Table of contents

Over the years, the field of bioprinting has attracted attention for its highly automated fabrication system that enables the precise patterning of living cells and biomaterials at pre-defined positions for enhanced cell-matrix and cell–cell interactions. Notably, vat polymerization (VP)-based bioprinting is an emerging bioprinting technique for various tissue engineering applications due to its high fabrication accuracy. Particularly, different photo-initiators (PIs) are utilized during the bioprinting process to facilitate the crosslinking mechanism for fabrication of high-resolution complex tissue constructs. The advancements in VP-based printing have led to a paradigm shift in fabrication of tissue constructs from cell-seeding of tissue scaffolds (non-biocompatible fabrication process) to direct bioprinting of cell-laden tissue constructs (biocompatible fabrication process). This paper, presenting a first-time comprehensive review of the VP-based bioprinting process, provides an in-depth analysis and comparison of the various biocompatible PIs and highlights the important considerations and bioprinting requirements. This review paper reports a detailed analysis of its printing process and the influence of light-based curing modality and PIs on living cells. Lastly, this review also highlights the significance of VP-based bioprinting, the regulatory challenges and presents future directions to transform the VP-based printing technology into imperative tools in the field of tissue engineering and regenerative medicine. The readers will be informed on the current limitations and achievements of the VP-based bioprinting techniques. Notably, the readers will realize the importance and value of highly-automated platforms for tissue engineering applications and be able to develop objective viewpoints towards this field.

This bioprinting roadmap features salient advances in selected applications of the technique and highlights the status of current developments and challenges, as well as envisioned advances in science and technology, to address the challenges to the young and evolving technique. The topics covered in this roadmap encompass the broad spectrum of bioprinting; from cell expansion and novel bioink development to cell/stem cell printing, from organoid-based tissue organization to bioprinting of human-scale tissue structures, and from building cell/tissue/organ-on-a-chip to biomanufacturing of multicellular engineered living systems. The emerging application of printing-in-space and an overview of bioprinting technologies are also included in this roadmap. Due to the rapid pace of methodological advancements in bioprinting techniques and wide-ranging applications, the direction in which the field should advance is not immediately clear. This bioprinting roadmap addresses this unmet need by providing a comprehensive summary and recommendations useful to experienced researchers and newcomers to the field.

Extrusion-based bioprinting is one of the leading manufacturing techniques for tissue engineering and regenerative medicine. Its primary limitation is the lack of materials, known as bioinks, which are suitable for the bioprinting process. The degree to which a bioink is suitable for bioprinting has been described as its 'printability.' However, a lack of clarity surrounding the methodologies used to evaluate a bioink's printability, as well as the usage of the term itself, have hindered the field. This article presents a review of measures used to assess the printability of extrusion-based bioinks in an attempt to assist researchers during the bioink development process. Many different aspects of printability exist and many different measurements have been proposed as a consequence. Researchers often do not evaluate a new bioink's printability at all, while others simply do so qualitatively. Several quantitative measures have been presented for the extrudability, shape fidelity, and printing accuracy of bioinks. Different measures have been developed even within these aspects, each testing the bioink in a slightly different way. Additionally, other relevant measures which had little or no examples of quantifiable methods are also to be considered. Looking forward, further work is needed to improve upon current assessment methodologies, to move towards a more comprehensive view of printability, and to standardize these printability measurements between researchers. Better assessment techniques will naturally lead to a better understanding of the underlying mechanisms which affect printability and better comparisons between bioinks. This in turn will help improve upon the bioink development process and the bioinks available for use in bioprinting.

Despite their outstanding potential and the success that has already been achieved with three-dimensional (3D) printed hydrogel scaffolds, there has been little investigation into their application in the regeneration of damaged or missing adipose tissue (AT). Due to their macroscopic shape, microarchitecture, extracellular matrix-mimicking structure, degradability and soft tissue biomimetic mechanical properties, 3D printed hydrogel scaffolds have great potential for use in aesthetic, structural and functional restoration of AT. Here, we propose a simple and cost-effective 3D printing strategy using gelatin-based ink to fabricate scaffolds suitable for AT engineering. The ink, successfully printed here for the first time, was prepared by mixing gelatin and methylenebisacrylamide (a crosslinker) to initiate the crosslinking reaction. The solution was then loaded into the cartridge (temperature T = 35 °C) of a pneumatic extrusion-based 3D printer and printed on a cooled surface (T = 4 °C) in the appropriate time window for ink printability as verified by rheological tests. Subsequently, the printed gelatin hydrogels were crosslinked at different temperatures to optimize their stability and fix the printed structure. The gelatin scaffolds crosslinked at 20 °C remained stable for 21 days at physiological temperature, with compressive mechanical properties mimicking those of AT (i.e. elastic modulus = 20 kPa). The 3D printed scaffolds showed no indirect cytotoxic effects on a 3T3-L1 pre-adipocyte cell line. Moreover, the printed scaffolds successfully promoted adhesion and proliferation of primary human pre-adipocytes, as demonstrated by LIVE/DEAD staining and Alamar Blue assay. The differentiation of primary human pre-adipocytes isolated from three different donors according to adipogenic phenotype was demonstrated by an increase in PPARγ gene expression detected by real-time PCR and accumulated lipid droplets stained by Oil Red O, thus proving the potential of the 3D printed gelatin hydrogels as scaffolds for AT engineering.

The current standard of care for patients with severe large-area burns consists of autologous skin grafting or acellular dermal substitutes. While emerging options to accelerate wound healing involve treatment with allogeneic or autologous cells, delivering cells to clinically relevant wound topologies, orientations, and sizes remains a challenge. Here, we report the one-step in situ formation of cell-containing biomaterial sheets using a handheld instrument that accommodates the topography of the wound. In an approach that maintained cell viability and proliferation, we demonstrated conformal delivery to surfaces that were inclined up to 45° with respect to the horizontal. In porcine pre-clinical models of full-thickness burn, we delivered mesenchymal stem/stromal cell-containing fibrin sheets directly to the wound bed, improving re-epithelialization, dermal cell repopulation, and neovascularization, indicating that this device could be introduced in a clinical setting improving dermal and epidermal regeneration.

