3D打印核/壳支架多生物响应微环境免疫应答引导促成骨和成血管研究

Osteogenesis and angiogenesis are two important coupled events in bone regeneration. It is difficult to repair bone defect in situations like critical-size defect, osteoporosis, and bone nonunion due to the lack of local blood supply. Bone regeneration is highly mediated by immune system. Therefore, it is meaningful for bone repair materials to promote osteogenesis and angiogenesis via osteoimmunomodulation in treating those hard-to-healing bone defects. Based on the previous studies on 3D printed calcium-free alginate scaffold, this project will prepare a core/shell PLGA/alginate scaffold via coaxial printing to build a multifunctional microenvironment. The surface charge of the shell will be regulated by grafting polylysine, and the controlled release of copper will by realized by the core/shell structure. MSCs and macrophages play an important role in bone regeneration and immune responses, respectively. The effects of signals in this elaborately designed microenvironment on MSCs in terms of osteogenesis and angiogenesis will be explored. Then the immune responses of macrophages to signals from the microenvironment will also be investigated. Finally, the performance of MSCs under the immune cues produced by macrophages will be studied to obtain scaffolds with desired microenvironment feature. The performance of the chosen scaffolds will be testified in vivo. To the end, a regulation model between microenvironment, immune responses, and bone regeneration will be built. This study will provide an inspiring reference for developing novel bone repair materials.

成骨与成血管密切关联，大段缺损、骨质疏松、骨不连等伴随的难愈合骨缺损治疗需要良好的血供环境推进成骨。免疫反应对骨再生过程影响显著，因此通过免疫引导促进成骨和成血管是骨再生材料攻克临床难题的重要方向，兼具科学意义和临床价值。本项目在无钙海藻酸3D打印研究基础上，采用同轴3D打印技术构建载铜PLGA/海藻酸核/壳支架，分别通过聚赖氨酸接枝和核/壳双调控模式实现表面电荷调控和铜离子可控缓释，获得多生物响应功能微环境。基于MSCs和巨噬细胞在骨再生和骨免疫中的关键作用，考察微环境表面电荷和铜离子释放对MSCs成骨和成血管功能的调控规律和机理，获得适配骨再生的微环境特征；研究微环境信号对巨噬细胞免疫应答的引导作用和机制，获得免疫友好型微环境特征；结合动物骨缺损模型，进一步探究微环境引导的免疫应答信号的促骨再生效应，建立“微环境-骨免疫-骨再生”精确调控模型，为新型骨再生材料研究提供重要的科学参考。

为了实现更有效的骨缺损再生治疗，本项目首先基于无钙海藻酸3D打印体系，采用同轴3D打印技术成功构建了具有核壳纤维结构的多孔支架（内核：PLGA或PLGA/CuO；外壳：海藻酸基水凝胶），实现对支架多孔结构的灵活调控。进一步通过在支架表面接枝聚赖氨酸以增强表面组织再生活性。系统研究发现聚赖氨酸接枝水平对表面Zeta电位、表面接触角、表面粗糙度、蛋白吸附、支架力学性能、支架溶胀性、支架降解性能、纤维多孔结构等都有显著影响。细胞学研究表明，聚赖氨酸接枝可以促进 BMSCs的黏附状态，进一步引入铜元素可以促进细胞的生长。巨噬细胞免疫应答实验表明聚赖氨酸修饰后对巨噬细胞的极化有一定调控作用。最后，以大鼠股骨缺损模型，结合影像学、组织学等方法发现，同轴支架具备比较好的骨再生特性；与细胞学规律不一致的是，表面接枝聚赖氨酸并未体现促成骨优势。整体而言，项目研究对于研制新型骨修复材料提供一定借鉴意义。

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