Strategies for Regenerative Vascular Tissue Engineering

Vascularization remains one of the key challenges in creating functional tissue‐engineered constructs for therapeutic applications. This review aims to provide a developmental lens on the necessity of blood vessels in defining tissue function while exploring stem cells as a suitable source for vascular tissue engineering applications. The intersections of stem cell biology, material science, and engineering are explored as potential solutions for directing vascular assembly.


Introduction
Regenerative medicine is a highly interdisciplinary field predicated on the promise of replacing or augmenting the function of diseased tissues through a variety of approaches. Here cells, biomaterial scaffolds, or their combinations can mediate a regenerative response. One of the major obstacles in achieving in situ restoration through tissue engineering approaches includes facilitating vascularization. [1] Without the support of blood vessels, organ function is restricted due to the lack of sufficient nutrient and oxygen exchange within the tissue microenvironment, which can limit tissue survival, proliferation, and differentiation events. Indeed, most tissues reside within 150 µm of a vascular bed, which is approximately the diffusion limit of oxygen. [2] To this end, vascular tissue engineering strategies either focus on creating prevascularized constructs for implantation or are designed to stimulate neovascularization at the transplantation site. Furthermore, a range of strategies is needed to facilitate vascularization across varied biological length scales including large diameter (>8 mm) and capillary (<1 mm) structures. [3] With these considerations in mind, tissue engineering approaches in regenerative medicine largely hinge on the ability to support vascularization, as reviewed elsewhere. [4] ECs from the brain and liver exhibit exclusive sets of markers that are distinct from other tissue-derived ECs. [21] Meanwhile, gene expression from adipose tissue, the diaphragm, heart, mammary gland, and skeletal muscle ECs appear to be more ubiquitous. [22] To this end, developing methods to obtain or differentiate stem cells to tissue-specific endothelium is needed to support the parenchyma of interest. In this review, we establish the developmental program that underlies vascular specification and explore sourcing considerations that drive vascular assembly for studying parenchymal structure. We will describe biomaterial and engineering approaches to coax the assembly of vasculature and postulate new techniques to control vascular interactions with parenchymal tissues for applications in regenerative medicine.

Development
Vasculogenesis in the mammalian embryo takes place in the extraembryonic yolk sac following gastrulation and is marked by the emergence of mesoderm-derived endothelial progenitor cells (EPCs). [23] EPC specification is initiated when mesodermal cells form angioblasts with the assistance of adjacent endoderm cells after gastrulation. [24] Angioblasts then join hematopoietic cells to form multiple aggregates called blood islands. The fusion of these blood islands eventually leads to the formation of the primary capillary plexus. During this process, lumens are formed within the construct and angioblasts start to differentiate from EPCs. The newly formed primary capillary plexus continues to mature through angiogenesis, an invasive proliferative and migratory process of the nascent vessel. During this time, sprouting branches form through a combination of ECM remodeling by matrix metalloproteinase (MMP) activity, secretion of new ECM proteins, and guided movement by filopodia protrusions as reviewed elsewhere. [25] As angiogenesis occurs concomitantly with the early development of the organism, most EPCs mature into ECs and establish arterial, venous, or lymphatic systems, while some undergo a transition towards hematopoietic or mesenchymal developmental lineages. [26] It is noteworthy that EPCs are not entirely derived from the angioblasts. Traditionally, EPCs are defined by the cell surface antigen expression, such as CD34, CD133, and vascular endothelial growth factor receptor 2 (VEGFR-2/FLK-1), [27] where two cell lineages conform to this test. One lineage possessing "endothelial cell colony-forming units" termed CFU-EC, which was later found to be a descendant of hematopoietic stem cells, is not capable of forming perfused vessels in vivo. In contrast, another lineage called endothelial colony-forming cells (ECFC) is isolated from the human peripheral blood, demonstrating the robust proliferative potential and vessel-forming activity in vivo. [28] Understanding the native developmental process is an important part of engineering biomimetic vascular tissues. Each of these developmental steps and the chemical and physical cues that mediate them are potential tools for recapitulating blood vessel development in vitro.
Analysis of fetal tissues reveals that EC maturation is heterogeneous during organ development, highlighting the importance of considering organ-specific vascularization for tissue engineering applications. Three months after gestation, ECs collected from the heart, liver, lungs, and kidneys displayed distinct marker expression patterns and localization. Upon further in vitro culture, ECs derived from those organs showed heterogeneity in network structure, barrier efficacy, angiogenic potential, and metabolism. [21] During the early organogenesis of the liver, for example, newly differentiated hepatocytes accumulate within the endodermal epithelium and then migrate inside the mesoderm derived septum transversum, which eventually gives rise to ECs. The surrounding ECs are proved to be indispensable for proper liver development, and depleting ECs during development or perturbing the expression of FLK-1 through fetal liver FLK-1 mutant embryos can lead to hepatic outgrowth defects. [16] Another example of the importance of vasculature during organ specification can be illustrated in the development of the pancreas. Pancreatic differentiation is initiated in the foregut endoderm, where the endodermal epithelium interacts with the endothelium of major blood vessels. Excision of the dorsal aorta causes a depletion in insulin secretion in Xenopus laevis. Additionally, deletion of pancreatic vascular endothelial growth factor A (VEGF-A) shifts the morphology of adjacent ECs to have more caveolae, highlighting the significance of large vessels and VEGF-A in pancreatic organogenesis. [29] Kidney development also requires vascular cues, and the presence of angioblasts and mesenchyme is crucial for inducing nephron differentiation. It has been established that Flk1 is mandatory for a stable mesenchymal paired box gene 2 (Pax2) expression level in the early kidney, thereby stimulating the expression of GDNF. In addition, VEGF-A secretion from the mesenchyme can also enhance the expression of Pax2 and GDNF, which in turn promotes the growth of the kidney. [9] This complex interplay between ECs, mesenchyme, and parenchymal tissue highlights the importance of the surrounding cell populations for EC and tissue development.
