仿生自组装构建生物响应性骨修复材料引发的免疫微环境时空调控巨噬细胞促进血管化的机制研究

Vascularization of bone repair materials is one of the most critical and urgent problems in the process of bone defect repair. The dynamic changes of macrophage phenotype and function play an important role in the process of vascularization of bone repair materials, and it is an effective way to promote the regeneration of blood vessels to endow bone repair materials with immunomodulatory properties, but how to achieve the precise and dynamic regulation of macrophages remains to be further studied. In this project, we propose the idea of constructing a bio-responsive bone repair scaffold that can trigger a suitable immune microenvironment, achieve favorable sequential activation of macrophages, and promote the generation of vascular networks. On the basis of previous studies, the oriented lamellar collagen / hydroxyapatite scaffolds were prepared by using the directional thermal induction assembly technology to simulate the natural bone structure, and then the bio-responsive double-layer coacervates loaded with IFN-γ and IL-4 macrophage regulatory factors were constructed in the space of scaffolds by layer by layer electrostatic self-assembly method. A suitable immune microenvironment was induced by regulating the spatial structure of scaffolds and the bio-responsive sequential release of macrophage regulatory factors. The effect of immune microenvironment on macrophage polarization and angiogenesis factor secretion and its key regulatory in angiogenesis were explored. Finally, the potential and theoretical mechanism of immune microenvironment regulating macrophages to promote vascularization in spatiotemporal were elucidated, and new strategies for the design of immune-regulated bone repair materials were provided.

骨修复材料的血管化是骨修复过程中亟待解决的关键问题之一。巨噬细胞表型和功能的动态变化在血管化过程中起着重要的作用，赋予骨修复材料免疫调节特性是促进血管再生的有效手段，但如何实现巨噬细胞的精准和动态调控有待进一步研究。本项目提出构建可引发适宜免疫微环境的生物响应性骨修复支架，精准调控有利的巨噬细胞顺序激活进而促进血管网络生成的设想。在前期研究基础上，模拟天然骨结构采用定向热诱导组装技术构建取向板层状胶原/羟基磷灰石支架，再以层层静电自组装方法在支架的空间内构建负载IFN-γ和IL-4巨噬细胞调控因子的生物响应性双层微液滴，通过调控支架空间结构及巨噬细胞调控因子生物响应性贯序释放引发适宜的免疫微环境。探究免疫微环境对巨噬细胞极化和血管生长因子分泌的影响规律及其在血管形成过程中所发挥的关键调节作用，阐明免疫微环境时空调控巨噬细胞促进血管化的潜能和理论机制，为免疫调控骨修复材料的设计提供新的策略。

骨修复材料的血管化是骨修复过程中亟待解决的关键问题之一。巨噬细胞表型和功能的动态变化在血管化过程中起着重要的作用，赋予骨修复材料免疫调节特性是促进血管再生的有效手段。本项目从仿生设计角度构建了可引发适宜免疫微环境的生物响应性骨修复支架，探究了支架引发的免疫微环境在血管网络形成过程中以及骨修复过程中所发挥的关键调节作用。主要研究内容包括：1、模拟天然骨结构采用定向热诱导组装技术及层层组装技术构建负载细胞因子的取向板层状胶原/羟基磷灰石支架；2、从细胞和分子水平深入研究了支架的取向板层状结构和细胞因子的响应性释放引发的免疫微环境对巨噬细胞行为的影响；3、构建了体内血管化模型，研究了生物响应性取向板层状支架在体内成血管能力，评估血管新生情况及新骨形成情况。.研究结果表明，采用定向热诱导自组装技术制备了不同孔径/层间距的Col/nHAP支架材料，并且采用层层自组装技术自备了负载不同生长因子的微液滴。巨噬细胞与支架共培养结果表明层间距较大的Col/nHAP支架能够更好的调控Mφs由M1表型向M2表型转变，并且促进促血管生成细胞因子的分泌。此外，与Col/nHAP支架相比，负载具有免疫调控作用细胞因子的支架抑制了促炎相关因子的分泌，同时促进了促修复相关因子的分泌，说明了生长因子的掺入能够促进M1型巨噬细胞向M2型巨噬细胞极化，并且人脐静脉内皮细胞（HUVEC）与Mφs共培养的上清液进行共培养时显示出杰出的细胞迁移和血管形成能力。动物实验结果表明，支架的取向板层状结构和细胞因子的负载展现出良好的血管生成和骨形成的能力。.天然骨仿生的生物响应性支架材料的构建为免疫调控骨修复材料的设计提供了新策略，对理解和明晰通过赋予骨修复材料免疫调节特性实现巨噬细胞的精准和动态调控进而促进血管化的相关机制具有重要的学术价值和科学意义。.