ABSTRACT Vascularization is one of the most challenging areas of tissue engineering research. Vascular engineering holds the key to counteracting cardiovascular diseases, which are the main

cause of death worldwide, and to performing prevascularization of regenerated in vitro tissues to improve implantation survival. Hydrogels have been thoroughly studied in this field due to

their mechanical properties and tissue-like characteristics, including their water content, biocompatibility, and efficient transport of nutrients and metabolites; these characteristics make

them applicable to vascular reconstruction. In this review, we focused on the fabrication of blood vessels using biofunctional hydrogels and compared natural and synthetic materials.

SUPPORT LUMEN FORMATION DURING ANGIOGENIC SPROUTING Article Open access 07 June 2021 PERFUSION AND ENDOTHELIALIZATION OF ENGINEERED TISSUES WITH PATTERNED VASCULAR NETWORKS Article 24 May

2021 INTRODUCTION Vascular diseases, such as atherosclerosis, are the most common direct or indirect causes of death in the world. Because of this, tissue engineering is important as an

innovative way to regenerate the vascular network in vivo. Vascular engineering also has an important application in the prevascularization of tissue-engineered grafts prior to implantation

for better integration into the host vasculature. In every engineered tissue, specifically for the vascular system, the challenge is that the elasticity of the tissue has to allow it to

resist physiological pressure to avoid leakage or development of aneurysms while also being well tolerated in vivo. The construction of Sparks’ mandrel was one of the early attempts in

engineering a vascular replacement [1], which was produced by subcutaneous implantation of a 5.1 mm diameter mandrel made of a loosely knitted Dacron (synthetic polymer polyethylene

terephthalate) in the affected person, which enable the production of autogenous conduits made from the patient’s own fibrous tissue. There are many concerns with this strategy, including

the occurrence of thrombosis and aneurysms. Therefore, this type of technology was not deemed effective for continued clinical use. In 1986, Weinberg and Bell reported the first formation of

a blood vessel in vitro; the vessel formation was accomplished through the use of collagen gels cast around a central mandrel supported by a Dacron mesh sleeve, which was surrounded by

fibroblasts that were seeded on the outer layer [2]. Although this engineered artery was not successfully implanted in vivo, due notably to its low burst power, it represented a significant

conceptual advancement that further improved the engineering of blood vessels by coculturing endothelial cells (ECs) and supporting cells. Using hydrogels for tissue engineering and

regenerative, medicine has become an incredibly productive research field owing to their mechanical properties and tissue-like characteristics, including water content, biocompatibility,

efficient transport of nutrients and metabolites, and suitability for biomedical implantation [3]. The list of tissue-based hydrogels seems almost endless, and it includes retina, ligament,

adipose, kidney, muscle, and blood vessels [4]. Generally, hydrogels consist of hydrophilic polymers synthesized by covalently connecting networks of natural or synthetic molecules. Hydrogel

materials have become useful for 3D vascular engineering scaffolds due to several of their features, including natural extracellular matrices (ECM) and viscoelastic properties [5]. To date,

many studies have been reported on the ECM-mimicking phenomena observed in biocompatible hydrogels based on natural or synthetic materials with different manufacturing techniques. Most of

these hydrogel systems were used for prevascularized tissue engineering by coculturing ECs with other supporting cells and were then used for in vivo implantation. Although there are

completely nonscaffolding strategies available, such as spheroid or cell sheet technology [6,7,8], most vascular engineering strategies require appropriate scaffolds that enable the

transport of cells or growth factors and provide a 3D blood vessel matrix. In this review, we focused on the different hydrogels used as scaffolds, including microfluidic systems, for the

tissue engineering and pharmaceutical fields. We summarized the recent progress in the development of in vitro blood capillary models and their detailed in vitro applications according to

the components and techniques used (Fig. 1). The review clarifies the strong contribution of natural and synthetic biofunctional hydrogels and suggests future aspects in this area.