Numerous soluble chemical factors participate in the regulation of vascular differentiation and have been identified from in vivo studies, informing approaches for building systems for vascular tissue engineering (Figure 1). Cellular signaling pathways render a systematic blueprint for neonatal development, consisting of a myriad of molecules and signaling pathways such as NOTCH, BMP, wingless, and integration 1 (WNT), TGF-β, fibroblast growth factor (FGF), insulin-like growth factor (IGF), and VEGF. These pathways impact mesoderm specification, EC formation and maturation, and morphogenic processes including vasculogenesis and angiogenesis. [30,31] Indian hedgehog (IHH) secreted from the yolk sac mediates the BMP pathway and VEGF-A is generated in the extraembryonic visceral endoderm, exerting influence on vasculogenesis by binding to the VEGF receptor 1 (Flt-1) and 2 (Flk-1). BMP4 or FGF2 deficiency leads to the arrest in mesoderm formation, and the overexpression of VEGF-A is embryonic lethal and impairs cardiogenesis, whereas insufficient VEGF-A jeopardizes embryonic vessel formation. [32] In early embryonic development, upstream pathways such as BMP, NOTCH, and WNT activate transcription factors including erythroblast transformation specific (ETS) translocation variant 2 (ETV2), which subsequently associates with other transcription factors including ovo-like zinc finger 2 (OVOL2), forkhead box C2 (FOXC2), and GATAbinding protein 2 (GATA2) to promote expression of essential EC differentiation genes. [33] Additional transcription factors www.advanced-bio.com that influence vasculogenesis include members of the forkhead box (FOX) family, namely FOXF1 and FOXO1, which modulate early EC differentiation and specific vessel formation. [34] Additional factors such as the Prospero homeodomain transcription factor (Prox1), are responsible for tissue specificity in endothelial development and drive lymphatic endothelial cell (LEC) specification. [35] In addition to the chemical cues that influence blood vessel assembly through regulating various signaling pathways, physical and mechanical properties provided by the ECM and blood flow are significant in driving vascular specification, morphogenesis, and remodeling. The ECM harbors instructive growth factors that render additional guidance for EC formation and maturation, while MMPs secreted by cells and distributed in the ECM facilitate vascular remodeling. [36][37][38] Blood flow introduces additional physical stimuli to the developing embryo in the form of laminar and oscillatory shear force. [39] Shear stress can modulate vascular organization, EC proliferation, and gene expression profiles in vascular cell populations. [40] EPCs preconditioned with shear stress compared to static culture conditions are quiescent, elongate, and orient in the direction of flow, and have an increased capacity to undergo capillary vessel formation when plated on collagen gels. [41] ECs can sense shear stress through tensor sensors governed by cell-cell junctions, cell-matrix interfaces, primary cilia, the glycocalyx, and a wide range of membrane-bound proteins and ion channels. [42][43][44][45][46][47] ECs are sensitive to both shear and pressure forces, where the administration of physiological relevant hydrostatic pressures (50 mmHg) drives EC proliferation. [48] Conversely, pathological pressures (200/100 mmHg at 85 per min) induce EC degeneration and apoptosis. [49] While ECs are exposed to various forces on their luminal side, the basolateral side provides additional instructive cues. The matrix stiffness of the endothelial niche in vivo ranges from 3 kPa to 9 MPa and influences EC morphology and fate determination. [50,51] Matrix compliance is interpreted through integrin-mediated bidirectional signaling between the cell and its extracellular niche. Integrins that engage with binding motifs in the ECM are tethered to intracellular proteins via complexes between focal adhesion, vinculin, and talin proteins. In addition, this mechanical engagement drives transcriptional programs via the Yes-associated protein (YAP) and the PDZbinding motif (TAZ) transcriptional coactivator. [52,53] Several in vitro approaches have been leveraged to mimic these interactions. For example, ECs cultured on compliant substrates adopt a round and less spread morphology, leading to fewer focal adhesions (FA) and larger traction forces. [54] Venous ECs can be derived while cultured on compliant substrates, whereas higher stiffness promotes the formation of arterial ECs. [55] Furthermore, LECs can be regulated to form lymphatic networks by tuning the matrix stiffness. Upregulation of key lymphatic markers such as Prox1, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and VEGFR-3 was detected when the LECs were cultured on a substrate with decreased stiffness. Stimulated by VEGF-C, these LECs can form cord-like structures by sensing the matrix through YAP/TAZ and releasing MMP-14. In addition to substrate mechanics, ECs are sensitive to topographical cues and display contact guidance when cultured on patterned surfaces, aligning with the orientation of the substrate features. [56] Collectively, input from mechanical and chemical cues in the extracellular niche contributes to vascular development and is an important design consideration when engineering vascular tissues.

Cell Sourcing
While mature vascular populations can be isolated from tissuespecific sources, their phenotypes are rapidly lost during in-vitro expansion, as cues from the native tissue Figure 1. Components of the vascular niche. The hierarchical vascular architecture is organized as networks of oxygen rich arteries (red) and oxygen poor veins (blue). Oxygen and nutrients are delivered to underlying tissues at capillary beds, which are small diameter endothelial networks supported by pericytes. Larger arteries and veins are mechanically and biochemically supported by fibroblasts and smooth muscle cells. Blood vessel lining endothelial cells (ECs) can be continuous, fenestrated, or discontinuous, depending on their resident tissue. These phenotypes determine the degree of humoral fluid exchange. The vasculature is influenced by an array of mechanical cues from the extracellular matrix including stiffness, viscoelasticity, and topographical cues from basement membrane proteins, as well as shear stress imposed by blood flow. In addition to the physical forces that stimulate the vasculature, additional signaling cues are introduced to vascular networks by various proteins in the 3D ECM.
www.advanced-bio.com cytoarchitecture are lost. [57] Sourcing from stem cells, which have the unique capacity of unlimited self-renewal and differentiation capacity, has the potential to address the limitations of primary cell sourcing. Early attempts at deriving a pool of regenerative cells for in vitro expansion relied on the isolation and expansion of EPCs which can be harvested from peripheral blood or through the spontaneous differentiation of stem cells as embryoid bodies. [58][59][60] However, due to their limited abundance in the blood and uncontrolled specification as embryoid bodies, the focus has shifted to exploring controlled directed differentiation approaches. Here, we highlight recent approaches for generating ECs from adult and human pluripotent stem cells (hPSCs). Finally, we visit how reprogramming technology allows the transdifferentiation of somatic cells to ECs [61] (Figure 2).

