Table of contents

Three-dimensional (3D) bioprinting is a promising technique for spatially patterning cells and materials into constructs that mimic native tissues and organs. However, a trade-off exists between printability and biological function, where weak materials are typically more suited for 3D cell culture but exhibit poor shape fidelity when printed in air. Recently, a new class of assistive materials has emerged to overcome this limitation and enable fabrication of more complex, biologically relevant geometries, even when using soft materials as bioinks. These materials include support baths, which bioinks are printed into, and sacrificial inks, which are printed themselves and then later removed. Support baths are commonly yield-stress materials that provide physical confinement during the printing process to improve resolution and shape fidelity. Sacrificial inks have primarily been used to create void spaces and pattern perfusable networks, but they can also be combined directly with the bioink to change its mechanical properties for improved printability or increased porosity. Here, we outline the advantages of using such assistive materials in 3D bioprinting, define their material property requirements, and offer case study examples of how these materials are used in practice. Finally, we discuss the remaining challenges and future opportunities in the development of assistive materials that will propel the bioprinting field forward toward creating full-scale, biomimetic tissues and organs.

The tumor microenvironment (TME) typically comprises cancer cells, tumor vasculature, stromal components like fibroblasts, and host immune cells that assemble to support tumorigenesis. However, preexisting classic cancer models like 2D cell culture methods, 3D cancer spheroids, and tumor organoids seem to lack essential TME components. 3D bioprinting offers enormous advantages for developing in vitro tumor models by allowing user-controlled deposition of multiple biomaterials, cells, and biomolecules in a predefined architecture. This review highlights the recent developments in 3D cancer modeling using different bioprinting techniques to recreate the TME. 3D bioprinters enable the fabrication of high-resolution microstructures to reproduce TME intricacies. Furthermore, 3D bioprinted models can be applied as a preclinical model for versatile research applications in the tumor biology and pharmaceutical industries. These models provide an opportunity to develop high-throughput drug screening platforms and can further be developed to suit individual patient requirements hence giving a boost to the field of personalized anti-cancer therapeutics. We underlined the various ways the existing studies have tried to mimic the TME, mimic the hallmark events of cancer growth and metastasis within the 3D bioprinted models and showcase the 3D drug-tumor interaction and further utilization of such models to develop personalized medicine.

Acute liver failure (ALF) is a rapidly progressive disease with high morbidity and mortality rates. Liver transplantation and artificial liver (AL) support systems, such as ALs and bioartificial livers (BALs), are the two major therapies for ALF. Compared to ALs, BALs are composed of functional hepatocytes that provide essential liver functions, including detoxification, metabolite synthesis, and biotransformation. Furthermore, BALs can potentially provide effective support as a form of bridging therapy to liver transplantation or spontaneous recovery for patients with ALF. In this review, we systematically discussed the currently available state-of-the-art designs and manufacturing processes for BAL support systems. Specifically, we classified the cell sources and bioreactors that are applied in BALs, highlighted the advanced technologies of hepatocyte culturing and bioreactor fabrication, and discussed the current challenges and future trends in developing next-generation BALs for large-scale clinical applications.

Recently developed modular bioassembly techniques hold tremendous potential in tissue engineering and regenerative medicine, due to their ability to recreate the complex microarchitecture of native tissue. Here, we developed a novel approach to fabricate hybrid tissue-engineered constructs adopting high-throughput microfluidic and 3D bioassembly strategies. Osteochondral tissue fabrication was adopted as an example in this study, because of the challenges in fabricating load bearing osteochondral tissue constructs with phenotypically distinct zonal architecture. By developing cell-instructive chondrogenic and osteogenic bioink microsphere modules in high-throughput, together with precise manipulation of the 3D bioassembly process, we successfully fabricated hybrid engineered osteochondral tissue in vitro with integrated but distinct cartilage and bone layers. Furthermore, by encapsulating allogeneic umbilical cord blood-derived mesenchymal stromal cells, and demonstrating chondrogenic and osteogenic differentiation, the hybrid biofabrication of hydrogel microspheres in this 3D bioassembly model offers potential for an off-the-shelf, single-surgery strategy for osteochondral tissue repair.

In vitro cancer models that can simulate patient-specific drug responses for personalized medicine have attracted significant attention. However, the technologies used to produce such models can only recapitulate the morphological heterogeneity of human cancer tissue. Here, we developed a novel 3D technique to bioprint an in vitro breast cancer model with patient-specific morphological features. This model can precisely mimic the cellular microstructures of heterogeneous cancer tissues and produce drug responses similar to those of human cancers. We established a bioprinting process for generating cancer cell aggregates with ductal and solid tissue microstructures that reflected the morphology of breast cancer tissues, and applied it to develop breast cancer models. The genotypic and phenotypic characteristics of the ductal and solid cancer aggregates bioprinted with human breast cancer cells (MCF7, SKBR3, MDA-MB-231) were respectively similar to those of early and advanced cancers. The bioprinted solid cancer cell aggregates showed significantly higher hypoxia (>8 times) and mesenchymal (>2–4 times) marker expressions, invasion activity (>15 times), and drug resistance than the bioprinted ductal aggregates. Co-printing the ductal and solid aggregates produced heterogeneous breast cancer tissue models that recapitulated three different stages of breast cancer tissue morphology. The bioprinted cancer tissue models representing advanced cancer were more and less resistant, respectively, to the anthracycline antibiotic doxorubicin and the hypoxia-activated prodrug tirapazamine; these were analogous to the results in human cancer. The present findings showed that cancer cell aggregates can mimic the pathological micromorphology of human breast cancer tissue and they can be bioprinted to produce breast cancer tissue in vitro that can morphologically represent the clinical stage of cancer in individual patients.

