Bureau and colleagues have recently examined non-woven PET fibers with surface-grafted PEG as potentially non-thrombogenic surfaces. The authors activated the surface of the PET with poly(vinyl amine) (PVAm), yielding pendant amine groups that were conjugated to carboxylic acid terminated PEG oligomers. The authors reported decreased platelet adhesion and activation with a 10% PEG solution grafting to the PVAm modified PET fibers when compared to knitted Dacron; however, the results were not superior to previously investigated carbon-coated ePTFE (Dimitrievska et al., 2011). In a prospective randomized multicenter study, Kapfer et al. demonstrated that in an extra-anatomical anterior tibial artery bypass model, carbon-impregnated ePTFE prostheses were not significantly better than standard ePTFE grafts in terms of graft patency or limb salvage (Kapfer et al., 2006). Similarly, Wang et al. also used PVAm to develop a surfactant polymer to coat ePTFE vascular grafts to prevent platelet adhesion and activation. The authors conjugated dextran to the PVAm backbone, which mimicked the polysaccharide-rich glycocalyx, generating a highly hydrated barrier, similar to the PEG-grafted PET fibers, inhibiting platelet adhesion and activation from both platelet-rich plasma and whole blood. Despite the promising in vitro results, the data from the proposed future study in a porcine model were never reported (Wang et al., 2009).

The highly hydrated surface of erythrocytes is also responsible for their non-thrombogenicity. The presence of the polar PC head groups of the lipid bilayer of the cell membrane are electrically neutral at physiologic pH while carrying both positive and negative charges. The zwitterionic nature of PC, resulting in the hydrated surface has shown limited protein and cell adhesion in vitro and thus has been explored as a non-thrombogenic coating on synthetic prostheses (Jordan and Chaikof, 2007). Yoneyama et al. demonstrated reduced thrombogenicity in an interpositional carotid rabbit model for up to 5 days with a segmented poly(etherurethane) (SPU)/2-methacryloyloxyethyl phosphorylcholine (MPC) polymer blend coating of Dacron prostheses (Yoneyama et al., 1998). Similarly, Chaikof and colleagues deposited a membrane-mimetic film onto the luminal surface of gelatin-impregnated ePTFE vascular prostheses and demonstrated reduced platelet adhesion after 1 h in a baboon femoral arteriovenous shunt model when compared to uncoated ePTFE grafts. The authors also observed a reduction in fibrinogen adhesion in the membrane-mimetic film coated graft; however, this reduction was not significant (Jordan et al., 2006; Jordan and Chaikof, 2007).

An alternative to the self-assembled phospholipid coating on MPC is the immobilization of merely the PC head group to the vascular prosthesis. Chevallier et al. functionalized the surface of ePTFE grafts through ammonia plasma treatment and then coupled PC to the resulting surface amino groups. The authors observed a concomitant decrease in platelet adsorption and activation and thrombogenicity index in the PC-functionalized grafts. Moreover, a significant reduction in neutrophil adhesion was observed, indicating a mitigation of the inflammatory response (Chevallier et al., 2005). In addition to the observed non-thrombogenicity of the PC, Chen et al. (1997) had previously demonstrated a reduction in neointimal hyperplasia with a PC coating of ePTFE grafts in a mongrel dog end-to-side carotid anastomoses model compared to virgin ePTFE grafts.

