Vasculogenic assembly of 3D capillary networks remains a promising approach to vascularizing tissue-engineered grafts, a significant outstanding challenge in tissue engineering and regenerative medicine. Current approaches for vasculogenic assembly rely on the inclusion of supporting mesenchymal cells alongside endothelial cells, co-encapsulated within vasculo-conducive materials such as low-density fibrin hydrogels. Here, we established a material-based approach to circumvent the need for supporting mesenchymal cells and report that the inclusion of synthetic matrix fibers in dense (>3 mg mL-1) 3D fibrin hydrogels can enhance vasculogenic assembly in endothelial cell monocultures. Surprisingly, we found that the addition of non-cell-adhesive synthetic matrix fibers compared to cell-adhesive synthetic fibers best encouraged vasculogenic assembly, proliferation, lumenogenesis, a vasculogenic transcriptional program, and additionally promoted cell-matrix interactions and intercellular force transmission. Implanting fiber-reinforced prevascularized constructs to assess graft-host vascular integration, we demonstrate additive effects of enhanced vascular network assembly during in vitro pre-culture, fiber-mediated improvements in endothelial cell survival and vascular maintenance post-implantation, and enhanced host cell infiltration that collectively enabled graft vessel integration with host circulation. This work establishes synthetic matrix fibers as an inexpensive alternative to sourcing and expanding secondary supporting cell types for the prevascularization of tissue constructs.

For single ventricle congenital heart patients, Fontan surgery is the final stage in a series of palliative procedures, bypassing the heart to enable passive flow of de-oxygenated blood from the inferior vena cava (IVC) to the pulmonary arteries. This circulation leads to severely elevated central venous pressure, diminished cardiac output, and thus numerous sequelae and premature mortality. To address these issues, we propose a bioprinted pulsatile conduit to provide a secondary power source for the Fontan circulation. A multiphysics computational framework was developed to predict conduit performance and to guide design prior to printing. Physics components included electrophysiology, cardiomyocyte contractility, and fluid-structure interaction coupled to a closed-loop lumped parameter network representing Fontan physiology. A range of myocardial contractility was considered and simulated. The initial conduit design with adult ventricular cardiomyocyte contractility values coupled to a Purkinje network demonstrated potential to reduce liver (IVC) pressure from 16.4 to 9.3 mmHg and increase cardiac output by 29%. After systematically assessing the impacts of contraction duration, fiber direction, and valve placement on conduit performance, we identified a favorable design that successfully reduces liver pressure to 7.3 mmHg and increases cardiac output by 38%, almost normalizing adverse hemodynamics in the lower venous circulation. Valves at the input and output of the conduit are essential to achieve these satisfactory results; without valves, performance is compromised. However, a potential drawback of the design is the elevation of superior vena cava (SVC) pressure, which varies linearly with liver pressure reduction.

The loss of organ function following traumatic injury is often irreversible and the demand for organ replacements continues to exceed supply. This discrepancy has driven the development of therapies and engineered tissues for the repair or replacement of damaged tissues. However, the survival of engineered tissues is constrained by the challenge of establishing a functional vasculature. Efforts have therefore focused on strategies that induce vascularization in tissue implants or stimulate vascular growth in recipients of the therapies. Here we discuss recent advances in vascular biology, biomaterials chemistry and 3D printing techniques for vascular patterning in engineered tissues. For tissue regeneration to be clinically viable, vascular formation must be guided across scales ranging from micrometres to millimetres through biological, chemical and physical approaches.

The development of perfusable and multiscale vascular networks remains one of the largest challenges in tissue engineering. As such, there is a need for the creation of customizable and facile methods to produce robustly vascularized constructs. In this study, secondarily crosslinkable (clickable) poly(ethylene glycol)-norbornene (PEGNB) microbeads were produced and evaluated for their ability to sequentially support suspension bioprinting and microvascular self-assembly towards the aim of engineering hierarchical vasculature. The clickable PEGNB microbead slurry exhibited mechanical behavior suitable for suspension bioprinting of sacrificial bioinks, could be UV crosslinked into a granular construct post-print, and withstood evacuation of the bioink and subsequent perfusion of the patterned void space. Endothelial and stromal cells co-embedded within jammed RGD-modified PEGNB microbead slurries assembled into capillary-scale vasculature after secondary crosslinking of the beads into granular constructs, with endothelial tubules forming within the interstitial space between microbeads and supported by the perivascular association of the stromal cells. Microvascular self-assembly was not impacted by printing sacrificial bioinks into the cell-laden microbead support bath before UV crosslinking. Collectively, these results demonstrate that clickable PEGNB microbeads are a versatile substrate for both suspension printing and microvascular culture and may be the foundation for a promising methodology to engineer hierarchical vasculature. STATEMENT OF SIGNIFICANCE: In this study, we leveraged and combined advances in microgel biomaterials, granular hydrogels, suspension bioprinting, and vascular biology to create relatively large volume (>500 mm3) vascularized constructs. We fabricated secondarily crosslinkable (clickable) poly(ethylene glycol)-norbornene (PEGNB) microbeads and demonstrated their ability to sequentially support suspension bioprinting and microvascular self-assembly towards the aim of engineering hierarchical vasculature. To the best of our knowledge, this is the first study that uses PEG microgels as supportive materials for bioprinting, and one of the first papers to document microvascular self-assembly within granular constructs. The combination of top-down and bottom-up approaches within a single construct represents a significant and innovative contribution that we believe will be of broad interest to the biomaterials and regenerative medicine communities.

