【作者】 王峰；

【导师】 牛忠英； 施生根；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2013， 博士

【摘要】 过去二十年，无疑是口腔种植技术迅猛发展的黄金期。种植牙已成为目前最理想和最重要的缺牙修复方式，越来越被广大缺牙患者所接受。然而在临床工作中，仍然有许多局部和全身因素会导致种植失败的发生，如骨质疏松、咬合负载过重、吸烟、糖尿病、牙周炎、自身免疫疾病等。在影响种植牙失败的诸多因素中，种植体周围发生细菌微生物引起的种植体周围炎和种植体周围粘膜炎往往是种植失败的主要原因。因此，牙种植体骨界面已成为种植体发生初期骨结合和种植成功的关键。同时，通过牙种植体骨界面的表面改性和修饰来促进种植体骨结合一直是国内外学者研究的热点和难点。本课题正是基于这一现状，受生物活性功能界面仿生学的启发，拟在牙种植体骨界面上多尺度下模拟构建出与自然牙槽骨组织相似的等级排列的微纳复合结构，从而使种植体获得更好的表面性能、亲水性、生物相容性、力学耦合，更有利于牙种植体发生初期骨结合，为临床上进一步提高种植牙成功率提供了新的思路和理论依据。研究目的：通过病理切片、SEM、AFM等多元化手段分析Wistar大鼠牙槽骨组织中微米级单位（哈弗氏管）和纳米级单位（胶原原纤维）的大小及各自的结构特点，得到体外牙种植体骨界面模拟构建的标准和自然参考模型。采用特定浓度下的双元酸处理和恒电位三电极氧化技术在体外Ti种植体表面模拟构建出与自然牙槽骨相似的等级排列的微纳复合结构，并对三种不同的Ti种植体骨界面（机械平滑面、微米级界面、微纳复合界面）作SEM、XRD、XPS等分析，比较三种不同Ti种植体骨界面表面特征及性状的差异；通过AFM观察分析三种不同Ti种植体骨界面的形貌差异及表面三维立体结构的差异；利用AFM的Tip动态扫描并力学耦合三种不同的Ti种植体骨界面，通过对三种不同表面接触力曲线的动态研究，比较三种不同的Ti种植体骨界面粘附功及力学性能的差异；用水接触角测量仪分析微纳复合构建后界面亲水性的变化。在体外通过SBF浸泡实验，观察三种不同的Ti种植体骨界面生物学活性和生物相容性的差异；分别在三种不同的Ti种植体表面作成骨细胞粘附培养，用SEM、AFM动态观察成骨细胞的粘附情况，通过荧光显微镜在相同培养时间节点上的成像及细胞计数，比较分析三种不同Ti种植体骨界面成骨细胞生物学行为和骨诱导性的差异。实验方法和结果：实验一：方法：将9周龄大的Wistar大鼠在腹膜内注射下麻醉，用环形取骨钻（配0.9%的无菌生理盐水冲洗降温）在大鼠左侧下颌角中心部位区取下一直径约为3mm的圆形骨块，置于10%的福尔马林溶液于4℃固定一周，然后修整标本边缘制样、包埋，制备30m厚的不脱钙硬组织切片，HE染色后用SEM、AFM观察分析其中微米级单位（哈弗氏管）和纳米级单位（胶原原纤维）的大小及各自的结构特点。结果：（1） Wistar大鼠自然牙槽骨中的微米级单位（即哈弗氏管）的大小在直径0.5-2m的范围内，SEM、AFM观察下显示为微坑状有序排列的结构。（2） Wistar大鼠自然牙槽骨中的纳米级单位（即胶原原纤维）的大小在直径60-80nm的范围内，SEM观察下显示为向一个固定方向延伸的似电纺丝样的结构，AFM观察下显示这些纳米级的胶原原纤维有序的嵌入了微米级的坑状结构中。实验二：方法：利用预先配置的特定浓度的HNO3和HF对Ti种植体表面作双元酸处理获得微米级结构，然后在此微米级结构表面通过恒电位三电极氧化技术构建垂直排列的TiO₂纳米管，这样就得到了等级排列的微纳复合界面。通过SEM、AFM观察分析微纳复合界面较机械平滑面、微米级界面的形态学差异，TiO₂纳米管及微坑状结构的大小、各自特点。利用AFM区域扫描分析三种不同Ti种植体骨界面（机械平滑面、微米级界面、微纳复合界面）表面粗糙度、平均峰谷高度的差异，利用AFM的Tip动态扫描及力学耦合分析三种不同Ti种植体骨界面粘附功及表面力学性能的差异。利用XRD、XPS分析Ti种植体骨界面微纳复合模拟构建后表面成分及元素的变化。利用静态水接触角测量仪分析三种不同Ti种植体骨界面亲水性的差异。结果：（1）本研究构建了一层厚度约为200nm的垂直排列的TiO₂纳米管嵌入微米级的坑状结构中，直径在30-50nm，与大鼠自然牙槽骨的胶原原纤维（纳米级单位，直径在60-80nm）大小接近。（2）三种不同Ti种植体骨界面表面粗糙度、平均峰谷高度均有统计学差异（P<0.05）（机械平滑面<微米级界面<微纳复合界面），三种不同Ti种植体骨界面的亲水性有统计学差异（P<0.05）（微纳复合界面>微米级界面>机械平滑面）。（3） XRD曲线分析显示三种不同Ti种植体骨界面上矿化晶体结构有明显差异，其中TiO₂、TiC的晶体峰差异明显；XPS分析显示三种不同界面的Ti、O元素峰有显著性差异（Ti元素：机械平滑面>微米级界面>微纳复合界面,O元素：机械平滑面<微米级界面<微纳复合界面）。（4） AFM上部Tip的动态扫描显示，三种不同Ti种植体骨界面的表面接触力曲线有显著差异，粘附功对比有统计学差异（P<0.05）（微纳复合界面<微米级界面<机械平滑面）。实验三：方法：将具有三种不同界面结构的厚度为1mm的纯Ti片分别置于SBF中浸泡7天、14天，利用SEM观察表面矿物质沉淀情况的差异，并用XPS、拉曼光谱分析证实该表面矿物质的HA样类似物的性质。将单位面积数量相等的成骨细胞接种、培养于具有三种不同界面结构的纯Ti片（边长为10mm，厚度为1mm）上，分别用SEM及Bio-AFM观察成骨细胞在2h、24h、48h后粘附状况的差异；然后在每个时间节点上用PBS冲洗掉每个样品上未粘附的成骨细胞，用DAPI染色固定，利用荧光显微镜成像及细胞计数，比较分析三组样本在每个时间节点上细胞粘附数量的差异。结果：（1） SBF浸泡14天后显示，三组样品表面矿物质沉淀情况有显著差异。机械平滑面上几乎看不见矿物质沉淀，微米级界面上可见一些小的、颗粒状散在分布的矿物质沉淀，而微纳复合界面上可见一层均匀一致的大的、球状颗粒物组成的矿物质沉淀层。拉曼光谱和XPS分析证实该矿物质沉淀物为HA样物质。（2） SEM和AFM观察分析显示三组不同样品在成骨细胞粘附培养2h、24h、48h后有显著性差异。机械平滑面上可见稀少的线状伪足粘附，微米级界面上可见一些的多边形细胞伪足粘附，而微纳复合界面可见大量的细胞核和细胞伪足同时更紧密的粘附。（3）在每个时间节点上（即细胞培养粘附2h、24h、48h后），三组不同样品表面荧光显微细胞计数显示有统计学差异（P<0.05）（机械平滑面<微米级界面<微纳复合界面）。研究结论：（1）本研究得到了大鼠自然牙槽骨中微米级单位（哈弗氏管）和纳米级单位（胶原原纤维）的大小及各自的结构特点，证实了多尺度结构的存在和有序排列。（2）本研究用特定浓度下的双元酸处理和恒电位三电极氧化技术在体外Ti种植体表面模拟构建出了与自然牙槽骨相似的等级排列的微纳复合结构，该技术简单易行、安全可靠、效果良好，适于下一步应用于临床研究和推广。（3）这种多尺度下模拟构建的微纳复合结构可以使Ti种植体获得更大的表面粗糙度、表面能，更理想的亲水性、力学特性，更低的粘附功，更好的生物学活性、生物相容性，更有利于成骨细胞粘附、生长，最终更有利于Ti种植体的初期骨结合。（4）这种拟生物的多尺度等级模拟构建，为牙种植体表面的改性及修饰提供了仿生学的新思考，也为临床上进一步研究提高种植牙成功率提供了新的思路和理论依据。更多还原

