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Abstract
To progress cardiac tissue engineering strategies closer to the clinic, thicker constructs are required to meet the functional need following a cardiac event. Consequently, pre-vascularization of these constructs needs to be investigated to ensure survival and optimal performance of implantable engineered heart tissue. The aim of this research is to investigate the potential of combining extrusion-based bioprinting (EBB) and melt electrowriting for the fabrication of a myocardial construct with a precisely patterned pre-vascular pathway. Gelatin methacryloyl (GelMA) was investigated as a base hydrogel for the respective myocardial and vascular bioinks with collagen, Matrigel and fibrinogen as interpenetrating polymers to support myocardial functionality. Subsequently, extrusion-based printability and viability were investigated to determine the optimal processing parameters for printing into melt electrowritten meshes. Finally, an anatomically inspired vascular pathway was implemented in a dual EBB set-up into melt electrowritten meshes, creating a patterned pre-vascularized myocardial construct. It was determined that a blend of 5% GelMA and 0.8 mg·ml−1 collagen with a low crosslinked density was optimal for myocardial cellular arrangement and alignment within the constructs. For the vascular fraction, the optimized formulation consisted of 5% GelMA, 0.8 mg·ml−1 collagen and 1 mg·ml−1 fibrinogen with a higher crosslinked density, which led to enhanced vascular cell connectivity. Printability assessment confirmed that the optimized bioinks could effectively fill the microfiber mesh while supporting cell viability (∼70%). Finally, the two bioinks were applied using a dual EBB system for the fabrication of a pre-vascular pathway with the shape of a left anterior descending artery within a myocardial construct, whereby the distinct cell populations could be visualized in their respective patterns up to D14. This research investigated the first step towards developing a thick engineered cardiac tissue construct in which a pre-vascularization pathway is fabricated within a myocardial construct.

Abstract
Abstract In living tissues, cells express their functions following complex signals from their surrounding microenvironment. Capturing both hierarchical architectures at the micro- and macroscale, and anisotropic cell patterning remains a major challenge in bioprinting, and a bottleneck towards creating physiologically-relevant models. Addressing this limitation, we introduced a novel technique, termed Embedded Extrusion-Volumetric Printing (EmVP), converging extrusion-bioprinting and layer-less, ultra-fast volumetric bioprinting, allowing to spatially pattern multiple inks/cell types. Light-responsive microgels were developed for the first time as bioresins (μResins) for light-based volumetric bioprinting, providing a microporous environment permissive for cell homing and self-organization. Tuning the mechanical and optical properties of gelatin-based microparticles enables their use as support bath for suspended extrusion printing, in which features containing high cell densities can be easily introduced. μResins can be sculpted within seconds with tomographic light projections into centimetre-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids enhanced differentiation of multiple stem/progenitor cells (vascular, mesenchymal, neural), otherwise not possible with conventional bulk hydrogels. As proof-of-concept, EmVP was applied to create complex synthetic biology-inspired intercellular communication models, where adipocyte differentiation is regulated by optogenetic-engineered pancreatic cells. Overall, EmVP offers new avenues for producing regenerative grafts with biological functionality, and for developing engineered living systems and (metabolic) disease models. This article is protected by copyright. All rights reserved

Abstract
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.

Abstract
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.

Abstract
Abstract For patients with soft tissue defects, repair with autologous in vitro engineered adipose tissue could be a promising alternative to current surgical therapies. A volume-persistent engineered adipose tissue construct under in vivo conditions can only be achieved by early vascularization after transplantation. The combination of 3D bioprinting technology with self-assembling microvascularized units as building blocks can potentially answer the need for a microvascular network. In the present study, co-culture spheroids combining adipose-derived stem cells (ASC) and human umbilical vein endothelial cells (HUVEC) were created with an ideal geometry for bioprinting. When applying the favourable seeding technique and condition, compact viable spheroids were obtained, demonstrating high adipogenic differentiation and capillary-like network formation after 7 and 14 days of culture, as shown by live/dead analysis, immunohistochemistry and RT-qPCR. Moreover, we were able to successfully 3D bioprint the encapsulated spheroids, resulting in compact viable spheroids presenting capillary-like structures, lipid droplets and spheroid outgrowth after 14 days of culture. This is the first study that generates viable high-throughput (pre-)vascularized adipose microtissues as building blocks for bioprinting applications using a novel ASC/HUVEC co-culture spheroid model, which enables both adipogenic differentiation while simultaneously supporting the formation of prevascular-like structures within engineered tissues in vitro.