Bone regeneration of critical-sized bone defects, bone fractures or joint replacements remains a significant clinical challenge. Although there has been rapid advancement in both the fields of bone tissue engineering and additive manufacturing, functional bone implants with rapid vascularization capacity to ensure osseointegration and long-term biological fixation in large bone defects remains limited in clinics. In this study, we developed an in vitro vascularized bone implant by combining cell-laden hydrogels with direct metal printed (DMP) porous titanium alloys (Ti–6Al–4V). A 5 wt% allylated gelatin (GelAGE), was utilized to co-encapsulate human mesenchymal stromal cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) to investigate concurrent osteogenic and vasculogenic performance. DMP macro-porous Ti–6Al–4V scaffolds were subsequently infused/enriched with cell-laden GelAGE to examine the feasibility to deliver cells and engineer vascular-like networks in the hybrid implant. Furthermore, as a proof of concept, a full-scale porous Ti–6Al–4V acetabular cup was impregnated with cell-laden hydrogel to validate the clinical potential of this strategy. The vasculogenic potential was evaluated by examining micro-capillary formation coupled with capillary network maturation and stabilization. Osteogenic differentiation was assessed via alkaline phosphatase activity as well as osteocalcin and osteopontin expression. Our results suggested that GelAGE supported HUVECs spreading and vascular-like network formation, along with osteogenesis of hMSCs. Titanium hybrid constructs with cell-laden hydrogel demonstrated enhanced osteogenesis with similar vasculogenic capability compared to the cell-laden hydrogel alone constructs. The full-scale implant with cell-laden hydrogel coating similarly showed cell distribution and spreading, implying the potential for further clinical application. Our study presents the feasibility of integrating bio-functional hydrogels with porous titanium implants to fabricate a vascularized hybrid construct with both mechanical support and preferable biological functionality (osteogenesis/vasculogenesis), which paves the way for improved strategies to enhance bone regeneration in complex large bone defects achieving long-term bone-implant fixation.

Microvasculature is essential for the exchange of gas and nutrient for most tissues in our body. Some tissue structures such as the meniscus presents spatially confined blood vessels adjacent to non-vascularized regions. In biofabrication, mimicking the spatial distribution of such vascular components is paramount, as capillary ingrowth into non-vascularized tissues can lead to tissue matrix alterations and subsequent pathology. Multi-material three-dimensional (3D) bioprinting strategies have the potential to resolve anisotropic tissue features, although building complex constructs comprising stable vascularized and non-vascularized regions remains a major challenge to date. In this study, we developed endothelial cell-laden pro- and anti-angiogenic bioinks, supplemented with bioactive matrix-derived microfibers (MFs) that were created from type I collagen sponges (col-1) and cartilage decellularized extracellular matrix (CdECM), respectively. Human umbilical vein endothelial cell (HUVEC)-driven capillary networks started to form 2 d after bioprinting. Supplementing cartilage-derived MFs to endothelial-cell laden bioinks reduced the total length of neo-microvessels by 29%, and the number of microvessel junctions by 37% after 14 d, compared to bioinks with pro-angiogenic col-1 MFs. As a proof of concept, the bioinks were bioprinted into an anatomical meniscus shape with a biomimetic vascularized outer and non-vascularized inner region, using a gellan gum microgel suspension bath. These 3D meniscus-like constructs were cultured up to 14 d, with in the outer zone the HUVEC-, mural cell-, and col-1 MF-laden pro-angiogenic bioink, and in the inner zone a meniscus progenitor cell (MPC)- and CdECM MF-laden anti-angiogenic bioink, revealing successful spatial confinement of the nascent vascular network only in the outer zone. Further, to co-facilitate both microvessel formation and MPC-derived matrix formation, we formulated cell culture medium conditions with a temporal switch. Overall, this study provides a new strategy that could be applied to develop zonal biomimetic meniscal constructs. Moreover, the use of ECM-derived MFs to promote or inhibit capillary networks opens new possibilities for the biofabrication of tissues with anisotropic microvascular distribution. These have potential for many applications including in vitro models of vascular-to-avascular tissue interfaces, cancer progression, and for testing anti-angiogenic therapies.

3D bioprinting offers an excellent opportunity to provide tissue-engineered cartilage to microtia patients. However, hydrogel-based bioinks are hindered by their dense and cell-restrictive environment, impairing tissue development and ultimately leading to mechanical failure of large scaffolds in vivo. Granular hydrogels, made of annealed microgels, offer a superior alternative to conventional bioinks, with their improved porosity and modularity. We have evaluated the ability of enzymatically crosslinked hyaluronic acid (HA) microgel bioinks to form mature cartilage in vivo. Microgel bioinks were formed by mechanically sizing bulk HA-tyramine hydrogels through meshes with aperture diameters of 40, 100 or 500 µm. Annealing of the microgels was achieved by crosslinking residual tyramines. Secondary crosslinked scaffolds were stable in solution and showed tunable porosity from 9% to 21%. Bioinks showed excellent rheological properties and were used to print different objects. Printing precision was found to be directly correlated to microgel size. As a proof of concept, freeform reversible embedding of suspended hydrogels printing with gelation triggered directly in the bath was performed to demonstrate the versatility of the method. The granular hydrogels support the homogeneous development of mature cartilage-like tissues in vitro with mechanical stiffening up to 200 kPa after 63 d. After 6 weeks of in vivo implantation, small-diameter microgels formed stable constructs with low immunogenicity and continuous tissue maturation. Conversely, increasing the microgel size resulted in increased inflammatory response, with limited stability in vivo. This study reports the development of new microgel bioinks for cartilage tissue biofabrication and offers insights into the foreign body reaction towards porous scaffolds implantation.