Pluripotent Stem Cells
hESCs were successfully separated from the inner cell mass of the blastocyst in 1998 but introduced ethical concerns related to their isolation. [62] However, somatic cells can be reprogrammed into human-induced pluripotent stem cells (hiPSC), circumventing the ethical conundrum of deriving stem cell populations through terminating fertilized embryos. [63] hiPSCs confer additional advantages for generating vascular sources for tissue regeneration in that they are readily accessible and can be derived from patient-specific cell sources for autologous transplant, mitigating the risk of immune rejection. Directed differentiation approaches, focused on the controlled administration of physical and chemical cues along the maturation process, have proven successful in efficient vascular specification from pluripotent populations. [64][65][66] With the ability to control exogenous cues during differentiation, hiPSCs can be directed to take on progenitor, [64] arterial, [54] venous, [65] lymphatic, [66] and other tissue-specific phenotypes such as those from the liver [67] and brain. [68] For example, hiPSCs can be converted to highly proliferative cord-blood endothelial colony-forming cells (CB-ECFCs), which can be expanded to over 10 8 cells per starting hiPSC. These cells form capillary-like structures when cultured on basement membrane mimics such as Matrigel and can repair hyperoxic retinal injuries and revascularize ischemic hindlimb regions upon implantation. [69] Derivation of tissuespecific vascular populations has served to be instrumental in reconstituting tissue function in vitro, as in the case of the Hemangioblasts give rise to progenitors with endothelial and hemopoietic differentiation potential. While CFU-Hill cells have angiogenic potential, they lack the proliferative, and vasculogenic potential of ECFCs. C) Tissue-specific vascular cells can be harvested from organs, but their capacity to expand and retain their phenotype is limited in vitro. D) Genetic engineering approaches, where somatic cells are transdifferentiated or reprogrammed to a vascular phenotype, allow the specification of "reset" endothelium, that can perform a variety of functions including vasculogenesis, integration with parenchymal tissues, and transcriptional adaption upon coculture.
blood-brain barrier (BBB). hiPSC derived pericytes, which support capillary structures in vivo, can also mediate tissue function through paracrine cues and have been shown to improve the barrier function of brain microvascular endothelial cells in coculture settings. [70] Directed differentiation approaches, with the addition of hypoxia, cyclic adenosine monophosphate (cAMP), and TGF-β stimulation were used to specify hiPSCs toward venous angioblasts, priming the population to undergo further maturation upon implantation in the neonatal liver. When engrafted, the progenitor populations were able to take on liver sinusoidal EC attributes, demonstrating morphological fenestrations, scavenger function, and a mature LSEC-like transcriptional profile. [67] With these approaches in hand, stem cell derived vasculature can be used for disease modeling and personalized drug discovery. Recent examples include deriving hiPSCs from pulmonary arterial hypertension (PAH) patients and differentiating them towards ECs to identify compounds that promote vascular survival. By combining high-throughput pharmaceutical assessment and transcriptomics, thousands of candidate molecules can be screened. With this approach AG1296, a tyrosine kinase inhibitor was shown to reduce features of PAH in an in vivo model. [71] Additionally, self-assembled hiPSC-derived vascular organoids, consisting of endothelial cells and pericytes, have been used to study vascular regeneration and disease mechanisms. Using vascular organoids, inflammatory factors including TNF-a, IL-6, and hyperglycemia were administered to vascular organoids in vitro. These drove injury through increasing BM deposition, mimicking features of inflamed vasculature found in type 1 diabetes patients. With this platform, Notch inhibition was found to alleviate matrix deposition associated with vascular inflammation. [69] While these techniques are useful for deriving vascular sources for studying development and de novo vascular assembly, strategies to integrate the resulting vasculature with other tissue sources are still needed. Approaches such as coculturing mesodermal progenitor cells (MPCs) with other tissue-specific sources such as neural progenitors can be a route for vascularization but controlling the subsequent architecture can be challenging. [72]

Endothelial Progenitor Cells
Mesodermal derived EPCs were a highly debated population of endothelial precursors that are now broadly defined as either CFU-ECs or ECFCs, based upon their vasculogenic potential. ECFCs, which can be isolated from the cord or peripheral blood, tissue-resident vascular populations of the umbilical cord, pulmonary artery, lung, and placenta, are of great interest in tissue engineering applications due to their ability to form perfusable blood vessels in vivo. [73][74][75][76][77] Nevertheless, the rarity of ECFCs makes it difficult to be isolated from the human body. It has been shown that adult ECFCs have reduced occurrence compared to their infant counterparts, which is corroborated by the fact that for an equal volume of blood, ECFC colony formation was increased 15-fold in cord blood in contrast to adult samples. [74] Only 0.017 ECFC colonies can be generated from one million blood mononuclear cells (MNCs) harvested from adult peripheral blood, much less than the 4.35 CFU-ECs colonies per million MNCs. [28] CFU-ECs on the other hand, lack vasculogenic properties but can take on features of various blood populations, including acquiring phagocytic macrophage activity and myeloid differentiation potential. [28] Transcriptome analysis of ECFCs has revealed that the expression pattern of HOX resembles those of microvascular endothelial cells, segregating them from a diverse variety of endothelial cell lines. [78] Owing to the close relationship between ECFCs and ECs, disease models have been developed to study endothelial dysfunction utilizing ECFCs. For instance, synthesis and storage of vWF can be profiled for ECs in individual patients with von Willebrand Disease by isolating their ECFCs. [79,80] Characterizing ECFCs from patients with Philadelphia-negative myeloproliferative neoplasms with thrombotic risk can be used as a biomarker as they harbor the Janus kinase 2 V617F mutation associated with this disease. [81] Similarly, gene expression analysis of ECFC from children with sickle cell anemia was shown to have an increased response to inflammatory stimuli compared to healthy individuals. [82] Finally, ECFCs isolated from patients with Hereditary Hemorrhagic Telangiectasia demonstrated depressed endoglin expression, hindered TGFβ signaling, and disorganized cytoskeleton and tube formation. [83] Collectively, ECFCs can serve as putative markers for an array of vascular diseases. In addition to their diagnostic potential, ECFCs are a potential cellular therapeutic source and can be applied for vascular repair. [84] ECFCs also exert paracrine effects on adjacent cells, promoting angiogenesis and tissue recovery. [85][86][87] By coupling EPCs with tissue engineering approaches, we can elucidate how biomaterial properties such as matrix stiffness drive ECFC network formation and vasculogenic behavior. [36,[88][89][90][91] Through integration with custom-made constructs, ECFCs can be tailored to regenerate particular tissues, as in the case of utilizing biphasic calcium phosphate/ BMP2 bone tissue engineering constructs to enhance in vivo osteogenesis after scaffold cellularization. [92] Although ECFCs show great clinical promise with a unique in vitro expansion potential, their rarity is a challenge for their widespread utilization clinically. With advances in techniques to derive ECFCs from iPSCs, and isolation from abundant sources such as white adipose tissue, their utilization as an autologous cell source for cell therapy is promising. [93,94]

