仿生层状聚乙二醇水凝胶的构建及其在心脏瓣膜组织工程中的应用

Heart valve disease is prevalent worldwide, and the number of patients requiring heart valve replacement significantly increases each year. Heart valve tissue engineering is appealing for providing solutions to the limitations of current artificial valve devices. In this project, biomimetic tri-layer PEG hydrogels will be fabricated for heart valve tissue engineering. First, small peptides, derived from natural proteins in the aortic valve leaflets, will be incorporated into poly(ethylene glycol) (PEG) polymer network to mimic the biological function of extracellular matrix (ECM) in leaflets. Through modulation of the composition of cell adhesion peptide RGDS and enzyme degradable peptides "PQ" (GGGPQGIWGQGK) in PEG network, a balance between de novo deposition of ECM and degradation of PEG hydrogels will be achieved. Moreover, by incorporation of bioactive peptides, e.g. RGDS or "AG73" (RKRLQVQLSIRT), into PEG network, valvular endothelial cells may be able to form a confluent layer on top of the modified PEG hydrogel, rendering the capability of thromboresistance and mediation of inflammation to the implant in vivo. Additionally, tri-layer PEG hydrogels will be fabricated, and the interface between two adjacent layers will be crosslinked by the photolithographic patterning using the two-photon microscope. Electrospun polycaprolactone fibers will be used to strengthen the tri-layers, which will be further modified to obtain the anisotropic mechanical property close to that of the aortic valve leaflet. Further, a synchronous multivalve bioreactor will be assembled to evaluate performance of artificial aortic valve leaflets from tri-layer PEG hdyrogels under the unidirectional fluid flow mimicking in vivo blood flow environment. Valvular cell function and behavior under normal static culture and dynamic culture in the bioreactor will be compared. The proposed research work will not only provide cues for current heart valve tissue engineering, but also shed light on valvular biology.

为满足日益增多的心脏瓣膜疾病患者对高性能人工瓣膜的需求，本项目将制备仿生层状聚乙二醇水凝胶并应用于心脏瓣膜组织工程，以解决现有人工瓣膜的诸多不足（如体内不能再生的问题）。我们将从瓣膜细胞外基质蛋白中提取活性多肽，将其嵌合到聚乙二醇水凝胶中，来模仿瓣膜细胞与基质蛋白间生化作用。通过优化多肽的组成，促进心脏瓣膜间质细胞在支架内分泌新的细胞外基质且对支架进行同步降解。并实现在多肽改性聚乙二醇水凝胶表层形成致密的瓣膜内皮细胞，以提高抗凝血和调节炎症反应功能。另外通过构建三层聚乙二醇水凝胶，使用层与层间交联增强以及纤维定向增强等方法，来仿制主动脉心脏瓣膜层状结构和各向异性的力学性能。最后使用多阀生物反应器模拟体内单向血流环境来评估所制备的人工心脏瓣膜的性能，并研究人工瓣膜中的间质细胞在静态培植和生物反应器中动态培植下的活性和功能差异。项目中的工作将为现今心脏组织工程和瓣膜生物学研究提供重要参考。

随着微创介入疗法的应用，生物瓣膜将广泛应用，然而生物瓣膜在体内服役时会出现钙化、或纤维结构破坏，故而服役寿命有限。新型高分子人工心脏瓣膜能够兼具机械瓣良好的机械性能以及生物瓣优越的血流动力学特性，且适合于介入瓣模型，因而备受关注成为研究热点之一。本项目主要目标是构建层状聚乙二醇—蛋白质纤维复合人工心脏瓣膜，模仿主动脉瓣膜的多级结构，实现与其相近的力学性能，进一步评价仿生人工心脏瓣膜的生物功能以及流体动力学特性。我们利用光交联聚乙二醇水凝胶与蛋白质纤维膜制成层状复合材料，进一步利用戊二醛化学交联制备成仿生人工心脏瓣膜，具有与人体瓣膜相似的成分和结构，以及相近的力学性能。其中蛋白质纤维膜取代人体瓣膜的纤维层和心室肌层，起到主要应力承载作用；而聚乙二醇水凝胶可以代替人体瓣膜的松质骨层，有效缓冲瓣膜的压缩变形，且能克服蛋白质纤维的拉伸应力集中问题。静电纺丝法可制备各向异性的丝素蛋白纤维，进一步制备的仿生层状人工心脏瓣膜也展示各向异性特征，平行于蛋白纤维方向弹性模量大约8.2-12.3MPa，垂直于纤维方向弹性模量大约1.4-2.8 MPa，与人体瓣膜力学性能（周向3-15MPa，径向1-2MPa）相近。体外体内实验结果证明聚乙二醇水凝胶的高分子网格能有效屏蔽水合离子和酶分子渗透，从而阻止其包覆的蛋白质纤维的钙化及酶解。动物体内实验结果显示仿生层状人工心脏瓣膜材料具有良好的生物相容性。上述结果显示利用聚乙二醇水凝胶与蛋白质纤维制备层状仿生人工心脏瓣膜，能克服现有机械瓣及生物瓣的不足，有望用于新型人工心脏瓣膜。本项目还建立了脉动流检测与有限元分析相结合体系，能够对人工心脏瓣膜的血流动力学性能进行有效评价，可进一步指导新型人工瓣膜的结构设计和产品研发。