磷酸和氟化钠对纯钛表面酸蚀后的渗氢效果以及对其生物学行为影响的探讨
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摘要
研究背景
口腔种植领域中,纯钛表面处理一直是学者们为提高种植体初期稳定性以及骨结合的关注焦点。目前钛表面处理工艺中最常用的方法包括多个途径：化学处理、物理处理、生物学处理等。其中化学处理中的酸蚀法是目前公认的可达到临床基本需求的常用方法。酸蚀的配方多种多样,但目前使用最广泛的方法为：49%H2SO4:19%HC1体积比为1：1时在60℃水浴中反应30分钟。此酸蚀方法实质为利用硫酸的强氧化性,在钛表面成一层致密的二氧化钛膜,而盐酸中的氯离子对该层氧化膜具有强亲和力,通过氯离子与氧化膜中的氧发生置换,进而形成可溶性的产物TiCl4-,达到对钛的腐蚀。因此,这种传统的酸蚀过程主要分为两步：第一：硫酸使钛表面发生还原反应,形成氧化膜；第二：盐酸使该氧化膜形成可溶性的氟化物,达到蚀刻效果。可见,硫酸和盐酸虽具有较强的腐蚀性能,但当用于纯钛的酸蚀,两者须发挥协同作用才可。该酸蚀配方可以去除钛材表面氧化膜,并达到临床上对粗糙度以及表面微孔形貌的要求。然而,由于硫酸及盐酸均为强酸,其在水溶液中可发生完全电离,溶液中存在大量游离氢离子,并大量渗透至钛材基体中,导致材料严重的渗氢,明显降低材料的机械性能,缩短材料使用寿命。另外,浓硫酸在加热条件下可以发生还原反应产生腐蚀性的二氧化硫气体,亦会对材料表面造成污染,不利于蛋白和细胞的吸附。因此,该传统配方在某些方面不具备理想酸蚀液的要求,需要寻找一种新的方法就这些缺点加以改变并提高。
在口腔种植应用中,常因患者咬合力过大,咬合间隙过紧,咬合侧向力大,种植上部结构冠根比例过大以及螺丝长期磨损致使其与基台不密合等原因,在反复应力作用下出现种植上部结构中螺丝的折断,此时断端螺丝多位于种植体内或基台内部。另外,当种植体采用硬度较低的纯钛材料,且种植体与周围骨结合良好时,由于上部结构设计的不合理而使种植体承受过大的扭力,如过长的悬臂梁设计,或者对于颈部设计较细窄的小直径种植体而言,当攻丝不足但仍使用较大力旋入种植体时,均可发生种植体的折断,尤以在骨质致密的下颌前牙区多见。折断的种植体和螺钉的取出均十分困难,需要使用专用器械及工具,且操作时间长,效果无法预计,给患者带来了额外的创伤以及巨大的精神负担,不利于良好医患关系的维持。因此,在对纯钛进行传统酸蚀的过程中,由于大量的游离氢离子以及钛本身对氢的敏感性,极易发生严重渗氢,其在持续缓慢的应力作用下导致种植部件发生断裂,这是氢脆的一类表现,是在临床工作中应该避免出现的。鉴于氢脆断裂对临床工作造成的诸多不利影响以及远期预后的不确定性,因此,寻找一种新的酸蚀方法代替这种传统酸蚀方法的要求则变得更加迫切。
约100年前,有学者发现当氢存在于金属中时,会降低金属的某些力学性能,因此,这种因渗氢导致的金属机械性能降低的现象被称为氢脆。氢脆可降低金属材料的弹性模量,抗拉强度,屈服强度,疲劳强度以及断裂韧性等,均可严重影响金属在长期持久应力下的使用寿命。氢对金属的损伤,通常分为6类：a)氢脆(包括延迟损伤和延迟断裂)。当氢在金属中局部达到一定饱和浓度时,可降低金属的延展性和韧性,严重时可导致延迟断裂的发生。b)氢腐蚀。金属中的氢在高温高压的条件下,可与其中的碳结合形成甲烷。当这些气体形成的压力达到某一临界值时,可导致晶界上产生微裂纹。c)氢化物。氢进入过渡元素及其合金的晶格中所形成的间隙型氢化物。d)微裂纹。若金属在冶炼过程中带入过量的氢,且这些氢不能扩散外逸,便会存留在材料缺陷处形成氢分子,后者使材料内部发生晶格畸变,甚至形成微裂纹。e)氢鼓泡。在硫化氢等腐蚀环境中使用的金属,或者对金属进行充氢的操作时,当扩散进入金属中的氢原子在非金属夹杂物中或者较大的缺陷处聚集并形成氢分子时,就可能出现氢鼓泡。f)流变性退化。氢进入金属后,可导致金属的韧性降低,合金在高温下的抗蠕变性能下降。
因此,采用一种新的酸蚀配方,在达到临床对于表面粗糙度和表面形貌要求的同时,尽可能降低对钛材的渗氢,以代替传统酸蚀方法,则变得十分有意义。曾有大量关于不同种类有机酸对纯钛进行酸蚀的研究,包括草酸、氢氟酸、磷酸、盐酸、硝酸等,但经理论分析发现,其中因盐酸为无机酸中的强酸,理论上无法减少氢离子的电离以降低渗氢量；草酸为有机酸中的强酸,其一级电离常数Kal=5.9×10-2,二级电离常数Ka2=6.4×10-5,从化学结构式可知,其未含有利于细胞分化以及成骨矿化的元素,为碳水化合物,可能在此方面不具备优势。硝酸具有强烈的钝化作用外,无法达到表面粗糙度要求,氢氟酸、磷酸均非强酸,不发生完全电离,理论上可降低对材料的渗氢量。然而,氢氟酸虽为弱酸,低浓度的氢氟酸即可与钛发生剧烈反应,不易控制,且有剧毒、强腐蚀性,易于对操作人员造成难愈合的溃疡,而与之相比较,磷酸为中强酸,低毒,且其中含有的磷元素在理论上可显著促进羟基磷灰石的形成,有利于种植体周围的成骨,且不具备氧化性。其不发生完全电离,易于发生一级电离,很难发生二级及三级电离,其电离常数均小于草酸者,因此游离氢离子的浓度明显低于硫酸和/或盐酸以及草酸者,因此其可能会减少游离氢离子渗入钛材内部的数量。然而,通过前期资料的整理以及预实验,发现在常温常态下磷酸无法对钛进行腐蚀。因此,有不少学者就磷酸与钛的反应以及磷酸处理后的钛表面的理化及生物学特性进行了研究,其中使两者发生反应的途径主要包括：a)热处理：处理温度界于180-800℃之间；以及b)电化学处理法：其实质包括阳极氧化反应,阳极腐蚀反应以及阴极还原反应。然而,经过本课题组前期预实验的探索,发现这些处理方法均不同程度忽略了高温下渗氢的发生以及临床应用中对粗糙度的要求,且需要复杂的设备仪器支持,无法达到研究目的。因此,通过大量观察、文献查阅以及预实验的深入,逐步发现并确定在常温下,在无机酸中加入酸性的氟化物,可以对纯钛发生腐蚀效应。本课题采用了氟化物中最常见的氟化钠与磷酸混合；并通过进一步对磷酸浓度以及氟化钠浓度的筛选,即分别使用6%,8%,10%,12%,16%,18%,25%,30%,35%,40%,46%,50%,55%以及62%磷酸与O.1mol/l NaF对钛在常温下进行酸蚀,发现35%,46%以及62%磷酸(浓度分别为3.57mol/l,4.62mol/1,6.32mol/l)在与氟化钠酸蚀钛的过程中,均可出现明显的腐蚀反应,并且通过对反应时间的筛选,发现该配方对钛的反应速度适宜,反应30分钟即可不同程度达到临床对钛片表面粗糙度的要求,且各组之间均有差异。另外,当改变氟化钠浓度时,可见钛片的表面形貌出现了稳定且规律的变化。通过初步检测渗氢量,发现经新酸蚀配方：磷酸+氟化钠处理后,钛材的渗氢量明显低于传统酸蚀处理者；另外,由于不同浓度的磷酸+氟化钠的配比,其可以达到高于传统酸蚀组的粗糙度,并且可能有磷、氟元素的同时存在,该配方可能会促进周围细胞对材料表面的吸附。综上所述,通过前期的理论研究以及预实验的结果,为消除关于磷酸热处理以及电化学处理导致的不利因素,本课题组期望该新酸蚀配方能够在常温下对纯钛处理后,与传统酸蚀方法相比,明显降低材料的渗氢量、达到临床所需的表面粗糙度,并且更有利于细胞外基质蛋白和成骨细胞的吸附。主要研究内容包括其处理后材料的表面形貌,粗糙度,表面化学成分及含量,材料内部的氢含量,氢分布以及在模拟体液中钛表面的磷酸钙类化合物的沉积,表面Zeta电位,对纤维连接蛋白和玻连蛋白的吸附能力以及对人成骨肉瘤来源的MG-63细胞的吸附以及对其形态影响的评估,为形成一种全新的材料表面处理方法,用以代替传统酸蚀方法,来避免传统酸蚀后材料严重的渗氢以及表面的污染,提供可靠的实验依据。
第一章磷酸和氟化钠对纯钛酸蚀后渗氢效果的影响
目的：研究一种新型的表面酸蚀配方：磷酸+氟化钠在常温下对纯钛进行酸蚀,与传统酸蚀方法进行比较,评估其处理后的钛材能否在达到临床需要的适宜表面粗糙度的同时,降低材料渗氢量,并具有较传统酸蚀方法更好的诱导磷酸钙盐沉积的能力。
方法：
(1)：样品处理：Ⅳ级纯钛板机加工后,在常温且无辅助设备的前提下,使用不同浓度配比的磷酸+氟化钠进行酸蚀,分组：磷酸浓度分别为3.57mmol/1,4.62mol/l和6.32mol/1时,分别为组A,组B以及组C；各组中,氟化钠浓度依照与磷酸的浓度比例逐渐递增,分别形成A1-A10亚组,B1-B10亚组和C1C6亚组。另用常规酸蚀方法：49%H2S04:19%HCl体积比为1：1时在60℃水浴中反应30分钟,酸蚀钛片,为对照组T。
(2)：表面形貌观察：使用扫描电子显微镜(Scanning electronic microscopy,SEM,HitachiS-3700N)观察表面形貌,以及仪器配用软件提供的标尺功能用来测量钛表面酸蚀凹坑的直径,每个坑进行10次测量后,取其平均值。
(3)：表面粗糙度检测：使用白光干涉仪(White light interferometer,Taylor HobsonCII)检测处理表面,包括表面粗糙度,Ssk(表面倾斜度：surface skewness)以及Sku(表面峰度：surface kurtosis)值,使用Talymap表面分析软件获得。
(4)：表面化学成分及含量分析：采用X射线衍射分析仪(X-ray diffractometry, XRD,Bruker,D8-advance)进行定性及半定量检测。同时,X射线光电子能谱(X-ray photoelectron spectroscopy,XPS,Kratos,Axis Ultra DLD)用于定量检测的样品处理后表面的化学成分：A1,B1和B10。数据由CasaXPS处理。
(5)：氢含量分析：使用惰气脉冲熔融热导仪根据ASTM E1447-1409(American Society of Testing Materials,ASTM)确定钛样品(美国力可Leco,TCH600)的氢含量。将样品置于坩埚中,高电流下除气,充入氦气,使样品中的氢以H2的形式释放,在催化剂作用下被转化为水,用红外线检测仪检测氢含量。
(6)：氢分布分析：根据ASTM E1504-1506,采用时间-飞行次级离子质谱仪(Time of flight secondary ion mass spectrometry,ToF-SIMS,2100TRIFT,Physical Electronics)检测处理后Ti试样横截面的氢元素分布。检测氢分布前,根据ASTME1078-09,先将各样品安装于横截面抛光仪(Cross-section polishing instrument日本电子株式会社JEOL, SM-09010)去除表面污染物并保证表面平整。随后,在真空度低于4×10-8Pa的条件下检测大小为200×200μm2和50×50pm2的试样横截面,测绘结果由Wincadence软件获得。
(7)：弹性模量的检测：按照IS07438-2005制备样品。使用万能材料试验机(Universal testing machine,Instron5565)进行三点弯曲试验和弹性模量的计算。弯曲试验在室温下进行；选择材料在处于弹性形变范围内的载荷进行检测(<800N)。
