Three dimensional (bio)printing of blood vessels: from vascularized tissues to functional arteries

Tissue engineering has emerged as a strategy for producing functional tissues and organs to treat diseases and injuries. Many chronic conditions directly or indirectly affect normal blood vessel functioning, necessary for material exchange and transport through the body and within tissue-engineered constructs. The interest in vascular tissue engineering is due to two reasons: (1) functional grafts can be used to replace diseased blood vessels, and (2) engineering effective vasculature within other engineered tissues enables connection with the host’s circulatory system, supporting their survival. Among various practices, (bio)printing has emerged as a powerful tool to engineer biomimetic constructs. This has been made possible with precise control of cell deposition and matrix environment along with the advancements in biomaterials. (Bio)printing has been used for both engineering stand-alone vascular grafts as well as vasculature within engineered tissues for regenerative applications. In this review article, we discuss various conditions associated with blood vessels, the need for artificial blood vessels, the anatomy and physiology of different blood vessels, available 3D (bio)printing techniques to fabricate tissue-engineered vascular grafts and vasculature in scaffolds, and the comparison among the different techniques. We conclude our review with a brief discussion about future opportunities in the area of blood vessel tissue engineering.


Introduction
Lifestyle changes and prolonged life expectancy have raised the incidence of various diseases and disorders with direct or indirect impacts on the functioning of surrounding structures like blood vessels, essential for nutrient and waste transport throughout the body [1].The prevalence of acute injuries and chronic disorders in vasculature health can provoke arterial disease or lead to organ failure, significant sources of morbidity and mortality that often require surgical intervention to treat them.Autografts are the gold standard for arterial disease, but they may lead to donor site infection and are not always available.Other practices include the use of auto, allo, xeno, and synthetic grafts.However, allo and xenografts have an increased chance of disease transmission and the need for immunosuppression.Synthetic grafts are also susceptible to clotting and rejection and usually do not function similarly to the native tissues.Allograft transplantation is the standard of care for organ failure.However, there is a significant shortage of organ donors worldwide [2,3].In 2015, ∼75% of applicants could not obtain a transplant in the US alone, whereas this figure for the UK in 2018 was ∼20% [4].Furthermore, the transplantation procedure is typically complicated by infection risks and the host rejection of the tissue [5].Even in successful cases, the patients often must use immunosuppressive drugs to combat organ rejection for their lifetime, which can have severe side effects.Tissue engineering was introduced to combat these challenges, looking to fabricate cellular constructs that offer functions comparable to the diseased or damaged tissues.To achieve this goal, the tissues should mimic the complex physical, biological, and chemical architecture of native tissues and organs [6,7].Vascular tissue engineering became a crucial target in tissue engineering practices because of the great clinical need for vascular grafts and the vital role of functional vasculature in the survival of any tissue-engineered construct.
The human vasculature is a remarkably complex network consisting of numerous blood vessels with a high variability in their sizes [8].The size of the blood vessels ranges from about 3-3.5 cm of the aorta to about 20 µm of the capillaries [9].The maximum distance between the capillaries should not exceed 200 µm to facilitate the transport of nutrients and waste products to and from the cells respectively [10].Thus, to make a functional organ with physiological similarities to human organs, all the properties of the blood vessels must be considered.Due to the diversity of required blood vessels, an array of techniques and strategies have been used for engineering biological alternatives.These strategies have focused on mimicking the tissue architecture, cell distribution, extracellular matrix (ECM) properties, and biomechanics [11].Weinberg and Bell [12] developed the first biological tissue-engineered blood arteries, consisting of intima, media, and adventitia, utilizing cultured mature smooth muscle cells and endothelial cells in bovine collagen gels.L'Heureux et al [13] fabricated the first tissue-engineered human blood artery without using any scaffold in 1998, which was reproduced for additional preclinical assessment using rat and mouse models in 2006 [14].With the recent advancements in material science and cell biology, three-dimensional (3D) (bio)printing has emerged as a promising tool that could overcome the limitations of traditional tissue engineering approaches [15].It derives its principles from the conventional 3D printing techniques and was first described in 1986 by Charles W Hull [16].3D printing is an additive manufacturing technique in which the substance is usuallyprinted successively in a layer-by-layer fashion to create a solid 3D structure [17,18].3D (bio)printing entails applying of 3D printing techniques to pattern biomaterials, cells, and biologically active substances in a controlled manner to influence tissue development [16].It has shown its potential utility in regenerative medicine to manufacture a range of transplantable tissues, including skin, cartilage, and bone [19].In (bio)printing, cells and biomaterials are integrated into scaffolds via a number of topdown and bottom-up techniques [20][21][22].In the topdown approach, cells are seeded into the scaffolds shaped in the form of biological structures, whereas in the bottom-up approach, the cells and the biomaterials are combined in a controlled manner to form the tissues [20,21].To date, various groups have worked on (bio)printing to develop vascularized tissues [23][24][25], skeletal muscles [26], cartilage [27][28][29][30][31], bone [32][33][34], cardiac tissue [35][36][37][38][39][40], liver [41][42][43][44], and blood vessels [24,[45][46][47][48]. Controlling the variables at both the microscopic and macroscopic levels is important to mimic the cellular environments in the human body.3D (bio)printing allows precise control of the cellular microenvironment variables and the fabricated construct geometry [49][50][51].By replicating the environmental conditions during the (bio)printing process, the physiological conditions of the living tissues can be recapitulated [52].
In this article, we review and discuss the challenges associated with blood vessel tissue engineering with the contexts of biological vascular grafts and vasculature in engineered tissues and organs.We will also discuss different (bio)printing approaches for fabricating artificial blood vessels.We will also highlight (bio)printing strategies that have been used for generating vasculature within tissue-engineered constructs to support their survival and integration with the host's body.Lastly, we discuss the challenges in (bio)printing blood vessels, the current advances in (bio)printing blood vessels, and what the future holds for this field.

Common vascular diseases
Aging, trauma, and chronic diseases are leading causes of damaged or dysfunctional blood vessels that cannot efficiently supply the required blood to the organ tissues [53,54].The most common bloodvessel-associated conditions are blood vessel narrowing or blockage and localized blood vessel weakening that can be caused by atherosclerosis and aneurysms.Atherosclerosis provokes the narrowing of the blood vessels due to the build-up of atheromatous plaque, which develops due to the slow accumulation of blood cells, cholesterol, fat, and other substances from our blood [55].Narrowing reduces the supply of oxygen-rich blood to tissues of vital organs in the body, potentially damaging them.Additionally, when plaques burst, blood clots form and may travel to other body parts and cause blockages of blood vessels in different organs.These blockages can manifest as stroke, heart attack, gangrene, and vascular dementia, leading causes of death and disability in the United States and worldwide [56].Aneurysms occur due to the localized weakening of the blood vessel wall because of medical conditions, genetic factors, and trauma.Aneurysms can lead to bulging, followed by the rupture of the weakened or injured blood vessels [57].To develop strategies to address the problems associated with the blood vessels, a brief understanding of the blood vessel composition and properties will be helpful.

