Bioengineered Vascular Scaffolds: The State of the Art

Decellularized tissue matrices

Due to a dearth of methods for de novo construction of a complex structure capable of imitating natural ECM, decellularized tissue matrices are currently considered an ideal scaffolding system due to their structural and mechanical similarity to native tissues and because they contain tissue-specific ECM proteins that remain after decellularization. Interestingly, Yoo and colleagues reviewed decellularization techniques and possible methods for using these decellularized matrices for whole organ engineering (17). Decellularized matrices have already been used for many tissue engineer applications. Kim et al developed a composite scaffold that was composed of a collagen matrix derived from decellularized porcine bladder submucosal matrix and synthetic poly(lactide-co-glycolide) (PLGA) polymer (18). This composite scaffold provides a microenvironment that facilitates osteogenic differentiation of amniotic fluid-derived stem cells. Furthermore, Choi et al investigated the interactions between the ECM environment and human corneal endothelial cells to improve cell proliferation and function (19). Following this marked path, vascular tissue engineering has experimented with new decellularized constructs. Thanks to studies like those just mentioned, which have investigated the interactions between the ECM environment and mesenchyme-derived cells, it has been possible to develop tubular decellularized matrices supporting the growth of a neointima with endothelial and smooth muscle cells (20). Decellularized arteries, from allogenic and xenogenic sources, are attractive scaffolds for tissue-engineered vascular grafts (TEVGS) thanks to their mechanical and biological properties (20). However, these natural scaffolds are limited by the lack of precise manufacturing control of the physical and mechanical properties as well as by problems with inflammation and calcification (21). The search for a viable off-the-shelf, small- diameter vascular graft that can match autograft performance in terms of mechanical properties, cell compatibility, and vascular healing has been the focus of many research efforts, but, to date, has remained an elusive target.

Electrospinning

Another approach in the development of biomimetic structures has been suggested by the textile industry. To date, electrospinning is a chemical engineering technique widely used as a fabrication method to generate nanofibers with diameters ranging from 100 nm or less to several micrometers, for various textile applications. Although many other scaffold fabrication technologies are routinely used in tissue engineering, only a few provide constructs with critical similarities to natural ECM like those that electrospinning can provide. Electrospinning has become a popular alternative fabrication method during the last two decades, as it can be applied to many disciplines and it is relatively simple and inexpensive. Shin and colleagues reviewed the current approaches to the development of electrospun nanofibers as a scaffold for tissue engineering applications (22). These electrospun scaffolds can also be functionalized by adding biochemical and mechanical cues to enhance cellular interactions for tissue engineering applications. Levorson et al described their work with fabrication and characterization of multi-scale electrospun scaffolds for cartilage regeneration (23). These scaffolds were able to maintain scaffold cellularity in serum-free conditions as well as aid the deposition of glycosaminoglycans. Xu et al presented a novel, controllable, dual protein delivery system through electrospun fibrous scaffolds with different hydrophilicities (24). Starting from these ideas, Zhang et al built up a double-layered electrospun membrane composed of chitosan hydrogel/poly(ethylene glycol)-b-poly(l-lactide-co-caprolactone) (PELCL) electrospun membrane loaded with VEGF (inner layer) and emulsion/PELCL loaded with PDGF (outer layer) to regulate proliferation of vascular endothelial cells and vascular smooth muscle cells (25). Only a rapid endothelialization along the lumen of grafts followed by proliferation of vascular smooth muscle cells around the exterior can prevent thrombosis and guarantee the right vascular compliance. Further applications of electrospinning technique are described in the following paragraphs.

Cell-seeded scaffolds

To improve biocompatibility features and promote remodeling and reabsorption, materials are usually seeded with human mesenchyme-derived cells. The first clinical application of a tissue-engineered vascular patch with pretreatment of human bone marrow cells, was reported by Shin'oka (26). Likewise, tissue-engineered biodegradable materials with autologous cell seeding before patch repair, where a bioreactor culture system was used, have been well documented as potential cardiovascular patches (27–33). Oh and Lee reviewed hydrophilization of synthetic biodegradable polymeric scaffolds for improving cell/tissue compatibility (34). This technique has been considered a simple and effective approach to achieve desirable in vitro cell culture and in vivo tissue regeneration within the synthetic polymeric scaffolds. In a very recent, cutting-edge study, polycaprolactone (PCL)/polylactide (PLA) and PCL/PLA/polyethylenglycol (PEG) electrospun small caliber vascular grafts and ePTFE grafts of the same size have been pre-coated with blood, gelatine or fibronectin and seeded with endothelial cells from human term placenta. The best results have been obtained with fibronectin-coated PCL/PLA/PEG grafts. Here, the number of attached viable cells was 78% to 81% higher than fibronectin pre-treated ePTFE grafts. Cells attached to PCL/PLA/PEG grafts appeared in physiological cobblestone morphology. Viability analysis showed a high cell viability of more than 98% (35).