COMPONENTS OF HYDROGELS Hydrogels need to act as scaffolds by providing a suitable environment for cell growth, which includes a sufficient supply of oxygen, nutrients and metabolic

substances, and the ability to remove cellular waste [9]. Hydrogels that follow these criteria can closely mimic a natural extracellular environment or tissue. The regeneration of complex

vascular tissues mimicking in vivo tissues is important for accurate drug screening models and long-term tissue implantation in pharmaceutical and medical applications, respectively.

Moreover, in vitro vascular tissue models would also contribute to a decrease in the use of animal experiments and avoid classic issues of species difference bias. Here, we will discuss the

different components of hydrogels that can be classified as natural, synthetic, or combined polymers, all of which are commonly used for engineered vascular tissue. NATURAL HYDROGELS

ALGINATE GELS Alginate consists of β-d-mannuronic acid (M) units and α-l-guluronic acid (G) units that are assembled as block copolymers with regions composed of either homogenous M or G

units or with alternating G and M units [10, 11]. Since alginate is not suitable for cell growth attachment, cells are unable to degrade it; therefore, alginate cannot be used alone and must

be modified with peptide (Fig. 2a) [12] or another adhesive ligand, such as RGD (arginine-glycine-aspartic acid), to enable vascular network formation. These modifications allowed the

generation of vessel-like structures with consistent quality that exhibit cell-friendly conditions [13, 14]. In addition, the ability of alginate to be easily dissolved into the process of

cell recovery makes it attractive for studying 3D cell–material interactions [15]. However, similar to other hydrogels, the physical properties of alginate hydrogels, such as its mechanical

strength, make them limited in terms of in vivo applications. FIBRIN GELS Fibrin is a natural polymer formed during wound healing [16] and hemostasis, which make it a potential biopolymer

for in vitro angiogenesis studies [17]. Furthermore, it promotes cell division, facilitates cell attachment, and migration along RGD adhesion sequences and encourages vascularization [18].

Fibrin hydrogels come from the enzymatic activation of fibrinogen by thrombin, which is followed by terminal factor XIIIa-mediated enzymatic crosslinking (Fig. 2b) [19]. These gels represent

suitable templates for endothelial and mesenchymal cells to easily form microcapillaries under appropriate coculture conditions [20]. Alternatively, Chen et al. used a coculture of human

umbilical vein endothelial cells (HUVECs) and fibroblasts to create a vascular network [21]. Another approach was used by Kolesky et al., who used sacrificial printing to generate a thick

(>1 cm) vascularized tissue construct with human fibroblasts and human mesenchymal stem cells (MSCs) in fibrin gels [22]. An interconnected vascular network was generated whose channels

were lined with HUVECs. However, for in vivo application, the relatively long time needed for ECs to develop into functional vessels (1–2 weeks), as well as fibrin gel dehydration and

degradation, limit their long-term cell culture usage. COLLAGEN GELS Collagen is a fibrous ECM protein involved in connective tissue construction. Type I collagen is the most common type of

collagen, and it is often used as a tissue engineering scaffold because of its ability to form hydrogels under physiological conditions. One of the earlier studies of vasculogenesis-driven

vascularization methods using a collagen matrix was done by Davis et al. [23], who embedded HUVECs and mesenchymal cells in such a matrix to demonstrate the formation of capillary network.

However, collagen suffers from some important drawbacks, including low stiffness, limited long-term stability, and batch-to-batch variability. It is also difficult to produce collagen

hydrogels with higher stiffness (>1 kPa) without extensive chemical crosslinking, which fundamentally alters the degradability of collagen fibrils. As a result, culturing cells in

collagen hydrogels for extended periods leads to significant contraction of the matrix. Deng et al. studied the ability of chitosan to improve the physical properties of the collagen

material and to enhance EC differentiation and angiogenesis [24]. However, the potential of a collagen–chitosan matrix in angiogenic therapy has not been tested. As an alternative to

collagen, gelatin (i.e., hydrolyzed collagen) is also widely used in tissue engineering. In one study, Park and Gerecht [25] combined gelatin with ferulic acid to form a hydrogel network via

oxygen consumption, inducing acute hypoxia. While seeded with endothelial progenitor cells, hypoxia accelerated vasculogenesis in vitro through, notably, the induction of the hypoxia

inducible factor-dependent increase of vascular endothelial growth factor and angiopoietin. This study demonstrated the fabrication of a vascular bed without the use of high-dose angiogenic

drugs or other perivascular cell types, which can have potential benefits, particularly for therapeutic applications based on angiogenesis-driven platforms. MATRIGEL Matrigel offers many of

the advantages of collagen and other natural hydrogels and has been used to study cell migration and angiogenesis [26]. It is a basement membrane-derived preparation extracted from

