多肽/微纳米多级结构支架调控MSCs分化及组装用于骨修复

The repair of massive bone defect, rebuilding its structure and renewing its function are great challenge for orthopedist. At present, it is difficulty to regulate the osteogenic differentiation and assembly of MSCs and guide the extracellular matrix and calcium compound to arrange into pattern mimicking compact bone structure at molecular, nanometer and micrometer scales simultaneously by using tissue engineering scaffold. In this study, soft lithography, phage display and phage self-assembly will be combined to solve the problem. First, soft lithography is used to prepare micro-grooved PLGA poles. The M13 phages express promoting osteogenic differentiation peptides though phage display. These phages modified with peptides self-assemble on the surface of PLGA poles and form nano-grooves. Collagen films mimicking periosteum are produced by replica technique. A collagen film mimicking periosteum is used to package a few nano/micro-grooved PLGA poles to form a 3D scaffold mimicking compact bone structure. We will characterized the physicochemical property of the 3D scaffold and study their influence on MSCs fate and assembly and pick the most optimal scaffold out. The effect of scaffold properties and peptides on MSCs osteogenic differentiation and assembly will be concluded, and correlative mechanism will be studied. The most optimal scaffold will be applied in animal assay to confirm the repair effect. This study will provide a good candidate for bone tissue engineering scaffold. The conclusion of the study and the revelation of related mechanism will guide the design of bone tissue engineering scaffold.

大块密质骨缺损修复及其结构和功能重建仍是骨科领域的难点。目前组织工程支架很难同时在分子、纳米及微米尺寸上调控间充质干细胞（MSCs）成骨分化及群体组装，使胞外基质及钙化物组装为类密质骨结构。本研究拟结合软光刻、噬菌体展示及噬菌体自组装构建3D支架解决上述问题。首先通过软光刻制得有沟槽结构的聚乳酸-羟基乙酸共聚物（PLGA）圆棒；用噬菌体展示获得表达促骨分化肽的M13噬菌体，将其组装到沟槽化PLGA圆棒表面形成取向纳米脊；用复模成型技术制得仿骨膜胶原膜；用该胶原膜包裹组装有噬菌体的PLGA圆棒制得仿密质骨3D支架。研究支架理化、促MSCs成骨分化等性质，获得最优支架。研究支架结构、多肽等对MSCs成骨分化、组装等的影响，阐明相关规律机理。将最优支架用于动物大块骨缺损修复实验，验证修复效果。本研究将为大块骨缺损修复提供候选支架，相关规律的总结、机制的揭示将为骨组织工程支架制备提供一定理论指导。

干细胞物理微环境对干细胞的行为和命运有着决定性作用，该方向的研究是再生医学、组织工程和体内生长发育机制理解的关键所在。围绕这一主题，尝试了5个体系以研究不同微纳拓扑结构、力学性能等对干细胞分化的影响。1）成功构建了微纳米多级有序仿密质骨物理微环境；2）构建了具有不同力学性能的丝素水凝胶，探明了不同弹性模量对MSCs粘附、形态的影响情况；3）初步明确了力学性质和表面拓扑结构调控NSCs分化的协同效应及规律；初步掌握了力学性质和拓扑结构对MSCs形态、粘附的协同作用规律；4）初步明确了一定力学性能的丝素水凝胶结合外泌体促进MSCs成软骨分化及修复体内软骨损伤的条件；5）构建了具有良好皮下促MSCs成骨的类骨羟基磷灰石/丝素复合膜，明确了相关工艺和条件； 6）明确了丝胶纳米凝胶调控MSCs成软骨分化的条件和相关机理。上述内容的完成，有望开发等离子体调控丝素表面矿化用于骨修复，丝素水凝胶包裹外泌体用于全程软骨损伤修复，体外干细胞促软骨分化纳米凝胶等多种临床治疗产品，为相关研究提供新的思路和理论基础。