【Abstract】 The past two decades is the rapid development period of oral implantologyundoubtedly. Dental implants has become the most ideal and important way to replace themissing teeth, which have been acceptable to more and more patients. However, failuresleading to dental implants’ removal still do occur. Several risk factors, such as loosetrabecular bone, excessive occlusal loading, tobacco use, diabetes, periodontitis,autoimmune disease, have been considered to contribute to implant failures. Among thesefactors, the peri-implantitis and peri-implant mucositis caused by bacteria microbialinfection is usually the most important reason of implant failures. Therefore, dentalimplant-bone interface is playing a key role in the osseointegration and survival of dentalimplants. Meanwhile, the surface modification and decoration of dental implants hasalways been the emphasis and difficulty of domestic and overseas scholars’ study. Thisaricle is precisely based on this background, the research was interested in design andbiomimetic fabrication of dental implant–bone interface with multiscaled surfacialarchitecture similar to hierarchical micro/nano structure in alveolar bone natively.Consequently, to acquire a Ti implant with a larger surface energy and roughness, a preferable hydrophilicity, a more adaptive mechanical property, a better bioactivity andbiocompatibity. This biomimetic fabrication on the surface of Ti implant can improve itsprimary osseointegration, and this bioinspired design idea can provide a novel thought andtheoretical basis to enhance the success rate of implanting.Study objectivesFirstly, we will analyze the size and structure characteristics of micro/nano unit inWistar rat’s alveolar bone through pathological section, SEM, AFM. Thus, we can acquirea standard and native reference model of dental implant-bone interface biomimeticfabrication.Secondly, a novel way that combined specific concentration binary acid treatmentwith constant potential anodizing will be used to fabricate a biomimetic micro/nanoarchitecture on Ti implant’s surface. Furthermore, we will utilize SEM, XRD and XPS toanalyze and compare three different Ti implant-bone interface （Smooth, Micro,Nano/micro）. Meanwhile, we observe and analyze three different Ti implant-boneinterface’s morphology and3D structure. In addition, three different Ti implant-boneinterface’s mechanical property and adhesion work is analyzed by surface contact forcecurve of AFM tip’s scanning. The dynamic change of hydrophilicity before and aftermicro/nano interface’s fabrication is measured by a statical goniometer.Finally, we observe three different Ti implant-bone interface’s bioactivity andbiocompatibility through SBF’s immersing experiment in vitro. Moreover, we utilize a cellculture and adhesion study to evaluate three different Ti implant-bone interface’sosteocompatibility. At each time point, a biological AFM and SEM were also used tocompare the osteoblastic cell morphology and response on three different Ti substratesurfaces. Then, at each time point, the fluorescence microphotograph of osteoblastic cellsadhesion on the three different Ti substrate specimens during2h,24h,48h of incubationis captured to analyze the discrepancy among them.Methods and Results:PartⅠ:Methods: The male SPF-level Wistar rats, age approximately9weeks were anaesthetized with an intraperitoneal