Cancer-associated cachexia (CAC) is a complex metabolic and behavioral syndrome with multiple manifestations that involve systemic inflammation, weight loss, and adipose lipolysis. It impacts the quality of life of patients and is the direct cause of death in 20%–30% of cancer patients. The severity of fat loss and adipose tissue remodeling negatively correlate with patients' survival outcomes. To address the mechanism of fat loss and design potential approaches to prevent the process, it will be essential to understand CAC pathophysiology through white adipose tissue models. In the present study, an engineered human white adipose tissue (eWAT) model based on three-dimensional (3D) bioprinting was developed and induced with pancreatic cancer cell-conditioned medium (CM) to mimic the status of CAC in vitro. We found that the CM induction significantly increased the lipolysis and accumulation of the extracellular matrix (ECM). The 3D eWATs were further vascularized to study the influence of vascularization on lipolysis and CAC progression, which was largely unknown. Results demonstrated that CM induction improved the angiogenesis of vascularized eWATs (veWATs), and veWATs demonstrated decreased glycerol release but increased UCP1 expression, compared to eWATs. Many unique inflammatory cytokines (IL-8, CXCL-1, GM-CSF, etc) from the CM were detected and supposed to contribute to eWAT lipolysis, UCP1 up-regulation, and ECM development. In response to CM induction, eWATs also secreted inflammatory adipokines related to the metastatic ability of cancer, muscle atrophy, and vascularization (NGAL, CD54, IGFBP-2, etc). Our work demonstrated that the eWAT is a robust model for studying cachectic fat loss and the accompanying remodeling of adipose tissue. It is therefore a useful tool for future research exploring CAC physiologies and developing potential therapies.

Porous Magnesium (Mg) is a promising biodegradable scaffold for treating critical-size bone defects, and as an essential element for human metabolism, Mg has shown sufficient biocompatibility. Its elastic moduli and yield strengths are closer to those of cortical bone than common, inert metallic implants, effectively reducing stress concentrations around host tissue as well as stress shielding. More importantly, Mg can degrade and be absorbed in the human body in a safe and controlled manner, thereby reducing the need for second surgeries to remove implants. The development of porous Mg scaffolds via conventional selective laser melting techniques has been limited due to Mg's low boiling point, high vapor pressures, high reactivity, and non-ideal microstructures in additively manufactured parts. Here we present an exciting alternative to conventional additive techniques: 3D weaving with Mg wires that have controlled chemistries and microstructures. The weaving process offers high throughput manufacturing as well as porous architectures that can be optimized for stiffness and porosity with topology optimization. Once woven, we dip-coat the weaves with polylactic acid to enhance their strength and corrosion resistance. Following fabrication, we characterize their mechanical properties, corrosion behavior, and cell compatibility in vitro, and we use an intramuscular implantation model to evaluate their in vivo corrosion behavior and tissue response.

Preparing a micropatterned elastomer film with characteristics that can simulate the mechanical properties, anisotropy, and electroactivity of natural myocardial tissues is crucial in cardiac tissue engineering after myocardial infarction (MI). Therefore, in this study, we developed several elastomeric films with a surface micropattern based on poly (glycerol sebacate) (PGS) and graphene (Gr). These films have sufficient mechanical strength (0.6 ± 0.1–3.2 ± 0.08 MPa) to withstand heartbeats, and the micropatterned structure also satisfies the natural myocardium anisotropy in the transverse and vertical. Moreover, Gr makes these films conductive (up to 5.80 × 10⁻⁷ S m⁻¹), which is necessary for the conduction of electrical signals between cardiomyocytes and the cardiac tissue. Furthermore, they have good cytocompatibility and can promote cell proliferation in H9c2 rat cardiomyocyte cell lines. In vivo test results indicate that these films have good biocompatibility. Notably, a film with 1 wt% Gr content (PGS–Gr1) significantly affects the recovery of myocardial function in rats after MI. This film effectively decreased the infarct size and degree of myocardial fibrosis and reduced collagen deposition. Echocardiographic evaluation showed that after treatment with this film, the left ventricular internal dimension (LVID) in systole and LVID in diastole of rats exhibited a significant downward trend, whereas the fractional shortening and ejection fraction were significantly increased compared with the control group. These data indicate that this electroactive micropatterned anisotropic elastomer film can be applied in cardiac tissue engineering.

Neuroblastoma is an extracranial solid tumor which develops in early childhood and still has a poor prognosis. One strategy to increase cure rates is the identification of patient-specific drug responses in tissue models that mimic the interaction between patient cancer cells and tumor environment. We therefore developed a perfused and micro-vascularized tumor-environment model that is directly bioprinted into custom-manufactured fluidic chips. A gelatin-methacrylate/fibrin-based matrix containing multiple cell types mimics the tumor-microenvironment that promotes spontaneous micro-vessel formation by embedded endothelial cells. We demonstrate that both, adipocyte- and iPSC-derived mesenchymal stem cells can guide this process. Bioprinted channels are coated with endothelial cells post printing to form a dense vessel—tissue barrier. The tissue model thereby mimics structure and function of human soft tissue with endothelial cell-coated larger vessels for perfusion and micro-vessel networks within the hydrogel-matrix. Patient-derived neuroblastoma spheroids are added to the matrix during the printing process and grown for more than two weeks. We demonstrate that micro-vessels are attracted by and grow into tumor spheroids and that neuroblastoma cells invade the tumor-environment as soon as the spheroids disrupt. In summary, we describe the first bioprinted, micro-vascularized neuroblastoma—tumor-environment model directly printed into fluidic chips and a novel medium-throughput biofabrication platform suitable for studying tumor angiogenesis and metastasis in precision medicine approaches in future.