Abstract
Abstract Despite advances in biomaterials engineering, a large gap remains between the weak mechanical properties that can be achieved with natural materials and the strength of synthetic materials. Here, we present a method for reinforcing an engineered cardiac tissue fabricated from differentiated iPSCs and an ECM-based hydrogel in a manner that is fully biocompatible. The reinforcement occurs as a post-fabrication step, which allows for the use of 3D printing technology to generate thick, fully cellularized, and vascularized cardiac tissues. After tissue assembly and during the maturation process in a soft hydrogel, a small, tissue-penetrating reinforcer is deployed, leading to a significant increase in the tissue's mechanical properties. The tissue's robustness is demonstrated by injecting the tissue in a simulated minimally invasive procedure and showing that the tissue is functional and undamaged at the nano-, micro-, and macro-scales. This article is protected by copyright. All rights reserved

Abstract
Bioprinting technologies are powerful new bioengineering tools that can spatially reproduce multiple microenvironmental cues in a highly controlled, tunable, and precise manner. In this study, microvalve bioprinting technology was successfully used to print in close proximity endothelial and tumor cells at higher concentrations than previously thought possible, while preserving their viability. We propose that the resulting multicellular models, bioprinted in a controlled extracellular matrix microenvironment, are well-suited to study endothelial and cancer cell crosstalk within a cancer niche. As proof of concept, microvalve bioprinting was applied to the bioengineering of a simplified glioblastoma model in which biological processes involved in tumor expansion, such as tumor cell invasion patterns, cell proliferation, and senescence could be easily visualized and quantified. In this model, U251 glioblastoma cells and primary human umbilical vein endothelial cells (HUVECs) exhibited good printability and high viability after printing. U251 cells formed physiologically relevant clusters and invasion margins, while HUVECs generated vascular-like networks when primary fibroblasts were added to the model. An oxidative stress mimicking the one encountered within a tumor microenvironment during radiotherapy or genotoxic chemotherapy was shown to both diminish endothelial cells proliferation and to increase their senescence. Results also suggested that stressed glioblastoma cells may alter normal endothelial cell proliferation but not impact their senescence. This data demonstrates the potential of microvalve bioprinting to fabricate in vitro models that can help decipher endothelial and tumor cell crosstalk, within controlled and modulable microenvironments, and can then be used to address critical questions in the context of cancer recurrence.

Abstract
Bone tissue engineering (BTE) has proven to be a promising strategy for bone defect repair. Due to its excellent biological properties, gelatin methacrylate (GelMA) hydrogels have been used as bioinks for 3D bioprinting in some BTE studies to produce scaffolds for bone regeneration. However, applications for load-bearing defects are limited by poor mechanical properties and a lack of bioactivity. In this study, 3D printing technology was used to create nano-attapulgite (nano-ATP)/GelMA composite hydrogels loaded into mouse bone mesenchymal stem cells (BMSCs) and mouse umbilical vein endothelial cells (MUVECs). The bioprintability, physicochemical properties, and mechanical properties were all thoroughly evaluated. Our findings showed that nano-ATP groups outperform the control group in terms of printability, indicating that nano-ATP is beneficial for printability. Additionally, after incorporation with nano-ATP, the mechanical strength of the composite hydrogels was significantly improved, resulting in adequate mechanical properties for bone regeneration. The presence of nano-ATP in the scaffolds has also been studied for cell-material interactions. The findings show that cells within the scaffold not only have high viability but also a clear proclivity to promote osteogenic differentiation of BMSCs. Besides, the MUVECs-loaded composite hydrogels demonstrated increased angiogenic activity. A cranial defect model was also developed to evaluate the bone repair capability of scaffolds loaded with rat BMSCs. According to histological analysis, cell-laden nano-ATP composite hydrogels can effectively improve bone regeneration and promote angiogenesis. This study demonstrated the potential of nano-ATP for bone tissue engineering, which should also increase the clinical practicality of nano-ATP.

