Engineering of the microenvironment to accelerate vascular regeneration

Abstract

Blood vessels are crucial for tissue development, functionality, and homeostasis and are typically a determinant in the progression of healing and regeneration. The tissue microenvironment provides physicochemical cues that affect cellular function, and the study of the microenvironment can be accelerated by engineering approaches capable of mimicking different aspects of the microenvironment. In this review, we introduce the major components of the vascular niche and focus on the roles of oxygen and the extracellular matrix. We demonstrate how vascular engineering approaches enhance our understanding of the microenvironment impact on the vasculature towards vascular regeneration and describe the current limitations and future directions toward clinical utilization.

Vascular Microenvironment: Using Engineering Approaches to Enhance Vascular Regeneration

Vasculature serves as a conduit to provide oxygen and nutrients to tissues throughout the body and clear away waste generated by the tissues. Blood vessels comprising the vascular system come in many sizes and provide various functions to support the body. The function of a given blood vessel influences the composition of each layer in the blood vessel, including the endothelial cells (ECs) in the tunica intima, vascular smooth muscle cells (vSMCs), and elastin fibers in the tunica media, connective tissue in the tunica adventitia, and pericytes throughout the vessel[1]. Arteries transport oxygen-rich blood from the heart, dealing with high pressures and shear stresses. Therefore, arteries have a smaller lumen with vSMCs and elastin fibers to handle high pressures[1, 2]. Arterioles are smaller vessels branching off arteries to help deliver blood to tissues. Capillaries are tiny vessels with thin walls comprised of ECs, allowing for the rapid exchange of oxygen and nutrients. Following blood flow through capillary beds, blood enters venules and then transitions to the veins. Veins transport deoxygenated blood to the heart in large volumes and possess valves that facilitate the unidirectional, low-pressure blood flow. To accommodate low-pressure flow, vein walls are thinner than arteries, lacking the elastic lamina[1]. The variations in vessel characteristics and morphologies create a complex and diverse vascular microenvironment comprised of multiple vascular cell types and tissue cells, biochemical cues, and mechanical forces resulting from blood flow and the extracellular matrix (ECM). The composition and functions of the ECM in the microenvironment greatly influence biological processes and cellular behavior. Additionally, the levels of oxygen present in the microenvironment influence cellular behavior. Specifically, hypoxia, low oxygen levels below 5%, occurs in various biological processes, such as development and ischemia, regulating cellular responses. Disruption of the vascular microenvironment can lead to damaged vasculature, thus severely impeding tissue homeostasis and regeneration. Therefore, it is essential to deepen the understanding of the influence each component of the microenvironment has on vascular function and regeneration.

In recent years, advances in engineering and biomolecular approaches have allowed to develop research strategies to elucidate the microenvironment’s role in vascular development, homeostasis, and regeneration. For example, the miniaturization of the vasculature system with recent developments in microfluidics and organ-on-a-chip technology has augmented the understanding of how shear stress exposure and oxygen gradients affect the vascular microenvironment[3, 4]. Additionally, endothelialized microfluidic devices and advanced ECM-mimicking biomaterials have been synthesized to create artificial environments in vitro, which recapitulate the native environment and allow an improved understanding of functional mechanisms[5–7]. This review introduces the roles of ECM and hypoxia in the microenvironment. It then discusses how engineering approaches have enhanced our understanding of the influence of hypoxia and ECM by engineering in vitro microenvironments and how these can be harnessed to regulate vascular morphogenesis and regeneration.