Stem cell spheroids are advanced building blocks to produce chondroid. However, the multi-step operations including spheroids preparation, collection and transfer, the following 3D printing and shaping limit their application in 3D printing. The present study fabricates an 'ALL-IN-ONE' bioink based on granular hydrogel to not only produce adipose derived stem cell (ASC) spheroids, but also realize the further combination of chondrocytes and the subsequent 3D printing. Microgels (6–10 μm) grafted with β-cyclodextrin (β-CD) (MG_β-CD) were assembled and crosslinked by in-situ polymerized poly (N-isopropylacrylamide) (PNIPAm) to form bulk granular hydrogel. The host-guest action between β-CD of microgels and PNIPAm endows the hydrogel with stable, shear-thinning and self-healing properties. After creating caves, ASCs aggregate spontaneously to form numerous spheroids with diameter of 100–200 μm inside the hydrogel. The thermosensitive porous granular hydrogel exhibits volume change under different temperature, realizing further adsorbing chondrocytes. Then, the granular hydrogel carrying ASC spheroids and chondrocytes is extruded by 3D printer at room temperature to form a tube, which can shrink at cell culture temperature to enhance the resolution. The subsequent ASC spheroids/chondrocytes co-culture forms cartilage-like tissue at 21 d in vitro, which further matures subcutaneously in vivo, indicating the application potential of the fully synthetic granular hydrogel ink toward organoid culture.

This research presents a novel testis-on-a-chip (ToC) platform. Testicular cells are enzymatically isolated from the seminiferous tubules of sexually immature mice, seeded in a methylcellulose gel and cultured in a microfluidic chip. The unique design sandwiches the soft methylcellulose between stiffer agar support gels. The cells develop into spheroids continuing to proliferate and differentiate. After seven weeks of culture the cells have over 95% viability. Confocal microscopy of the developed spheroids reveals a structure containing the various stages of spermatogenesis up to and including meiosis II: premeiotic, meiotic and post-meiotic germ cells. The spheroid structure also contains the supporting Sertoli and peritubular cells. The responsiveness of the system to the addition of testosterone and retinoic acid to the culture medium during the experiment was also investigated. As a benchmark, the ToC is compared to a conventional three-dimensional methylcellulose cell culture system in a well plate. Analysis via fluorescence-activated cell sorting shows more haploid cells in the chip as compared to the plates. Immunofluorescence staining after seven weeks of culture shows more differentiated cells in the chip as compared to the well plate. This demonstrates the feasibility of our platform as well as its advantages. This research opens new horizons for the study and realization of spermatogenesis in-vitro. It can also enable the implementation of microfluidic technologies in future therapeutic strategies for pre-pubertal male fertility preservation and adults with maturation arrest. Lastly, it can serve as a platform for drug and toxin testing.

Tissue biomanufacturing aims to produce lab-grown stem cell grafts and biomimetic drug testing platforms but remains limited in its ability to recapitulate native tissue mechanics. The emerging field of soft robotics aims to emulate dynamic physiological locomotion, representing an ideal approach to recapitulate physiologically complex mechanical stimuli and enhance patient-specific tissue maturation. The kneecap's femoropopliteal artery (FPA) represents a highly flexible tissue across multiple axes during blood flow, walking, standing, and crouching positions, and these complex biomechanics are implicated in the FPA's frequent presentation of peripheral artery disease. We developed a soft pneumatically actuated (SPA) cell culture platform to investigate how patient-specific FPA mechanics affect lab-grown arterial tissues. Silicone hyperelastomers were screened for flexibility and biocompatibility, then additively manufactured into SPAs using a simulation-based design workflow to mimic normal and diseased FPA extensions in radial, angular, and longitudinal dimensions. SPA culture platforms were seeded with mesenchymal stem cells, connected to a pneumatic controller, and provided with 24 h multi-axial exercise schedules to demonstrate the effect of dynamic conditioning on cell alignment, collagen production, and muscle differentiation without additional growth factors. Soft robotic bioreactors are promising platforms for recapitulating patient-, disease-, and lifestyle-specific mechanobiology for understanding disease, treatment simulations, and lab-grown tissue grafts.

Carbon nanotubes (CNTs) have attracted increasing attention in the field of peripheral nerve tissue engineering due to their unique structural and physical characteristics. In this study, a novel type of aligned conductive scaffolds composed of polycaprolactone (PCL) and CNTs were fabricated via electrospinning. Utilizing mussel-inspired polydopamine (PDA) surface modification, brain-derived neurotrophic factor (BDNF) was loaded onto PCL/CNT fibrous scaffolds to obtain PCL/CNT-PDA-BDNF fibrous scaffolds capable of the sustained release of BDNF over 28 d. Schwann cells were cultured on these scaffolds, and the effect of the scaffolds on peripheral nerve regeneration in vitro was assessed by studying cell proliferation, morphology and the expressions of myelination-related genes S100, P0 and myelin basic protein. Furthermore, the effect of these scaffolds on peripheral nerve regeneration in vivo was investigated using a 10 mm rat sciatic nerve defect model. Both the in vitro and in vivo results indicate that PCL/CNT-PDA-BDNF fibrous scaffolds effectively promote sciatic nerve regeneration and functional recovery. Therefore, PCL/CNT-PDA-BDNF fibrous scaffolds have great potential for peripheral nerve restoration.