Engineering functional cellular tissue components holds great promise in regenerative medicine. Collagen I, a key scaffolding material in bodily tissues, presents challenges in controlling its assembly kinetics in a biocompatible manner in vitro, restricting its use as a primary scaffold or adhesive in cellular biofabrication. Here we report a collagen fabrication method termed as tunable rapid assembly of collagenous elements that leverages macromolecular crowding to achieve the instant assembly of unmodified collagen. By applying an inert crowder to accelerate the liquid-gel transition of collagen, our method enables the high-throughput creation of physiological collagen constructs across length scales-from micro to macro-and facilitates cell self-assembly and morphogenesis through the generation of tunable multiscale architectural cues. With high biocompatibility and rapid gelation kinetics, the tunable rapid assembly of collagenous elements method also offers a versatile bioprinting approach for collagen over a wide concentration range, enabling the direct printing of cellular tissues using pH-neutral, bioactive collagen bioinks and achieving both structural complexity and biofunctionality. This work broadens the scope of controllable multiscale biofabrication for tissues across various organ systems using unmodified collagen.

By monitoring brain neural signals, neural recorders allow for the study of neurological mechanisms underlying specific behavioural and cognitive states. However, the large brain volumes of non-human primates and their extensive range of uncontrolled movements and inherent wildness make it difficult to carry out covert and long-term recording and analysis of deep-brain neural signals. Here we report the development and performance of a stealthy neural recorder for the study of naturalistic behaviours in non-human primates. The neural recorder includes a fully implantable wireless and battery-free module for the recording of local field potentials and accelerometry data in real time, a flexible 32-electrode neural probe with a resorbable insertion shuttle, and a repeater coil-based wireless-power-transfer system operating at the body scale. We used the device to record neurobehavioural data for over 1 month in a freely moving monkey and leveraged the recorded data to train an artificial intelligence model for the classification of the animals' eating behaviours.

Tissue engineering aims to develop tissue constructs as models or substitutes for native tissues. For organ-level biological studies and regenerative medicine applications, it is essential to fabricate tissue constructs with physiologically relevant cell densities (on the order of 10 million to 1 billion cells·ml-1), large size (centimeter scale and larger), and a controllable geometry to guide tissue maturation. State-of-the-art biofabrication methods, however, struggle to simultaneously meet all of these demands. The recently proposed acoustic holographic assembly (AHA) method shows promise, as it is compatible with culture media and enables the contactless, label-free, and volumetric assembly of biological cells in a predefined geometry within few minutes. Here we present an AHA biofabrication scheme designated for fabricating cell-dense, centimeter-scale, and arbitrarily-shaped tissue constructs using a compact benchtop instrument compatible with a biolab environment. We demonstrate the assembly of C2C12 myoblasts in gelatin methacryloyl (GelMA) into large and asymmetric branch-shaped constructs, which are rapidly formed with an average cell density of 40 million cells·ml-1and a local density of up to 260 million cells·ml-1. Featuring a high viability of 90.5 ± 4.3%, the assembled cell constructs are observed to grow within the GelMA hydrogel under perfusion over five days. Further, we show how AHA can-in a single step-assemble cells into layered and three-dimensional geometries inside standard cell culture labware. It can therefore help obtain engineered tissue constructs with structural and functional characteristics seen in more complex native tissues.

Blood vessels are hierarchical microchannels that transport nutrients and oxygen to different tissues and organs, while also eliminating metabolic waste from the body. Disorders of the vascular system impact both physiological and pathological processes. Conventional animal vascular models are complex, high-cost, time-consuming, and low-validity, which have limited the exploration of effective in vitro vascular microsystems. The morphologies of micro-scaled tubular structures and physiological properties of vascular tissues, including mechanical strength, thrombogenicity, and immunogenicity, can be mimicked in vitro by engineering strategies. This review highlights the state-of-the-art and advanced engineering strategies for in vitro vascular microsystems, covering the domains related to rational designs, manufacturing approaches, supporting materials, and organ-specific cell types. A broad range of biomedical applications of in vitro vascular microsystems are also summarized, including the recent advances in engineered vascularized tissues and organs for physiological and pathological study, drug screening, and personalized medicine. Moreover, the commercialization of in vitro vascular microsystems, the feasibility and limitations of current strategies and commercially available products, as well as perspectives on future directions for exploration, are elaborated. The in vitro modeling of vascular microsystems will facilitate rapid, robust, and efficient analysis in tissue engineering and broader regenerative medicine towards the development of personalized treatment approaches.