(8)：磷酸钙盐的定性及定量分析：按要求配制模拟体液,将具有代表性表面形貌的处理组B1,B4,B10以及传统酸蚀组T试样,分别置于37℃的模拟体液中,每组试样静置20天。用扫描电子显微镜和X射线光电子质谱仪观察并检测试样表面沉淀物的分布以及沉淀物的成分、比例。
结果：新酸蚀配方处理后试样的表面粗糙度在0.47±0.03μm到0.87±0.05pm之间；且随氟化钠浓度的升高,其表面形貌呈规律性变化：由圆弧凹坑状逐渐过渡至尖锐棱角状；其表面有磷、氟元素共同附着；氢含量在30.9±3.8ppm至86.9±6.0ppm之间,且在此范围内,随着氢含量的升高,其表面覆盖的磷酸钙盐的面积亦增加；氢元素主要分布于钛材的晶格点阵上；磷酸钙盐的覆盖面积高于传统酸蚀组T(F=4.507,P=0.010),亚组B1、B4沉积物的钙磷比例更接近羟基磷灰石者,,且沉积部位无选择性。传统酸蚀处理组T的表面粗糙度为0.59±0.04μ m；氢含量为287±16.5ppm,其与磷酸各处理组间均具有明显的差异(前者F=10.349,P<0.001；后者F=221.945,P<0.001)；氢元素同时分布于材料的晶格点阵间隙上和其边界之间；其磷酸钙盐仅沉积于酸蚀形成的凹坑内。
结论：新酸蚀配方在达到临床粗糙度的需求同时,明显降低了氢含量；且氢元素主要分布于晶胞内部,理论上可降低氢脆导致的”沿晶断裂”的几率；可以通过改变各组分浓度比来获得不同特点的表面形貌；其表面同时附着的磷、氟元素以及其表面形貌规律性的转化,可能影响了磷酸钙盐的沉积。
第二章纯钛经磷酸和氟化钠处理后对其表面蛋白吸附以及成骨细胞初期附着的影响
目的：研究经磷酸和氟化钠处理后试样的表面电荷性质,及其在动物体内对纤维连接蛋白以及玻连蛋白的吸附能力,并考察其对人骨肉瘤细胞MG-63的吸附以及对其形态的影响。
方法：
(1)：样品处理：Ⅳ级纯钛板按照检测仪器及实验条件的要求加工成一定形状的钛片。在常温且无辅助设备前提下,使用不同浓度配比的磷酸+氟化钠进行酸蚀,选择具有代表性表面形貌的亚组B1,B4以及B10与传统酸蚀组T进行比较。
(2)：试样表面Zeta电位检测：使用固体表面Zeta电位分析仪进行Zeta电位分析。采用Helmholtz-Smoluchowski模型进行检测。在可调间隙样品池中,分别将各实验组两个大小完全相同的样品测试面相对放置,间隔0.1mm-0.2mm。用电解液氯化钾(浓度为1×10-6mol/l)校准,绘制工作曲线,检测样品表面平整度。随后,检测待测样品在碱性和酸性环境中携带的电荷种类及数目,并绘制样品在不同pH条件下的表面电位曲线。所得数据用Attract软件进行分析。
(3)：试样表面对纤维连接蛋白(Fibronectin, FN)及玻连蛋白(、Vitronectin, VN)吸附能力的检测：SD大鼠,雄性,8-10周龄,180-220g,16只。将SD大鼠左右股骨髓腔内各植入一枚试样,静置30分钟。截断股骨,取出试样,去离子水充分洗涤。试样置入SDS蛋白洗脱液(50mM/L Tris,2%SDS,5%β-巯基乙醇),震荡15min。1000转/分离心15分钟,吸去上清液。使用western blot对目标蛋白进行半定量检测。首先使用BCA试剂盒测定蛋白浓度,-20℃保存备用。SDS-PAGE胶(Sodium lauryl sulfate-Polyacrylamide gelelectrophoresis)制作好后,加样,100V下进行浓缩胶电泳,20min；140V下分离胶中电泳,40min。转膜,并将膜浸入封闭液中4℃过夜；将膜取出放入目标蛋白一抗(1ug/ml,按要求稀释后)中,4℃封闭过夜；再将膜转入目标蛋白二抗(1：5000)中,用化学发光法显影。
(4)：试样表面对人骨肉瘤细胞MG-63的早期吸附：SD大鼠,雄性,8-10周龄,180-220g,32只。将SD大鼠左右股骨髓腔内各植入一枚处理后试样,静置30分钟。将股骨截断,取出试样,去离子水充分洗涤。4个处理组B1,B4,B10以及T,各有4个平行样品。获得处理样品后,分别置入96孔板,将已培养稳定且生长达到80%接触率的MG-63细胞冲洗,消化,离心,重悬后,细胞计数,并按1×105个/cm2的密度接种于钛片表面。将已接种细胞的钛片置于37℃,体积分数5%CO2的培养箱内常规孵化培养,分别检测在6h,12h,18h和24h时的细胞粘附量,以及细胞形态的变化。每到观察时间点,采用3%-4%多聚甲醛室温下固定细胞30分钟；再将0.1%Triton X-100加入96孔板中500μL/孔,静置3分钟,以增加细胞通透性；避光条件下中加入100微升/孔肌动蛋白绿色荧光鬼笔环肽工作液,在室温下对细胞肌动蛋白F-actir进行避光染色30分钟。细胞计数软件Tanon colon对显微镜下每视野的细胞数定量并计算,每个试样获取5个视野范围内的细胞数目,将获得数据进行单因素方差统计学分析。
结果：所有检测试样表面Zeta电位均为负值,其中亚组B10的Zeta电位绝对值最小,与其他各组有明显差异(F=70.719,P<0.001),B1者最大,B4与组T者相近。亚组B10对纤连蛋白吸附能力最强,与其他组有明显差异(F=116.692,P<0.001),B1者最低,B4及组T对纤连蛋白吸附能力相近。B10组者对玻连蛋白吸附能力亦为最强,组T者高于B4者,与B10者相近,B1仍为最低(F=13.937,P=0.002)。对MG-63的吸附数量,各组表现与对纤连蛋白吸附能力结果一致(F=701.542,P<0.001)。新酸蚀配方中磷氟元素的同时附着可能更有利于加快接种细胞粘附初期的成熟速度,使其表面吸附的MG-63细胞铺展较快。传统酸蚀组T则可能因表面硫元素的存在,细胞铺展慢,成熟速度慢。
结论：高表面粗糙度以及完全呈尖锐棱角状的表面形貌可以获得低的Zeta电位绝对值,则材料表面最易发生对周围蛋白以及细胞的吸附,如亚组B10。低表面粗糙度以及呈圆弧凹坑状的表面形貌则获得高的Zeta电位绝对值,此类表面不易发生吸附聚集,如亚组B1。而组T可能因表面具有次级微孔结构有利于其对蛋白及细胞的吸附。新酸蚀配方中磷氟元素的同时附着可能有利于加快接种细胞初期的成熟速度；而传统酸蚀配方中残留的硫元素则不具备此作用。
Background
In the area of oral implantology, studying of titanium surface treatment is always a focus of scholars who aimed to improve the initial implant stability and osseointegration.The most commonly used surface treatment methods of titanium currently include several channels:chemical treatments, physical treatments and biological treatments. Wherein the chemical etching method of treatment is now recognized as a basic approach to reach clinical needs. Etching recipes varied, but the most widely used method is:49%H2SO4:19%HCl1:1by volume in the reaction for30minutes at60℃in water bath. In the traditional method, H2SO4, as a strong oxidant, oxidizes the surface of the Ti substrate into TiO2. The strong affinity of the chloride ions to the oxide film in HCl leads to the replacement of oxygen with chloride ions, producing the soluble product TiCl4-. That is, this combination performs faceting by first forming an oxide film and then corroding it, and the achieved surface topographies depend on the construction of the oxide film to a great extent. Therefore, sulfuric acid or hydrochloric acid, although with strong acidity, can't perform etching for pure titanium alone, and the two acids need to work together. The tradiational etch combination can remove titanium oxide film and meet the clinical requirements of surface roughnes. However, due to both sulfuric acid and hydrochloric acid, which can occur completely ionized in aqueous solution, and a large number of free hydrogen ions exist, which can penetrate to itanium matrix, resulting in severe hydrogen embrittlement for titanium material, and reducing its mechanical properties. Meanwhile, concentrated sulfuric acid under heating could produce sulfur dioxide as a corrosive gas which induces contamination of the surface. Therefore, the tradiational etching solution does not poccess the ideal requirements in some areas, a new way to deal with the shortcoming is needed.
