotropic response time for realizing high-speed EIW is developed. In this system, the interactions between particle additive and three representative polymeric hydrogels enable the resulting nanocomposites to demonstrate different rheological behaviors. Accordingly, the interaction models for the nanocomposites are established, which are subsequently validated by macroscale rheological measurements and advanced microstructure characterization techniques. Filament formation mechanisms in the particle-hydrogel interactive baths are comprehensively investigated at high printing speeds. To demonstrate the effectiveness of the proposed high-speed EIW method, an anatomic-size human kidney construct is successfully printed at 110 mm s-1, which only takes ∼4 h. This work breaks the printing speed barrier in current EIW and propels the maximum printing speed by at least 10 times, providing an efficient and promising solution for organ reconstruction in the future.

The architectures of hydrogels fabricated with stereolithography (SLA) 3D printing systems have played various roles in bioengineering applications. Typically, the SLA systems successively illuminated light to a layer of photo-crosslinkable hydrogel precursors for the fabrication of hydrogels. These SLA systems can be classified into point-scanning types and digital micromirror device (DMD) types. The point-scanning types form layers of hydrogels by scanning the precursors with a focused light, while DMD types illuminate 2D light patterns to the precursors to form each hydrogel layer at once. Overall, SLA systems were cost-effective and allowed the fabrication of hydrogels with good shape fidelity and uniform mechanical properties. As a result, hydrogel constructs fabricated with the SLA 3D printing systems were used to regenerate tissues and develop lab-on-a-chip devices and native tissue-like models.

Extrusion-based embedded 3D bioprinting, where bioinks and biomaterials are extruded within a support bath, has greatly expanded the achievable tissue architectures and complexity of biologic constructs that can be fabricated. However, significant scientific, engineering, and process-related challenges remain to recreate the full anatomic structure and physiologic function required for many therapeutic applications. This perspective explores the future advances in extrusion-based embedded 3D bioprinting that could address these challenges, paving the way for clinical translation of bioprinted tissues.

Direct ink writing is a 3D printing method that is compatible with a wide range of structural, elastomeric, electronic, and living materials, and it continues to expand its uses into physics, engineering, and biology laboratories. However, the large footprint, closed hardware and software ecosystems, and expense of commercial systems often hamper widespread adoption. This work introduces a compact, low-cost, multimaterial, and high-throughput direct ink writing 3D printer platform with detailed assembly files and instructions provided freely online. In contrast to existing low-cost 3D printers and bioprinters, which typically modify off-the-shelf plastic 3D printers, this system is built from scratch, offering a lower cost and full customizability. Active mixing of cell-laden bioinks, high-throughput production of auxetic lattices using multimaterial multinozzle 3D (MM3D) printing methods, and a high-toughness, photocurable hydrogel for fabrication of heart valves are introduced. Finally, hardware for embedded multinozzle and 3D gradient nozzle printing is developed for producing high-throughput and graded 3D parts. This powerful, simple-to-build, and customizable printing platform can help stimulate a vibrant biomaker community of engineers, biologists, and educators.

Bone tissue engineering aims to address critical-sized defects by developing biomimetic scaffolds that promote repair and regeneration. This study introduces a material extrusion-based embedded bioprinting approach to fabricate dense cellular constructs within methacrylated hyaluronic acid (MeHA) hydrogels enhanced with bioactive microparticles. Composite matrices containing human bone allograft or tricalcium phosphate (TCP) particles were evaluated for their rheological, mechanical, and osteoinductive properties. High cell viability (>95%) and uniform strand dimensions were achieved across all bioprinting conditions, demonstrating the method's ability to preserve cellular integrity and structural fidelity. The inclusion of bone or TCP particles did not significantly alter the viscosity, crosslinking kinetics, or compressive modulus of the MeHA hydrogels, ensuring robust mechanical stability and shape retention. However, bone allograft particles significantly enhanced osteogenic differentiation of human mesenchymal stem cells (hMSCs), as evidenced by increased alkaline phosphatase (ALP) activity and calcium deposition. Notably, osteogenesis was observed even in basal media, with a dose-dependent response to bone particle concentration, highlighting the intrinsic bioactivity of allograft particles. This study demonstrates the potential of combining embedded bioprinting with bioactive matrices to create dense, osteoinductive cellular constructs. The ability to induce osteogenesis without external growth factors positions this platform as a scalable and clinically relevant solution for bone repair and regeneration.

Thrombosis, a major cause of morbidity and mortality worldwide, presents a complex challenge in cardiovascular medicine due to the intricacy of clotting mechanisms in living organisms. Traditional research approaches, including clinical studies and animal models, often yield conflicting results due to the inability to control variables in these complex systems, highlighting the need for more precise investigative tools. This review explores the evolution of in vitro thrombosis models, from conventional polydimethylsiloxane (PDMS)-based microfluidic devices to advanced hydrogel-based systems and cutting-edge 3D bioprinted vascular constructs. We discuss how these emerging technologies, particularly vessel-on-a-chip platforms, are enabling researchers to control previously unmanageable factors, thereby offering unprecedented opportunities to pinpoint specific clotting mechanisms. While PDMS-based devices offer optical transparency and fabrication ease, their inherent limitations, including non-physiological rigidity and surface properties, have driven the development of hydrogel-based systems that better mimic the extracellular matrix of blood vessels. The integration of microfluidics with biomimetic materials and tissue engineering approaches has led to the development of sophisticated models capable of simulating patient-specific vascular geometries, flow dynamics, and cellular interactions under highly controlled conditions. The advent of 3D bioprinting further enables the creation of complex, multi-layered vascular structures with precise spatial control over geometry and cellular composition. Despite significant progress, challenges remain in achieving long-term stability, incorporating immune components, and translating these models to clinical applications. By providing a comprehensive overview of current advancements and future prospects, this review aims to stimulate further innovation in thrombosis research and accelerate the development of more effective, personalized approaches to thrombosis prevention and treatment.

