Wnt与Notch信号协同调控钙磷植入体界面MSCs骨向分化并靶向促进其骨整合的作用及机制研究

Calcium phosphate ceramic is an ideal implanted material which can be used to replace the injured hard tissues and organs, but the key point, which needs to be solved immediately, is how to form stable osseointegration in both physiological and pathological conditions. Using the given signal factors to regulate MSCs to aggregate, colonize, proliferate and differentiate into osteoblasts in the interface of bone- ceramic is worthy of exploration. Many findings obtained from detailed analyses of mice having mutations of Wnt or Notch signaling molecules have confirmed that Wnt and Notch signaling has potential roles in bone remodeling. There are two pathways of Wnt signaling: β-catenindependent canonical and independent non-canonical pathways. Wnts act on osteoblast precursor cells and promote their differentiation into mature osteoblasts through the β-catenindependent canonical pathway. Wnt proteins are palmitoylated and glycosylated ligands that play a central role in the development of hard tissues. .To date, many studies have shown that some Notch pathway genes such as Lfng, Hes1, Hes5, and Hes7 have been involved in several aspects of bone formation and bone resorption and provide strong evidence that the Notch proteins are necessary for blood and vessel formation. So Wnt and Notch Signal pathway may be the two main modulators which are related to bone growth and metabolism. In recent years, many researchers have found that the two pathways have direct or indirect connections, such as synergy or antagonist, and the connections play a critical role in regulating the cell ossification and bone formation. And many researchers suggest that the balance between Notch and Wnt signaling determines whether to make or break new vessel connections. However the certain role of the two pathways and the cross-talk between them are still not clear. Our project is about to put the titanic implants, on which nHA/PA or HA has been flame plated, into the femur bone of the female SD rats or ovariectomized (OVX) SD rats. By meas of some techniques，such as gene suppression or gene subtraction, RNAi、SiRNA、Westen blot、Flow Cytometer, RT-PCR and so on，we could explore the expression of the key gene and protein, and its relationship with the aggregation, proliferation and differentiation of MSCs. Besides, how and in which part the pathways regulate the osseointegration would be studied.Also, with the application of key factors, the WNT and NOTCH pathway could be intervened to develop its synergy effect for the synosteosis.

钙磷复合物是目前较理想的可植入性硬组织替代材料，在生理或病理状态下有效促进植入体与主体骨形成稳定的骨整合是急需解决的主要问题，运用关键信号因子靶向调控MSCs在钙磷-骨界面上募集、增殖、骨向分化是值得探索的关键路径之一。Wnt和Notch通路是骨组织生长发育和代谢相关的主要调控信号。近年发现：两条通路之间存在着许多直接或间接的串话机制，可表现为互相协同或拮抗的耦合效应，其交互串话机制在成骨细胞系增殖、分化，改建中均起着重要作用。本课题拟将多孔nHA/PA、HA表面喷涂的钛桩植入正常及骨质疏松的SD雌性大鼠股骨，并运用流式细胞仪，RT-PCR，基因抑制消减、RNAi、Westen blot等技术探索通路中关键基因及蛋白的表达及其与MSCs增殖、分化、凋亡的对应关系，探索二通路对钙磷-骨整合的调控方式及作用环节，并运用关键信号因子靶向干预Wnt与Notch通路，发挥协同作用，有效促钙磷骨整合。

临床上，很多患者由于先天畸形、创伤和疾病所致的骨、关节和牙缺失、缺损或病变需要植入或置换硬组织替代材料。钙磷类植入体是目前及今后研发骨、牙、关节等硬组织替代材料的一种主要趋势。骨髓间充质干细胞（BMSCs）向成骨细胞分化的过程受到多种信号通路的调控。作为保守、经典的信号通路，Wnt和Notch信号通路在骨组织的生长发育、骨代谢及骨重建领域发挥着重要作用，可调控BMSCs成骨分化过程。因此，本研究将多孔nHA/PA、HA 表面喷涂的钛桩植入正常及骨质疏松的SD雌性大鼠股骨，针对Wnt和Notch信号转导通路靶向促进BMSCs在钙磷植入体表面骨向分化的机制进行了探索，为靶向促进BMSCs在植入体表面的骨整合及相应组织工程化骨的临床应用提供实验依据。.方法：1.利用阳极氧化法制备表面存有TiO2纳米孔的钛植入体，利用组织学、显微CT、生物力学等检测方法研究植入体表面TiO2纳米化修饰对植入体骨整合的影响。.2.SD大鼠骨髓间充质干细胞的体外培养与成骨向诱导。.3.成骨向分化的大鼠BMSCs与喷涂羟基磷灰石/聚酰胺（nHA/PA）的表面纳米化改性钛片体外复合培养，检测骨整合效果.4.通过Jagged-1体外上调Notch通路，将BMSCs与nHA喷涂的钛片在体外复合培养，观察Jagged-1对钙磷植入体表面BMSCs成骨向分化的作用.5.引入基质胶（Matrigel）作为缓释载体，观察缓释Jagged-1对钛种植体周边骨整合的影响.6.植入体羟基磷灰石涂层在骨整合中对Notch信号通路的影响.7.将BMSCs与两种磷酸钙（CaP）生物陶瓷材料复合培养。检测CaP陶瓷诱导BMSCs成骨分化过程中成骨相关因子及Wnt和Notch信号通路相关因子的表达情况；同时研究了培养基中加入Wnt通路抑制剂DKK1后相关因子的表达情况。.结果：1.植入体表面的纳米化改性显著改善植入体的骨整合。.2.Jagged-1可以通过激活Notch通路，调控BMSCs向成骨方向分化，增强其在钙磷植入体表面的早期粘附、伸展、增殖和迁移，促进BMSCs在钙磷植入体表面上的骨整合。.3.建立了由Matrigel介导的Jagged-1缓释系统，延长了Jagged-1的释放时间，并且有效促进了钙磷植入体表面的骨整合。.4.羟基磷灰石涂层是Notch信号通路的一种强激活剂, 提高了种植体的界面稳定性，促进了骨整合的