Abstract
The development of small-caliber tissue-engineered vascular grafts (sTEVGs) presents several challenges, including achieving balanced endothelialization, facilitating smooth muscle cell infiltration, preventing leakage, and ensuring anti-thrombogenic properties, while maintaining mechanical strength sufficient to withstand physiological pressures, surgical handling, and suturing. Here, we present a multi-layered polycaprolactone (PCL)-based sTEVG using a combination of electrospinning and 4-axis printing, providing precise control over scaffold porosity, fiber alignment, and tunable mechanical properties. To improve biocompatibility and hemocompatibility, the PCL nanofibers were functionalized with sulfated polysaccharides purified from the marine invertebrate Holothuria tubulosa, which significantly enhanced endothelialization and provided strong anti-thrombogenic properties. The inner layer of tightly aligned electrospun nanofibers supported rapid formation of a mature endothelium, while preventing graft leakage even at supraphysiological pressure (>1100 mmHg). The middle layers, combining circumferential electrospun nanofibers and 4-axis printed microfibers, increased scaffold porosity, and promoted adhesion, orientation and infiltration of human coronary artery smooth muscle cells (HCASMCs), facilitating functional tunica media formation. The outer layer of randomly oriented electrospun nanofibers contributed significantly to the mechanical properties of the graft, namely elasticity, toughness, burst pressure, and resistance to physiological vessel pressures, thus mimicking the tunica adventitia. The customizable four-layered graft integrates structural and biological cues to address key limitations of sTEVGs, representing a valuableoff-the-shelf alternative to autologous grafts.