3D bioprinting technology offers significant advantages in the fabrication of tissue and organ structures by allowing precise layer-by-layer patterning of cells and various biomaterials. However, conventional bioinks exhibit poor mechanical properties, which limit their use in the fabrication of large-scale vascularized tissue constructs. To address these limitations, recent studies have focused on the development of rapidly crosslinkable bioinks through chemical modification. These enable rapid crosslinking within minutes, offering substantial advantages for engineering large-scale tissue constructs. Nevertheless, challenges remain in the production of constructs that fully incorporate the complex vascular networks inherent to native tissues. Recently, embedded bioprinting technique, which involves the direct writing of bioink into a support bath that provides physical support, has gained significant attention for enabling the freeform fabrication of 3D structures. This method has been extensively studied and offers the advantage of fabricating structures ranging from tissue constructs with simple vascular channels to complex structures containing multiscale vascular networks. This review presents an overview of the various materials utilized in embedded bioprinting and elucidates the rheological properties of these materials. Furthermore, it examines the current research trends in the biofabrication of vascularized tissue constructs using embedded bioprinting techniques, along with their associated limitations. The review concludes by proposing areas for future improvement, specifically addressing material and biofabrication approaches as well as bioprinting systems.

Despite significant advances in bioprinting technology, current hardware platforms lack the capability for process monitoring and quality control. This limitation hampers the translation of the technology into industrial GMP-compliant manufacturing settings. As a key step towards a solution, we developed a novel bioprinting platform integrating a high-resolution camera forin-situmonitoring of extrusion outcomes during embedded bioprinting. Leveraging classical computer vision and image analysis techniques, we then created a custom software module for assessing print quality. This module enables quantitative comparison of printer outputs to input points of the computer-aided design model's 2D projections, measuring area and positional accuracy. To showcase the platform's capabilities, we then investigated compatibility with various bioinks, dyes, and support bath materials for both 2D and 3D print path trajectories. In addition, we performed a detailed study on how the rheological properties of granular support hydrogels impact print quality during embedded bioprinting, illustrating a practical application of the platform. Our results demonstrated that lower viscosity, faster thixotropy recovery, and smaller particle sizes significantly enhance print fidelity. This novel bioprinting platform, equipped with integrated process monitoring, holds great potential for establishing auditable and more reproducible biofabrication processes for industrial applications.

Linking the macroscopic flow properties and nanoscopic structure is a fundamental challenge to understanding, predicting, and designing disordered soft materials. Under small stresses, these materials are soft solids, while larger loads can lead to yielding and the acquisition of plastic strain, which adds complexity to the task. In this work, we connect the transient structure and rheological memory of a colloidal gel under cyclic shearing across a range of amplitudes via a generalized memory function using rheo-X-ray photon correlation spectroscopy (rheo-XPCS). Our rheo-XPCS data show that the nanometer scale aggregate-level structure recorrelates whenever the change in recoverable strain over some interval is zero. The macroscopic recoverable strain is therefore a measure of the nano-scale structural memory. We further show that yielding in disordered colloidal materials is strongly heterogeneous and that memories of prior deformation can exist even after the material has been subjected to flow.

Nature uses fibrous structures for sensing and structural functions as observed in hairs, whiskers, stereocilia, spider silks, and hagfish slime thread skeins. Here, we demonstrate multi-nozzle printing of 3D hair arrays having freeform trajectories at a very high rate, with fiber diameters as fine as 1.5 µm, continuous lengths reaching tens of centimeters, and a wide range of materials with elastic moduli from 5 MPa to 3500 MPa. This is achieved via 3D printing by rapid solvent exchange in high yield stress micro granular gel, leading to radial solidification of the extruded polymer filament at a rate of 2.33 μm/s. This process extrudes filaments at 5 mm/s, which is 500,000 times faster than meniscus printing owing to the rapid solidification which prevents capillarity-induced fiber breakage. This study demonstrates the potential of 3D printing by rapid solvent exchange as a fast and scalable process for replicating natural fibrous structures for use in biomimetic functions.

Fabricating tubular hydrogel models with arbitrary structural complexity and controllable diameters using an ultrafast, facile yet universal method is desirable for vascular prototypes yet still a great challenge. Herein, inspired by the denaturing ability of spider silks, a novel strategy to induce complexation via applying highly concentrated alkali into a polyvinyl alcohol/ionic liquid (PVA/IL) solution, i.e., alkali-induced molecular aggregation (AMA), is proposed to achieve such purpose. This strategy enables the rapid and facile fabrication of tubular hydrogel architectures with tunable diameters, controllable thicknesses, and excellent mechanical performance with a tensile strength of up to 1.1 MPa and stretchability exceeding 600%. Importantly, this novel strategy combined with 3D printing facilitates the rapid fabrication of a variety of precise tubular hydrogel models with connected cavity structures which are difficult to achieve using current methods. This ultrafast solidification strategy could also be extended to various alkalis, cations and anions to build different hydrogels, showcasing its versatility and universality. Hence, this strategy can be pioneering to rapidly fabricate complex three-dimensional and hollow enclosed hydrogel models for simulating endovascular interventional therapy.