Blood vessel composition, structure, and mechanical properties
Cellular and extracellular composition, properties, and structure of blood vessels will vary depending on their size, role, and location across the circulatory system.Hence, all these aspects should be considered when designing tissue-engineered vascular grafts and scaffolds with integrated vasculature.The vascular wall consists of cells and the ECM is dominantly made of collagen, proteoglycans, and elastin [58].Blood vessels are composed of three concentric layers: the inner, middle, and outer layers [59,60].As can be seen from figure 1, the innermost layer, tunica intima, is composed of non-thrombogenic monolayer endothelial cells [61,62].These endothelial cells prevent and modulate platelet activity and thrombus formation by secreting particular chemicals such as nitric oxide [63,64].The outer layer, tunica adventitia, contains a collagenous ECM, fibroblast cells, and some elastin which provide the mechanical support and elasticity for resistance against rupture [62].A dense population of circumferentially arranged smooth muscle cells makes up the middle layer, called the tunica media, which is separated from the tunica intima by an internal elastic lamina [62].Smooth muscle cells synthesize the elastin molecules that are incorporated into elastic fibers, which are then arranged into concentric rings of elastic lamellae around the arterial media in elastic arteries such as the aorta, the brachiocephalic, common carotid, subclavian, and iliac arteries [65].The artery's elastic lamellae helps arteries to maintain their structure and stretch during systole and recoil and maintain blood flow during diastole [66].Smooth muscle cells regulate blood flow by controlling the dilatation and contraction of blood vessels in a physiological state [67,68].
Depending on the blood pressure and the location of the vessel within the circulatory system, blood vessels can be classified into three types: arteries, which carry blood from the heart to other organs; veins, which carry blood towards the heart; and capillaries, that distribute blood within tissues [69].The arteries have flexible thick walls (diameters varying from 0.6 to 16 mm) to sustain the high blood pressure and the continuous expansion and contraction due to pressure changes caused by the heart pumping.Since veins have to sustain lower blood pressure, they have thinner walls that lack the structures of the arteries.Capillaries (with diameters in the range of 9 µm) are formed by just a single layer of endothelial cells surrounded by pericytes.This organization facilitates the transport and diffusion of nutrients and oxygen to the adjacent cells.
The mechanical properties of the blood vessels are largely determined by their ECM composition [70].Properties like elasticity, viscoelasticity, deformability, and tensile stiffness are critical for the efficient functioning of the tissues.For example, the adventitia of the human coronary artery has higher tensile strength values (1430 ± 604 kPa circumferential and 1300 ± 692 kPa longitudinal) as they are mainly composed of type I and III collagen, whereas the high deformability is due to the proteoglycans [71].The elastic property is obtained from the elastin fibers and the viscoelasticity is obtained by both collagen and elastin [72].

Tissue engineering strategies for engineering blood vessels and vascularized tissues
As discussed above vascular diseases are frequent and the need for engineered tissue is growing.Therefore, numerous research groups have dedicated efforts towards discovering practical solutions.These efforts can be divided broadly into engineering vascular grafts and generating effective vasculature in tissueengineered scaffolds.Comprehensive reviews of the conventional strategies, biomaterials, and cells can be found elsewhere [61,[74][75][76].Here, we briefly highlight some of the important practices and examples.
A common practice in tissue engineering is the use of electrospun scaffolds, also used in engineering vascular grafts [77].Electrospinning creates planar scaffolds made of nano to micron-sized filaments [78].The filaments can be made from various polymers and proteins, and their organization can be controlled by the strategy that is used for collecting them [79].The sheet could be either collected and then rolled to form cylindrical conduits, or the scaffold could be directly spun onto a cylindrical collector with the correct dimension and then removed [80].Electrospun scaffolds are mechanically robust, suturable, and can be easily implanted [80,81].However, in the acellular form, they are porous, and blood leakage is probable.Although, electrospun sheets can be coated with hydrogels that support cell ingrowth (figure 2(A)) [82].Acellular scaffolds can also be thrombogenic.Nevertheless, to overcome this barrier the scaffolds can be endothelialized and kept under perfusion to let the endothelial layer be matured [83].
A widely explored are in scaffold fabrication is the introduction of vasculature in biological scaffolds.Micromolding is another common practice that can be applied to fabricate cylindrical structures, templates with surface topography similar to vascular networks, or complex shapes with embedded channels.For example, a temporary cylindrical structure can be inserted into a construct and removed post-crosslinking of the surrounding materials.Researchers showed a method where a metallic needle was coated layer by layer into different biomaterials carrying different cell lines, and then the needle was removed to create a perfusable channel [84].This strategy, however, can only be used to generate parallel channels, which do not fully recapitulate the vascular network organization in our body.In another interesting study, the researchers utilized a 3D printer to deposit hydrogel fibers later coated with a gelatin methacryloyl (GelMA) scaffold.After GelMA crosslinking, the fibers were manually removed, leaving behind more complex structures [85].Also, researchers have used microfabrication to recreate vasculature surface topography and create more biomimetic vasculature molds that could be used to cast biomaterials and manufacture scaffolds with embedded channels [86].Another very interesting study used a decellularized leaf as a mold with biomimetic structure to generate a culture of hepatocytes [87].They later perfused the channels with endothelial cells to create endothelialized vasculature.
Micromolding has also been used to create vasculature from sacrificial material, then covered by the scaffolding material [88].The sacrificial materials are then removed by applying the suitable stimuli to leave channels.This strategy was used to generate gelatin-based structures later embedded into scaffolds.The gelatin structure was dissolved after incubating the construct at 37 • C [88].Photolithography has also been used for engineering vascular niches with designed patterns covered with scaffolding materials.For instance, Kazemzadeh Narbat created endothelial patterns using photolithography and then covered them with GelMA carrying mesemchymal stem cells (MSCs) to form vascularized bone models (figure 2(B)) [89].MSCs in the construct differentiated to bone lineage.However, MSCs interfaced with endothelial cells differentiated to smooth muscle cells to stabilize the endothelial capillaries.
A relevant property of endothelial cells is their tube-forming and self-assembly characteristics.Researchers have tried to utilize this property and direct it to generate vasculature.Microfabricated tools have also been used for engineering patterns of proteins and cells on surfaces.These patterns could lead the adhesion of endothelial cells to form vasculature within the constructs.In a remarkable example, Rezaei Nejad et al used microfabricated microfluidic stamps to create temporary channels with a biomimetic design on a substrate [90].The channels were perfused with endothelial cells, and then the constructs were covered by scaffolds made of various hydrogels.They observed that the endothelial cells sprout faster from the smaller channels, and some of the formed sprouts were tubular (figures 2(C)-(E)).An important example showcasing the possibility of self-organization in endothelial cells for capillary formation can be observed in microfluidic systems.In these systems, endothelial cells are cultured within a channel that is perfused with culture media, while a parallel channel, separated by a hydrogel layer, is perfused with culture media containing biological factors or stimulants.The generated stimulant gradient directs endothelial cells to form organized capillaries [91,92].In one study, Kim et al fabricated two channels seeded by endothelial cells separated by a fibrin hydrogel carrying fibroblasts.The results showed that endothelial cells sprouted into the gel and the co-culture formed stabilized vessels [93].
Despite the approach selected to develop vascular constructs, it is noteworthy that exogenous growth factors and cytokines can stimulate endothelial cells' angiogenesis and vascularization.Several studies have reported strategies to deliver biological factors and demonstrated their benefits in enhancing vascularization and overcoming vascular problems.Important examples of these factors include vascular endothelial growth factor (VEGF), IGF-1, PDGF, and platelet rich plasma [94][95][96][97].
Overall, the vasculature formed by the selfassembled endothelial cells are typically capillaries.The process is slow, and it might not be sufficient to support the survival of cells in large constructs.On the other hand, the prefabricated vasculature can supply oxygen and nutrients to the cells within the tissue but lack the biomimetic architecture observed in the circulatory system.Vascular networks are nonplanar structures that develop in multiple directions.Although these studies did not achieve a fully structured and functional multilayered vascular network, they highlight some key aspects to consider in further investigations, such as the need to create antithrombogenic structures with perfusable channels, the relevance of surface topography, the need for an expedited biofabrication process, as well as the chances to expand the range of biomaterials used as direct bioinks or as sacrificial structures.3D printing and (bio)printing technologies have been considered a solution to overcome this barrier.