The vasculature plays a critical role in homeostasis and health as well as in the development and progression of a wide range of diseases, including cancer, cardiovascular diseases, metabolic diseases (and their complications), chronic inflammatory diseases, ophthalmic diseases, and neurodegenerative diseases. As such, the growth of the vasculature mediates normal development and physiology, as well as disease, when pathologically induced vessels are morphologically and functionally altered owing to an imbalance of angiogenesis-stimulating and angiogenesis-inhibiting factors. This review offers an overview of the angiogenic process and discusses recent findings that provide additional interesting nuances to this process, including the roles of intussusception and angiovasculogenesis, which may hold promise for future therapeutic interventions. In addition, we review the methodology, including those of in vitro and in vivo assays, which has helped build the vast amount of knowledge on angiogenesis available today and identify important remaining knowledge gaps that should be bridged through future research.

Cells sense physical cues from their environment and convert them into biochemical responses through mechanotransduction. Unlike solid tumours, the role of such forces in haematological cancers is underexplored. In this context, immune cells experience dynamic mechanical stimuli as they migrate, extravasate and home to specific tissues. Understanding how these forces shape B-cell function and malignancy represents a groundbreaking area of research. This review examines the key mechanosensory pathways and molecules involved in lymphocyte mechanotransduction, beginning with mechanosensory proteins at the plasma membrane, followed by intracellular signal propagation through the cytoskeleton, eventually highlighting the nucleus as a 'signal actuator'. Subsequently, we cover some measurement approaches and advanced systems to investigate tumour biomechanics, highlighting their application in the context of B cells. Finally, we focus on the implications of mechanobiology in leukaemia, identifying molecules involved in B-cell malignancies that could serve as potential 'mechano-targets' for personalised therapies. This review emphasises the need to understand how lymphocytes generate, sense and respond to mechanical stimuli, which could open avenues for future biomedical innovations. Impact statement Our review is particularly valuable in highlighting the underexplored role of mechanobiology in B cell function and malignancies, while also discussing emerging techniques that can advance this research area. It bridges mechanotransduction, immunology, and cancer biology in a way that will be of interest to researchers across these three main fields.

Blood vessel formation is a necessary part of bone tissue regeneration. MSCs-sEVs play a vital role in the in vivo bone regeneration strategy. However, natural MSCs-sEVs suffer from limited blood vessel formation potency, which makes it difficult to induce vascularized bone regeneration. Here, sEVs derived from magnesium cation-activated DPSCs (Mg2+-EVs) are purified and found to have superior potential in promoting endothelial cell migration and angiogenesis, as well as BMSC proliferation and osteogenesis. The beneficial effects of Mg2+-EVs could be attributed to the enrichment of miR-451a and the subsequent regulation and activation of AKT/eNOS signaling pathways. On this basis, Mg2+-EVs are delivered on β-TCP-modified GelMA scaffolds for slow release and better bioavailability. The rat cranial defect model verifies that GelMA/β-TCP with Mg2+-EVs has enhanced potential of inducing vascularized bone regeneration. The present study provides a cation-activated strategy to modulate the cargos and contents of MSC-derived sEVs, obtaining desirable vascular promotion and bone regeneration potential. Furthermore, the developed β-TCP-modified delivery scaffolds represent a promising strategy for efficient loading and slow-release delivery of sEVs for clinical translation.

Three-dimensional printing of microchannel networks mimicking native vasculature provides essential functions for biomedical applications. However, developing a highly "adaptable" technique - that can adjust to diverse materials choices, high shape accuracy, and broad size ranges - for producing physiologically responsive vasculature remains challenging. Here, an innovative hydrogen bond re-orientation (HyBRO) strategy for microchannel network fabrication is reported. By identifying interfacial instability of sacrificial material (SM) during embedding as a core limitation, this strategy prints the SM into an optimal "nonsolvent" to shape the desirable channel structure. In this process, the nonsolvent instantaneously switches the SM from forming hydrogen bonds with exterior water to forming interior linkages inside it. This transition protects the SM from external solvent "erosion" upon re-exposure to embedding material, inhibiting deformation. Consequently, this approach enables the creation of accurate (>90%), multiscale (10-fold), hierarchical microchannel networks, accommodating accurate printing of a wide range of ink materials - extending from typical hydrophilic polymers into non-typical hydrophobic ones. Further biological tests demonstrate that HyBRO-produced vasculature recapitulates not only essential endothelial barrier function but also delicate ion-channel responses to varying shear stresses, highlighting its potential for engineering physiologically responsive vasculature in broad applications.