In oral implant applications, due to the patient heavy bite occlusal force, tight occlusal clearance, lateral bite force, the proportion of crown and root structure is too large and wear of upper screw resulting in close contact with the base, in the slow and repeated stress on the superstructure, break induces the fracture of the screw, and the end of the screws were always located inside the base or in the implants. On one hand, when the implants made of titanium materials with a lower hardness, such as grade II, and osseointegration is good and the upper part of the implant is unreasonable which bears too much torque, may cause the implant fracture occurred. On another hand, the small diameter implants with narrow neck are likely to fracture near the weak point, when a large force was used to screw the implant into the bone, especially in the anterior mandiblewith high bone density.It is difficult to remove the broken implants and screws, which require the special equipments and tools, and the operation for a long time, the effect can not be expected, exerting patients with additional trauma and huge mental burden, is not conducive to maintaining a good doctor-patient relationship. Therefore, in the process of titanium etching, since a large number of free hydrogen ions exist, titanium sensitivity to hydrogen and severe long-term stress leads to fracture of implants, which is a kind of hydrogen embrittlement performance and should be avoided.Given the many adverse effects of hydrogen embrittlement caused to the clinical work, therefore, it is even more urgent to find a new etching method instead of the traditional etching methods should be proposed.
About100years ago, researchers found that when the metal in the presence of hydrogen will reduce some of the mechanical properties of the metal, so that hydrogen embrittlement is known due to the mechanical properties of metal reducedsince the hydrogen permeation. Hydrogen embrittlement can reduce many properties of metallic material, such as elastic modulus, tensile strength, yield strength, fatigue strength and fracture toughness, etc.,can seriously affect the life span of the metal under the long-lasting metal stress. Damageto metal caused by hydrogen embrittlement is usually divided into six categories:a) hydrogen crisp (including delayed damage and delayed fracture) when the hydrogen in the metal reached a certain saturation concentration locally, can reduce the ductility and toughness of metals, in severe cases which can lead to delayed fracture occurs.b) hydrogen corrosion。Metal hydrogen underhigh temperature and high pressure will turn into methane since react with carbon. When the value of gas pressure reaches a certain critical level, the internal stress will lead to the generation of micro-cracks on the grain boundaries.c) hydride. Gap hydrides are formed when hydrogen into the transition element in the alloy latticed) microcracks. If an excess of hydrogen entered into the metal in the smelting process, these atoms can not diffuse and escape, and are retained to form hydrogen molecules in the material defects, which will occur the lattice distortion, and even the micro-cracks.e) hydrogen bubbling. Metal works in corrosive environments such as hydrogen sulfide, or for the hydrogen charging operation, when the hydrogen diffusion into the metal with largedefects and exist as non-metallic inclusions may cause hydrogen blistering.f) rheologydegradation. After the hydrogen atoms enter into the metal, the metal can lead to a decrease in toughness of the alloy as high temperature creep resistance decreases.
Therefore, involving a new etching recipe, while reaching clinical requirements of proper surface roughness and surface morphology as much as possible, to reduce the infiltration of titanium hydrogen, becomes very necessary.There was many a studies about the different kinds of organic acid etching of titanium researches, including oxalic acid, hydrofluoric acid, phosphoric acid, hydrochloric acid, nitric acid, etc..But by theoretical analysis, hydrochloric acid and oxalic acid, in theory, can not reducehydrogen ions; nitric acid has a strong passivative effect, can not reach the requirement of surface roughness.Hydrofluoric acid, phosphoric acid are not strong acid, in theory, can reduce the amount of hydrogen ions permeating into materials. However, although the hydrofluoric acid is a weak acid, a low concentration of hydrofluoric acid can react violently with the titanium, and it is difficult to control with highly toxic, corrosive ability, easy to cause heal ulcers to the operators. Phosphoric acid with moderate strength acidity and low toxicity can significantly promote the formation of hydroxyapatite, and is conducive to bone formation around the implant, and do not have oxidation, be absence of the oxidation. It is prone to cause the first stage ionization, but difficult to initiate the secondor thethird ionization. This phenomenon indicates that in the phosphoric acid solution, the concentration of free hydrogen ions is significantly lower than those of sulfuric acid, and therefore may reduce the number of free hydrogen ions into the interior part of the titanium material. However, pure phosphoric acid at room temperature can not etch the titanium. Therefore, there are many scholars who worked on physicochemical and biological characteristics of the phosphoric acid and phosphoric acid reaction with the titanium, in which the reaction occurs within the two approaches:including hydrothermal (180-800℃) and electrochemical treatments, which include anodic oxidation, anodic corrosion and cathodic reduction. However,some of these studies failed to estimate the adverse impact of the hydrothermal treatment, which could aggravate the permeation of hydrogen into Ti substrates. Other studies lead to a considerably limited faceting effect that cannot satisfy the roughness requirement. Through our investigation, we discovered that Ti could be considerably facetted by a combination of inorganic acid and acid fluoride. Therefore, a large of pre-preliminary experiments found that adding the acid fluoride to an inorganic acid, the corrosive effects may occur on the titanium at room temperature. A further concentration of the sodium phosphate had been screened, using6%,8%,10%,12%,16%,18%,25%,30%,35%,40%,46%,50%,55%and62%phosphoric acid respectively and0.1mol/1NaF acid etching of titanium, found that35%,46%and62%phosphoric acid(the concentration of3.57mol/1,4.62mol/1,6.32mol/1) and sodium fluoride in the process of etching the titanium, the corrosion reaction occurs with proper reaction speed, and the surface roughness reach the clinical requirements of titanium when reaction processes for30minutes, and there are differences between the three groups.Furthermore, by varying the concentration of sodium fluoride, the surface topography of the titanium shows stable changes. In summary, through theoretical studies and the results of the pre-experiment, to eliminate the negative factors on heat treatment and electrochemical treatment, our group expect the new etch recipe, compared to conventional etching method, could significantly reduces the amount of hydrogen permeability material, achieve the desired surface roughness clinical, and be more conducive to the adsorption of extracellular matrix proteins and osteoblasts. We primarily focus on the performance of this combination with respect to the surface roughness, the hydrogen content permeating into the Ti substrate, calcium phosphates deposition ability, zeta potential, adhesive ability with FN, VN and MG-63. This study investigates the surface topography, roughness, surface chemistry, hydrogen content, hydrogen distribution and growth ability in simulated body fluid (SBF) of titanium samples treated using various methods, and adhesive ability with FN, VN and MG-63are performed and detected by western blot and immunohistochemisitry. We hope that this study will produce a novel surface with a morphology, composition and hydrogen content that is appropriate for dental implantation.
CHAPTER ONE Phosphoric acid and sodium fluoride:a novel etching combination on hydrogen embrittlement of titanium
Objective:We investigate whether a novel and inexpensive etching method, H3PO4+NaF, on titanium, could obtain both a lower hydrogen content and superior calcium phosphates deposition performance, while achieving proper surface roughness, in comparison with the traditional etching method.
