钛表面微纳层级结构促MC3T3-E1细胞黏附增殖及载药潜力评价  

Micro/nano hierarchical structure of titanium surface promotes MC3T3⁃E1 adhesion proliferation and drug loading potential evaluation

作　　者：丁彤 王健辉 肖智勇 武腾飞[5] 杨瑟飞 DING Tong;WANG Jianhui;XIAO Zhiyong;WU Tengfei;YANG Sefei(Medical School of Chinese PLA,Beijing 100853,China;Department of Stomatology,the First Medical Center,Chinese PLA General Hospital,Beijing 100853,China;Institute of Pharmacology and Toxicology,Beijing 100850,China;State Key Laboratory of Toxicology and Medical Countermeasures,Beijing 100850,China;Avic Changcheng Institute of Metrology&Measurement,Beijing 100095,China)

机构地区：[1]中国人民解放军医学院,北京100853 [2]中国人民解放军总医院第一医学中心口腔科,北京100853 [3]军事科学院军事医学研究院毒物药物研究所,北京100850 [4]抗毒药物与毒理学国家重点实验室,北京100850 [5]航空工业北京长城计量测试技术研究所,北京100095

出　　处：《口腔疾病防治》2023年第8期543-551,共9页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金面上项目(62175229)。

摘　　要：目的探讨钛表面微纳层级结构对MC3T3-E1细胞黏附增殖的影响和载药潜力的评价,为钛表面选区改性并载药缓释提供参考。方法根据钛表面处理方式将纯钛样本(直径10 mm,厚2.5 mm)随机分为:抛光组(T)、阳极氧化组(TO)和微纳层级结构组(FTO)。T组仅做抛光处理;TO组采用阳极氧化技术处理;FTO组采用飞秒激光蚀刻联合阳极氧化技术处理。用扫描电镜(scanning electron microscopy,SEM)观察3种表面形貌,接触角测量其表面的润湿性,X射线能谱仪(energy dispersive spectroscopy,EDS)分析表面化学成分;激光共聚焦显微镜(confocal laser scanning microscope,CLSM)测量FTO结构深度和表面粗糙度;免疫荧光染色、CCK-8和茜苏红染色半定量分析评估MC3T3-E1细胞在各组样本表面的黏附增殖分化;应用冷冻干燥法加载重组人源骨形成蛋白-2(recombinant human bone morphogenetic proteins-2,rhBMP-2),酶联免疫吸附反应(enzyme-linked immunosorbent assey,ELISA)评估不同表面结构的载药潜力。结果SEM观察到T组钛板表面可见方向一致且均匀的抛光痕迹;TO组表面为纳米级蜂窝状的二氧化钛(titanium dioxide,TiO2)纳米管结构;FTO组形成直径约为100 nm的规则有序的微纳层级结构。FTO组的接触角最小,为32°±1.7°,润湿性最好;一级结构圆孔平均深度为93.6μm,粗糙度1.5~2μm。TO组和FTO组含氧量占比多,提示TiO2形成。FTO组表面细胞增殖最显著(P<0.001),细胞黏附表面积最大(P<0.05)。FTO组加载rhBMP-2后缓慢释放14 d,且能促进细胞外基质矿化(P<0.001)。结论钛表面微制备的纳米层级结构有促进MC3T3-E1细胞黏附增殖和成骨分化的作用,有载药潜力,是钛表面处理的新方式。Objective To investigate the effect of micro/nano hierarchical structures on the adhesion and prolifera⁃tion of MC3T3⁃E1 cells,evaluate the drug delivery potential of titanium surfaces,and provide a reference for the modifi⁃cation of selected areas of titanium surfaces to enhance drug delivery and slow drug release.Methods Pure titanium samples(10 mm in diameter and 2.5 mm in thickness)were randomly divided into a polished group(T),anodized group(TO),and micro/nano hierarchical structure group(FTO)according to the surface treatment of the titanium.The T group was polished,the TO group was treated with anodic oxidation technology,and the FTO group was treated by femtosec⁃ond laser etching combined with anodic oxidation technology.The three surface morphologies were observed by scan⁃ning electron microscopy(SEM),the wettability of the surface was measured by the contact angle,and the surface chemi⁃cal composition was analyzed by X⁃ray energy dispersive spectroscopy(EDS).The depth of the FTO structure and the surface roughness were measured by confocal laser scanning microscopy(CLSM).MC3T3⁃E1 cell adhesion proliferation and differentiation on the surface of each group of samples was assessed by immunofluorescence staining,CCK⁃8,and semiquantitative analysis of Alizarin staining.A freeze⁃drying method was applied to load recombinant human bone mor⁃phogenetic protein⁃2(rhBMP⁃2),and an enzyme⁃linked immunosorbent assay(ELISA)was used to assess the drug⁃load⁃ing potential of different surface structures.Results SEM revealed that the surface of T group titanium plates showed uniform polishing marks in the same direction.The surface of the TO group was a nanoscale honeycomb⁃like titanium di⁃oxide(TiO2)nanotube structure,and the FTO group formed a regular and ordered micro/nano layered structure.The con⁃tact angle of the FTO group was the smallest at 32°±1.7°.Its wettability was the best.The average depth of the first⁃level structure circular pores was 93.6μm,and the roughness wa

关 键 词：钛 激光 表面处理 阳极氧化 微纳层级结构 细胞黏附 细胞增殖 细胞分化 骨形成蛋白-2 药物载体 药物释放 

分 类 号：R78[医药卫生—口腔医学]