生物组织工程血管的体外构建及其生物力学特性的研究

目的:探索生物组织工程血管(TEBV)的体外构建,观察血管壁形成过程中血管平滑肌细胞的变化,检测TEBV的生物力学特性,为构建理想的临床血管替代物提供资料。方法采用酶消化法制备猪颈总动脉脱细胞支架,再种植犬胸主动脉的平滑肌细胞,体外培养4周,分别于2周和4周取材作形态结构观察和平滑肌肌动蛋白-α(SMA)、金属蛋白酶-2(MMP-2)及血小板衍生生长因子-AA(PDGF-AA)的免疫组化染色并作图象分析。将幼年犬和成年犬的胸主动脉作对照。用INSTRON1122型万能电子强力仪检测TEBV的断裂强度、应力应变关系和松弛试验。结果体外培养2周和4周,血管平滑肌细胞大量增殖并迁移至支架全层,细胞间出现桥粒、缝隙连接。TEBV培养2周和4周,SMA的表达高于幼年犬,但明显低于成年犬;PDGF-AA的表达低于幼年犬,但明显高于成年犬;MMP-2高于成年犬,培养4周时MMP-2的表达接近幼年犬。TEBV的应力应变曲线呈典型的粘弹性特征,松弛应力和断裂强度均与生理血管相似。结论生物组织工程血管体外构建中平滑肌细胞分泌PDGF-AA、SMA的表达逐渐减少,平滑肌细胞由收缩表型逐渐转变为合成表型,大量增殖和分泌胶原,有细胞连接形成;同时MMP-2分泌增加,细胞发生迁移,重建TEBV的管壁结构,且血管壁的生物力学特性逐渐接近于生理血管。

Reconstruction of complete biological tissue engineered blood vessel wall in vitro and test for biomechanics characteristic of the vessel

Objective To reconstruct the complete biological tissue engineered blood vessels(TEBV)in vitro,the biological change of vascular smooth muscle cells(VSMC) planted on the scaffolds of TEBV and the biomechanics characteristic of the TEBV were investigated. Methods The decellularised scaffolds were obtained from swine common carotid arteries by enzyme digestion. VSMC isolated from dog thoracic aorta were seeded onto the inner surface of scaffolds and they were cultured in vitro for 4w. The change of reconstruction TEBV was checked by histological staining and transmission electron microscopy (TEM) observation. The expressions of smooth muscle-alpha-actin (SMA), platelet-derived growth factor- AA(PDGF-AA) and metalloproteinases-2(MMP-2) in TEBV were also detected by immunohistochemistry staining and image analysis. The thoracic aorta of immature dogs and adult dogs were separately chosen as the controls. The biomechanics characteristic of the TEBV, including rupture intensity, the relation ship of stress and strain, and the loose experiment of TEBV were all tested by INSTRON 1122 electron mechanics test system. Results VSMC gradually proliferated a lot and migrated into scaffolds. There were desmosome and gap conjunction of cells in TEBV cultured for 2 or 4w. The expression level of SMA was higher in TEBV cultured for 2 or 4w than that of the immature dog vessels, but significantly higher than that of the adult dog vessels. The expression level of MMP-2 was higher in TEBV cultured for 2 or 4w TEBV was similar to the one of the immature dog. The stress-strain curve of TEBV showed the characteristic viscoelasticity and the value of strain rate,loosen stress, and rupture intensity of TEBV were similar to that of physiological blood vessels. Conclusions With expressing of more PDGF-AA and less SMA and secreting of more MMP-2, VSMC in complete biological TEBV transformed from contractile phenotype to synthetic phenotype and proliferated and migrated rapidly into scaffolds, which resulted in the TEBV wall remodeling and the biomechanics characteristic similar to that of physiological blood vessels.