bFGF预载脱细胞支架构建小口径人工血管的研究

目的目前临床使用的小口径（〈5cm）人工血管因生物相容性差、远期通畅率低，效果不理想。拟通过在脱细胞血管支架表面预载bFGF，制备一种新型的小口径人工血管。方法采用去污剂一酶消化法制备犬颈动脉脱细胞支架，将bFGF预载在经肝素固化（肝素固化组）和未固化的（单脱细胞组）脱细胞支架表面，ELISA法检测结合的bFGF量及体外释放情况。通过与犬BMSCs体外复合培养1～5d，观察bFGF预载肝素固化脱细胞支架（bFGF预载组）和未固化的脱细胞支架（未预载组），以及各自空白对照组细胞生长情况。取8只杂交犬，切断并剪下颈总动脉造成约5cm缺损，随机选取一侧，将预载组支架行端端吻合于缺损中，作为实验侧；将未预载组支架以同样方法植入对侧，作为对照侧。术后8周取材行DSA、HE染色观察移植效果。结果犬颈动脉经脱细胞后大体形态完好、细胞基本去除、纤维结构完整。肝素固化组支架表面结合的bFGF量与bFGF反应浓度成正相关，与相同反应浓度下单脱细胞组比较差异有统计学意义（P〈0．05）；肝素固化组在浓度为100ng／mL下结合的bFGF可在体外持续释放20d。bFGF预载支架促进BMSCs增殖，MTT显示BMSCs在两组支架表面均可黏附生长，复合培养1、2d两组差异无统计学意义（P〉0．05）：3～5d，bFGF预载组支架表面细胞增殖活性明显高于未预载组（P〈0．01）。异体犬颈动脉移植后8周，实验侧支架均通畅，且有细胞覆盖内膜及浸润管壁，而对照侧通畅率仅为12．5％（1／8），闭塞的移植物腔内均为血栓形成，未见细胞覆盖。结论对同种异体血管脱细胞支架表面进行bFGF预载，初步获得一种具有良好生物相容性和通畅性的生物人工血管，其远期通畅率及生物安全性仍待进一步评价。

CONSTRUCTING A SMALL-DIAMETER DECELLULARIZED VASCULAR GRAFT PRE-LOADED WITH bFGF

OBJECTIVE: To design a novel small-caliber vascular graft using a decellularized allogeneic vascular scaffold pre-loaded with bFGF. METHODS: The decellularized canine common carotid were obtained by a detergent-enzymatic procedure, then the scaffolds were covalently linked with heparin and pre-loaded with bFGF, the amount of binding bFGF and releasing curve were assayed by ELISA. Canine BMSCs expanded in vitro were seed on the scaffolds to observe the effects of binding bFGF on proliferation. Both bFGF pre-loaded and non-pre-loaded decellularized grafts were implanted in canines as carotid artery interposition for 8 weeks, the patency was examined by digital subtraction angiography and histological method. RESULTS: Histology and electron microscopic examination of the decellularized scaffolds showed that cellular components were removed completely and that the extracellular matrix structure remained intact. The amount of binding bFGF positively related to the concentration of bFGF. There was a significant difference in the amount of binding bFGF between two different scaffolds throughout all bFGF concentrations (P < 0.05), and up to 100 ng/mL, the local and sustained release of bFGF from the heparin treated scaffolds were assayed up to 20 days. Additionally, MTT test showed the bFGF-preloaded scaffolds significantly enhanced the proliferation of seeded BMSCs in vitro compared with non-bFGF-preloaded scaffolds at 3 days after seeding and thereafter (P < 0.01). Furthermore, in vivo canine experiments revealed that all 8 bFGF-pre-loaded scaffolds remained patent after 8 weeks of implantation, and host cell lined the lumen and populated the wall. Only 1 non-bFGF-pre-loaded scaffold was patent, and the other 7 grafts were occluded because of thrombsus formation. CONCLUSION: This study provides a new strategy to develop a small diameter vascular graft with excellent biocompatibility and high patency rate.