【作者】 马晓妮；

【导师】 徐欣； 朱瑞富；

【作者基本信息】 山东大学 ， 口腔种植学（专业学位）， 2014， 博士

【摘要】 生物材料是一个多学科交叉的前沿研究领域,是国民经济和社会发展中的重要科技问题,已成为国内外研究的热点之一。作为生物材料的典型代表,牙种植体材料成为目前临床和科研最为关注的焦点领域。种植体的表面处理方式对于提高种植成功率起着至关重要的作用。目前市场上各类产品的种植体表面处理方式多种多样,不同的处理方法均具有其各自的优缺点,但其共同的目标是形成粗糙表面,使其具有更好的骨结合能力和生物活性。种植体表面特性是直接影响骨整合过程骨结合率的主要因素之一,对于提高种植体的成功率有十分重要的意义和临床价值。多年来学者们也一直在努力找寻最佳种植体表面处理方式,以达到最佳的种植体-骨结合界面。早期学者提出的机械表面处理的钛种植体有植入早期难与组织形成化学结合、骨结合形成慢、愈合期较长等诸多缺点。故目前口腔种植体表面处理方式的研究热点是采用不同处理工艺改变种植体表面的物理和化学性质,促进种植体早期形成骨结合,从而保证种植体的长期功能。纯钛种植体表面微弧氧化复合喷砂酸蚀处理,是指通过改变表面处理工艺条件设计,调整氧化膜的结构、元素组成及生物特性,从而实现膜层的最佳的生物学效果。该技术还具有工艺简单、经济环保等特点,值得进一步论证并应用于临床广泛推广。为此本文拟在以下几方面开展研究：材料和方法：第一部分：不同表面处理纯钛试样的制备及理化性能研究喷砂：把一定颗粒度的Al203磨粒用1010型普压干式喷砂机高速喷射至打磨制备的TA2纯钛试样表面,影响喷砂纯钛基体表面性能的主要工艺参数有：磨粒尺寸为200μm,气体压力为0.9bar,气体流速为210m/min,喷射角度为70。,喷口距离为15cm。酸蚀：用氢氟酸HF和盐酸HCl的混合酸溶液腐蚀TA2纯钛基体表面,影响酸蚀处理表面性能的主要工艺参数有：氢氟酸浓度为40%,盐酸浓度为38%,混合溶液中HF和HCl比例为6：4,腐蚀溶液总体积为40ml,酸蚀温度为25℃,酸蚀时间为100min。微弧氧化：是采用较高的工作电压,将工作区域由普通的阳极氧化法的法拉第区域引入到高压放电区域,在铝、镁、钛等金属表面原位生成氧化物陶瓷膜的方法。其中电极电压为500V,电极频率为550Hz,氧化温度小于40℃,氧化时间为18min,电解液中CH3COO)2Ca·H20浓度为30mmol/L,为10mmol/L, NaOH为8mmol/L,由于微弧氧化的形成是非常复杂的过程,受多方面因素的影响和调控。本课题前期实验已优化出一套工艺参数,本实验在此工艺参数的基础上结合喷砂与酸蚀处理,将实验对象分为：单纯微弧氧化、喷砂结合微弧氧化、酸蚀结合微弧氧化、喷砂加酸蚀结合微弧氧化四组,并对各组处理表面分别从表面形貌、能谱分析、膜层厚度、表面粗糙度、膜层结合强度等方面加以表征分析。第二部分：不同表面处理纯钛试样的体外细胞相容性研究将TA2纯钛用数控线切割机床CNC加工成圆柱形试样。加工为高度3.0mm,底面直径为8.0mm。采用200～1000#的金相砂纸将样本逐级打磨,样本经打磨后将其置于60-80℃氢氧化钠溶液进行去油处理,浸泡时间为5min。采用去离子水将试样清洗干净,并置于盐酸及硝酸混合溶液中浸泡10～15s,充分去除金属表明陈旧的氧化层,然后采用去离子水将金属置于常温下干燥。将样品分为如下四组：单纯微弧氧化、喷砂结合微弧氧化、酸蚀结合微弧氧化、喷砂加酸蚀结合微弧氧化。此四组试样分别与ST2细胞系共培养,通过SEM观察、细胞毒性检测、ALP活性检测、骨钙素含量检测等,分析细胞的增殖、粘附、分化情况,对比分析四组不同表面处理的纯钛种植体生物相容性。第三部分：不同表面处理纯钛试样的动物体内生物学评价将TA2纯钛用数控线切割机床CNC加工成圆柱形试样。加工为高度10.0mm,底面直径为3.0mm。采用200～1000#的金相砂纸将样本逐级打磨,样本经打磨后将其置于60-80℃氧化钠溶液进行去油处理,浸泡时间为5min。采用去离子水将试样清洗干净,并置于盐酸及硝酸混合溶液中浸泡10-15s,充分去除金属表明陈旧的氧化层,然后采用去离子水将金属置于常温下干燥。将样品分为如下四组：单纯微弧氧化、喷砂结合微弧氧化、酸蚀结合微弧氧化、喷砂加酸蚀结合微弧氧化。分别将纯钛试样植入新西兰大白兔双侧股骨内,在愈合4w和8w时取出标本,通过顶出试验、骨与种植体接触率,计算骨种植体周围骨量及骨质测量,评价喷砂酸蚀后微弧氧化处理种植体骨结合强度的情况,并从组织形态计量学上研究喷砂酸蚀后微弧氧化处理种植体的促成骨特性。