【作者】 王敬旭；

【导师】 周磊；

【作者基本信息】 南方医科大学 ， 外科学， 2014， 博士

【摘要】 研究背景与目的自上世纪中期口腔种植技术出现以来,种植牙修复以其无需损伤邻牙、咀嚼功能恢复好、舒适美观等优点,在众多修复技术中脱颖而出,为越来越多的患者所接受。种植牙修复已成为治疗牙列缺损,恢复牙颌系统功能的最理想方法之一。良好的骨结合是牙种植体成功修复的基础和前提,而表面处理是影响骨结合的一个重要因素。目前口腔种植医学领域应用最广泛的种植体材料是钛及钛合金,钛及钛合金具有优良的机械性能和生物相容性,但是钛金属属于生物惰性材料,植入体内后形成功能性修复需要较长的愈合时间,且植入体周围组织反应呈非特异性和随机性的,与骨组织界面主要是机械锁合方式的物理整合,缺乏有效的化学结合。为了使钛种植材料获得更好的生物相容性,促进更迅速的骨整合,其表面处理一直是口腔种植的研究热点之一。种植体表面处理指用物理和化学的方法,制备出粗糙的表面,使之具有更好的生物黏附力、表面亲水性、骨组织亲和性和适宜的电势能等。根据其处理方式可大致归类为以下三种：表面加成法、表面减成法和其他方法。加成法表面处理技术包括：阳极氧化法、钛浆喷涂法、羟基磷灰石涂层；减成法表面处理技术包括：表面喷砂法、表面酸蚀法、表面喷砂后加酸蚀法；其他方法包括激光融蚀、微弧氧化等。种植体表面经过处理,可显著改变其表面性能,从而促进骨结合的形成,提高种植的成功率。种植体表面粗化处理后,其表面张力增加,对成骨细胞的吸附、分化、扩增以及界面的早期骨结合均具有积极作用,对软骨细胞的分化和扩增也产生积极影响,现已普遍应用于临床。研究表明微米级表面形貌能增加骨结合面积、稳定细胞外基质蛋白支架及血凝块,为成骨细胞提供稳定的增殖分化微环境；纳米级表面形貌模仿天然细胞生长环境,可以促进蛋白的吸附、成骨细胞的附着和增殖分化。同时具微米和纳米的表面形态已成为未来种植体表面处理的发展趋势。大量的研究表明非涂层的种植体表面处理相对于涂层的表面处理方法更有优势,是目前种植体表面处理的主流。当前国际上使用最为广泛的种植体表面是喷砂酸蚀(Sand Blast and Acid Etching, SLA)表面,因其良好的机械生物相容性和骨引导性已经得到了临床实践的验证。处理技术首先是用大的Al2O3颗粒在特定的压力和时间控制下,通过高速气流对种植体表面进行粗化处理,常用的喷砂材料是直径25μm～250μm的Al2O3颗粒,大颗粒喷砂能在种植体表面形成10～30μm凹坑,这些凹坑具有促成骨的作用；酸蚀技术也广泛运用于临床种植体的表面改性,而经过酸蚀后,种植体表面获得大量的微孔(大小主要为1～3μm),另外酸蚀技术降低了喷砂处理后的种植体表面污染,酸蚀处理清除部分镶嵌的喷砂颗粒,消除了异种元素的污染,使种植体表面清洁,形态较均一、规则,表面积相对增大,表面钛离子的溶出速度降低,种植骨结合形成率增加。然而喷砂加酸蚀的表面处理方法也存在一定的缺点：如喷砂操作的不可控性,其形成的孔坑大小不确定可直接影响种植体表面的成骨效能；此外,SLA的表面处理技术后的种植体表面会嵌留下一些不利于骨结合的颗粒如Al2O3等。激光具有处理准确、效率高和可控性强等优点而广泛地用于金属表面处理。近年来,随着激光加工和口腔种植学的不断进步,激光表面处理新技术逐渐被引进种植体表面处理的领域。与喷砂、酸蚀等传统的种植体表面处理技术不同的是,激光表面处理是一种非接触性的清洁技术,其具有准确可控、高度重复性、高效率性等特点,它能在纯钛的表面产生独特的表面形貌和表面物质构成,还能增加钛表面的耐腐蚀性等。采用激光对种植体进行表面处理,可在其表面融蚀出大小形态均匀的孔洞,不会污染种植体的表面,并且不会在种植体表面生成新的杂质。研究表明,激光蚀刻联合酸蚀可增加骨细胞的吸附,提高骨结合效果。Cho等在兔子胫骨内植入激光融蚀后表面为凹坑的种植体,以光滑面种植体做对照,可显著增加扭力,增加骨接触面积,更利于骨细胞生长。随着纳米技术的发展,金属材料的表面结构研究也进入了纳米时代。二氧化钛(TiO2)纳米管是一种在钛金属表面加工出的纳米级多孔状结构,是由大量与材料表面相垂直的纳米级管状结构组成,可有效的增加种植体表面积,增强表面能。2001年,Gong等首次报道采用钛为基底,以电化学的阳极氧化方法在氢氟酸电解液中制出了均匀有序的TiO2纳米管,这样制备的TiO2纳米管以均匀整齐的阵列排列,并以肖特基势垒和金属钛导电基底直接相连,结合很牢固,呈现出极高的有序性和极低的团聚性,具有较大的表面积、较强的吸附能力和较高的量子效应,能产生特异的纳米效应。近来的研究发现TiO2纳米管在生物医学领域也有潜在的应用价值。KarlaS在体外实验观察到纳米管可促进牛血管上皮细胞的伸展、迁移。Seimghan等的研究发现在具有纳米管形貌的材料表面,成骨细胞的细胞突可伸到纳米管内,提高成骨细胞的增殖达300%-400%。在动物体内实验中,Kubota发现大鼠股骨纳米管种植体显示出优良的骨再生效果。这些研究表明TiO2纳米管结构能促进成骨细胞在种植材料上初期的黏附、增殖,并可促进骨组织的修复再生。纳米结构较微米结构更能促进成骨细胞的生长,其原因在于①纳米结构有仿生作用,骨无机矿物的直径为纳米级；②纳米结构增加种植体表面积,可为骨细胞黏附提供更多的位点；③细胞的突触可以伸入纳米管结构,成为锚点,从而促进细胞的黏,附和生长；④纳米结构能吸附细胞外基质蛋白,改变蛋白的构象,促进受体识别后细胞的黏附和生长。研究证实纳米结构可以诱导类骨磷灰石在材料与组织界面形成,同时纳米结构的材料表面对蛋白分子有较强的吸附作用,纳米级的蛋白质能够特异性的“识别”纳米级的表面结构,黏附后的蛋白分子构型和活性会较之前发生改变。沉积于材料表面的类骨磷灰石及黏附于材料表面的蛋白质、多肽、细胞因子等可以通过细胞表面的配体位点、离子通道等将材料信号转化成为细胞可识别的生物信号,传导至细胞内,启动第二信号通路,从而促进骨髓基质细胞/成骨细胞的聚集、黏附、增殖、分化、细胞骨架铺展及分泌基质和矿化一系列反应。Kubota的研究表明TiO2纳米管的促进骨组织生长的效果优于羟基磷灰石。羟基磷灰石具有骨诱导性,这表明TiO2纳米管具有与之相似的作用,说明TiO2纳米管在牙种植体的临床应用上具有很大的应用潜能。结合多种方法对种植体表面进行加工是目前种植体表面处理研究的趋势,例如将微弧载银和非载银、喷砂酸蚀与微弧氧化等综合处理方法相互比较骨结合效果和表面活性,还有学者将种植体表面先喷砂酸蚀,再加激光处理来观察其表面生物效应；Prodanov L在喷砂酸蚀处理与单纯激光处理的对比性研究发现,两者均获得了良好的骨结合效果。尽管目前已经有不少的有关喷砂酸蚀和激光的研究,但仍未见有对激光酸蚀加纳米管和喷砂酸蚀的对比性研究的报道,究竟激光酸蚀结合纳米管(laser/acid/nanotubes modifying,LAN)处理是否比喷砂加酸蚀这一经典的表面处理方法更好的效果?本课题组前期已经对激光酸蚀和喷砂酸蚀的表面进行对比性研究,发现两者具有相似的引导骨再生效应和生物相容性。本次的研究拟在激光酸蚀的基础上,结合纳米管处理的方法,再与喷砂酸蚀的表面在理化性质及动物实验方面进行对比,对这两种表面处理方法作进一步的研究,为寻求更好的种植体表面处理技术奠定基础。方法1.实验种植体的制备：根据预先确定的表面处理方法及相关实验参数,制备出符合实验标准的种植体。2.形貌及理化性质的检测：通过扫描电子显微镜、BMT Experi3D表面形貌仪分别对喷砂酸蚀(SLA)和激光酸蚀加纳米管(LAN)这两种表面的形貌特征、粗糙度进行分析,评估这两处理组表面的理化性能特征的异同。