促血管祖细胞募集的小口径血管支架构建与原位再生研究

To construct the intact endothelial cell (EC) and smooth muscle cell (SMC) layers plays the critical role in the development of small-diameter vascular grafts. The traditional in vitro tissue engineering (TE) approach requires harvest the cells from the patient and extensive culture period, which limits its clinical applicability. To overcome these limitations, an in situ TE approach was proposed and became the research hotspot, in which endogenous progenitor cells are recruited by biomimetic scaffolds for in situ blood vessel regeneration. For mimicking the architecture of a native vessel, we have recently developed a bi-layered tubular scaffold by using a two-step thermally induced phase separation (TIPS) approach. The scaffold incorporates a microporous nanofibrous inner layer which allows ECs adhesion and growth, and a macroporous nanofibrous outer layer to facilitate SMCs infiltration and ingrowth, which may be used as a suitable scaffold to create small diameter vascular grafts in situ. Stromal cell derived factor-1α (SDF-1α) is a potent factor for vascular progenitor cells (VPCs) recruitment in vivo, and platelet-derived growth factor-BB (PDGF-BB) can promote vascular maturity by the recruitment of SMCs and stimulating cell proliferation. In this study, we will fabricate a new bi-layered tubular scaffold with optimal pore size, mechanical and biodegradable properties for in situ blood vessel regeneration, and SDF-1α and PDGF-BB are then immobilized on the inner and outer layers by using heparin as a molecular linker, respectively. We will investigate the effects of the immobilized SDF-1α and PDGF-BB on the recruitment and proliferation of VPCs both in vitro and in vivo, and focus on the roles of the released growth factors in the remodeling of both EC and SMC layers in the vascular grafts. The results of this study may provide new insight into the in situ small-diameter blood vessel regeneration.

完整的内皮层与平滑肌层是小口径血管再生的关键。传统的体外构建方法受限于细胞来源和培养费时等问题，利用仿生支架诱导血管原位再生成为当前的研究热点。针对小口径血管的结构仿生，我们最近研制出一种具有双相孔径结构的双层纳米纤维血管支架，其内层的微孔结构便于血管内皮细胞的粘附生长，外层的大孔结构利于平滑肌细胞的渗透长入，适合用于小口径血管的原位构建。基质细胞衍生因子-1α（SDF-1α）能够在体内募集血管祖细胞，而血小板衍生生长因子（PDGF-BB）可促进平滑肌细胞的募集和增殖。本项目拟进一步开发具有最佳孔径、力学和降解性能的双层小口径血管支架，并采用肝素固定技术分别在支架内、外层固定SDF-1α和PDGF-BB。通过体内、外实验评价生长因子释放对促进血管祖细胞募集和分化的影响。特别是通过血管原位再生研究，探明生长因子释放对血管内皮层和平滑肌层重建的影响，为小口径血管的原位再生提供新的思路。

心血管疾病（CVD）是人类的第一大杀手，严重威胁着患者的生命安全。血管旁路移植术是目前治疗CVD的主要方法之一，自体血管是临床上手术治疗CVD的黄金标准，但是来源有限，限制了它们的广泛应用。以膨化聚四氟乙烯（ePTFE）和涤纶（Dacron）等不可降解合成高分子材料构建的血管移植物在大口径（内径 ≥ 6 mm）血管移植应用中已经取得了良好的治疗效果，但是在小口径（内径 < 6 mm）血管移植应用中效果不佳。组织工程的发展为小口径血管移植物的构建提供了新的解决思路。热致相分离（TIPS）是一种能够制备仿生纳米纤维支架的方法，该方法操作简单，不需要特殊设备，在组织工程支架制备中具有良好的应用前景。本项目采用TIPS技术，根据天然血管的分层结构特点，构建同时满足内皮细胞（ECs）和平滑肌细胞（SMCs）功能性生长的双层纳米纤维血管支架，然后对制备的血管支架进行理化性能评价和体内、外血管修复潜能研究。本项目主要开发了两步相分离技术构建具有仿生天然血管结构和功能的双层纳米纤维血管支架，并在内外层分别负载生长因子SDF-1α和PDGF-BB，研究了生长因子对血管内皮层和中膜层重建的影响。本项目为小口径血管支架的构建以及原位再生研究提供了新的方法和思路。