Multipotent Mesenchymal Stromal Cells
Multipotent mesenchymal stromal cells (MSCs) are found in bone marrow and fat and have been shown to directly contribute to various lineages including the vasculature. [95] While the addition of growth factors such as VEGF alone is insufficient to induce an endothelial phenotype, treatment with shear stress enables bone marrow derived MSCs to form capillary-like structures on Matrigel, with increased expression of the platelet endothelial cell adhesion molecule (PECAM-1/ CD31). [96] Blood vessels recruit support cells as they mature, eventually becoming co-populated with α-SMA-expressing cells such as pericytes and smooth muscle cells. Adipose microvascular endothelial cells excel at rapidly generating intricate vascular structures with increased network complexity and vessel alignment when cocultured with MSCs. Compared to EC www.advanced-bio.com coculture with human neonatal dermal fibroblasts (HNDFs), coculture with MSCs improved vascular maturation after 14 d of coculture. This was indicated by a higher ratio of oriented vessels and higher expression of α-smooth muscle actin (α-SMA). [97] Mesenchymal angioblasts, bipotent stem cells that have MSC and endothelial differentiation potential, can be directed from hESCs through coculture with OP9 stromal cells. The addition of both FGF2 and BMP4 can direct the stem cell derivates toward a vascular fate and direct the outgrowth of endothelial cells as vascular tubes when grown on Matrigel. [98] MSCs also influence EC specification through paracrine cues and when cultured with EPCs can augment their expression of CD31 and vWF. Additionally, stimulating endothelial cells with MSC-derived miRNA exosomes can promote angiogenesis in both in vitro and in vivo settings. [98] MSCs provide various means to develop and mature endothelial cells, particularly through coculture and paracrine support. These cells are important to consider for the development of mature vascular models as they serve as a biomimetic enhancement of endothelial vessel formation/maturation. While coculture may be effective, there are certain limitations involved related to cellular competition for resources and culture space. Conditions for coculture might not be feasible for models, and methods involving gene editing which do not require the use of support cells can serve as a viable alternative. [99]

Cellular Reprogramming through Gene Editing Tools
Reprogramming somatic cells via gene editing is an additional tool that can be used to generate vascular cell populations. Chemically modified mRNA (modRNA) technology has been used recently to enhance the durability of synthetic RNA, enabling its delivery to cells for protein expression. To this end, a synthetic modRNA was developed to activate the expression of transcription factor E26 transformation specific (ETS) variant 2 (ETV2) at high levels in MPCs independent of the existence of VEGF. [100] ETV2 is an important transcription factor that plays a role in vascular cell fate decisions and lumen formation during gestation. Expression of ETV2 is turned off at later stages of development, and it is not presented in mature ECs. This approach was implemented for reproducible differentiation of various hiPSC lines into human-induced ECs (h-iECs), amenable to anastomosis and perfusion when implanted in vivo. It has been reported that reactivation of ETV2 in mature human ECs by lentiviral transduction also restores their capacity to form robust vascular networks in vitro. These so-called resetting vascular ECs (R-VEC) not only can form capillary plexi but are physiologically responsive to perfusion and align under shear stress, permitting the transport of heparinized human blood. In addition, these cells can adopt tissue-specific transcriptional profiles, integrating with healthy and diseased organoids. [101] Functional ECs can also be forged by targeted ETV2 reprogramming in fibroblasts. The transduced ETV2 works concomitantly with endogenous FoxC2, inducing endothelial markers indicating EC development (FLI1, ERG, TAL1) and function (EGFL7, vWF). The converted cells can assemble as vascular cords on Matrigel and integrate with host vasculature upon transplantation. [102] hPSC models of endothelial cells of the BBB have been developed, but transcriptional analysis suggests that their identities mirror epithelial cells rather than specialized endothelium. Overexpression of ETS transcription factors in these stem cell populations reprograms them to take on transcriptional and functional features of brain endothelium. [103] Overall, reprogramming approaches provide the ability to create tissue-specific ECs or cells that can take on transcriptional properties of the surrounding tissue. [101] Genetic reprogramming of progenitor cells is yet another avenue for controlling endothelial cell development without reliance on growth factors or support cells.
It is established that primary endothelial cell progenitors are inherently difficult to source and expand due to their limited abundance, unspecified specification, and propensity for phenotypic instability. Mature populations also incur challenges upon isolation, with limitations in expansion and phenotype. [57] These challenges are addressed using several EC-sourcing approaches, ranging from growth factor-mediated directed differentiation to multipotent stromal cell support and transcriptional reprogramming. While EC sourcing is critical for vascular tissue engineering, it is not the complete picture. Biomaterials provide another level of control over the vascular niche, which impacts vascular tissue development. The following section outlines the importance of the ECM in vascularization and describes several biomaterials which are leveraged for niche-ECM recapitulation.

Biomaterial Approaches to Coax Vascularization
The vascular system is governed by the extracellular milieu, a multicellular community comprised of instructive growth factors, cytokines, and ECM that stores chemical and physical cues that mediate tissue proliferation, homeostasis, and differentiation. Approaches to recapitulate features of the native ECM harness biomaterials that are natural, including various polysaccharides (e.g., alginate, hyaluronic acid, fibrin, chitosan, chitin, cellulose, heparin, dextran, agarose) and proteins (e.g., collagen, gelatin), or synthetic (e.g., poly(ethylene glycol), poly(lactide-co-glycolide) acid, poly(e-caprolactone), polyisocyanopeptide). These have been leveraged to study vascular morphogenesis in 3D structures and lineage specification. Although natural polymers have the advantages of bioactivity and biocompatibility and are suitable options for evaluating parameters that inform vascular morphogenesis and function in vitro, the inability to independently tune ligand density, mesh size and stiffness makes it difficult to disentangle their contributions. In contrast, synthetic polymers can be functionalized by a large array of bio-orthogonal chemistries, permitting controlled degradation kinetics and presentation of binding motifs and growth factors.