Method:
(1):Pretreatment of samples:Ti plate of grade IV has been processed, then were treated with different concentrations of H3PO4+NaF at ambient temperature without auxiliary implementations, as group A, B and C, when the concentrations of phosphoric acid were3.57mol/1,4.62mol/1and6.32mol/1respectively. Samples were treated by the traditional method as sulfuric acid and hydrochloric acid at60℃for30min(group T).The samples were then maintained in simulated body fluid with pH7.4for10days and20days at37℃.
(2):Observation of surface morphology:The surface morphologies were examined using a scanning electron microscope (SEM, HitachiS-3700N), The Scaleplate feature of the software provided with the instrument was used to measure the diameters of the round pits;10measurements were performed for each pit, and the values were averaged.
(3):Detection of surface roughness:The surface roughnesses were then determined through white-light interferometry(Taylor Hobson CII).The surface roughness, and the values of Ssk and Sku were obtained using Talymap surface analysis software.
(4):Detection of surfacecompositions and contents:XRD patterns of sub-groups Al, B1, B10, and Cl and of group T were obtained by X-ray diffractometry (Bruker, D8-advance). X-ray photoelectron spectroscopy (Kratos, Axis Ultra DLD) was used to quantify the P and F contents of Al, B1and B10. The obtained data were processed by Casa XPS.
(5):Detection of hydrogen contents:Inert gas fusion thermal conductivity analysis was used to determine the hydrogen contents in the Ti samples (Leco, TCH600) according to ASTM E1447-09. The samples were put into the graphite crucible. The air remaining in the graphite crucible was degassed under40A under a helium atmosphere. The hydrogen in the sample was released in the form of H2and converted into water by rare earth.
(6):Detection of hydrogen distribution:The hydrogen distributions in the transverse sections of sub-group B1and group T were detected by time-of-flight secondary ion mass spectrometry (ToF-SIMS, Model2100Trift, Physical Electronics, USA) according to ASTM E1504-06. Before the measurement, the samples were prepared by cross-section polishing (JEOL, SM-09010) according to ASTM E1078-09to prevent contamination and to ensure smoothness. The H mapping results were obtained using Wincadence software.
(7):Detection of elastic modulus:Universal material testing machine (Instron5565) were involved to examine the elastic modulus of samples treated by different etching methods. Specifically, the bending test was limited within the elastic deformation range (0-800N) at ambient temperature.
(8):Hydroxyapatite deposition on Ti plate:Simulated body fiulde were prepared according the reference. The HA deposition abilities of different etching combinations were evaluated by immersing the samples, as B1, B4, B10and T, into SBF for20days. Scanning electron microscopy (SEM, Hitachi S-3700N) and X-ray photoelectron spectroscopy (Kratos, Axis Ultra DLD) was used to observe the morphology and to quantify the Ca and P contents of B1,B4,B10and T.
Results:The surface roughnesses of groups A, B, and C are in the range of0.47±0.03μm to0.87±0.05μm. The surface topography switches from small round profile to deep V-shape groove dramatically as increasing the concentration of NaF. P and F attach onto the surface synchronously.The hydrogen contents of new groups are in the range of30.9±3.8ppm to86.9±6.0ppm, and that of group T is287±16.5ppm. The distribution of hydrogen atoms were mainly inside of the grains of titanium. Calcium salt coverage areas are larger than that of traditional etching group T (F=4.507, P=0.010), the calcium phosphorus ratio of the subgroup B1, B4are closer to those of hydroxyapatite, with non-selective deposition site. The surface roughness of group T was0.59±0.04μm; hydrogen content of287±16.5ppm, show the significant differences (former:F=10.349, P<0.001; latter:F=221.945, P<0.001);hydrogen distribution concentrate on the boundaries between the crystal lattice material, and only calcium salt deposited in the etch pits.
Conclusion:Meeting withthe roughness of clinical needs, the novel etching combination could significantly reducing the hydrogen content as well. The hydrogen is mainly located inside the titanium grains, which could reduce the probability of intergranular fracture caused by hydrogen embrittlement theoretically. By changing the concentration ratio of the components,it could obtain different characteristics of the surface topography. The synchronous attachments of phosphorous and fluorine onto the surface and regularity of its surface morphology transformation may affect the deposition of calcium phosphates.
CHAPTER TWO Influence of titanium treated by phosphoric acid and sodium fluoride on the early adsorption of protein and osteoblasts
Objective:To study the nature of the surface charge of the samples treated by phosphoric acid and sodium fluoride, and the early adsorption capacity to fibronectin and vitronectin as well as to human osteosarcoma cells MG-63.
Methods:
(1):Pretreatment of samples.Ti plate of grade IV has been processed according to experimental conditions and instrumentation requirements, then were treated with different concentrations of H3PO4+NaF at ambient temperature without auxiliary implementations, and treated by the traditional method as sulfuric acid and hydrochloric acid at60℃for30min.We randomly selected subgroup B1, B4and B10with conventional acid group T for comparison.
(2):Zeta potential sample surface test:Use a solid surface zeta potential analyzer for analysis with Helmholtz-Smoluchowski model.Two identical samples of each experimental group were put into the adjustable gap in the sample cell, face-to-face, spacing0.1mm-0.2mm. KCl electrolyte was used to(at a concentration of1×10-6mol/1) calibrate curve and test sample surface flatness.Then detect the charge and its numberon the sample surface in acidic and basic environments, and the surface potential of the sample curves plotted at different pH conditions.Data obtained were analyzed by the Attract software.
(3):Sample surface detection of fibronectin and vitronectin adsorption capacity: SD rats, male,8-10weeks old,180-220g,16.Only one sample was implanted in one femoral bone marrow cavitiesof each leg.Sample was allowed to maintaine for30minutes.Truncated femur samples were washed with deionized water sufficiently, aiming to wash off the loosely attached proterins. Then samples were washed by SDS solution (50mM/L Tris,2%SDS,5%β-mercaptoethanol), vibrated for15min, centrifugedfor15minutes with1000r/min, the supernatant was moved, and stroed in ultra-low temperature refrigerator.Western blot was used for the semi-quantitative detection of the target protein. Firstly,we determined the total protein concentration with the BCA kit,-20℃to save for backup.Wash the gel with SDS-PAGE running buffer and load samples into gel.Run gel at100V for20min through the stacking part of the gel andturn the volts up to140V for40min after the proteins have gone through the stack and are migrating through the resolvinggel. And the membrane was immersed in blocking solution overnight at4℃. The target protein antibodies (1μg/ml, according to the requirements of dilution) were added,4℃closed overnight. Then the film was transferred to the target protein secondary antibody(1:5000), and developed by chemiluminescence.
(4):Early adsorption ability of samples surfacesto human osteosarcoma cells MG-63:Subgroup B1, B4and B10with conventional acid group T were randomly selected for comparison.SD rats, male,8-10weeks old,180-220g,32.Only one sample was implanted in one femoral bone marrow cavitiesof each leg.Sample was allowed to maintaine for30minutes.Truncated femur samples were washed with deionized water sufficiently.Four treatment groups B1, B4, B10and T, have four parallel samples respectively. Samples obtained were placed in96-well plates, and MG-63cells with80%contact rate were rinsed, digestion, centrifugation, resuspended in cell count, and then were seeded as a density of1×105/cm2onto the titanium sheet surface. Samples were placed in37℃, the volume fraction in the conventional5%CO2incubator incubation culture, were detected cell adhesion capacity, as well as changes in cell morphology at6h,12h,18h and24h. Cells were fixed using3%-4%paraformaldehyde at room temperature for30minutes; then0.1%Triton X-100was added96-500μL/hole,allowed to stand for3minutes,to increase cell permeability. F-actin green fluorescent phalloidin working fluid was added100μl/well in the dark conditionat room temperature, stained for30minutes in the dark.Quantitative software Tanon colon of cell number was used to calculate cell number in per field of view under the microscope, and for each sample to obtain the number of5fields of view, the data was performed one-way ANOVA statistical analysis.
Results:Zeta potential of all test sample surfaces are negative, showing significant difference(F=70.719,P<0.001), those of subgroups B10and B1are the lowest and the highest absolute value, respectively, and that of B4is similar to that of group T. Subgroup B10poccessed the highest adsorption capacities of fibronectin and vitronectins, showing significant difference with other groups(former:F=116.692, P<0.001; latter:F=13.937, P=0.002). While, the capacities of B1are the lowest, B4and group T on fibronectin adsorption capacity are similar, but the group T on vitronectin adsorption capacity is better than that of B4, and is similar to that of B10. The results of MG-63cells'number on each group are consistent with those of fibronectin adsorption ability of each group(F=701.542, P<0.001),.Maybe the amount of phosphate and fluorine attached onto H3PO4group are benefit for the MG-63cellsspread speed, and their mature are faster than that of other groups.Samples treated by traditional etching method as group T is contaminated by sulfur, whose cell spreading and maturation are slow.
Conclusion:The high surface roughness and surface morphology with sharp edges and cusps induce the sample surfaceto poccess a low absolute value of zeta potential. Therefore, the surface adsorption of proteins and cells in surrounding are the most feasible, such as the subgroup B10.The sample surface with the round pits obtain high absolute value of zeta potential, less prone to guide the adsorption with surrounding substance, such as the subgroup B1. due to a secondary pore structure of the surface, group T may haveaffecting the adsorption of proteins and cells. Samples treated by new etch recipe with attached phosphate element and fluorine element may be beneficial to accelerate the speed of cell mature. The residual sulfur of traditional etching formulations not possessesthis effect.