Abstract
Bone tissue engineering scaffolds have made significant progress in treating bone defects in recent decades. However, the lack of a vascular network within the scaffold limits bone formation after implantation in vivo. Recent research suggests that leonurine hydrochloride (LH) can promote healing in full-thickness cutaneous wounds by increasing vessel formation and collagen deposition. Gelatin and Sodium Alginate are both polymers. ATP is a magnesium silicate chain mineral. In this study, a Gelatin/Sodium Alginate/Nano-Attapulgite composite hydrogel was used as the base material first, and the Gelatin/Sodium Alginate/Nano-Attapulgite composite polymer scaffold loaded with LH was then created using 3D printing technology. Finally, LH was grafted onto the base material by an amide reaction to construct a scaffold loaded with LH to achieve long-term LH release. When compared to pure polymer scaffolds, in vitro results showed that LH-loaded scaffolds promoted the differentiation of BMSCs into osteoblasts, as evidenced by increased expression of osteogenic key genes. The results of in vivo tissue staining revealed that the drug-loaded scaffold promoted both angiogenesis and bone formation. Collectively, these findings suggest that LH-loaded Gelatin/Sodium Alginate/Nano-Attapulgite composite hydrogel scaffolds are a potential therapeutic strategy and can assist bone regeneration.

Abstract
Abstract 4D Biofabrication – a pioneering biofabrication technique – involves the automated fabrication of 3D constructs that are dynamic and show shape-transformation capability. Although current 4D biofabrication methods are highly promising for the fabrication of vascular elements such as tubes, the fabrication of tubular junctions is still highly challenging. Here, for the first time, a 4D biofabrication-based concept for the fabrication of a T-shaped vascular bifurcation using 3D printed shape-changing layers based on a mathematical model is reported. The formation of tubular structures with various diameters is achieved by precisely controlling the parameters (e.g. crosslinking time). Consequently, the 3D printed films show self-transformation into a T-junction upon immersion in water with a diameter of a few millimeters. Perfusion of the tubular T-junction with an aqueous medium simulating blood flow through vessels shows minimal leakages with a maximum flow velocity of 0.11 m s–1. Furthermore, human umbilical vein endothelial cells seeded on the inner surface of the plain T-junction show outstanding growth properties and excellent cell viability. The achieved diameters are comparable to the native blood vessels, which is still a challenge in 3D biofabrication. This approach paves the way for the fabrication of fully automatic self-actuated vascular bifurcations as vascular grafts.

Abstract
Background:The two ends of arteriovenous graft (AVG) are anastomosed to the upper limb vessels by surgery for hemodialysis therapy. However, the size of upper limb vessels varies to a large extent among different individuals.Methods:According to the shape and size of neck vessels quantified from the preoperative computed tomography angiographic scan, the ethylene-vinyl acetate (EVA)-based AVG was produced in H-shape by the three-dimensional (3D) printer and then sterilized. This study investigated the function of this novel 3D-printed AVG in vitro and in vivo.Results:This 3D-printed AVG can be implanted in the rabbit’s common carotid artery and common jugular vein with ease and functions in vivo. The surgical procedure was quick, and no suture was required. The blood loss was minimal, and no hematoma was noted at least 1 week after the surgery. The blood flow velocity within the implanted AVG was 14.9 ± 3.7 cm/s. Additionally, the in vitro characterization experiments demonstrated that this EVA-based biomaterial is biocompatible and possesses a superior recovery property than ePTFE after hemodialysis needle cannulation.Conclusions:Through the 3D printing technology, the EVA-based AVG can be tailor-made to fit the specific vessel size. This kind of 3D-printed AVG is functioning in vivo, and our results realize personalized vascular implants. Further large-animal studies are warranted to examine the long-term patency.