Adhesive tissue engineering scaffold (ATES) devices can be secured on tissues by relying on their intrinsic adhesive properties, hence, avoiding the complications such as host tissue/scaffold damage that are associated with conventional scaffold fixation methods like suturing or bioglue. This study introduces a new generation of three-dimensional (3D) bioprinted ATES systems for use as cardiac patches to regenerate the adult human heart. Tyramine-modified methacrylated hyaluronic acid (HAMA-tyr), gelatin methacrylate (GelMA), and gelatin were used to create the hybrid bioink formulation with self-adhesive properties. ATESs were bioprinted and further modified to improve the adhesion properties. In-depth characterization of printing fidelity, pore size, mechanical properties, swelling behavior, as well as biocompatibility was used to create ATESs with optimal biological function. Following in vitro testing, the ATESs were tested in a mouse model of myocardial infarction to study the scaffold adhesive strength in biological milieu. The method developed in this study can be used to manufacture off-the-shelf ATESs with complex cellular and extracellular architecture, with robust potential for clinical translation into a variety of personalized tissue engineering and regenerative medicine applications.

Protein-based hydrogels have great potential to be used as bioinks for biofabrication-driven tissue regeneration strategies due to their innate bioactivity. Nevertheless, their use as bioinks in conventional 3D bioprinting is impaired due to their intrinsic low viscosity. Using embedding bioprinting, a liquid bioink is printed within a support that physically holds the patterned filament. Inspired by the recognized microencapsulation technique complex coacervation, crystal self-healing embedding bioprinting (CLADDING) is introduced based on a highly transparent crystal supporting bath. The suitability of distinct classes of gelatins is evaluated (i.e., molecular weight distribution, isoelectric point, and ionic content), as well as the formation of gelatin-gum arabic microparticles as a function of pH, temperature, solvent, and mass ratios. Characterizing and controlling this parametric window resulted in high yields of support bath with ideal self-healing properties for interaction with protein-based bioinks. This support bath achieved transparency, which boosted light permeation within the bath. Bioprinted constructs fully composed of platelet lysates encapsulating a co-culture of human mesenchymal stromal cells and endothelial cells are obtained, demonstrating a high-dense cellular network with excellent cell viability and stability over a month. CLADDING broadens the spectrum of photocrosslinkable materials with extremely low viscosity that can now be bioprinted with sensitive cells without any additional support.

The aim of this study was to develop a composite thermosensitive hydrogel for bone regeneration applications. This hydrogel consisted of chitosan, alginate and hydroxyapatite, and was loaded with zoledronic acid as a model drug. The feasibility of three-dimensional (3D) printing of the thermosensitive hydrogel using the extrusion based technique was investigated. The 3D printing technique called Freeform Reversible Embedded Suspended Hydrogel (FRESH) printing was employed for this purpose. To characterize the composite hydrogels, several tests were conducted. The gelation time, rheological properties, and in vitro drug release were analyzed. Additionally, the cell viability test on human osteosarcoma MG-63 cells for the composite hydrogel was assessed using an MTT assay. The results of the study showed that the zoledronic acid-loaded composite thermosensitive hydrogel was successfully printed using the FRESH 3D printing technique which was not possible otherwise i.e., by using traditional 3D printing techniques. Further examination of the printed constructs using a Scanning Electron Microscope revealed the presence of porous and layered structures. The gelation times of the composite thermosensitive hydrogel was determined to be 10 and 20 min, respectively for scaffolds with and without HA, indicating the successful formation of the gel within a reasonable time to the FRESH technique. The flow behavior of the hydrogel was found to be pseudoplastic, following a non-Newtonian flow pattern with Farrow's constant (N) values of 1.708 and 1.853 for scaffolds with and without hydroxyapatite, respectively. In terms of drug release, scaffolds prepared with and without hydroxyapatite reached nearly 100% of zoledronic acid release in 360 h and 48 h, respectively. The cell viability test on human osteosarcoma MG-63 cells using MTT assay has shown increased cell viability % in the case of composite hydrogel, indicating biocompatibility of the scaffold. Overall, this study successfully developed a composite thermosensitive hydrogel loaded with zoledronic acid for bone regeneration applications and was 3D printed using the FRESH 3D printing technique. The results of this study provide valuable insights into the potential use of this composite hydrogel for future biomedical applications.

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of the epithelia comprising the ductal network of the pancreas. During disease progression, PDAC tumors recruit fibroblasts that promote fibrosis, increasing local tissue stiffness and subjecting epithelial cells to increased compressive forces. Previous in vitro studies have documented cytoskeletal and nuclear adaptation following compressive stresses in two-dimensional (2D) and three-dimensional (3D) environments. However, a comparison of the responses of normal and tumor-derived ductal epithelia to physiologically relevant confinement remains underexplored, especially in 3D organoids. Here we control confinement with an engineered 3D microenvironment composed of Matrigel mixed with a low yield stress granular microgel. Normal and tumor-derived murine pancreas organoids (normal and tumor) were cultured for 48 h within this composite 3D environment or in pure Matrigel to investigate the effects of confinement on morphogenesis and lumen expansion. In confinement, tumor organoids (mT) formed a lumen that expanded rapidly, whereas normal organoids (mN) expanded more slowly. Moreover, a majority of normal organoids in more-confined conditions exhibited an inverted apicobasal polarity compared to those in less-confined conditions. Tumor organoids exhibited a collective "pulsing" behavior that increased in confinement. These pulses generated forces sufficient to locally overcome the yield stress of the microgels in the direction of organoid expansion. Normal organoids more commonly exhibit unidirectional rotation. Our in vitro microgel confinement platform enabled the discovery of two distinct modes of collective force generation in organoids that may shed light on the mutual interactions between tumors and the microenvironment. These insights into in vitro dynamics may deepen our understanding of how the confinement of healthy cells within a fibrotic tumor niche disrupts tissue organization and function in vivo.