Instead of preventing platelet adhesion and activation through highly hydrophilic surfaces, some researchers have adopted the approach of controlled protein adsorption to inhibit coagulation. Albumin induces less platelet adsorption than other plasma proteins such as fibrinogen, which binds to the platelet GP IIb/IIIa receptor via arginine–glycine–aspartic acid (RGD) sequences (Zaidi et al., 1996; Jordan and Chaikof, 2007; Srokowski et al., 2011). Despite albumin’s rapid adsorption to the surface of a synthetic material, more thermodynamically favored proteins replace albumin on the surface (Andrade and Hlady, 1987). Given the susceptibility of albumin to be displaced by larger proteins including fibrinogen, the promotion of albumin immobilization on the surface of vascular prostheses has been investigated. Choi et al. evaluated polylactide coated ePTFE grafts as a mediator of protein adsorption. The authors observed a preferential adsorption of albumin over fibrinogen on the surface of the coated ePTFE grafts compared to both unmodified and modified ePTFE grafts. The modified grafts were rendered more hydrophilic via chemical treatment, yielding an increase in surface energy. Despite the preferential adsorption of albumin, the polylactide surfaces induced greater blood cell adhesion than both control grafts suggesting a thrombogenic nature of the polylactide surface (Choi et al., 2005). In effort to mitigate the thrombogenicity of a secondary material, albumin was immobilized to the surface of knitted Dacron grafts through glutaraldehyde crosslinking. In this study, Kottke-Marchant et al. observed reduced platelet adhesion and aggregation, as well as reduced leukocyte adhesion with the crosslinked albumin-coated, knitted Dacron grafts when compared to unmodified Dacron. In addition, the release of fibrinopeptide A, an indicator of fibrin formation, was also reduced in the albumin-coated knitted Dacron (Kottke-Marchant et al., 1989). Although promising in vitro, no difference was observed in clinical outcomes between the coated and uncoated grafts in arterial bypass grafts for aortoiliac disease (Bearn et al., 1993; Al-Khaffaf and Charlesworth, 1996).

Like albumin, elastin has also inhibited platelet aggregation (Baumgartner et al., 1976; Barnes and Maclntyre, 1979); however, the mechanism is not fully understood. Despite the initial studies of the antagonistic effect of elastin on platelet aggregation, few studies have evaluated the extracellular matrix (ECM) protein for passivation of vascular prostheses. Woodhouse et al. have tested elastin-like peptides (ELPs) as coatings of vascular graft materials including PET (Mylar™), poly(tetrafluoroethylene/ethylene) copolymer (Tefzel™), and a poly(carbonate urethane) (Corethane™). The authors passively coated all three test materials with a recombinant human elastin peptide and demonstrated a reduction in platelet activation in all cases. Moreover, fibrin accretion and thrombus formation on the surface of a polyurethane catheter was retarded in a New Zealand white rabbit model (Woodhouse et al., 2004). In a follow-up study, Woodhouse and colleagues reported platelet adhesion and fibrinogen adsorption on Mylar™ coated with three different ELPs. The authors observed a reduction in fibrinogen adsorption in all ELP-coated films; however, only the longer polypeptide chains exhibited decreased platelet adhesion (Srokowski et al., 2011). In a related study, an elastin-mimetic triblock protein polymer was evaluated by Chaikof and colleagues as a non-thrombogenic coating of impregnated ePTFE vascular grafts. They too observed inhibition of platelet adhesion and fibrin deposition in a baboon arteriovenous shunt model (Jordan et al., 2007). In addition to the non-thrombogenicity of ELPs and proteins, Ito et al. examined the effect of coacervated α-elastin in preventing intimal hyperplasia. The authors observed a dose dependent decrease in smooth muscle cell proliferation in vitro with crosslinked coacervated α-elastin, suggesting a potential reduction of intimal hyperplasia in vivo (Ito et al., 1998a). The authors repeated the study using tritiated thymidine to monitor cell proliferation and despite evidence suggesting that tritiated thymidine may result in cell arrest and apoptosis (Hu et al., 2002), the authors reported increased endothelial cell proliferation at a coacervated α-elastin concentration of 0.1 mg/mL (Ito et al., 1998b).

As active members of both anti-coagulant and anti-platelet mechanisms, endothelial cells (ECs) are integral in regulating hemostasis (Verhamme and Hoylaerts, 2006). Therefore, much research has been focused on mimicking the anti-thrombogenic features of the endothelial cell surface. Immobilization of anti-coagulant molecules thrombomodulin and heparin onto the surface of synthetic materials to directly inhibit thrombus formation has been studied. Mitigation of thrombus formation can be achieved through a number of coagulation or fibrinolytic pathways.

