Self-Healing Tissue Engineered Vascular Grafts with Immobilized Biological Cues Exhibit Efficient Endothelialization and Patency

Introduction: Cardiovascular and end-stage renal diseases (ESRD) are major cause of mortality in the United States, with over 26 million adult suffer from coronary artery disease, receiving more than 370,000 bypass grafting operation annually, and over 800,000 ESRD patients, with approximately 71% undergoing hemodialysis. Hemodialysis patients receiving arteriovenous fistula constitute only 15.1% due to long waiting time, mismatch compliance between vein and artery, comorbid diseases such diabetes, aging and repeated access failure. The growing demand for clinically-suitable alternative vessels drives the advancement of next-generation vascular grafts to be used as bypass vessels or hemodialysis shunts. Patients with ESRD require life-long hemodialysis graft that are robust and maintain their mechanical properties without causing infection and clotting. Early-cannulation of arteriovenous grafts is hampered by high risk of bleeding post-cannulation, pseudoaneurysm, intimal hyperplasia, thrombosis, and hematoma. Consequently, patients rely on a tunneled dialysis catheter for several weeks until the grafts are coated with host cells and extracellular matrix molecules that promote maturation and minimize clinical risks. Substantial progress in bypass surgery procedures has been achieved using synthetic, native, and tissue engineering vascular grafts (TEVG), improving, in part, the patency rate and reducing graft failure. Native blood vessels exhibit innate self-healing capability, enabling restoration of their original structure after damage. Thus, there is unmet need for conferring self-healing behavior to vascular grafts to reduce the risk of bleeding upon early cannulation and to preserve mechanical integrity after multiple cannulations.

Materials and

Methods: A Self-Healing Tissue Engineered Vessel (SH-TEV) was developed by electrospinning a rationally designed autonomous self-healing polyurethane, named PU-DAA, around a tube of a natural biomaterial, small intestinal submucosa (SIS), followed by functionalization of the luminal surface with vascular endothelial growth factor (VEGF) to promote endothelialization. Linear PU-DAAx was developed by thermal condensation of soft segment, PTMG, with isophorone diisocyanate in the presence of ferric acetylacetonate as catalyst to form the prepolymer. Further chain extension with different molar ratios of [DAA-diol hard segment]/[PTMG soft chain] (1.0, 1.5, and 2.0) resulted in PU-DAA1.0, PUDAA1.5, PU-DAA2.0, respectively. The self-healing capability of PU-DAAx was assessed by mechanical test under dry and wet conditions at different times to evaluate restoration of mechanical integrity after damage. Coating the polymer film (160 µm thickness) with poly-L-lysine, heparin and H2R5 sequentially supported spreading and proliferation of human dermal fibroblasts. The SH-TEV tube (1 cm length × 2 mm inner diameter) was functionalized at the luminal surface with heparin sulfate via EDC/NHS chemistry to reduce thrombogenicity. Then, VEGF was immobilized on the lumen via the heparin-binding domain to promote endothelialization. Afterwards, SIS-TEVs and SH-TEVs were implanted as interposition aortic graft in rats using end-to-end interrupted suture technique. The self-healing capability of transplanted SH-TEVs was evaluated by measuring the bleeding time, after puncturing with 26-gauge needle multiple times.

Results, Conclusions, and Discussions: Engineering SH-TEVs was achieved by electrospinning of autonomous self-healing PU-DAA around a biomaterial SIS tube, followed by immobilization of heparin, VEGF, and H2R5 at the luminal surface. The design of PU-DAAx relied on the synergy between reversible H-bonds and dynamic oxime bonds to confer self-healing capability, toughness, and strength to the grafts. To achieve efficient self-healing capability of PU-DAA, the ratio of [DAA]/[PTMG] was optimized to minimize the self-healing time, while maintaining mechanical strength (Fig. A). Unlike PU-DAA1.0 and PU-DAA2.0, PU-DAA1.5 exhibited high strength (3.92 ±0.09 MPa), toughness (22.45±1.99 MJ/m3), and fast autonomous self-healing (86.44±6.65 % after 12 hr) under physiological conditions. DSC analysis of PU-DAAx showed a Tg value in the range of -26.6 to 6.4 °C, while SAXS analysis of demonstrated formation of short-range order structures as indicated by appearance of small peak at lower q values ( < 1 A°). The results were corroborated with XRD analysis which revealed a broad peak at 22° characteristic to amorphous PU-DAAx, and absence of any crystalline diffraction peaks. The self-healing layer supported attachment, spreading and proliferation of fibroblasts, suggesting potential interactions with host cells upon transplantation. Indeed, when SH-TEVs were implanted into the rat aorta for 4 weeks (Fig. B, C), they remained patent without any thrombosis (100% animal survival and 100% graft patency), formed native-aorta-like structure. Specifically, the grafts contained multiple cell layers in the lumen (Fig. D), with the top layer of eNOS-expressing endothelial markers, and several underlying layers of calponin-expressing smooth muscle cells (Fig. E). Most notably, the grafts showed extraordinary self-healing ability upon puncturing with various size needles (hemostatic time < 40 sec). Unlike the majority of grafts reported in the literature, no anti-platelet treatment was used to improve the graft performance post-implantation. Overall, the results demonstrated the self-healing capability and patency of the acellular SH-TEVs and their potential applications as hemodialytic vascular access grafts.

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