TiO2纳米管负载淫羊藿苷对骨质疏松下钛种植体骨结合的作用及分子机理研究

Patients with osteoporosis is prone to suffering from decreased primary stability and delay bone bonding during implant treatment.This may impair the successful rate in implant surgery. To increase implant's early stability so as to achieve early osseointegration is one of the key problems in dental implantology. Icariin has the osteoinductivity, could promote osteogenisis, angiogenesis and inhibit bone absorption, and is effective on curing osteoporosis, without adverse effect. In the present research, we combined the modern TiO2 nanotube preparation technology and traditional Chinese herb icariin to establish the new implant surface which could promote the implant osseointegration. We constructed the TiO2 nanotube by anodic oxidation, then loaded icariin onto nanotube. This subject studies the release rules of the icariin loaded TiO2 nanotube surface of titanium implant through surface characterization and drug delivery test. We further cocultured the icariin loaded TiO2 nanotube titanium implant material with bone marrow mesenchymal stem cells to examine its bioactivity in vitro and further implanted into rabbit tibia to examine its bone bonding ability in vivo. The proliferation, differentiation effect, molecular signaling pathway and network change in icariin loaded TiO2 nanotube titanium implant material are examined. In addtion, the high throughput Gene Chip technology was combined with bioinformatics method, to screen the osteogenesis-related and osteoclast-related gene expression, signal pathway and molecular network. In the present study, we explore the underlying molecular mechanism of TiO2 tube and icariin to promote the osteogenic activity respectively and their synergistic action in vitro and osseointegration in vivo. The results will provide theoretic basis for improve the osseointegration of titanium implant under osteoporosis situation.

骨质疏松易造成种植体初期稳定性降低和延迟骨结合，影响种植成功率，是种植临床工作难点。淫羊藿苷具有骨诱导性，可促进成骨、血管生成，抑制骨质吸收，治疗骨质疏松效果明确，无毒副作用。本研究拟通过阳极氧化方法在纯钛表面制备TiO2纳米管，在此基础上负载淫羊藿苷。通过试件表征和药物释放实验研究纳米管内淫羊藿苷加载及释放规律；采用骨髓间充质干细胞体外培养实验探讨纳米管和淫羊藿苷单独作用与协同作用下对骨髓间充质干细胞增殖、分化和信号通路及分子网络的影响；经由骨质疏松兔模型研究纳米管和淫羊藿苷单独与协同作用下诱导骨-种植体界面骨结合的分子机制。进一步采用基因芯片和生物信息学手段，全面快速分析TiO2纳米管负载淫羊藿苷对种植体表面成骨、破骨相关基因表达的影响，筛选并验证关键性的成骨信号通路，探讨TiO2纳米管负载淫羊藿苷的分子作用机制，为骨质疏松条件下种植体表面改性提供新思路和理论基础。

骨质疏松易造成种植体初期稳定性降低和延迟骨结合，影响种植成功率，本项目研究TiO2纳米管负载治疗骨质疏松的药物淫羊藿苷后对骨质疏松下钛种植体骨结合的作用及分子机理。项目分两部分：第一部分为纳米管表面加载淫羊藿苷的研究。采用阳极氧化的方法，不同电压条件下（5V和18V）在纯钛表面制备 TiO2 纳米管并负载淫羊藿苷。管径随电压加大而增大；纳米管形貌有效增加亲水性。药物释放实验显示，大管径的纳米管的药物加载量更大，药物释放达到5天。体外细胞培养实验的SEM、CCK-8、AO/EB细胞活性和碱性磷酸酶（ALP）活性结果显示，淫羊藿苷和大管径的纳米管协同促进成骨细胞的粘附、活性、增殖和分化。Real-time PCR检测提示，成骨相关基因OPN、OCN和Osterix的表达水平均呈现18V纳米管负载淫羊藿苷＞18V纳米管＞纯钛组的趋势。Western blot结果提示，成骨相关蛋白OPN及Runx-2的表达量与上述基因表达趋势一致。通过KEGG数据库进行Pathway分析后发现，成骨相关的差异基因主要分布于MAPK、PI3K-AKT、cGMP-PKG 通路，ICA可能通过MAPK/ERK通路发挥效应。利用雌激素受体抑制剂后细胞培养的RT-PCR和Western blot检测结果均显示，抑制剂显著减弱淫羊藿苷的成骨分化作用。提示MAPK/ERK通路是淫羊藿苷调节成骨细胞分化成骨的重要通路之一。大鼠骨质疏松模型结果显示，TiO2纳米管及淫羊藿苷均有刺激新骨生成的作用，而18V纳米管负载淫羊藿苷组新生骨的形成更加明显。为进一步增强淫羊藿苷的缓释效果，第二部分引入新型的变性溶菌酶预处理方法在钛表面加载透明质酸和壳聚糖层层自组装负载淫羊藿苷的涂层，达到了两周的缓释周期。细胞实验显示改性后的表面更加促进成骨细胞粘附、增殖、分化，动物实验证实，加载淫羊藿苷的钛种植体周围可见更多的新骨生成。本项目为骨质疏松条件下种植体表面改性提供新思路和理论基础。