Silk fibroin single-layer and two-layer tubular scaffolds for small diameter blood vessel regeneration

Bypass surgeries are commonly performed to allow the peripheral or coronary revascularization. To date, the clinical approach for the replacement of small diameter blood vessels (inner diameter (ID) < 6 mm) is the use of autografts, despite the fact that they may not be the optimal solution. Tissue engineering has become a promising approach for vascular regeneration. Although vascular tissue engineering has reached promising results in clinical trials, tissue-engineered vascular grafts (TEVGs) exhibit some drawbacks, such as the regeneration of nonfunctional endothelium, a mismatch between the mechanical proprieties of grafts and natural blood vessels, long manufacturing times. Among available materials for fabrication of TEVGs, natural polymers exhibit good biological performances but usually lack the mechanical properties necessary for in vivo implantation. In contrast, silk fibroin (SF) excels for its peculiar mechanical properties and high biocompatibility. The present PhD thesis aims to design, fabricate and characterize innovative scaffolds based on SF for the regeneration of small diameter blood vessels able to overcome the limits of the autografts. Nanostructured electrospun SF (ES-SF) tubular scaffolds with 1.5 mm ID were successfully fabricated for the first time (Fig. 1A, B); in fact, 1.5 mm ES-SF tubes are novel SF scaffolds, not yet reported in the literature. Scanning electronic microscope (SEM) analysis showed a homogeneous and random fiber distribution in the nanometric range. ES-SF morphology allowed for the in vitro adhesion and growth of primary porcine smooth muscle cells (SMCs). Specifically, SMCs seeded on external surface of the ES-SF tubular scaffold were able to migrate inside tubes, reaching the lumen and demonstrating an appropriate scaffold porosity for cell migration. The results obtained by the in vitro characterization appear promising for the investigated final application. ES-SF tubes were mechanically characterized, exhibiting appropriate mechanical performance for the specific application. In fact, ES-SF tubes showed higher ultimate tensile strength (UTS) in circumferential direction than anterior descending human coronary arteries and higher strain at break (εb) in both directions than human saphenous veins, the gold standard for arterial bypass grafts. Furthermore, the suture retention strength (SRS) was similar to human grafts and the burst pressure (BP), calculated by rearranging the Laplace’s law, was higher than the upper physiological pressures, but lower than native human saphenous veins. ES-SF tubes were evaluated in vivo in a rat model in the short period. Acellular ES-SF tubes were implanted in the abdominal aorta of Lewis rats by end-to-end anastomosis. After 7 days, rats exhibited no signs of acute thrombosis and occlusion and the graft lumen showed the absence of aneurismal dilatation and apparent intimal hyperplasia. ES-SF tubes allowed the in vivo regeneration of a vessel-like structure similar to the native blood vessels, specifically inducing the elastin regeneration that only few TEVGs described in literature are able to promote. The in vivo favorable interaction between host cells and ES-SF tubes may be due to the combination of SF peculiar properties and the ES technique that allows for the fabrication of porous structures with nanofibers and high surface to volume ratio. These preliminary results showed that the ES-SF tubes would be promising off-the-shelf scaffolds for the regeneration of small diameter blood vessels. To better mimic the native structure of blood vessels, novel two-layer SF scaffolds were developed by the innovative combination of two techniques, the ES and the gel spinning (GS). In particular, two-layer SF tubular scaffolds consisted of an ES-SF tube coated with a GS-SF layer, specifically the ES layer acted as the tunica intima and the GS layer mimicked the tunica media. To enhance the endothelial cell adhesion and proliferation, the lumen of the two-layer SF tubes was functionalized with the RGD sequences by an innovative combination of carbodiimide and diazonium coupling to enhance the efficiency of the RGD functionalization. Two-layer SF tubes were mechanically characterized demonstrating similar or higher mechanical properties than native blood vessels. Specifically, two-layer SF tubes showed higher UTS in circumferential direction than anterior descending human coronary arteries and higher εb than human saphenous veins in both directions. Furthermore, the SRS of two-layer SF tubes is in the range of that of human grafts and the two-layer SF tubes demonstrated a BP similar to native human saphenous veins. Two-layer SF tubes were seeded in the lumen and on the outer surface with primary human aortic endothelial cells and primary human aortic smooth muscle cells, respectively. Cell-seeded scaffolds were in vitro cultured for 7 days in a perfusion bioreactor. The results confirmed the biocompatibility of the two-layer SF tubes and their ability to support adhesion and growth of primary human cells. The developed single-layer ES-SF and two-layer ES-SF/GS-SF tubes exhibited promising performance for small diameter blood vessel regeneration, in terms of morphological, mechanical and biological behavior.