Recently, decellularized extracellular matrix-based bio-ink (dECM bio-ink) derived from animal organs is attracting attention because of its excellent biocompatibility. However, its poor 3D printability and weak mechanical properties remain a challenge. Here, we developed a new dECM bio-ink with enhanced 3D printability and mechanical properties. dECM micro-particles of about 13.4 μm in size were prepared by decellularizing a porcine liver followed by freeze-milling. The new bio-ink, named as dECM powder-based bio-ink (dECM pBio-ink), was prepared by loading the dECM micro-particles into a gelatin mixture. The usefulness of the dECM pBio-ink was evaluated by assessing its mechanical properties, printability, and cytocompatibility. The results showed that its mechanical properties and 3D printability were greatly improved. Its elastic modulus increased by up to 9.17 times that of the conventional dECM bio-ink. Micro-patterns with living cells were successfully achieved with 93% cell viability. Above all, the new bio-ink showed superior performance in stacking of layers for 3D printing, whereas the conventional bio-ink could not maintain its shape. Finally, we demonstrated that the dECM pBio-ink possessed comparable cytocompatibility with the conventional dECM bio-ink through in vitro tests with endothelial cells and primary mouse hepatocytes.

Printing is a promising method to reduce the cost of fabricating biomedical devices. While there have been significant advancements in direct-write printing techniques, non-contact printing of biological reagents has been almost exclusively limited to inkjet printing. Motivated by this lacuna, this work investigated aerosol jet printing (AJP) of biological reagents onto a nonfouling polymer brush to fabricate in vitro diagnostic (IVD) assays. The ultrasonication ink delivery process, which had previously been reported to damage DNA molecules, caused no degradation of printed proteins, allowing printing of a streptavidin-biotin binding assay with sub-nanogram ml⁻¹ analytical sensitivity. Furthermore, a carcinoembryogenic antigen IVD was printed and found to have sensitivities in the clinically relevant range (limit of detection of approximately 0.5 ng ml⁻¹ and a dynamic range of approximately three orders of magnitude). Finally, the multi-material printing capabilities of the aerosol jet printer were demonstrated by printing silver nanowires and streptavidin as interconnected patterns in the same print job without removal of the substrate from the printer, which will facilitate the fabrication of mixed-material devices. As cost, versatility, and ink usage become more prominent factors in the development of IVDs, this work has shown that AJP should become a more widely considered technique for fabrication.

After surgical resection for a bone tumor, the uncleared bone tumor cells can multiply and cause recurrence of the bone tumor. It is worthwhile to design a scaffold that kills the remaining bone tumor cells and repairs bone defects that were given rise to by surgical resection. Additionally, it is extremely important to consider the function of angiogenesis in the process of bone regeneration because the newly formed blood vessels can offer the nutrients for bone regeneration. In this work, a novel metal-organic framework Cu-TCPP nanosheets interface-structured β-tricalcium phosphate (TCP) (Cu-TCPP-TCP) scaffold was successfully prepared through integrating a 3D-printing technique with an in-situ growth method in a solvothermal system. Owing to the excellent photothermal effect of Cu-TCPP nanosheets, Cu-TCPP-TCP scaffolds that were illuminated by near-infrared (NIR) light demonstrated photothermal performance, which was well regulated through varying the contents of Cu-TCPP nanosheets, and the ambient humidity and power density of NIR light. When cultured with osteosarcoma cells, Cu-TCPP-TCP scaffolds killed a significant quantity of osteosarcoma cells through released heat energy after exposure to NIR light with power density 1.0 W cm⁻² and duration 10 min. Similarly, Cu-TCPP-TCP scaffolds ablated subcutaneous bone tumor tissues on the backs of naked mice and suppressed their growth because of the heat energy transformed from NIR light. In-vitro studies found that Cu-TCPP-TCP scaffolds ably supported the attachments of both human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs), and significantly stimulated expressions of osteogenesis differentiation-related genes in HBMSCs and angiogenesis differentiation-related genes in HUVECs. After implanting Cu-TCPP-TCP scaffolds into the bone defects of rabbits, they effectively promoted bone regeneration. Thus, the integration of the bone-forming bioactivity of TCP scaffolds with the photothermal properties of Cu-TCPP nanosheets and angiogenesis activity of Cu ions makes Cu-TCPP-TCP scaffolds multifunctional, representing a new horizon to develop biomaterials for simultaneously curing bone tumors and repairing bone defects.

The mammalian retina contains multiple cellular layers, each carrying out a specific task. Such a controlled organization should be considered as a crucial factor for designing retinal therapies. The maintenance of retinal layered complexity through the use of scaffold-free techniques has recently emerged as a promising approach for clinical ocular tissue engineering. In an attempt to fabricate such layered retinal model, we are proposing herein a unique inkjet bioprinting system applied to the deposition of a photoreceptor cells (PRs) layer on top of a bioprinted retinal pigment epithelium (RPE), in a precise arrangement and without any carrier material. The results showed that, after bioprinting, both RPE and PRs were well positioned in a layered structure and expressed their structural markers, which was further demonstrated by ZO1, MITF, rhodopsin, opsin B, opsin R/G and PNA immunostaining, three days after bioprinting. We also showed that considerable amounts of human vascular endothelial growth factors (hVEGF) were released from the RPE printed layer, which confirmed the formation of a functional RPE monolayer after bioprinting. Microstructures of bioprinted cells as well as phagocytosis of photoreceptor outer segments by apical RPE microvilli were finally established through transmission electron microscopy (TEM) imaging. In summary, using this carrier-free bioprinting method, it was possible to develop a reasonable in vitro retina model for studying some sight-threatening diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP).

