Research On Tissue Regeneration Of Small Caliber Regenerated Silk Fibroin Vascular Scaffold After Implantion Into Animal

Clinically,there is an increasing demand for vascular replacement in the field of blood vessel reformation,and artificial blood vessels provide an alternative strategy for autologous transplantation.However,small caliber(less than 6 mm in diameter)blood vessels are often associated with the risk of thrombosis and early damage.Although very promising research results have been obtained,they have not been implemented in specific clinical applications.Previously,we have explored a technique for preparing a three-dimensional small-diameter vascular stent designed from a silk fabric as a basic framework composite regenerated silk fibroin.The scaffold has a three-layered biomimetic structure(three layers of inner,middle and outer membranes)similar to an arterial vessel,and the structure and properties of the vascular scaffold can be adjusted by changing the preparation parameters.In the early stage,the silk fibroin small-diameter vascular scaffold has been implanted into animals for cell-shaped research and patency observation.The results show that the silk fibroin scaffold keeps long-term patency after implantation,and can well support the adhesion,spreading and proliferation of vascular endothelium and smooth muscle cells.This silk fibroin small-bore vascular scaffold is biodegradable and ultimately hopes to induce autovascular remodeling in situ.The vascular tissue function,especially the mechanical properties,is the key to the success of vascular reconstruction.The mechanical pi operties of blood vessels depend on the extracellular matrix.Elastic fiber and collagen fiber are important components that impart vascular mechanical properties.The self-assembly and arrangement of two protein fibers.It is a sign of rejuvenation of revascularization.Therefore,this paper focuses on the tissue regeneration and maturation ability of silk fibroin small-caliber vascular stents implanted in animals.First,the raw silk is degummed,dissolved,dialyzed and concentrated to obtain a silk fibroin solution,and the composite silk fibroin fabric is obtained by a freeze-drying method using a self-made mold to obtain a three-layered small-diameter vascular scaffold.Rabbits were used as recipients,and silk fibroin small-diameter vascular scaffolds were used as vascular prostheses.Rabbit carotid arteries were implanted by end-to-end anastomosis.The animals were sacrificed at the set time points,the materials were taken out,and foreign bodies were treated and stored.From the SEM photograph and the HE staining picture,it can be seen that when the scaffold is implanted for one month,the whole tube wall has a sandwich structure,new tissue is formed on the inner and outer sides of the tube wall,and the new tissue is arranged in an orderly circumferential direction;At the time of month,the morphological structure of the entire wall is similar to that of one month,and the area of the regenerated tissue is widened.However,due to the degradation of the regenerated silk fibroin porous material,some vacancies appear inside the scaffold;at six months,the fibroin porous structure in the tube wall basically disappeared,the new tissue covered the entire wall;one week to three months after implantation,there was no obvious orderly arrangement in the scaffold,mainly extracellular matrix deposition.Six months after the implantation,the regenerated tissue began to be ordered and distributed.The fiber structure was gradually aggregated into large fiber bundles from the initial small fibers,and further entangled and bent.From the analysis of immunohistochemical photos,elastin,collagen type I and collagen type III began to accumulate in the scaffold space at the beginning(one week)of vascular stent implantation;with the passage of time,three protein expression levels Continued to increase to nine months,then after a dynamic balance.Collagen type I is most secreted,elastin is second,and collagen type ? is the least.RT-PCR quantitative analysis showed that the expression of elastin showed a dynamic change throughout the time period after implantation.At the initial stage of implantation(one week),the relative expression level of mRNA of traumatic elastin was significantly higher than that of autologous blood vessels;one week to three months of implantation,the expression level of elastin decreased significantly with the deposition of extracellular matrix in the scaffold.After three months,the silk fibroin material was significantly degraded,and there were vacancies inside the scaffold,the proliferation activity of smooth muscle cells and the secretion capacity of extracellular matrix were improved.Therefore,the elastin expression ability was significantly increased at the time of implantation for six months.With the orderly self-assembly and maturation of the regenerative tissue,the level of elastic expression was significantly down-regulated at the time of implantation for nine months.The dynamic changes in the expression capacity of collagen type I and collagen type III were similar to those of elastic fibers,but collagen type III remained at a high level of expression after six months of implantation.From the WVG staining and Masson staining pictures,the distribution of elastic fibers appeared on the inner and outer sides of the tube wall after one month of implantation;at three months,the inner and outer elastic fibers became continuous and curved,and with the extension of the implantation time,it is more orderly and regular,and gradually spreads from the sides to the inside.After six months of implantation,an irregular fibrous purple distribution appeared evenly on the inside of the scaffold.At nine months,the elastic fibers were further ordered,and the thickness and distribution of the elastic fibers inside the stent were uniform.The formation and distribution of collagen fibers are similar to those of elastic fibers.The outer collagen fibers are thicker and the inner collagen fibers are finer and more tortuous.The lysyl oxidase like 1 that promotes the self-assembly of elastic fibers and collagen fibers accumulates in the scaffold and maintains equilibrium after three months of implantation,but the expression level is up-regulated after six months,especially at nine months.In conclusion,the silk fibroin small-diameter vascular stent used in this study can obtain good tissue regeneration after implantation in animals,indicating that the stent has a good application prospect in clinical practice.