Common (bio)printing techniques in blood vessel tissue engineering
3D (Bio)printing strategies commonly used for tissue engineering can be broadly categorized into four main types, depending on the mechanism by which the bioinks or biomaterials inks are deposited to form the constructs [16,[98][99][100][101].These are namely (i) inkjet, (ii) extrusion, (iii) laser-based, and (iv) stereolithography [101].In the following subsections, these different methods along with their subtypes are discussed.Figure 3 provides a schematic representation of the different (bio)printing techniques.

Inkjet
Inkjet (bio)printing is one of the oldest (bio)printing techniques.Inkjet bioprinters work by depositing droplets of the bioinks to form the desired structure the same way commercial inkjet printers work [98,102,103].They have the advantage of being low cost due to their similarities in structure with commercial printers, good resolution, and high printing speed with considerably high cell viability (generally from 80 to 90%) [104][105][106][107][108].However, there are also some drawbacks to these inkjet bioprinters, specifically in their inability to handle materials with high viscosity (>15 mPa s −1 ) and high cell density (>1 × 10 6 cells ml −1 ), which may clog the printing nozzle head [16,103,[109][110][111].As a result, biological materials must be liquid with low viscosity to form droplets.Given this aspect, one typical disadvantage is the difficulty of stacking 3D solid structures without fast crosslinking processes, so quick crosslinking methods, such as chemical or UV mechanisms, are necessary immediately after deposition.On the other hand, while using low-viscosity materials as the bioink, the printed structure has inferior mechanical strength compared to the replicated tissue [112].An added disadvantage is the imparting thermal and mechanical stresses on the cells because of the small diameter of the nozzle, which adversely impacts cell viability [113].Various physical phenomena can generate droplets in inkjet 3D (bio)printers.The most common ones include thermally and piezoelectrically actuated nozzles.
A thermal inkjet bioprinter comprises a microheating element (made of a thin film resistor) that heats the ink chamber in the inkjet printhead and a nozzle that ejects the bioink [112].When a short electric pulse is applied to the resistor, heat is generated and forms bubbles of picolitre (pl) volume [103].This generated bubble upon expansion bursts with the removal of heat.These forces of expansion and implosion cause the ink droplets to flow out.The droplet volume can vary from 10 to 150 pl [114] and depends on the current pulse frequency, the induced temperature gradient, and the bioink viscosity [115].The thermal stresses induced in the cells due to the heating of the bioink can affect cell viability [116].However, studies have shown that this frequent high heating, which produces localized temperature increases from 200 • C to 300 • C, does not affect the biological molecule stability, including the DNA [117,118], the cell viability, and the functionality of the printed mammalian cells after printing is completed [106,119].As the duration of this heating is very short (∼2 µs), there is a little increase in temperature in the printer head [120], and little to no damage to the cells takes place.Other advantages of thermal inkjet printers are their rapid printing speed, good resolution, affordable cost, and easy availability of the printers.Cell viability was 89% as stated by Cui et al [107], and in most of the studies, it varies between 70 and 90% [121,122].Some drawbacks of thermal bioprinters include poor directionality of droplet deposition, uneven droplet size, irregular geometric shapes, and nozzle clogging issues [16].
In Piezoelectric printers, an acoustic wave or a shape-changing physical component induces changes in pressure inside the ink pool, forcing the bioink out and forming the droplets [113,123,124].In the method utilizing a physical component, a shift in the voltage pulses induces the ejection of the bioink.When the voltage is applied, the crystal produces quick shape change responses and compresses the bioink contact with the transducer.The bioink is ejected by a pressure pulse caused by the ink chamber's size changes [123,125].In acoustic (bio)printing, the acoustic force in the local region forms bioink drops, where a microhole is built to allow for drop jetting [116].This technique can be highly controlled by changing different parameters like the time for application of the wave, its frequency, and the amplitude.The droplet diameter varies from 3 to 200 µm and uses frequencies from 1 Hz to 10 KHz [112].In some studies that have used encapsulated cells with acoustic (bio)printing, cell viability was from 80% to 90% [126].The throughput is very high with values reaching almost 100 000 drops per second [127].With no involvement of a dispensing nozzle, there are no clogging issues or shear stresses imparted to the cellladen bioink and no cellular functionality changes.Due to the use of multiple ejectors, different cell types and materials can be printed simultaneously [128].Researchers have used 3D acoustic tweezers that utilize standing surface acoustic wave technology to manipulate single cells or cell assemblies to make 2D or 3D patterns [129].Piezoelectric (bio)printing uses a voltage of roughly 15-25 kHz [105,114,130] that can be detrimental to cell viability, as cell membranes may get disrupted.However, there is no involvement of temperature increases as in the case of thermal or laser-based printers.Issues related to nozzle clogging can be avoided, and a wider variety of inks can be used than in thermal inkjets [131,132].Although the cell membranes and the biomolecular structures may be adversely affected by the vibration frequencies in piezoelectric bioprinters, some studies report high post-printing cell viability of more than 90% by using fibroblast cells [132][133][134].The piezoelectric printing technique was also successfully used to fabricate scaffold-free hollow zigzag tubes using Fibroblasts (3T3 cells) [135].