Engelbreth-Holm-Swarm mouse sarcoma tumors, and it comprises laminin, type IV collagen, entactin, and various other constituents, including proteoglycans and growth factors [26]. Despite all

its extracellular components, which are important for vasculature formation, Matrigel has significant drawbacks, including the fact that it is derived from mouse tumors, has diverse

composition, and batch-to-batch variability, which in turn restricts its use as a research tool in basic science [27]. HYALURONIC ACID GELS Hyaluronic acid–based hydrogels are also widely

used in natural hydrogels. For instance, when hyaluronic acid is chemically modified with fibronectin, it promotes EC binding, thereby resulting in vascularization that is better than that

of nonmodified hydrogels [28]. Another example of a hyaluronic acid–based hydrogel was reported by Kageyama et al., who integrated hydrazide-modified gelatin with aldehyde-modified HA to

produce in situ cross-linkable hydrogels that could rapidly fabricate perfusable vascular-like constructs [29]. The modified HA hydrogel was also applied to in vivo transplantation, and it

exhibited anastomosis with the host vasculature [30,31,32,33]. Human-induced PSCs differentiated into early vascular cells were also seeded onto synthetic hyaluronic acid–based hydrogel

(acrylated HA) and produced vascular networks with lumens and pericytes (PCs) surrounding the ECs [32]. Thus, modified hyaluronic acid hydrogels have great potential for engineering

clinically relevant vascular tissues. However, chemical modifications can cause toxicity to cells and subsequently affect angiogenesis when applied in vivo. Although there are many

advantages to natural hydrogels, their major drawbacks include their poor mechanical properties, which are generally difficult to control and tune, in addition to their potential to

influence immunogenic reactions [34]. Therefore, synthetic polymers have been explored and developed to solve this problem. SYNTHETIC HYDROGEL Synthetic polymer materials are also good

candidates for use in vascular tissue engineering. The properties needed for these materials include biocompatibility, biodegradation, and nonimmunogenicity. Synthetic polymers that have

been used for vascular tissue engineering include polycaprolactone, poly-l-lactic acid (PLLA), polyglycolic, acid and their mixtures. When using these materials, the mechanical strength of

the resulting graft will also depend on the new ECM proteins produced by seeded cells [35]. To combine their cell-instructive and biocompatible features with desired mechanical properties,

synthetic hydrogels can be tuned by chemical modifications or combined with natural ECM components to capitalize on the benefits of both [36]. POLY(ETHYLENE GLYCOL) GELS Several studies have

shown that coculture of endothelial and mesenchymal cells in PEG hydrogels is a promising approach for the formation of 3D prevascularized scaffolds with fully defined properties

[37,38,39,40]. PEG can be modified with various functional groups and can form hydrogels via different types of polymerization techniques, such as chain-growth, step-growth, or mixed-mode

[41], giving the user more design flexibility than is available with other gels (Fig. 2c). POLY(LACTIC-CO-GLYCOLIC ACID) PLGA AND POLYGLYCEROL SEBACATE (PGS) GELS Another example of

synthetic hydrogels is PLGA and PGS, which have been used to fabricate complex structures with microchannels and have been seeded with endothelial and hepatocyte cells [42]. However, their

application has been limited because of the biodegradable nature of the scaffold, making it difficult to achieve uniform cell seeding in the scaffold. POLYDIMETHYLSILOXANE (PDMS) GELS

