RESOURCES

Abstract
Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral
gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-g-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bonemarrow-derived mesenchymal stemcells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization andmineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs.

Abstract
Bone tissue engineering strategies that recapitulate the developmental process of endochondral ossification offer a promising route to bone repair. Clinical translation of such endochondral tissue engineering strategies will require overcoming a number of challenges, including the engineering of large and often anatomically complex cartilage grafts, as well as the persistence of core regions of avascular cartilage following their implantation into large bone defects. Here 3D printing technology is utilized to develop a versatile and scalable approach to guide vascularisation during endochondral bone repair. First, a sacrificial pluronic ink was used to 3D print interconnected microchannel networks in a mesenchymal stem cell (MSC) laden gelatin-methacryloyl (GelMA) hydrogel. These constructs (with and without microchannels) were next chondrogenically primed in vitro and then implanted into critically sized femoral bone defects in rats. The solid and microchanneled cartilage templates enhanced bone repair compared to untreated controls, with the solid cartilage templates (without microchannels) supporting the highest levels of total bone formation. However, the inclusion of 3D printed microchannels was found to promote osteoclast/immune cell invasion, hydrogel degradation, and vascularisation following implantation. In addition, the endochondral bone tissue engineering strategy was found to support comparable levels of bone healing to BMP-2 delivery, whilst promoting lower levels of heterotopic bone formation, with the microchanneled templates supporting the lowest levels of heterotopic bone formation. Taken together, these results demonstrate that 3D printed hypertrophic cartilage grafts represent a promising approach for the repair of complex bone fractures, particularly for larger defects where vascularisation will be a key challenge.

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
The critical shortage in organ donors is a problem facing patients and health care systems worldwide. The most promising solution to this crisis is the artificial production of organs and tissues, known as tissue engineering. Significant advances in this field have led to the commercial production of artificial skin and cartilage, but limitations remain in the production of thicker tissues (i.e., >200 μm thickness). The challenge of producing thicker tissues relates to the inability to establish and maintain a vascular system, which is the basic requirement for artificially producing any vital organ. Given the importance of these structures, a major scientific effort is underway to better understand how to reproduce a vascular system. This review presents the most recent advances in the manufacturing of vascular-like structures, especially techniques involving additive manufacturing, or 3D and 4D printing. This review starts with a primer on the classification, composition, and mechanical proprieties of blood vessels, offering the reader a better understanding of the challenges involved in the artificial production of vessels. The review then discusses the methodologies, technologies, and materials used and available for the manufacturing of the vascular system.

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
Recent progress in bioprinting has made possible the creation of complex 3D intestinal constructs, including vascularized villi. However, for their integration into functional units useful for experimentation or implantation, the next challenge is to endow them with a larger-scale, anatomically realistic vasculature. In general, the perfusion of bioprinted constructs has remained difficult, and the current solution is to provide them with mostly linear and simply branched channels. To address this limitation, here we demonstrated an image analysis-based workflow leading through computer-assisted design from anatomic images of rodent mesentery and colon to the actual printing of such patterns with paste and hydrogel bioinks. Moreover, we reverse-engineered the 2D intestinal image-derived designs into cylindrical objects, and 3D-printed them in a support hydrogel. These results open the path towards generation of more realistically vascularized tissue constructs for a variety of personalized medicine applications.

Abstract
Recent progress in bioprinting has made possible the creation of complex 3D intestinal constructs, including vascularized villi. However, for their integration into functional units useful for experimentation or implantation, the next challenge is to endow them with a larger-scale, anatomically realistic vasculature. In general, the perfusion of bioprinted constructs has remained difficult, and the current solution is to provide them with mostly linear and simply branched channels. To address this limitation, here we demonstrated an image analysis-based workflow leading through computer-assisted design from anatomic images of rodent mesentery and colon to the actual printing of such patterns with paste and hydrogel bioinks. Moreover, we reverse-engineered the 2D intestinal image-derived designs into cylindrical objects, and 3D-printed them in a support hydrogel. These results open the path towards generation of more realistically vascularized tissue constructs for a variety of personalized medicine applications.

Abstract
Recent progress in bioprinting has made possible the creation of complex 3D intestinal constructs, including vascularized villi. However, for their integration into functional units useful for experimentation or implantation, the next challenge is to endow them with a larger-scale, anatomically realistic vasculature. In general, the perfusion of bioprinted constructs has remained difficult, and the current solution is to provide them with mostly linear and simply branched channels. To address this limitation, here we demonstrated an image analysis-based workflow leading through computer-assisted design from anatomic images of rodent mesentery and colon to the actual printing of such patterns with paste and hydrogel bioinks. Moreover, we reverse-engineered the 2D intestinal image-derived designs into cylindrical objects, and 3D-printed them in a support hydrogel. These results open the path towards generation of more realistically vascularized tissue constructs for a variety of personalized medicine applications.