Macrophages are key players of the innate immune system that can roughly be divided into the pro-inflammatory M1 type and the anti-inflammatory, pro-healing M2 type. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates a proper healing and subsequent regeneration. One promising strategy to drive macrophage polarization by biomaterials is precise control over biomaterial geometry. For regenerative approaches, it is of particular interest to identify geometrical parameters that direct human macrophage polarization. For this purpose, we advanced melt electrowriting (MEW) towards the fabrication of fibrous scaffolds with box-shaped pores and precise inter-fiber spacing from 100 μm down to only 40 μm. These scaffolds facilitate primary human macrophage elongation accompanied by differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 μm. These new findings can be important in helping to design new biomaterials with an enhanced positive impact on tissue regeneration.

Mounting evidence supports the importance of the intestinal epithelial barrier and its permeability both in physiological and pathological conditions. Conventional in vitro models to evaluate intestinal permeability rely on the formation of tightly packed epithelial monolayers grown on hard substrates. These two-dimensional models lack the cellular and mechanical components of the non-epithelial compartment of the intestinal barrier, the stroma, which are key contributors to the barrier permeability in vivo. Thus, advanced in vitro models approaching the in vivo tissue composition are fundamental to improve precision in drug absorption predictions, to provide a better understanding of the intestinal biology, and to faithfully represent related diseases. Here, we generate photo-crosslinked gelatine methacrylate (GelMA)—poly(ethylene glycol) diacrylate (PEGDA) hydrogel co-networks that provide the required mechanical and biochemical features to mimic both the epithelial and stromal compartments of the intestinal mucosa, i.e. they are soft, cell adhesive and cell-loading friendly, and suitable for long-term culturing. We show that fibroblasts can be embedded in the GelMA-PEGDA hydrogels while epithelial cells can grow on top to form a mature epithelial monolayer that exhibits barrier properties which closely mimic those of the intestinal barrier in vivo, as shown by the physiologically relevant transepithelial electrical resistance (TEER) and permeability values. The presence of fibroblasts in the artificial stroma compartment accelerates the formation of the epithelial monolayer and boosts the recovery of the epithelial integrity upon temporary barrier disruption, demonstrating that our system is capable of successfully reproducing the interaction between different cellular compartments. As such, our hydrogel co-networks offer a technologically simple yet sophisticated approach to produce functional three-dimensional (3D) in vitro models of epithelial barriers with epithelial and stromal cells arranged in a spatially relevant manner and near-physiological functionality.

Biophysical cues robustly direct cell responses and are thus important tools for in vitro and translational biomedical applications. High throughput platforms exploring substrates with varying physical properties are therefore valuable. However, currently existing platforms are limited in throughput, the biomaterials used, the capability to segregate between different cues and the assessment of dynamic responses. Here we present a multiwell array (3 × 8) made of a substrate engineered to present topography or rigidity cues welded to a bottomless plate with a 96-well format. Both the patterns on the engineered substrate and the well plate format can be easily customized, permitting systematic and efficient screening of biophysical cues. To demonstrate the broad range of possible biophysical cues examinable, we designed and tested three multiwell arrays to influence cardiomyocyte, chondrocyte and osteoblast function. Using the multiwell array, we were able to measure different cell functionalities using analytical modalities such as live microscopy, qPCR and immunofluorescence. We observed that grooves (5 μm in size) induced less variation in contractile function of cardiomyocytes. Compared to unpatterned plastic, nanopillars with 127 nm height, 100 nm diameter and 300 nm pitch enhanced matrix deposition, chondrogenic gene expression and chondrogenic maintenance. High aspect ratio pillars with an elastic shear modulus of 16 kPa mimicking the matrix found in early stages of bone development improved osteogenic gene expression compared to stiff plastic. We envisage that our bespoke multiwell array will accelerate the discovery of relevant biophysical cues through improved throughput and variety.

Smart nanofibrillar constructs can be a promising technological solution for many emerging and established fields, facilitating the potential impact of nano-scale strategies to address relevant technological challenges. As a fabrication technique, electrospinning (ESP) is relatively well-known, accessible, economic, and fast, though until now has shown limitation over control and design of the fibrillar constructs which can be produced. Here, we introduce 'Tandem Electrospinning' (T-ESP), a novel technique able to create increasingly complex patterns of fibrous polymeric constructs on a micro and nano-scale. Modifying a standard ESP configuration results in a focusing electric field that is able to spatially define the deposition pattern of multiple polymer jets simultaneously. Additional spatially defined heterogeneity is achieved by tuning polymer solution properties to obtain a gradient of fiber alignment. Heterogeneous fibrous meshes are created with either random, aligned, or a divergent fiber patterns. This approach holds potential for many fields, with application examples shown for tissue engineering and separation technologies. The technique outlined here provides a rapid, versatile, and accessible method for polymeric nanofabrication of patterned heterogeneous fibrous constructs. Polymer properties are also shown to dictate fiber alignment, providing further insight into the mechanisms involved in the electrospinning fabrication process.

During the bioprinting processes that employ either pneumatic or screw-driven mechanisms, living cells are subject to process-induced forces, which may cause cell injury or damage. However, the similarities and differences between these two mechanisms have not been discovered and documented in terms of process-induced forces and cell damage. In this paper, we examined the process-induced forces, including hydrostatic pressure, shear stress, extensional stress, and tensile/compressive forces that the cells experienced during the bioprinting processes by means of these two mechanisms; we also experimentally investigated the process-induced cell damage (featured by the rupture of the cell membrane) under various printing conditions or factors, including the volumetric flow rates, cell types, bioink solutions, needle types and sizes, and printing head-movement speeds. On this basis, we correlated the percent of cell damage to the process-induced forces, which were considered mainly responsible for the rupture of the cell membrane. Our results illustrate that compared to the pneumatic bioprinting process, the screw-driven bioprinting process generally induces more cell damage, varying with the printing conditions. This study, for the first time, discovers the similarities and differences between the pneumatic and screw-driven bioprinting processes and further demonstrates their merits and demerits for bioprinting in terms of printing-process control, process-induced forces, and cell damage.