口腔种植领域中,纯钛表面处理一直是学者们为提高种植体初期稳定性以及骨结合的关注焦点。目前钛表面处理工艺中最常用的方法包括多个途径：化学处理、物理处理、生物学处理等。其中化学处理中的酸蚀法是目前公认的可达到临床基本需求的常用方法。酸蚀的配方多种多样,但目前使用最广泛的方法为：49%H2SO4:19%HC1体积比为1：1时在60℃水浴中反应30分钟。此酸蚀方法实质为利用硫酸的强氧化性,在钛表面成一层致密的二氧化钛膜,而盐酸中的氯离子对该层氧化膜具有强亲和力,通过氯离子与氧化膜中的氧发生置换,进而形成可溶性的产物TiCl4-,达到对钛的腐蚀。因此,这种传统的酸蚀过程主要分为两步：第一：硫酸使钛表面发生还原反应,形成氧化膜；第二：盐酸使该氧化膜形成可溶性的氟化物,达到蚀刻效果。可见,硫酸和盐酸虽具有较强的腐蚀性能,但当用于纯钛的酸蚀,两者须发挥协同作用才可。该酸蚀配方可以去除钛材表面氧化膜,并达到临床上对粗糙度以及表面微孔形貌的要求。然而,由于硫酸及盐酸均为强酸,其在水溶液中可发生完全电离,溶液中存在大量游离氢离子,并大量渗透至钛材基体中,导致材料严重的渗氢,明显降低材料的机械性能,缩短材料使用寿命。另外,浓硫酸在加热条件下可以发生还原反应产生腐蚀性的二氧化硫气体,亦会对材料表面造成污染,不利于蛋白和细胞的吸附。因此,该传统配方在某些方面不具备理想酸蚀液的要求,需要寻找一种新的方法就这些缺点加以改变并提高。
在口腔种植应用中,常因患者咬合力过大,咬合间隙过紧,咬合侧向力大,种植上部结构冠根比例过大以及螺丝长期磨损致使其与基台不密合等原因,在反复应力作用下出现种植上部结构中螺丝的折断,此时断端螺丝多位于种植体内或基台内部。另外,当种植体采用硬度较低的纯钛材料,且种植体与周围骨结合良好时,由于上部结构设计的不合理而使种植体承受过大的扭力,如过长的悬臂梁设计,或者对于颈部设计较细窄的小直径种植体而言,当攻丝不足但仍使用较大力旋入种植体时,均可发生种植体的折断,尤以在骨质致密的下颌前牙区多见。折断的种植体和螺钉的取出均十分困难,需要使用专用器械及工具,且操作时间长,效果无法预计,给患者带来了额外的创伤以及巨大的精神负担,不利于良好医患关系的维持。因此,在对纯钛进行传统酸蚀的过程中,由于大量的游离氢离子以及钛本身对氢的敏感性,极易发生严重渗氢,其在持续缓慢的应力作用下导致种植部件发生断裂,这是氢脆的一类表现,是在临床工作中应该避免出现的。鉴于氢脆断裂对临床工作造成的诸多不利影响以及远期预后的不确定性,因此,寻找一种新的酸蚀方法代替这种传统酸蚀方法的要求则变得更加迫切。
约100年前,有学者发现当氢存在于金属中时,会降低金属的某些力学性能,因此,这种因渗氢导致的金属机械性能降低的现象被称为氢脆。氢脆可降低金属材料的弹性模量,抗拉强度,屈服强度,疲劳强度以及断裂韧性等,均可严重影响金属在长期持久应力下的使用寿命。氢对金属的损伤,通常分为6类：a)氢脆(包括延迟损伤和延迟断裂)。当氢在金属中局部达到一定饱和浓度时,可降低金属的延展性和韧性,严重时可导致延迟断裂的发生。b)氢腐蚀。金属中的氢在高温高压的条件下,可与其中的碳结合形成甲烷。当这些气体形成的压力达到某一临界值时,可导致晶界上产生微裂纹。c)氢化物。氢进入过渡元素及其合金的晶格中所形成的间隙型氢化物。d)微裂纹。若金属在冶炼过程中带入过量的氢,且这些氢不能扩散外逸,便会存留在材料缺陷处形成氢分子,后者使材料内部发生晶格畸变,甚至形成微裂纹。e)氢鼓泡。在硫化氢等腐蚀环境中使用的金属,或者对金属进行充氢的操作时,当扩散进入金属中的氢原子在非金属夹杂物中或者较大的缺陷处聚集并形成氢分子时,就可能出现氢鼓泡。f)流变性退化。氢进入金属后,可导致金属的韧性降低,合金在高温下的抗蠕变性能下降。
因此,采用一种新的酸蚀配方,在达到临床对于表面粗糙度和表面形貌要求的同时,尽可能降低对钛材的渗氢,以代替传统酸蚀方法,则变得十分有意义。曾有大量关于不同种类有机酸对纯钛进行酸蚀的研究,包括草酸、氢氟酸、磷酸、盐酸、硝酸等,但经理论分析发现,其中因盐酸为无机酸中的强酸,理论上无法减少氢离子的电离以降低渗氢量；草酸为有机酸中的强酸,其一级电离常数Kal=5.9×10-2,二级电离常数Ka2=6.4×10-5,从化学结构式可知,其未含有利于细胞分化以及成骨矿化的元素,为碳水化合物,可能在此方面不具备优势。硝酸具有强烈的钝化作用外,无法达到表面粗糙度要求,氢氟酸、磷酸均非强酸,不发生完全电离,理论上可降低对材料的渗氢量。然而,氢氟酸虽为弱酸,低浓度的氢氟酸即可与钛发生剧烈反应,不易控制,且有剧毒、强腐蚀性,易于对操作人员造成难愈合的溃疡,而与之相比较,磷酸为中强酸,低毒,且其中含有的磷元素在理论上可显著促进羟基磷灰石的形成,有利于种植体周围的成骨,且不具备氧化性。其不发生完全电离,易于发生一级电离,很难发生二级及三级电离,其电离常数均小于草酸者,因此游离氢离子的浓度明显低于硫酸和/或盐酸以及草酸者,因此其可能会减少游离氢离子渗入钛材内部的数量。然而,通过前期资料的整理以及预实验,发现在常温常态下磷酸无法对钛进行腐蚀。因此,有不少学者就磷酸与钛的反应以及磷酸处理后的钛表面的理化及生物学特性进行了研究,其中使两者发生反应的途径主要包括：a)热处理：处理温度界于180-800℃之间；以及b)电化学处理法：其实质包括阳极氧化反应,阳极腐蚀反应以及阴极还原反应。然而,经过本课题组前期预实验的探索,发现这些处理方法均不同程度忽略了高温下渗氢的发生以及临床应用中对粗糙度的要求,且需要复杂的设备仪器支持,无法达到研究目的。因此,通过大量观察、文献查阅以及预实验的深入,逐步发现并确定在常温下,在无机酸中加入酸性的氟化物,可以对纯钛发生腐蚀效应。本课题采用了氟化物中最常见的氟化钠与磷酸混合；并通过进一步对磷酸浓度以及氟化钠浓度的筛选,即分别使用6%,8%,10%,12%,16%,18%,25%,30%,35%,40%,46%,50%,55%以及62%磷酸与O.1mol/l NaF对钛在常温下进行酸蚀,发现35%,46%以及62%磷酸(浓度分别为3.57mol/l,4.62mol/1,6.32mol/l)在与氟化钠酸蚀钛的过程中,均可出现明显的腐蚀反应,并且通过对反应时间的筛选,发现该配方对钛的反应速度适宜,反应30分钟即可不同程度达到临床对钛片表面粗糙度的要求,且各组之间均有差异。另外,当改变氟化钠浓度时,可见钛片的表面形貌出现了稳定且规律的变化。通过初步检测渗氢量,发现经新酸蚀配方：磷酸+氟化钠处理后,钛材的渗氢量明显低于传统酸蚀处理者；另外,由于不同浓度的磷酸+氟化钠的配比,其可以达到高于传统酸蚀组的粗糙度,并且可能有磷、氟元素的同时存在,该配方可能会促进周围细胞对材料表面的吸附。综上所述,通过前期的理论研究以及预实验的结果,为消除关于磷酸热处理以及电化学处理导致的不利因素,本课题组期望该新酸蚀配方能够在常温下对纯钛处理后,与传统酸蚀方法相比,明显降低材料的渗氢量、达到临床所需的表面粗糙度,并且更有利于细胞外基质蛋白和成骨细胞的吸附。主要研究内容包括其处理后材料的表面形貌,粗糙度,表面化学成分及含量,材料内部的氢含量,氢分布以及在模拟体液中钛表面的磷酸钙类化合物的沉积,表面Zeta电位,对纤维连接蛋白和玻连蛋白的吸附能力以及对人成骨肉瘤来源的MG-63细胞的吸附以及对其形态影响的评估,为形成一种全新的材料表面处理方法,用以代替传统酸蚀方法,来避免传统酸蚀后材料严重的渗氢以及表面的污染,提供可靠的实验依据。
第一章磷酸和氟化钠对纯钛酸蚀后渗氢效果的影响
目的：研究一种新型的表面酸蚀配方：磷酸+氟化钠在常温下对纯钛进行酸蚀,与传统酸蚀方法进行比较,评估其处理后的钛材能否在达到临床需要的适宜表面粗糙度的同时,降低材料渗氢量,并具有较传统酸蚀方法更好的诱导磷酸钙盐沉积的能力。
方法：
(1)：样品处理：Ⅳ级纯钛板机加工后,在常温且无辅助设备的前提下,使用不同浓度配比的磷酸+氟化钠进行酸蚀,分组：磷酸浓度分别为3.57mmol/1,4.62mol/l和6.32mol/1时,分别为组A,组B以及组C；各组中,氟化钠浓度依照与磷酸的浓度比例逐渐递增,分别形成A1-A10亚组,B1-B10亚组和C1C6亚组。另用常规酸蚀方法：49%H2S04:19%HCl体积比为1：1时在60℃水浴中反应30分钟,酸蚀钛片,为对照组T。
(2)：表面形貌观察：使用扫描电子显微镜(Scanning electronic microscopy,SEM,HitachiS-3700N)观察表面形貌,以及仪器配用软件提供的标尺功能用来测量钛表面酸蚀凹坑的直径,每个坑进行10次测量后,取其平均值。
(3)：表面粗糙度检测：使用白光干涉仪(White light interferometer,Taylor HobsonCII)检测处理表面,包括表面粗糙度,Ssk(表面倾斜度：surface skewness)以及Sku(表面峰度：surface kurtosis)值,使用Talymap表面分析软件获得。
(4)：表面化学成分及含量分析：采用X射线衍射分析仪(X-ray diffractometry, XRD,Bruker,D8-advance)进行定性及半定量检测。同时,X射线光电子能谱(X-ray photoelectron spectroscopy,XPS,Kratos,Axis Ultra DLD)用于定量检测的样品处理后表面的化学成分：A1,B1和B10。数据由CasaXPS处理。
(5)：氢含量分析：使用惰气脉冲熔融热导仪根据ASTM E1447-1409(American Society of Testing Materials,ASTM)确定钛样品(美国力可Leco,TCH600)的氢含量。将样品置于坩埚中,高电流下除气,充入氦气,使样品中的氢以H2的形式释放,在催化剂作用下被转化为水,用红外线检测仪检测氢含量。
(6)：氢分布分析：根据ASTM E1504-1506,采用时间-飞行次级离子质谱仪(Time of flight secondary ion mass spectrometry,ToF-SIMS,2100TRIFT,Physical Electronics)检测处理后Ti试样横截面的氢元素分布。检测氢分布前,根据ASTME1078-09,先将各样品安装于横截面抛光仪(Cross-section polishing instrument日本电子株式会社JEOL, SM-09010)去除表面污染物并保证表面平整。随后,在真空度低于4×10-8Pa的条件下检测大小为200×200μm2和50×50pm2的试样横截面,测绘结果由Wincadence软件获得。
(7)：弹性模量的检测：按照IS07438-2005制备样品。使用万能材料试验机(Universal testing machine,Instron5565)进行三点弯曲试验和弹性模量的计算。弯曲试验在室温下进行；选择材料在处于弹性形变范围内的载荷进行检测(<800N)。
