基于基因工程改造的可打印生物活性类丝弹性蛋白促进血管化骨再生的作用及其机制研究

Silk-based materials have outstanding mechanical properties, good biocompatibility as well as biodegradability, which show a promising application prospect in bone regeneration. However, single molecular composition and poor stimuli responsive units make it difficult to print alone to meet the individual design requirements for bone repair scaffold material, and lack the ability to induce osteoblast differentiation. In this study, we attempt to construct silk-elastin-like proteins (SELPs) with rapid stimuli responsiveness and ionic controlled release by genetic engineering through combining silk protein module with elastin module and ionic binding peptides, which can be used as "Bio Ink" for 3D hollow channel scaffold construction. We also aim to elucidate the relationships between the molecular module composition of SELPs and its three-dimensional printability as well as ion release efficiency，to investigate whether or how active ion components act on the osteogenesis and vascularization of BMSCs，and to explore the influence of active ion/three-dimensional hollow channel microenvironment on survival and potentials of loaded BMSCs. Finally, we decide to comprehensively evaluate the role of active ions and three-dimensional tube structures in vascularized bone regeneration in the bone defect site. This project not only enriches the theory and practice in the field of protein biomaterials, but also provides new vascularization strategies for bone tissue engineering.

丝蛋白基材料具有优异的力学性能和良好的生物相容性、生物可降解性，在骨再生中展示了较好的应用前景。然而由于丝蛋白分子组成单一且缺少刺激响应性单元，导致其单独无法进行打印制备以满足个性化骨修复支架材料的需求，并且自身缺乏成骨诱导性能。本项目拟通过基因工程手段在丝蛋白模块基础上，融合弹性蛋白和离子结合肽段，构建兼具快速刺激响应性和离子控释功能的类丝弹性蛋白聚合物（SELPs），并作为“生物墨水”用于三维管道结构支架打印。拟阐明SELPs分子模块组成与其离子释放效率以及三维可打印性之间的关系，研究活性离子对BMSCs的成骨成血管刺激调控作用及其相关的分子调控机制，探讨活性离子/三维管道微环境对负载BMSCs的存活及转归的影响。最后在体内骨缺损区原位评价离子和管道结构协同诱导血管化骨再生的效果。本项目的实施不仅可以丰富蛋白生物材料领域的理论和实践，还将为骨再生的血管化调控提供新的思路和策略。

血管化是制约大块骨缺损修复的关键问题，活性离子成分以其成本低廉、使用方便、稳定性高等优点，在血管化骨再生中展现出良好的应用前景。本项目围绕支架材料的离子结合与缓释及其血管化效果开展研究：探索了通过重组类丝弹性蛋白（SELPs）分子设计，实现SELPs的铜离子特异性结合，并将该聚合物作为生物墨水用于三维管道结构打印，显著提升组织在材料内部的浸润效果；构建三维打印聚多巴胺-丝蛋白的铜肽缓释体系，缓释的铜肽除提高负载骨髓干细胞增殖速率外，还可以通过调节间充质干细胞旁分泌、巨噬细胞M2向极化，促进VEGF、BMP2、TGF-β等因子分泌，形成有利于骨组织再生微环境，并通过大鼠临界大小颅骨缺损模型评价，证明该支架具有更好的血管化骨再生效果。综上所述，本项目的实施，为活性离子的控释及血管化骨组织修复材料设计提供了新的思路。