Pneumonia is one of the most common infectious diseases worldwide. The influenza virus can cause severe epidemics, which results in significant morbidity and mortality. Beyond the virulence of the virus itself, epidemiological data suggest that bacterial co-infections are the major cause of increased mortality. In this context, Staphylococcus aureus represents a frequent causative bacterial pathogen. Currently available models have several limitations in the analysis of the pathogenesis of infections, e.g. some bacterial toxins strongly act in a species-specific manner. Human 2D mono-cell culture models often fail to maintain the differentiation of alveolus-specific functions. A detailed investigation of the underlying pathogenesis mechanisms requires a physiological interaction of alveolus-specific cell types. The aim of the present work was to establish a human in vitro alveolus model system composed of vascular and epithelial cell structures with cocultured macrophages resembling the human alveolus architecture and functions. We demonstrate that high barrier integrity maintained for up to 14 d in our model containing functional tissue-resident macrophages. We show that flow conditions and the presence of macrophages increased the barrier function. The infection of epithelial cells induced a high inflammatory response that spread to the endothelium. Although the integrity of the epithelium was not compromised by a single infection or co-infection, we demonstrated significant endothelial cell damage associated with loss of barrier function. We established a novel immune-responsive model that reflects the complex crosstalk between pathogens and host. The in vitro model allows for the monitoring of spatiotemporal spreading of the pathogens and the characterization of morphological and functional alterations attributed to infection. The alveolus-on-a-chip represents a promising platform for mechanistic studies of host-pathogen interactions and the identification of molecular and cellular targets of novel treatment strategies in pneumonia.

Bone is a highly vascularized tissue, in which vascularization and mineralization are concurrent processes during skeletal development. Indeed, both components should be included in any reliable and adherent in vitro model platform for the study of bone physiology and pathogenesis of skeletal disorders. To this end, we developed an in vitro vascularized bone model, using a gelatin-nanohydroxyapatite (gel-nHA) three-dimensional (3D) bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold, which underwent osteogenic differentiation for 2 weeks. Then, we included lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted scaffold macropores to form a capillary-like network during 2 more weeks of culture. We tested three experimental conditions: condition 1, bone constructs with HUVECs cultured in 1:1 osteogenic medium (OM): endothelial medium (EM); condition 2, bone constructs without HUVECs cultured in 1:1 OM:EM; condition 3: bone construct with HUVECs cultured in 1:1 growth medium:EM. All samples resulted in engineered bone matrix. In conditions 1 and 3, HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even when OM was removed under condition 3, we observed clear osteogenesis, which was notably accompanied by upregulation of osteopontin, vascular endothelial growth factor, and collagen type I. These findings indicate that we have successfully realized a bone model with robust vascularization in just 4 weeks of culture and we highlighted how the inclusion of endothelial cells more realistically supports osteogenesis. The approach reported here resulted in a biologically inspired in vitro model of bone vascularization, simulating de novo morphogenesis of capillary vessels occurring during tissue development.

Multi-material 3D printing technologies that resolve features at different lengths down to the microscale open new avenues for regenerative medicine, particularly in the engineering of tissue interfaces. Herein, extrusion printing of a bone-biomimetic ceramic ink and melt electrowriting (MEW) of spatially organized polymeric microfibres are integrated for the biofabrication of an osteochondral plug, with a mechanically reinforced bone-to-cartilage interface. A printable physiological temperature-setting bioceramic, based on α-tricalcium phosphate, nanohydroxyapatite and a custom-synthesized biodegradable and crosslinkable poloxamer, was developed as bone support. The mild setting reaction of the bone ink enabled us to print directly within melt electrowritten polycaprolactone meshes, preserving their micro-architecture. Ceramic-integrated MEW meshes protruded into the cartilage region of the composite plug, and were embedded with mechanically soft gelatin-based hydrogels, laden with articular cartilage chondroprogenitor cells. Such interlocking design enhanced the hydrogel-to-ceramic adhesion strength >6.5-fold, compared with non-interlocking fibre architectures, enabling structural stability during handling and surgical implantation in osteochondral defects ex vivo. Furthermore, the MEW meshes endowed the chondral compartment with compressive properties approaching those of native cartilage (20-fold reinforcement versus pristine hydrogel). The osteal and chondral compartment supported osteogenesis and cartilage matrix deposition in vitro, and the neo-synthesized cartilage matrix further contributed to the mechanical reinforcement at the ceramic-hydrogel interface. This multi-material, multi-scale 3D printing approach provides a promising strategy for engineering advanced composite constructs for the regeneration of musculoskeletal and connective tissue interfaces.

Hydrogels are very popular in biomedical areas for their extraordinary biocompatibility. However, most bio-hydrogels are too brittle to perform micro/nanofabrication. An effective method is cast molding; yet during this process, many defects occur as the excessive demolding stress damages the brittle hydrogels. Here, we propose a brand-new damage-free demolding method and a soft ultrafine fiber mold (SUFM) to replace the traditional mold. Both mechanical and finite element analysis (FEA) reveal that SUFMs have obvious advantages especially when the contact area between hydrogel and mold gets larger. By means of a high-resolution 3D printing called electrohydrodynamic (EHD) printing, SUFMs with various topological structures can be achieved with the fiber diameter ranging from 500 nm to 100 μm, at a low cost. Microfluidics and cell patterns are implemented as the demonstration for potential applications. Owing to the tiny scale of microstructures and the hydrophilicity of hydrogels, significant capillary effect occurs which can be utilized to deliver liquid and cells autonomously and to seed cells into those ultrafine channels evenly. The results open up a new avenue for a wider use of hydrogels in biomedical devices, tissue engineering, hydrogel-based microfluidics and wearable electronics; the proposed fabrication method also has the potential to become a universal technique for micro/nanofabrication of brittle materials.

