RANKL-RANK-OPG系统促进磷酸钙/聚乳酸-羟基乙酸共聚物微球复合骨水泥降解作用的探索研究

calcium phosphate based materials are widely used as synthetic bone substitutes in bone reconstructive surgery because of their osteoconductivity , beside this, injectable calcium phosphate cement(CPC) were proposed due to its excellent handling properties, the degradation rate can also be improved if poly(lactic-co-glycolic acid)(PLGA) microsphere was introduced. the drug carrier ability also make the formulation more promising.however, low degradation of the materials still present, also the molevular mechanism of acceleration on degradation is still not fully understood todate. .In this project,Osteoclastogenic factors will be screen for the acceleration of the degradation, since the implant biomaterials should be replaced by new bone, by mimic the bone metobolic process mediated by osteoclast. osteoclast and macrophage share the same progenitor, which is regulated by the pathway of RANKL-RANK-OPG, therefore, the crosstalk between monocyte, macrophage and osteoblast,the impact of RANKL-RANK-OPG system to the degradation of CPC should be thouroughly investigated.In this project, in vitro as well as in vivo effect of RANKL-RANK-OPG system during CPC/PLGA degradation will be investigated, osteoclastogenesis related cells will expose to the environment of CPC/PLGA and their degradation product as well as animal will be implanted the materials via injection. on the other hand, varis osteoclastogenic cytokine such as soluble RANKL, PEG2,IL-4.IL-13 will be incorporated into CPC/PLGA formulation to accelerate the degradation. the interaction between materials interface with the host will be investigated.

本研究结合RANKL-RANK-OPG调控轴系统在骨代谢中的调控机理，探讨其促进磷酸钙/聚乳酸-羟基乙酸共聚物微球骨水泥（CPC/PLGA）降解的可能性. 研究从组织学,细胞学以及分子生物学层面上分别了解RANKL-RANK-OPG环路对磷酸钙类骨水泥降解过程的作用，主要通过在体外实验考察骨水泥及其降解产物对单核细胞，巨噬细胞，破骨细胞间的作用，了解RANKL,OPG的表达变化和相互影响，同时在体内实验中进行RANKL-RANK-OPG环路中各种细胞进行鉴别定位，以及血清学，分子生物学的表达检测。最后利用可溶性RANKL、前列腺素2、白介素IL-4,13等促破骨细胞分化因子，分别加入CPC, PLGA内进行载药，以形成RANGKL-RANK-OPG失衡的微环境，在体外,体内实验了解该新型材料降解性能的变化以及生物学效应，为进一步研制具有良好降解性能的磷酸钙支架材料提供科学依据。

本研究从优化磷酸钙骨水泥（Calcium Phosphate Cement, CPC）多孔结构入手，系统总结了CPC孔隙连通率的降解性微球粒径、混合比例的最佳参数，分别探索了CPC加入全血成分、多种成分以及多种形式的制孔结构以及添加外源性RANKL因子等条件下，CPC/PLGA 复合材料的降解成骨作用。研究发现：1、①提出了一个表征孔隙连通的量化指标——孔隙连通指数，孔隙连通指数越高，孔隙连通性越好。②球体连通指数随两种微球粒径比的增大而增大，粒径比大于 0.7 时， 球体连通指数更高，连通性更好。③将微球与 CPC 进行复合，随着微球体积比的增大，复合物的孔隙率增加，但孔隙率的增加与微球粒度无关。2、CPC/PLGA复合物中加入全血成分可以促进骨的再生，全血成分达到复合物20%的质量比例获得良好的降解成骨效果。3、将ChS颗粒与CPC进行复合，可显著增加CPC的孔隙率，CPC/ChS颗粒复合物具有良好的骨修复能力及生物相容性。4、以静电纺丝PLGA纳米纤维膜的形式和CPC进行复合，可以有效地改善复合物的机械韧性，材料具有良好的骨传导性及生物相容性。5、CPC／PLGA复合物浸提液能够上调RAW264．7细胞上RANK因子的表达，在细胞增殖早期促进成骨细胞上RANKL 因子表达上调以及促进破骨前体细胞破骨向分化和炎症因子TNF-α、 IL-1、 IL-6的表达，表明CPC/PLGA复合材料降解成骨性能的改善和成骨细胞／破骨细胞的早期偶联启动密切相关。