Although PDMS is not cytotoxic, cells cannot be cultured within them because it is not permeable to water. However, Yu et al. built PDMS-based microfluidic chips coated with the ionic

complementary peptide EAR16-II so that the surface of PDMS interacted strongly in a fashion that was similar to that of proteins [43]. Thus, nonspecific protein adsorption was minimized, and

the biocompatibility of these microfluidic chips was improved. Another class of synthetic hydrogels used for microfluidic fabrication are self-assembling peptide hydrogels. They have been

introduced as possible biomaterials for tissue engineering applications due to their high biocompatibility, biodegradability, tailorable bioactive properties, and the ability to be

self-organized into nanofibrous structures that imitate the natural ECM [44]. SEMISYNTHETIC HYDROGEL GELATIN METHACRYLOYL (GELMA) GELS GelMA is also used as a vascularization scaffold that

can produce anastomoses between microcapillaries and murine hosts through in vitro prevascularization or subcutaneous injection [45, 46]. Another example of a gelatin-based hybrid approach

involved electrospinning with PLLA. By this method, homogeneous vessel-like tubular scaffolds could be formed with good physical and biological properties [47]. PEG-BASED HYBRID GELS A

photocross-linkable collagen-PEG hybrid material in which the mechanical properties could be increased independently of the material density has been developed [48]. Within this collagen-PEG

hydrogel, microcapillary networks can be generated. In addition, PEG was also modified via photoinitiator-induced crosslinking with fibrinogen. PEGylated fibrinogen hydrogels with different

PEG concentrations exhibited altered network structures, which could influence cell growth and the morphology of cells [49]. In another study, peptide-modified PEG hydrogel was used to

provide a cell culture system in three dimensions that could support cell adhesion, cell migration, and vascular formation [37, 50, 51]. HEPARIN-BASED HYBRID GELS Hybrid hydrogels built by

the crosslinking of GAG heparin and PEG (Fig. 2d) can host microcapillary networks when biologically modified to be sensitive to RGD and matrix metalloproteinase activity [52, 53]. Moreover,

in contrast to HA hydrogels, the advantage of heparin-based hydrogels is that the binding affinities of a variety of growth factors are enabled through the high abundance of negatively

charged sulfate groups. METHODS FOR ENGINEERING VASCULAR HYDROGELS Vascular engineering can be performed using static or dynamic systems. However, the systems result in different cellular

responses and sensitivities toward several factors, such as mechanical stimuli and biological environment, that in turn affect the vascular structure. These systems will provide an

understanding of the influence of biomolecules and of the application of shear stress on the regulation of morphology, reorganization, alignment, differentiation, and remodeling of ECs,

specifically as they related to capillary formation [54]. This information opens new possibilities for the construction of novel biomaterial-based hydrogels to be used as tissue models

and/or in tissue regeneration. STATIC METHODS THERMORESPONSIVE CULTURE PLATE Using a cell sheet technique, vascularized tissue was formed through the stacking of a single monolayer sheet of

ECs along with the cell sheets of interest (sandwich fashion) (Fig. 3a) and culturing them on a temperature-responsive material to create a cell–ECM matrix. Prior to preparation, nanometer

scale poly(N-isopropylacrylamide) was coated on polystyrene tissue culture surfaces to facilitate attachment and detachment due to the hydrophobic and hydrophilic conditions at higher (37 

°C) or lower temperature (<25 °C) [55]. Thick tissue can be formed in vitro by compiling several stacked cell sheets to form a functional vascularized tissue. Even though the development

of vascularized tissue might occur as a cell sheet, the main challenge with this technique is the integrity and low mechanical strength of the stacked cell sheet tissue, which requires

further investigation. LAYER-BY-LAYER ASSEMBLY A cell accumulation technique was used to construct a blood capillary model through the seeding of HUVECs with fibronectin–gelatin (FN-G)

nanofilms onto the layers of FN-G-coated NHDFs with a sandwich culture method (Fig. 3b). After culture, observation with confocal laser scanning microscope revealed that a tubular structure

in the tissues was formed by a very dense and homogeneous network that looked similar to in vivo blood capillaries. In addition, clear lumen structures were present in many regions of these

blood capillaries. From this technique, the two factors promoting vascularization were (1) the expression of angiogenic factors secreted from NHDFs and (2) the 3D microenvironment [56].