Hydrogels serve as an ECM substitute and are a class of 3D networks of polymer chains that, due to their hydrophilic nature, have high water absorption and swelling capacity. Shear-thinning hydrogels, which show viscous flow behavior under force have immense potential in various biomedical applications including injectable biomaterials, drug delivery, 3D bioprinting, and tissue regeneration. Multiple cells can be encapsulated within these hydrogels and released in a www.advanced-bio.com controlled manner for cell therapy or studying the interaction between cells and their microenvironment. Matrigel, derived from Engelbreth-Holm-Swarm tumor cells, is a complex natural extracellular matrix extract that is used for a variety of applications. This includes serving as a surface coating to support stem cell expansion, in addition to supporting organoid assembly in 3D culture. When used as a substrate, Matrigel can also induce the rapid self-assembly of ECs into capillary-like structures and has been routinely used to investigate stimulators and inhibitors of angiogenesis. [104] However, Matrigel is inherently variable, contains xenogenic content due to its natural origins, and has limited therapeutic potential. Poly(ethylene glycol) (PEG) hydrogels have been used in a variety of biomedical applications and can serve as a cellular scaffold, with customizable adhesive, mechanical, and degradation properties. Eight-arm PEG-norbornene hydrogels engineered to contain linear or cyclic RGD peptides were used to test the role of mechanical stiffness and VEGF sequestration on the angiogenic potential of HUVECS and iPSC derived ECs. [105] With this platform, a thin hydrogel array with combinatorial variation in the parameters was used to disentangle the relative contribution of binding motif presentation, VEGF concentration, and stiffness. From this screen, a single condition was found to support prolonged HUVEC network assembly, but multiple conditions were able to support iPSC-EC networks for more than 48 h. PEG hydrogels have also been used to induce in vivo angiogenesis, a key strategy in promoting the survival of tissueengineered grafts. Four-arm PEG hydrogels functionalized with RGD and tethered VEGF improve the survival of extrahepatic-transplanted islet grafts. These gels increase vascular recruitment and islet cell engraftment and function compared to typical intraportal transplantation techniques. [106] The use of a PEG hydrogel here provides a clinically relevant means to induce tissue vascularization in a functional tissue graft. Another in vivo approach leveraged hyaluronic acid (HA) hydrogels containing fibronectin fragments for integrin engagement. Hydrogels with different fibronectin segments were implanted subcutaneously and induced vessel sprouting and infiltration within the hydrogel. Gels containing α3/α5β1 integrins promoted organized vessel structures, while the αvβ3 integrin-containing gels gave rise to unevenly distributed vessel clumps. [107] While this approach does not apply hydrogel technology to directly implant a parenchymal tissue graft, it does take a deeper look at the mechanisms which drive vessel recruitment and assembly. Integrin engagement underpins in vivo vascularization by mediating vessel morphology and distribution, two important factors for recreating native vasculature.
Hyaluronic acid is a biodegradable hydrophilic glycosaminoglycan, composed of alternating units of glucuronic acid and N-acetyl-D-glucosamine that form hydrogels. HA has been studied for many biomedical applications including dermal fillers, wound dressings, and tissue regeneration. Chen et al. developed an injectable HA hydrogel by guest-host interactions of b-cyclodextrin-modified HA (CD-HA) and adamantane-modified HA (Ad-HA) to have shear-thinning properties. EPC-derived-extracellular vesicles were encapsulated within these hydrogels and delivered into a rat model of ischemic myocardium for the study of myocardial hemodynamics. HA hydrogels were found to enhance myocardial maintenance, and the delivery of the extracellular vesicles increased angiogenic activity. Similarly, HA hydrogels have been shown to guide the proliferation and differentiation of human cardiospherederived cells into ECs and drive their formation into vascularlike networks via TGFB-1 and CD105 signaling. Specifically, VEGF-containing-hydrogels injected into the ischemic muscles of rats promoted local blood flow in the muscle and demonstrated the ability to treat the ischemic legs in patients with peripheral arterial disease. Histological assays and treadmill tests evaluated the efficacy of VEGF-containing HA hydrogels in treating rats with ischemic limbs. [108] Results showed that running distance and blood flow were significantly improved compared to non-treated controls. HA hydrogels also promote the self-assembly of stem cell-derived vascular networks and support vascular engraftment. [109] Likewise, HA hydrogels with reversible dynamic bonds have been developed with adamantane and β-cyclodextrin guest-host interactions. These shearthinning hydrogels are effective for increasing cell retention and promoting vasculogenesis in the myocardium after infarction. Using these shear-thinning HA hydrogels in the heart permitted localized and targeted therapies leading to improved cardiac function, enhanced vasculogenesis, and limited adverse remodeling. [110] HA-collagen hydrogels with an interpenetrating network, created by crosslinking HA with dynamic bonds using hydrazone and collagen, mimic the viscoelastic and fibrillar properties of tissue ECM. The hydrogels exhibited stress relaxation properties which were found to increase ECFC spreading, realignment of collagen fibers, as well as the formation of focal adhesions that indicate mechano-regulation. Stress relaxation leads directly to physical remodeling in hydrogels that mimic native ECM microenvironments. [111] Granular HA hydrogels composed of microgels were developed by modifying HA with norbornene groups (Nor-HA) and then crosslinking with dithiol cross-linkers. Flow-focused microfluidic technology was used to fabricate photocrosslinked Nor-HA microgels. Granular hydrogels are porous due to the interstitial spaces between the microgels, as opposed to bulk hydrogels. The porosities enabled cells and blood vessels to invade the hydrogel, enhancing its ability to integrate with host tissue. Endothelial cell invasion was studied in the synthesized HA hydrogels embedded with spheroids. Spheroids embedded in Nor-HA hydrogels that had been crosslinked with an MMPdegradable crosslinker revealed lumens with high sprout branching levels. [112] Complex microchannels have been created within HA hydrogels using embedding bioprinting. The hydrogels were formed by modifying HA using guest-host interactions with β-cyclodextrin (β-CD-HA) and adamantane (Ad) (Ad-HA). Ad-HA modified with norbornene (Nor) to produce AdNor-HA, allowed RGD peptides to be introduced for adhesion, as well as covalent crosslinking with thiol-ene click chemistry. An endothelial cell layer formed confluent monolayers on the complex microchannels, mimicking the various blood vessel shapes. Angiogenic factors cause these cells to degrade the hydrogels with proteases to form sprouts in the hydrogel. These systems are useful for the development of vascular grafts with specific geometries that can trigger or suppress angiogenesis in the treatment of diseases such as cancer and ischemic disease. [113] www.advanced-bio.com Dextran (Dex) and gelatin (Gtn) have been applied in hydrogel systems, taking advantage of multiple properties for specific vascular applications. Gelatin crosslinked with modified dextran (Gtn-I-odex) hydrogels were fabricated such that their dynamic imine chemistry would allow for re-gelation after the cessation of shear stress. These gels termed self-healing, provide an ideal platform for the delivery of EPCs via injection to promote the regeneration of host vasculature. [114] Gelatin, selected for its optimal cell adhesive properties, blended with imine-containing dextran, forms dynamic networked hydrogels. These, in comparison to methacrylate-functionalized static hydrogels, promote vessel assembly and BM deposition through focal adhesion kinase signaling. In addition, these dynamic gels increase endothelial colony-forming cell clustering and microvessel formation when implanted in vivo. [115] Similarly, pliable dextran hydrogels implanted in the skin were found to improve endothelial cell recruitment to a wound site following full-thickness burns. The resultant neovascularization after seven days improved blood flow to the affected area and subsequently improved dermal regeneration compared to non-hydrogel treated wounds. [116] The functionality and chemical flexibility of Gtn-Dex systems make them powerful candidates for improving vessel recruitment and assembly. Gelatin/ dextran hydrogels with dynamic networks were also developed. These hydrogels with dynamic networks were formed using acyl hydrazone and imine as dynamic covalent bonds, while nondynamic network hydrogels were created using methacrylates. On multialdehyde-modified dextran (Dex-CHO), aldehydes were crosslinked by acyl hydrazone and imine bonds, whereas on modified gelatin with adipic acid dihydrazide (Gtn-ADH), they were crosslinked by modified acyl hydrazide and amino groups. The results indicated that dynamic hydrogel networks result in increased interaction between matrix RGD sites and integrins on the cell surface, causing a faster rate of vascular morphogenesis than hydrogels without dynamic networks. The findings suggest that dynamic hydrogels facilitated the formation of thicker and longer vessels as well as open lumen than nondynamic hydrogels. [115] These approaches highlight the wide-ranging potential for the use of hydrogel platforms for in vivo vascular regeneration. Across the aforementioned biomaterials, there is a variety of tunable parameters of which these techniques take advantage. These range from chemical properties such as degradability and polymer backbone, to biological properties including signaling molecules and adhesion peptides, and physical properties such as stiffness and elasticity (Figure 3). While biomaterials often succeed at mimicking aspects of the native vascular niche, there are still levels of spatial control and architectural mimicry that are not achieved using hydrogel-only platforms. Engineered solutions such as bioprinting, microfluidics, and spheroid technology leverage some aspects of these biomaterial approaches in combination with inventive techniques meant to recreate a more complete vascular system (cytoarchitecture, spatial organization, perfusion, 3D orientation). The following sections highlight recent advances in engineered solutions for vascularization.