injection of2%pentobarbital sodium. Then, we cut a roundalveolar bone block adjacent to the angle of mandible by a surgical micromotor andirrigated with0.9%sterile saline solution. The alveolar bone sample was then harvestedand fixed in10%buffered formalin for1week at4℃. Subsequently, the bone sampleswere embedded in polyester resin and mounted in a sawing microtome. Thus, we obtaineda30m-thick undecalcified section, which was stained with hematoxylin and eosin stain.Finally, we utilize SEM and AFM to analyze the size and feature of the micro and nanounit.Results:（1）Wistar rats’micro units （haversian canals） can be observed clearly in the pathologicalsection with hematoxylin and eosin stain （the diameter range from0.5m to2m）.Through SEM and AFM analysis, the microscale pit-like structure was aligned inorder.（2）Wistar rats’ nano units （collagen fibril molecules） can be seen imbedding on themicropits by AFM （the diameter range from60nm to80nm）. The tightly packedcollagen fibril molecules were stretched in the one direction similar toelectrospinning by SEM.PartⅡ:Methods: We utilize a binary acid （specific concentration HNO3&HF） treatment to builda microscale structure on Ti implant’s surface. Then, the vertical aligned TiO₂nanotubeswere fabricated on micro-treated Ti substrate surface by a three-electrodes’ anodization.Thus, a hierarchical aligned micro/nano structure can be fabricated on Ti implant’s surface.A morphology difference on nano/micro Ti implant’s surface can be observed by SEM andAFM as compared to smooth and micro counterpart. The diameter and characteristics ofTiO₂nanotubesµpits were analyzed by SEM. The roughness and averagepeak-to-valley height of three different Ti implant-bone interfaces were analyzed by AFMdomain scanning. We utilize a dynamic AFM tip contact scanning to analyze and couplethree different Ti implant-bone interfaces, which can acquire relative data （adhesion workand mechanical property）. Chemical characteristics and function groups on the three different Ti implant’s surfaces were also surveyed by XRD and XPS analysis. A staticalgoniometer was used to compare the discrepancy among three different Ti implant-boneinterfaces’s hydrophilicity.Results:（1）Our study has successfully fabricated a200nm-height TiO2nanotubes on micropits’sstructure. Their diameter （range from30nm to50nm） was similar to nano units （rangefrom60nm to80nm） in Wistar rats’ alveolar bone.（2）Three different Ti implant-bone interfaces’ roughness and average peak-to-valleyheight has a significant difference （P<0.05）（Smooth<Micro<Nano/micro）. Thehydrophilicity among three different Ti implant-bone interfaces also has a significantdifference （P<0.05）（Nano/micro>Micro> Smooth）.（3） XRD curves showed that mineralized crystal structure on three different Tiimplant-bone interfaces has a obvious discrepancy, which the peaks of TiO2and TiCin particular. XPS curves also demonstrated a significant difference in the peaks of Tiand O （Ti: Smooth>Micro>Nano/micro, O: Smooth<Micro<Nano/micro）.