The surfaces of ePTFE prostheses have been modified to minimize pro-coagulation or anti-fibrinolytic mechanisms or conversely promote anti-coagulation and pro-fibrinolytic pathways. Thrombomodulin, an endothelial cell surface glycoprotein, strongly binds with thrombin forming a complex that inhibits the pro-coagulant properties of thrombin. Thrombomodulin also activates the anti-coagulant protease protein C, which further hinders thrombus formation (Li et al., 2000). Li et al. initially demonstrated that a soluble recombinant human thrombomodulin dose dependently retarded α-thrombin-induced porcine arterial smooth muscle cell proliferation in vitro (Li et al., 2000). In a follow-up study, the authors demonstrated a reduction in neointimal hyperplasia in immobilized recombinant human thrombomodulin coated ePTFE stent grafts (Wong et al., 2008).

Heparin, a glycosaminoglycan that inhibits both thrombin and activated factors IX, X, XI, and XII, which are involved in the conversion of prothrombin to thrombin (Lin et al., 2004) has been extensively investigated. In addition to its anti-coagulation properties, heparin has been shown to reduce smooth muscle cell proliferation and thus possesses the capability to inhibit intimal hyperplasia (Clowes and Karnowsky, 1977). Lin et al. demonstrated the reduction in neointimal hyperplasia at both the proximal and distal anastomoses on heparin-coated ePTFE grafts in a baboon arteriovenous shunt model, as shown in Figure 1 (Lin et al., 2004). In a canine carotid artery interposition model, the end-point covalent attachment of heparin using the Carmeda^® BioActive Surface (CBAS) technology to an expanded polytetrafluoroethylene graft compared favorably to a bare ePTFE graft. In a 2 h acute study, all five heparin-immobilized grafts remained patent exhibiting strong thromboresistance, while 60% of the control grafts occluded. In a longer-term study, 13 of 16 control grafts had occluded as early as 3 days, yet all CBAS–ePTFE remained patent. Following 180 days in the end-to-end anastomoses, seven CBAS–ePTFE grafts had occluded, while the three control grafts initially patent at 3 days remained patent or partially patent suggesting the heparin-immobilization reduced early graft thrombosis. Although, there was no statistical loss of heparin function at 12 weeks, the decreasing trend between weeks 2 and 12 suggests that at 180 days, the increase in occluded CBAS–ePTFE grafts may have resulted from a loss in heparin bioactivity (Begovac et al., 2003). In an ex vivo study, Heyligers et al. demonstrated a reduction in fibrinopeptide A on the heparin-immobilized grafts, suggesting an inhibition of fibrin deposition. Scanning electron microscopy revealed no platelet aggregation on the surface of the CBAS–ePTFE grafts confirming the thromboresistant nature of the heparinized ePTFE grafts (Heyligers et al., 2006). The promise that the heparin-bonded vascular graft could prevent early graft thrombosis through anti-coagulation and anti-platelet mechanisms led to its commercialization (Propaten^® W. L. Gore & Associates, Inc., Flagstaff, AZ, USA) and subsequent use as a peripheral vascular graft in humans.

In addition to being a part of the anti-coagulation and anti-platelet mechanisms, ECs also maintain hemostasis by providing a blood-compatible lining (Mason et al., 1977). In the absence of an adherent and coherent endothelium along the luminal surface of a synthetic graft, late stage thrombosis may still occur despite anti-coagulation therapy. Consequently, research has also focused on improving endothelial cell growth, retention, and confluency on the luminal surface of synthetic grafts. Due to electrostatic and hydrophobic interactions with ePTFE vascular grafts, incomplete endothelialization is typically encountered (Lu et al., 2013). Williams et al. have recently studied the effect of conjugated mouse laminin type 1 on endothelialization and neovascularization of ePTFE grafts as interpositional aortic grafts in rats. The presence of red cells, leukocytes, fibrin, and platelets in the control grafts suggested a highly thrombogenic surface, while the laminin type 1 conjugated grafts exhibited a complete lining of cells, indicative of an intact endothelium as shown in Figure 2. The authors also observed neovascularization in the interstices of the ePTFE grafts in the laminin-conjugated grafts (Williams et al., 2011).