3.实验种植体的植入：选择Beagle犬作为SLA和LAN表面对比性研究的实验动物,按随机分配的原则,分别在其双侧下颌骨和胫骨植入这两种表面的植体,每侧下颌骨共植入5枚种植体,编号从前往后编号为1-5号,胫骨编号从上往下编号为1-4号,每种处理组各4枚。4.组织学观察：将带种植体的骨组织标本制作成不脱钙的骨组织切片,按实验目的行甲苯胺蓝染色观察分析,评估骨结合的生长速率和计算BIC%、BA%。5.测量记录好BIC%(Bone-to-implant Contact,BIC)和骨面积BA%(Bone Area)等数据,进行统计分析,得出实验结论。统计学处理实验数据以均数±标准差(x±s)来表示,样本量以n表示,应用SPSS v16.0软件(SPSS Inc.,Chicago, USA)进行统计分析。当满足正态分布且方差齐性时,采用两独立样本t检验或单因素方差来进行整体的均数比较；当不满足正态分布或方差不齐齐性时,采用两独立样本t’检验或秩和检验。假设检验为双侧检验,检验水准为0.05。当P>0.05时,被认为无统计学显著差异；当P<0.05时,被认为具有统计学显著差异。结果1.通过查阅大量国内外文献及结合本实验的研究目的,生产出合格的实验种植体,然后根据预先设定的表面处理方法及相关实验参数,制备出较好的两种种植体表面：激光酸蚀加纳米管(LAN)表面和喷砂加酸蚀(SLA)表面。2.两种表面理化性质检测结果：喷砂加酸蚀组和激光酸蚀加纳米管组均具有良好的二级粗糙结构,LAN表面的粗糙度大于SLA表面的粗糙度(LAN: Ra=8.19±0.09μm;SLA:Ra=2.09±0.13μm),两者具有统计学差异(P<0.05)；LAN的表面仍可见孔洞周围存留突起的熔融物,但酸蚀能酸洗去激光表面的碳物质；喷砂加酸蚀表面可见尖锐的边缘,其中仍有散在的一些三氧化二铝颗粒。3.SLA组和LAN组相比,LAN组骨结合和新骨生成效果更好。在2周和4周的胫骨BIC%、BA%统计学差异有显著性(P<0.05)；在4周和8周的颌骨BIG%、BA%统计学差异有显著性(P<0.05),在颌骨和胫骨的即刻种植均获得良好的骨结合效果。结论激光酸蚀联合纳米管与喷砂加酸蚀的表面均具有二级粗糙的表面,前者较后者干洁而规则,这两种表面均具有良好的生物相容性和良好的骨组织引导性,SLA组和LAN组相比,LAN组骨结合和新骨生成效果更好。在2周和4周的胫骨、4周和8周的颌骨BIC%、BA%统计学差异有显著性(P<0.05)。激光酸蚀加纳米管是一种可行的、效果较好的表面处理方法,具有较好的应用前景。更多还原

【Abstract】 Background and Objective:Since the middle of last century, dental implant has been accepted by more and more patients. Dental implant has the advantages of no damage to adjacent teeth, chewing function recovered well, comfortable and beautiful. Dental implant has become one of the most ideal method for restoration of dental system function.Osseointegration between dental implant and bone is the basis for successful repair, and surface treatment is one of the important factors influencing osseointegration. Now, titanium is the most widely used implant materials for excellent mechanical properties and biocompatibility. But the titanium is bio inert metal, functional reconstruction needs long time to heal after implantation. The tissue around the implant reaction is non-specific and randomness, interface with the bone tissue is mainly physical integration of mechanical locking mode, lack of effective chemical combination. In order to improve titanium implant compatibility and ossointegration, the surface treatment has been a hotspot of research in implant.The implant surface treatment is to make the surface roughness with physical and chemical methods. Implant with rough surface has the biological adhesion, surface hydrophilicity, bone affinity and potential suitable property. The implant surface treatment includes surface additive, subtractive method and other methods. Surface additive including the:anodic oxidation method, titanium plasma sprayed hydroxyapatite coating method. Subtractive surface including the:sand blasting, acid etching, sandblasting and etching.Rough implant surface can significantly change the surface properties, so as to promote the formation of osseointegration, improve the success rate of planting. The implant surface roughening treatment has a positive effect to the surface tension increases, osteoblast differentiation, amplification and early bone interface, chondrocyte differentiation and expansion. Studies show that micron surface can increase the bone bonding area, stable extracellular matrix protein scaffolds and blood clot, provide stable micro