Bioprinting for vascular and vascularized tissue biofabrication

Abstract

Bioprinting is a promising technology to fabricate design-specific tissue constructs due to its ability to create complex, heterocellular structures with anatomical precision. Bioprinting enables the deposition of various biologics including growth factors, cells, genes, neo-tissues and extra-cellular matrix-like hydrogels. Benefits of bioprinting have started to make a mark in the fields of tissue engineering, regenerative medicine and pharmaceutics. Specifically, in the field of tissue engineering, the creation of vascularized tissue constructs has remained a principal challenge till date. However, given the myriad advantages over other biofabrication methods, it becomes organic to expect that bioprinting can provide a viable solution for the vascularization problem, and facilitate the clinical translation of tissue engineered constructs. This article provides a comprehensive account of bioprinting of vascular and vascularized tissue constructs. The review is structured as introducing the scope of bioprinting in tissue engineering applications, key vascular anatomical features and then a thorough coverage of 3D bioprinting using extrusion-, droplet- and laser-based bioprinting for fabrication of vascular tissue constructs. The review then provides the reader with the use of bioprinting for obtaining thick vascularized tissues using sacrificial bioink materials. Current challenges are discussed, a comparative evaluation of different bioprinting modalities is presented and future prospects are provided to the reader.

Statement of Significance

Biofabrication of living tissues and organs at the clinically-relevant volumes vitally depends on the integration of vascular network. Despite the great progress in traditional biofabrication approaches, building perfusable hierarchical vascular network is a major challenge. Bioprinting is an emerging technology to fabricate design-specific tissue constructs due to its ability to create complex, heterocellular structures with anatomical precision, which holds a great promise in fabrication of vascular or vascularized tissues for transplantation use. Although a great progress has recently been made on building perfusable tissues and branched vascular network, a comprehensive review on the state-of-the-art in vascular and vascularized tissue bioprinting has not reported so far. This contribution is thus significant because it discusses the use of three major bioprinting modalities in vascular tissue biofabrication for the first time in the literature and compares their strengths and limitations in details. Moreover, the use of scaffold-based and scaffold-free bioprinting is expounded within the domain of vascular tissue fabrication.

Introduction

Three-dimensional (3D) printing technology has witnessed rapid strides from being considered a futuristic technology, as it promises to revolutionize clinical practice by making available customized equipment, organ models for surgical practices, organ replacement parts, and drugs, at a pace not witnessed before [1], [2], [3], [4], [5]. Though initially this technology was envisioned to provide surgical models and prototypes, scientists have quickly advanced the technology to produce tissue constructs for regenerative medicine; however, large-scale adaptation has still been limited due to the anatomical complexity and compositional (cellular and extracellular) diversity, and vascularization of native tissues [6]. In this respect, though significant advances have been made to recreate the geometric complexity, the concern of developing vascularization within tissue constructs still haunts scientists as the holy grail of tissue engineering [7], [8]. In this context, bioprinting emerges as a promising method for fabrication of vascular constructs and vascularized tissues, general concept of which is described schematically in Fig. 1 and through the paper.

Bioprinting can be defined as the spatial patterning of living cells and other biologics by stacking and assembling them using a computer-aided layer-by-layer deposition approach for fabrication of living tissue and organ analogs for tissue engineering, regenerative medicine, pharmacokinetic, cancer research [9] and other biological studies [10]. With the emergence of bioprinting in medical and pharmaceutical research [11], the substantial evolution in bioprinter designs have also taken place with successful commercialization of several bioprinters, a comprehensive review on which has been presented recently [12]. Briefly, according to their working mechanism, bioprinting technologies can be classified into three major modalities including extrusion-based bioprinting (EBB), droplet-based bioprinting (DBB) and laser-based bioprinting (LBB) [10], [13], many of which have been used for vascular or vascularized tissue fabrication. However, before examining the potential of bioprinting to create artificial blood vessels, a brief account of the anatomy of blood vessels are presented to define the target tissue of bioprinting.