Microwell arrays have emerged as three-dimensional substrates for cell culture due to their simplicity of fabrication and promise for high-throughput applications such as 3D cell-based assays for drug screening. To date, most microwells have had cylindrical geometries. Motivated by our previous findings that cells display 3D physiological characteristics when grown in the spherical micropores of monodisperse foam scaffolds (Lee et al 2013 Integr. Biol.5 1447–55 and Lin et al 2011 Soft Matter7 10010–6), here we engineered novel microwells shaped as spherical caps with obtuse polar angles, yielding narrow apertures. When used as bare substrates, these microwells were suitable for culturing cell spheroids; the narrow apertures sterically hindered unattached cultured cells from rolling out of microwells under agitation. When only the walls of the microwell were conjugated with extracellular matrix proteins, cells remained confined in the microwells. Epithelial cells proliferated and burst out of the aperture, and cell polarity was oriented based on the distribution of extracellular matrix proteins in the microwells. Surprisingly, single fibroblast cells in spherical wells of various diameters (40–100 μm) underwent cell-cycle arrest, while cells in circular cylindrical microwells continued to proliferate. Spatial confinement was not sufficient to cause cell-cycle arrest; however, confinement in a constant negative-curvature microenvironment led to cell-cycle arrest. Overall, these investigations demonstrate that this spherical microwell substrate constitutes a novel basic research tool for elucidating how cells respond to dimensionality and microenvironment with radii of curvature at the cellular length scale.

Current practices in drug development have led to therapeutic compounds being approved for widespread use in humans, only to be later withdrawn due to unanticipated toxicity. These occurrences are largely the result of erroneous data generated by in vivo and in vitro preclinical models that do not accurately recapitulate human physiology. Herein, a human primary cell- and stem cell-derived 3D organoid technology is employed to screen a panel of drugs that were recalled from market by the FDA. The platform is comprised of multiple tissue organoid types that remain viable for at least 28 days, in vitro. For many of these compounds, the 3D organoid system was able to demonstrate toxicity. Furthermore, organoids exposed to non-toxic compounds remained viable at clinically relevant doses. Additional experiments were performed on integrated multi-organoid systems containing liver, cardiac, lung, vascular, testis, colon, and brain. These integrated systems proved to maintain viability and expressed functional biomarkers, long-term. Examples are provided that demonstrate how multi-organoid 'body-on-a-chip' systems may be used to model the interdependent metabolism and downstream effects of drugs across multiple tissues in a single platform. Such 3D in vitro systems represent a more physiologically relevant model for drug screening and will likely reduce the cost and failure rate associated with the approval of new drugs.

Cellular therapies play an important role in tendon tissue engineering, with tenocytes being the most prominent and potent cell population available. However, for the development of a rich extracellular matrix tenocyte-assembled tendon equivalent, prolonged in vitro culture is required, which is associated with phenotypic drift. Recapitulation of tendon tissue microenvironment in vitro with cues that enhance and accelerate extracellular matrix synthesis and deposition, whilst maintaining tenocyte phenotype, may lead to functional cell therapies. Herein, we assessed the synergistic effect of low oxygen tension (enhances extracellular matrix synthesis) and macromolecular crowding (enhances extracellular matrix deposition) in human tenocyte culture. Protein analysis demonstrated that human tenocytes at 2% oxygen tension and with 50 μg ml⁻¹ carrageenan (macromolecular crowder used) significantly increased synthesis and deposition of collagen types I, III, V and VI. Gene analysis at day 7 illustrated that human tenocytes at 2% oxygen tension and with 50 μg ml⁻¹ carrageenan significantly increased the expression of prolyl 4-hydroxylase subunit alpha 1, procollagen-lysine 2- oxoglutarate 5-dioxygenase 2, scleraxis, tenomodulin and elastin, whilst chondrogenic (e.g. runt-related transcription factor 2, cartilage oligomeric matrix protein, aggrecan) and osteogenic (e.g. secreted phosphoprotein 1, bone gamma-carboxyglutamate protein) trans-differentiation markers were significantly down-regulated or remained unchanged. Collectively, our data clearly illustrates the beneficial synergistic effect of low oxygen tension and macromolecular crowding in the accelerated development of tissue equivalents.

Laser bioprinting is a term that refers to a group of laser-based techniques for printing living cells with high precision and good viability. Most of these techniques are based on modifications of the standard laser induced forward transfer technique (LIFT). When it comes to printing living material, direct laser irradiation should be avoided, therefore an indirect LIFT technique comprising an energy absorption layer should be used. This work presents a blister actuated-LIFT (BA-LIFT) technique which uses a commercial polyimide tape as a uniform energy absorption layer. To increase the potential of the technique for cell selection and printing accuracy, we take advantage of the high optical transmission of the polyimide layer to implement an in-line fluorescence and conventional imaging vision system coaxial with the laser path. With this system and using the appropriate staining methodology it is possible to track and identify different cell types, selecting those to be transferred and tracking cell survival both in the donor and acceptor substrates. We studied the BA-LIFT printability map for sodium alginate and methylcellulose hydrogels in the fluence range from 6.1 to 2.0 J cm⁻² together with the cell viability assessment measured by our fluorescence system. The study has revealed that less concentrated, therefore less viscose hydrogel shows better results with lower fluences, whereas hydrogels with higher concentrations present better results at higher fluences. Also, at low fluences 98 % of cell viability was obtained, besides both primary cells and cell lines keep their integrity, proliferating and functional activity. The technique was tested by tracking and targeting mouse hematopoietic progenitor stem cells transferred to assess colony forming units; moreover, natural killer cells were isolated and its activation in a stimulation media was tracked with the fluorescence system.

The effect of disturbed flow profiles on the endothelium have been studied extensively in systemic vasculature, but less is known about the response of the blood-brain barrier (BBB) to these flow regimes. Here we investigate the effect of disturbed flow on the integrity of the BBB using a three-dimensional, perfusable bifurcation model consisting of a co-culture of endothelial cells with mural and glial cells. Experimental flow patterns predicted by computational fluid dynamics mimic in vivo flow regimes, specifically the presence of a recirculation zone immediately downstream of the bifurcation. Dextran permeability assays and immunostaining with markers for tight junctions show that barrier disruption is significantly greater in areas of disturbed flow compared to fully developed regions downstream of the bifurcation. Probing crosstalk between cell types suggests that disturbed flow causes barrier breakdown independent of endothelial-mural and endothelial-glial interaction. Overall, disturbed flow-induced disruption of the blood-brain barrier suggests that flow-mediated mechanisms may contribute to vascular pathologies in the central nervous system.