The Extracellular Matrix and the Vascular Microenvironment

The ECM is a complex, three-dimensional (3D) assembly comprised of glycosaminoglycans (GAGs), proteoglycans, and fibrous proteins such as collagens, elastin, and fibronectin[8, 9]. The major types of GAGs in the ECM are hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratin sulfate, heparan sulfate, and heparin[10]. GAGs provide structure and assist cell hydration, growth, proliferation, and migration[10]. Proteoglycans are glycosylated proteins with GAG side chains[11]. Aggrecan, perlecan, decorin, and syndecans are examples of proteoglycans, and they predominately aid structural support and cell adhesion[11]. For fibrous proteins, collagen is a triple helix glycoprotein made of α-chains that also provide structure to tissues[12]. There are numerous types of collagens important in vascular homeostasis. Collagens I and III are significant components of the interstitial matrix in the tunica intima, tunica media, and tunica adventitia, providing tensile strength to support large vessel integrity and stability[13–15]. Collagens IV, XV, and XVIII are found in the basement membrane, facilitating filtration and resistance within blood vessels[13, 16]. Elastin, a hydrophobic protein produced by vSMCs in the tunica media and fibroblasts in the tunica adventitia, makes up elastic fiber networks responsible for large artery elasticity and is primarily found in the tunica media as well as the internal and external elastic membrane[17, 18]. Additionally, fibronectin is a dimer glycoprotein bonded by a disulfide bridge, and it mediates vascular cell adhesion, regulates vessel remodeling, and is predominately found in the tunica adventitia[19, 20].

Within the vascular microenvironment, there are two predominant types of ECM, basement membranes and interstitial matrix[21]. In the vasculature, the basement membrane is predominately comprised of nidogens, collagen IV, and laminins. The interstitial matrix contains various collagens, glycoproteins, and proteoglycans[21]. Properties of the vascular ECM principally depend on ECM composition and variations in matrix fiber density. Furthermore, the ECM generates forces that impact vascular cell morphology and function. vSMCs need to adjust the production of ECM proteins in response to changes in ECM tensional forces to maintain the necessary stiffness for blood flow[22]. Moreover, ECs and vSMCs experience stress and strain from the surrounding ECM. The physical forces experienced through the ECM drive mechanical signaling in the microenvironment, such as microvascular network formation responding to matrix compression[23]. In addition to mechanical signaling, signaling stemming from cell-cell and cell-matrix adhesion, often facilitated by integrins, influences vascular cell growth, behavior, morphology, and functionality[24, 25]. Furthermore, signaling that results in ECM degradation through cell-derived matrix metalloproteinases enhances angiogenesis[26, 27]. In summary, the ECM within the vascular microenvironment influences vascular cell growth, function, and regeneration, highlighting the need to elucidate the various mechanisms underlying those influences (Table 1).

Table 1.