Application of inkjet (bio)printers in vascular tissue engineering.
Figure 4 shows several examples of fabricated constructs using inkjet printing.Cui and Boland used human microvascular endothelial cells (HMVEC) with fibrin as a bioink for fabricating microvasculature constructs using a modified HP Deskjet 500 printer as shown in figure 4(A) [23].In their study, they precisely fabricated fibrin channels of the order of microns by using a thermal-based inkjet printer.During the printing process, it was found that HMVEC cells, when combined with fibrin, multiplied and arranged themselves in the channels, resulting in the production of adherent linings.Zheng et al developed a biofabrication strategy using highresolution electrohydrodynamic inkjet to create cellembedded microvascular constructs as shown in figure 4(B) [136].The hierarchical and branching designs were printed using a sacrificial ink made of a temperature-sensitive biomaterial, and then the structure was cast with a gelatin-based hydrogel, with and without cells.After crosslinking, the fugitive ink was removed, leaving complex hollow channels.Two cell types, human dermal fibroblasts (HDF) and human umbilical vein endothelial cells (HUVECs), were successfully co-cultured up to 21 d in this model.The microchannels created were within 30−60 µm ideal for recreating blood vessels at the capillary level.Nakamura and Henmi employed an inkjetbased (bio)printing technique to develop miniature 3D tube-like structures [48].Constructs were produced by depositing sodium alginate in a magnetically stirred bath containing a calcium chloride solution.The constructs had 100 µm diameter and 40 µm wall thickness.Nakamura et al used sodium alginate droplets to form homogenously sized microgel beads when they were deposited into a CaCl 2 solution [137].These microgel beads produced the tubular 3D constructs with diameters ranging from 50 µm to 1000 µm.They used a similar approach to produce constructs using HeLa cells in Na-alginate gel [138].These constructs are comprised of lines, planes, laminated structures, and tubes with a diameter of 200 µm.Xu et al used an inkjet bioprinter to fabricate complex 3D zigzag tubular constructs without the use of any scaffolds [135].Zigzag overhanging tubular structures were successfully made using Fibroblasts (3T3 cells), as shown in figure 4(C).Using the proposed printing settings for optimum droplet formation, the viability of 3T3 cells after printing was found to be over 82% (or 93% when the control effect was considered) after an incubation period of 72 h.
Inkjet bioprinting shows promise in reproducing the structural shape of thin vascular networks and larger blood vessels that can be used as vascular grafts.However, most of these studies are proofs of concept and use bioinks that do not fully resemble vasculature's mechanical properties and composition.In most cases, the successful prints use fibrous tissue where cells are randomly seeded if used.The main disadvantage of inkjet (bio)printing is the current limited range of bioinks compatible with this approach.This constrains its application for engineering vascular grafts, especially in multi-layered structures requiring different cell types.Inkjet bioprinters were one of the earliest used bioprinters, but recent advances in bioprinting techniques like extrusion bioprinting have taken over inkjet-based methods.Extrusion (bio)printing will be discussed in the following sub-section.