Micro Electro Mechanical Systems (MEMS) and microfluidic devices have found numerous applications in the industrial sector. However, they require a fast, cost-effective and reliable manufacturing process in order to compete with conventional methods. Particularly, at the sub-micron scale, the manufacturing of devices are limited by the dimensional complexity. A proper bonding and stiction prevention of these sub-micron channels are two of the main challenges faced during the fabrication process of low aspect ratio channels. Especially, in the case of using flexible materials such as polydimethylsiloxane (PDMS). This study presents a direct laser microfabrication method of sub-micron channels using an infrared (IR) ultrashort pulse (femtosecond), capable of manufacturing extremely low aspect ratio channels. These microchannels are manufactured and tested varying their depth from 0.5 μm to 2 μm and width of 15, 20, 25, and 30 μm. The roughness of each pattern was measured by an interferometric microscope. Additionally, the static contact angle of each depth was studied to evaluate the influence of femtosecond laser fabrication method on the wettability of the glass substrate. PDMS, which is a biocompatible polymer, was used to provide a watertight property to the sub-micron channels and also to assist the assembly of external microfluidic hose connections. A 750 nm depth watertight channel was built using this methodology and successfully used as a blood plasma separator (BPS). The device was able to achieve 100% pure plasma without stiction of the PDMS layer to the sub-micron channel within an adequate time. This method provides a novel manufacturing approach useful for various applications such as point-of-care devices.

Systematic analysis of the extrusion process in 3D bioprinting is mandatory for process optimization concerning production speed, shape fidelity of the 3D construct and cell viability. In this study, we applied numerical and analytical modeling to describe the fluid flow inside the printing head based on a Herschel–Bulkley model. The presented analytical calculation method nicely reproduces the results of Computational Fluid Dynamics simulation concerning pressure drop over the printing head and maximal shear parameters at the outlet. An approach with dimensionless flow parameter enables the user to adapt rheological characteristics of a bioink, the printing pressure and needle diameter with regard to processing time, shear sensitivity of the integrated cells, shape fidelity and strand dimension. Bioinks consist of a blend of polymers and cells, which lead to a complex fluid behavior. In the present study, a bioink containing alginate, methylcellulose and agarose (AMA) was used as experimental model to compare the calculated with the experimental pressure gradient. With cultures of an immortalized human mesenchymal stem cell line and plant cells (basil) it was tested how cells influence the flow and how mechanical forces inside the printing needle affect cell viability. Influences on both sides increased with cell (aggregation) size as well as a less spherical shape. This study contributes to a systematic description of the extrusion-based bioprinting process and introduces a general strategy for process design, transferable to other bioinks.

The stiffness and topography of a cell's extracellular matrix (ECM) are physical cues that play a key role in regulating processes that determine cellular fate and function. While substrate stiffness can dictate cell differentiation lineage, migration, and self-organization, topographical features can change the cell's differentiation profile or migration ability. Although both physical cues are present and intrinsic to the native tissues in vivo, in vitro studies have been hampered by the lack of technological set-ups that would be compatible with cell culture and characterization. In vitro studies therefore either focused on screening stiffness effects in cells cultured on flat substrates or on determining topography effects in cells cultured onto hard materials. Here, we present a reliable, microfabrication method to obtain well defined topographical structures of micrometer size (5–10 μm) on soft polyacrylamide hydrogels with tunable mechanical stiffness (3–145 kPa) that closely mimic the in vivo situation. Topographically microstructured polyacrylamide hydrogels are polymerized by capillary force lithography using flexible materials as molds. The topographical microstructures are resistant to swelling, can be conformally functionalized by ECM proteins and sustain the growth of cell lines (fibroblasts and myoblasts) and primary cells (mouse intestinal epithelial cells). Our method can independently control stiffness and topography, which allows to individually assess the contribution of each physical cue to cell response or to explore potential synergistic effects. We anticipate that our fabrication method will be of great utility in tissue engineering and biophysics, especially for applications where the use of complex in vivo-like environments is of paramount importance.

In this paper we investigate the application of a direct writing technique for printing conductive patterns onto a biocompatible electrospun-pyrolysed carbon-fibre-based substrate. The result is a first study towards the production of bio-electrical scaffolds that could be used to enhance the promotion of efficient connections among neurons for in vitro studies in the field of neural tissue engineering. An electrospinning process is employed for production of the materials derived from the precursor polyacrylonitrile, in which the embedding of carbon nanotubes (CNTs) is also investigated. Subsequently, the methodology of research into suitable parameters for the printed electronics, using a commercial silver nanoparticle (Ø_{avg,particle size} ∼ 100 nm) ink, is described. The results show values of 2 Ω cm for the resistivity of the carbon-fibre materials and conductive printed lines of resistance ∼50 Ω on glass and less than ∼140 Ω on carbon-fibre samples. Biocompatibility results demonstrate the possibility of using electrospun-pyrolysed mats, also with embedded CNTs, as potential neural substrates for spatially localized electrical stimulation across a tissue. In addition, the data concerning the potential toxicity of silver suspensions are in accordance with the literature, showing a dose-dependent behaviour. This work is a pioneering feasibility study of the use of the flexible and versatile printed electronic approach, combined with engineered biocompatible substrates, to realize integrated bio-electrical scaffolds for in vitro neural tissue engineering applications.

The field of bioprinting has made significant recent progress towards engineering tissues with increasing complexity and functionality. It remains challenging, however, to develop bioinks with optimal biocompatibility and good printing fidelity. Here, we demonstrate enhanced printability of a polymer-based bioink based on dynamic covalent linkages between nanoparticles (NPs) and polymers, which retains good biocompatibility. Amine-presenting silica NPs (ca. 45 nm) were added to a polymeric ink containing oxidized alginate (OxA). The formation of reversible imine bonds between amines on the NPs and aldehydes of OxA lead to significantly improved rheological properties and high printing fidelity. In particular, the yield stress increased with increasing amounts of NPs (14.5 Pa without NPs, 79 Pa with 2 wt% NPs). In addition, the presence of dynamic covalent linkages in the gel provided improved mechanical stability over 7 d compared to ionically crosslinked gels. The nanocomposite ink retained high printability and mechanical strength, resulting in generation of centimeter-scale porous constructs and an ear structure with overhangs and high structural fidelity. Furthermore, the nanocomposite ink supported both in vitro and in vivo maturation of bioprinted gels containing chondrocytes. This approach based on simple oxidation can be applied to any polysaccharide, thus the widely applicability of the method is expected to advance the field towards the goal of precision bioprinting.