The structure of blood vessels is characterized by a concentric layer arrangement; with cellular and non-cellular composition of each layer showing distinct variations (see Fig. 1). The intima, or the innermost layer, contains a monolayer of endothelial cells (ECs) and forms a tight barrier between the vessel lumen and vessel wall. This layer provides non-thrombogenicity and resistance to infections. The basement membrane forms the next layer and is composed of Type IV collagen and laminin. This layer is followed by elastin layer, also known as internal elastic lamina [14]. After intimal layer, the medial layer is composed of Type I and III collagen, along with smooth muscle cells (SMCs). These cells possess coordinated contractile properties, which cause vessel contraction or dilation [15]. Herein, collagen fibers and SMCs are in spiral arrangement along the vessel axis. The medial layer is quite thickened in large arteries and may also contain nervous supply. Further, the medial layer is circumvented by external elastic lamina. The outermost layer is the adventitia, which is composed of fibroblasts rooted on a loose collagen matrix. These layers continually interact with each other in remodeling and perfusion of organs they supply [16]. It is worthwhile to note that while larger vasculature can be identified as separate anatomical entities, micro-vessels are structurally and functionally part of the tissue they supply. The microvasculature is composed of three types of vessels namely arterioles, capillaries, and venules, which form a network architecture. The arteries branch down to arterioles, which are typically 10–200 μm in diameter (average lumen diameter being 30 μm) composed of the three tunic layers as found in macro-vessels, but with reduction in thickness of these layers. The endothelial lining of tunica intima is intact, while in tunica media, only to one or two smooth muscle cell layers are found. The tunica externa also becomes thinner compared to arteries. The arterioles are richly innervated by sympathetic nerves, because of which they can control of blood volume or pressure. The arterioles continue into the capillaries, and the connecting arterioles are referred to as meta-arterioles. Metarterioles act as precapillary sphincters and lack the true tunica media structures, wherein a single smooth muscle cell encircles the metarteriole-capillary. Capillaries are the part of microscopic vessels with lumen diameter of approximately 5–10 μm. In the capillary walls, endothelial layer is surrounded by a basement membrane and generally does not contain any smooth muscle, though pericytes are present to stabilize the wall structure. This structural feature allows reduction in the distance of diffusion for solutes to reach the cells of the tissue. In addition, capillary structure of different tissues may allow different solute permeability, and accordingly capillaries are classified as continuous, fenestrated or sinusoid capillaries. Found in muscle, skin, lung, central nervous system, basement membrane is continuous and intercellular clefts between adjacent endothelial cells have tight junctions. The fenestrated capillaries contain porous structures and are seen in exocrine glands, renal glomeruli, and intestinal mucosa while capillaries with large intercellular gaps are observed in liver, spleen and bone marrow. Sinusoidal capillaries are flattened with incomplete basement membranes and large gaps between cells. Capillaries arising out of a single metarteriole reunite and empty into a venule, which is generally 8–100 μm in diameter. Such multiple venules join to form veins. The venules walls contain an endothelium, a few muscle cells and elastic fibers in the middle layer, and a thin tunica externa composed of connective tissue fibers [17], [18].

Since the last two decades, it has been agreed that it is essential to recreate the hierarchical vascular network which can ensure steady perfusion of implant-injury sites for successful engineering of most complex tissues [19]; however, this issue has persisted as a major limitation over years on generating engineered tissues of clinically-relevant volumes and complexities [20], [21]. Apart from providing diffusion and mass transport of nutrients, substantial evidences also suggest that integration of vascular network also plays a pivotal role in governing tissue formation [22], [23]. Additionally, micro-vascularized tissue constructs would significantly improve clinical outcome with respect to innervations and complete functional recovery, as indicated by revelation of several cross-talk mechanisms between vascular promoting factors, endothelial cells and nerve cells [24], [25], [26], [27]. Except for tissues, such as avascular cartilage or cornea, wherein vascularization may cause inflammatory reactions [28], engineered constructs with thickness of more than a millimeter does not remain viable without vascularization [29]. Currently, tissue constructs of greater than 200 μm in thickness (the diffusion limit of oxygen from nearest capillaries under in vivo conditions) have little chance of subsequent clinical success in most complex tissues [30]. In highly vascularized tissues, such as liver, kidney, lungs, spleen, heart, pancreas, or thyroid, formation of new blood vessels becomes essential for a tissue to grow beyond the diffusion limit [31], [32]. Since most present tissue engineering strategies are evaluated in much smaller volumes than the requirements of larger clinical reconstruction, successful scaling is essential in order to ensure their clinical applicability. For example, in case of liver tissue, there exists a huge demand for tissue construct of clinically-relevant volumes (i.e., about 500 cm³) required of implantable liver with the cell density and functionality being close to native liver tissue [33], [34], [35].

Vascular tissue fabrication via the tissue engineering route comprises several approaches depending upon the function required. For one, vascular tissues are required for coronary vessel surgeries. Patients suffering from cardiovascular diseases like cerebrovascular disease, coronary heart disease, deep vein thrombosis, and peripheral arterial disease, which are associated with vessel blockages and require long-term (life expectancy >2 years) revascularization are principally indicated for vascular replacements like coronary arterial bypass grafting. However, synthetic vascular grafts have satisfactory results documented only for large-diameter (>8 mm) such as aortoiliac or medium-diameter (6–8 mm) carotid or common femoral artery arteries replacements. Their performance in small-caliber vessels (<6 mm) like coronary, infrainguinal or infrageniculate arteries reconstruction remains poorer compared to the gold standard autologous grafts harvested from internal thoracic or radial artery, and saphenous vein (SV). Thus the Food and Drugs Administration (FDA) does not approve the synthetic biomaterial based small caliber vascular grafts (internal diameter <5 mm) due to poor patency rates though autologous grafting may not be suitable for a large number of patients due to poor quality of donor site. These failures are usually due to intimal hyperplasia, thrombosis or infections of the graft. Absence of healthy endothelial cell layer, diameter mismatch, synthetic material surface properties and compliance mismatch with the native tissue are contributory factors to all these mechanisms [36], [37]. Bioprinting with suitable endothelial cell source into the diameters required is thus an attractive approach to engineer small diameter blood vessels and has been pursued.