Alternatively, Kibbe and colleagues mechanically coated the luminal nodes and fibrils of ePTFE with poly(1,8-octanediol citrate) (POC) and implanted the grafts in a porcine carotid model. The authors demonstrated increased endothelial cell growth; however, confluency was only reached at 10 days. Moreover, the improved growth rate did not limit neointimal hyperplasia nor were the authors able to confirm that the neoendothelium was functional (Kibbe et al., 2010). Hoshi et al. took it a step further by covalently immobilizing heparin to the shear thinned POC-coated ePTFE vascular grafts. The authors demonstrated reduced whole blood clotting and platelet adhesion of the heparin immobilized POC–ePTFE grafts when compared to both POC–ePTFE and ePTFE grafts. In addition, the heparin conjugated grafts supported both endothelial cell and blood outgrowth endothelial cell adhesion as evidenced by expressions of von Willebrand factor (vWf) and vascular endothelial cadherin (Hoshi et al., 2013). The authors also claimed that SMCs cultured on the heparin–POC–ePTFE grafts showed increased expression of α-actin and decreased cell proliferation, but the smooth muscle α-actin (SMαA) expression was only examined qualitatively. The cell proliferation data showed that the heparin conjugated POC–ePTFE grafts resulted in a decreased SMC proliferation rate only when compared to two-dimensional cell culture on tissue culture polystyrene (TCPS) and not when compared to POC–ePTFE grafts (Hoshi et al., 2013). Although this data does not suggest that intimal hyperplasia may be reduced due to decreased SMC with the heparin immobilized POC-coated ePTFE grafts, Hoshi et al. did demonstrate the combined benefit of anti-coagulant therapeutic delivery and improved endothelialization.

In a study by Lu et al., the authors coated 6 mm diameter ePTFE vascular grafts with an anti-CD133 antibody-functionalized heparin/collagen multilayer. Poly(ethyleneimine) (PEI) was electrostatically bound to the surface of the negatively charged ePTFE graft. Alternating immersion in heparin and collagen solutions generated a layer-by-layer self-assembled multilayered graft, which was then crosslinked with glutaraldehyde in the presence of anti-CD133 to immobilize the antibody to the surface of the vascular graft. The authors chose to immobilize CD133 to the surface of the graft as CD133 is a cell surface antigen expressed on endothelial progenitor cells, which induced rapid endothelialization in a 7 day porcine carotid artery transplantation model. In addition to the in situ endothelialization, the authors reported significantly decreased platelet adhesion in the heparin/collagen coated ePTFE grafts. Despite the promise of the reported work, future studies will need to address two issues – appropriately sized ePTFE grafts (<6 mm) and longer in vivo studies to ensure no late stage thrombosis occurs due to a loss of functional endothelium (Lu et al., 2013).

Flameng and colleagues recently coated stem cell homing factor (SDF-1α) on the surface of Gelsoft™(Vascutek Ltd., Inchinnan, Scotland) and Polymaille C (Pérouse Laboratories, Ivry le Temple, France), which are both knitted polyester vascular grafts pre-coated with collagen. The authors examined SDF-1α as it acts as a chemoattractant for hematopoietic stem cells while inducing the recruitment of endothelial progenitor cells. The authors observed an increase in endothelialization from 27 ± 4 to 48 ± 4% with the SDF-1α coating compared to vascular grafts alone in an ovine model. Concurrently, they also observed a decrease in intimal hyperplasia (De Visscher et al., 2012).

One approach in designing tissue-engineered vascular grafts (TEVGs) is through the implantation of an “off-the-shelf” acellular vascular scaffold. In vivo tissue engineering of vascular grafts necessitates the host’s cells to infiltrate and populate a non-thrombogenic three-dimensional porous matrix, while concomitantly preventing blood leakage through the graft. As stated, the scaffold must also remain compliant to preserve the necessary hemodynamic environment, yet provide sufficient burst pressure and suture retention strengths. Given the extensive list of pre-requisites of a tissue-engineered vascular graft, numerous approaches have been adopted.