Abstract
Abstract Bioelastomers have been extensively used in biomedical applications due to their desirable mechanical strength, tunable properties, and chemical versatility; however, 3D printing bioelastomers into microscale structures has proven elusive. Herein, a high throughput omnidirectional printing approach via coaxial extrusion is described that fabricated perfusable elastomeric microtubes of unprecedently small inner diameter (350-550 μm) and wall thickness (40-60 μm). The versatility of this approach was shown through the printing of two different polymeric elastomers, followed by photocrosslinking and removal of the fugitive inner phase. Designed experiments were used to tune the dimensions and stiffness of the microtubes to match that of native ex vivo rat vasculature. This approach afforded the fabrication of multiple biomimetic shapes resembling cochlea and kidney glomerulus and afforded facile, high-throughput generation of perfusable structures that can be seeded with endothelial cells for biomedical applications. Post-printing laser micromachining was performed to generate numerous micro-sized holes (5-20 μm) in the tube wall to tune microstructure permeability. Importantly, for organ-on-a-chip applications, the described approach took only 3.6 minutes to print microtubes (without microholes) over an entire 96-well plate device, in contrast to comparable hole-free structures that take between 1.5 to 6.5 days to fabricate using a manual 3D stamping approach. This article is protected by copyright. All rights reserved

Abstract
Bioprinting is a biofabrication technology which allows efficient and large-scale manufacture of 3D cell culture systems. However, the available biomaterials for bioinks used in bioprinting are limited by their printability and biological functionality. Fabricated constructs are often homogeneous and have limited complexity in terms of current 3D cell culture systems comprising multiple cell types. Inspired by the phenomenon that hydrogels can exchange liquids under the infiltration action, infiltration-induced suspension bioprinting (IISBP), a novel printing technique based on a hyaluronic acid (HA) suspension system to modulate the properties of the printed scaffolds by infiltration action, was described in this study. HA served as a suspension system due to its shear-thinning and self-healing rheological properties, simplicity of preparation, reusability, and ease of adjustment to osmotic pressure. Changes in osmotic pressure were able to direct the swelling or shrinkage of 3D printed gelatin methacryloyl (GelMA)-based bioinks, enabling the regulation of physical properties such as fiber diameter, micromorphology, mechanical strength, and water absorption of 3D printed scaffolds. Human umbilical vein endothelial cells (HUVEC) were applied as a cell culture model and printed within cell-laden scaffolds at high resolution and cell viability with the IISBP technique. Herein, the IISBP technique had been realized as a reliable hydrogel-based bioprinting technique, which enabled facile modulation of 3D printed hydrogel scaffolds properties, being expected to meet the scaffolds requirements of a wide range of cell culture conditions to be utilized in bioprinting applications.

Abstract
Hydroxyapatite (HA) is a promising scaffold material for the treatment of bone defects. However{,} the lack of angiogenic properties and undesirable mechanical properties (such as fragility) limits the application of HA. Nanoattapulgite (ATP) is a nature-derived clay mineral and has been proven to be a promising bioactive material for bone regeneration due to its ability to induce osteogenesis. In this study{,} polyvinyl alcohol/collagen/ATP/HA (PVA/COL/ATP/HA) scaffolds were printed. Mouse bone marrow mesenchymal stem/stromal cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) were used in vitro to assess the biocompatibility and the osteogenesis and vascularization induction potentials of the scaffolds. Subsequently{,} in vivo micro-CT and histological staining were carried out to evaluate new bone formation in a rabbit tibial defect model. The in vitro results showed that the incorporation of ATP increased the printing fidelity and mechanical properties{,} with values of compressive strengths up to 200% over raw PC-H scaffolds. Simultaneously{,} the expression levels of osteogenic-related genes and vascularization-related genes were significantly increased after the incorporation of ATP. The in vivo results showed that the PVA/COL/ATP/HA scaffolds exhibited synergistic effects on promoting vascularization and bone formation. The combination of ATP and HA provides a promising strategy for vascularized bone tissue engineering.

