骨诱导性钙磷陶瓷与宿主局部微环境间交互应答机制及骨再生效应的研究

Osteoinductive porous calcium phosphate ceramics are regarded as a novel class of biomimetic bone substitute with biological activity. After implantation the host organism is used as bioreactor to construct tissue-engineered bone shows a relatively good therapeutic effect in the reconstruction of bone defect. However, the mechanism of interactive response at the implant/bone interface of the local microenvironment and induction bone tissue regeneration is still unclear. And its clinical application is limited by defects including osteogenesis inadequate , poor osseointegration , difficulty in shape controling. Our project is about to implant porous scaffolds, fabricated by rapid prototyping technology, which is matching the host bone shape, structure and mechanical properties, in experiment animals to repair the extreme bone defect on the basis a self-developed calcium phosphate ceramic. By means of some techniques，such as genome science and proteomics techniques, we could explore the pivotal signaling pathway and action segment regulate the proliferation and differentiation of MSCs in the interface of bone-ceramic. Besides, the key regulation factors would be selected by flow cytometry, gene suppression, gene subtraction, RNAi, Westen blot and so on. And the role of the drug-loading pattern targeting the selected factors in local microenvironment would be studied. To evaluate the promoting effect of bone regeneration and bone integration and achieve a breakthrough in the field of core technologies of construction of tissue engineered bone in vivo.

骨诱导性多孔钙磷陶瓷是一类具有生物活性的仿生骨替代材料，植入后以宿主机体作为生物反应器构建组织工程化骨，用于修复骨缺损取得了较好的治疗效果。然而，陶瓷材料植入后在接触界面局部微环境与宿主发生交互应答并诱发骨再生的调控机制尚未阐明，还存在成骨不足，骨整合不良，形态难于精确控制等问题限制了它的临床应用。本项目在自主研发的骨诱导性钙磷陶瓷基础上，拟通过快速原型技术制备出与宿主骨外形、结构和力学性能相匹配的多孔支架，植入动物体内整复极限骨缺损，并运用基因组学和蛋白组学等技术探索调控MSCs,在陶瓷-宿主骨界面上增殖、骨向分化等应答的关键信号通路及作用环节，并运用流式细胞、基因抑制消减、RNAi、Westen blot等技术筛选出关键信号调控因子，并探索靶向应用所选因子的缓控载药方式,作用于细胞或支架局部微环境,评价其促进骨再生及骨整合的实际效果，实现在体内构建组织工程骨领域中关键核心技术的突破。

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。骨诱导钙磷陶瓷作为目前及今后研发骨、牙、关节等硬组织的一种主要生物替代材料受到广泛关注。钙磷陶瓷植入后可以在接触界面局部微环境内与宿主发生交互应答，启动新骨形成、并促进主体骨有效长入多孔支架，形成良好的骨整合，然而此生物应答并诱发骨再生的调控机制尚未得到阐明。因此，本研究在自主研发骨诱导性钙磷陶瓷基础上，针对骨诱导钙磷陶瓷促进BMSCs在植入体表面成骨向分化及宿主微环境与钙磷陶瓷的交互作用机制进行了探索，为干细胞的骨向分化与信号调控、特殊生物活性材料的表面改性、成骨因子及其信号通路与抑制破骨作用药物的靶向治疗及相应组织工程化骨的临床应用提供实验依据。. 方法：1. 研究骨诱导钙磷陶瓷材料对BMSCs成骨分化的影响及骨整合效果。. 2. 检测及比较四种不同比例的骨诱导钙磷陶瓷对BMSCs成骨分化的调控作用。. 3. 用基因芯片技术研究磷酸钙陶瓷材料体内外诱导大鼠BMSCs成骨分化过程中miRNAs表达谱的变化。. 4. 制备聚多巴胺/BMP-2功能化修饰的钙磷陶瓷/聚己内酯复合支架检测其体内外促进BMSCs成骨向分化及成骨相关因子的表达情况。. 5. 应用促红细胞生成素/多巴胺接枝处理双相骨诱导钙磷陶瓷，检测其体内外成骨成血管情况及相关成骨成血管基因的表达。. 结果：1. 不同比例的骨诱导钙磷陶瓷材料均可以有效促进BMSCs成骨分化及提高骨整合效果。. 2. 骨诱导钙磷陶瓷材料的骨整合经历了一系列复杂的生物学过程，其中miRNAs起到了重要的作用。. 3. 成功构建了聚多巴胺/BMP-2功能化修饰的钙磷陶瓷/聚己内酯复合支架加速骨缺损的修复。. 4. 成功应用促红细胞生成素/多巴胺接枝处理双相骨诱导钙磷陶瓷，促进了体内外的成骨及成血管过程。