Bilayer vascular grafts separately composited with nitric oxide-releasing keratin conjugates and hydrogen sulfide-releasing heparin conjugates

Gasotransmitters such as nitric oxide (NO) and hydrogen sulfide (H₂S) play crucial roles in various physiological and pathological processes, including angiogenesis, vascular homeostasis, thrombosis, inflammation, and remodeling. In addition to playing their respective roles, these two gasotransmitters act synergistically to regulate physiological pathways. This study designed and fabricated bilayer tissue-engineered vascular grafts with respective dual NO and H₂S release capability for vascular cell regulation according to the spatiotemporal regulation strategy. Keratin/methacrylated arginine conjugate (KMA) was prepared and then electrospun with poly(ε-caprolactone) (PCL) with NO release potential, serving as the inner layer of grafts. For the outer layer of grafts with H₂S release capability, heparin/4-aminobenzothioamide conjugate (HAT) was synthesized and then coaxially electrospun with PCL. These two conjugates could retain keratin's good biocompatibility and heparin's anticoagulation nature. The bilayer grafts selectively promoted the proliferation of HUVECs and inhibited the abnormal proliferation of HUASMCs. More importantly, the release of NO and H₂S can stimulate the secretion of the other, thus resulting in a synergistic effect. In addition, these grafts exhibited antibacterial, antioxidant, and anti-inflammatory properties. Furthermore, the grafts could modulate macrophage polarization toward the M2 phenotype. In rat models with abdominal aorta replacement for 1 month of implantation, the grafts facilitated rapid endothelialization with enhanced anticoagulant and anti-calcification properties. These findings suggest that these bilayer grafts are promising candidates for small-diameter tissue-engineered vascular grafts.