Abstract
For 3D bioprinted tissues to be scaled-up to clinically relevant sizes, effective prevascularisation strategies are required to provide the necessary nutrients for normal metabolism and to remove associated waste by-products. The aim of this study was to develop a bioprinting strategy to engineer prevascularised tissues in vitro and to investigate the capacity of such constructs to enhance the vascularisation and regeneration of large bone defects in vivo. From a screen of different bioinks, a fibrin-based hydrogel was found to best support human umbilical vein endothelial cell (HUVEC) sprouting and the establishment of a microvessel network. When this bioink was combined with HUVECs and supporting human bone marrow stem/stromal cells (hBMSCs), these microvessel networks persisted in vitro. Furthermore, only bioprinted tissues containing both HUVECs and hBMSCs, that were first allowed to mature in vitro, supported robust blood vessel development in vivo. To assess the therapeutic utility of this bioprinting strategy, these bioinks were used to prevascularise 3D printed polycaprolactone (PCL) scaffolds, which were subsequently implanted into critically-sized femoral bone defects in rats. Microcomputed tomography (µCT) angiography revealed increased levels of vascularisation in vivo, which correlated with higher levels of new bone formation. Such prevascularised constructs could be used to enhance the vascularisation of a range of large tissue defects, forming the basis of multiple new bioprinted therapeutics.
Statement of Significance
This paper demonstrates a versatile 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into a bone tissue engineering strategy to improve vascularisation in a rat femoral defect model.

Abstract
A functional vascular supply is a key component of any large-scale tissue, providing support for the metabolic needs of tissue-remodeling cells. Although well-studied strategies exist to fabricate biomimetic scaffolds for bone regeneration, success rates for regeneration in larger defects can be improved by engineering microvascular capillaries within the scaffolds to enhance oxygen and nutrient supply to the core of the engineered tissue as it grows. Even though the role of calcium and phosphate has been well understood to enhance osteogenesis, it remains unclear whether calcium and phosphate may have a detrimental effect on the vasculogenic and angiogenic potential of endothelial cells cultured on 3D printed bone scaffolds. In this study, we presented a novel dual-ink bioprinting method to create vasculature interwoven inside CaP bone constructs. In this method, strands of a CaP ink and a sacrificial template material was used to form scaffolds containing CaP fibers and microchannels seeded with vascular endothelial and mesenchymal stem cells (MSCs) within a photo-crosslinkable gelatin methacryloyl (GelMA) hydrogel material. Our results show similar morphology of growing vessels in the presence of CaP bioink, and no significant difference in endothelial cell sprouting was found. Furthermore, our initial results showed the differentiation of hMSCs into pericytes in the presence of CaP ink. These results indicate the feasibility of creating vascularized bone scaffolds, which can be used for enhancing vascular formation in the core of bone scaffolds.

Abstract
Self-assembling bioinks offer the possibility to biofabricate with molecular precision, hierarchical control, and biofunctionality. For this to become a reality with widespread impact, it is essential to engineer these ink systems ensuring reproducibility and providing suitable standardization. We have reported a self-assembling bioink based on disorder-to-order transitions of an elastin-like recombinamer (ELR) to co-assemble with graphene oxide (GO). Here, we establish reproducible processes, optimize printing parameters for its use as a bioink, describe new advantages that the self-assembling bioink can provide, and demonstrate how to fabricate novel structures with physiological relevance. We fabricate capillary-like structures with resolutions down to ∼10 µm in diameter and ∼2 µm thick tube walls and use both experimental and finite element analysis to characterize the printing conditions, underlying interfacial diffusion-reaction mechanism of assembly, printing fidelity, and material porosity and permeability. We demonstrate the capacity to modulate the pore size and tune the permeability of the resulting structures with and without human umbilical vascular endothelial cells. Finally, the potential of the ELR-GO bioink to enable supramolecular fabrication of biomimetic structures was demonstrated by printing tubes exhibiting walls with progressively different structure and permeability.

