二氧化钛纳米管阵列应用于牙科种植体表面改性的生物力学性能分析与优化

Recent fundamental research on dental implantation was focused on implant surface modification for rapid and improved osseointegration. Since the initial fate of host tissues and cells is directly affected by nanoscale surface roughness. Nano-engineering modification including titania nanotube (TNT) may be beneficial to augment the bioactivity of Ti based implants. Notably, as-anodized TNT is very brittle, which can easily be peeled off by mechanical bending. Furthermore, for the dental implant setting, which is continuously under load bearing in bone micro-environment, the stable mechanical properties of bioactive TNT are essential to ensure long term success, which remains underexplored. Highlighting this research gap, fabrication optimization and post-treatments to augment their mechanical properties are critical for integration into the current implant market. In the previous study, we elaborate on augmenting the stability of therapeutic nanotubes on anodized dental implants by optimizing the electrochemical anodization setup. We first fabricated robust TNT on dental implant abutment and screw. Thus, based on the results we reported previously, the present project will incorporate various strategies including physical treatment, chemical modification and biological coating to augment mechanical stability, as well as advanced implantation studies were performed. TNT modified implants were inserted ex vivo and in vivo to determine if the nanotubes survive the mechanical handling, implantation surgery and the bone contact itself. Furthermore, the osteogenic effect was evaluated using histology methods. Aiming at promoting the successful translation of TNT from research to clinical testing and integration into the commercial implant market.

通过种植体表面改性获得快速稳定的骨结合是当今种植领域的研究热点之一。阳极氧化生成的二氧化钛纳米管（TNT）阵列具有促进早期成骨的特性，但仍有机械强度不足、与钛基底的结合力弱、易脱落等不足。因此，通过优化阳极氧化反应体系与后期改性处理增强TNT阵列机械性能是实现其临床应用的关键。本项目拟以前期优化的、可增强TNT阵列力学性能的反应体系和参数为基础，进一步采用物理（退火）、化学（F-沉积）和生物学（覆盖多肽涂层）方法改性TNT阵列，观察其微结构、力学性能、与基底结合强度的改变；通过体外培养骨髓基质干细胞以及小型猪牙槽骨块种植手术，观察细胞在材料表面黏附生长与功能分化情况，分析TNT阵列的机械稳定性和成骨性能；建立小型猪顶骨种植模型，采用组织学和生物力学的方法研究材料的骨整合效果和机械稳定性。本项目的完成有望构建出机械性能强、成骨活性好的表面涂层，为TNT阵列应用于牙种植体表面改性提供实验依据。

种植修复的长期成功与软组织良好的生物学封闭密切相关。研究证明阳极氧化法形成的二氧化钛纳米管（TNT）阵列具有促进早期成骨的特性，但对其周围软组织/细胞的研究相对较少，且TNT阵列仍有机械强度不足，与钛基底结合力弱等不足。如何优化与改性TNT阵列以增强其机械性能，同时促进良好的软、硬组织结合具有重要的临床意义。本项目优化阳极氧化反应条件制备TNT阵列、通过聚多巴胺薄膜（PDA）中间层将D-amino生物多肽连接于TNT阵列，研究其对TNT阵列机械力学性能的影响；体外探究对牙龈成纤维细胞（HGFs）以及种植体表面早期定殖菌和种植体周围炎致病菌生物学行为的影响；兔股骨种植实验评价种植体表面结构的机械稳定性和骨结合效果。使用扫描电镜（SEM）观察见阳极氧化电压设置为60V，反应10分钟，形成的纳米孔沿钛表面微米级沟槽有序排列，结构完整；原子力显微镜（AFM）观察见PDA包被后材料表面有点状颗粒，D-amino多肽接枝组表面颗粒变大；激光共聚焦显微镜（CLSM）观察发现D-amino接枝后材料表面出现红色荧光； X射线光电子能谱分析（XPS）结果显示PDA包被后出现N1S峰，且C1S峰升高，D-amino多肽接枝后出现S2P峰，且N1S峰升高；纳米压痕实验结果显示PDA-D-amino改性的TNT阵列（PDA-D-amino-TNT）强度显著增强；纳米划痕实验结果显示PDA-D-amino-TNT与钛基底的结合强度显著增强。使用活死菌染色法和SEM观察培养24小时的变形链球菌和具核梭杆菌在材料表面的粘附情况，可见PDA-D-amino-TNT表面两种细菌的粘附数量显著减少；采用菌落计数法评估对 S.m和 F.n两种细菌抗菌率分别为81.52%和71.7%。 CCCK-8实验测定细胞增殖的数量（1、2、3、4天），培养第3天和第4天，HGFs在PDA-D-amino-TNT表面增殖明显；CLSM观察细胞整合素ß1和黏着斑蛋白的表达显示PDA-D-amino-TNT表面蛋白分布更为广泛。兔股骨植入种植体24后取出SEM观察植体表面TNT结构完整，植入过程未发生机械损害；术后4周、8周 Micro-CT评估种植体周围骨结合情况。综上，D-amino可借助PDA中间层稳定接枝于TNT阵列，显著增强TNT阵列机械性能，提高软、硬组织相容性，为该材料进一步应用提供良好的实验基础。