Cancer continues to be a leading cause of mortality in modern societies; therefore, improved and more reliable in vitro cancer models are needed to expedite fundamental research and anti-cancer drug development. Here, we describe the use of a miniaturized continuous stirred tank reactor (mCSTR) to first fabricate and mature cancer spheroids (i.e. derived from MCF7 cells, DU145 cells, and a mix of MCF7 cells and fibroblasts), and then to conduct anti-cancer drug assays under continuous perfusion. This 3 ml mCSTR features an off-center agitation system that enables homogeneous chaotic laminar mixing at low speeds to support cell aggregation. We incubated cell suspensions for 3 d in ultra-low-attachment plates to allow formation of discoid cell aggregates (∼600 µm in diameter). These cell aggregates were then transferred into mCSTRs and continuously fed with culture medium. We characterized the spheroid morphology and the expression of relevant tumor biomarkers at different maturation times for up to 4 weeks. The spheroids progressively increased in size during the first 5–6 d of culture to reach a steady diameter between 600 and 800 µm. In proof-of-principle experiments, we demonstrated the use of this mCSTR in anti-cancer drug testing. Three drugs commonly used in breast cancer treatment (doxorubicin, docetaxel, and paclitaxel) were probed at different concentrations in MCF7-derived spheroids. In these experiments, we evaluated cell viability, glucose consumption, spheroid morphology, lactate dehydrogenase activity, and the expression of genes associated with drug resistance (ABCB1 and ABCC1) and anti-apoptosis (Bcl2). We envision the use of this agitated system as a tumor-on-a-chip platform to expedite efficacy and safety testing of novel anti-cancer drugs and possibly in personalized medicine applications.

The integration of three-dimensional (3D) bioprinted scaffold's structure and function for critical-size bone defect repair is of immense significance. Inspired by the basic component of innate cortical bone tissue—osteons, many studies focus on biomimetic strategy. However, the complexity of hierarchical microchannels in the osteon, the requirement of mechanical strength of bone, and the biological function of angiogenesis and osteogenesis remain challenges in the fabrication of osteon-mimetic scaffolds. Therefore, we successfully built mimetic scaffolds with vertically central medullary canals, peripheral Haversian canals, and transverse Volkmann canals structures simultaneously by 3D bioprinting technology using polycaprolactone and bioink loading with bone marrow mesenchymal stem cells and bone morphogenetic protein-4. Subsequently, endothelial progenitor cells were seeded into the canals to enhance angiogenesis. The porosity and compressive properties of bioprinted scaffolds could be well controlled by altering the structure and canal numbers of the scaffolds. The osteon-mimetic scaffolds showed satisfactory biocompatibility and promotion of angiogenesis and osteogenesis in vitro and prompted the new blood vessels and new bone formation in vivo. In summary, this study proposes a biomimetic strategy for fabricating structured and functionalized 3D bioprinted scaffolds for vascularized bone tissue regeneration.

Bioceramics are widely used in bone tissue repair and regeneration due to their desirable biocompatibility and bioactivity. However, the brittleness of bioceramics results in difficulty of surgical operation, which greatly limits their clinical applications. The spicules of the marine sponge Euplectella aspergillum (Ea) possess high flexibility and fracture toughness resulting from concentric layered silica glued by a thin organic layer. Inspired by the unique properties of sponge spicules, flexible bioceramic-based scaffolds with spicule-like concentric layered biomimetic microstructures were constructed by combining two-dimensional (2D) bioceramics and 3D printing. 2D bioceramics could be assembled and aligned by modulating the shear force field in the direct ink writing (DIW) of 3D printing. The prepared spicules-inspired flexible bioceramic-based (SFB) scaffolds differentiated themselves from traditional 3D-printed irregular particles-based bioceramic-based scaffolds as they could be adaptably compressed, cut, folded, rolled and twisted without the occurrence of fracture, significantly breaking through the bottleneck of inherent brittleness of traditional bioceramic scaffolds. In addition, SFB scaffolds showed significantly enhanced in vitro and in vivo bone-forming bioactivity as compared to conventional β-tricalcium phosphate (β-TCP) scaffolds, suggesting that SFB scaffolds combined both of excellent mechanical and bioactive characteristics, which is believed to greatly promote the bioceramic science and their clinical applications.

Scaffold-based regenerative strategies that emulate physical, biochemical, and mechanical properties of the native extracellular matrix (ECM) of the region of interest can influence cell growth and function. Existing ECM-mimicking scaffolds, including nanofiber (NF) mats, sponges, hydrogels, and NF-hydrogel composites are unable to simultaneously mimic typical composition, topography, pore size, porosity, and viscoelastic properties of healthy soft-tissue ECM. In this work, we used cryoelectrospinning to fabricate 3D porous scaffolds with minimal fibrous backbone, pore size and mechanical properties similar to soft-tissue connective tissue ECM. We used salivary glands as our soft tissue model and found the decellularized adult salivary gland (DSG) matrix to have a fibrous backbone, 10–30 μm pores, 120 Pa indentation modulus, and ∼200 s relaxation half time. We used elastin and alginate as natural, compliant biomaterials and water as the solvent for cryoelectrospinning scaffolds to mimic the structure and viscoelasticity of the connective tissue ECM of the DSG. Process parameters were optimized to produce scaffolds with desirable topography and compliance similar to DSG, with a high yield of >100 scaffolds/run. Using water as solvent, rather than organic solvents, was critical to generate biocompatible scaffolds with desirable topography; further, it permitted a green chemistry fabrication process. Here, we demonstrate that cryoelectrospun scaffolds (CESs) support penetration of NIH 3T3 fibroblasts 250–450 µm into the scaffold, cell survival, and maintenance of a stromal cell phenotype. Thus, we demonstrate that elastin-alginate CESs mimic many structural and functional properties of ECM and have potential for future use in regenerative medicine applications.

