以靜電紡絲製備角蛋白/蠶絲蛋白支架在血管組織工程的應用

Abstract

心血管疾病造成的死亡率歷年來一直居高不下，而繞道手術是臨床常用的一種治療心血管疾病方式。到目前為止，仍未發現適合用於動脈繞道手術的材料。因此本研究以靜電紡絲的方法將蠶絲中的絲素蛋白與人類頭髮中的角蛋白製備成多孔隙纖維管，以利血管組織工程移植物的發展。 首先將絲素蛋白(F)與角蛋白(K)分別以9:1 (FK91)、8:2 (FK82)、7:3 (FK73)的重量比混合均勻後，以靜電紡絲噴成纖維膜。藉由掃描式電子顯微鏡可得知所得之纖維膜具有多孔且互相連通的結構，且F、FK91、FK82、FK73纖維膜的平均直徑隨著角蛋白的含量增加而降低。接著進一步以拉伸試驗機測得F、FK91、FK82、FK73纖維膜的最大拉伸強度亦隨蛋白的含量增加而降低。藉由親/疏水性測試可得知F、FK91、FK82、FK73纖維膜的潤濕性因角蛋白的含量增加而提升。 由體外試驗結果得知纖維膜的細胞貼附能力與其潤濕性一致，且其對於人類臍帶靜脈內皮細胞不具有毒性。免疫螢光染色與即時聚合酶鏈鎖反應分析亦證實了纖維膜並不影響細胞的表型。根據纖維膜的最大拉伸強度、細胞貼附能力與微觀結構，本研究認為FK82與血管相似，可用於後續動物植入實驗。藉由微電腦斷層掃瞄、組織學染色分析得知將大鼠主動脈內皮細胞種在FK82之多孔纖維管(FK82C)，並移植至大鼠腹主動脈一個月後，大鼠並未發生再狹窄與鈣化的情況，綜合以上結果顯示其具有血管組織工程的材料的潛力。

Cardiovascular diseases (CVD) are one of the leading causes of death and bypass surgery is one of the common treatment options for the critical CVD patients. There is no ideal material available for arterial bypass surgery. Herein, fibroin and keratin porous fibrotic tube was prepared by electro-spinning and proposed for tissue-engineered vascular graft (TEVG). The purified fibroin (F) and keratin (K) was mixed in different weight ratios of 9:1 (FK91), 8:2 (FK82), and 7:3 (FK73) to obtain membrane. The SEM images revealed that the electro-spinned membranes have fibrous interconnected porous structure. The average diameter and the ultimate tensile strength (UTS) of the developed membrane decreased with the increased keratin content. The wettability of the developed membrane increased with keratin content. The wettability results were in agreement with that of the cell adhesion to the electro-spinning membranes. The attached HUVECs on the developed membranes showed no cytotoxicity. The immunocytochemistry staining and qPCR analysis showed that, the phenotype of keratin-fibroin membrane was not altered. The results of ultimate tensile strength, cell adhesion and microstructure revealed that FK82 is similar to that of native vessels and could be used in vivo test. The rat aorta endothelial cells were seeded in FK82 porous fibrous tube (FK82C) and then implanted to the rat abdominal aorta for one month. FK82C vascular graft could not be restenosis and calcification. Therefore, it could be considered as a potential material for TEVG.