Advancing three-dimensional (3D) tissue constructs is central to creating in vitro models and engineered tissues that recapitulate biology. Materials that are permissive to cellular behaviors, including proliferation, morphogenesis of multicellular structures, and motility, will support the emergence of tissue structures. Granular hydrogels in which there is no interparticle cross-linking exhibit dynamic properties that may be permissive to such cellular behaviors. However, designing granular hydrogels that lack interparticle cross-linking but support cellular self-organization remains underexplored relative to granular systems stabilized by interparticle cross-linking. In this study, we developed a polyethylene glycol-based granular hydrogel system, with average particle diameters under 40 μm. This granular hydrogel exhibited bulk stress-relaxing behaviors and compatibility with custom microdevices to sustain cell cultures without degradation. The system was studied in conjunction with cocultures of endothelial cells and fibroblasts, known for their spontaneous network formation. Cross-linking, porosity, and cell-adhesive ligands (such as RGD) were manipulated to control system properties. Toward supporting cellular activity, increased porosity was found to enhance the formation of cellular networks, whereas RGD reduced network formation in the system studied. This research highlights the potential of un-cross-linked granular systems to support morphogenetic processes, like vasculogenesis and tissue maturation, offering insights into material design for 3D cell culture systems.

Cell-laden bioprinting is a promising biofabrication strategy for regenerating bioactive transplants to address organ donor shortages. However, there has been little success in reproducing transplantable artificial organs with multiple distinctive cell types and physiologically relevant architecture. In this study, an omnidirectional printing embedded network (OPEN) is presented as a support medium for embedded 3D printing. The medium is state-of-the-art due to its one-step preparation, fast removal, and versatile ink compatibility. To test the feasibility of OPEN, exceptional primary mouse hepatocytes (PMHs) and endothelial cell line-C166, were used to print hepatospheroid-encapsulated-artificial livers (HEALs) with vein structures following predesigned anatomy-based printing paths in OPEN. PMHs self-organized into hepatocyte spheroids within the ink matrix, whereas the entire cross-linked structure remained intact for a minimum of ten days of cultivation. Cultivated HEALs maintained mature hepatic functions and marker gene expression at a higher level than conventional 2D and 3D conditions in vitro. HEALs with C166-laden vein structures promoted endogenous neovascularization in vivo compared with hepatospheroid-only liver prints within two weeks of transplantation. Collectively, the proposed platform enables the manufacture of bioactive tissues or organs resembling anatomical architecture, and has broad implications for liver function replacement in clinical applications.

Engineering biomimetic tissue implants with human induced pluripotent stem cells (hiPSCs) holds promise for repairing volumetric tissue loss. However, these implants face challenges in regenerative capability, survival, and geometric scalability at large-scale injury sites. Here, we present scalable vessel-integrated muscle-like lattices (VMLs), containing dense and aligned hiPSC-derived myofibers alongside passively perfusable vessel-like microchannels inside an endomysium-like supporting matrix using an embedded multimaterial bioprinting technology. The contractile and millimeter-long myofibers are created in mechanically tailored and nanofibrous extracellular matrix-based hydrogels. Incorporating vessel-like lattice enhances myofiber maturation in vitro and guides host vessel invasion in vivo, improving implant integration. Consequently, we demonstrate successful de novo muscle formation and muscle function restoration through a combinatorial effect between improved graft-host integration and its increased release of paracrine factors within volumetric muscle loss injury models. The proposed modular bioprinting technology enables scaling up to centimeter-sized prevascularized hiPSC-derived muscle tissues with custom geometries for next-generation muscle regenerative therapies.

All-aqueous embedded 3D printing, which involves extruding inks in an aqueous bath, has emerged as a transformative platform for the freeform fabrication of 3D constructs with precise control. The use of a supporting bath not only enables the printing of arbitrarily designed 3D constructs but also broadens ink selection for various soft matters, advancing the wide application of this technology. This review focuses on recent progress in the freeform preparation of 3D constructs using all-aqueous embedded 3D printing. It begins by discussing the significance of ultralow interfacial tension in all-liquid embedded printing and highlights the fundamental concepts and properties of all-aqueous system. The review then introduces recent advances in all-aqueous embedded 3D printing and clarifies the key factors affecting printing stability and shape fidelity, aiming to guide expansion and assessment of emerging printing systems used for various representative applications. Furthermore, it proposes the potential scope and applications of this technology, including in vitro models, cytomimetic microreactors, and soft ionic electronics. Finally, the review discusses the challenges facing current all-aqueous embedded 3D printing and offers future perspectives on possible improvements and developments.