The feasibility of creating a completely biodegradable vascular regenerating guide directly in vivo, without chemical and cellular preconditioning in vitro, is of great interest. This approach may overcome the main problems related to present techniques. Tissue-engineered grafts currently being investigated require an extended period of preparation, and thus they cannot be used in emergency situations (36–38). Furthermore, the prolonged duration of culture increases the risk of infection and raises costs in terms of personnel, equipment, and materials needed. In TEVGS autologous cell seeding and culture or growth with bioreactors before the operation is usually necessary to improve their antithrombogenicity and performance. Many strategies have been attempted to facilitate regeneration of autologous tissue on tissue-engineered materials. A major method is preoperative cellular manipulation (26, 39, 40). Kaushal and coworkers (39) demonstrated functional small diameter neovessels produced with endothelial progenitor cells. Another effective method is the use of a bioreactor (39, 41, 42). Campbell and associates (43) suggested that the host's own peritoneal cavity is an effective bioreactor and showed that it is feasible to grow vascular prostheses within it. Also, Hoerstrup and colleagues (42) demonstrated the growth capacity of cell-seeded TEVGs grown in vitro for 21 days under biomimetic conditions.

Although the utility of these pretreatments is fully recognized, their short comings, including invasiveness, risk of contamination, and the need for preparation time, have led to hesitation in their clinical use. For these reasons, Torikai et colleagues (44) used in situ cellularization as an alternative method, more suitable for clinical application because of its ready availability (33, 45). The patch they designed was composed of three layers: the interior (luminal side) was composed of knitted PGA compounded with collagen microsponge, the middle layer was PCL, and the exterior was composed of woven PLA. This PGA/PLA graft showed evidence of good in situ cellularization, including relatively early endothelialization and population with functioning SMCs, after implantation into porcine aorta. The graft was durable enough to withstand high-pressure conditions for at least 12 postoperative months, despite the presence of a residual PLA layer, and also demonstrated the potential to acquire intrinsic physiologic vascular function. Considering the established use of artificial grafts for aortic surgery and the need for small-diameter prostheses, the complete and early endothelialization demonstrated by their TEVG was meaningful, particularly in the setting of small diameter vascular structures, such as peripheral arteries. The limitations of study were the lack of regenerated elastin and the residual PLA layers.

Notwithstanding these encouraging results, the ex vivo cell-seeding procedure is complicated, invasive, expensive, and can cause contaminations. To overcome these problems, the last few years have marked a substantial paradigm shift in design criteria for modern synthetic biomaterials, and materials equipped with molecular cues mimicking certain aspects of the structure or function of natural extracellular microenvironments have quickly been developed (46–50).

Synthetic biodegradable polymeric scaffolds

Takahashi et al developed a novel tissue-engineered patch made of PGA/PLA that showed constructive in situ remodeling by site-specific host cells without prior ex vivo cell seeding (51). Unfortunately, PGA can induce a local inflammatory response following patch repair (52). More recently, Giammona et al have reported the in vivo application on a murine animal model of two electrospun biodegradable materials, specifically designed to create tubular structures. In one case a α, β-Poly [(N-hydroxyethyl)-dl-aspartamide] (PHEA)-PLA mixture was co-spun with silk fibroin (Fibro-PHEA-PLA) by a parallel electrospinning process to obtain a scaffold with two different polymeric fibers. In the other case, PHEA-PLA was mixed with PCL (PCL-PHEA-PLA) to obtain a hybrid fibers scaffold. The in vitro assay showed fibroblast colonization in both materials. Histopathological findings showed that after implantation a neutrophilic reaction associated to colliquative necrosis was predominant, particularly for PCL-PHEA-PLA. Fibro-PCL-PHEA caused a non-organized stromal reaction (53). Pitarresi et al have improved PHEA-PLA by producing a new copolymer named a, b-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-D, L-aspartamide-graft-polylactic acid (PHEA-EDA-g-PLA) as a starting material for producing a fibrillar scaffold for vascular regeneration (54). Thanks to the presence of free amino and hydroxyl groups in PHEA-EDA-g-PLA, the electrospun scaffold has been bound to heparin, a known natural anticoagulant molecule (55). Through electrostatic interaction, heparin is also able to retain growth factors like basic fibroblast growth factor (bFGF) (56–58). For this reason, the presence of heparin in avascular grafts could prevent thrombosis phenomena and improve cell attachment and proliferation. In this case, the scaffolds obtained have been able to allow the formation of new endothelial tissue, making them of potential use for the production of bioengineered blood vessels (54). Due to their inflammatory potential many researchers have abandoned synthetic polymers and have tested vascular patches entirely made of a benzyl ester of hyaluronic acid (HA).