Walpoth and colleagues recently evaluated a bilayered biodegradable electrospun polycaprolactone (PCL) scaffold as a vascular graft. The authors prepared the bilayered PCL graft by electrospinning two different concentrations of PCL to obtain a nanofibrous luminal surface, and a microfibrous adventitial side (de Valence et al., 2012). The microfibrous matrix was designed to allow a dense infiltration of the SMCs, while the nanofibrous structure was designed to allow the ECs to bridge the gaps to form a confluent monolayer to prevent blood leakage. In a 4-week bilateral porcine carotid artery model, the authors observed a marginal improvement in graft patency – 78 versus 67% for the PCL and ePTFE vascular grafts, respectively. An increase in neoendothelialization from 58 to 86% was also observed in the PCL grafts. Despite the increase in endothelialization, neointima, and thrombus formation were not improved with the PCL electrospun vascular grafts (Mrówczyński et al., 2014). In a long-term follow-up study, the authors compared the patency of the bilayered PCL vascular graft against the ePTFE prosthesis in a Sprague-Dawley rat aorta replacement study. At a median of 16.5 months, the rats were euthanized, and the grafts were excised and examined. Graft patency in the PCL grafts was 100%, while only 67% of ePTFE grafts remained patent. Although, there was a reduction in calcification and a concomitant improvement in cell infiltration and graft compliance with the PCL grafts when compared to ePTFE grafts, the PCL graft compliance was markedly less than the native aorta (Mugnai et al., 2013).

In a progressive study, Wang et al. (2014) examined the effect of PCL fiber diameter and pore size on arterial regeneration in a rat abdominal aorta model. All grafts remained patent through 28 days, with one thicker-fiber (5–6 μm) graft becoming occluded at 100 days (80% graft patency). Of all patent grafts, the luminal surface was smooth and free of platelets and thrombi, with a stable inner diameter suggesting no measurable restenosis. Moreover, significant cell infiltration was observed as early as 7 days in the thicker-fiber graft with both hematoxylin and eosin (H&E) and 4′,6-diamidino-2-phenylindole (DAPI) staining when compared to the thinner-fiber (0.7 μm) graft. Of the infiltrating cells, SMαA-positive cells were observed at 7 days around the connective tissue, but had migrated toward the luminal surface at 14 days. In addition, two of three explanted grafts exhibited a dense medial layer of smooth muscle myosin heavy chain (SMMHC)-positive cells at 28 days suggesting the generation of mature smooth muscle, which contracted in response to vasoconstrictors potassium chloride (KCl) and adrenaline. Although complete endothelialization was not yet achieved at 28 days, vWf immunostaining revealed a coherent layer of ECs along the luminal surface of the graft at day 100, which underwent acetylcholine-induced relaxation following vasoconstriction. Given the functional smooth muscle and endothelium, histological staining with H&E, Masson’s trichrome, and Verhoeff-van Gieson (VVG) confirmed the initiation of vascular remodeling, as neotissue was observed in the lumen with ECM deposition within the graft. Although the elastin within the explanted graft did not exhibit the degree of undulation of the native aorta, the fibers were oriented circumferentially as part of as sub-endothelial layer. Despite the secretion of collagen and elastin, the circumferential strength of the explanted grafts mirrored that of the pre-implanted graft, while the magnitude of the contractile response of the explanted grafts were far less than the native aorta indicating little PCL matrix degradation (Wang et al., 2014). Given that arterial remodeling is expected to be slower in humans compared to rats (Swartz and Andreadis, 2013), the stiffer PCL (Young’s modulus ~ 21 MPa) vascular graft (Wang et al., 2014) could persist further mitigating vascular remodeling, while concomitantly limiting elastin synthesis (Crapo and Wang, 2010).