Free-standing tissue structures tethered between pillars are powerful mechanobiology tools for studying cell contraction. To model interfaces ubiquitous in natural tissues and upgrade existing single-region suspended constructs, we developed Suspended Tissue Open Microfluidic Patterning (STOMP), a method to create multi-regional suspended tissues. STOMP uses open microfluidics and capillary pinning to pattern subregions within free-standing tissues, facilitating the study of complex tissue interfaces, such as diseased-healthy boundaries (e.g., fibrotic-healthy) and tissue-type interfaces (e.g., bone-ligament). We observed altered contractile dynamics in fibrotic-healthy engineered heart tissues compared to single-region tissues and differing contractility in bone-ligament enthesis constructs compared to single-tissue periodontal ligament models. STOMP is a versatile platform - surface tension-driven patterning removes material requirements common with other patterning methods (e.g., shear-thinning, photopolymerizable) allowing tissue generation in multiple geometries with native extracellular matrices and advanced four-dimensional (4D)- materials. STOMP combines the contractile functionality of suspended tissues with precise patterning, enabling dynamic and spatially controlled studies.

Skeletal muscle's elegant protein-based architecture powers motion throughout the animal kingdom, with its constituent actomyosin complexes driving intra- and extra-cellular motion. Classical motors and recently developed soft actuators cannot match the packing density and contractility of individual muscle fibers that scale to power the motion of ants and elephants alike. Accordingly, the interdisciplinary fields of robotics and tissue engineering have combined efforts to build living muscle actuators that can power a new class of robots to be more energy-efficient, dexterous, and safe than existing motor-powered and hydraulic paradigms. Doing so ethically and at scale─creating meter-scale tissue constructs from sustainable muscle progenitor cell lines─has inspired innovations in biomaterials and tissue culture methodology. We weave discussions of muscle cell biology, materials chemistry, tissue engineering, and biohybrid design to review the state of the art in soft actuator biofabrication. Looking forward, we outline a vision for meter-scale biohybrid robotic systems and tie discussions of recent progress to long-term research goals.

Additive manufacturing (AM) has revolutionized biomedical applications by enabling personalized designs, intricate geometries, and cost-effective solutions. This progress stems from interdisciplinary collaborations across medicine, biomaterials, engineering, artificial intelligence, and microelectronics. A pivotal aspect of AM is the development of materials that respond to stimuli such as heat, light, moisture, and chemical changes, paving the way for intelligent systems tailored to specific needs. Among the materials employed in AM, polymers have gained prominence due to their flexibility, synthetic versatility, and broad property spectrum. Their adaptability has made them the most widely used material class in AM processes, offering the potential for diverse applications, including surgical tools, structural composites, photovoltaic devices, and filtration systems. Despite this, integrating multiple polymer systems to achieve multifunctional and dynamic performance remains a significant challenge, highlighting the need for further research. This review explores the foundational principles of AM, emphasizing its application in tissue engineering and medical technologies. It provides an in-depth analysis of polymer systems, besides inorganic oxides and bioinks, and examines their unique properties, advantages, and limitations within the context of AM. Additionally, the review highlights emerging techniques like rapid prototyping and 3D printing, which hold promise for advancing biomedical applications. By addressing the critical factors influencing AM processes and proposing innovative approaches to polymer integration, this review aims to guide future research and development in the field. The insights presented here underscore the transformative potential of AM in creating dynamic, multifunctional systems to meet evolving biomedical and healthcare demands.

Heart failure (HF) affects over 64 million people globally, marked by high incidence and mortality rates. Accurate measurements of myocardial contractility are crucial for evaluating cardiac pathomechanisms and monitoring disease progression. Hydrogel sensing devices, known for their flexibility, programmable structures, biocompatibility, and cell adhesion, are ideal for studying cardiac function, minimizing disruption to cardiomyocytes, and supporting long-term culture and monitoring. These platforms, while employing diverse detection principles to accurately measure cell contractility, still face challenges in achieving long-term stability and durability. This review summarizes current methods for monitoring cardiomyocyte contractility, emphasizes the significant impact of substrate mechanical properties on cellular function, and explores recent advances in hydrogel-based platforms for monitoring cell contraction forces. It also discusses the technical challenges and future prospects for measuring cardiac systolic function with these devices.

Cardiac organoids offer sophisticated 3D structures that emulate key aspects of human heart development and function. This review traces the evolution of cardiac organoid technology, from early stem cell differentiation protocols to advanced bioengineering approaches. We discuss the methodologies for creating cardiac organoids, including self-organization techniques, biomaterial-based scaffolds, 3D bioprinting, and organ-on-chip platforms, which have significantly enhanced the structural complexity and physiological relevance of in vitro cardiac models. We examine their applications in fundamental research and medical innovations, highlighting their potential to transform our understanding of cardiac biology and pathology. The integration of multiple cell types, vascularization strategies, and maturation protocols has led to more faithful representations of the adult human heart. However, challenges remain in achieving full functional maturity and scalability. We critically assess the current limitations and outline future directions for advancing cardiac organoid technology. By providing a comprehensive analysis of the field, this review aims to catalyze further innovation in cardiac tissue engineering and facilitate its translation to clinical applications.

