2.1. Choice of Materials
There are many different types of materials that can be used in vascular tissue engineering. Hydrogels, most commonly defined as systems of three-dimensional (3D), physically or chemically bound polymer networks that entrap water in the intermolecular space [
24], are the most commonly used form of materials for this purpose, mostlyis because of their inherent property of structurally and biochemically mimicking the extracellular matrix (ECM). Their physical and chemical properties can be optimized by additives (e.g., nanofibrillated cellulose (NFC)) [
25] to achieve optimal material properties for a specific application; additionally, different types can also be combined to form hybrid formulations with the same aim [
9,
26]. The ideal biomaterial should enable the formation of new blood vessels without toxic effects, induce the growth of vessels similar to native ones, and provide adequate mechanical support to the growing tissue. For example, scaffold stiffness is one of the most important tissue engineering parameters, namely, ECM stiffness has been shown to influence cell behavior, including adhesion, proliferation, migration, differentiation, signaling, and apoptosis [
27,
28,
29]. By adjusting the concentration of, for example, alginate and carboxymethyl cellulose (CMC) from 0.1 to 8%, hydrogels with a varying stiffness from 0.1 kPa to 90 kPa can be produced [
30,
31], which is well within the range of soft tissues, such as striated muscles, or skin [
32,
33]. One of the most important parameters in the search for ideal materials for engineering specific tissues is their biodegradability. The latter should match the rate at which cells can replace the artificial ECM. Moreover, the degradation products should not negatively impact the developing tissue [
2,
34]. In addition to determining cellular behavior, the materials also influence the feasibility and strategies of fabrication [
2].
Ink and bioink (ink containing live cells) components are typically divided into the following two categories: naturally derived and synthetic materials [
9,
35]. A number of naturally derived materials can be used for this purpose, which can be broadly categorized as either polysaccharides or proteins [
9]. They are derived from a biological source, either animals (mammalian or nonmammalian), plants, or algae [
2,
9]. Naturally derived materials, especially proteins, can vary from batch to batch, increasing the variability of experimental results. Nevertheless, they exhibit superior biocompatibility and other characteristics that stimulate tissue development, such as proangiogenicity [
9]. In addition to using purchased, defined materials, ECM materials can also be sourced from cells cultured in vitro [
22]. Many cell types, especially fibroblasts, can deposit different ECM materials such as collagen, elastin, and fibronectin. The main advantage of this method is that this ECM has a native composition and properties that can induce physiologically relevant cell behavior [
22,
36,
37].
Thus, the use of naturally derived source materials or even fully decellularized ECMs sets the standard for tissue engineering applications [
38]. The targeted standard are artificially constructed ECM substitutes that allow more precise control and reproducibility. Some key input materials are discussed below.
Collagen is a commonly used material for microvessel engineering [
1,
9,
22]. Type I collagen-based biomaterials have been shown to provide a suitable environment for angiogenesis. Therefore, it can stimulate the binding endothelial cell-surface integrins α
1β
1 and α
2β
2 via the GFPGER amino acid sequence of the collagen fibril. Moreover, ECs can degrade and invade the collagen matrix via metalloproteinases (MMPs) to establish vascular networks. This is mediated by collagen I-integrin interaction [
1,
9]. The macroscopic mechanical properties of the ECM and the behavior of the embedded cells depend on the microscopic orientation and fibrillar thickness of the collagen gel. Various techniques, such as electrospinning, stretching, and microfluidics, have been explored to control these properties [
1,
9,
22].
Fibrin, one of the main components of the blood-clotting cascade, has also been used for microvascular tissue engineering due to its intrinsic angiogenic properties [
22]. It is polymerized using fibrinogen and thrombin solutions and has been shown to promote cell migration, proliferation, and matrix synthesis [
39] and facilitate successful vasculogenesis [
40,
41,
42]. Cui et al. studied a bioink composed of human microvascular endothelial cells (HMVECs) and fibrin, for microvasculature construction. They precisely fabricated micron-sized fibrin channels using a drop-on-demand polymerization. Using this approach, they produced well aligned and straight fibrin fiber structures appropriate for cell seeding and microvasculature fabrication [
39]. However, fibrin-only inks are generally poorly printable and have poor mechanical stability [
9].