Summary of ECM and Hypoxia studies for Vascular Regeneration

ECM/Hypoxia	Modeling Tools	Cell Type/Animal Model	Conclusions for Vascular Regeneration	Citation
ECM & Hypoxia	3D on-chip device with hypoxia-inducing buffer	Human umbilical vein endothelial cells (HUVECs) & human lung fibroblasts (hLFs)	Short-term hypoxia facilitated angiogenic characteristics, while long-term hypoxia led to disruption of formed lumens	3
Hypoxia	Microfluidic device with oxygen scavenger in adjacent channel	Endothelial colony forming cell-derived endothelial cells (ECFC-ECs) & Normal hLFs	Spatial and temporal control of oxygen in microfluidic devices can model vascular behavior	4
ECM	Stiffening hydrogel	Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs)	ECM stiffening resulted in activation of FAK and dissociation of β-catenin from VECad-mediated adherens junctions	7
ECM	Hydrogel undergoing mechanical loading	Microvascular fragments from rats	Immediate loading inhibited angiogenesis while delayed loading enhanced networks	23
ECM	Dynamic and nondynamic crosslinks in hydrogels	Human ECFCs (hECFCs)	Dynamic hydrogels lead to the activation of FAK and metalloproteinase expression in ECFCs	28
ECM	Electrospinning and enzymatic crosslinking of gelatin-hydroxyphenylpropionic acid hydrogel	HUVECs	Fibrous hydrogel supported cell adhesion, proliferation, and spreading along with demonstrating biodegradability	29
ECM	Multifunctional hydrogel	HUVECs & db/db congenital diabetic mice	Engineered hydrogel accelerated wound healing, down-regulated TNF-α, and up-regulated CD31 and α-SMA in vivo	32
ECM	Gel-MA hydrogel with photografting	HUVECs & Human adipose-derived stem cells (hASCs)	Photo-grafted patterns facilitated hASC alignment and migration along the pattern	33
ECM	Gel-MA hydrogel with microcavities inside	Human bone marrow stromal cells (hMSCs) & HUVECs	Co-cultures displayed improved vascularization, and hMSCs were suggested to differentiate towards pericytes	34
ECM	Electrospinning of a collagen and hyaluronic acid-based poly(l-lactide-co-ε-caprolactone) hydrogel	HUVECs & Adipose tissue-derived MSCs	Xenogeneic-free scaffold with small pore size and high water uptake supported angiogenesis and cellular proliferation	35
ECM	Collagen hydrogel with FGF-2, platelet-derived growth factor-BB, or VEGF added	Laminin+ cells from brain slices	FGF-2 facilitated the migration and proliferation of laminin+ cells in collagen hydrogel	37
ECM	Hydrogels with RGD gradients and elastic modulus gradients	HUVECs & Human umbilical arterial smooth muscle cells (HUASMCs)	Gradients present in hydrogels influence cellular behavior	40
ECM	PEG hydrogel supplemented with protease-sensitive and cell-adhesive peptides	Human umbilical cord blood-endothelial progenitor cells (hCB-EPCs)	PEG-based hydrogels can support microvessel formation when modified with peptides that facilitate degradation and adhesion	41
ECM	Fibronectin hydrogel with VEGF and BMP2 added	HUVECs & HDFs	The hydrogel can release and uptake GFs to aid in angiogenesis	42
ECM	3D printed hydrogel with interconnected channels and macro pores	HUVECs, fibroblasts, & balb/c female mice	Interconnected channels and macro pores have the potential to promote wound healing	46
ECM	3D printed, gradient-structured hydrogel with various pore sizes	BMSC & male New Zealand white rabbits	Cartilage repair and blood vessel ingrowth are mediated by HIF1α/FAK axis activation and influences by pore size	47
ECM	Micropatterned surface on hydrogel	ECs, SMCs, & New Zealand white rabbits	Micropatterns influence cellular distribution and can assist with vascularization	48
ECM	Micropatterned surface	vSMCs	Micropatterns influence vSMC behavior and morphology	50
ECM	Patterned fibronectin surface	hEPCs	Patterning influenced chains and tubular structure formations to enhance formation of vascular structures	52
ECM	Heparin-like polymers added to silicone surfaces	HUVECs & HUVSMCs	Surface polymers and patterning can enhance or restrict cell responses to the surface	57
ECM	3D printed cell-fibrin strips anchored to PCL frame	HUVECs & HNDFs	YAP activity played a regulatory role in vessel formation with no branches	58
Hypoxia	Hypoxia incubator	OBs & OECs	Short-term hypoxia promotes vascularization through balancing angiogenic and antiangiogenic factors	72
ECM & Hypoxia	Fibrinogen hydrogel with varied HA, uniform oxygen, and oxygen gradients in microfluidics	HUVECs	Provides 3D system to study various oxygen conditions and demonstrating EC networks align along gradients while adding HA to the system can help network formation	90
Hypoxia	Microfluidic device with oxygen gradient	HUVECs	Oxygen gradients guide EC migration	91
ECM & Hypoxia	Oxygen gradient in hydrogel	ECFCs	Clustering is controlled through cell-cell interaction regulators such as vascular cell adhesion molecule 1	95
ECM & Hypoxia	Hypoxic hydrogels	ECFCs	Cluster-based vasculogenesis is regulated by genes associated with oxidative stress and cAMP signaling in hypoxia	96
Hypoxia	Hypoxic chambers	hiPSC-ECs, human retinal endothelial cells, and OIR mouse model	SDF1a/CXCR4 axis in hiPSC-ECs supports revascularization of ischemic retina	97

Hydrogels to Mimic the Extracellular Matrix to Recapitulate the Vascular Microenvironment