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 Conventional synthetic vascular grafts require ongoing anticoagulation, and autologous venous grafts are often not available in elderly patients. This review highlights the development of bioartificial vessels replacing brain-dead donor- or animal-deriving vessels with ongoing immune reactivity. The vision for such bio-hybrids exists in a combination of biodegradable scaffolds and seeding with immune-neutral cells, and here different cells sources such as autologous progenitor cells or stem cells are relevant. This kind of in situ tissue engineering depends on a suitable bioreactor system with elaborate monitoring systems, three-dimensional (3D) visualization and a potential of cell conditioning into the direction of the targeted vascular cell phenotype. Necessary bioreactor tools for dynamic and pulsatile cultivation are described. In addition, a concept for design of vasa vasorum is outlined, that is needed for sustainable nutrition of the wall structure in large caliber vessels. For scaffold design and cell adhesion additives, different materials and technologies are discussed. 3D printing is introduced as a relatively new field with promising prospects, for example, to create complex geometries or micro-structured surfaces for optimal cell adhesion and ingrowth in a standardized and custom designed procedure. Summarizing, a bio-hybrid vascular prosthesis from a controlled biotechnological process is thus coming more and more into view. It has the potential to withstand strict approval requirements applied for advanced therapy medicinal products.

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
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.

Abstract
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.

Abstract
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.

Abstract
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.

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
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
The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples  of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.

Abstract
The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples  of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.

Abstract
The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples  of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.

Abstract
Since the emergence of regenerative medicine and tissue engineering more than half a century ago, one obstacle has persisted: the in vitro creation of large-scale vascular tissue (>1 cm3) to meet the clinical needs of viable tissue grafts but also for biological research applications. Considerable advancements in biofabrication have been made since Weinberg and Bell, in 1986, created the first blood vessel from collagen, endothelial cells, smooth muscle cells and fibroblasts. The synergistic combination of advances in fabrication methods, availability of cell source, biomaterials formulation and vascular tissue development, promises new strategies for the creation of autologous blood vessels, recapitulating biological functions, structural functions, but also the mechanical functions of a native blood vessel. In this review, the main technological advancements in bio-fabrication are discussed with a particular highlights on 3D bioprinting technologies. The choice of the main biomaterials and cell sources, the use of dynamic maturation systems such as bioreactors and the associated clinical trials will be detailed. The remaining challenges in this complex engineering field will finally be discussed.

Abstract
The survival and function of thick tissue engineered implanted constructs depends on pre-existing, embedded, functional, vascular-like structures that are able to integrate with the host vasculature. Bioprinting was employed to build perfusable vascular-like networks within thick constructs. However, the improvement of oxygen transportation facilitated by these vascular-like networks was directly quantified. Using an optical fiber oxygen sensor, we measured the oxygen content at different positions within 3D bioprinted constructs with and without perfusable microchannel networks. Perfusion was found to play an essential role in maintaining relatively high oxygen content in cell-laden constructs and, consequently, high cell viability. The concentration of oxygen changes following switching on and off the perfusion. Oxygen concentration depletes quickly after pausing perfusion but recovers rapidly after resuming the perfusion. The quantification of oxygen levels within cell-laden hydrogel constructs could provide insight into channel network design and cellular responses.

Abstract
3D bioprinting is a technology that supports fabrication of biomimetic tissues with complex architecture. It has application in drug discovery, tissue development, and regenerative medicine. The aim of this study was to create a blood vessel model correlating properties of collagen-hyaluronic acid hydrogel with bioprinter parameters such as speed rate, pressure, number of layers, nozzle diameter, and temperature. The blood vessel model was created using BioCAD software and bioprinted by extrusion technology using collagen-hyaluronic acid hydrogel. We analyzed the water uptake, enzymatic degradation and morphology by scanning electron microscopy and after staining with Hematoxylin and Eosin (H&E) and Trichromic Masson dyes. The results showed that the blood vessel constructs have 2.46 mm (±0.41) mean diameter, 1.4 mm (±0.10) mean thick wall, and 2.8 mm (±0.05) mean height which is appropriate with the model created in the BioCAD software. The optimal parameters for these constructs were: 1.1 bar pressure, 1mm/sec speed rate, 18°C temperature, 0.2 mm nozzle diameter, and 10 numbers of layers. Increasing hydrogel weight by 22% at 2 hours after immersion in PBS suggesting that is hydrophilic. Furthermore, decreasing by up to 47.2% in the presence of collagenase (50 μg/ml) shows that is biodegradable. H&E and Trichromic Masson staining showed that collagen-hyaluronic acid hydrogel organized in a network with pores dimension that could support cells growth and differentiation. In conclusion, our scaffold mimics the blood vessel structure, further experiment will be addressed for study the biocompatibility of these scaffold with mesenchymal stem cells.

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.