Free-standing tissue structures tethered between pillars are powerful mechanobiology tools for studying cell contraction. To model interfaces ubiquitous in natural tissues and upgrade existing single-region suspended constructs, we developed Suspended Tissue Open Microfluidic Patterning (STOMP), a method to create multiregional suspended tissues. STOMP uses open microfluidics and capillary pinning to pattern subregions within free-standing tissues, facilitating the study of complex tissue interfaces, such as diseased-healthy boundaries (e.g., fibrotic-healthy) and tissue-type interfaces (e.g., bone-ligament). We observed altered contractile dynamics in fibrotic-healthy engineered heart tissues compared to single-region tissues and differing contractility in bone-ligament enthesis constructs compared to single-tissue periodontal ligament models. STOMP is a versatile platform - surface tension-driven patterning removes material requirements common with other patterning methods (e.g., shear-thinning, photopolymerizable) allowing tissue generation in multiple geometries with native extracellular matrices and advanced 4D materials. STOMP combines the contractile functionality of suspended tissues with precise patterning, enabling dynamic and spatially controlled studies.

Creating perfusable channels within engineered tissues is crucial for the development of large-scale tissues. Unfortunately, existing technologies have not achieved uniformly distributed, perfusable networks on a large scale. To overcome this, we developed a method using a hollow fiber bioreactor (HFB) equipped with an array of closely packed semipermeable hollow fibers that function as artificial circulation systems, ensuring uniform nutrient and oxygen distribution throughout the tissue. Furthermore, the HFB includes microfabricated anchors for promoting cell alignment. When using active perfusion, biofabricated centimeter-scale chicken muscle tissue exhibited an elevated level of marker protein expression and sarcomere formation throughout the tissue, along with improved texture and flavor. In addition, a robotic-assisted fiber threading system was developed to achieve efficient assembly of the HFBs. Future full automation of this approach may revolutionize both the cultured meat industry and the tissue engineering field, which aims to create large-scale, tissue-engineered organs.

Cardiac tissue lacks regenerative capacity, making heart transplantation the primary treatment for end-stage heart failure. Engineered cardiac tissues developed through three-dimensional bioprinting (3DBP) offer a promising alternative. However, reproducing the native structure, cellular diversity, and functionality of cardiac tissue requires advanced cardiac bioinks. Major obstacles in CTE (cardiac tissue engineering) include accurately characterizing bioink properties, replicating the cardiac microenvironment, and achieving precise spatial organization. Optimizing bioink properties to closely mimic the extracellular matrix (ECM) is essential, as deviations may result in pathological effects. This review encompasses the rheological and electromechanical properties of bioinks and the function of the cardiac microenvironment in the design of functional cardiac constructs. Furthermore, it focuses on improving the rheological characteristics, printability, and functionality of bioinks, offering valuable perspectives for developing new bioinks especially designed for CTE.

3D bioprinting approaches offer highly versatile solutions to replicate living tissue and organ structures. While current bioprinting approaches can generate desired shapes and spatially determined patterns, the material selection for embedded bioprinting has remained limited, as it has relied on the use of viscous, shear-thinning, or liquid-like solid materials to create shape controlled constructs, which could then be modified downstream via multi-step processes. We here explore aqueous two-phase system stabilized 3D bioprinting of low viscous materials in combination with supramolecular complexation to fabricate intricate, perfusable engineered constructs that are both mechanically and chemically tunable in a single-step manner. To this end, we introduce Dex-TAB as a highly versatile backbone, that allows for mechanical and chemical tuning during as well as after printing. To showcase the printability as well as spatial chemical modification and mechanical tunability of this material, ejectability, and local/gradual or bulk functionalized interconnected tube shaped constructs were generated. Subsequently, we demonstrated that these functionalized channels could be printed directly into a syringe containing crosslinkable polymer solution, which upon ejection forms pre-patterned perfusable constructs. In short, we report that ATPS enabled low viscous 3D bioprinting can produce highly functional and even potentially minimally invasive injectable yet functionalized and perfusable constructs, which offers opportunities to advance various biofabrication applications.

Microvasculature is essential for the continued function of cells in tissue and is fundamental in the fields of tissue engineering, organ repair and drug screening. However, the fabrication of microvasculature is still challenging using existing strategies. Here, we developed a general PRINting Cell Embedded Sacrificial Strategy (PRINCESS) and successfully fabricated microvasculatures using degradable DNA biolubricant. This is the first demonstration of direct cell printing to fabricate microvasculature, which eliminates the need for a subsequent cell seeding process and the associated deficiencies. Utilizing the shear-thinning property of DNA hydrogels as a novel sacrificial, cell-laden biolubricant, we can print a 70 μm endothelialized microvasculature, breaking the limit of 100 μm. To our best knowledge, this is the smallest endothelialized microvasculature that has ever been bioprinted so far. In addition, the self-healing property of DNA hydrogels allows the creation of continuous branched structures. This strategy provides a new platform for constructing complex hierarchical vascular networks and offers new opportunity towards engineering thick tissues. The extremely low volume of sacrificial biolubricant paves the way for DNA hydrogels to be used in practical tissue engineering applications. The high-resolution bioprinting technique also exhibits great potential for printing lymphatics, retinas and neural networks in the future.

