Sheath-core SF/PCL Fibers And Bilayered Vascular Scaffolds For Regeneration Of Small-diameter Vessels

When human blood vessels cannot work normally because of arteriosclerosis, thrombus, dilapidation or aging of vascular, implantation of vascular prostheses was necessary. Small-diameter artery(less than 6 mm) had not ideal substitute due to easy thrombosis in the clinical. The ideal way to treat vascular disease was in vivo angiogenesis which was based on the scaffolds. Therefore, design of vascular scaffolds was the urgent need of clinical. Silk fibroin(SF) fibers which prepared by electrospinnig were of good potential for scaffolds of vascular regeneration. If its mechanical properties were further regulated, the ability to regulate cell behavior and guide newly tissue formation was further enhanced, it could meet the needs for regeneration of small-diameter blood vessels. In this study, based on the coaxial electrospinning, the poly(ε-caprolactone)(PCL) was used as core layer to enhance the mechanical properties of SF fibers. The relationship between electrospinning conditions and the covering rate of core-sheath structure fibers(C-SF/PCL) was studied. The blood vessel cells were cultured on the C-SF/PCL fiber mats to study the influence of fiber surface composition and the structure of fiber mats to the behavior of cells. After cross-linking heparin, the blood and cells compatibility to the C-SF/PCL fiber mats were investigated. On these basis, the bilayered vascular scaffold was prepared by coaxial electrospinning, the activity and mechanism of scaffold for regeneration of vesels was evaluated in vivo.Firstly, the coaxial electrospinning was used to prepare the sheath-core structured C-SF/PCL fibers, and the covering rate of fibers was controlled by adjusting the volatilization rate of core or sheath solvents. results show that the core-sheath fibers with high covering rate were prepared when the core solvent volatilization rate slightly higher than the sheath. The C-SF/PCL fiber integrated the mechanical properties of PCL and bioactivity of SF. And the cells on the C-SF/PCL fiber scaffolds were similar to pure SF. C-SF/PCL fiber had more advantages than single or hybrid components in scaffold of vascular regeneration.Secondly, C-SF/PCL and polyethylene oxide(PEO) fibers were oriented collected by mandrel at the same time, and then leaching with water to prepare fiber mats with large pore(the average pore area was about 525 μm2). Results show that the fiber mats had different tensile properties on the transverse and longitudinal directions due to the effect of fiber orientation. And the mechanical properties of fiber mats could be regulated by compositing oriented and disordered fiber mats. The smooth muscle cells were cultured on this fiber mat, and the results show that cells could grow along the direction of the fibers orientation and migrated into the mat due to the large pore.Then, heparin was covalent bonded to the C-SF/PCL fibers by mediating of carbodiimide(EDC), and the amount was about 9 wt. %. In the first day, heparin had a sudden release, then the stable release. The release of accumulation of 14 days was about 34%. Platelet adhesion and hemolysis rate(1.9%) showed that the fiber mats after grafting heparin were of anticoagulant activity. Compared with fiber mats without heparin, endothelial cells could better adhesion and proliferation, and smooth muscle cells were obviously inhibit on the grafting heparin fiber mat. The fiber mats grafted with heparin were suitable for the scaffolds of vascular regeneration, it promote the growth of endothelial cells and inhibit the growth of smooth muscle cell proliferation, therefore guarantee the stability of the organizational environment in vivo.Further, small-diameter scaffolds were prepared by mandrel collecting and water leaching. The diameter of scaffold was about 2 mm, the wall thickness was about 650 μm, composed of two layers of fibers, the inner was compact, uniform, disordered arrangement of fibers, and the outer was loose, uniform, directional arrangement of fibers, thus the scaffold have functional partition. The breaking tensile strength(about 5.6 MPa of radial direction, about 3.6 MPa of axial direction), breaking elongation(about 88% of radial direction, and about 140% of axial direction), suture strength(1.8 N) and blasting pressure(3.4 MPa) were higher than that of natural blood vessels, which could meet the clinical requirements of the mechanical properties of scaffolds.Finally, the 2 cm long scaffolds were successfully replaced of the rabbit carotid artery by end to end. The wound of animals was healing well. Scaffold could withstand the impact of the blood flow and maintain patency in the experimental period. The scaffold could merge with the surrounding tissue. HE staining showed that the scaffold in vivo had no obvious inflammatory cells infiltration, immune response caused by material and slight inflammatory reaction. Blood vessel cells could migrate in the outer layer of the scaffold to build blood vessels. Weigert and Masson staining results showed that the collagen and elastic fibers of biylaered scaffold were obviously higher than that of single-layered scaffold with the time of repairing, which illustrated that the bilayered scaffold could promote the regeneration of the vascular tissue. Compared with single-layer scaffold, CD31(PECAM 1), SMC α-actin of the bilayered scaffold had higher positive expression. The bilayered scaffold created a better environment for vascular tissue repair, cell migration, adhesion, growth and function, promoting the activity of vascular regeneration and reconstruction.In conclusion, the C-SF/PCL fiber was prepared by coaxial electrospinning, which integrated the good mechanical properties of PCL and the biological activity of SF. And then small-diameter tubular scaffold with double structure was constructed by C-SF/PCL fibers. Research showed that the scaffold could promote the activity of vascular tissue repair and regeneration, provided a new material for small blood vessels.