Extracellular Matrix Composition

There is much to be discovered about the functions and influence of growth factors, proteins, and proteoglycans within the complex composition of the ECM in the vascular environment. While 2D monolayer cell cultures provide insight into the behaviors and function of vascular cells, they do not faithfully recapitulate the complexity found in vivo. Cell culture in 3D allows for more complex and accurate modeling of the vascular niche. Synthetic polymers and chemical modifications to natural polymers are common tools to engineer the ECM in vitro, as they have many tunable characteristics to mimic the natural ECM. When constructing ECM-mimicking hydrogel biomaterials to study the vascular system, collagen, fibrin, gelatin, hyaluronic acid (HA), and chitosan are commonly used for hydrogel fabrication through nucleation, electrospinning, enzymatic or light-based crosslinking methods (Figure 1) [28–31]. Modeling vascularization in vitro has been accomplished by modifying collagen, gelatin, and other hydrogels backbones by adding functional groups to the chemical structure, such as collagen or gelatin with methacrylic anhydride, gelatin with adipic acid dihydrazide, and gelatin with hydroxyphenyl propionic acid [7, 28, 29, 32–35]. Additionally, ECM-mimicking hydrogels have been modified to incorporate components of the microenvironment. For example, growth factors (GF) and adhesion/degradation peptides have been included in hydrogel fabrication to study the impact of each component in vitro and more closely model the native tissue. GFs are molecules within the body that elicit various cell responses such as proliferation[36, 37]. When modeling the vascular niche in vitro using hydrogels and engineering approaches, vascular endothelial growth factor (VEGF) has been incorporated into hydrogels to modulate and influence EC behavior when the cells are encapsulated in the hydrogel[38]. EC migration was facilitated by fibroblast growth factor 2 (FGF-2) when encapsulated within a collagen hydrogel that served as an ECM-mimic, demonstrating that FGF-2 functions as an angiogenic factor[37]. In wound healing models, methacryloyl grafted gelatin (Gel-MA) mixed with chitosan-catechol and further modified with polypyrrole-based nanoparticles and histatin-1 reduced inflammatory tumor necrosis factor α (TNF-α) as well as promoted transforming growth factor β1 (TGF-β1) and accelerated wound healing [32]. HA-based hydrogels with TGF-β1 incorporated during cell encapsulation promoted cell proliferation and vascular-like network formation modulated by CD105 and TGF-β receptor 2 co-activity[39]. Another aspect of the ECM impacting the vasculature is adhesion and degradation sites. When exposed to a gradient of the immobilized, fibronectin-derived cell adhesion peptide RGD in poly(ethylene) glycol monoacrylate macromer, vascular sprouting from spheroids comprised of human umbilical vein ECs and human umbilical arterial smooth muscle cells was observed with longer sprouts directed toward the higher concentration of RGD[40]. Degradation sites added to hydrogels to facilitate matrix remodeling by vascular cells, such as matrix metalloproteinase-sensitive peptides incorporated into hydrogel synthesis, have been shown to support microvessel formation along with evidence of lumen formation[41]. Furthermore, glycoprotein-based hydrogels such as fibronectin- and fibrin-containing biomaterials have been developed to model vascular morphogenesis due to their structural features binding to GFs, support of EC growth, promoting microvessel formation, and regulation of coagulation protein[42–45]. Engineering biomimetic hydrogels to recapitulate the complexity of the ECM composition of the ECM demonstrates the impact it has on facilitating vascular assembly and regeneration (see Clinician’s Corner).

Figure 1. Engineering Hydrogels to Model ECM for Vascular Regeneration.

Schematic illustration of the various engineering approaches utilized to create ECM-mimicking hydrogels for modeling and facilitating vascular regeneration. While a given technique is depicted as influencing one aspect of ECM modeling, such as structure, mechanics, or composition, a given approach often influences more than one aspect of ECM modeling. Created using Biorender.com.

Clinician’s Corner.

• Given the central role of blood vessels in organ function, an improved understanding of the microenvironment’s role in regulating vascular growth and morphogenesis is necessary to advance regenerative medicine.