Leukemia patients undergo chemotherapy to combat the leukemic cells (LCs) in the bone marrow. During therapy not only the LCs, but also the blood-producing hematopoietic stem and progenitor cells (HSPCs) may be destroyed. Chemotherapeutics targeting only the LCs are urgently needed to overcome this problem and minimize life-threatening side-effects. Predictive in vitro drug testing systems allowing simultaneous comparison of various experimental settings would enhance the efficiency of drug development. Here, we present a three-dimensional (3D) human leukemic bone marrow model perfused using a magnetic, parallelized culture system to ensure media exchange. Chemotherapeutic treatment of the acute myeloid leukemia cell line KG-1a in 3D magnetic hydrogels seeded with mesenchymal stem/stromal cells (MSCs) revealed a greater resistance of KG-1a compared to 2D culture. In 3D tricultures with HSPCs, MSCs and KG-1a, imitating leukemic bone marrow, HSPC proliferation decreased while KG-1a cells remained unaffected post treatment. Non-invasive metabolic profiling enabled continuous monitoring of the system. Our results highlight the importance of using biomimetic 3D platforms with proper media exchange and co-cultures for creating in vivo-like conditions to enable in vitro drug testing. This system is a step towards drug testing in biomimetic, parallelized in vitro approaches, facilitating the discovery of new anti-leukemic drugs.

Coaxial bioprinting of hydrogel tubes has tremendous potential in the fabrication of highly complex large-scale vascularized structures, however, constructs with bioinks of simultaneous weak printability and perfusable networks have not been reported. Here, we report a coaxial printing method in which double-channel filaments are three-dimensional (3D) extrusion-bioprinted using a customized dual-core coaxial nozzle. The filament in one channel can perform core/shell role and the other channel can play a role in perfusion. These parallel channels within filaments are separated by an interval wall of alginate, whose thickness (∼50 μm) is beneficial to supplement nutrients via perfusion. Different cell-laden hydrogels of weak mechanics were used to test the adaptability and perfusability of our method, and the results showed that dynamic perfusion maintained higher viability and functions than static culture. By combining with a bioprinter, 8-layer perfusable double-channel constructs were fabricated, and the cell viabilities gradually decreased with the reduction in nutrients and oxygen in the downstream medium. Furthermore, the double-channel filaments were tested as a platform to mimic dynamic functions between cells through sequential perfusion by using Mouse insulinoma 6 (Min6) and Hepatocellular carcinoma (HepG2) as the model cells. These results demonstrated the insulin secreted by Min6 upstream simulated and increased the uptake of glucose by the downstream HepG2 cells. In conclusion, our study provided evidence for the probability of all-in-one fabrication of 3D double-channel perfusable constructs with high simplicity, expansibility, and versability. Our strategy has significant potential for building large-scale tissue constructs for applications in tissue engineering, possibly even in drug screening and regenerative medicine.

Three-dimensional (3D) bioprinting of self-supporting stable tissue and organ structure is critically important in extrusion-based bioprinting system, especially for tissue engineering and regenerative medicine applications. However, the development of self-standing bioinks with desired crosslinking density, biocompatibility, tunable mechanical strength and other properties like self-healing, in situ gelation, drug or protein incorporation is still a challenge. In this study, we report a hydrogel bioink prepared from alginate (Alg) and hyaluronic acid (HA) crosslinked through multiple crosslinking mechanisms, i.e. acyl-hydrazone, hydrazide interactions and calcium ions. These Alg-HA gels were highly dynamic and shear-thinning with exceptional biocompatibility and tunable mechanical properties. The increased dynamic nature of the gels is mainly chemically attributed to the presence of acyl-hydrazone bonds formed between the amine groups of the acyl-hydrazide of alginate and the monoaldehyde of the HA. Among the different combinations of Alg-HA gel compositions prepared, the A5H5 (Alginate-acyl-hydrazide:HA-monoaldehyde, ratio 50:50) gel showed a gelation time of ∼60 s, viscosity of ∼400 Pa s (at zero shear rate), high stability in various pH solutions and increased degradation time (>50 days) than the other samples. The A5H5 gels showed high printability with increased post-printing stability as observed from the 3D printed structures (e.g. hollow tube (∼100 layers), porous cube (∼50 layers), star, heart-in, meniscus and lattice). The scanning electron microscopy analysis of the 3D constructs and hydrogels showed the interconnected pores (∼181 µm) and crosslinked networks. Further, the gels showed sustained release of 5-amino salicylic acid and bovine serum albumin. Also, the mechanical properties were tuned by secondary crosslinking via different calcium concentrations. In vitro assays confirmed the cytocompatibility of these gels, where the 3D bioprinted lattice and tubular (∼70 layers) constructs demonstrated high cell viability under fluorescence analysis. In in vivo studies, Alg-HA gel showed high biocompatibility (>90%) and increased angiogenesis (threefolds) and reduced macrophage infiltration (twofold decrease), demonstrating the promising potential of these hydrogels in 3D bioprinting applications for tissue engineering and regenerative medicine with tunable properties.