Extrusion-based (bio)printing
Extrusion is one of the most common (bio)printing techniques [139].This (bio)printing method includes a dispenser, an electric component that positions the piston, and a third component that controls the temperature.The dispenser is programmed to move in x, y, and z directions.The printing bed supports the structure being printed, and the host control system commands the positioning of the nozzle, the dispensing volume of the bioink, and the temperature of the dispenser.A printing resolution of 100-150 µm can be obtained based on the internal nozzle diameter [140].The cell viability post-printing usually ranges between 40 to 98%.Parameters that affect the bioink printability include the contact angle, printing speed, flow characteristics, melting temperature, complex modulus, gelation kinetics, and the ability of the bioink to crosslink [120,[141][142][143][144][145][146][147][148].
During the printing process, the syringe or barrel is filled with the bioink or biomaterial ink which is then extruded by mechanical forces (generated by either hydraulic, pneumatic, or a screw mechanism) from the nozzle onto the printing bed to form the structure in a layer-by-layer fashion [149][150][151][152][153]. For printing bioinks of high viscosities, screw systems are utilized instead of hydraulic or pneumatic-based systems.However, there are concerns about cell damage in the screw-based systems due to the high shear stresses applied to the cell-laden bioink [154].
While other printing techniques are confined to printing hydrogel polymers with suspended cells, extrusion (bio)printing may dispense a wide range of biomaterials and cells, including native and synthetic hydrogel polymers, cell aggregates, and decellularized ECM.Printing of biomaterials with densities similar to those of the physiological cells is feasible by using the extrusion method [155].Largescale scaffolds have also been produced by using the extrusion (bio)printing technique [140].In one example, a whole multi-layered artery was formed with one of the layers being extruded from a printing nozzle [156].Large arterial grafts can be bioprinted by depositing fiber in a circular fashion.The circumferential organization of the filaments mimics the architecture of tunica layer in blood vessels.
Compared to inkjet and laser-based (bio)printing (LBB), the printing resolution in extrusion (bio)printing is typically beyond 100 µm.This is because of the restrictions of high mechanical forces induced in the nozzle generated by the forces required to drive the bioink through the nozzle.Furthermore, the printability of hydrogels is strongly influenced by their crosslinking ability and/or printing settings; biomaterials with a poor crosslinking speed may not be suitable for printing due to challenges in generating 3D structures.In addition, needle clogging with biomaterial solution is a concern in extrusion (bio)printing that can cause biomaterial deposition to stop completely, compromising the integrity of the final scaffold structure.Because of the delay in pressurized gas volume in pneumatic systems and the high complexity of electromagnetically driven systems, the screw-based mechanical dispensing systems may be preferred as they allow more direct control over material flows.
Micro-extrusion printers use inks with viscosities lying in the range of 30 mPa•s to over 6 × 10 7 mPa•s [157].Materials with higher viscosities are generally used to support the structure under the print, while materials with low viscosities are used to provide favorable conditions to maintain proper functionality and viability.
One important strategy in generating vasculature within scaffolds is to 3D print sacrificial materials.Sacrificial materials can be co-printed, embedded, or printed and then covered with the scaffolding material.The sacrificial materials that have been widely used include sugar, gelatin, alginate, and Pluronic F127.In a notable study, Miller et al created networks of cylindrical constructs employing carbohydrate glass as sacrificial templates in ECM prepolymer containing live cells using extrusion (bio)printing [24].These carbohydrate glass networks were lined with endothelial cells and perfused with high-pressure pulsatile blood flow.This vascular fabrication method is compatible with a large variety of cells and crosslinking techniques along with artificial and natural extracellular matrices.Thus, this method permits autonomous regulation of the network structure, the extravascular tissue as well as the endothelialization.To demonstrate the cell viability of the perfused network architecture, they showed that the channels maintained the metabolic activity of rat hepatocyte cells in artificial tissue constructs.In another study, sugar-based materials were used to create temporary stents that facilitated vascular anastomosis [158].The sugar-based composition was rendered anti-thrombogenic by incorporating sodium citrate into the composition.
Wu et al used the technique of omnidirectional printing to fabricate 3D microvasculature networks in hydrogel, as shown in figure 5(A) [159].Initially, the sacrificial ink was deposited into a gel reservoir to form branched networks.After photopolymerization of the reservoir and chemical cross-linking of the fugitive ink with the photocurable hydrogel matrix, the ink was liquified and washed out under vacuum to expose the microvascular channels in the solidified gel matrix.Chang et al used extrusion (bio)printing for producing constructs in collagen gels that permitted the remodeling of vessels after the formation of newer ones [160].Multiple microvascular constructs were printed onto a single electrospun fibrinogen mat by first dispensing F127-PBS followed by dispensing Rat Fat Microvessel Fragments-collagen into the formed channels, as shown in figure 5(B).Shengjie et al produced a cell-encapsulated hydrogel construct with vascular networks using a novel bi-nozzle extrusion technique that served as a conduit for mass exchange shown in figure 5(C) [161].[160] printed constructs in collagen gels that permitted restructuring of vessels.Reproduced with permission from [160].© 2012 American Heart Association, Inc. (C) Shengjie et al [161] developed vascular network using bi nozzle extrusion method.Reproduced with permission from [161].© 2009, © SAGE Publications.(D) Lee et al [162] produced fluidic channels in collagen scaffold.[162] John Wiley & Sons.Copyright © 2009 Wiley Periodicals, Inc. (E) Xu et al [47] fabricated blood vessels in thick tissues that were prevascularized with cell-layers.Reproduced from [47].CC BY 4.0.
Lee et al used a thermal extrusion-based (bio)printing technique to produce 3D hydrogel constructs containing fluidic channels [162].Grooved collagen layers were initially formed which were filled with fugitive gelatin, as shown in figure 5(D).Upon setting of the collagen, the gelatin was washed out by heating to produce the vasculature in the collagen scaffold.The diameter of the channels ranged from a micrometer to a millimeter, and the constructs maintained their integrity even under a hydrostatic pressure of 13.8 kPa.They reported a high post-printing cell viability of HDF; 95% ± 2.3%.They tried to anatomically mimic the structure of the liver by using two concentric nozzles.The inner nozzle was filled with adipose-derived stromal cells (ADSC) and gelatin/alginate/fibrinogen hydrogel.Hepatocytes were combined with gelatin/alginate/chitosan and placed surrounding the inner layer by using a second nozzle to form the outer layer.ADSC with endothelial growth factors were found to differentiate into endothelial-like cells.In addition, they noted that the albumin secretion of the hepatocytes was increased after two weeks of culture.In another study, Byambaa et al used gelatin to fabricate vascularized bone constructs [163].They printed VEGF conjugated GelMA fibers as the cell-carrying scaffold and then printed gelatin fibers in between to create perfusable vessels.VEGF concentration was tailored to create a gradient towards the scaffold periphery directly cell sprouting [163].They showed that endothelial cells lined the vessels effectively, and the presence of the vessel enhanced cell viability and improved bone differentiation.It was also observed that MSCs at the interface of the endothelial cells differentiated to smooth muscle cells to stabilize the vessels.
Xu et al fabricated blood vessels in thick tissues that were prevascularized with cell layers using support scaffolds shown in figure 5(E) [47].The vascularized tissues were made up of a decellularized ECM (dECM), cells, and vessels of varying sizes and diameters with branching networks.They used Pluronic F127 (PF 127) as the sacrificial material for producing the varying-diameter channels.Upon setting of the fugitive Pluronic F127, it was washed away, producing the hollowed-out channels.These hollowed-out channels were then attached with HUVECs.An interesting aspect of their study was their use of different cell types for different layers of blood vessels.To form the adventitia, media, and intima, HDF-neonatal (HDF-n), human aortic vascular smooth muscle cells (HA-VSMCs), and HUVECs were respectively used.It was found that the cell viability and the structural integrity of the constructs was maintained 48 h postprinting.They also reported that the elastic modulus of the printed construct was similar to that of the natural aorta.
In addition to the use of sacrificial materials, (bio)printing using microfluidic nozzles to produce the core-shell flows in which only the shell is crosslinked leads to the formation of hollow fibers.In an important study, Jia et al used this strategy to fabricate pre-vascularized constructs of GelMA and alginate [164].This strategy for formation of GelMAbased constructs was previously demonstrated by Tamayol et al [165].Jia et al showed that endothelial cells expressed tight junctions and the channels were perfusable.
However, the studies discussed above mainly focused on the fabrication of single-layered blood vessels.Gao et al took this to another level by developing 3D hydrogel-based tubular structures with varying channel sizes, ranging from a micrometer to a millimeter, by using extrusion (bio)printing, as shown in figure 6(A) [166].These fabricated channels can be used to mimic the blood vessel microenvironments.Fibroblasts and smooth muscle cells were loaded into hollow alginate constructs that were partially crosslinked and then extruded using a coaxial nozzle around a rotating rod template.The inner walls of the constructs were lined with endothelial cells.Structures encapsulated with L929 mouse fibroblast cells had about 90% cell survivability after one week of printing.Zhou et al developed novel twocell layered blood vessels of small diameters, as shown in figure 6(B) [46].The fabricated blood vessels consisted of vascular smooth muscle cells (VSMC) in the outer wall of the vessel, whereas vascular endothelial cells (VEC) filled the inner lumen.This was then kept in a solution of CaCl 2 for complete crosslinking.After that, F-127 was leached out to form a hollow cylindrical construct.Bioink-laden VEC was then injected into the inner section of the construct to form the final structure.The printed constructs had 0.3 mm wall thickness with the diameter of the lumen being 1 mm.In another study, Maiullari et al developed multicellular cardiac tissue constructs consisting of induced pluripotent cell-derived cardiomyocytes (iPSC-CMs) and HUVECs, as shown in figure 6(C) [37].Initially, the iPSC-CMs cells were seeded into hydrogel matrices composed of PEG-Fibrinogen and alginate and then these were extruded using a custommade printing head into CaCl 2 for crosslinking of the constructs.Cell viability post two weeks of culture was assessed between 80%-90%.Zhang et al used Human Umbilical Vein Smooth Muscle cells (HUVSMCs) incorporated into sodium alginate to print coaxial vascular conduits [167].HUVSMCs suspended with alginate and CaCl 2 solutions were distributed through the coaxial nozzle's sheath and core parts to form cylindrical constructs.Vascular conduits larger than 80 cm in length were printed and had an average conduit and lumen diameter of 1449 ± 27 µm and 990 ± 16 µm, respectively.After prolonged in-vitro culture, the cell viability in the constructs was found to be around 84 ± 1%.Using HUVECs and human smooth muscle cells (HMSCs) with the bioinks of GelMA, alginate, and eight-arm poly(ethylene-glycol)acrylate with a tripentaerythritol core, Pi et al fabricated multi-layered co-axial tubular structures, as shown in figure 6(D) [168].They constructed tubular urothelial tissue structures with the use of human bladder smooth muscle cells and human urothelial cells.Vascular tissue constructs were fabricated with HUVECs and HMSCs.The viability of the vascular cells, HUVECs, and hSMCs was found to be between 85% and 97% after two weeks of culture.They also showed that these constructs can be perfused with nutrients to foster cell growth and proliferation.Figure 6(E) shows the vascular grafts that were developed by comprising both the endothelial cells and smooth muscle cells by Gao et al [169].A triple-coaxial nozzle was used for printing the constructs.The middle and the outermost nozzle layers were used for printing bioinks containing HUVEC Figure 6.Blood vessels fabricated using extrusion (bio)printing.(A) Gao et al fabricated hydrogel channels with diameters varying from micrometer to millimeter [166].Reprinted with permission from [166].Copyright (2017) American Chemical Society.(B) Zhou et al developed novel two-cell layered blood vessels [46].Reprinted with permission from [46].Copyright (2020) American Chemical Society.(C) Maiullari et al constructed multicellular cardiac tissue networks [37].Reproduced from [37].CC BY 4.0.(D) Pi et al made tubular urothelial tissue structures using human bladder smooth muscle cells and human urothelial cells [168].[168] John Wiley & Sons.© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.(E) Gao et al fabricated multicellular vascular grafts using a triple-coaxial-nozzle [169].Reprinted from [169], with the permission of AIP Publishing (F) Jia et al developed perfusable vascular constructs using a coaxial nozzle [164].Reprinted from [164], Copyright (2016), with permission from Elsevier.and HAoSMC, while through the innermost nozzle, Pluronic F127 containing Ca 2+ was extruded.They implanted this graft in rat abdominal aorta and found that the implant showed good host integration, and tissue maturation when observed over a period of three weeks.In the study conducted by Jia et al, authors directly fabricated perfusable vascular constructs using a coaxial nozzle as shown in figure 6(F) [164].They used a mixture of bioinks, GelMA, sodium alginate, and 4-arm poly (-ethylene glycol)-tetra-acrylate (PEGTA).This bioink mixture was first chemically crosslinked with CaCl 2, followed by photo cross-linking of GelMA and PEGTA to stabilize the vascular constructs.They found that the mechanical properties of the constructs can be tuned by changing the percentage composition of PEGTA.The bioink also facilitated the proliferation of the seeded endothelial and stem cells in the constructs.The cell viability seven days post-printing was found to be greater than 80%.
Extrusion bioprinting overcame a significant drawback of inkjet bioprinting in vascular tissue engineering, expanding the range of bioinks that can be used to construct vascular structures.The research presented above exhibited that several bioinks compatible with extrusion bioprinting may display tunable mechanical and rheological properties, as well as bioactivity, because of their potential to be conjugated with chemical moieties and growth factors to increase cell adhesion, spreading, and proliferation, enhancing vascularization in bioprinted constructs [161].Also, the discussed studies showed that extrusion (bio)printing is one of the most suitable (bio)printing techniques in terms of the use of higher bioink density, printing speed, and cell viability.Although blood vessel diameters may vary between the millimeter to the micrometer scale, most constructs using extrusion (bio)printing have limited resolution and are unable to replicate the whole range of blood vessel diameters on the micrometer scale.Thus, an important limitation of extrusion-based (bio)printing is the inability for engineering fully biomimetic 3D vasculature.Besides, since most bioinks used for (bio)printing are hydrogel, they are not suturable and thus the vascular anastomosis needed for their implantation is very challenging.Recently, there have been efforts to use sealants and adhesives to overcome this barrier.In the next section two other (bio)printing techniques, LBB and stereolithography (bio)printing, will be discussed.