environment for the proliferation and differentiation of osteoblasts; Nano surface structures, which mimic the natural cell growth environment, can promote the protein adsorption, bone cell attachment and proliferation and differentiation. The surface including micro and nano structure has become the future development trend of implant.A large number of studies show that non coating surface treatment compared with the method of implant surface treatment coating has more advantages, is currently the mainstream of body surface treatment plant.the Sandblasted and Acid-etched (SLA) surface has been widely used in implant surface treatment, and its mechanical biocompatibility has been recognized. The holes with diameter10μm-30μm in the implant surface can be obtained by the large particles blasting, these holes were good for the bone formation. Meanwhile, a large number of pores were formed by the acid-etched method. To some extent, titanium implant surface was also cleaned, that would increase their surface hydrophilicity. Surface treatment method of sand blasting and acid etching also has some disadvantages:such as not controlled blasting operations, the formation of the pit size uncertainty can directly affect the implant surface osteogenic efficiency; In addition, SLA surface treatment of implant surface technology can be embedded to some particles such as Al2O3which has unfavorable on osseointegration.Laser is widely used in metal surface treatment for its processing accuracy, high efficiency and strong controllability. With the development of laser technology and oral implantology, laser technology are introduced into the implant surface treatment field. Laser surface treatment technology is different sandblasting and etching. Laser surface treatment is a non-contact cleaning technology. Research shows that the laser combined etching increases the absorption of bone cells, improve the bone bonding effect. Cho planted implant treated with laser, increased the bone contact area, more conducive to the growth of bone cells.With the development of nanotechnology, study on the surface structure of metal materials into the nanometer era. Titanium dioxide (TiO2) nanotubes is a nanometer titanium metal surface processing of porous structure, is composed of nanoscale tubular structures by surface and perpendicular to the material, can effectively increase the planting area of body surface, enhanced surface energy. In2001, Gong first reported the titanium substrate using anodic oxidation, electrochemical method in hydrofluoric acid electrolyte prepared uniformly ordered TiO2nanotube, so preparation of TiO2nanotube array arranged in neat uniform, and the Schottky barrier and titanium metal conductive substrate directly connected, with very strong, showing a highly ordered and the low aggregation, quantum effect has large surface area, strong adsorption capacity and higher, nano effect can produce a specific. Recent studies found that TiO2nanotubes have potential applications in biomedical field. KarlaS in vitro experiment observed nanotube can promote the migration of stretch, bovine vascular epithelial cells. The study of Seimghan found in the surface material has the