Hyaluronic acid scaffolds

HA can be processed to obtain several types of devices such as tubes, membranes, nonwoven fabrics, gauzes, and sponges. All these scaffolds are highly biocompatible. In the human body they do not elicit any adverse reactions and are reabsorbed by the host tissues (59). HA is the only nonsulphated glycosaminoglycan of ECM. Despite its simple structure, HA is involved in a great number of biological functions, such as cell proliferation and migration, morphogenesis, wound healing, inflammation, angiogenesis, and tumor growth (60–64). An important biological role is related to HA oligosaccharides that stimulate cytokine secretion and endothelial cell proliferation (60–64). Moreover, recent studies indicate that HA may inherently stimulate endothelialization, favoring the recruitment of endothelial cells and endothelial progenitor cells (EPCs) (43, 45). EPCs can migrate and adhere inside HA-based biomaterials, maintain their preendothelial phenotype, and express angiogenic factors, especially within the first week of growth (63, 65). These results indicate that HA-based biomaterials could be promising candidates as a vehicle for EPCs for regenerative medicine applications. They may also overcome problems related to current vascular implant materials that insufficiently recruit endothelial cells to form a normally functional and confluent endothelium, a key challenge to reinstating vascular homeostasis at the surgical site.

An Italian team obtained stimulating results in testing vascular prostheses entirely made of HA in rat and pig experimental models (65–68). In this case, human vascular cells, such as endothelial cells (62) and smooth muscle cells (SMCs) (63), were grown in vitro on benzylic esters of hyaluronic acid (namely HYAFF-11 biomaterials) constructs to develop new tissue-engineered vascular substitutes. Previous in vivo studies (65–68) confirmed that HYAFF tubes could sequentially orchestrate the vascular regeneration events needed for very small artery reconstruction. HYAFF-11 was shown to be very well tolerated and to elicit no adverse reactions in clinical practice (62, 63, 69). More recently, Du et al have fabricated well-aligned nanofibrous scaffolds with PCL using a high-speed rotating collector. They then modified those surfaces with HA and studied the synergistic effect of the scaffolds on the in vitro behavior of the endothelial cells. The results of adhesion and the morphology of human umbilical vein endothelial cells (HUVECs) showed that the HA-coating aligned PCL (HA-aPd) nanofibrous scaffolds could highly promote attachment and guide the HUVECs bipolar spread with the parallel, aligned nanofibers. Furthermore, HUVECs on the HA-aPd formed a confluent monoendothelial cell layer and exhibited superior protein expression levels of von Willebrand factor. This study suggested that the combination of aligned nanostructure and HA modification was more capable of promoting the regeneration of functional endothelium for vascular tissue engineering than the use of either individually (70).

HA is also often mixed with other ECM polymers to better reproduce the complexity and biological role of the natural ECM. In some studies, HA has been combined with collagen fibers, showing advantages such as the enhancement of cell migration and division compared to either material on its own (71, 72). Another potential material applicable to TEVG field is HA-EDA-g-α-elastin copolymer. In this case HA was functionalized with EDA and the resulting derivative was reacted with α-elastin, a water-soluble protein that maintains the main biological performance of native elastin. The presence of unreacted amino groups in HA-EDA-g-α-elastin allow chemical crosslinking with ethylene glycol diglycidyl ether (EGDGE) and the production of porous scaffolds. The authors evaluated attachment, viability, and proliferation of primary rat dermal fibroblasts and umbilical artery smooth muscle cells, obtaining good results. Furthermore, the prepared scaffold showed good water affinity, a resistance in simulated physiological fluid, and an appropriate susceptibility to hydrolysis by hyaluronidase. Undoubtedly, thanks to the presence of HA and α-elastin, a potential application of the investigated scaffolds could be dermal or vascular regeneration, since both components are abundant in these tissues where they play important biological functions (73).