(8)：磷酸钙盐的定性及定量分析：按要求配制模拟体液,将具有代表性表面形貌的处理组B1,B4,B10以及传统酸蚀组T试样,分别置于37℃的模拟体液中,每组试样静置20天。用扫描电子显微镜和X射线光电子质谱仪观察并检测试样表面沉淀物的分布以及沉淀物的成分、比例。
结果：新酸蚀配方处理后试样的表面粗糙度在0.47±0.03μm到0.87±0.05pm之间；且随氟化钠浓度的升高,其表面形貌呈规律性变化：由圆弧凹坑状逐渐过渡至尖锐棱角状；其表面有磷、氟元素共同附着；氢含量在30.9±3.8ppm至86.9±6.0ppm之间,且在此范围内,随着氢含量的升高,其表面覆盖的磷酸钙盐的面积亦增加；氢元素主要分布于钛材的晶格点阵上；磷酸钙盐的覆盖面积高于传统酸蚀组T(F=4.507,P=0.010),亚组B1、B4沉积物的钙磷比例更接近羟基磷灰石者,,且沉积部位无选择性。传统酸蚀处理组T的表面粗糙度为0.59±0.04μ m；氢含量为287±16.5ppm,其与磷酸各处理组间均具有明显的差异(前者F=10.349,P<0.001；后者F=221.945,P<0.001)；氢元素同时分布于材料的晶格点阵间隙上和其边界之间；其磷酸钙盐仅沉积于酸蚀形成的凹坑内。
结论：新酸蚀配方在达到临床粗糙度的需求同时,明显降低了氢含量；且氢元素主要分布于晶胞内部,理论上可降低氢脆导致的”沿晶断裂”的几率；可以通过改变各组分浓度比来获得不同特点的表面形貌；其表面同时附着的磷、氟元素以及其表面形貌规律性的转化,可能影响了磷酸钙盐的沉积。
第二章纯钛经磷酸和氟化钠处理后对其表面蛋白吸附以及成骨细胞初期附着的影响
目的：研究经磷酸和氟化钠处理后试样的表面电荷性质,及其在动物体内对纤维连接蛋白以及玻连蛋白的吸附能力,并考察其对人骨肉瘤细胞MG-63的吸附以及对其形态的影响。
方法：
(1)：样品处理：Ⅳ级纯钛板按照检测仪器及实验条件的要求加工成一定形状的钛片。在常温且无辅助设备前提下,使用不同浓度配比的磷酸+氟化钠进行酸蚀,选择具有代表性表面形貌的亚组B1,B4以及B10与传统酸蚀组T进行比较。
(2)：试样表面Zeta电位检测：使用固体表面Zeta电位分析仪进行Zeta电位分析。采用Helmholtz-Smoluchowski模型进行检测。在可调间隙样品池中,分别将各实验组两个大小完全相同的样品测试面相对放置,间隔0.1mm-0.2mm。用电解液氯化钾(浓度为1×10-6mol/l)校准,绘制工作曲线,检测样品表面平整度。随后,检测待测样品在碱性和酸性环境中携带的电荷种类及数目,并绘制样品在不同pH条件下的表面电位曲线。所得数据用Attract软件进行分析。
(3)：试样表面对纤维连接蛋白(Fibronectin, FN)及玻连蛋白(、Vitronectin, VN)吸附能力的检测：SD大鼠,雄性,8-10周龄,180-220g,16只。将SD大鼠左右股骨髓腔内各植入一枚试样,静置30分钟。截断股骨,取出试样,去离子水充分洗涤。试样置入SDS蛋白洗脱液(50mM/L Tris,2%SDS,5%β-巯基乙醇),震荡15min。1000转/分离心15分钟,吸去上清液。使用western blot对目标蛋白进行半定量检测。首先使用BCA试剂盒测定蛋白浓度,-20℃保存备用。SDS-PAGE胶(Sodium lauryl sulfate-Polyacrylamide gelelectrophoresis)制作好后,加样,100V下进行浓缩胶电泳,20min；140V下分离胶中电泳,40min。转膜,并将膜浸入封闭液中4℃过夜；将膜取出放入目标蛋白一抗(1ug/ml,按要求稀释后)中,4℃封闭过夜；再将膜转入目标蛋白二抗(1：5000)中,用化学发光法显影。
(4)：试样表面对人骨肉瘤细胞MG-63的早期吸附：SD大鼠,雄性,8-10周龄,180-220g,32只。将SD大鼠左右股骨髓腔内各植入一枚处理后试样,静置30分钟。将股骨截断,取出试样,去离子水充分洗涤。4个处理组B1,B4,B10以及T,各有4个平行样品。获得处理样品后,分别置入96孔板,将已培养稳定且生长达到80%接触率的MG-63细胞冲洗,消化,离心,重悬后,细胞计数,并按1×105个/cm2的密度接种于钛片表面。将已接种细胞的钛片置于37℃,体积分数5%CO2的培养箱内常规孵化培养,分别检测在6h,12h,18h和24h时的细胞粘附量,以及细胞形态的变化。每到观察时间点,采用3%-4%多聚甲醛室温下固定细胞30分钟；再将0.1%Triton X-100加入96孔板中500μL/孔,静置3分钟,以增加细胞通透性；避光条件下中加入100微升/孔肌动蛋白绿色荧光鬼笔环肽工作液,在室温下对细胞肌动蛋白F-actir进行避光染色30分钟。细胞计数软件Tanon colon对显微镜下每视野的细胞数定量并计算,每个试样获取5个视野范围内的细胞数目,将获得数据进行单因素方差统计学分析。
结果：所有检测试样表面Zeta电位均为负值,其中亚组B10的Zeta电位绝对值最小,与其他各组有明显差异(F=70.719,P<0.001),B1者最大,B4与组T者相近。亚组B10对纤连蛋白吸附能力最强,与其他组有明显差异(F=116.692,P<0.001),B1者最低,B4及组T对纤连蛋白吸附能力相近。B10组者对玻连蛋白吸附能力亦为最强,组T者高于B4者,与B10者相近,B1仍为最低(F=13.937,P=0.002)。对MG-63的吸附数量,各组表现与对纤连蛋白吸附能力结果一致(F=701.542,P<0.001)。新酸蚀配方中磷氟元素的同时附着可能更有利于加快接种细胞粘附初期的成熟速度,使其表面吸附的MG-63细胞铺展较快。传统酸蚀组T则可能因表面硫元素的存在,细胞铺展慢,成熟速度慢。
结论：高表面粗糙度以及完全呈尖锐棱角状的表面形貌可以获得低的Zeta电位绝对值,则材料表面最易发生对周围蛋白以及细胞的吸附,如亚组B10。低表面粗糙度以及呈圆弧凹坑状的表面形貌则获得高的Zeta电位绝对值,此类表面不易发生吸附聚集,如亚组B1。而组T可能因表面具有次级微孔结构有利于其对蛋白及细胞的吸附。新酸蚀配方中磷氟元素的同时附着可能有利于加快接种细胞初期的成熟速度；而传统酸蚀配方中残留的硫元素则不具备此作用。
Background
In the area of oral implantology, studying of titanium surface treatment is always a focus of scholars who aimed to improve the initial implant stability and osseointegration.The most commonly used surface treatment methods of titanium currently include several channels:chemical treatments, physical treatments and biological treatments. Wherein the chemical etching method of treatment is now recognized as a basic approach to reach clinical needs. Etching recipes varied, but the most widely used method is:49%H2SO4:19%HCl1:1by volume in the reaction for30minutes at60℃in water bath. In the traditional method, H2SO4, as a strong oxidant, oxidizes the surface of the Ti substrate into TiO2. The strong affinity of the chloride ions to the oxide film in HCl leads to the replacement of oxygen with chloride ions, producing the soluble product TiCl4-. That is, this combination performs faceting by first forming an oxide film and then corroding it, and the achieved surface topographies depend on the construction of the oxide film to a great extent. Therefore, sulfuric acid or hydrochloric acid, although with strong acidity, can't perform etching for pure titanium alone, and the two acids need to work together. The tradiational etch combination can remove titanium oxide film and meet the clinical requirements of surface roughnes. However, due to both sulfuric acid and hydrochloric acid, which can occur completely ionized in aqueous solution, and a large number of free hydrogen ions exist, which can penetrate to itanium matrix, resulting in severe hydrogen embrittlement for titanium material, and reducing its mechanical properties. Meanwhile, concentrated sulfuric acid under heating could produce sulfur dioxide as a corrosive gas which induces contamination of the surface. Therefore, the tradiational etching solution does not poccess the ideal requirements in some areas, a new way to deal with the shortcoming is needed.
