高频振动刺激Wnt/β-catanin信号通路促进骨再生的研究

Bone tissue engineering strategy has been intensively explored to augment bone regeneration. However, challenges of bone regeneration are still confronted with critical size defects (CSD) of bone. The healing of bony trauma (e.g. peri-implant bone due to implant cavity preparation, fracture) depends on the interaction between (1) the osteogenic cell, (2) the osteoinductive stimulus and (3) the osteoconductive matrix scaffold. In recent years, the interest has risen in the use of tissue engineering scaffold to facilitate osteogenesis. A popular strategy is to expand progenitor cells ex vivo and then to seed them onto biodegradable scaffolds (with/without growth factors that stimulate osteogenic differentiation), followed by implantation into the site of the bone defect. In this project, we plan to use a well-designed combination of tissue engineering and biomechanical loading to offer a possible solution for the challenges mentioned abvove.The molecular mechanism of bone cell responding to the biomechanical loading was also exploered. For tissue engineering, we aim to fabricate a novel porous PLGA/HA composite scaffolds and to bio-activate the scaffolds with mesenchymal stem cells (MSCs). For biomechanical loading, we aim to apply high-frequency vibration loading which have recently been suggested to be a potent non-invasive anabolic signal. By use of the CSD model of rat calvaria, the effect of MSCs bio-activated scaffolds, high-frequency vibration loading and their combination on bone regeneration can be assessed. For the molecular mechanism, the Wnt/β-catanin signal pathway was specificly studied in vitro. To identify the corresponding factor(s) of Wnt/β-catanin signal pathway in response to loading, the antagonists/RNAi of the signal pathway were applied in cells with/without loading stimulation. The research flow consists of in vitro and in vivo parts. In the in vitro part of the project, MSCs are isolated, expanded，characterized and loaded with high-frequency vibration. The osteogenic potential and Wnt/β-catanin factors were assessed. During the meantime, we fabricate a novel porous PLGA/HA composite scaffold, followed by bio-activating with MSCs. In the in vivo part, the CSD modle in rat calvaria is established and treated with MSCs bio-actived PLGA/HA scaffold and HF-LM loading. The bone regeneration in CSD area are assessed by means of RT-PCR, molecular biology and (immuno-)histology.

在重度骨缺损(CSD)的情况下，如何取得理想的骨再生是目前的一大临床难题。本课题旨在根据骨缺损愈合的三要素（即成骨性细胞，成骨传导性基质支架和成骨诱导刺激），从当前科研的新进展和本课题组的前期试验结果出发，利用组织工程技术，构建MSCs作为种子细胞的生物活性支架修复骨缺损，施加高频振动刺激，促进骨再生，分析成骨标志性蛋白和基因的表达变化，揭示其内在机理。使用定向受体抑制及RNA干扰手段研究高频振动刺激Wnt/β-catanin信号通路的效应因子，探索高频振动促进骨再生的分子机制。申请人在前期研究中发现高频振动刺激能够促进种植体周围的骨形成和种植体骨整合，已经具备了初步的实验基础和数据支持。本课题将为严重骨缺损情况下的骨再生和种植体骨整合的生物学基础提供新的线索，为寻找利用人工手段促进骨再生，加速种植体骨界面形成开拓新的途径。

骨组织再生的关键性要素是间充质干细胞募集至患处并分化为成骨细胞。Wnt/β-catanin信号通路是募集间充质干细胞，使其向成骨细胞分化，修复骨损伤的重要分子机制。在细胞膜表面SDF-1/CXCR4的配体-受体结合机能决定着间充质干细胞的归巢和迁移。本项目着眼于干细胞在骨再生修复中的作用及其机制，联合应用细胞、组化、分子生物学等技术，研究发现：（1）BMP2/Foxc2的协调作用是牙囊干细胞（SCAP）成骨向分化的重要作用机制，（2）使用细胞渗透性抑制剂对骨髓间充质干细胞（BMSCs）进行预处理，可以有效促进CXCR4在干细胞的表达，显著提高干细胞的迁移效应；（3）采用磁珠分选的策略，从BMSCs中优选出CXCR4高表达的干细胞亚群，同样可以明显提高干细胞的归巢和迁移；（4）Sema3A/Nrp1炎症信号轴对于破骨细胞活性和根尖病变区骨质破坏范围呈负相关的调节作用。本系列的研究发现，为实现骨再生的精准、快速修复提供了理论依据，具有很好的科学意义及潜在的应用前景。受本项目资助已发表研究论文7篇，其中SCI论文4篇（最高IF为3.99）中文核心杂志3篇。培养博后1名和硕士研究生9名，其中3名研究生已毕业。项目投入73万，支出60.502万，各项支出基本与预算相符。剩余经费12.498万，计划用于本项目研究的后续支出。

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