针对骨质疏松条件的miRNAs缓释钛种植体制备和骨结合能力与分子机制研究

How to create the ideal osseointegration under the condition of osteoporosis with disordered balance of bone metabolism turns into an urgent problem for the tianium implants. Recently, we have reported that MAO Ti surfaces can actually serve as a good drug loading and delivering platform. Considering the outstanding ability of antimiR-138 and miR-34a which work respectively through promoting osteogenesis and suppressing osteoclastogenesis to positively regulate bone turnover, we plan to develop chitosan/antimiR-138 and chitosan/miR-34a loading MAO coating on dental implant by covalently binding and layer-by-layer self-assembly. The novel coating is hoped to be firmly combined and long-lastingly release antimiR-138 and miR-34a in a controlled mode, finally leading to faster and better osseointegration in osteoporotic condition. After fabrication, the surface properties of the miRNAs loading MAO Ti surfaces will be well characterized, including stability and release rate. In vitro experiments will be conducted to observe the effect of the miRNAs loading MAO Ti surfaces on bone mesenchymal stem cell and osteoclast precursor cells functions, especially osteogenic differentiation and osteoclast differentiation. Then the in vivo osseointegration of the miRNAs loading MAO Ti surfaces will be observed in osteoporotic rats to determine the sample that generates the best osseointegration in the osteoporotic condition. Further, the influence of the long-lasting effect of the two factors of promoting osteogenesis and suppressing osteoclastogenesis on bone turnover related signal pathways will be inspected to deeply understanding their biological effect and further guide the structure optimization of the miRNAs loading MAO Ti surfaces. The miRNAs loading MAO Ti surfaces, combining the dual effects of antimiR-138 and miR-34a, are very promising to generate good in vivo osseointegration in the osteoporotic condition. The technique reported here can be also extended to other biomaterials.

如何在骨质疏松（OP）这一骨代谢失衡条件下形成理想骨结合是牙科钛种植体亟待解决的问题。最近我们发现微弧氧化处理钛表面具有优秀的载药能力，同时考虑到antimiR-138与miR-34a可分别通过促成骨和抑破骨正向调节骨代谢平衡，本课题提出联合采用共价结合和自组装多层复合技术，在微弧氧化处理钛表面构建稳固结合且可同时缓释壳聚糖/antimiR-138和壳聚糖/miR-34a的涂层以在OP患者体内获得长效骨结合能力。对涂层中miRNA活性和释放进行系统表征，体外观察该涂层对间充质干细胞和破骨前体细胞功能特别是成骨和破骨分化的影响，体内观察在OP动物模型内的骨结合情况，筛选出最佳的涂层构成。深入探索持续作用的促成骨和抑破骨双因素共同对骨代谢相关信号通路的作用以指导优化种植体构建。该新型涂层有望在OP患者体内获得更好的种植体骨结合，同时为其它生物材料的设计提供借鉴。

项目背景：牙科种植技术已成为牙列缺损或缺失的重要修复方法之一。骨质疏松病人其特殊的骨质条件进行种植修复时风险较大，失败率较普通人群高。具有较强成骨能力钛种植体的生物功能化有望进一步促进种植体表面的骨结合，尤其是在特定骨缺损及骨质条件如骨质疏松等的情况下意义尤为突出。.主要研究内容：我们首先对壳聚糖的分子量、脱乙酰度和与miRNA的N/P进行了筛选。采用硅烷戊二醛偶联法在微弧氧化钛表面构建稳固钛-壳聚糖涂层。以壳聚糖-miRNA (CS-miRNA)复合物和透明质酸钠（HA）分别为带正电和负电的聚电解质，采用层层自组装在微弧氧化钛-壳聚糖涂层表面制备聚电解质多层涂层。首先采用动态接触角测量和扫描电镜对涂层的堆积过程进行分析。采用核糖苷定量法测定miRNA的加载和释放情况。将间充质干细胞（MSCs）接种于CS-antimiR-138/HA聚合电解质功能化的微孔钛表面，观察其体外转染效率和细胞毒性。检测了CS-antimiR-138/HA 聚合电解质功能化微孔钛植入物的MSCs的体外成骨分化和体内骨整合情况。构建并检测CS-miR-34a/HA 聚合电解质涂层对破骨细胞前体细胞的影响。构建CS-antimiR-138和CS-miR-34a混合涂层并分析体外促MSCs成骨分化能力。.重要结果和数据：筛选出壳聚糖最优分子量为10KDa、脱乙酰度为95%，miRNA的N/P 比例为60:1。表面润湿性在PEMs形成过程中交替变化。CS-miRNA纳米颗粒沿MAO表面均匀分布。miRNA的负载量随着层数的增加而增加。miRNA持续释放超过2周。体外转染结果显示，壳聚糖/antimiR-138纳米颗粒被细胞高效吸收，下调细胞内miR-138表达，但细胞毒性不明显。CS-antimiR-138/HA 聚合电解质功能化的微孔钛表面增强了MSCs在其上的成骨分化，增强了碱性磷酸酶、胶原分泌和细胞外基质矿化。在大鼠模型中，它显著增强了体内骨结合。CS-miR-34a/HA 聚合电解质涂层有效抑制破骨前体细胞的破骨分化。CS-antimiR-138和CS-miR-34a混合涂层有效促MSCs成骨分化。.科学意义：CS-miRNA/HA 聚合电解质涂层对于种植体表面成骨具有积极地促进作用，能加速骨结合。这一成果对骨组织工程修复材料的研制具有积极地意义。