组织工程血管培养生物反应器出口端加载阻力的应力刺激形成系统的设计与验证

Design and verification of Luo-Ye pump-based stress formation for cultivation of tissueengineered blood vessel

目的改进组织工程血管(TEBV)培养应力形成系统,增强平滑肌细胞分泌的刺激作用。方法在生物反应器出口外侧加装阻力气泵,构建新的TEBV体外三维培养体系;通过压力导丝监测生物反应器内不同点压力变化,获得应力-时间变化曲线;按动态培养中是否添加阻力分成改进组和对照组,并设静态培养组,对动脉平滑肌细胞(VSMC)进行四周的三维培养;终止培养后采用HE染色、masson染色、α-SMA免疫组化染色及电子显微镜进行组织学检测,并运用软件对管壁成分进行半定量分析。结果压力监测数据及拟合曲线显示,添加阻力后可明显加大动力源"罗叶泵"每搏的应力刺激;培养结果显示改进组新生组织中VSMC及胶原纤维密布于管壁全层,细胞分布及密度、胶原纤维含量及排列、α-SMA表达均显著优于对照组及静态组。结论改进的系统应力刺激作用明显得到增强并可进一步促进VSMC分泌功能。

Objective To improve Luo-Ye pump-based stress-forming system and optimize the stimulating effect on smooth muscle cells during cultivation of tissue-engineered blood vessels（TEBV）.Methods A new Luo-Ye pump-based TEBV 3D culture system was developed by adding an air pump to the output of the bioreactor.A pressure guide wire was used to measure the stress at different points of the silicone tube inside the TEBV bio-reactor,and fitting curves of the stress changes over time was created using Origin 8.0 software.The TEBVs were constructed by seeding vascular smooth muscle cells（VSMCs） isolated from human umbilical artery on polyglycolic acid（PGA） and cultured under dynamic conditions with 40 mm Hg resistance（improved group）,dynamic conditions without resistance（control group） or static condition（static group）for 4 weeks.The harvested TEBVs were then examined with HE staining,masson staining,α-SMA immunohistochemical staining,and scanning and transmission electron microscopy with semi-quantitative analysis of collagen content and α-SMA expression.Results The measured stress values and the fitting curves showed that the stress stimuli from the Luo-Ye pump were enhanced by adding an air pump to the output of the bioreactor.Histological analysis revealed improved VSMC density,collagen content and α-SMA expression in the TEBVs constructed with the improved method as compared with those in the control and static groups.Conclusion Adding an air pump to the Luo-Ye pump significantly enhances the stress stimulation in the TEBV 3-D culture system to promote the secretion function of VSMCs.