• Mimicking the microenvironment for in vitro studies can aid in understanding the mechanisms governing vascular behavior. This, in turn, can be harnessed to develop targeted therapeutics because advances in biomaterials and biofabrication allow for the precise engineering of various vascular microenvironment features to recapitulate complex interactions.

• The extracellular matrix greatly influences cell behaviors in diseased and healthy states, and it can provide insight into the mechanisms responsible for the behaviors and what drives them.

• The role of hypoxia must be considered during therapeutic approaches, and it is necessary to balance the various oxygen levels necessary to facilitate appropriate treatments.

• Engineering-modified biomaterials and vascular tissue constructs can be incorporated into treatments to improve wound healing.

Extracellular Matrix Structure

The structures and topography of the ECM within the vascular microenvironment influence vascular cell behavior and orientation. When designing hydrogels to mimic the ECM, the ultrastructure, including pores, connecting pores, fibrillar structures, and fibrillar orientation, are essential when guiding cellular responses in the intact hydrogel. 3D bioprinting has been used to print gradients in pore sizes and interconnected microchannels, with the former being shown to facilitate vascularization in a cartilage regeneration model[46, 47]. In addition to pore size, patterning is important to recapitulate vascular structure or the ECM’s micro- and nano-scale features. Patterning can be accomplished through techniques such as physical patterning or chemical patterning. Physical patterning through micropatterning creates intricate and tunable surfaces upon which vascular cells can grow[48]. Patterning channels for EC growth in vascular tissue models have been accomplished through sacrificial templates, which create network-like patterns cast in a hydrogel. The patterned channels can be lined with ECs and perfused with culture media[49]. Micropatterning has been shown to influence vascular cell orientation and spatial organization[50–52]. Micropatterning resulted in increased elongation and cell alignment in vSMCs, which indicated the vSMCs are of the healthy, contractile phenotype like vSMCs found in vivo[50, 53]. EC morphology also responds to micropatterning, aligning in the direction of contact-guidance patterns in a concave shape resulting from micropatterned cylinders[51]. Micropatterning techniques have also provided insight into the mechanisms driving vSMC behavior. vSMCs exposed to micropatterning expressed more smooth muscle myosin heavy chain and α-actin at low passage number, and vSMCs were less responsive to transforming growth factor β (TGFβ) regardless of passage number due to a decrease in TGFβ receptor 1, implicating a possible mechanism responsible for the contractile phenotype in vSMCs[50]. Engineering vascular tissue with vSMCs with the contractile phenotype would provide the correct phenotype necessary to support vascular regeneration because the generation of synthetic vSMCs characteristic of pathological conditions is undesirable. Additionally, micropatterning and spatial patterning within vessel constructs can result in biomimetic small pulmonary arteries and ECs, vSMCs, and fibroblasts which attain an anatomically correct patterning in vitro[54, 55]. Chemical patterning in hydrogels creates patterned surfaces with various chemistries to assess how cells and proteins interact with the different surface chemistries[56]. For example, heparin-like homopolymers modified to express sulfonate-containing sodium 4-vinylbenzenesulfonate supported EC adhesion to the polymer surface with higher rates of VEGF absorption, while homopolymers modified to display glycol-containing 2-(methacrylamido)glucopyranose demonstrated decrease EC attachment and lower VEGF uptake[57]. ECM structure significantly affects vascular cell morphology and function within the vascular microenvironment and can be engineered to deepen the understanding of the mechanisms driving successful vascular regeneration (Table 1).