Engineering 3D tissue-like constructs for applications such as regenerative medicine remains a major challenge in biomedical research. Recently, self-healing, viscoplastic fluids have been introduced as suspension media to allow lower viscosity, water-rich bioinks to be printed within them for the fabrication of more biomimetic structures. Here, we present gellan gum granular gels produced through the application of shear during gelation, as a candidate suspension medium. We demonstrate that these granular gels exhibit viscoplasticity over a wide range of temperatures, permitting their use for 3D bioprinting of filaments and droplets at low (4°C) as well as physiological temperatures. These granular gels exhibit very low yield stresses (down to 0.4 Pa) which facilitated printing at print speeds up to 60 mm.s-1. Furthermore, we demonstrate the printing of cell-laden droplets maintained over 7 days to show the potential for multiple days of cell culture, as well as the fabrication of hydrogel features within a crosslinkable version of the suspension medium containing granular gellan gum and gelatine-methacryloyl. The combination of ease of preparation, high printing speed, wide temperature tolerance, and crosslinkability makes this gellan gum sheared through cooling-induced gelation an attractive candidate for suspended biofabrication.

In the body, capillary beds fulfill the metabolic needs of cells by acting as the sites of diffusive transport for vital gasses and nutrients. In artificial tissues, replicating the scale and complexity of capillaries has proved challenging, especially in a three-dimensional context. In order to better develop thick artificial tissues, it will be necessary to recreate both the form and function of capillaries. Here we demonstrate a top-down method of patterning hydrogels using sacrificial templates formed from thermoresponsive microfibers whose size and architecture approach those of natural capillaries. Within the resulting microchannels, we cultured endothelial monolayers that remain viable for over three weeks and exhibited functional barrier properties. Additionally, we cultured endothelialized microchannels within hydrogels containing fibroblasts and characterized the viability of the co-cultures to demonstrate this approach's potential when applied to cell-laden hydrogels. This method represents a step forward in the evolution of artificial tissues and a path towards producing viable capillary-scale microvasculature for engineered organs.

In embedded 3D printing (EMB3D), a nozzle extrudes continuous filaments inside of a viscoelastic support bath. Compared to other extrusion processes, EMB3D enables softer structures and print paths that conform better to the shape of the part, allowing for complex structures such as tissues and organs. However, strategies for high-quality dimensional accuracy and mechanical properties remain undocumented in EMB3D. This work uses computational fluid dynamics simulations in OpenFOAM to probe the underlying physics behind two processes: deformation of the printed part due to nearby nozzle motion and fusion between neighboring filaments during printing. Through simulations, we disentangle yielding from viscous dissipation, and we isolate interfacial tension effects from rheology effects, which are difficult to separate in experiments. Critically, these simulations find that disturbance and fusion are controlled by the flow of support fluid around the nozzle. To avoid part deformation, the nozzle must remain far from existing parts during non-printing moves, moreso when traveling next to the part than above the part and especially when the interfacial tension between the ink and support is non-zero. Additionally, because support can become trapped between filaments at zero interfacial tension, the spacing between filaments must be tight enough to produce over-printing, or printing too much material for the designed space. In non-Newtonian fluids, spacings for vertical walls must be even tighter than spacings for horizontal planes. At these spacings, printing a new filament sometimes creates and sometimes mitigates shape defects in the old filament. While non-zero ink-support interfacial tensions produce better inter-filament fusion than zero interfacial tension, interfacial tension also produces shape defects. Slicing algorithms that consider these unique EMB3D defects are needed to improve mechanical properties and dimensional accuracy of bioprinted constructs.

Recent explorations of bioengineering have generated new concepts and strategies for the processing of soft and functional materials. Droplet assembly techniques can address problems in the construction of extremely soft architectures by expanding the manufacturing capabilities using droplets containing liquid or hydrogels including weak hydrogels. This Perspective sets out to provide a brief overview of this growing field, and discusses the challenges and opportunities ahead. The study highlights the recent key advances of materials and architectures from hitherto effective droplet-assembly technologies, as well as the applications in biomedical and bioengineering fields from artificial tissues to bioreactors. It is envisaged that these assembled architectures, as nature-inspired models, will stimulate the discovery of biomaterials and miniaturized platforms for interdisciplinary research in health, biotechnology, and sustainability.

4D printing of hydrogels is an emerging technology used to fabricate shape-morphing soft materials that are responsive to external stimuli for use in soft robotics and biomedical applications. Soft materials are technically challenging to process with current 4D printing methods, which limits the design and actuation potential of printed structures. Here, a simple multi-material 4D printing technique is developed that combines dynamic temperature-responsive granular hydrogel inks based on hyaluronic acid, whose actuation is modulated via poly(N-isopropylacrylamide) crosslinker design, with granular suspension bath printing that provides structural support during and after the printing process. Granular hydrogels are easily extruded upon jamming due to their shear-thinning properties and their porous structure enables rapid actuation kinetics (i.e., seconds). Granular suspension baths support responsive ink deposition into complex patterns due to shear-yielding to fabricate multi-material objects that can be post-crosslinked to obtain anisotropic shape transformations. Dynamic actuation is explored by varying printing patterns and bath shapes, achieving complex shape transformations such as 'S'-shaped and hemisphere structures. Furthermore, stepwise actuation is programmed into multi-material structures by using microgels with varied transition temperatures. Overall, this approach offers a simple method to fabricate programmable soft actuators with rapid kinetics and precise control over shape morphing.