Another commonly used example of protein-based hydrogels is Matrigel
®, a tradename for an ECM mixture that contains many factors, including laminin, nidogen, collagen IV, and heparan sulfate proteoglycans (perlecan). It is secreted by mouse tumor cells and resembles the complex basement membrane environment found in many normal tissues [
43,
44]. With its excellent proangiogenic properties, Matrigel
® has been designated as the standard substrate material in EC tube formation assay and in vivo angiogenesis tests. It can also be used as a supplement to other materials while retaining its proangiogenic effect. This was demonstrated in 2016 in a study by McCoy et al. in which lumenized angiogenic sprouting of human cerebral ECs was significantly improved when collagen hydrogel was supplemented with 2% (
v/
v) Matrigel
® [
1,
45]. Schumann et al. showed that preincubating mesenchymal stem cells (MSCs) in Matrigel
® presents a promising approach to develop rapid microvascular growth in tissue engineering constructs since the microvascular capillary-like structures developed exceptionally fast [
46].
Gelatin methacryloyl (GelMA) is a semisynthetic hydrogel. It is based on a naturally derived material—gelatin—and contains methacrylate and methacrylamide groups. The mechanical properties of the gel can be fine-tuned by adjusting the degree of methacrylation. The bioactivity and tunability, as well as great mechanical stability of GelMA, make it an excellent candidate for direct bioprinting of microvasculature [
9,
47]. Chen et al. evaluated the use of GelMA in microvascular tissue engineering in a study in which they cocultured endothelial colony-forming cells (ECFCs) and MSCs and showed that ECFCs assembled into capillary-like networks [
47]. However, the lack of MSCs in the culture and increased methacrylation degree negatively impacted the generation of capillary-like networks [
47].
Alginate, a hydrophilic linear polysaccharide, as well as gelatin, chitosan, dextran, agarose, and hyaluronic acid also showed to have proangiogenic properties when modified with different functional groups [
2,
22,
48]. For example, when an unmodified hyaluronic acid hydrogel was used, less successful vascularization was achieved, compared to a fibronectin-supplemented hyaluronic acid-based hydrogel [
22,
49]. Similarly, using hybrid alginate–chitosan microcapsule scaffolds for providing support and guiding alignment of human umbilical vein endothelial cells (HUVECs) resulted in vascular-like network formation [
48].
A variety of other materials have been studied for microvascular tissue engineering. Due to their limited success for this purpose, the review does not include more detail in this regard. Some of these are polyethylene glycol (PEG) and propylene glycol diacetate (PGDA), polyglycolic acid (PGA), polycaprolactone (PCL), poly-L-lactic acid (PLLA), and elastin [
1,
9,
22].
2.2. Choice of Cell Source
ECs are the main cell type for microvascular tissue engineering. They make up the inner lining of blood vessels and display inherent angiogenic behavior. Different types of ECs are commonly used in vascular tissue engineering. Among them, HUVECs, HMVECs, and induced pluripotent stem cell-derived endothelial cells (iPSC–ECs) are most commonly used. Vascular endothelial cells exhibit broad phenotypic heterogeneity, which is related to the tissue of their origin [
50,
51] and is a consequence of various (patho)physiological factors such as chemical (e.g., growth factors, hormones, cytokines) and mechanical cues [
52]. For example, HMVECs can be categorized into adipose-tissue-derived, liver-tissue-derived, cardiac-tissue-derived, lung-tissue-derived, and dermal-tissue-derived subtypes. Although derived from different tissues, these subtypes of ECs share common markers, such as vWF and CD31 [
1,
9]. Evidence suggests that most of the phenotypic variability of ECs can be explained by environmental factors. Though epigenetic factors also seem to play a role to some extent, their impact is likely diluted through prolonged culturing [
50]. Since the in vivo microenvironment remains difficult to be recapitulated to the full extent in in vitro settings, the morphology and function of ECs can differ significantly between the in vitro and in vivo studies [
53]. The apparent plasticity of cells presents an opportunity for novel in vitro models. The use of a few cell types and appropriate environmental cues may cause the appropriate phenotype expression, as Nolan et al. showed that the cells’ microenvironment phenotypically and functionally “educates” ECs [
54].