In oral implant applications, due to the patient heavy bite occlusal force, tight occlusal clearance, lateral bite force, the proportion of crown and root structure is too large and wear of upper screw resulting in close contact with the base, in the slow and repeated stress on the superstructure, break induces the fracture of the screw, and the end of the screws were always located inside the base or in the implants. On one hand, when the implants made of titanium materials with a lower hardness, such as grade II, and osseointegration is good and the upper part of the implant is unreasonable which bears too much torque, may cause the implant fracture occurred. On another hand, the small diameter implants with narrow neck are likely to fracture near the weak point, when a large force was used to screw the implant into the bone, especially in the anterior mandiblewith high bone density.It is difficult to remove the broken implants and screws, which require the special equipments and tools, and the operation for a long time, the effect can not be expected, exerting patients with additional trauma and huge mental burden, is not conducive to maintaining a good doctor-patient relationship. Therefore, in the process of titanium etching, since a large number of free hydrogen ions exist, titanium sensitivity to hydrogen and severe long-term stress leads to fracture of implants, which is a kind of hydrogen embrittlement performance and should be avoided.Given the many adverse effects of hydrogen embrittlement caused to the clinical work, therefore, it is even more urgent to find a new etching method instead of the traditional etching methods should be proposed.
About100years ago, researchers found that when the metal in the presence of hydrogen will reduce some of the mechanical properties of the metal, so that hydrogen embrittlement is known due to the mechanical properties of metal reducedsince the hydrogen permeation. Hydrogen embrittlement can reduce many properties of metallic material, such as elastic modulus, tensile strength, yield strength, fatigue strength and fracture toughness, etc.,can seriously affect the life span of the metal under the long-lasting metal stress. Damageto metal caused by hydrogen embrittlement is usually divided into six categories:a) hydrogen crisp (including delayed damage and delayed fracture) when the hydrogen in the metal reached a certain saturation concentration locally, can reduce the ductility and toughness of metals, in severe cases which can lead to delayed fracture occurs.b) hydrogen corrosion。Metal hydrogen underhigh temperature and high pressure will turn into methane since react with carbon. When the value of gas pressure reaches a certain critical level, the internal stress will lead to the generation of micro-cracks on the grain boundaries.c) hydride. Gap hydrides are formed when hydrogen into the transition element in the alloy latticed) microcracks. If an excess of hydrogen entered into the metal in the smelting process, these atoms can not diffuse and escape, and are retained to form hydrogen molecules in the material defects, which will occur the lattice distortion, and even the micro-cracks.e) hydrogen bubbling. Metal works in corrosive environments such as hydrogen sulfide, or for the hydrogen charging operation, when the hydrogen diffusion into the metal with largedefects and exist as non-metallic inclusions may cause hydrogen blistering.f) rheologydegradation. After the hydrogen atoms enter into the metal, the metal can lead to a decrease in toughness of the alloy as high temperature creep resistance decreases.
Therefore, involving a new etching recipe, while reaching clinical requirements of proper surface roughness and surface morphology as much as possible, to reduce the infiltration of titanium hydrogen, becomes very necessary.There was many a studies about the different kinds of organic acid etching of titanium researches, including oxalic acid, hydrofluoric acid, phosphoric acid, hydrochloric acid, nitric acid, etc..But by theoretical analysis, hydrochloric acid and oxalic acid, in theory, can not reducehydrogen ions; nitric acid has a strong passivative effect, can not reach the requirement of surface roughness.Hydrofluoric acid, phosphoric acid are not strong acid, in theory, can reduce the amount of hydrogen ions permeating into materials. However, although the hydrofluoric acid is a weak acid, a low concentration of hydrofluoric acid can react violently with the titanium, and it is difficult to control with highly toxic, corrosive ability, easy to cause heal ulcers to the operators. Phosphoric acid with moderate strength acidity and low toxicity can significantly promote the formation of hydroxyapatite, and is conducive to bone formation around the implant, and do not have oxidation, be absence of the oxidation. It is prone to cause the first stage ionization, but difficult to initiate the secondor thethird ionization. This phenomenon indicates that in the phosphoric acid solution, the concentration of free hydrogen ions is significantly lower than those of sulfuric acid, and therefore may reduce the number of free hydrogen ions into the interior part of the titanium material. However, pure phosphoric acid at room temperature can not etch the titanium. Therefore, there are many scholars who worked on physicochemical and biological characteristics of the phosphoric acid and phosphoric acid reaction with the titanium, in which the reaction occurs within the two approaches:including hydrothermal (180-800℃) and electrochemical treatments, which include anodic oxidation, anodic corrosion and cathodic reduction. However,some of these studies failed to estimate the adverse impact of the hydrothermal treatment, which could aggravate the permeation of hydrogen into Ti substrates. Other studies lead to a considerably limited faceting effect that cannot satisfy the roughness requirement. Through our investigation, we discovered that Ti could be considerably facetted by a combination of inorganic acid and acid fluoride. Therefore, a large of pre-preliminary experiments found that adding the acid fluoride to an inorganic acid, the corrosive effects may occur on the titanium at room temperature. A further concentration of the sodium phosphate had been screened, using6%,8%,10%,12%,16%,18%,25%,30%,35%,40%,46%,50%,55%and62%phosphoric acid respectively and0.1mol/1NaF acid etching of titanium, found that35%,46%and62%phosphoric acid(the concentration of3.57mol/1,4.62mol/1,6.32mol/1) and sodium fluoride in the process of etching the titanium, the corrosion reaction occurs with proper reaction speed, and the surface roughness reach the clinical requirements of titanium when reaction processes for30minutes, and there are differences between the three groups.Furthermore, by varying the concentration of sodium fluoride, the surface topography of the titanium shows stable changes. In summary, through theoretical studies and the results of the pre-experiment, to eliminate the negative factors on heat treatment and electrochemical treatment, our group expect the new etch recipe, compared to conventional etching method, could significantly reduces the amount of hydrogen permeability material, achieve the desired surface roughness clinical, and be more conducive to the adsorption of extracellular matrix proteins and osteoblasts. We primarily focus on the performance of this combination with respect to the surface roughness, the hydrogen content permeating into the Ti substrate, calcium phosphates deposition ability, zeta potential, adhesive ability with FN, VN and MG-63. This study investigates the surface topography, roughness, surface chemistry, hydrogen content, hydrogen distribution and growth ability in simulated body fluid (SBF) of titanium samples treated using various methods, and adhesive ability with FN, VN and MG-63are performed and detected by western blot and immunohistochemisitry. We hope that this study will produce a novel surface with a morphology, composition and hydrogen content that is appropriate for dental implantation.
CHAPTER ONE Phosphoric acid and sodium fluoride:a novel etching combination on hydrogen embrittlement of titanium
Objective:We investigate whether a novel and inexpensive etching method, H3PO4+NaF, on titanium, could obtain both a lower hydrogen content and superior calcium phosphates deposition performance, while achieving proper surface roughness, in comparison with the traditional etching method.