3D Printing
Conventional tissue fabrication methods do not mimic the complete complexity, geometry, and mechanical properties of human tissues. Biomimicry in engineered systems requires multi-pronged approaches for creating structural features of the matrix in tandem with orienting cells appropriately. The goal Figure 3. Biomaterial approaches to control vascular assembly. A wide range of biomaterial hydrogels with various natural and synthetic polymer compositions such as PEG, fibrin, collagen, and dextran have been developed. The properties of these hydrogels can be classified into chemical, mechanical, and biological properties. A) Chemical properties include using various types of polymers with different molecular weights and polymer concentrations for the synthesis of hydrogels with different chemical structures with optimized cross-linking density, degradability, and porosity. B) Mechanical properties such as stiffness, viscoelasticity, and stress relaxation can be tuned. C) The presence of signaling pathways such as WNT, NOTCH, and the addition of adhesion peptides, as well as growth factors to biomaterials, help to modulate cell-material interactions to promote the biological properties of hydrogels with the ability to control vascular assembly.
www.advanced-bio.com of some common approaches is to create channel networks embedded in the engineered tissue using direct printing of vascular structures or indirect printing of channel molds using sacrificial materials. [117] As a manufacturing tool, 3D printing can be used to generate tissues with controlled cytoarchitectures that are scalable for potential clinical use. Particularly, 3D bioprinting techniques can use bioinks composed of sacrificial materials, growth factors, cells, and ECM analogs to create embedded vascular architectures. The development of fugitive inks or sacrificial materials can be printed into filament networks within bulk hydrogel scaffolds and removed to form microfluidic channels that can be subsequently seeded with vascular cells. For instance, Miller et al. demonstrated the ability to fabricate vascular structures in a range of natural and synthetic scaffolds using a water-soluble sugar ink, showing that vascularized conduits sustain liver and kidney function. [118] In a similar approach, Debbi et al. 3D printed sacrificial polydimethylsiloxane (PDMS) molds and salt-leaching methods to fabricate porous and aligned microchannel scaffolds composed of poly(l-lactic acid)/poly(lactide co glycolide) copolymers for axonal repair, creating a hierarchical vascular tissue containing dental pulp stem cells and human adipose microvascular ECs. [117] Multi-material 3D bioprinting has been used to fabricate thick vascularized tissues with complex architectures (≥1 cm) which can be perfused on a chip for more than 8 weeks. Specifically, cell-laden inks based on a mixture of gelatin and fibrinogen, cross-linked by transglutaminase and thrombin, were shown to support different cell types MSCs, HUVECs, and HNDFs. [119] Bio-orthogonal chemistry, such as the addition of adhesion ligands (e.g., RGD) in 3D-printed structures can promote the incorporation of host blood vessels using synthetic matrices. For example, 3D printed microchannels within a hydrogel were developed by printing shear-thinning and self-healing hydrogel inks within a support hydrogel. Here, hydrogel sacrificial inks made from HA were modified with guest-host pairs of adamantane (Ad) (Ad-HA) and β-cyclodextrin (β-CD). These materials were extruded in support hydrogels made of RGD functionalized Ad-HA modified with norbornene (Nor) (AdNor-HA). [120] In a different study, heterogeneous hepatic lobules composed of ECs, hepatic cells, and a lumen were bioprinted to demonstrate the importance of architectural positioning in supporting hepatic and vascular function, evidenced by enriched secretion of urea and albumin, and protein levels of CD31 compared to monoculture models. [121] Additionally, multimaterial bioprinting, containing cell-laden gelatin methacryloyl and fibrin bioinks, can be used to fabricate multiscale liver-like tissues with perfusable vascular structures. By vascularizing the liver tissue in this manner, the hepatic function could not only be sustained in vitro but could support hepatic tissue in vivo, when surgically anastomosed to the host vasculature for up to 4 d. [122] To investigate the role of vascular architecture in wound healing, biodegradable 3D-printed vascular fibrin patches were fabricated and implanted in hind limb ischemic models. The degree of blood flow restoration was evaluated as a function of pre-vascularized geometry. Briefly, sacrificial sugar filaments were embedded in fibrin gels, evacuated, and seeded with ECs. Vascular patches with three different channel patterns, namely grid (Grid VP), parallel (Par VP), and smaller diameter parallel structures (Sm_D VP) were assessed. Via Doppler imaging, which measures blood prefusion, implants with Grid VP patterns were less effective in tissue revascularization compared to Par VPs after 5 d. Notably, Par VP implants exhibited the highest amount of blood flow and were able to repair ischemic injury compared to other geometric configurations. [123] In a similar study, implantable gelatin hydrogels consisting of perfusable microchannel networks were created to enhance angiogenesis in ischemic injury models. Using temperature-responsive (N-isopropylacrylamide) (PNIPAM) as a sacrificial mold, the channel networks were able to induce host vascular ingrowth and perfusion upon implantation. [124]

Microfluidic Models of Vascular Assembly
