Bilayer vascular grafts separately loaded with sodium copper chlorophyllin and keratin-based hydrogen sulfide donor with pro-endothelialization, anti-thrombogenicity, anti-inflammation, and anti-calcification properties.

The clinical utility of small-diameter vascular grafts (SDVGs) remains limited due to thrombosis, inflammation, and intimal hyperplasia, which compromise long-term patency. Mimicking the structures and functions of blood vessels, bilayer SDVGs with gradient pore sizes were fabricated, thereby preventing the infiltration of vascular smooth muscle cells (VSMCs) into the inner layer. Additionally, sodium copper chlorophyllin (SCC) was embedded in the inner layer, potentially generating NO from endogenous donors in the blood and regulating vascular cells. Keratin-based H2S donor of KSN was synthesized and then electrospun with poly(L-lactide-co-ε-caprolactone) (PLCL) to serve as the outer layer of the grafts. The bilayer grafts promoted rapid endothelialization by selectively enhancing the adhesion, proliferation, and migration of vascular endothelial cells (VECs) while inhibiting those of VSMCs. More importantly, the released NO and H2S synergistically enhanced the anti-thrombotic, anti-inflammatory, and anti-calcification properties of the grafts. Furthermore, the bilayer grafts maintained the contractile phenotype of VSMCs and polarized macrophages toward the M2 phenotype. The grafts achieved patency with negligible intimal hyperplasia and calcification in the rat abdominal aorta replacement models for 1 month of implantation. The grafts modulated VECs via the PI3K-AKT signaling pathway, focal adhesion, apoptosis, and regulation of actin cytoskeleton, while regulating VSMCs through gap junction, ECM-receptor interaction, adherens junction, focal adhesion, and PI3K-AKT signaling pathway. These bilayer grafts with rapid endothelialization, antithrombogenicity, anti-inflammation, and anti-calcification properties are promising candidates for tissue-engineered SDVGs.