Method:
(1):Pretreatment of samples:Ti plate of grade IV has been processed, then were treated with different concentrations of H3PO4+NaF at ambient temperature without auxiliary implementations, as group A, B and C, when the concentrations of phosphoric acid were3.57mol/1,4.62mol/1and6.32mol/1respectively. Samples were treated by the traditional method as sulfuric acid and hydrochloric acid at60℃for30min(group T).The samples were then maintained in simulated body fluid with pH7.4for10days and20days at37℃.
(2):Observation of surface morphology:The surface morphologies were examined using a scanning electron microscope (SEM, HitachiS-3700N), The Scaleplate feature of the software provided with the instrument was used to measure the diameters of the round pits;10measurements were performed for each pit, and the values were averaged.
(3):Detection of surface roughness:The surface roughnesses were then determined through white-light interferometry(Taylor Hobson CII).The surface roughness, and the values of Ssk and Sku were obtained using Talymap surface analysis software.
(4):Detection of surfacecompositions and contents:XRD patterns of sub-groups Al, B1, B10, and Cl and of group T were obtained by X-ray diffractometry (Bruker, D8-advance). X-ray photoelectron spectroscopy (Kratos, Axis Ultra DLD) was used to quantify the P and F contents of Al, B1and B10. The obtained data were processed by Casa XPS.
(5):Detection of hydrogen contents:Inert gas fusion thermal conductivity analysis was used to determine the hydrogen contents in the Ti samples (Leco, TCH600) according to ASTM E1447-09. The samples were put into the graphite crucible. The air remaining in the graphite crucible was degassed under40A under a helium atmosphere. The hydrogen in the sample was released in the form of H2and converted into water by rare earth.
(6):Detection of hydrogen distribution:The hydrogen distributions in the transverse sections of sub-group B1and group T were detected by time-of-flight secondary ion mass spectrometry (ToF-SIMS, Model2100Trift, Physical Electronics, USA) according to ASTM E1504-06. Before the measurement, the samples were prepared by cross-section polishing (JEOL, SM-09010) according to ASTM E1078-09to prevent contamination and to ensure smoothness. The H mapping results were obtained using Wincadence software.
(7):Detection of elastic modulus:Universal material testing machine (Instron5565) were involved to examine the elastic modulus of samples treated by different etching methods. Specifically, the bending test was limited within the elastic deformation range (0-800N) at ambient temperature.
(8):Hydroxyapatite deposition on Ti plate:Simulated body fiulde were prepared according the reference. The HA deposition abilities of different etching combinations were evaluated by immersing the samples, as B1, B4, B10and T, into SBF for20days. Scanning electron microscopy (SEM, Hitachi S-3700N) and X-ray photoelectron spectroscopy (Kratos, Axis Ultra DLD) was used to observe the morphology and to quantify the Ca and P contents of B1,B4,B10and T.
Results:The surface roughnesses of groups A, B, and C are in the range of0.47±0.03μm to0.87±0.05μm. The surface topography switches from small round profile to deep V-shape groove dramatically as increasing the concentration of NaF. P and F attach onto the surface synchronously.The hydrogen contents of new groups are in the range of30.9±3.8ppm to86.9±6.0ppm, and that of group T is287±16.5ppm. The distribution of hydrogen atoms were mainly inside of the grains of titanium. Calcium salt coverage areas are larger than that of traditional etching group T (F=4.507, P=0.010), the calcium phosphorus ratio of the subgroup B1, B4are closer to those of hydroxyapatite, with non-selective deposition site. The surface roughness of group T was0.59±0.04μm; hydrogen content of287±16.5ppm, show the significant differences (former:F=10.349, P<0.001; latter:F=221.945, P<0.001);hydrogen distribution concentrate on the boundaries between the crystal lattice material, and only calcium salt deposited in the etch pits.
Conclusion:Meeting withthe roughness of clinical needs, the novel etching combination could significantly reducing the hydrogen content as well. The hydrogen is mainly located inside the titanium grains, which could reduce the probability of intergranular fracture caused by hydrogen embrittlement theoretically. By changing the concentration ratio of the components,it could obtain different characteristics of the surface topography. The synchronous attachments of phosphorous and fluorine onto the surface and regularity of its surface morphology transformation may affect the deposition of calcium phosphates.
CHAPTER TWO Influence of titanium treated by phosphoric acid and sodium fluoride on the early adsorption of protein and osteoblasts
Objective:To study the nature of the surface charge of the samples treated by phosphoric acid and sodium fluoride, and the early adsorption capacity to fibronectin and vitronectin as well as to human osteosarcoma cells MG-63.
Methods:
(1):Pretreatment of samples.Ti plate of grade IV has been processed according to experimental conditions and instrumentation requirements, then were treated with different concentrations of H3PO4+NaF at ambient temperature without auxiliary implementations, and treated by the traditional method as sulfuric acid and hydrochloric acid at60℃for30min.We randomly selected subgroup B1, B4and B10with conventional acid group T for comparison.
(2):Zeta potential sample surface test:Use a solid surface zeta potential analyzer for analysis with Helmholtz-Smoluchowski model.Two identical samples of each experimental group were put into the adjustable gap in the sample cell, face-to-face, spacing0.1mm-0.2mm. KCl electrolyte was used to(at a concentration of1×10-6mol/1) calibrate curve and test sample surface flatness.Then detect the charge and its numberon the sample surface in acidic and basic environments, and the surface potential of the sample curves plotted at different pH conditions.Data obtained were analyzed by the Attract software.
(3):Sample surface detection of fibronectin and vitronectin adsorption capacity: SD rats, male,8-10weeks old,180-220g,16.Only one sample was implanted in one femoral bone marrow cavitiesof each leg.Sample was allowed to maintaine for30minutes.Truncated femur samples were washed with deionized water sufficiently, aiming to wash off the loosely attached proterins. Then samples were washed by SDS solution (50mM/L Tris,2%SDS,5%β-mercaptoethanol), vibrated for15min, centrifugedfor15minutes with1000r/min, the supernatant was moved, and stroed in ultra-low temperature refrigerator.Western blot was used for the semi-quantitative detection of the target protein. Firstly,we determined the total protein concentration with the BCA kit,-20℃to save for backup.Wash the gel with SDS-PAGE running buffer and load samples into gel.Run gel at100V for20min through the stacking part of the gel andturn the volts up to140V for40min after the proteins have gone through the stack and are migrating through the resolvinggel. And the membrane was immersed in blocking solution overnight at4℃. The target protein antibodies (1μg/ml, according to the requirements of dilution) were added,4℃closed overnight. Then the film was transferred to the target protein secondary antibody(1:5000), and developed by chemiluminescence.
(4):Early adsorption ability of samples surfacesto human osteosarcoma cells MG-63:Subgroup B1, B4and B10with conventional acid group T were randomly selected for comparison.SD rats, male,8-10weeks old,180-220g,32.Only one sample was implanted in one femoral bone marrow cavitiesof each leg.Sample was allowed to maintaine for30minutes.Truncated femur samples were washed with deionized water sufficiently.Four treatment groups B1, B4, B10and T, have four parallel samples respectively. Samples obtained were placed in96-well plates, and MG-63cells with80%contact rate were rinsed, digestion, centrifugation, resuspended in cell count, and then were seeded as a density of1×105/cm2onto the titanium sheet surface. Samples were placed in37℃, the volume fraction in the conventional5%CO2incubator incubation culture, were detected cell adhesion capacity, as well as changes in cell morphology at6h,12h,18h and24h. Cells were fixed using3%-4%paraformaldehyde at room temperature for30minutes; then0.1%Triton X-100was added96-500μL/hole,allowed to stand for3minutes,to increase cell permeability. F-actin green fluorescent phalloidin working fluid was added100μl/well in the dark conditionat room temperature, stained for30minutes in the dark.Quantitative software Tanon colon of cell number was used to calculate cell number in per field of view under the microscope, and for each sample to obtain the number of5fields of view, the data was performed one-way ANOVA statistical analysis.
Results:Zeta potential of all test sample surfaces are negative, showing significant difference(F=70.719,P<0.001), those of subgroups B10and B1are the lowest and the highest absolute value, respectively, and that of B4is similar to that of group T. Subgroup B10poccessed the highest adsorption capacities of fibronectin and vitronectins, showing significant difference with other groups(former:F=116.692, P<0.001; latter:F=13.937, P=0.002). While, the capacities of B1are the lowest, B4and group T on fibronectin adsorption capacity are similar, but the group T on vitronectin adsorption capacity is better than that of B4, and is similar to that of B10. The results of MG-63cells'number on each group are consistent with those of fibronectin adsorption ability of each group(F=701.542, P<0.001),.Maybe the amount of phosphate and fluorine attached onto H3PO4group are benefit for the MG-63cellsspread speed, and their mature are faster than that of other groups.Samples treated by traditional etching method as group T is contaminated by sulfur, whose cell spreading and maturation are slow.
Conclusion:The high surface roughness and surface morphology with sharp edges and cusps induce the sample surfaceto poccess a low absolute value of zeta potential. Therefore, the surface adsorption of proteins and cells in surrounding are the most feasible, such as the subgroup B10.The sample surface with the round pits obtain high absolute value of zeta potential, less prone to guide the adsorption with surrounding substance, such as the subgroup B1. due to a secondary pore structure of the surface, group T may haveaffecting the adsorption of proteins and cells. Samples treated by new etch recipe with attached phosphate element and fluorine element may be beneficial to accelerate the speed of cell mature. The residual sulfur of traditional etching formulations not possessesthis effect.
引文
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