The advent of microfluidic technology has enabled the investigation of chemical and physical factors that influence vascular assembly. These systems are suitable for mechanistically evaluating cellular processes that mediate endothelial barrier function and can be designed to study multitissue interactions. [47,[125][126][127][128][129][130][131] Recent iterations of these tools have been used to study a range of vascular functions including wound healing and multicellular interactions between stem cell-derived tissues. [132,133] Combining the tunability of synthetic hydrogel scaffolds with microfluidic gradient systems can be used to investigate how matrix properties such as adhesiveness and degradation control angiogenic sprouting potential. Methacrylated dextran functionalized with varying concentrations of adhesive peptide CGRGDS was evaluated on its role in tailoring angiogenic sprouting events. RGD immobilized at a concentration of 0.15 × 10 -9 m compared to 6 × 10 -3 m induced increased sprouting multicellularity, high degradable structures, and promoted lumen formation. [134] Microfluidic devices can also be fabricated for control over matrix stiffness and topography. This was achieved by forging circular microchannels with a set of diameters and a range of material rigidity based on the ratio of the reagents. This model was tested with HUVECs for their responses under shear flow, yet its potential lies beyond the study of vasculature formation. [135] For instance, keratinocytes and dermal fibroblasts were introduced to either side of two microchannels, and angiogenic sprouts were observed at the interface. The release of cytokines and growth factors from injured keratinocytes could also drive angiogenic sprouting events. This study reveals a novel application to investigate the effects of potential cosmetic irritants on the skin and serves as a potential alternative to traditional animal models. [136] Building upon an existing PDMS-based microfluidic platform, two perfusable vascular structures were created between self-assembled microvessels, containing HUVECs and dermal fibroblasts, by incorporating 300 µm diameter needles as sacrificial molds. After casting fibrin gels around the molds, the needles were removed and subsequently seeded with ECs to create hierarchical vessel structures amenable to perfusion. After establishing that a ratio of 3 million HUVECs per mL to 6 million fibroblasts per mL drives optimal vasculogenesis with sustained barrier function, Song et.al, leveraged an inducible Casp9 transgene to study the role of temporal stromal support in vascular assembly and homeostasis. Removal of the fibroblasts, through chemical induced dimerization at different time points, showed that initial stromal/EC interaction is pivotal in driving vascular assembly, but dispensable post vasculogenesis. Furthermore, tricultures within the microfluidic device, consisting of iCasp-9 fibroblasts, endothelial cells, and primary hepatocytes were similarly appraised upon fibroblast removal. Vascularized hepatocyte-laden devices secreted urea and albumin, even in the absence of fibroblasts, supporting the observation that transient stromal interactions are sufficient to sustain parenchymal tissue function. [137,138] In addition to supporting hepatic function, engineered fluidic devices have been used to study the role of vascular architecture in sustaining parenchymal tissue function.
In a similar PDMS-based device, Musah et al. showed that the crosstalk between ECs and stem cell-derived podocytes can drive kidney maturation events in vitro. These devices are comprised of two layers, separated by a semi-permeable membrane that allows biomimicry of tissue interfaces within the body. In addition to containing this degree of separation, these two-layer microfluidic devices can be mechanically actuated to introduce cyclic strain and fluid shear stress. In this format, hiPSC derived podocytes were cultured in proximity to primary glomerular endothelial cells. HiPSC-derived podocytes cultured under 10% mechanical strain upregulated markers of podocyte maturation in the presence of fluid shear stress compared to controls that only were exposed to fluid flow. Furthermore, without the presence of endothelial cells, hiPSC-derived podocytes were unable to phenotypically mature and perform differential clearance of insulin and albumin. [139] The crosstalk between vascular endothelium and kidney maturation was also explored in an engineering device amenable to fluid flow. In a millifluidic chip, Homan et al. showed that the addition of superfusion flow, in developing stem cell-derived kidney organoids, promotes the codifferentiation of EPCs which contributes to tubular epithelial maturation. [140] Furthermore, LECs can be seeded into PDMS-based and collagen-coated microfluidic devices, forming perfusable lymphatic vessels, which reveal the lymphatic junction morphogenesis. Bolstered by this model, integrin α5 was identified as a regulator of lymphatic barrier function, paving the way for further study in lymphatic vessels in fluid homeostasis, host immunity, and cancer. [141] Collectively, these studies highlight how fluidic environments can be used to study vascular morphogenesis. Additionally, these engineered systems shed new light on the role of epithelial-endothelial tissue interfaces in parenchymal maturation and reflect the importance of dynamic microenvironments in vascular tissue engineering and organoid maturation.

Micromolding Techniques
Spheroid assembly reconstitutes the three-dimensionality of dense tissues by encouraging homotypic cell-cell interactions, and enriching tissue-specific gene expression, cytokine production, and signaling pathways compared to 2D planar culture. [142] Spheroids have been used in a range of tissue engineering applications, and can be fabricated via various conventional (e.g., spinning flask, hanging drop, rotating vessel, micropatterned mold, and nonadherent surface) and advanced (e.g., microfluidics, water-in-water emulsions, and hydrogels) methods. Their flexible fabrication methods allow them to be fabricated independently or embedded in a variety of hydrogels, either by 3D printing, electrospinning, or shrinkage techniques, to create intricate 3D structures. However, cells at the interior of spheroids, with diameters ranging from 400 to 600 µm, are diffusion-limited and necrosis results from a lack of oxygen, nutrients, or waste transport. [143] To address these restrictions, oxygen can be artificially supplied through fiber injection, but vascularization techniques can sustain large-scale tissue aggregates.