nanotube morphology, osteoblast cell process can be extended to the nanotubes, increase osteoblast proliferation up to300%-400%.Nano structure can promote the growth of osteoblasts better than Micron structure, the reason is that the nano structure bionic function, bone mineral diameter of nanometer level; The nano structure increase the planting area, can provide more sites for the bone cell adhesion; Cell synapse can extend into the nanotube structure, become the anchor point, thus promote cell adhesion, adhesion and growth; The nano structure can absorb the extracellular matrix protein, the conformational change of protein, promote cell attachment and growth of receptor recognition. Research confirmed that the nano structure can induce the formation of bone like apatite in materials and tissue interface, and the surface nanostructure materials has stronger adsorption to the protein molecule, nanoscale protein to specific "recognition" nano surface structure, protein molecular conformation and activity of adhesion process of change than before. The bone like apatite deposited on the surface and the protein, polypeptide, cytokines,adhesion on the surface could start the second signal pathways s through cell surface ligand sites, ion channels, thereby promoting the bone marrow stromal cells and osteoblasts aggregation, adhesion, proliferation, differentiation, cytoskeletal spreading and secretion of matrix and mineralization series of reactions.The Kubota study showed that the TiO2nanotubes promoting effect is better than that of hydroxyapatite bone tissue growth. Hydroxyapatite has osteoinductive, suggesting that the TiO2nanotubes have a similar effect.The clinical application of TiO2nanotube implants in the teeth has the great application potential.Combined with a variety of processing methods on the implant surface is the growing trend of surface treatment, such as the recent reported in sandblasting etching and micro-arc oxidation, micro-arc containing silver and non containing silver and other integrated approach between the surfactant and bone effects.Prodanov’s study was the latest research in laser implant surface treatment reports, there was the comparative study of laser-treated with the Sandblasted/acid-etched, both of them obtained good osseointegration. It provided timely experimental basis and theoretical basis for further study in laser surface treatment.Although there has been research on sandblast and acid etching and laser many, but still no comparative study of laser etching and sandblasting etching of the nanotubes and reports. Whether the osseointegration of laser/acid/nanotubes modifying(LAN) surface had the some effect as or better than the Sandblasted/acid-etched surface was to be determined yet.In addition, our research group had been done comparative study about Laser-treated/acid-etched surface and the acid-etched surface, we found that two surface had the same biocompatibility and osteoconduction. The study was intended to changed some conditions of combinating with nanotubes,and then made a comparative study with the sandblasted/acid-etched surface by animal experiments, so as to provid experimental basis and theoretical basis for in-depth