LBB
Another advanced (bio)printing method is LBB, which uses lasers to deposit the cells in a highly controlled manner.LBB can be divided into two major divisions: laser-guided, and laser-induced.In the laser-guided (bio)printing approach, the laser beams can be used as optical tweezers for trapping and guiding the cells into a receiving substrate to form the desired structure.This process is made possible by the differences in the cell and the cell media's refractive indices [170,171].
The more commonly used LBB technique is laserinduced (bio)printing.It is comprised of a source of pulsed laser, a targeting system, a target plate, and a substrate collecting base [172].A gold or titanium ribbon is also included for providing support to the donor layer.The upper layer of the ribbon absorbs the laser's energy and gets heated up.The bottom part of the ribbon, layered with the bioink, absorbs the heat energy from the top layer.A bioink bubble is formed during the process which induces shock waves pushing the bioink out of the ribbon onto the collector slide.The printing resolution of LBB is higher than the other (bio)printing techniques, with resolutions reaching up to around 1 µm [173].Printing resolution depends on the energy of the laser beam being focused (laser fluence), the frequency of the pulse, the viscosity of the bioink, the substrate wettability, and the distance between the absorbing ribbon and the collector plate.The printing speed is a function of the laser energy, the ribbon material, and the bioink properties.Furthermore, increasing the laser pulse rate or merging several laser beams can boost fabrication speed.As this is a nozzle-free printing technique, there are no nozzle clogging issues.Bioinks with viscosities ranging from 1 to 300 mPa•s can be easily used in this method [174][175][176].The use of cell-laden bioink densities of up to 108 cells ml −1 have been reported in literature [120].
LBB has high deposition rates with cell viability of more than 90% [177].The cell viability is considerably high as there are no mechanical stresses induced in the cells.Individual cells can be placed within an intended location of 5.6 ± 2.5 µm [178].In addition, printing different cell types is also possible with LBB [179].The droplet volumes can be varied from 10 pl to 7000 pl by controlling the bioink viscosity and the layer thickness on the ribbon [120].
LBB has also been used for the sintering of sacrificial materials.In an important study, Kinstlinger et al used laser sintering to form biomimetic vascular architectures from sugar powder [180,181].The sugar constructs were then covered by a hydrogel.Upon sacrificing the sugar structures, perfusable constructs with biomimetic vasculature were formed that could effectively transport oxygen and nutrients throughout the construct.Wu and Ringeisen used a LBB technique to produce constructs replicating the branching pattern of the vessels using HUVECs and HUVSMCs [182].They initially printed HUVECs to form a layer followed by its incubation for 24 h and then HUVSMCs were printed over this incubated structure to obtain the final constructs.Constructs of the size of a micrometer diameter range were fabricated using this approach.Xiong et al used freeform laser printing to build alginate-based bifurcated cellular constructs similar to vascular structures as shown in figure 7(A) [183].The researchers used a 2% alginate-based fibroblast suspension as bioink and a 2% calcium chloride and cell culture medium as crosslinking and support solution.The cell viability of the constructs was 68.1% and 70.8% immediately after printing and after one day of incubation, respectively.By varying the printing parameters like frequency, cell density, and laser beam energy, Guillotin et al fabricated different organized microscale structures, as shown in figure 7(B) [184].Rabbit carcinoma cell line B16, and human umbilical vein endothelial cell line Eahy92 were used, with the bioinks Na-alginate, glycerol, Matrigel, and DMEM for cell suspension.Meyer et al used stereolithography (SL) and multiphoton polymerization to fabricate tubes and bifurcated tube systems using α,w-polytetrahydrofuranether-diacrylate (PTHF-DA) resins.Tubular structures with diameters less than 2 mm were fabricated, as shown in figure 7(C) [185].
The main advantages of laser-based (bio)printers in blood vessel tissue engineering include: a greater degree of control on material deposition to obtain branched and complex structures like vascular  [183].Reproduced from [183].© IOP Publishing Ltd.All rights reserved.(B) Guillotin et al [184] made two layered multicellular microscale structures.Reprinted from [184], Copyright (2010), with permission from Elsevier.(C) Meyer et al [185] developed bifuracated constructs using α,w-polytetrahydrofuranether-diacrylate resins.Reproduced from [185].CC BY 4.0.networks and substantial cell viability at different cell densities.However, compared to other 3D (bio)printing methodologies, LBB presents some challenges for developing vascular tissue-engineered constructs.LBB is disadvantageous from an economic point of view and requires more time to print the constructs.Hence, the production of large blood vessels with this technique will be limited.To reduce printing times, there is a demand for new biomaterials compatible with the properties of distinct blood vessel structures previously discussed.Although LBB is a nozzle-free strategy, cell damage may occur due to the heat generated from the lasers.Furthermore, LBB involves a considerable number of process parameters, so obtaining a favorable setting might be a tedious process.Finally, there are also chances of contamination due to the use of a metallic ribbon, and due to the heating of the bioink involved during printing, only a limited number of bioinks can be appropriately used [172,186].