Extracellular Matrix Mechanics

Polymer composition and crosslinking affect ECM mechanical properties and guide vascular cell behavior. Synthesizing hydrogels of varying stiffness can be accomplished through various methods, such as 3D printing, chemically crosslinking hydrogel precursors, and photo crosslinking polymer precursors. For example, 3D printing of a fibrin-EC-fibroblast mixture in a confined area resulted in tensile loading. As a result, Yes-associated protein (YAP), an essential mechanotransducer, activity was shown to play a role in the subsequent vessel formation[58]. Hydrogel stiffness is also adjustable through covalently crosslinking of the polymer precursors [59]. Modifying the hydrogel polymer precursors before crosslinking can be harnessed to modulate hydrogel stiffness and mechanical properties[28, 59]. Hydrogel precursors can be modified to support vascularization by reducing undesirable reactions between the added functional groups and the cells encapsulated within the hydrogels, such as with elastin-like proteins crossed with strain-promoted azide-alkyne cycloaddition, and increase stiffness[59]. Vascular cells have responded to changes in hydrogel mechanical properties in vitro. With hydrogels stiffening, as the vascular ECM would through aging, EC contractility increased, and β-catenin dissociated from vascular endothelial cadherin-mediated cell junctions, ultimately disrupting vascular networks[7]. Additionally, ECM stiffening was demonstrated to regulate EC barrier integrity through focal adhesion kinase (FAK) activity, and heterogeneous ECM stiffening also disrupted barrier integrity[60, 61]. Another example of vascular cells responding to changes in ECM mechanics can be observed in durotaxis, the migration of cells toward a stiffer substrate, which was observed in vSMCs when cultured on a mechanical gradient surface coated with fibronectin but not on a gradient coated with laminin[62]. Furthermore, the release of vSMC podosomes can be stimulated by cellular contact with a physical barrier[63]. Viscoelastic hydrogels promote EC contractility-mediated integrin clustering leading to matrix remodeling, thereby enabling EC morphogenesis[28]. ECM mechanics are an important consideration when investigating vascular diseased states and regeneration, for they are influenced by ECM composition and significantly impact vascular cell behavior in the vascular microenvironment. The vascular ECM is balanced through mechanical cues, biochemical cues, ECM degradation, ECM protein deposition, microstructure, and ultrastructure (Table 1). Unraveling the nuances responsible for that balance will provide greater insight into creating therapeutics for vascular regeneration.

Oxygen and the Vascular Microenvironment

Oxygen is essential for the growth and function of every tissue within the body, and the vasculature is no exception. Oxygen’s role in and influence on the vascular microenvironment depends on the needs and state of the tissue. For example, during human embryogenesis, the early stages of vasculogenesis occur in a hypoxic environment, with oxygen levels below 5%, because maternal blood is unavailable to the fetus[64]. As oxygen tension rises over the next few weeks during development, branching angiogenesis occurs and later gives rise to nonbranching angiogenesis[64]. In adulthood, blood oxygen saturation levels are typically between 95% and 100%. Additionally, oxygen levels within each adult tissue will depend on the tissue vascularization and metabolic activity, yet tissue oxygen levels range from 1% to 11%[65]. Following development, hypoxia is often associated with diseased and injured states, such as ischemic tissue resulting from myocardial infarctions and the development of tumor environments[66, 67]. In certain cancers, hypoxia is a common characteristic, resulting in signaling that enhances endothelial permeation into tumors, tumor growth, and metastasis[68–71]. However, decreasing oxygen levels for a short time can augment blood vessel formation during vascular regeneration in wound healing following injury [72]. Yet, tissue regeneration requires balance, and facilitating oxygen delivery following ischemia can also improve angiogenesis[73]. Oxygen signaling in the vascular microenvironment is regulated by transcription factors, hypoxia-inducible factors (HIFs), and reactive oxygen species (ROS). HIFs are comprised of α- and β-subunits, and the α-subunits respond to decreases in oxygen levels and bind to β-subunits[74, 75]. HIFs then stimulate the expression of genes responsible for the proliferation, apoptosis, anaerobic metabolism, vascular tone, inflammation, and angiogenesis through TIMP-1, Tie-2, EGF, and VEGF expression, primarily through HIF-1α[76–83]. ROS, by-products of cellular metabolism, help maintain cellular function. For example, H₂O₂, an endogenous ROS, is necessary for EC function at low oxygen levels in the body[84]. Hypoxia and oxygen gradients are critical players in the vascular microenvironment. Understanding the nuances of the signaling pathways in the vascular microenvironment influenced by hypoxia and oxygen gradients is essential to enhance vascular regeneration further (see Clinician’s Corner).