Of all the EC types, HUVECs have been the most studied. This is also the reason why the vast majority of microvascular models use HUVECs as the main cell type. However, since ECs are heterogeneous throughout the body, both in terms of physiology and functionality, the use of specific endothelial subtypes may be more suitable for specific tissue engineering applications [
17,
55].
Coculture
ECs alone are not sufficient for long-term vascular tissue culture—several additional supporting cell types are required that play a role in microvascular tissue engineering (see and ) [
1], which include the following:
Figure 3. A histological image of different vessel types’ cross sections. The image shows the difference between different vessel walls—single layer wall of the capillary with only endothelial cells and occasional pericytes (not seen in this image), and multiple layer walls of arterioles and venules, which have many layers of muscle cells lining the endothelium [
58]. For better visualization, the typical vessel diameters are as follows: arterioles (smallest, precapillary arteries), <100 μm; capillaries, 5–40 μm; venules (smallest, postcapillary veins), 10–200 μm [
59]. Reprinted (adapted) from Creative Commons Attribution License CC BY-SA 4.0.
-
Mural cells line the endothelium. These include vessel-associated cell types, such as pericytes and vascular smooth muscle cells (vSMCs). Vascular smooth muscle cells are found predominantly on larger vessels, including arterioles and venules. Pericytes, on the other hand, are also found in capillaries. They provide mechanical support to ECs and manage the diameter of vessels and, according to recent studies, also regulate the permeability of the vessels [
1,
9]. Furthermore, they support angiogenic EC migration via MMP secretion, regulate endothelial permeability, and contribute to basement membrane formation [
1,
56,
57];
-
MSCs secrete growth factors and thus promote blood vessel formation through angiogenesis. They are also the progenitor cells that can differentiate into both ECs and vSMCs (vSMCs are not directly associated with microvasculature as they only appear in larger diameter vessels) [
9];
-
Fibroblasts also secrete many proangiogenic growth factors. Their main function is to secrete ECM proteins to reinforce the mechanical structure and promote the vascular network and lumen formation [
9].
shows a histological image of different vessel types. See for a schematic cross-sectional comparison.
Cell–cell interactions play a major role in regulating vascularization development and coculture of different cell types. For example, a combination of ECs with fibroblasts has been shown to enhance angiogenesis in vitro [
2].
When HUVECs were cultured alone, tube-like structures formed but quickly started to regress. In contrast, when HUVECs and MSCs were cocultured, MSCs were shown to migrate toward HUVECs and supported the formation and maturation of vascular networks [
2,
60]. Additionally, higher levels of endothelialization were observed in similar studies [
61]. Chen et al. used ECs in coculture with hepatocytes. They showed that HUVECs started to form 3D capillary-like structures, whereas no such formation was observed when HUVECs were cultured alone [
2,
62].
Darland and D’Amore reported that when they cocultured HUVECs with fibroblasts or other stromal cells, capillary-like structures self-assembled without the addition of exogenous factors. They also showed that EC sprouting was robust in the presence of cocultured fibroblasts and intercellular lumens formed within 4–5 days. However, in the absence of fibroblasts, no vessels formed, and most cells died after 4–5 days [
2,
63]. Stromal cells, besides providing support for growth, were also found to wrap around ECs and take on a pericyte-like behavior [
2,
28,
64]. Many studies have also shown that fibroblasts secrete soluble angiogenic growth factors such as vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-β), and platelet-derived growth factor (PDGF), to name a few [
63,
65,
66,
67,
68]. However, Marsano et al. studied VEGF overexpression in cardiac tissue patches and found that the therapeutic window of VEGF does not depend on the total VEGF in the tissue but rather on its concentration in the microenvironment around each producing cell since VEGF remains tightly bound to ECM [
69,
70,
71]. They showed that localized high VEGF expression is sufficient to cause the formation of angiomas (vascular tumors). To prevent such processes, they controlled the distribution of VEGF by delivering monoclonal populations of transduced myoblasts, in which every cell produced the same amount of VEGF. This way, stable and functional angiogenesis was induced over a wide range of VEGF expression levels [
69].
An important parameter in the preparation of cocultures is the ratio of ECs to tissue-specific cells, such as MSCs. The use of too many ECs in relation to tissue-specific cells decreases neovascularization of the graft, according to researchers [
7,
72]. To date, no ratio has been accepted as optimal as it depends on tissue type and graft size [
7].