To further improve the interaction of PCL with both ECs and SMCs, recent work has focused on incorporating tripeptides to direct cell behavior. The cell adhesive motif RGD has been incorporated into a PCL-functionalized vascular graft. A naphthalene conjugated hexapeptide phenylalanine–phenylalanine–glycine–arginine–glycine–aspartic acid (FFGRGD) self-assembled on the surface of the hydrophobic PCL yielding pendant RGD handles, which have previously been shown to provide improved hydrophilicity, cell attachment, and spreading. In an arteriovenous shunt model in female New Zealand White rabbits, Zheng et al. observed reduced platelet adhesion, improved patency, and smooth muscle cell infiltration and greater endothelialization with the RGD-functionalized PCL grafts. Although the authors reported improved smooth muscle cell infiltration and vasoactivity, this did not result in a thicker smooth muscle tissue, nor was endothelialization complete even after a 4-week implantation (Zheng et al., 2012). In a similar study, the tripeptide cysteine–alanine–glycine (CAG) was electrospun with PCL fibers into a vascular graft and implanted in a Sprague-Dawley rat carotid artery model. Kuwabara et al. previously noted that the CAG tripeptide improved endothelialization, while it retarded smooth muscle cell proliferation. Upon excision at 1, 2, and 4 weeks, the authors reported improved endothelialization at all time points and virtually complete endothelialization had occurred within 2 weeks. The presence of vWf was demonstrated at 1 week, while the production of endothelial nitric oxide synthase (eNOS) was detected as early as 1 week, demonstrating functional endothelium. The authors also demonstrated reduced SMαA expression at 6 weeks in the CAG-modified PCL graft, suggesting a potential to reduce intimal hyperplasia (Kuwabara et al., 2012); however, SMαA is a contractile phenotype marker protein and may not give a true representation of SMC proliferation.

Although these studies investigated the ability of PCL-modified vascular grafts to either improve patency, endothelialization, or SMC infiltration, only the study by Wang et al. fully examined neovascularization in vivo. Matsumura et al. had previously engineered a vascular graft consisting of polyglycolide knitted fibers and an l-lactide and ε-caprolactone copolymer sponge reinforced with glycolide and ε-caprolactone copolymer monofilaments, but only recently applied the biodegradable scaffold as a cell-free graft in a canine inferior vena cava (IVC) model. Immunohistological studies revealed endothelialization (factor VIII-positive) and SMC proliferation (SMαA) in the TEVG at 1 and 2.5 months with elastic and collagenous fibers also observed at 24 months. The authors reported no significant differences in the hydroxyproline, elastin, and calcium contents between the vascular graft and the native IVC up to 24 months follow-up. Moreover, the elastic modulus of the graft reached that of the native IVC at 2.5 months and continued to mirror the IVC up to 24 months following implantation (Matsumura et al., 2012). In a follow-up study, the authors examined their vascular graft in a canine pulmonary artery model. Again, they reported no significant differences in hydroxyproline and elastin content between the left pulmonary artery and the TEVG; however, significant calcification was observed with the TEVG at 12 months. Although the authors noted a lack of stenosis and thrombosis in the vasculature of any of the animals, the tubular scaffolds were 8 mm in diameter (Matsumura et al., 2013) and may not replicate the hemodynamics of a small-diameter blood vessel.

In another study examining neovascularization, Wang and colleagues implanted a poly(glycerol sebacate) (PGS) vascular graft supported with an outer PCL sheath in the abdominal aorta of Lewis rats. The sheath was electrospun around the tubular PGS graft to strengthen the graft, allowing it to be sutured in place, while preventing blood loss through the highly porous PGS network. The authors selected PGS because it is elastomeric, allowing for effective transduction of mechanical stimulation, while degrading rapidly in vivo, hence minimizing the host response to a foreign body. At 3 months, the authors observed significant SMC infiltration into the remodeled graft wall; however, SMαA expression was localized away from the luminal surface as shown in Figure 3 (Wu et al., 2012). The authors also demonstrated significant ECM production at 90 days. Verhoeff’s, Masson’s trichrome and safranin O staining revealed the presence of elastin, collagen, and glycosaminoglycans in the neoartery, nearing the ECM protein content of the native aorta, as shown in Figure 4 (Wu et al., 2012). The considerable ECM protein content also had a significant impact on the mechanical properties. The neoartery was tough yet compliant with a burst pressure approaching that of the aorta and greater than the saphenous vein. Given the promising results, future studies with large animals more representative of the human vasculature are proposed.