Controlling Hypoxia to Recapitulate the Vascular Microenvironment

Oxygen and Vascular Regeneration in Microfluidic and Organ-on-Chip Devices

Modeling hypoxia to understand its impact on vascular processes requires tools that recreate the levels of oxygen tissues experience in vivo (Figure 2). To accomplish this, hypobaric chambers and incubators, along with inhibiting/stimulating hypoxia-induced signaling pathways, have been utilized[85, 86]. While these methodologies mimic hypoxia, there is a lack of control over the perinuclear oxygen levels and gradients. Control over and measure of pericellular oxygen levels drives the need to engineer more tunable and controllable culture conditions to study the impact of hypoxia[87]. Microfluidic and organ-on-chip devices provide highly tunable systems to model hypoxia in vitro. Microfluidic devices are small chips or platforms with chambers and channels allowing for precise control of liquids at the microscale. The miniaturization of physiologically-relevant cell culture systems in microfluidic devices helps model aspects of the vasculature microenvironment, such as oxygen gradients and shear stress in 2D and 3D. Modeling native vasculature through microfluidics begins during the device fabrication stage, as the material to fabricate the microfluidics influences oxygen availability inside the device[88]. Consideration of the oxygen permeability of material permits the regulation and monitoring of oxygen supply to cells within microfluidic devices[88]. Other important considerations highlighted through microfluidic modeling of the vasculature are cell density and cell type composition within devices. Given the miniaturized nature, high cell densities lead to rapid consumption of available oxygen, where various cell types consume oxygen at different rates, with larger cells often consuming oxygen quicker[88, 89]. Recent microfluidics models utilize 3D culture systems to recapitulate the vasculature microenvironment by incorporating shear stress and supporting cell types into the model. To accomplish this, gels supporting 3D cell culture have been incorporated into microfluidic devices[3, 4]. Following the shift to 3D culture, spatial and temporal control of oxygen was accomplished through sodium sulfate flow in a microfluidic device channel adjacent to a chamber supporting EC and fibroblast growth to demonstrate how averaged physiological levels of oxygen, around 5%, better support vessel sprouting and angiogenesis than hypoxic conditions, <5% oxygen[4]. Additionally, oxygen levels can be uniform or form an oxygen gradient within the microfluidic platform[90, 91]. When cultured under an oxygen gradient, EC networks are more prevalent in 5% oxygen gradients, and ECs migrate parallel to the gradients toward the low oxygen condition[90, 91]. Microfluidic devices have also been utilized to mimic the tumor microenvironment and suggest increased EC permeability in hypoxic conditions[92]. When considering vascular regeneration, microfluidic and organ-on-chip devices are engineered to create repeatable injury in vitro to study EC behavior during wound healing[93, 94]. Interestingly, in devices where only ECs are present, ECs can migrate to close the wound, yet when fibroblasts are present at an equal to or greater number, fibroblasts preferentially migrate into the open wound while ECs are left on the wound periphery[93, 94]. While microfluidic and organ-on-chip devices do not fully capture all the nuances of the vascular microenvironment, they provide a physiologically relevant model of the vascular microenvironment by incorporating hypoxia, shear stress, and multiple cell types in 3D, permitting a greater understanding of vascular cell behavior during regeneration.

Figure 2. Engineering Approaches to Recapitulate Hypoxia.

(left) Microfluidic devices have been utilized to incorporate hypoxic conditions in studying vascular regeneration. (right) Hydrogels with oxygen gradients and constant hypoxia have also been employed for studying the effects of varying oxygen levels during vascularization. Created using Biorender.com.

