微流控技术制备装载Ephrin-B2的微球并复合三维纳米纤维支架在血管再生中的应用

The main reason for the long-term failure of small-diameter vascular grafts is due to the thrombus formation in the early phase followed by a continuous and excessive tissue ingrowth in the chronic phase. To solve this problem, we propose to fabricate silk fibroin Ephrin-B2-delivering microspheres using microfluidic devices and entrap the microspheres within tubular sulfated silk fibroin nanofibrous scaffolds by electrospinning. In addition, the inner surface of the tubular scaffolds is immobilized with CD34 antibody. We hypothesize that the entrapping microspheres between nanofibers do not have an adverse impact on mechanical properties, and that the composite would exhibit sustained release of Ephrin-B2. The tubular scaffold can capture the circulating endothelial cells (ECs) and endothelial progenitor cells (EPCs) directly from the blood stream and support the cells attachment, growth and differentiation to accelerate self-endothelialization of the vascular grafts. More importantly, the releasing Ephrin-B2 can promote smooth muscle cells (SMCs) adhesion, spreading, and directional migration during blood vessel wall assembly. We will further evaluate in vitro and in vivo endothelialization and blood vessel wall remodeling of the vascular grafts using the vascular bioreactor developed by our group and created animal model. Therefore, the aim of this study is to improve the long-term patency of small diameter vascular grafts and provide reliable experimental data for its clinic application.

如何防止急性血栓形成、提高小口径人造血管的远期通畅率一直是人造血管研究的难点和热点问题。本项目在申请人前期研究工作的基础上，拟采用微流控技术制备装载Ephrin-B2的丝素蛋白微球，利用静电纺丝技术将其均匀分散于硫酸化丝素蛋白纳米纤维支架中，制备具有“微－纳”复合结构的三维纳米纤维支架，并在管状支架的内壁接枝CD34抗体。希望该小口径人造血管具备CD34+细胞的捕获能力，能够提高内皮细胞和内皮祖细胞在人造血管内壁的黏附率，增强内皮细胞的增殖、迁移并诱导内皮祖细胞向内皮细胞分化，促进小口径人造血管植入体内后的自发性内皮化，迅速在其内壁形成完整的内皮细胞层；同时促进血管平滑肌细胞向支架内部迁移生长，重塑血管壁。旨在提高小口径人造血管的远期通畅率，为其最终的临床应用提供可靠的实验依据。

小口径人造血管植入体内后如何防止急性血栓形成，提高远期通畅率已经成为人造血管研究的难点和热点问题。为了提高人造血管的抗血栓性能和远期通畅率，加速血管的内皮化是解决这一问题的重要途径之一。静电纺丝制备的小口径人造血管的孔径和纤维直径对内皮细胞的黏附和增殖存在一定的影响。传统方法制备的微球不够均匀、结构差异较大、方法重复性差，采用微流控技术制备的微球粒径均匀且容易操控。因此，本研究以丝素蛋白作为分散相，油溶液作为连续相，通过微流控技术制备了单分散丝素蛋白微滴，然后对微滴进行固化处理，成功制备出了具有良好细胞相容性且粒径均匀的丝素蛋白微球，并将该微球与静电纺丝纳米纤维复合，通过不同数量以及不同粒径微球的加入对纳米纤维支架的孔径进行调控，探索并制备出有利于内皮化的具有“微－纳”复合结构的三维纳米纤维支架，为构建小口径人造血管提供了新的思路。此外，我们还探索了丝素蛋白微球在光动力学治疗的潜在应用价值。