The aggregation of immortalized, cadaveric, or stem cellderived hepatocytes can improve in vitro viability and functionality and encourage vascularization upon implantation compared to single-cell encapsulates. [144][145][146][147] Similarly, directing vascular assembly before implantation can be obtained. In this regard, a vascularized hepatic structure was developed by culturing hepatocyte and ECs aggregates within 3D-printed channels and the external chamber, respectively. The 3D engineered structure was sutured in mice, and host engraftment and albumin production were improved. [148] As an example of the beneficial properties of microwell technology from an aggregate technology standpoint, we have highlighted a noteworthy study by Stevens et al. [149] Here, microwell technology was used for aggregation, containing a mixture of normal human dermal fibroblasts (HDFs) and human hepatocytes. Similarly, microtissue molding was used for building patterned endothelial cord constructs from ECs. Hepatic cellular aggregates were coencapsulated with the EC cords in fibrin hydrogels and implanted ectopically into mice. By combining hepatocyte aggregates with normal HDFs, a sixfold increase in human albumin production was observed compared to aggregates composed of only hepatocytes. Along the same vein, culturing hepatocytes in aggregates and patterning of stromal cells in the vicinity of hepatocytes in cellular aggregates were evaluated with separate endothelial cords close but without contact with hepatic aggregates in fibrin hydrogels. These aggregates have been shown to lead to hepatocyte transplantation with improved function in comparison with different cell arrangements. Overall, the combination of ECs, human hepatocytes, and stromal cells produced liver tissue grafts that could remain viable and functional upon transplantation. [149] Micropatterned electrospun fibrous constructs containing ECs, hepatocytes, and fibroblasts were seeded in spatial coculture configurations, mimicking the cytoarchitecture of hepatic lobules. Hepatocytes were organized as dense polyhedral spheroids with an average diameter of 80 to 100 µm, polarized to contain bile canaliculi structures. When cocultured with either ECs or fibroblasts, the hepatocytes increased urea synthesis, albumin secretion, and cytochrome P-450 expression compared to hepatocytes cultured alone. The ECs exhibited enhanced proliferation and capillary tube formation in the coculture configuration, suggesting that controlled cellular interactions can direct parenchymal tissue function. [150] These micropatterning strategies can also be applied to hydrogel systems for delivering microtissues with defined architectures for a variety of tissue engineering applications. For instance, microcontact honeycomb patterns made from printing temperature-responsive www.advanced-bio.com polyethylene oxide hydrogels were able to organize HUVECs and HDFs as vascular networks. By leveraging the temperature sensitivity of this synthetic scaffold, the geometry of the vascular structures could be sustained upon retrieval and was shown to maintain its honeycomb structure after implantation. [151] Overall, rapidly evolving micropatterning methods are important approaches for the development of 3D microvascular networks which can support parenchymal function through juxtracrine and paracrine cues, mediated tissue self-assembly, and improved vessel formation in vivo.

Conclusions and Future Directions
Here, we highlight the importance of cell sourcing in building vascularized tissue constructs for applications in regenerative medicine and describe innovative approaches to control vascular organization. Engineering methods are constantly evolving, but simultaneous control of developmental cues such as mechanics, signaling pathways, cellular organization, hypoxia, metabolism, and ECM for vascular development to direct organ-specific vasculature in the future is key. Prevascularized structures with proangiogenic molecules can facilitate scaffold vascularization and stimulate angiogenesis. However, these structures suffer from anastomotic limitations with the host vascular network, which must be addressed to function in the implanted structures. Biomaterials, both synthetic and natural, provide a customizable means to mimic the properties of the native niche in a chemical, biological, and physical manner. Many of these biomaterial hydrogels have tunable properties and can work in tandem to generate complex, dynamic matrices such as dextran and gelatin. Significant work has demonstrated the capabilities of such gels to promote angiogenesis in vivo, but biomaterials themselves do not fully satisfy the architectural and cellular complexity of the native vascular environment. Engineering technologies that illicit better control over the complex factors which govern vascularization in the in vivo environment are emerging in the field of tissue engineering. 3D printing, for example, can be employed as a promising scalable approach capable of fabricating hydrogels with functional and hierarchical vascular architectures. In summary, ECs can be used to explore future applications that support tissue formation and reveal important clues into developmental pathways by being used in organ morphogenesis models. Moreover, the future of stem cell research should involve linking tissuespecific EC to in vivo developmental models to determine how various organs and tissues mature. The application of vascular engineering extends beyond the generation of large vessels to generate microvascular networks, for example, the potential of vasculature to perform research on organs and tissues ex vivo. In this regard, deep research and breakthroughs in vascular and stem cell engineering, biology as well as biomaterials are required in the future to motivate and provide hope for new treatments and strategies. Moreover, biomaterials that mimic diseased tissue's dynamic signaling are being developed. There is a great potential for applications of hybrid polymer-based vascular tissue engineering alternatives to study vascular diseases and therapeutic drugs. Particularly, studies focused on physicochemical properties of hydrogels, will impact healthcare. Future research should entail the incorporation of vasculature or provide conditions for mimicking vasculature to function properly for long-term efficacy. Insight gained from mechanistic studies will facilitate the development of innovative biomaterials for modulating the immune system, stimulating tissue regeneration, as well as improving drug delivery methods. Thus, the advancements and future potential of biomaterials make them ideal for use as delivery tools as well as model platforms for immunomodulation therapies. Importantly, stem cell derived ECs can be used to explore future applications that support tissue formation and reveal important clues into developmental pathways by being used with organoid models. Vascular tissue engineering strategies can also be applied for therapeutic applications, and disease modeling leverging various methods including organoids, organ-on-a-chip technology, and drug screening systems. For regeneration applications, these approaches can improve vascular grafts for the surgical treatment of diabetic ulcers and can be utilized for skin/cardiac patches, and stents, for wound healing and cardiovascular repair. In conclusion, we have introduced a plethora of examples demonstrating progress in vascular tissue engineering and anticipate that these approaches can be applied for regeneration, therapeutics, and improve in vitro models of tissue development and homeostasis (Figure 4). . Integrative approaches to vascularizing organs for regenerative medicine applications. In vitro vascularization strategies, including the use of microfluidics, biomaterials, and 3D printing, permit basic science investigation of vascular function for elucidating mechanisms of homeostasis and disease. Tissue-engineered vasculature, forged through these in vitro approaches, can be leveraged for translational research. Some examples include using patient-specific vascular constructs to screen drugs, and integration with other parenchymal tissues. Engineered vasculature when applied for regenerative medicine, can accelerate wound healing, stent cellularization, and support tissue engraftment.  Patrick Barhouse served as the lab manager in Dr. Smith's group during its development. Patrick oversaw administrative duties and facilitated stem cell tissue engineering projects, specifically investigating a 3D differentiation platform for developing vascular organoids. Having graduated from the University of Minnesota with a degree in Biomedical Engineering, Patrick has studied a wide range of concepts critical for tissue engineering. Through his emphasis on cell and tissue engineering, Patrick studied biomaterials, bioprinting, microfluidics, transport phenomena, and cell biology. Patrick is currently working towards his medical degree at Brown University and plans to continue stem cell research throughout medical school.

Quinton Smith is a Howard Hughes Medical Institute Hanna Gray
Fellow and an assistant professor in the Department of Chemical and Biomolecular Engineering at the University of California, Irvine. His research is focused on the development of engineering technologies that control stem cell lineage specification and assembly for therapeutic application. He hopes that these approaches will lead to new fundamental discoveries related to tissue morphogenesis and mechanobiology.