Several cartilage and bone organoids have been developed in vitro and in vivo using adult mesenchymal stromal/stem cells (MSCs) or pluripotent stem cells (PSCs) to mimic different phases of endochondral ossification (ECO), as one of the main processes driving skeletal development and growth. While cellular and molecular features of growth plate-like structures have been observed through the generation and in vivo implantation of hypertrophic cartilage tissues, no functional analogue or model of the growth plate has yet been engineered. Herein, after a brief introduction about the growth plate architecture and function, we summarize the recent progress in dissecting the biology of the growth plate and indicate the knowledge gaps to better understand the mechanisms of its development and maintenance. We then discuss how this knowledge could be integrated with state-of-art bioengineering approaches to generate a functional in vitro growth plate model.

The design of neural tissue models with architectural and biochemical relevance to native tissues opens the way for the fundamental study and development of therapies for many disorders with limited treatment options. Here, we systematically review the most recent literature on 3D bioprinted neural models, including their potential for use in drug screening. Neural tissues that model the central nervous system (CNS) from the relevant literature are reviewed with comprehensive summaries of each study, and discussion of the model types, bioinks and additives, cell types used, bioprinted construct shapes and culture time, and the characterization methods used. In this review, we accentuate the lack of standardization among characterization methods to analyze the functionality (including chemical, metabolic and other pathways) and mechanical relevance of the 3D bioprinted constructs, and discuss this as a critical area for future exploration. These gaps must be addressed for this technology to be applied for effective drug screening applications, despite its enormous potential for rapid and efficient drug screening. The future of biomimetic, 3D printed neural tissues is promising and evaluation of the in vivo relevance on multiple levels should be sought to adequately compare model performance and develop viable treatment options for neurodegenerative diseases, or other conditions that affect the CNS.

Advances in biofabrication have enabled the generation of freeform perfusable networks mimicking vasculature. However, key challenges remain in the effective endothelialization of these complex, vascular-like networks, including cell uniformity, seeding efficiency, and the ability to pattern multiple cell types. To overcome these challenges, we present an integrated fabrication and endothelialization strategy to directly generate branched, endothelial cell-lined networks using a diffusion-based, embedded 3D bioprinting process. In this strategy, a gelatin microparticle sacrificial ink delivering both cells and crosslinkers is extruded into a crosslinkable gel precursor support bath. A self-supporting, perfusable structure is formed by diffusion-induced crosslinking, after which the sacrificial ink is melted to allow cell release and adhesion to the printed lumen. This approach produces a uniform cell lining throughout networks with complex branching geometries, which are challenging to uniformly and efficiently endothelialize using conventional perfusion-based approaches. Furthermore, the biofabrication process enables high cell viability (>90%) and the formation of a confluent endothelial layer providing vascular-mimetic barrier function and shear stress response. Leveraging this strategy, we demonstrate for the first time the patterning of multiple endothelial cell types, including arterial and venous cells, within a single arterial-venous-like network. Altogether, this strategy enables the fabrication of multi-cellular engineered vasculature with enhanced geometric complexity and phenotypic heterogeneity.

Generation of in vitro tissue models with serially perfused hierarchical vasculature would allow greater control of fluid perfusion throughout the network and enable direct mechanistic investigation of vasculogenesis, angiogenesis, and vascular remodeling. In this work, we have developed a method to produce a closed, serially perfused, multiscale vessel network fully embedded within an acellular hydrogel, where flow through the capillary bed is required prior to fluid exit. We confirmed that the acellular and cellular gel-gel interface was functionally annealed without preventing or biasing cell migration and endothelial self-assembly. Multiscale connectivity of the vessel network was validated via high-resolution microscopy techniques to confirm anastomosis between self-assembled and patterned vessels. Lastly, using a simple acrylic cassette and fluorescently labeled microspheres, the multiscale network was demonstrated to be perfusable. Directed flow from inlet to outlet mandated flow through the capillary bed. This method for producing closed, multiscale vascular networks was developed with the intention of straightforward fabrication and engineering techniques so as to be a low barrier to entry for researchers who wish to investigate mechanistic questions in vascular biology. This ease of use offers a facile extension of these methods for incorporation into organoid culture, organ-on-a-chip (OOC) models, and bioprinted tissues.

Transplantable engineered organs could one day be used to treat patients suffering from end-stage organ failure. Yet, producing hierarchical vascular networks that sustain the viability and function of cells within human-scale organs remains a major challenge. Sacrificial templating has emerged as a promising biofabrication method that could overcome this challenge. Here, we explore and evaluate various strategies and materials that have been used for sacrificial templating. First, we emphasize fabrication approaches that use highly biocompatible sacrificial reagents and minimize the duration that cells spend in fabrication conditions without oxygen and nutrients. We then discuss strategies to create continuous, hierarchical vascular networks, both using biofabrication alone and using hybrid methods that integrate biologically driven vascular self-assembly into sacrificial templating workflows. Finally, we address the importance of structurally reinforcing engineered vessel walls to achieve stable blood flow in vivo, so that engineered organs remain perfused and functional long after implantation. Together, these sacrificial templating strategies have the potential to overcome many current limitations in biofabrication and accelerate clinical translation of transplantable, fully functional engineered organs to rescue patients from organ failure.

