丝素支架微纳层级结构的构筑及其介导的促血管化研究

The vascularization of tissue engineering scaffolds is critically important in ensuring successful engraftment. The strategy of cells seeding into scaffolds has emerged as a promising concept for enhancing rapid vascularization, however, how to activate cell behavior for improving vascularization remains a key challenge. Our previous researches have showed that the construction of three-dimensional micro/nano hierarchy architecture in the pore space of porous silk fibroin scaffolds significantly promoted cell adhesion and proliferation, and enhanced dermal regeneration. In this program, silk fibroin scaffolds with controllable three dimensional micro/nano fibrous structures are firstly prepared based on the freezing-induced silk fibroin assembly technology and further study of assembly mechanism. Functional cells are seeded into these scaffolds and then used to promote the vascularization of regenerated dermal tissue based on the hypothesis that the micro/nano fibers can be used as instructive cues to direct cellular responses. The effect and further regulating mechanism of the three dimensional micro/nano fibrous structures in the pore space of scaffolds on the vasculogenesis ability of different functional cells will be clarified. Meanwhile, the regularity and main mechanism of the vascularization of micro/nano structures-mediated seeding cells will be elaborated. The results of these studies can provide new insight into the cell-material interaction, and will also afford alternative ways and fundamental theory basis for improving vascularization of biomaterials.

血管化是组织工程支架移植成功与否的关键，亟待研究和解决。应用种子细胞是实现组织工程产品快速血管化的可行途径，但如何调控细胞行为以促进其血管化能力是迫切需要解决的关键问题。本项目前期的研究发现，在丝素支架的孔空间内进一步构筑微纳层级上的三维纤维网络结构，能显著促进细胞的黏附、增殖及真皮组织的重建。本项目立足于微纳纤维对细胞的引导和调控作用，基于冷冻诱导丝素蛋白向微纳纤维组装的策略，深化其组装和调控机制，进一步实现对丝素支架孔空间内三维微纳纤维结构的调控，结合促血管化的功能性细胞，促进真皮支架的血管化能力。阐明支架孔空间内构筑的三维微纳纤维结构对不同功能性细胞成血管能力的影响及控制机制，揭示其介导种子细胞促血管化的作用规律和主要机制。以上研究既是对细胞与材料相互作用这一基本科学问题的拓展和深化，也将为组织工程支架材料的促血管化研究提供新的思路和理论指导。

血管化是组织工程及再生医学领域面临的关键问题。应用种子细胞是实现组织工程产品快速血管化的可行途径，但如何调控细胞行为以促进其血管化能力是迫切需要解决的关键问题。本项目针对该问题，立足于微纳纤维对细胞的引导和调控作用，建立了冷冻诱导丝素蛋白向微纳纤维组装的策略，实现了具有微纳层级结构的多孔支架的构建。通过体外培养血管内皮细胞和脐带沃顿胶间充质干细胞，揭示了支架内微纳纤维结构对细胞的引导作用。结果证实，支架内微纳层级结构的构筑不仅促进了细胞的粘附和增殖，而且能够使细胞在多孔支架中以3D方式迁移，从而构建了促进移植细胞生长的3D微环境。在体内，以间充质干细胞分化的内皮细胞和间充质干细胞作为种子细胞加载到支架上并作为真皮替代物植入到大鼠皮下，检测和分析结果表明，支架内的微纳纤维网络显著显着促进了胶原沉积、生长因子（VEGF和bFGF）表达和新生血管形成。在种子细胞和微纳纤维的协同作用下，支架在植入7天后就形成大量的新生血管，从而实现了支架的快速血管化。本项目的研究结果表明，仿生微纳纤维结构设计是增强种子细胞促血管化能力的有效策略，从而为解决组织工程血管化问题和构建快速血管化的新型支架材料提供了重要的科学依据。