Suspension bath-based 3D bioprinting (SUB3BP) is effective in creating engineered vascular structures. The transfer of oxygen and nutrients via engineered vascular networks is necessary for tissue or organ survival and integration following transplantation. Existing SUB3BP techniques face challenges in fabricating hierarchical structures with multicellular organization, including issues related to suspension bath removal, restricted material choices, and low accuracy. A next-generation SUB3BP technique that is removal-free and multicellular is presented. A simple, storable, stable, and scalable starch hydrogel design leverages the diverse spectrum of hydrogels available for use in SUB3BP. Starch granules (8.1 µm) create vascular structures with minimal surface roughness (2.5 µm) that simulate more natural vessel walls compared to prior research. The development of cells and organoids, as well as the bioprinting of multicellular skin models with vasculature, demonstrates that starch suspension baths eliminate the removal process and have the potential for fabricating artificial tissue with a hierarchical structure and multicellular distribution.

Particle surface chemistry and internal softness are two fundamental parameters in governing the mechanical properties of dense colloidal suspensions, dictating structure and flow, therefore of interest from materials fabrication to processing.

Here, we modulate softness by tuning the crosslinker content of poly(N-isopropylacrylamide) microgels, and we adjust their surface properties by co-polymerization with polyethylene glycol chains, controlling adhesion, friction and fuzziness. We investigate the distinct effects of these parameters on the entire mechanical response from restructuring to complete fluidization of jammed samples at varying packing fractions under large-amplitude oscillatory shear experiments, and we complement rheological data with colloidal-probe atomic force microscopy to unravel variations in the particles' surface properties.

Our results indicate that surface properties play a fundamental role at smaller packings; decreasing adhesion and friction at contact causes the samples to yield and fluidify in a lower deformation range. Instead, increasing softness or fuzziness has a similar effect at ultra-high densities, making suspensions able to better adapt to the applied shear and reach complete fluidization over a larger deformation range. These findings shed new light on the single-particle parameters governing the mechanical response of dense suspensions subjected to deformation, offering synthetic approaches to design materials with tailored mechanical properties.

Embedded 3D printing (EMB3D) is an additive manufacturing technique that enables complex fabrication of soft materials including tissues and silicones. In EMB3D, a nozzle writes continuous filaments into a support bath consisting of a yield stress fluid. Lack of fusion defects between filaments can occur because the nozzle pushes support fluid into existing filaments, preventing coalescence. Interfacial tension was previously proposed as a tool to drive interfilament fusion. However, interfacial tension can also drive rupture and shrinkage of printed filaments. Here, we evaluate the efficacy of interfacial tension as a tool to control defects in EMB3D. Using polydimethylsiloxane (PDMS)-based inks with varying amounts of fumed silica and surfactant, printed into Laponite in water supports, we evaluate the effect of rheology, interfacial tension, print speeds, and interfilament spacings on defects. We print pairs of parallel filaments at varying orientations in the bath and use digital image analysis to quantify shrinkage, rupture, fusion, and positioning defects. By comparing disturbed filaments to printed pairs of filaments, we disentangle the effects of nozzle movement and filament extrusion. Critically, we find that capillary instabilities and interfilament fusion scale with the balance between support rheology and interfacial tension. Less viscous supports and higher interfacial tensions lead to more shrinkage and rupture at all points in the printing process, from relaxation after writing, to disturbance of the line, to writing of a second line. It is necessary to overextrude material to achieve interfilament fusion, particularly at high support viscosities and low interfacial tensions. Finally, fusion quality varies with printing orientation, and writing neighboring filaments causes displacement of existing structures. As such, specialized slicers are needed for EMB3D that consider the tighter spacings and orientation-dependent spacings necessary to achieve precise control over printed shapes.

Granular hydrogels, formed by jamming microgels suspension, are promising materials for three-dimensional bioprinting applications. Despite their extensive use as support materials for embedded bioprinting, the influence of the particle's physical properties on the macroscale viscoelasticity on one hand and on the printing performance on the other hand remains unclear. Herein, we investigate the linear and nonlinear rheology of κ-carrageenan granular hydrogel through small- and large-amplitude oscillatory shear measurements. We tuned the granular hydrogel's properties by changing the stiffness (soft, medium, stiff) and the packing density of the individual microgels. Characterizations in the linear viscoelasticity regime revealed that the storage modulus of granular hydrogels is not a simple function of microgel stiffness and depends on the microgel packing density. At larger strains, increasing the microgel stiffness reduced the energy dissipation of the granular beds and increased the solid-fluid transition point. To understand how the different rheological properties of granular support materials influence embedded bioprinting, we examined the printing fidelity and cellular filament shrinkage within the granular beds. We found that microgels with low packing density diminished the printing quality, while stiff microgels promoted filament roughness. In addition, we found that highly packed stiff microgels significantly reduced the postprinting contraction of cellular filaments. Overall, this work provides a comprehensive knowledge of the rheology of granular hydrogels that can be used to rationally design support beds for bioprinting applications with specific characteristics.

