脉动式张应力环境下培养组织工程血管的初步实验

Incubation of tissue engineered blood vessels under pulsatile stress ： a pilot study

目的探讨脉动式张应力环境下，在生物反应器内进行组织工程血管（TEBV）培养的可行性。方法从人脐动脉获取血管平滑肌细胞（VSMC），经体外培养扩增后接种到聚乙醇酸（PGA）支架并置于生物反应器内进行三维培养。实验组利用气动式左心循环辅助泵对VSMC施加模拟人体内血管环境（压力120mmHg，1mmHg=0．133kPa，频率60次／rain）的脉动式张应力作用。对照组无张力环境下进行三维培养。2周后收获TEBV分别行扫描电镜（SEM）检测、组织切片HE染色和Masson染色。结果实验组TEBV外观色泽鲜亮，具有一定厚度和弹性，能自我维持管腔形状。SEM显示实验组TEBV管壁表面光滑平整，横切面有丰富的细胞外基质（ECM），ECM分布均匀，把PGA完全包裹。HE染色管壁均匀致密，ECM及细胞的排列具有一定方向性，其中含有未降解的PGA碎片。Masson染色管壁胶原纤维丰富。对照组TEBV外观色泽暗淡，管壁薄，弹性差，从支架取下后管腔塌陷。SEM显示管壁表面欠光滑，横切面上ECM含量少，大部分PGA没有被包裹。组织切片染色管壁结构疏松，ECM和胶原纤维含量较少，层次感差。结论在生物反应器内的脉动式张应力环境下，可培养出具有良好形态结构的组织工程血管样组织。

Objective To explore the feasibility of incubation of tissue engineered blood vessels （TEBV,） subjected to pulsatile stress in a bioreactor. Methods The vascular smooth muscle ceils （VSMCs） isolated from human umbilical artery were, following in vitro incubation, seeded on polyglycolic acid （PGA） scaffold and placed into the bioreactor for three-dimensional culture. The pulsatile stress was simulated by using a gas-driven left ventricular assisted pump mimicking the human cardiovascular internal environment （pressure： 120 mm Hg, 1 mm Hg=0.133 kPa, frequency： 60 beats/min）. The control group was cultured without pulsatile stress. This was followed by harvesting of TEBVs for subsequent scanning electron microscopy （SEM）, HE staining and Masson staining at week 2. Results The TEBVs in the experimental group appeared bright-colored, with considerable thickness and flexibility sufficient to maintain the lumen architecture. As revealed by SEM, the internal wall lining the TEBV was smooth, with abundant extracellular matrix （ECM） that was evenly distributed and totally surrounded the PGA in the cross-section. HE staining showed a dense uniform vascular wall with well-oriented ECM and cells juxtaposed against undegraded PGA fragments. An abundance of collagen fibers lining the vascular wall was evidenced by Masson staining. In thecontrol group, the dim-colored TEBV was characterized by its thin wall with poor flexibility that collapsed instantaneously following removal from the scaffold. SEM revealed a rough surface with little ECM by which PGA was not mostly surrounded. Histopathology staining showed a loose architecture characterized by unremarkable ECM and collagen fibers lining the vascular wall. Conclusion TEBV-like tissue with an ideal morphology can be cultured under the pulsatile stress in a bioreaetor.