Stereolithography
Stereolithography (SLA) uses a reservoir containing the photocurable resin, an X-Y guided laser control, and a Z-axis control stage or platform.Through the bottom of a reservoir tray, the resin is treated with light in the range of ultraviolet (10-400 nm) or visible light (380-750 nm) [187].There are two commonly used approaches in SLA, the bottom-up and the top-down approach.In the bottom-up approach, the resins at the bottom of the reservoir are cured first, followed by the layers above.This is accomplished by incremental downward movement of the Z-axis control combined with the in-plane movement of the X-Y control.In the top-down approach, the curing takes place in reverse order.The printing resolution is manipulated by changing the sintering laser focus and energy.Cell viability was found to be enhanced when the scaffolds were first printed, followed by the incorporation of cells.However, direct incorporation of cellular components during the printing of vessels resulted in reduced cell viability because of the laser beams short wavelength [187].Some studies have shown the possibility of DNA damage when using high-intensity light sources [188].The printing resolution of SLA is comparatively higher than the extrusion-based printing techniques.This is advantageous for the formation of complex tubular structures with variable diameters [189,190].Being a maskless technique, substantial time and cost are saved during the fabrication of the scaffolds [191].In one interesting study, researchers have shown the potential application of widely available light projectors customized for low-cost (bio)printing with resolutions reaching (around 50 µm) and with high cell viability post-printing (around 85%) [192].One of the most prominent examples of SLA-based fabrication of vasculature was reported by Grigoryan et al They utilized a free radical absorbing material to enhance the resolution of the fabricated constructs and demonstrated impressive biomimetic vasculature formed within photocroslinkable hydrogels [193].In particular, they fabricated an in vitro model of lung alveoli with its vasculature and demonstrated that the The comparison of the concentration of oxygen carried by RBCs when the gas channel is perfused with oxygen and nitrogen shows that the bivascular system can model blood oxygenation in lungs.From [193].Reprinted with permission from AAAS.construct can be mechanically actuated and deformed similar to a human lung as shown in figure 8.
Yeleswarapu et al [194] fabricated scaffold-free tubular constructs using a stereolithography-based 3D printing approach with the help of caprine esophagus muscle dECM hydrogel.The L929 mouse fibroblasts which were embedded in the hydrogel were found to differentiate well into myofibroblasts.They noted a significant increase in the mechanical stability of the fabricated construct after seven days of culture.SLA is an emerging technology, which offers the possibility of fabrication of 3D biomimetic constructs for tissue-engineered applications.However, SLA is limited to photocrosslinkable materials.While photocrosslinking has been widely used in tissue engineering, it is still considered controversial due to the possibility of DNA damage causing abnormal cellular function.Methods like stereolithographybased (bio)printing provide greater printing resolution compared to extrusion (bio)printing but at an increased component cost.Another important limitation is their inability of multi-material printing, desirable for the biofabrication of multi-layered vasculature.However, in a recent study, Miri et al combined a microfluidic system and an SLA platform to achieve multi-material SLA (bio)printing [190].Nevertheless, similar with inkjet and LBB techniques, with stereolithography the biomaterials available to produce biomimetic vascular constructs are sparse.
In the above sections, we presented an overview of the different (bio)printing techniques employed for the fabrication of artificial blood vessels.As observed, the printed constructs are primarily affected by the printing techniques used as well as the parameters associated with them, we summarized a comparison between these parameters in table 1.From this table, it can be concluded that when the bioink used is to be of high viscosity, extrusion (bio)printing will be the preferred choice, similarly when we are concerned with cell viability, LBB will be the favored method.In the same way, the other printing parameters like droplet size, printing speed, throughput, spatial resolution, etc. can be appropriately selected.When choosing a (bio)printing technique, we may have a general idea of the possible outcomes of the designed constructs guided by their technical limitations.However, as presented, other factors like the addition of growth factors, the choice of cell sources, mechanical stimulation, and surface topography may help to recreate an adequate physiological environment and further enhance vascularization in the (bio)printed constructs [96,160,161,163,195,196].