Oxygen in Hydrogels

When considering hypoxia in 3D hydrogels, monitoring oxygen levels and gradients inside the ECM-mimicking hydrogels allows for cell culture conditions that further recapitulate the native tissue environment and provide insight into the mechanisms driving cell behavior (Figure 2) [95–97]. For example, by culturing hydrogels containing retinal ECs or ECs derived from human pluripotent stem cells in hypoxic conditions (1% O₂), it was discovered that revascularization of the ischemic retina is supported through the stromal cell-derived factor-1a/CXCR4 axis[97]. Controlling oxygen to model the vascular microenvironment can occur by fabricating hydrogels that consume oxygen during polymerization resulting in hypoxic gradients inside the gels[95, 96, 98]. Previously, hypoxic conditions in hydrogels have been controlled via laccase-mediated reaction converting oxygen into dihydrogen monoxide[98]. In these hypoxia-inducible hydrogels, endothelial progenitors formed vascular networks through stabilizing HIFs, which did not occur in nonhypoxic hydrogels[98]. Building upon these tunable oxygen gradients in hydrogels, materials can be layered to create discretized oxygen gradients, allowing for a more advanced cell culture system to recapitulate the vascular microenvironment[95]. Such tight oxygen regulation allows for mechanistic investigations of the cellular and biochemical processes underlying vascular homeostasis and regeneration. For example, hypoxic hydrogel culture systems enabled the modeling and subsequent characterization of the mechanisms underlying endothelial progenitor cell clustering in vitro[95, 96]. More specifically, endothelial progenitor cells cultured in hypoxic conditions demonstrated clustering regulated by genes that respond to oxidative stress, and cyclic adenosine monophosphate (cAMP) signaling and clustering were also promoted by ROS produced independently of HIF[95, 96]. Each component of the vascular microenvironment influences the behavior and function of the others. So, while it is challenging to engineer systems that decouple one microenvironmental aspect from another, engineering 3D tissue constructs allow us to investigate how a change in one microenvironmental component influences another. Using the 3D hypoxic hydrogel system, it was found that hypoxia induces matrix metalloprotease-mediated hydrogel degradation required for endothelial progenitor clustering[96] and network formation[98]. Hypoxic levels and gradients greatly influence the mechanisms governing the functions within the vascular microenvironment. Engineering systems in the lab to mimic biologically relevant conditions permits further exploration and understanding of that environment to enhance vascular regeneration.

Concluding Remarks

Engineering approaches have enhanced our understanding of the vascular microenvironment’s roles in health and disease (Figure 3), yet there is much to consider moving forward (see Outstanding Questions). Current limitations include creating in vitro systems that support the inclusion of immune components and tissue-specific cells without conceding too much control over the microenvironment, thus further increasing the complexity of the systems. Increasing the complexity of in vitro systems may permit a better understanding of the microenvironment but compromise the translatability to the clinic with the loss of control over the overall system. Future research directions include creating tailored systems for studying specific diseases and sex-linked differences. Engineering tissue-specific vasculature to allow for the different functions of the vasculature in the brain, eye, heart, liver, and other organs within the body is an additional future direction.

Figure 3. Examples of ECM and Oxygen Level Modeling.

Schematic and data from A. Schnellmann et al. modeling ECM stiffening that lead to compromised microvascular phenotype. B. Sayer et al. modeling ECM patterning with high-resolution photographing, modulating endothelial sprout migration, C. Shih et al. demonstrating endothelial cell migration and proliferation under an oxygen gradient using microfluidics, and D. Blatchley et al. illustrating how low oxygen environments mediate endothelial cell cluster formation. Schematics and results were slightly modified for formatting and clarity. Schematics Created using Biorender.com.

Outstanding Questions.

• How can ECM-mimicking biomaterials be used to create and investigate tissue-specific vascular constructs in vitro?

• How can the complexity of vascular tissue constructs be increased in vitro in a controllable manner?

• How can future therapeutics treating vascular diseases be more efficacious following an improved understanding of the disease mechanisms?

Highlights.

• The vascular microenvironment is a complex environment of numerous components, with the extracellular matrix and oxygen playing critical roles in regulating cellular processes.

• Advances in engineering and biomolecular approaches have allowed to develop research strategies to delineate the role of the microenvironment in vascular development, growth, and regeneration.

• Biomaterials enable a deeper understanding of the biological mechanisms at play in health and disease states, and the enhanced knowledge can be used to develop therapeutic approaches to diseases.

• Controlling and understanding the oxygen levels in diseased and healthy tissues can drive forward therapeutics by enhancing the knowledge of the mechanisms driving vascular cell behavior to support vascular regeneration.