Improving the proliferation of probiotics (ca. Bifidobacterium) and inhibiting the growth of pathogenic bacteria (ca. Escherichia coli) is crucial for human health. This study demonstrates the fabrication of core–shell structure fibers using electrohydrodynamic 3D printing to help improve gastrointestinal tract microbial content. These fibers have various geometries and are capable of encapsulating stachyose into cellulose acetate (shell layer) and loading proteoglycan into polyacrylic resin II (core layer). The impact of membrane geometry on drug release behavior and the effect of exchanging the loading site on physicochemical properties of the resulting fibers were studied. The printed fibrous membranes possess a biphasic drug release profile in simulated intestinal fluid with a burst release within the first 12 h and a slower sustained release up to 72 h. The speed order priority for drug release rate of the printed membrane was whole-circle > semi-circle > square. Moreover, the membranes exhibit good biocompatibility on L929 cells and excellent improvement effects on Bifidobacterium bifidum, combining inhibition effects on Escherichia coli. In summary, the dual-drug fibrous membranes presented here and their precision-fabricated patterns pave a new direction for improving the gastrointestinal tract microbial ecosystem health in the human body.

Extracellular matrices (ECMs) are dynamically altered and remodeled during tissue development. How the dynamic remodeling of ECM affects stem cell functions remains poorly understood due to the difficulty of obtaining biomimetic ECMs. In this study, stepwise osteogenesis-mimicking ECM-deposited hybrid meshes were prepared by culturing human mesenchymal stem cells (hMSCs) in poly (lactic-co-glycolic acid) (PLGA)-collagen hybrid meshes and controlling the stages of the osteogenesis of hMSCs. Three types of hybrid mesh mimicking the ECMs that were secreted from stem cell stage of hMSCs (SC-ECM), early stage (EO-ECM) and late stage (LO-ECM) osteogenesis of hMSCs were prepared. The stepwise osteogenesis-mimicking ECM deposited PLGA-collagen hybrid meshes showed different ECM compositions associated with the stage of osteogenesis. Their effects on the osteogenic differentiation of hMSCs differed. EO-ECM scaffold increased and LO-ECM scaffold moderately promoted the osteogenic differentiation of hMSCs. However, SC-ECM scaffold inhibited the osteogenic differentiation of hMSCs. The novel PLGA-collagen-ECM hybrid meshes will provide useful tools for stem cell culture and tissue engineering.

The meniscus has critical functions in the knee joint kinematics and homeostasis. Injuries of the meniscus are frequent, and the lack of a functional meniscus between the femur and tibial plateau can cause articular cartilage degeneration leading to osteoarthritis development and progression. Regeneration of meniscus tissue has outstanding challenges to be addressed. In the current study, novel Entrapped in cage (EiC) scaffolds of 3D-printed polycaprolactone (PCL) and porous silk fibroin were proposed for meniscus tissue engineering. As confirmed by micro-structural analysis the entrapment of silk fibroin was successful, and all scaffolds had excellent interconnectivity (≥99%). The EiC scaffolds had more favorable micro-structure compared with the PCL cage scaffolds by improving the pore size while keeping the interconnectivity almost the same. When compared with the PCL cage, the entrapment of porous silk fibroin into the PCL cage decreased the high compressive modulus in a favorable matter in the wet state thanks to the silk fibroin's high swelling properties. The in vitro studies with human stem cells or meniscocytes seeded constructs, demonstrated that the EiC scaffolds had superior cell adhesion, metabolic activity, and proliferation compared to the PCL cage scaffolds. Upon subcutaneous implantation of scaffolds in nude mice, all groups were free of adverse incidents, and mildly invaded by inflammatory cells with neovascularization, while the EiC scaffolds showed better tissue infiltration. The results of this work indicated that the EiC scaffolds of PCL and silk fibroin are favorable for meniscus tissue engineering, and the findings are encouraging for further studies using a larger animal model.

To achieve a three-dimensional (3D) microenvironment for complex tissue regeneration is a great challenge when developing biomaterials as artificial extracellular matrix (ECM) with properties similar to that of native tissue. Polysaccharide-based hydrogel shows great potential as ECM in the regeneration of damaged tissues or reconstruction of organs, demonstrating properties similar to those of native ECM. Extrusion 3D printing of cell-free or cell-loaded hydrogel ink has led to a more sophisticated fabrication of the desired compositions and architectures for tissue engineering applications. The development of stable cell-free and cell-loaded hydrogel inks with optimal physicochemical properties and biocompatibility is also a major concern in direct-write extrusion-based 3D printing. In this study, carboxylated cellulose nanocrystals (cCNCs) were prepared using ammonium persulfate, where transmission electron microscopy, Fourier-transform infrared spectroscopy, and x-ray diffraction analyses confirmed their successful preparation. Further, the effect of cCNCs (–COOH) and/or xanthan gum (XG) (–COOH) was evaluated on the rheological behavior of the sodium alginate (SA) hydrogel matrix. The incorporation of cCNCs and XG manipulated the flow and shear-thinning behavior of the hydrogel inks, thereby improving the printing ability. The results showed good rheological properties, post-printing fidelity, and dynamic mechanical properties under compression of the developed hydrogel inks. Furthermore, good viability of the human skin fibroblast (CCD-986Sk) cells on bulk hydrogels (hydrogel inks) was observed, as demonstrated by both qualitative and quantitative cell analyses. The use of cCNCs and XG in SA hydrogel inks provides a primary insight for further improvement in designing 3D bioprintable hydrogel inks.