Abstract
Bone tissue engineering (TE) has the potential to transform the treatment of challenging musculoskeletal pathologies. To date, clinical translation of many traditional TE strategies has been impaired by poor vascularisation of the implant. Addressing such challenges has motivated research into developmentally inspired TE strategies, whereby implants mimicking earlier stages of a tissue’s development are engineered in vitro and then implanted in vivo to fully mature into the adult tissue. The goal of this study was to engineer in vitro tissues mimicking the immediate developmental precursor to long bones, specifically a vascularised hypertrophic cartilage template, and to then assess the capacity of such a construct to support endochondral bone formation in vivo. To this end, we first developed a method for the generation of large numbers of hypertrophic cartilage microtissues using a microwell system, and encapsulated these microtissues into a fibrin-based hydrogel capable of supporting vasculogenesis by human umbilical vein endothelial cells (HUVECs). The microwells supported the formation of bone marrow derived stem/stromal cell (BMSC) aggregates and their differentiation toward a hypertrophic cartilage phenotype over 5 weeks of cultivation, as evident by the development of a matrix rich in sulphated glycosaminoglycan (sGAG), collagen types I, II, and X, and calcium. Prevascularisation of these microtissues, undertaken in vitro 1 week prior to implantation, enhanced their capacity to mineralise, with significantly higher levels of mineralised tissue observed within such implants after 4 weeks in vivo within an ectopic murine model for bone formation. It is also possible to integrate such microtissues into 3D bioprinting systems, thereby enabling the bioprinting of scaled-up, patient-specific prevascularised implants. Taken together, these results demonstrate the development of an effective strategy for prevascularising a tissue engineered construct comprised of multiple individual microtissue “building blocks,” which could potentially be used in the treatment of challenging bone defects.

Abstract
When considering regenerative approaches, the efficient creation of a functional vasculature, that can support the metabolic needs of bioengineered tissues, is essential for their survival after implantation. However, it is widely recognized that the post-implantation microenvironment of the engineered tissues is often hypoxic due to insufficient vascularization, resulting in ischemia injury and necrosis. This is one of the main limitations of current tissue engineering applications aiming at replacing significant tissue volumes. Here, we have explored the use of a new biomaterial, the cell-assembled extracellular matrix (CAM), as a biopaper to biofabricate a vascular system. CAM sheets are a unique, fully biological and fully human material that has already shown stable long-term implantation in humans. We demonstrated, for the first time, the use of this unprocessed human ECM as a microperforated biopaper. Using microvalve dispensing bioprinting, concentrated human endothelial cells (30 millions ml−1) were deposited in a controlled geometry in CAM sheets and cocultured with HSFs. Following multilayer assembly, thick ECM-based constructs fused and supported the survival and maturation of capillary-like structures for up to 26 d of culture. Following 3 weeks of subcutaneous implantation in a mice model, constructs showed limited degradative response and the pre-formed vasculature successfully connected with the host circulatory system to establish active perfusion.This mechanically resilient tissue equivalent has great potential for the creation of more complex implantable tissues, where rapid anastomosis is sine qua non for cell survival and efficient tissue integration.

Abstract
The field of bioprinting has shown a tremendous development in recent years, focusing on the development of advanced in vitro models and on regeneration approaches. In this scope, the lack of suitable biomaterials that can be efficiently formulated as printable bioinks, while supporting specific cellular events, is currently considered as one of the main limitations in the field. Indeed, extracellular matrix (ECM)-derived biomaterials formulated to enable printability and support cellular response, for instance via integrin binding, are eagerly awaited in the field of bioprinting. Several bioactive laminin sequences, including peptides such as YIGSR and IKVAV, have been identified to promote endothelial cell attachment and/or neurite outgrowth and guidance, respectively. Here, we show the development of two distinct bioinks, designed to foster vasculogenesis or neurogenesis, based on methacrylated collagen and hyaluronic acid (CollMA and HAMA, respectively), both relevant ECM-derived polymers, and on their combination with cysteine-flanked laminin-derived peptides. Using this strategy, it was possible to optimize the bioink printability, by tuning CollMA and HAMA concentration and ratio, and modulate their bioactivity, through adjustments in the cell-active peptide sequence spatial density, without compromising cell viability. We demonstrated that cell-specific bioinks could be customized for the bioprinting of both human umbilical vein cord endothelial cells (HUVECs) or adult rat sensory neurons from the dorsal root ganglia, and could stimulate both vasculogenesis and neurite outgrowth, respectively. This approach holds great potential as it can be tailored to other cellular models, due to its inherent capacity to accommodate different peptide compositions and to generate complex peptide mixtures and/or gradients.