Another material garnering attention as a vascular biomaterial is the elastomeric poly(ester urethane)urea (PEUU) derived from PCL diol, 1,4-diisocyanatobutane and putrescine in part due to its mechanical properties, as it has a burst pressure strength and suture retention strength approaching that of the ITA (Nieponice et al., 2008). Further to their original study, Vorp, Wagner, and colleagues have covalently attached MPC to the luminal surface of electrospun PEUU conduits to impart anti-thrombogenicity. The vascular grafts were then implanted in the abdominal aorta of rats and examined upon sacrifice at 4, 8, 12, and 24 weeks. The authors demonstrated a significant reduction in platelet adhesion in the PMA-conjugated PEUU grafts, with a corresponding patency rate of 92% compared to just 40% in the uncoated controls at 8 weeks. In the occluded grafts, 90% were attributed to acute thrombosis, while the remaining 10% failed due to intimal hyperplasia. The PMA-immobilized PEUU grafts also yielded aligned collagen and elastin fibers, both necessary precursors in neovascularization. Moreover, the authors also demonstrated an intact endothelium as evidenced by the continuity in vWf immunostaining between the native aorta and the electrospun PEUU graft. The authors also reported significant changes to the dynamic compliance and ultimate tensile strength of the functionalized PEUU grafts upon excision. An initial increase in the dynamic compliance and concomitant decrease in ultimate tensile strength was observed at 4 weeks and believed to be the result of acute degradation of the nodes of the electrospun fibers (Soletti et al., 2011).

Although the synthetic grafts may be easier to handle, suture, scale up, and sterilize, while yielding more reproducible properties, some researchers believe that the high modulus and tensile strength exhibited by fibroin, a component of spider silk, is equally viable as a vascular scaffold material. Catteneo et al. electrospun fibroin into 1.5 mm diameter tubular grafts that were implanted in the abdominal aorta of Lewis rats in end-to-end anastomoses. Upon explantation at 7 days, the constructs were infiltrated with SMCs and lined with ECs as evidenced by the immunostaining of SMαA and vWf, respectively. The authors also reported the presence of elastin at 7 days, which by volume density, was approximately 50% of native vessels. Despite the evidence of complete endothelialization, the graft was only 1.5 cm in length (Cattaneo et al., 2013). In a similar study, Lovett et al. gel spun aqueous fibroin solutions to yield 2 cm silk tubular scaffolds and implanted them in the abdominal aorta of Sprague-Dawley rats. Silk fibroin (SF) grafts remained patent up to 4 weeks implantation, while acute thrombosis in all PTFE constructs was observed in 24 h. The recruitment of vascular cells from the adjacent native aorta resulted in a medial smooth muscle layer and complete endothelialization as confirmed by SMαA and factor VIII staining. Further to the in vivo studies, the hemocompatibility of the SF grafts were also studied in vitro. A reduction in thrombin and fibrinogen adsorption was observed compared to PTFE and although an increase in platelet adhesion was seen from incubation of platelet-rich plasma on SF films, the platelets were deemed non-activated by their non-spreading morphology (Lovett et al., 2010). In a complementary study, Yagi et al. prepared three kinds of knitted double-raschel silk fiber grafts coated with crosslinked SF again with an internal diameter of 1.5 mm, but with a length of 7 cm. The grafts were also implanted in the abdominal aorta of Sprague-Dawley rats. Upon excision at 8 weeks, gross observation of the vascular constructs revealed patency of the most elastic and flexible SF tubular graft tested, attributed to a reduction in intimal hyperplasia. Immunohistological studies revealed SMC infiltration, resulting in smooth muscle tissue thickness similar to that of the native rat aorta; however, complete endothelialization was not achieved, in particular at the midpoint of the vascular graft following 8 weeks implantation (Yagi et al., 2011). Given that incomplete endothelialization was observed, along with growing smooth muscle tissue, longer studies are necessary to ensure intimal hyperplasia does not result in occlusion of the vascular grafts.