Drug delivery systems (DDSs) are increasingly important in ensuring drug safety and enhancing therapeutic efficacy. Micro/nano-technology has been utilized to develop DDSs for achieving high stability, bioavailability, and drug efficiency, as well as targeted delivery; meanwhile, 3D printing technology has made it possible to tailor DDSs with diverse components and intricate structures. This review presents the latest research progress integrating 3D printing technology and micro/nano-technology for developing novel DDSs. The technological fundamentals of 3D printing technology supporting the development of DDSs are presented, mainly from the perspective of different 3D printing mechanisms. Distinct types of DDSs leveraging 3D printing and micro/nano-technology are analyzed deeply, featuring micro/nanoscale materials and structures to enrich functionalities and improve effectiveness. Finally, we will discuss the future directions of 3D-printed DDSs integrated with micro/nano-technology, focusing on technological innovation and clinical application. This review will support interdisciplinary research efforts to advance drug delivery technology.

In recent years, neural organoids derived from human pluripotent stem cells (hPSCs) have offered a transformative pre-clinical platform for understanding central nervous system (CNS) development, disease, drug effects, and toxicology. CNS vasculature plays an important role in all these scenarios; however, most published studies describe CNS organoids that lack a functional vasculature or demonstrate rudimentary incorporation of endothelial cells or blood vessel networks. Here, we review the existing knowledge of vascularization during the development of different CNS regions, including the brain, spinal cord, and retina, and compare it to vascularized CNS organoid models. We highlight several areas of contrast where further bioengineering innovation is needed and discuss potential applications of vascularized neural organoids in modeling human CNS development, physiology, and disease.

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.

Advancements in bioprinting technology are driving the creation of complex, functional tissue constructs for use in tissue engineering and regenerative medicine. Various methods, including extrusion, jetting, and light-based bioprinting, have their unique advantages and drawbacks. Over the years, researchers and industry leaders have made significant progress in enhancing bioprinting techniques and materials, resulting in the production of increasingly sophisticated tissue constructs. Despite this progress, challenges still need to be addressed in achieving clinically relevant, human-scale tissue constructs, presenting a hurdle to widespread clinical translation. However, with ongoing interdisciplinary research and collaboration, the field is rapidly evolving and holds promise for personalized medical interventions. Continued development and refinement of bioprinting technologies have the potential to address complex medical needs, enabling the development of functional, transplantable tissues and organs, as well as advanced in vitro tissue models.

Natural tissues are composed of diverse cells and extracellular materials whose arrangements across several length scales-from subcellular lengths1 (micrometre) to the organ scale2 (centimetre)-regulate biological functions. Tissue-fabrication methods have progressed to large constructs, for example, through stereolithography3 and nozzle-based bioprinting4,5, and subcellular resolution through subtractive photoablation6-8. However, additive bioprinting struggles with sub-nozzle/voxel features9 and photoablation is restricted to small volumes by prohibitive heat generation and time10. Building across several length scales with temperature-sensitive, water-based soft biological matter has emerged as a critical challenge, leaving large classes of biological motifs-such as multiscalar vascular trees with varying calibres-inaccessible with present technologies11,12. Here we use gallium-based engineered sacrificial capillary pumps for evacuation (ESCAPE) during moulding to generate multiscalar structures in soft natural hydrogels, achieving both cellular-scale (<10 µm) and millimetre-scale features. Decoupling the biomaterial of interest from the process of constructing the geometry allows non-biocompatible tools to create the initial geometry. As an exemplar, we fabricated branched, cell-laden vascular trees in collagen, spanning approximately 300-µm arterioles down to the microvasculature (roughly ten times smaller). The same approach can micropattern the inner surface of vascular walls with topographical cues to orient cells in 3D and engineer fine structures such as vascular malformations. ESCAPE moulding enables the fabrication of multiscalar forms in soft biomaterials, paving the way for a wide range of tissue architectures that were previously inaccessible in vitro.

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.

The development of perfusable and multiscale vascular networks remains one of the largest challenges in tissue engineering. As such, there is a need for the creation of customizable and facile methods to produce robustly vascularized constructs. In this study, secondarily crosslinkable (clickable) poly(ethylene glycol)-norbornene (PEGNB) microbeads were produced and evaluated for their ability to sequentially support suspension bioprinting and microvascular self-assembly towards the aim of engineering hierarchical vasculature. The clickable PEGNB microbead slurry exhibited mechanical behavior suitable for suspension bioprinting of sacrificial bioinks, could be UV crosslinked into a granular construct post-print, and withstood evacuation of the bioink and subsequent perfusion of the patterned void space. Endothelial and stromal cells co-embedded within jammed RGD-modified PEGNB microbead slurries assembled into capillary-scale vasculature after secondary crosslinking of the beads into granular constructs, with endothelial tubules forming within the interstitial space between microbeads and supported by the perivascular association of the stromal cells. Microvascular self-assembly was not impacted by printing sacrificial bioinks into the cell-laden microbead support bath before UV crosslinking. Collectively, these results demonstrate that clickable PEGNB microbeads are a versatile substrate for both suspension printing and microvascular culture and may be the foundation for a promising methodology to engineer hierarchical vasculature.