Conclusions and future directions
(Bio)printing has emerged as a promising technology for creating extremely complex, functional, and predictive preclinical models to aid in the resolution of the critical issue of organ shortage for patients.Bioprinters have been used for the fabrication of whole blood vessel grafts.Once proper imaging modality is used, such efforts enable the production of grafts with identical geometrical features to the diseased tissue.In addition, it is expected that through the use of patient derived cells, cellular graft with minimal adverse immune response be generated to enhance the chance of integration and lower the risk of graft rejection.However, to use such grafts in clinical practice, the bioinks and bioprinters as well as imaging modalities should be present in the operation room.In addition, the production speed should be fast enough not to require significantly longer anesthesia time for the patients.Aside from the fabrication of arterial grafts, to make a functional organ that can be transplanted in the patients, the development of vasculature and micro-capillaries is very important.To meet the increased demand for organ recipients, there is a need to make generalized organs that can be accepted by the recipients.The role of rapid prototyping comes into the picture here, and there is a need to develop newer (bio)printing techniques.Newer and advanced bioinks capable of carrying multiple cells can increase the structural integrity and viability of the printed constructs.Lastly, to better optimize the various parameters used in the printing process and to increase the cell viability of the printed structures, the role of artificial intelligence (AI) and specifically machine learning (ML) can be explored [208].AI can be used to correlate input parameters such as bioink density, viscosity, nozzle diameter, part geometries, and types of cells with output parameters such as cell viability, mechanical properties, and geometrical accuracies along the (bio)printing process chain.Optimizing the various parameters can be reduced to the problem of optimization in higher dimensions.Another important challenge is the anastomosis of the tissue engineered constructs to the host circulatory system.This means that either the formed vasculature should be mature enough to withstand suturing or reinforcing materials or systems should be used to effectively connect them to the host circulatory system.Post implantation, blood clotting inside the vasculature of the new tissue could be detrimental to its survival and function.Clotting possibility can increase by imperfection in the architecture of the blood vessels.Most of current bioprinting tools struggle to generate hollow structures without significant imperfections.Therefore, new printing technologies or hybrid methods that could create structures without significant imperfections are needed.
The focus of the current review article was on different (bio)printing techniques used for fabricating artificial blood vessels and generating vasculature in tissue engineered scaffolds.There are new (bio)printing techniques like 4D (bio)printing which have not been discussed in this review.4D (bio)printing involves time as a fourth dimension, whereby the functionality and shape of the printed construct changes with time upon application of external stimulus [209].4D (bio)printing is expected to play an important role in generating pre-vascularized scaffolds The resolution of most bioprinters is not high enough to generate microcapillaries and therefore a proper strategy for the formation of multiscale vasculature is to generate them by the endothelial cells lining larger capillaries form by (bio)printing and creating an environment to direct their sprouting patterns to create capillaries.
Another growing area of (bio)printing is in situ or in vivo (bio)printing.In this strategy, the scaffolds are directly printed within the patient's body [210].This approach allows for the creation of scaffolds that seamlessly fit the defect and would adhere to the tissue without the need for sutures or staples [94,211].Most of the in vivo printed scaffolds lack vasculature and there are a few examples that demonstrated in situ (bio)printing of pre-vascularized scaffolds.However, even in those examples, the vasculature was not connected to the host circulatory system.It is expected that researchers find strategies for in situ printing of vascular grafts and connecting in situ printed scaffolds to the host circulatory system.One strategy could be to use materials carrying vasculogenic factors or possessing porosity to facilitate vascular ingrowth [212][213][214].
Another important area of improvement would be on the biomaterial ink development.An (bio)printing allows the production of patient specific architectures, biomaterials can also be prepared to be personalized.This could be achieved through the use of high throughput screening of various biomaterial composition and studying their interaction with the patient's cells.
One important area that requires special attention to make tissue engineered constructs translational is regulatory process.Most researchers disregard the challenges of regulatory process for products that include various cells and therapeutics.Therefore, it is important to identify strategies to inform these researchers about the methods or techniques that could face fewer regulatory hurdles [101].

Figure 1 .
Figure 1.Components of the human circulatory system.(A) The structure of arteries, capillaries, and venous walls, Reproduced from [61].CC BY 4.0.(B) Changes in the composition of blood vessels based on their location in the circulatory system; Reproduced from [73].CC BY 4.0.

Figure 2 .
Figure 2. Selected tissue engineering practices for engineering vascular tissues.(A) A PCL electrospun scaffold covered by collagen.The overall structure of the engineered graft (i) and micrographs of different layers (ii) are shown.SEM images of cross-sectional (iii) outer layer, (iv) entire, and (v) interface between outer and inner layers of bilayered scaffolds.Reprinted from [82], Copyright (2010), with permission from Elsevier.(B) A two-step photolithography methodology to create vascularized bone models (i), micropatterned gels containing HUVECs/hMSCs (ii), and randomly distributed cells on unpatterned constructs (iii).[89] John Wiley & Sons.© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.(C) The microfluidic stamping process for creating patterns of endothelial cells and covering them with scaffolding materials.(D), (E) Endothelial cells sprouted into Matrigel and collagen scaffolds, however, the organization of cells covered by different scaffolds were different.[90] John Wiley & Sons.© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 8 .
Figure 8. Entangled vasculature and biomimetic constructs fabricated using stereolithography.The structures are made of 20% polyethylene glycol diacrylate.(A)-(D) Various architectures bivascular designs fabricated using the strategy.The digital files and the actual fabricated structures are shown.(E), (F) Oxygen perfusion from one of the vessels and red blood cell (RBC) perfusion from another blood vessel.(G)The comparison of the concentration of oxygen carried by RBCs when the gas channel is perfused with oxygen and nitrogen shows that the bivascular system can model blood oxygenation in lungs.From[193].Reprinted with permission from AAAS.

Table 1 .
Comparison of the parameters of the different (bio)printing techniques.