Apart from fabrication of vascular grafts, vascularization is also endeavored to be improved for blood circulation by fabrication of a vascular microcirculatory network at affected regions of other tissues, encourage vessel in-growth into implanted constructs from the host, or to generate new circulation system at the local site through stem cell therapy [38]. In human embryos as well as adult tissues, new vascular networks are formed by two distinct mechanisms known as vasculogenesis and angiogenesis. The former refers to de novo generation of blood vessel from vascular progenitor cells or the capillary plexus formation from circulating EPCs while angiogenesis is used to indicate sprouting of new vessels blood vessels via extension or remodeling from pre-existing capillaries. The process of angiogenesis can occur through elongation, inosculation, intussusception, or sprouting. Amongst them, intussusception involves bifurcation of existing lumen while sprouting is an outcome of multistep processes involving first the resorption of basement membrane, followed by infiltration and growth of ECs, lumen organization and subsequent formation of vessel with the addition of pericytes. The process of inosculation is important for making connection between the vascular networks transplanted and host microcirculatory network. In the context of tissue engineering, it is observed that EPCs can be expanded ex vivo and transplanted to augment neovascularization for engineering of vascular structures. Further, EPC recruitment can be enhanced by stromal-derived factor-1, and granulocyte colony stimulating factor, amongst others [39], [40], [41], [42]. Thus, vasculogenesis-driven mechanism of vascular tissue engineering can be adopted for bioprinting.

Angiogenesis promoting strategies have focused on either in-growth of newly formed capillaries inside implanted constructs from the host or to achieve a rapid blood supply within engineered constructs by stimulating inosculation with the help of pre-vascularized constructs [29], [43]. After implantation, the pre-vascularized channels within the constructs develop interconnections with neighboring tissue vessel network to get perfused rapidly. In other approaches, angiogenesis promoting agents like vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF), either alone or in co-delivery forms are often employed [44], [45]. The recruitment of circulating stem cells into ischemic sites by targeted activation of concerned pathways or by cell seeding into the constructs are other approaches for achieving vascularization. In this respect, Tasso et al. have reported that mesenchymal stem cells (MSCs) seeded into porous cubes resulted in homing of pericyte-like cells or circulating endothelial progenitor cells (EPC) inside engineered constructs after implantation. However, teratoma formation or tumorogenicity of various cell sources, including stem cells, remains unaddressed for standardization of tissue constructs [46]. Other potential sources for vascular cells are adult bone marrow, adipose tissue, hair follicle (HF), umbilical cord (UC), and muscles. Amongst them, the adipose derived stem cells (ADSC) are shown to differentiate into multiple cell types of vascular tissue including smooth muscle cells on inducted by transforming growth factor-beta 1 (TGF-β1) or bone morphogenetic protein-4 (BMP-4) and found to be a rich source of endothelial cells. Provided the fact that adipose tissue is routinely harvested during liposuction procedures, they constitute a promising source for vascular tissue engineering. On the other hand, hair follicles and umbilical cords can also be obtained without invasive procedures and the various stem cells isolated from these sources have shown promising differentiation potential into vascular tissue [47]. Further, newer biological understanding indicates that cellular crosstalk is involved in angiogenesis as well as organ remodeling process [48]. An example is the cross-interaction between ECs, osteoblasts, osteoclasts and osteo-progenitors, which are mediated by secretion of soluble factors and gap junction proteins like connexin 43. Though it can be concluded that various cells types have been suitably engineered to form microcirculatory networks in vitro as well as implanted in vivo, further comparative assessment of more dynamic properties for extended time periods of intended application like extent of vascularization, remodeling, and regression, would be required [49].

For tissues at clinically-relevant volumes, availability of cell sources becomes an important criterion along with current methods of cell expansion to obtain industrial scale cell numbers [50]. It may be noted here that many vascular network forming cell sources (i.e., human umbilical vein endothelial cells (HUVECs)) used in tissue engineering research, are not clinically available and hence more comparative studies are needed with EPCs or MSCs. These have turned the attention to explore alternative sources of like autologous circulating EPCs, induced pluripotent stem cells (iPSCs) and postnatal stem cells, which are being continually experimented to produce stable and mature 3D capillary network [51], [52]. It is further imperative to note that different tissue engineering techniques have been applied to fabricate blood vessels including traditional methods like solvent-casting [53], particulate leaching [54], gas foaming [55], fiber bonding [56], [57], and phase separation [58], [59], while electrospinning and self-assembly are also explored to obtain nano-fibrous scaffolds [60], [61]. These techniques offer sparse control over pore size and shape while also lacking in precision requirement of target architectures. To overcome these limitations, rapid prototyping techniques have been introduced such as vat photopolymerization [62], [63], material extrusion [64], [65], and sheet lamination [66]. Over conventional or any other techniques, they offer the advantage of high level of scaffold-to-scaffold consistency and the ability to cater to patient specific geometries [67]. Amongst them, the principal advantage of bioprinting lies in the ability to directly fabricate heterocellular tissue constructs with ease of modulation of cell densities. This advantage is driving researchers to explore 3D bioprinting techniques at deeper scales to solve the vascularization problem in tissue engineering [68].