Due to the increasing aging population and the high probability of sport injury among young people nowadays, it is of great demand to repair/regenerate diseased/defected osteochondral tissue. Given that osteochondral tissue mainly consists of a subchondral layer and a cartilage layer which are structurally heterogeneous and mechanically distinct, developing a biomimetic bi-phasic scaffold with excellent bonding strength to regenerate osteochondral tissue is highly desirable. Three-dimensional (3D) printing is advantageous in producing scaffolds with customized shape, designed structure/composition gradients and hence can be used to produce heterogeneous scaffolds for osteochondral tissue regeneration. In this study, bi-layered osteochondral scaffolds were developed through cryogenic 3D printing, in which osteogenic peptide/β-tricalcium phosphate/poly(lactic-co-glycolic acid) water-in-oil composite emulsions were printed into hierarchically porous subchondral layer while poly(D,L-lactic acid-co-trimethylene carbonate) water-in-oil emulsions were printed into thermal-responsive cartilage frame on top of the subchondral layer. The cartilage frame was further filled/dispensed with transforming growth factor-β1 loaded collagen I hydrogel to form the cartilage module. Although the continuously constructed osteochondral scaffolds had distinct microscopic morphologies and varied mechanical properties at the subchondral zone and cartilage zone at 37 °C, respectively, the two layers were closely bonded together, showing excellent shear strength and peeling strength. Rat bone marrow derived mesenchymal stem cells (rBMSCs) exhibited high viability and proliferation at both subchondral- and cartilage layer. Moreover, gradient rBMSC osteogenic/chondrogenic differentiation was obtained in the osteochondral scaffolds. This proof-of-concept study provides a facile way to produce integrated osteochondral scaffolds for concurrently directing rBMSC osteogenic/chondrogenic differentiation at different regions.

Cancer is the leading cause of mortality worldwide, and lung cancer is the most malignant. However, the high failure rate in oncology drug development from in vitro studies to in vivo preclinical models indicates that the modern methods of evaluating drug efficacies in vitro are not reliable. Traditional 2D cell culture has proved inadequate to mimic real physiological conditions. Current 3D cell culture methods do not represent the delicate structure of lung alveoli. To mimic lung alveoli structure, a cell-containing enzyme-crosslinked gelatin microbubble scaffold was produced by mixing surfactant-containing gelatin solution with microbial transglutaminase (mTGase)-mixed A549 cell suspension in a four-channel flow-focusing microfluidic device. With uniform pore size of about 100 μm in diameter, this gelatin microbubble scaffold resembled the lung alveoli in structure and in mechanical properties with good biocompatibility. Effective gemcitabine concentration required to induce cell death in microbubble scaffolds was significantly higher than in 2D culture together with a longer treatment time. Cell death mechanisms were confirmed to be gemcitabine-induced cell apoptosis through Western blotting and real-time polymerase chain reaction. H&E staining and TUNEL assay showed rounded cells with DNA damage in drug-treated scaffolds. Taken together, the cell-containing microbubble scaffolds successfully mimicked lung alveoli in structure and cellular responses after gemcitabine treatment were similar to clinical regimen of treating lung carcinoma. The microbubble scaffold is promising to facilitate anticancer drug discovery by providing more accurate preclinical predictions.

The fabrication technique determines the physicochemical and biological properties of scaffolds, including the porosity, mechanical strength, osteoconductivity, and bone regenerative potential. Biphasic calcium phosphate (BCP)-based scaffolds are superior in bone tissue engineering due to their suitable physicochemical and biological properties. We developed an indirect selective laser sintering (SLS) printing strategy to fabricate 3D microporous BCP scaffolds for bone tissue engineering purposes. The green part of the BCP scaffold was fabricated by SLS at a relevant low temperature in the presence of epoxy resin (EP), and the remaining EP was decomposed and eliminated by a subsequent sintering process to obtain the microporous BCP scaffolds. Physicochemical properties, cell adhesion, biocompatibility, in vitro osteogenic potential, and rabbit critical-size cranial bone defect healing potential of the scaffolds were extensively evaluated. This indirect SLS printing eliminated the drawbacks of conventional direct SLS printing at high working temperatures, i.e. wavy deformation of the scaffold, hydroxyapatite decomposition, and conversion of β-tricalcium phosphate (TCP) to α-TCP. Among the scaffolds printed with various binder ratios (by weight) of BCP and EP, the scaffold with 50/50 binder ratio (S4) showed the highest mechanical strength and porosity with the smallest pore size. Scaffold S4 showed the highest effect on osteogenic differentiation of precursor cells in vitro, and this effect was ERK1/2 signaling-dependent. Scaffold S4 robustly promoted precursor cell homing, endogenous bone regeneration, and vascularization in rabbit critical-size cranial defects. In conclusion, BCP scaffolds fabricated by indirect SLS printing maintain the physicochemical properties of BCP and possess the capacity to recruit host precursor cells to the defect site and promote endogenous bone regeneration possibly via the activation of ERK1/2 signaling.

How to pack materials into well-defined volumes efficiently has been a longstanding question of interest to physicists, material scientists, and mathematicians as these materials have broad applications ranging from shipping goods in commerce to seeds in agriculture and to spheroids in tissue engineering. How many marbles or gumball candies can you pack into a jar? Although these seem to be idle questions they have been studied for centuries and have recently become of greater interest with their broadening applications in science and medicine. Here, we study a similar problem where we try to pack cells into a spherical porous buckyball structure. The experimental limitations are short of the theoretical maximum packing density due to the microscale of the structures that the cells are being packed into. We show that we can pack more cells into a confined micro-structure (buckyball cage) by employing acoustofluidic activation and their hydrodynamic effect at the bottom of a liquid‐carrier chamber compared to randomly dropping cells onto these buckyballs by gravity. Although, in essence, cells would be expected to achieve a higher maximum volume fraction than marbles in a jar, given that they can squeeze and reshape and reorient their structure, the packing density of cells into the spherical buckyball cages are far from this theoretical limit. This is mainly dictated by the experimental limitations of cells washing away as well as being loaded into the chamber.