Arterial disorders such as atherosclerosis, thrombosis, and aneurysm pose significant health risks, necessitating advanced interventions. Despite progress in artificial blood vessels and animal models aimed at understanding pathogenesis and developing therapies, limitations in graft functionality and species discrepancies restrict their clinical and research utility. Addressing these issues, bioengineered arterial equivalents (AEs) with enhanced vascular functions have been developed, incorporating innovative technologies that improve clinical outcomes and enhance disease progression modeling. This review offers a comprehensive overview of recent advancements in bioengineered AEs, systematically summarizing the bioengineered technologies used to construct these AEs, and discussing their implications for clinical application and pathogenesis understanding. Highlighting current breakthroughs and future perspectives, this review aims to inform and inspire ongoing research in the field, potentially transforming vascular medicine and offering new avenues for preclinical and clinical advances.

Tissue-engineered skin substitutes (TESS) emerged as a new therapeutic option to improve skin transplantation. However, establishing an adequate and rapid vascularization in TESS is a critical factor for their clinical application and successful engraftment in patients. Therefore, several methods have been applied to improve the vascularization of skin substitutes including (i) modifying the structural and physicochemical properties of dermal scaffolds; (ii) activating biological scaffolds with growth factor-releasing systems or gene vectors; and (iii) developing prevascularized skin substitutes by loading scaffolds with capillary-forming cells. This review provides a detailed overview of the most recent and important developments in the vascularization strategies for skin substitutes. On the one hand, we present cell-based approaches using stem cells, microvascular fragments, adipose tissue derived stromal vascular fraction, endothelial cells derived from blood and skin as well as other pro-angiogenic stimulation methods. On the other hand, we discuss how distinct 3D bioprinting techniques and microfluidics, miRNA manipulation, cell sheet engineering and photosynthetic scaffolds like GelMA, can enhance skin vascularization for clinical applications. Finally, we summarize and discuss the challenges and prospects of the currently available vascularization techniques that may serve as a steppingstone to a mainstream application of skin tissue engineering.

Blood and lymphatic vessels in the body are central to molecular and cellular transport, tissue repair, and pathophysiology. Several approaches have been employed for engineering microfabricated blood and lymphatic vessels in vitro, yet traditionally these approaches require specialized equipment, facilities, and research training beyond the capabilities of many biomedical laboratories. Here we present xurography as an inexpensive, accessible, and versatile rapid prototyping technique for engineering cylindrical and lumenized microvessels. Using a benchtop xurographer, or a cutting plotter, we fabricated modular multi-layer poly(dimethylsiloxane) (PDMS)-based microphysiological systems (MPS) that house endothelial-lined microvessels approximately 260 μm in diameter embedded within a user-defined 3-D extracellular matrix (ECM). We validated the vascularized MPS (or vessel-on-a-chip) by quantifying changes in blood vessel permeability due to the pro-angiogenic chemokine CXCL12. Moreover, we demonstrated the reconfigurable versatility of this approach by engineering a total of four distinct vessel-ECM arrangements, which were obtained by only minor adjustments to a few steps of the fabrication process. Several of these arrangements, such as ones that incorporate close-ended vessel structures and spatially distinct ECM compartments along the same microvessel, have not been widely achieved with other microfabrication strategies. Therefore, we anticipate that our low-cost and easy-to-implement fabrication approach will facilitate broader adoption of MPS with customizable vascular architectures and ECM components while reducing the turnaround time required for iterative designs.

Three-dimensional (3D) bioprinting has emerged with potential for creating functional 3D tissues with customized geometries. However, the limited availability of bioinks capable of printing 3D structures with both high-shape fidelity and desired biological environments for encapsulated cells remains a key challenge. Here, we present a 3D bioprinting approach that uses universal fugitive network bioinks prepared by loading cells and hydrogel precursors (bioink base materials) into a 3D printable fugitive carrier. This approach constructs 3D structures of cell-encapsulated hydrogels by printing 3D structures using fugitive network bioinks, followed by cross-linking printed structures and removing the carrier from them. The use of the fugitive carrier decouples the 3D printability of bioinks from the material properties of bioink base materials, making this approach readily applicable to a range of hydrogel systems. The decoupling also enables the design of bioinks for the biological functionality of the final printed constructs without compromising the 3D printability. We demonstrate the generalizable 3D printability by printing self-supporting 3D structures of various hydrogels, including conventionally non-3D printable hydrogels and those with a low polymer content. We conduct preprinting screening of bioink base materials through 3D cell culture to select bioinks with high cell compatibility. The selected bioinks produce 3D constructs of cell-encapsulated hydrogels with both high-shape fidelity and high cell viability and proliferation. The universal fugitive network bioink platform could significantly expand 3D printable bioinks with customizable biological functionalities and the adoption of 3D bioprinting in diverse research and applied settings.