ORGANOID SYSTEMS In 2019, organoid blood vessels (Fig. 3c) were successfully constructed using human pluripotent stem cells (PSCs); the blood vessels contained ECs and PCs that

self-assembled into capillary tissues, but they also contained Matrigel and collagen I as a matrix. Organoid human blood vessels also have displayed good potential for use in transplantation

methods, since they have been shown in mice to establish a stable and complete vascular tree, including arteries, arterioles, and venules [57]. DYNAMIC METHODS MICROFLUIDIC SYSTEMS The

effectiveness of microfluidic systems in vasculature formation relies heavily on the mechanical and biological properties of the materials being used (Fig. 3d) [58]. For instance, EC

monolayers show poor barrier function in terms of transporting biomolecules, oxygen, and nutrients when grown into micropatterns using synthetic materials such as poly(methyl methacrylate),

poly(dimethyl siloxane), silicon, polycarbonate, polyvinyl chloride, polystyrene, PLGA, and poly(glycerol sebacate). This is because they show poor biodegradation, barrier function, and

biocompatibility and provoke cytotoxicity and inflammatory responses in vivo [59]. Therefore, researchers have used silk fibroin, Matrigel, collagen type I, and fibrin to form 3D vascular

networks [60]. However, while a coculture of HUVECs and MSCs in a collagen gel/microfluidic system reduced capillary formation, it stabilized the newly formed capillaries [61]. In addition,

research in this direction has not advanced far because these techniques are time-consuming and could cause significant cell damage during fabrication. 3D bioprinting represents the most

recent trend in fabricating vascular tissues using a top–down approach, where generating patterns of biomaterials and cells at a high 3D resolution is possible [62]. Zhang et al. used an

extrusion-based 3D bioprinter to create chitosan–alginate tubes (with diameters as low as 200 μm) and variable pathways within alginate hydrogels [63]. However, this technique yielded low

viability of cells after 12 h of media perfusion post printing. To address this problem, researchers have developed a thermal-based bioprinter to create a sacrificial template serving as a

lattice. In one study (Fig. 3e), carbohydrate glass filaments were used. The filaments were formed in 3D-shaped networks that were subsequently released at 110 °C. Then, they were packed in

agarose polymers loaded with primary rat hepatocytes and fibroblast cells. Finally, the dissolved filament was released by cell culture media. The result of this technique revealed that

vascularized formation still showed metabolic function of primary hepatocytes, such as maintaining high albumin secretion and urea synthesis [64]. Nonetheless, the application of

vascularization from this technique may be limited due to the hygroscopic behavior of carbohydrates and the difficulty associated with high temperature dispensing. In one study, Heintz et

al. used a structure that was derived from scanning in vivo cerebral cortex vasculature to pattern a photodegradable material [65] with channels as small as 3 µm in diameter (which is

similar to the smallest scale of capillary vessels). However, there were issues in perfusing the smallest channels with HUVECs without clogging the channels. In addition, the shortcomings of

this technique, such as long fabrication time, laser-induced cell damage, and low scalability for large constructs, limit its application in tissue vascularization. MICROCHANNEL SYSTEMS

Bertassoni et al. used molded channels based on GelMA and PEG hydrogels with agarose rods, and after removing the rods, the channels were seeded with a suspension of ECs [66]. However, this

technique has limitations due to the difficulty of placing perfusion needles in a narrow microchannel. In another study, gold rods coated with oligopeptides and ECs were used for

endothelialization. After hydrogel formation, ECs were transferred from the gold bars to the hydrogel electrochemically, subsequently forming endothelialized channels [67]. However, in such

systems, oscillatory shear stress can cause cell disruption and may hinder fluid flow. These data indicate that the combination of high oscillation shear stress and high oxygen content

inhibited angiogenesis. In another study, the channels in hydrogels were created by laser-based photoablation, which allows high spatiotemporal control over the channel design and saves

labor [68]. Overall, in vivo applications of these techniques are yet to be realized. APPLICATION OF ENGINEERED HYDROGEL VASCULATURE In vitro models of vascularized tissues under

physiological conditions are needed to study diseases related to disrupted vascular function. As a result, both cancer biology and tissue engineering have become rapidly growing research

topics involving in vitro screening assays for 3D cancer models. Because angiogenesis is one of the characteristics of cancer, vascularization is also incorporated in 3D models of cancer. As

an example, a study conducted by Nashimoto et al. involved the formation of a perfusable vascular network in spheroids containing the human breast cancer cell line MCF-7 using a

microfluidic platform (Fig. 4a) [69]. This method produces a condition similar to what is observed in vivo by directing the angiogenic secretion of spheroid cancer cells to ECs in

microchannels, while fibrin gels acted as an ECM. An in vitro microvascular model of the human blood–brain barrier was also developed (Fig. 4b) by coculturing human iPSC–ECs, human brain