（4） The contact force curves of AFM tip scanning on three different Ti implant-boneinterfaces showed a obvious difference. The adhesion work among them has asignificant difference （P<0.05）（Nano/micro<Micro<Smooth）.PartⅢ:Methods: The bioactivity of three different Ti substrate surfaces was assessed through theimmersion of each sample into a polypropylene tube containing30ml of SBF at37C for7and14days. The difference of mineral deposits on three different Ti implant’s surfacewas analyzed by SEM, and we utilize XPS and Raman spectroscopy to affirm that thesemineral deposits were HA kind material. Then, we utilize a cell culture and adhesion studyto evaluate three different Ti implant’s osteocompatibility. At each time point （after2h,24h, and48h）, a Bio-AFM and SEM were used to observe osteoblasts’ adhesion on eachspecimen. Meanwhile, at each prescribed time point, the nonadherent cells were removedby rinsing with PBS. Cells were fixed and stained with DAPI. The cell numbers in five random fields were counted under a fluorescence microscope, and analyzed differenceamong three different Ti implant’s surfaces.Results:（1）After immersing in SBF for14days, a obvious difference in HA’s formation can beobserved between three different Ti substrate surfaces. On the smooth Ti substratesample, we can hardly observe mineral deposits. However, some small, granular,nonuniform mineral particles’ deposits were observed obviously on the micro-treatedTi substrate sample. Furthermore, we can observe a uniform tightly aligned minerallayer consisting of large, global particles on the nano/micro Ti substrate sample.（2）Three different Ti implant’s surface displayed a remarkable difference in SEM andAFM images after culturing and adhering osteoblasts for2h,24h,48h. On smooth Tisubstrate surface, the osteoblastic cells were highly elongated, presenting a rod-likeshape. However, the cells on micro-treated Ti samples were flattened and had morefilopodia, showing polygonal shape. Meanwhile, on nano/micro samples’ surfacerevealed that cell nucleus and several cell pseudopodia adhered together more closelyon it.（3） At each time point （after culturing for2h,24h,48h）, the cell number adhered onthree different interfaces have a significant difference （P<0.05）（Smooth<Micro<Nano/micro）.Conclusions:（1） Our study acquired the data about the size and structure characteristics of thenanoscaleµscale units in rat’s native alveolar bone. Thus, our results haveconfirmed that the multiscale hierarchical structures were existed and aligned orderlyin oral cavity.（2） Our study successfully obtained a novel way to biomimetic fabricate a nano/microarchitecture on Ti implant’s surface. Furthermore, this bioinspired structure is veryresemblant to hierarchical structure in native alveolar bone. This innovativetechnique has many superiority （such as simplicity, safety, excellent effectiveness）,which can apply to clinical research in near future. （3） This multiscale biomimetic fabrication on Ti implant’s surface can provide Tiimplant with a larger surface energy and roughness, a preferable hydrophilicity, amore adaptive mechanical property and adhesion work, a better bioactivity andbiocompatibity, a superior attachment and growth capacity of osteoblasts.（4） This biomimetic multiscale fabrication creates a new consideration to dentalimplant’s surface modification from bionics. In addition, this study will also providedental clinicians with a ingenious idea and theory to enhance the success rate ofdental implantation.更多还原