Organoids are powerful models of tissue physiology, yet their applications remain limited due to their relatively simple morphology and high organoid-to-organoid structural variability. To address these limitations we developed a soft, composite yield-stress extracellular matrix that supports optimal organoid morphogenesis following freeform 3D bioprinting of cell slurries at tissue-like densities. The material is designed with two temperature regimes: at 4 °C it exhibits reversible yield-stress behavior to support long printing times without compromising cell viability. When transferred to cell culture at 37 °C, the material cross-links and exhibits similar viscoelasticity and plasticity to basement membrane extracts such as Matrigel. We first characterize the rheological properties of MAGIC matrices that optimize organoid morphogenesis, including low stiffness and high stress relaxation. Next, we combine this material with a custom piezoelectric printhead that allows more reproducible and robust self-organization from uniform and spatially organized tissue "seeds." We apply MAGIC matrix bioprinting for high-throughput generation of intestinal, mammary, vascular, salivary gland, and brain organoid arrays that are structurally similar to those grown in pure Matrigel, but exhibit dramatically improved homogeneity in organoid size, shape, maturation time, and efficiency of morphogenesis. The flexibility of this method and material enabled fabrication of fully 3D microphysiological systems, including perfusable organoid tubes that experience cyclic 3D strain in response to pressurization. Furthermore, the reproducibility of organoid structure increased the statistical power of a drug response assay by up to 8 orders-of-magnitude for a given number of comparisons. Combined, these advances lay the foundation for the efficient fabrication of complex tissue morphologies by canalizing their self-organization in both space and time.

Microextrusion-based 3D bioprinting into support baths has emerged as a promising technique to pattern soft biomaterials into complex, macroscopic structures. It is hypothesized that interactions between inks and support baths, which are often composed of granular microgels, can be modulated to control the microscopic structure within these macroscopic-printed constructs. Using printed collagen bioinks crosslinked either through physical self-assembly or bioorthogonal covalent chemistry, it is demonstrated that microscopic porosity is introduced into collagen inks printed into microgel support baths but not bulk gel support baths. The overall porosity is governed by the ratio between the ink's shear viscosity and the microgel support bath's zero-shear viscosity. By adjusting the flow rate during extrusion, the ink's shear viscosity is modulated, thus controlling the extent of microscopic porosity independent of the ink composition. For covalently crosslinked collagen, printing into support baths comprised of gelatin microgels (15-50 µm) results in large pores (≈40 µm) that allow human corneal mesenchymal stromal cells (MSCs) to readily spread, while control samples of cast collagen or collagen printed in non-granular support baths do not allow cell spreading. Taken together, these data demonstrate a new method to impart controlled microscale porosity into 3D printed hydrogels using granular microgel support baths.

Granular hydrogels, which are formed by densely packing microgels, are promising materials for bioprinting due to their extrudability, porosity, and modularity. However, the multidimensional parameter space involved in granular hydrogel design makes material optimization challenging. For example, design inputs such as microgel morphology, packing density, or stiffness can influence multiple rheological properties that govern printability and the behavior of encapsulated cells. This review provides an overview of fabrication methods for granular hydrogels, and then examines how important design inputs can influence material properties associated with printability and cellular responses across multiple scales. Recent applications of granular design principles in bioink engineering are described, including the development of granular support hydrogels for embedded printing. Further, the paper provides an overview of how key physical properties of granular hydrogels can influence cellular responses, highlighting the advantages of granular materials for promoting cell and tissue maturation after the printing process. Finally, potential future directions for advancing the design of granular hydrogels for bioprinting are discussed.

Printing human tissues and organs replete with biomimetic vascular networks is of growing interest. While it is possible to embed perfusable channels within acellular and densely cellular matrices, they do not currently possess the biomimetic architectures found in native vessels. Here, coaxial sacrificial writing into functional tissues (co-SWIFT) is developed, an embedded bioprinting method capable of generating hierarchically branching, multilayered vascular networks within both granular hydrogel and densely cellular matrices. Coaxial printheads are designed with an extended core-shell configuration to facilitate robust core-core and shell-shell interconnections between printed branching vessels during embedded bioprinting. Using optimized core-shell ink combinations, biomimetic vessels composed of a smooth muscle cell-laden shell that surrounds perfusable lumens are coaxially printed into granular matrices composed of: 1) transparent alginate microparticles, 2) sacrificial microparticle-laden collagen, or 3) cardiac spheroids derived from human induced pluripotent stem cells. Biomimetic blood vessels that exhibit good barrier function are produced by seeding these interconnected lumens with a confluent layer of endothelial cells. Importantly, it is found that co-SWIFT cardiac tissues mature under perfusion, beat synchronously, and exhibit a cardio-effective drug response in vitro. This advance opens new avenues for the scalable biomanufacturing of vascularized organ-specific tissues for drug testing, disease modeling, and therapeutic use.

Fibroblast growth factor 23 (FGF23) plays a crucial role in managing renal phosphate and the synthesis of 1,25(OH)2-vitamin D3, which is essential for bone homeostasis. Developing robust in vitro systems to study FGF23-regulating mechanisms is crucial for advancing our knowledge and identifying potential therapeutic targets. The traditional in vitro 2D culture system results in relatively low expression of FGF23, complicating further exploration of its regulatory mechanisms and potential therapeutic targets. Herein, we reported a high-throughput approach to generate preosteoblastic cell spheroids with enhanced FGF23 production. For this purpose, murine preosteoblast cell line (MC3T3-E1) was cultured in our previously reported nonadherent microwells (200 µm in diameter, 148 µm in depth, and 100 µm space in between) and self-assembled into spheroids with a diameter of 92.3 ± 15.0 µm after 24 h. Compared with monolayer culture, the MC3T3-E1 spheroids showed a significant upregulation of FGF23 in both gene and protein levels after 24 h of serum-free induction. RNA sequencing and western blotting analysis further suggested that the enhanced FGF23 production in MC3T3-E1 spheroids was attributed to the activation of the parathyroid hormone (PTH)/PTH1R signaling pathway. Impressively, inhibition of PTH signaling through small molecular inhibitors or short hairpin RNA targeting PTH1R effectively reduced FGF23 production. In summary, the current study revealed the efficacy of the high-throughput formation of preosteoblast cell spheroid in stimulating FGF23 expression for mechanistic studies. Importantly, our findings highlight the potential of the current 3D spheroid system for target identification and drug discovery.

