应力微环境调控骨膜来源细胞生物学行为以及影响干细胞招募的机制研究

When a fracture occurs, the periosteum will suffer damage or separation, and the internal stress state of the periosteum tissue will change dramatically. The regeneration ability of the periosteum after fracture may be related to the change of stress state after the damage of the periosteum, and this change of stress state may activate the static state of the mechanical sensitive periosteum-derived cells (PDCs) in the periosteum, and further trigger the endogenous stem cell recruitment to reach the damaged part and repair the damaged bone tissue. However, the effect of mechanical stimulation on the biological behavior of PDCs in vitro is mostly focused on the use of silica gel membrane as substrate, which is difficult to simulate the real mechanical microenvironment of PDCs in natural periosteum and the effect of the stress release of periosteum on the biological behaviors of PDCs. This project intends to decellularize the periosteum of New Zealand white rabbit tibia, obtain natural acellular periosteal tissue, explore the periosteal prestress release of PDCs proliferation, migration, differentiation, growth factors release, as well as the release of the periosteum tissue after fracture of the stromal cell derived factor -1 (SDF-1) is regulated by the prestress release of periosteum tissue. The effects of prestress releasing of periosteum on the biological behavior of PDCs and the recruitment mechanism of endogenous stem cells and the importance of prestress in bone tissue engineering scaffolds based on periosteum regeneration were studied.

当骨折发生时，骨膜会出现受损或分离，骨膜组织的内在应力状态会发生巨大变化。骨折后骨膜的再生能力可能与骨膜受损后应力状态的改变有关，这种应力状态改变可能会将骨膜内力学敏感骨膜来源细胞(PDCs)从静息状态激活，并进一步触发内源性干细胞招募到达损伤部分，参与修复受损骨组织。但目前体外研究力学刺激对PDCs生物学行为的影响还多集中在使用硅胶膜作为基底，难以模拟PDCs在天然骨膜中的真实力学微环境以及骨折时骨膜应力释放对PDCs的生物学行为的影响。本项目拟对兔胫骨骨膜进行脱细胞处理，获得天然脱细胞骨膜组织，探索骨膜预应力释放对PDCs增殖、迁移、分化、因子释放，以及骨折后骨膜组织释放的基质细胞衍生因子-1 (SDF-1)是否受骨膜组织预应力释放的调控。通过研究骨折后骨膜预应力释放对PDCs生物学行为的影响和在体干细胞招募的机制以及明确预应力在基于骨膜再生的骨组织工程支架中的重要意义。

临界尺寸的骨修复依旧是临床的主要挑战之一，而骨损伤一般伴随着骨膜的撕裂以及骨膜组织内部应力微环境的改变，我们从基质力学的角度去探究骨膜预应力的释放对骨膜来源细胞(PDCs)的生物学影响及募集周围干细胞的机制。在本研究中，我们通过脱细胞的方式处理得到具有真实细胞外基质成分、结构以及力学性能的天然脱细胞骨膜，种植上来自大鼠颅骨骨膜的原代骨膜来源细胞，来搭建天然细胞外基质力学研究平台。对脱细胞骨膜进行20%的形变拉伸力突然释放，模拟骨损伤时骨膜破碎，骨膜组织内部应力微环境的改变。对细胞膜上力学感受器Intergrin β1和力学敏感蛋白Yes-相关蛋白(Yes-associated protein, YAP)进行免疫荧光染色，发现Intergrin β1-YAP信号通路被激活，其上清液中基质衍生因子SDF-1α急剧上升。即骨膜预应力释放产生的机械刺激被PDCs表面的力学感受器Intergrin β1感受到，向细胞内机械传导路径发出信号，刺激YAP的入核表达，调控SDF-1α的分泌。骨膜预应力释放后七天，我们对早期的成骨标志物骨钙蛋白(OCN)，骨桥蛋白(OPN)以及碱性磷酸酶(ALP)的表达进行检测发现其含量均比对照组高。表明基质力学的刺激能够在一定程度上影响新骨生成的速度，此外，将该上清液用于体外培养PDCs，发现处于增殖期的PDCs明显增多，原代PDCs会更多地向成骨细胞分化，细胞划痕实验表明骨髓间充质干细胞(BMSCs)会向高浓度的SDF-1α区域迁移，即骨膜预应力释放，对周围PDCs在增殖、迁移以及分化上产生趋向于加速骨修复方向的积极生物学影响。