Attempts to minimize intimal hyperplasia from SF scaffolds have been explored through the incorporation of heparin. Zhu et al. prepared heparin-loaded, porous scaffolds through a modified freeze-drying approach, which permitted their fabrication into tubular grafts. The authors demonstrated near complete release of heparin within 3 days corresponding to a reduction in SMC metabolic activity in vitro at 4 and 7 days. The ability of the SF scaffolds to facilitate tissue integration in vivo was evaluated upon excision of subcutaneously implanted tubular grafts in Sprague-Dawley rats. At 2 weeks, H&E staining revealed initial tissue integration in the heparin-loaded grafts as evidenced by an increased number of capillaries; however, at 4 weeks, the number of capillaries decreased suggesting potential resorption of the de novo blood vessels. Although SMC metabolic activity was retarded at 4 and 7 days following the rapid release of heparin, there was no statistical difference in metabolic activity at 14 days (Zhu et al., 2014), suggesting a slower release profile or covalent attachment of heparin may be needed to prevent long-term intimal hyperplasia and ultimately graft occlusion.

Despite some promising results from foreign biomaterials, other researchers have proposed that clinical success will incorporate all fundamental features of the vascular wall. Kumar et al. have recently incorporated both fibrillar collagen type 1 and an elastin-like polypeptide as a vascular graft. Collagen fibrils were prepared from monomeric type 1 collagen extracted from rat tail tendon before the ELP was coated on the surface of the collagen and rolled to generate a luminal ELP layer on multiple fibrillar collagen layers. Initial studies revealed reduced platelet adhesion in the ELP-coated vascular graft when compared to the uncoated collagen graft control. Histologically, the 2-week rat aorta interposition study revealed little neointima formation, and continuous vWf staining indicative of an intact endothelium. Despite the promising short-term in vivo data, the burst pressure strength and suture retention strength still remain a fraction of that of the ITA (Kumar et al., 2013).

In effort to improve the mechanical properties of a vascular graft yet still incorporate the fundamental features of the vascular wall, research has focused on decellularized arterial conduits, which have demonstrated similar burst pressure strength, suture retention strength, and compliance to native blood vessels (Wilshaw et al., 2012; Sheridan et al., 2013; Xiong et al., 2013). Recently, Hwang et al. harvested abdominal aorta from Sprague-Dawley rats, which were evaluated as infrarenal allografts. The decellularized implants appeared thinner with the elastic fibers of the medial layer exhibiting less undulation when compared to the native aorta. Following 8 weeks implantation, intimal thickening due to increased collagen fibers and SMαA-positive cells was observed. Although vWf staining revealed the presence of endothelial-like cells on the luminal surface of the implanted grafts, the expression was noticeably less than the native aorta (Hwang et al., 2011). In a related study, Akhyari and co-workers harvested rat aortae, which the authors decellularized and coated with fibronectin. The coated vessels were implanted systemically in Wistar rats for up to 8 weeks. Explanted fibronectin-coated grafts exhibited improved endothelialization and medial recellularization compared to uncoated controls. Despite the acceleration in endothelial cell recruitment, a concomitant increase in local hyperplasia was still observed (Assmann et al., 2013).

Similarly, Niklason and colleagues harvested human aortae and then decellularized the constructs to produce human tissue-engineered vessels (hTEV). The decellularized hTEVs exhibited burst strength pressures similar to the saphenous vein, yet still inferior to the native ITA. The grafts were implanted in an end-to-end anastomoses in the abdominal aorta of female nude mice. Upon excision of the grafts at 6 weeks, there was evidence of neointima and neotissue formation on the decellularized grafts. Although cells did not fully infiltrate the decellularized graft, both elastin and SMCs were prevalent. In addition, vWf staining demonstrated the recruitment of ECs to the luminal surface of the vascular graft. The authors acknowledge that larger animal studies are needed and that these studies also need to examine the potential for late stage stenosis following neotissue or neointima formation (Quint et al., 2012).

While some researchers have adopted the approach of in vivo tissue engineering, where cells are recruited into the biodegradable scaffold from adjacent tissue, others propose that seeded cells serve as the “building blocks of neotissue.” In this regard, there are two unique approaches to enhance tissue regeneration and remodeling: cell sheet self-assembly and cell sodding of biodegradable scaffolds.