可注射microRNA活化水凝胶对炎症期巨噬细胞及干细胞的时序一体化调控及促骨修复研究

Inflammation phase plays a crucial role in bone healing-process. Promotion of fracture healing can be achieved by modulation of behavior of either macrophages or stem cells which are involved in this stage. The present proposal to improve bone fracture healing by sequential and integrative modulation of behavior of macrophages and stem cells is therefore raised. On the foundation of our previous works that microRNA (miRNA) induces osteogenic differentiation of stem cells and miRNA/nanocapsule induces bone formation in vivo, we design a miRNA activated injectable hydrogel based on miRNA/nanocapsule and a MMP sensitive peptide modified hyaluronic acid through Michael addition. MiRNA/nanocapsules are sequentially released through a diffusion and a matrix metalloproteinase responsive degradation mechanism according to the time sequence of molecular events at inflammation stage to achieve effective transfection of macrophages and stem cells by miRNA. M1-M2 polarization of macrophage is modulated to enhance angiogenesis, and osteogenic differentiation of stem cells is further induced to facilitate fracture repair. The relationships between injectable hydrogel structure and transfection efficiency, principles of sequential delivery, modulation mechanism of miRNAs to inflammatory and osteogenic signaling pathway, as well as bone healing will be investigated systematically. Strategy based on inflammation phase modulation to promote bone fracture healing will be provided and new theoretical basis and technology for material design and gene therapy for bone repair improvement will also be put forward.

炎症期在骨愈合过程中具有关键作用，对炎症期巨噬细胞和干细胞调控均可促进骨的愈合进程，本课题据此提出巨噬细胞和干细胞时序一体化调控促骨愈合的思路，在课题组前期miRNA纳米微囊体内诱导成骨等工作的基础上，通过Michael加成原位凝胶技术，构建以透明质酸、miRNA微囊为基础的可注射miRNA活化水凝胶。按照炎症期细胞事件顺序，通过物理扩散和MMP酶响应性降解两种机制时序释放miRNA纳米微囊，实现巨噬细胞和干细胞的高效miRNA转染。通过促进巨噬细胞极化表型转化促进血管新生，进一步诱导干细胞成骨分化促进骨愈合。系统研究凝胶结构设计与miRNA转染效率、时序投递规律、miRNA对炎症反应及促骨形成信号通路的调节机制与促骨愈合的关系，同时深入探讨炎症期巨噬细胞和干细胞行为与血管化和成骨分化及与骨愈合之间的相互影响，为骨修复提供立足于炎症期调控的新策略，为材料设计和基因治疗提供新的理论依据和技术

为实现microRNA对巨噬细胞、干细胞的有效调控，构建可高效转染目的细胞的纳米粒子载体是关键。本项目通过合理选择单体和交联剂，利用原位自由基聚合法合成了负载microRNA的纳米粒子。结果显示，该microRNA纳米粒子的小尺寸和弱正电荷使其可高效转染巨噬细胞和干细胞（摄取率达到80%以上），酸降解交联剂使其可在弱酸性溶酶体中释放基因。..按照骨愈合自然进程利用基因对炎症期的巨噬细胞及干细胞进行时序一体化干预，是促进骨愈合的新策略，可响应此天然进程的分子信号以时序释放不同种类microRNA的体系对该策略的实现以及快速有效促骨愈合意义重大。本项目利用MMP7敏感多肽通过Micheal加成反应构建了MMP7酶敏感可注射水凝胶，并模拟骨修复进程中MMP7酶在适当时间点出现的情况进行释放研究。结果显示，凝胶在此模拟进程中成功实现了对所负载的两种纳米粒子的时序释放，累计释放量与凝胶组成、组分含量及交联程度密切相关。..从凝胶中释放出的microRNA纳米粒子对巨噬细胞分型以及干细胞成骨分化的调控，对促进骨愈合至关重要。本项目选择miRNA-155和miRNA-21，研究microRNA纳米粒子在体外对细胞行为的调控。结果显示，miRNA-155纳米粒子可有效促进巨噬细胞向M1型极化，miRNA-21纳米粒子则可调控巨噬细胞向M2表型转换，并可通过抑制PTEN，激活PI3K/AKT信号通路，上调β-catenin调控RUNX-2，促进干细胞的成骨分化。此外，两种microRNA纳米粒子由凝胶体系释放后，其功能性仍得以很好保持。..本项目还通过建立大鼠体内胫骨平台骨缺损模型，对microRNA纳米粒子及凝胶体系的体内促骨早期愈合效果进行了研究。结果显示，miRNA-21对巨噬细胞和干细胞的体内调控与体外调控效果一致，凝胶组分对成骨不具备明显的干预性，microRNA活化凝胶组在1周和4周时都表现出明显优于对照组的骨量和骨质，具有较好的促进骨愈合的效果。