Research On Patency And Reconstruction Of Small Caliber Silk Fibroin Tubular Scaffold After Replacement Of Rabbit Common Carotid Artery

There is an increasing need for vascular prosthesis in the field of surgical revascularization.Artificial grafts provide an alternative strategy for autologous tissue,however,small caliber(diameter < 6 mm)vascular scaffolds are associated with a high probability of thrombosis and early failure.In spite of promising results,tissue engineering vascular graft is not yet a clinical reality owing to the complexity of this approach.Previously,we took regenerated silk fibroin(SF)as perfusion solution and the silk fibroin fiber woven mesh as the basic framework to develop a 3-dimensional small caliber tubular scaffold.This tubular graft possessed three layers that was alike to the native vascular structure(tunica intima,tunica media and tunica externa).The pore structure and mechanical properties could be optimized through changing the fabricated parameters.To investigate the mechanism of tissue reconstruction after replacing the defect vessels with the small caliber SF tubular vascular scaffolds,the co-culture of vascular cells on the regenerated SF materials had been conducted.The feasibility of replacement between vessels and the small caliber SF tubular scaffolds was the main research in this paper.Employing end-to-end anastomosis surgery,the scaffold was implanted into rabbit common carotid artery as vascular prosthesis.After implantation,the long term patency of scaffolds were conducted at first.Then the endothelialization,cell adhesion and infiltration,and the expression of cell function factors in the scaffolds were researched.In addition,the tissue reconstruction of the implanted SF tubular scaffold was evaluated.Firstly,the patency of the implanted scaffolds were evaluated using anatomical observation,color doppler ultrasound and angiography at the given time.The results suggested that the scaffold was still unblocked and the blood flow in diastolic and systolic had no significant difference with native artery after implanted 15 months.Then,the endothelialization of the implanted scaffold was researched by scanning electron microscope(SEM).The results showed that the distinct endothelium layer was observed on the scaffold lumen after implanted 1 month.And above 3 months after implantation,the observed endothelial cell morphology was similar to native artery.Immunohistochemical studies were performed for vascular endothelia growth factor(VEGF)and platelet endothelial cell adhesion molecule-1(PECAM-1/ CD31)and immunofluorescent staining for VEGF to evaluate the endothelial cell function.The positive staining of an endothelial cell layer on the lumen area was confirmed after implantation 1 month,and it showed a similar pattern to those of native common carotid artery after 3 months.Moreover,haematoxylin-eosin(HE)staining results also proved that one layer of cells on the lumen was consist of endothelium with a spindle shape.To evaluate the cell infiltration and biodegradation of materials,HE staining was conducted to the explanted SF scaffolds and the result was obtained using inverted fluorescence microscope.The scaffold was infiltrated by a great deal of cells after implantation 2 weeks,and was almost fulfilled with cells after 3 months.The infiltrated or proliferative cells distributed equably in the scaffold after implantation 6 months.Besides,immunohistochemical studies were performed for ?-smooth muscle actin(?-SMA)and smooth muscle myosin heavy chain(SM-MHC)and immunofluorescent staining for ?-SMA to locate and evaluate the smooth muscle cells.The results indicated that the expression level of ?-SMA was lower initial stage of implantation.After implantation more than 6 months,the ?-SMA positive expression area showed a similar pattern to those of native common carotid artery.The expression level of cells in the implanted tubular scaffold was analysed with real-time fluorescent quantitative PCR.Results indicated that: the expression level of VEGF was higher than that of native artery during the implanting time.The expression level of ?-SMA was higher after implantation 2 weeks and then became lower until 12 months(40 %).However,the expression level of CD31 and SM-MHC was a bit lower than that of native artery.The expression level of the endothelial functional factors(VEGF and CD31)was superior to that of smooth muscle cells.