基于热诱导相分离技术一体化构建组织工程带瓣血管的实验研究

Artificial valved conduits are the guarantee of successful operation in children with severe congenital heart disease with pulmonary atresia / absence. However, its application is limited due to poor mechanics, valve calcification and complicated preparation techniques. We verified the feasibility and superiority of integrated construction of valved conduits by thermally induced phase separation (TIPS) previously. This project intends to improve the 3D mold design to achieve integrated construction of valved conduits with different mechanical requirements (PU valve, PLLA / PLCL wall) based on the digital hemodynamics. Explore the relationship between the ratio of PLLA to PLCL and the pore size of composite materials. SI-ATRP method for PEGMA modification to improve the hydrophilicity of PU to increase cell adhesion. Use PEG hydrogel to construct a three-dimensional cell culture network and perform dynamic cell culture by loading FGF-2, stiffening under the best elastic modulus and simulating physiological condition to accelerate the proliferation of valve interstitial cells (VICs), maintain the resting phenotype and guide the extracellular matrix remodeling, try to solve the valve calcification caused by the activation of VICs. Finally perform the orthotopic transplantation in large animals. The implementation of this project is expected to strongly promote the transformation of tissue-engineered valved conduits and save a large number of children with severe congenital heart disease.

人工带瓣血管是罹患肺动脉瓣闭锁/缺如等严重先心病患儿手术成功的保障，但其应用有限，归因于力学性能不佳、瓣膜钙化失功以及制备工艺繁琐。我们先期验证了基于热诱导相分离技术一体化构建人工带瓣血管的可行性和优越性。本项目拟依据数字化血流动力学，优化3D模具结构，一体化构建满足不同力学需求的仿生带瓣血管（PU瓣膜、PLLA/PLCL管壁）；研究PLLA与PLCL的配比与复合材料孔径的关系，采用SI-ATRP方法对PU材料进行PEGMA修饰提高亲水性增加细胞粘附；利用PEG水凝胶构建细胞培养三维网络，通过搭载FGF-2因子、最佳弹性模量下成胶、模拟生理条件进行细胞动态培养，促进瓣膜间质细胞（VICs）增殖、维持VICs表型、引导细胞外基质重塑，试图解决因VICs活化导致的瓣膜钙化等问题；最后行大动物体内原位移植。本项目实施预期将有力地推动组织工程带瓣血管向临床转化，拯救大量罹患严重先心病患儿的生命。

人工带瓣血管是罹患肺动脉瓣闭锁/缺如等严重先心病患儿手术成功的保障，但其应用有限，归因于力学性能不佳、瓣膜钙化失功以及制备工艺繁琐。我们先期验证了基于热诱导相分离技术一体化构建人工带瓣血管的可行性和优越性。我们通过CAD软件设计出改进后的金属模具三维实体模型图，并在探索水凝胶材料制备带瓣血管的过程中，采用两步法制备了多酚增韧PEG-蛋白基水凝胶。通过材料筛选，我们以聚L-丙交酯-已内酯（PLCL）和丝素蛋白（SF）为材料来源，并参考目前商用的IMPRA®人工血管结构(由膨体聚四氟乙烯制备而成)，通过采取共轭静电纺丝方法，最终制备出一种力学性能良好、生物相容性优异，能初步满足动脉系统应用的载肝素（Hep）的生物可降解单层人工血管。在此基础上，我们进一步对带瓣血管的瓣膜部分进行了探索，我们独辟蹊径，运用相应的技术手段，以聚乙烯醇（PVA）气凝胶、聚（L-丙交酯-共-己内酯）（PLCL）纳米纤维和褪黑激素开发了一种兼具外科缝合性和抗纤维化抗炎症的生物补片，具备进一步加工成瓣膜材料并实际应用的潜力，以期为后续的瓣膜制备成型提供参考。下一步我们会进一步将前期制备的生物可降解单层人工血管与生物补片相结合，最终制备出理想的组织工程带瓣血管。