Elastin into scaffolds: the keystone of TEVGs

A major structural element of arterial walls is elastic fiber, which endows vessels with the critical property of elastic recoil (74). In arteries, elastin dictates tissue mechanics at low strains before stiffer collagen fibers are engaged. It confers elasticity, preventing dynamic tissue creep by stretching under load and recoiling to its original configuration after the load is released. In addition to mechanical responsiveness, elastin is a potent autocrine regulator to vascular SMC activity, and this regulation is important for preventing fibrocellular pathology. Finally, elastin degradation associated with matrix metalloproteinase (MMP) activity is a cell-mediated process, observed in almost all types of vascular calcification. Elastin peptides associated with transforming growth factor-β could induce osteogenic gene expression in SMCs, possibly via elastin laminin receptor (ELR) signaling (13, 74). Their presence in a vascular graft would greatly enhance design and patency; furthermore, elastic fibers also influence vascular cell behavior through direct interaction and by regulation of growth factor activation. Elastin knockout studies and clinical observations have revealed the essential regulatory function of elastin during artery development. In the absence of extracellular elastin accumulation, smooth muscle proliferation leads to arterial stenosis (75–79). Thus, to ensure appropriate mechanical function and prevent serious complications, successful artery replacements must incorporate an elastic component.

Approaches to date have included building tissue replacements on an elastin scaffold isolated from cadaveric tissues, supplying soluble tropoelastin to a cell culture, or designing biocompatible synthetic elastic polymers (13). One of the most promising approaches was described by l'Heureux et al (80–82), who developed a completely autologous technique called sheet-based tissue engineering. Dermal fibroblasts are obtained from a small skin biopsy and grown in conditions that promote an engineered complete vessel, ready to be implanted in six months. This approach is time consuming, with total production time at ∼24 weeks. It is conceivable that in previous techniques (26, (83–86), the change from two-dimensional to three-dimensional culture conditions utilizing porous scaffolds may have contributed to a loss in elastin biosynthesis by SMCs. It is now known that the scaffold must provide the appropriate chemistry for SMCs to secrete elastin. Few scaffolds are able to promote elastin biosynthesis.

One of the most promising ECM-derived polymers is hyaluronan, as discussed earlier. In vitro studies with gel preparations of hyaluronan (87, 88) have confirmed elastogenesis in neonatal SMCs. In 2012, McKenna et al (89) electrospun small-diameter vascular grafts containing recombinant human tropoelastin (rTE), the monomer unit of elastin, that, when cross-linked, mimics native elastin fibers. The material provided both the compliance and support of endothelial cell adhesion for a component of a vascular graft biomaterial, but it lacked the tensile strength to support in vivo arterial pressures. The authors concluded that, for vascular graft applications, the tensile strengths of the prTE scaffolds should be further increased by reinforcement or co-spinning with either collagen or synthetic materials. Interestingly, Bashur et al (90) have reviewed many important issues related to elastin-based TEVGs. Starting from a description of the current challenges that exist in the field of elastic matrix engineering, specifically for vascular applications, they continue by introducing the specific targets and processes that impact elastogenesis, and conclude with a review of current progress toward achieving robust elastogenesis in TEVGs aimed at clinical use.

Three-dimensional (3D) printed vessels

Several rapid prototyping techniques and materials, from designs as simple as a single channel (91), to designs that recreate the complex geometries of vascular pathways (92), and even scaffolds based on complex collagen forms (93), have been used to achieve a vasculature reconstruction that is as natural as possible. Currently, solid freeform fabrication (SFF) as well as inverted colloidal crystal (ICC) fabrication or the newly emerging multi-photon absorption polymerization (MAP), provide precise controllability over 3D matrix architecture at a micro-scale level. Limited material selection and lack of submicro-scale structural resolution are the major shortcomings of these techniques. Bioprinting (94), in which cells and matrix are deposited dropwise, has been developed over the past decade but also is a slow, serial process with limitations on print resolution, materials, and cells.

In contrast to these methods, 3D sacrificial molding provides an intriguing alternative (95–97). Proof-of-concept studies have shown that a network of channels can be fabricated by creating a rigid 3D lattice of filaments, casting the lattice into a rubber or plastic material, and then sacrificing the lattice to reveal a microfluidic architecture in the bulk material. However, 3D sacrificial molding of perfusable channels has so far required the use of cytotoxic organic solvents or processing conditions for either removing the sacrificial filaments or casting the surrounding material, and thus could not be accomplished with aqueous-based ECMs or in the presence of living cells. In their work, Miller et al (98) describe a biocompatible sacrificial material - a simple glass made from mixtures of inexpensive and readily available carbohydrates - and a means to 3D print the material to facilitate the rapid casting of patterned vascular networks in engineered tissues. In this case, they used the material as a cytocompatible sacrificial template in engineered tissues containing primary rat hepatocytes to generate cylindrical networks that could be lined with endothelial cells and perfused with blood under high-pressure pulsatile flow. Unfortunately, it was difficult to achieve the cellular densities of native tissues (approximately 10-500 million cells per mL), but it demonstrated an avenue for building and studying such tissue mimics, in which the vasculature appears not to constrain the design space for the tissue itself, allowing for arbitrary cell types, matrices, and their patterning.