Abstract
Abstract Bioprinting of unmodified soft extracellular matrix into complex 3D structures has remained challenging to fabricate. Herein, we established a novel process for the printing of low-viscosity hydrogel by using a unique support technique to retain the structural integrity of the support structure. We demonstrated that this process of printing could be used for different types of hydrogel, ranging from fast crosslinking gelatin methacrylate to slow crosslinking collagen type I. In addition, we evaluated the biocompatibility of the process by observing the effects of the cytotoxicity of L929 and the functionality of the human umbilical vein endothelium primary cells after printing. The results show that the bioprinted construct provided excellent biocompatibility as well as supported cell growth and differentiation. Thus, this is a novel technique that can be potentially used to enhance the resolution of the extrusion-based bioprinter.

Abstract
Combating necrosis, by supplying nutrients and removing waste, presents the major challenge for engineering large three-dimensional (3D) tissues. Previous elegant work used 3D printing with carbohydrate glass as a cytocompatible sacrificial template to create complex engineered tissues with vascular networks (Miller et al. 2012, Nature Materials). The fragile nature of this material compounded with the technical complexity needed to create high-resolution structures led us to create a flexible sugar-protein composite, termed Gelatin-sucrose matrix (GSM), to achieve a more robust and applicable material. Here we developed a low-range (25–37˚C) temperature sensitive formulation that can be moulded with micron-resolution features or cast during 3D printing to produce complex flexible filament networks forming sacrificial vessels. Using the temperature-sensitivity, we could control filament degeneration meaning GSM can be used with a variety of matrices and crosslinking strategies. Furthermore by incorporation of biocompatible crosslinkers into GSM directly, we could create thin endothelialized vessel walls and generate patterned tissues containing multiple matrices and cell-types. We also demonstrated that perfused vascular channels sustain metabolic function of a variety of cell-types including primary human cells. Importantly, we were able to construct vascularized human noses which otherwise would have been necrotic. Our material can now be exploited to create human-scale tissues for regenerative medicine applications.
Statement of Significance
Authentic and engineered tissues have demands for mass transport, exchanging nutrients and oxygen, and therefore require vascularization to retain viability and inhibit necrosis. Basic vascular networks must be included within engineered tissues intrinsically. Yet, this has been unachievable in physiologically-sized constructs with tissue-like cell densities until recently. Sacrificial moulding is an alternative in which networks of rigid lattices of filaments are created to prevent subsequent matrix ingress. Our study describes a biocompatible sacrificial sugar-protein formulation; GSM, made from mixtures of inexpensive and readily available bio-grade materials. GSM can be cast/moulded or bioprinted as sacrificial filaments that can rapidly dissolve in an aqueous environment temperature-sensitively. GSM material can be used to engineer viable and vascularized human-scale tissues for regenerative medicine applications.

Abstract
Vascularization is one major obstacle in bioprinting and tissue engineering. In order to create thick tissues or organs that can function like original body parts, the presence of a perfusable vascular system is essential. However, it is challenging to bioprint a hydrogel-based three-dimensional vasculature-like structure in a single step. In this paper, we report a new hydrogel-based composite that offers impressive printability, shape integrity, and biocompatibility for 3D bioprinting of a perfusable complex vasculature-like structure. The hydrogel composite can be used on a non-liquid platform and is printable at human body temperature. Moreover, the hydrogel composite supports both cell proliferation and cell differentiation. Our results represent a potentially new vascularization strategy for 3D bioprinting and tissue engineering.