基于细胞片层构建TiO2纳米棒仿骨复合体逐层调控骨免疫效应及机制研究

Biomaterials for bone repair play a key role in critical-sized bone defect recovery. Bone implants initiated immune responses when they implanted in vivo. However, biomaterials which could not induce a good immune response and lack of vascularization result in failure of the implant, becoming one of the hot issues. Our previous study suggested that interrod space of TiO2 nanorods-patterned could regulate osteoimmune response by modulating macrophage polarization. Based on the previous study, in the project, we want to figure out which interrod space could induce good osteoimmune responses. Based on cell sheet culture engineering which controlled by temperature and pH dual response systems to obtain cell sheet about macrophages, mesenchymal stem cells, and vascular endothelial cells respectively and co-culture these with ECM. Then cell sheets are assembled as multicoated cell sheet and cultured on TiO2 nanorods-patterned so that to construct biomimetic bone grafts, applied to repair critical-sized bone defects. In this project, we want to study the effects and mechanism of layer-by-layer regulation of biomimetic bone grafts in osteogenesis, angiogenesis and bone repair. This project will provide a new idea and evidence for critical-sized bone defect repair and biomimetic bone grafts construction.

骨植入材料在临界骨缺损的修复与再生中发挥着关键作用，但其进入体内引发的免疫反应不易调控，骨缺损部位形成纤维包囊、缺少血管生成等原因易导致植入失败。课题前期研究提示二氧化钛纳米棒阵列涂层的不同棒间距可调控骨免疫反应中巨噬细胞极化。本项目拟在前期研究基础上，考察二氧化钛纳米棒阵列涂层的棒间距对巨噬细胞极化和自噬的调控规律，明确能诱导良好免疫反应的棒间距；以此棒间距的二氧化钛纳米棒阵列涂层为研究主体，基于温度和pH双重响应型细胞片层技术，将巨噬细胞、间充质干细胞及血管内皮细胞片层在二氧化钛纳米棒阵列涂层表面行多层培养，构建仿骨复合体，应用此仿骨复合体以逐层调控的方式进行骨缺损修复。本项目拟阐明多细胞片层及二氧化钛纳米棒阵列涂层形成的仿骨复合体在骨免疫反应中逐层调控间充质干细胞的成骨分化及内皮细胞血管生成的效应和机制，为骨缺损修复和仿骨复合体构建提供理论依据。

针对钛基骨植入体存在的免疫反应不易调控及植入后腐蚀疲劳断裂导致植入失败等临床问题，本项目从钛基植入材料表面涂层设计角度出发，在纯钛表面构建二氧化钛纳米棒阵列涂层，提升钛基体抗腐蚀疲劳效力的同时，使其具备免疫调控能力和促成骨效力，最终实现骨与植入体整合。表面机械研磨处理（SMAT）可在钛基表面得到纳米晶层与其底层区域的晶粒变形协同提高钛基体腐蚀疲劳效力。在SMAT钛上水热生长金红石TiO2纳米棒阵列涂层。这种复合钛基体可以克服直接水热生长纳米棒阵列涂层导致的腐蚀疲劳强度降低，从而显著提升钛基体的腐蚀疲劳性能，延长其服役时间。本项目研究了二氧化钛纳米棒阵列涂层及其棒间距对巨噬细胞极化的调控效应及机制，明确了能诱导良好免疫反应的棒间距：二氧化钛纳米棒阵列涂层可促进巨噬细胞由早期促炎M1型及时向抑炎M2型转变；将巨噬细胞及骨髓间充质干细胞共培养，极化后的巨噬细胞分泌的细胞因子通过促进骨髓间充质干细胞的迁移和成骨分化，从而促进体内骨整合。适宜尺度的纳米棒间距可在粘附初期激活巨噬细胞中FG-integrin αMβ2-IKK-NF-κB信号通路，而后转变为激活FN-integrin α5β1-PI3K-Akt1信号通路，从而调控巨噬细胞由M1向M2表型的转换。本项目从植入体力学完整性和巨噬细胞免疫调控等方面阐明了二氧化钛纳米棒阵列涂层调控骨免疫的效应和机制，有望克服钛基骨植入体临床应用瓶颈，使其满足功能性及长期服役的要求。在本项目支持下，项目负责人发表SCI论文（影响因子>10）2篇，参加学术会议3次。