PCs, and astrocytes using a microfluidic device with fibrin gel in the central region [70, 71]. In another study, perfusable blood/lymph capillary networks were fabricated in microplates to

enable screening of the drug irinotecan (an anticancer drug for metastatic liver or pancreatic cancers) and gemcitabine (an anticancer drug for colon cancer) [72]. However, these methods are

subject to certain limitations, such as being time-consuming and requiring complicated techniques that cannot be translated into high-throughput assays. We recently fabricated vascular

tissue models for use in chemical probe screening (Fig. 4c) to identify a specific live-blood vessel imaging probe [73]. Nonetheless, the critical key to enabling the fabrication of in vitro

vascular tissue would be the use of shear stress or biomechanical force to regulate EC functions. The importance of shear stress in vascular ECs has been discussed in many reviews

[74,75,76,77], and it plays a part in regulating EC proliferation, migration, and morphological orientation. In vivo blood flow is the main source of shear stress in the vascular luminal

surface; in comparison, with in vitro vascular tissue, the source of the shear stress comes from the culture medium flow applied [78]. Thus, microfluidic systems seem to be the best platform

for reproducing physiological biomechanical force and shear stress from the culture medium flow. However, there are limitations in the microfluidic systems where shear stress provided by

the cell culture medium is lower than that of in vivo blood flow. This is due to the Newtonian nature of the fluid, which is predominantly water, in the culture medium providing low

viscosity in fluid dynamics, whereas blood is a non-Newtonian fluid that has higher viscosity in fluid dynamics [79]. CONCLUSIONS This review summarizes biofunctional hydrogels and

methodologies for the in vitro fabrication of blood capillaries. Various components are used by researchers in the manufacture of vascular engineering. Certainly, hydrogels that use natural

components are better than other forms of hydrogels because they are similar in nature to ECM. In the case of engineered hydrogel vasculature, fibrin is one of the most suitable components

because it is also involved in the blood clotting process, which helps to promote the development of new blood vessels. However, the main drawback of the fibrin hydrogel is its low

mechanical stability. To overcome this problem, fibrin hydrogels can be manipulated through the addition of other natural components, such as collagen fiber, which would act as a filler and

thus strengthen the mechanical properties of hydrogels. The early approaches described in the engineering of vascular models were mainly designed to mimic physiological and pathological

tissue functions. The engineered vascular models could then be used in the development of novel vascular drugs via the integration of highly reproducible vascular models in high-throughput

platforms [79, 80]. The issues of species-related false positive and negative results would be avoided as tissue vessel models are constructed with human cells; they, therefore, would also

represent interesting alternatives to preclinical animal models. Critically, the future development of vascularized tissues is dependent on hydrogel material that is compatible with

cell-specific functions as well as manufacturing technology. To achieve this goal, collaboration between cell biologists, bioengineers, and surgeons is required to integrated knowledge from

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acknowledge financial support from the Grant-in-Aid for Scientific Research (B) (17H02099), the JST Mirai-Program (18077228), the Bilateral Joint Research Projects of the JSPS and an AMED

Grant (JP18be0304207). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka, 565-0871, Japan Muhammad Asri

Abdul Sisak & Michiya Matsusaki * Joint Research Laboratory (TOPPAN) for Advanced Cell Regulatory Chemistry, Graduate School of Engineering, Osaka University, Osaka, 565-0871, Japan

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Sisak, M.A., Louis, F. & Matsusaki, M. In vitro fabrication and application of engineered vascular hydrogels. _Polym J_ 52, 871–881 (2020). https://doi.org/10.1038/s41428-020-0331-z

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