Many in vitro models of neural physiology utilize neuronal networks established on two-dimensional substrates. Despite the simplicity of these 2D neuronal networks, substrate stiffness may influence cell morphology, network interactions and how neurons communicate and function. With this perspective, three-dimensional (3D) gel encapsulation is a powerful to recapitulating aspects of in vivo features, yet such an approach is often limited in terms of the level of resolution and feature size relevant for modelling aspects of brain architecture. Here, we report 3D bioplotting of rat primary cortical neural cells using a hydrogel system comprising gelatin norbornene (GelNB) and poly (ethylene glycol) dithiol (PEGdiSH). This bioink benefits from a rapid photo-click chemistry, yielding eight-layer crosshatch neural scaffolds and a filament width of 350 µm. The printability of this system depends on hydrogel concentration, printing temperature, extrusion pressure and speed. These parameters were studied via quantitative comparison between rheology and filament dimensions to determine the optimal printing conditions. Under optimal conditions, cell viability of bioprinted primary cortical neurons at day 1 (68 ± 2%) and at day 7 (68 ± 1%) were comparable to the 2D control group (72 ± 7%). The present study relates material rheology and filament dimensions to generate compliant free-standing neural constructs through bioplotting of low-concentration GelNB-PEGdiSH, which may provide a step forward to study 3D neuronal function and network formation.

Current techniques for the generation of cell-laden microgels are limited by numerous challenges, including poorly uncontrolled batch-to-batch variations, processes that are both labor- and time-consuming, the high expense of devices and reagents, and low production rates; this hampers the translation of laboratory findings to clinical applications. To address these challenges, we develop a droplet-based microfluidic strategy based on metastable droplet-templating and microchannel integration for the substantial large-scale production of single cell-laden alginate microgels. Specifically, we present a continuous processing method for microgel generation by introducing amphiphilic perfluoronated alcohols to obtain metastable emulsion droplets as sacrificial templates. In addition, to adapt to the metastable emulsion system, integrated microfluidic chips containing 80 drop-maker units are designed and optimized based on the computational fluid dynamics simulation. This strategy allows single cell encapsulation in microgels at a maximum production rate of 10 ml h⁻¹ of cell suspension while retaining cell viability and functionality. These results represent a significant advance toward using cell-laden microgels for clinical-relevant applications, including cell therapy, tissue regeneration and 3D bioprinting.

An ideal wound dressing for full-thickness wound regeneration should offer desirable biocompatibility, adequate mechanical properties, barrier function, and cellular regulation. Here, a bilayer scaffold resembling the hierarchical structure of human skin was developed using silk fibroin and sodium alginate. The upper membrane was prepared through casting and functioned as the epidermis, whereas the lower porous scaffold was prepared by freeze-drying and mimicked extracellular matrix structures. The membrane had nonporous structure, desirable mechanical properties, moderate hydrophilic surface, and suitable water vapor transmission rate, whereas the porous scaffold revealed 157.61 ± 41.67 µm pore size, 86.10 ± 3.60% porosity, and capability of stimulating fibroblast proliferation. The combination of the two structures reinforced the tensile strength by five-fold and provided protection from wound dehydration. A suitable degradation rate reduced potential administration frequency. Furthermore, an in vivo rabbit full-thickness wound healing test demonstrated that the bilayer scaffold facilitated wound closure, granulation tissue formation, re-epithelialization and skin component transition towards normal skin by providing a moist wound environment, advancing the inflammation stage, and stimulating angiogenesis. Collectively, as an off-the-shelf and cell-free wound dressing with single topical administration, the bilayer scaffold is a promising wound dressing for full-thickness wound regeneration.

Multicellular agglomerates in form of irregularly shaped or spherical clusters can recapitulate cell–cell interactions and are referred to as microtissues. Microtissues gain increasing attention in several fields including cardiovascular research. Cardiac microtissues are evolving as excellent model systems for drug testing in vitro (organ-on-a-chip), are used as tissue bricks in 3D printing processes and pave the way for improved cell replacement therapies in vivo. Microtissues are formed for example in hanging drop culture or specialized microwell plates; truly scalable methods are not yet available. In this study, a novel method of encapsulation of cells in poly-N-isopropylacrylamid (PNIPAAm) spheres is introduced. Murine induced pluripotent stem cell-derived cardiomyocytes and bone marrow-derived mesenchymal stem cells were encapsulated in PNIPAAm by raising the temperature of droplets formed in a microfluidics setup above the lower critical solute temperature (LCST) of 32 °C. PNIPAAM precipitates to a water-insoluble physically linked gel above the LCST and shrinks by the expulsion of water, thereby trapping the cells in a collapsing polymer network and increasing the cell density by one order of magnitude. Within 24 h, stable cardiac microtissues were first formed and later released from their polymer shell by washout of PNIPAAm at temperatures below the LCST. Rhythmically contracting microtissues showed homogenous cell distribution, age-dependent sarcomere organizations and action potential generation. The novel approach is applicable for microtissue formation from various cell types and can be implemented into scalable workflows.

Mechanical loading has been shown to influence various osteogenic responses of bone-derived cells and bone formation in vivo. However, the influence of mechanical stimulation on the formation of bone organoid in vitro is not clearly understood. Here, three-dimensional (3D) bioprinted human mesenchymal stem cells-laden graphene oxide composite scaffolds were cultured in a novel cyclic-loading bioreactors for up to 56 d. Our results showed that mechanical loading from day 1 (ML01) significantly increased organoid mineral density, organoid stiffness, and osteoblast differentiation compared with non-loading and mechanical loading from day 21. Importantly, ML01 stimulated collagen I maturation, osteocyte differentiation, lacunar-canalicular network formation and YAP expression on day 56. These finding are the first to reveal that long-term mechanical loading is required for the formation of 3D bioprinted functional osteocyte bone organoids. Such 3D bone organoids may serve as a human-specific alternative to animal testing for the study of bone pathophysiology and drug screening.