This study explores the bioprinting of a smooth muscle cell-only bioink into ionically crosslinked oxidized methacrylated alginate (OMA) microgel baths to create self-supporting vascular tissues. The impact of OMA microgel support bath methacrylation degree and cell-only bioink dispensing parameters on tissue formation, remodeling, structure and strength was investigated. We hypothesized that reducing dispensing tip diameter from 27 G (210μm) to 30 G (159μm) for cell-only bioink dispensing would reduce tissue wall thickness and improve the consistency of tissue dimensions while maintaining cell viability. Printing with 30 G tips resulted in decreased mean wall thickness (318.6μm) without compromising mean cell viability (94.8%). Histological analysis of cell-only smooth muscle tissues cultured for 14 d in OMA support baths exhibited decreased wall thickness using 30 G dispensing tips, which correlated with increased collagen deposition and alignment. In addition, a TUNEL assay indicated a decrease in cell death in tissues printed with thinner (30 G) dispensing tips. Mechanical testing demonstrated that tissues printed with a 30 G dispensing tip exhibit an increase in ultimate tensile strength compared to those printed with a 27 G dispensing tip. Overall, these findings highlight the importance of precise control over bioprinting parameters to generate mechanically robust tissues when using cell-only bioinks dispensed and cultured within hydrogel support baths. The ability to control print dimensions using cell-only bioinks may enable bioprinting of more complex soft tissue geometries to generatein vitrotissue models.

Engineering functional tissues and organs remains a fundamental pursuit in bio-fabrication. However, the accurate constitution of complex shapes and internal anatomical features of specific organs, including their intricate blood vessels and nerves, remains a significant challenge. Inspired by the Matryoshka doll, here a new method called "Intra-Embedded Bioprinting (IEB)" is introduced building upon existing embedded bioprinting methods. a xanthan gum-based material is used which served a dual role as both a bioprintable ink and a support bath, due to its unique shear-thinning and self-healing properties. IEB's capabilities in organ modeling, creating a miniaturized replica of a pancreas using a photocrosslinkable silicone composite is demonstrated. Further, a head phantom and a Matryoshka doll are 3D printed, exemplifying IEB's capability to manufacture intricate, nested structures. Toward the use case of IEB and employing an innovative coupling strategy between extrusion-based and aspiration-assisted bioprinting, a breast tumor model that included a central channel mimicking a blood vessel, with tumor spheroids bioprinted in proximity is developed. Validation using a clinically-available chemotherapeutic drug illustrated its efficacy in reducing the tumor volume via perfusion over time. This method opens a new way of bioprinting enabling the creation of complex-shaped organs with internal anatomical features.

Analogous of pixels to two-dimensional pictures, voxels-in the form of either small cubes or spheres-are the basic building blocks of three-dimensional objects. However, precise manipulation of viscoelastic bio-ink voxels in three-dimensional space represents a grand challenge in both soft matter science and biomanufacturing. Here, we present a voxelated bioprinting technology that enables the digital assembly of interpenetrating double-network hydrogel droplets made of polyacrylamide/alginate-based or hyaluronic acid/alginate-based polymers. The hydrogels are crosslinked via additive-free and biofriendly click reaction between a pair of stoichiometrically matched polymers carrying norbornene and tetrazine groups, respectively. We develop theoretical frameworks to describe the crosslinking kinetics and stiffness of the hydrogels, and construct a diagram-of-state to delineate their mechanical properties. Multi-channel print nozzles are developed to allow on-demand mixing of highly viscoelastic bio-inks without significantly impairing cell viability. Further, we showcase the distinctive capability of voxelated bioprinting by creating highly complex three-dimensional structures such as a hollow sphere composed of interconnected yet distinguishable hydrogel particles. Finally, we validate the cytocompatibility and in vivo stability of the printed double-network scaffolds through cell encapsulation and animal transplantation.

The lymphatic system maintains tissue fluid homeostasis and it is involved in the transport of nutrients and immunosurveillance. It also plays a pivotal role in both pathological and regenerative processes. Lymphatic development in the embryo occurs by polarization and proliferation of lymphatic endothelial cells from the lymph sacs, that is, lymphangiogenesis. Alternatively, lymphvasculogenesis further contributes to the formation of lymphatic vessels. In adult tissues, lymphatic formation rarely occurs under physiological conditions, being restricted to pathological processes. In lymphvasculogenesis, progenitor cells seem to be a source of lymphatic vessels. Indeed, mesenchymal stem cells, adipose stem cells, endothelial progenitor cells, and colony-forming endothelial cells are able to promote lymphatic regeneration by different mechanisms, such as direct differentiation and paracrine effects. In this review, we summarize what is known on the diverse stem/progenitor cell niches available for the lymphatic system, emphasizing the potential that these cells hold for lymphatic tissue engineering through 3D bioprinting and their translation to clinical application.