study of the laser/acid/nanotubes modifying, to seek a better implant surface treatment technology eventually was also the experimental target.Methods and materials1. Preparation of laboratory samples:based on foreign literature and research purposes, using precision CNC machine tools to produce experiment implant; By surface treatment method, according to pre-determined and related experimental parameters, prepared two good implant surfaces:the laser/acid/nanotubes modifying surface and the sandblasted/acid-etched surface.2. The test of physical and chemical properties:measure the morphology, roughness, elemental composition with scanning electron microscopy, BMT Experi3D surface topography instrument, respectively, to assess the physical and chemical properties of SLA and LAN surfaces.3. Making implant bone tissue sections and measurement of experimental: After decalcification, the anatomy of the rabbit tibia were made into paraffin section for HE staining observing; The bone tissues with implant were used to made sections of undecalcified, osseointegration growth rate,theBA%and the BIC%was assessment and calculated by the fluorescence labeling and staining.4. The BA%and BIC%data, statistical analysis obtained experimental results.Statistical analyses:The experimental data are expressed as mean±standard deviation (SD). Statistical analysis was carried out by SPSS(?) vl6.0software (SPSS Inc., Chicago, USA). For the single factor data, when meet the normal distribution and homogeneity of variance, using two independent sample t test and single factor variance to the overall mean comparison; When the data not meet the normal distribution or variance not neat homogeneous, using two independent sample t’test or rank sum test; For two factors of single variable data with normal distribution and homogeneity of variance, to analyse the main effect and interaction by analysis of variance of factorial design. Hypothesis test for two-sided test, the test level was0.05, probabilities (P)>0.05was considered to be no statistically significant; Probabilities (P)<0.05was considered to be statistically significant.Results: 1. Through access to a large number of domestic and foreign literatures and combined the purpose of the present study, using precision CNC machine tools produced some experimental implant. According to pre-set surface treatment methods and experimental parameters, two different implant surfaces were made: Laser/acid/nanotubes modifying surface and Sandblasted/acid-etched surface.2. Results of the physical and chemical nature detecting:both of SLA and LAN obtainted two rough structure, the surface roughnesses of LAN was bigger than the SLA (LAN:Ra=8.09±0.09μm; SLA:Ra=2.09±0.13μm)(P<0.05). A few oxide aluminum particles was still existed.3. Both groups obtained good osseointegration. tThe BA%and BIC%of LAN was better than the SLA (P<0.05).Conclusion:The surface treatment methods of the laser/acid/nanotubes modifying and the sandblasted/acid-etched could produce the rough surface, the former was more cleaner and more uniform than the latter, but both of them had good biocompatibility and promoted a good implant osseointegration, the LAN was better than SLA.更多还原