Randomly oriented type I collagen (COL1) fibers in the extracellular matrix are reorganized by biophysical forces into aligned domains extending several millimeters and with varying degrees of fiber alignment. These aligned fibers can transmit traction forces, guide tumor cell migration, facilitate angiogenesis, and influence tissue morphogenesis. To create aligned COL1 domains in microfluidic cell culture models, shear flows have been used to align thin COL1 matrices (<50 µm in height) in a microchannel. However, there has been limited investigation into the role of shear flows in aligning 3D hydrogels (>130 µm). Here, we show that pure shear flows do not induce fiber alignment in 3D atelo COL1 hydrogels, but the simple addition of local extensional flow promotes alignment that is maintained across several millimeters, with a degree of alignment directly related to the extensional strain rate. We further advance experimental capabilities by addressing the practical challenge of accessing a 3D hydrogel formed within a microchannel by introducing a magnetically coupled modular platform that can be released to expose the microengineered hydrogel. We demonstrate the platform's capability to pattern cells and fabricate multi-layered COL1 matrices using layer-by-layer fabrication and specialized modules. Our approach provides an easy-to-use fabrication method to achieve advanced hydrogel microengineering capabilities that combine fiber alignment with biofabrication capabilities.

The heart is an essential organ for animals and humans. With the increased availability of pluripotent stem cells, the use of three-dimensional cardiac tissues consisting of cultured cardiomyocytes in in vitro drug evaluation has been widely studied. Several models have been proposed for the realization of the pump function, which is the original function of the heart. However, there are no models that simulate the human circulatory system using cultured cardiac tissue. This study shows that a dome-shaped cardiac tissue fabricated using the cell sheet stacking technique can achieve a heart-like pump function and circulate culture medium, there by mimicking the human circulatory system. Firstly, human induced pluripotent stem cells were differentiated into autonomously beating cardiomyocytes, and cardiomyocyte cell sheets were created using temperature-responsive culture dishes. A cardiomyocyte sheet and a human dermal fibroblast sheet were stacked using a cell sheet manipulator. This two-layered cell sheet was then inflated to create a dome-shaped cardiac tissue with a base diameter of 8 mm. The volume of the dome-shaped cardiac tissue changed according to the autonomous beating. The stroke volume increased with the culture period and reached 21 ± 8.9 μl (n = 6) on day 21. It also responded to β-stimulant and extracellular calcium concentrations. Internal pressure fluctuations were also recorded under isovolumetric conditions by dedicated culture devices. The peak heights of pulsatile pressure were 0.33 ± 0.048 mmHg (n = 3) under a basal pressure of 0.5 mmHg on day 19. When the tissue was connected to a flow path that had check valves applied, it drove a directional flow with an average flow rate of approximately 1 μl s⁻¹. Furthermore, pressure–volume (P–V) diagrams were created from the simultaneous measurement of changes in pressure and volume under three conditions of fluidic resistance. In conclusion, this cardiac model can potentially be used for biological pumps that drive multi-organ chips and for more accurate in vitro drug evaluation using P–V diagrams.

In recent decades, 3D in vitro cultures of primary human hepatocytes (PHHs) have been increasingly developed to establish models capable of faithfully mimicking main liver functions. The use of 3D bioprinting, capable of recreating structures composed of cells embedded in matrix with controlled microarchitectures, is an emergent key feature for tissue engineering. In this work, we used an extrusion-based system to print PHH in a methacrylated gelatin (GelMa) matrix. PHH bioprinted in GelMa rapidly organized into polarized hollow spheroids and were viable for at least 28 d of culture. These PHH were highly differentiated with maintenance of liver differentiation genes over time, as demonstrated by transcriptomic analysis and functional approaches. The cells were polarized with localization of apico/canalicular regions, and displayed activities of phase I and II biotransformation enzymes that could be regulated by inducers. Furthermore, the implantation of the bioprinted structures in mice demonstrated their capability to vascularize, and their ability to maintain human hepatic specific functions for at least 28 d was illustrated by albumin secretion and debrisoquine metabolism. This model could hold great promise for human liver tissue generation and its use in future biotechnological developments.

Embedded freeform writing addresses the contradiction between the material printability and biocompatibility for conventional extrusion-based bioprinting. However, the existing embedding mediums have limitations concerning the restricted printing temperature window, compatibility with bioinks or crosslinkers, and difficulties on medium removal. This work demonstrates a new embedding medium to meet the above demands, which composes of hydrophobically modified hydroxypropylmethyl cellulose and Pluronic F-127. The adjustable hydrophobic and hydrophilic associations between the components permit tunable thermoresponsive rheological properties, providing a programmable printing window. These associations are hardly compromised by additives without strong hydrophilic groups, which means it is compatible with the majority of bioink choices. We use polyethylene glycol 400, a strong hydrophilic polymer, to facilitate easy medium removal. The proposed medium enables freeform writing of the millimetric complex tubular structures with great shape fidelity and cell viability. Moreover, five bioinks with up to five different crosslinking methods are patterned into arbitrary geometries in one single medium, demonstrating its potential in heterogeneous tissue regeneration. Utilizing the rheological properties of the medium, an enhanced adhesion writing method is developed to optimize the structure's strand-to-strand adhesion. In summary, this versatile embedding medium provides excellent compatibility with multi-crosslinking methods and a tunable printing window, opening new opportunities for heterogeneous tissue regeneration.