结果：第一部分：不同表面处理纯钛试样的制备与表征表面粗糙度：纯钛基体的表面粗糙度Ra值是0.2μm。而经过微弧氧化工艺过程的处理表面,其Ra值变成0.8μm。经过微弧氧化处理后表面粗糙度的增大则主要是微弧氧化膜上孔洞出现引起的。喷砂纯钛基体微弧氧化膜的微观粗糙度Ra为1.4μm,粗糙度略大于只经过微弧氧化处理的样品。酸蚀纯钛基体微弧氧化处理的试样微弧氧化膜微观粗糙度Ra测定为1.7gmm,也比只经过微弧氧化处理的样品粗糙度大。而经过喷砂+酸蚀双重处理的纯钛基体微弧氧化膜的粗糙度达到了2.2μm,表明喷砂和酸蚀处理对微弧氧化膜的粗糙度都有了显著增加,表观上则表现为氧化膜上较多均匀的孔洞的出现。膜层厚度：单纯微弧氧化的样品微弧氧化膜厚度为37.2μm；喷砂纯钛基体微弧氧化膜稍薄,为28.3μm；而酸蚀纯钛基体微弧氧化膜厚度能达到40.2μm；经过喷砂+酸蚀双重处理的纯钛基体微弧氧化膜厚度最厚,为43.1μm。不同处理对样品表面产生不同影响,因此得到不同厚度的氧化膜结合强度：单纯微弧氧化处理的试样氧化膜载荷为20.7N。而喷砂试样氧化膜载荷较未处理的试样稍大,为23.2N,酸蚀基体微弧氧化膜临界载荷达到27.9N,即酸蚀纯钛基体微弧氧化膜结合强度高于喷砂和未处理试样的结合强度。而经过喷砂和酸蚀处理的微弧氧化膜临界载荷最高,为31.2N。接触角：测试结果如下,蒸馏水液滴在未处理纯钛基体微弧氧化膜表面的接触角是82.9°,喷砂纯钛试样微弧氧化膜表面的接触角是76.7°,在酸蚀试样微弧氧化膜表面的接触角是69.3°,喷砂和酸蚀双处理试样微弧氧化膜的表面接触角为63.2°。第二部分：不同表面处理纯钛试样的体外细胞相容性研究细胞毒性检测：根据MTT法测定的OD值计算相对增殖率(RGR),细胞相对增殖率RGR%=OD试验组/OD阴性对照组X100%.根据不同时期各组RGR值进行细胞毒性分级(CTS)。由本实验结果可知：四组改性后纯钛种植体浸提液与阴性对照组比较均无差异显著(P>0.05),CTS分级均为0级,说明本研究所采用的纯钛种植体无毒性,符合《医疗器械生物学评价国家标准GB/T16886.1—2001》中对材料细胞毒性的要求。细胞粘附结果：扫描电镜2500倍视野下可见：单纯微弧氧化组ST2细胞平铺于多孔钛种植体表面,细胞呈长梭形或多边形,伪足扁平,铺于表面；酸蚀+微弧氧化试样组：细胞多呈长梭形,细胞体位于酸蚀的一级空洞内,两端伪足呈扁平样附着于微弧氧化形成的微型空洞内；喷砂+微弧氧化试样组：细胞多呈不规则多边形,平铺于处理后的粗糙表面,伪足和触角突出附着在微弧氧化形成的二级空洞内；喷砂+酸蚀+微弧氧化试样组：可见细胞呈不规则形,铺展良好,胞体嵌入喷砂+酸蚀形成的一级空洞内,多个细胞突起深入微弧氧化形成的类骨小梁样结构内,牢固附着。细胞增殖结果：根据ST2接种到试样表面1、3、5、7天试样表面的细胞数量变化差异可了解试样对细胞增殖功能的影响。实验结果可见：随时间的增加,各组细胞计数均增高；同一时间点,微弧氧化组、喷砂+微弧氧化组、酸蚀+微弧氧化组及喷砂+酸蚀+微弧氧化组OD呈现逐渐升高趋势,除喷砂+微弧氧化组、酸蚀+微弧氧化组外,组间差异显著(P<0.05)。细胞分化结果：培养3天后,微弧氧化组、喷砂+微弧氧化组、酸蚀+微弧氧化组ALP活性差异无显著性(P>0.05),喷砂+酸蚀+微弧氧化组细胞ALP活性升高,组间比较差异显著(P<0.05)。培养5天后,微弧氧化组、喷砂+微弧氧化组、酸蚀+微弧氧化组ALP活性差异无显著性(P>0.05),喷砂+酸蚀+微弧氧化组细胞数量组ALP活性升高,组间比较差异显著(P<0.05)。培养7天后,趋势同前。第三部分：不同表面处理纯钛试样的动物体内生物学评价影像学检查：X线片检查结果显示：种植体植入方向垂直于股骨表面,种植体两端侧穿骨壁。同时,植入区域未发生骨折等异常现象。顶出实验：在4周、8周测试时间点,骨-种植体界面抗剪切强度表现为D组>B组>C组>A组,D组骨-种植体界面抗剪切强度最高,较其它各组间差异有统计学意义(P<0.05),试验组中B组与C组差异无统计学意义(P>0.05),A组骨-种植体界面抗剪切强度最低,较其它各组间差异有统计学意义(P<0.05)组织学检查：硬组织切片实验发现,酸蚀+微弧氧化组与组喷砂+微弧氧化组)钛种植体周围骨结合情况相近,两组的BIC分别为58.74±6.57%、51.09±6.39%,两组之间无明显区别。单纯微弧氧化组中BIC明显降低,为30.13±6.14%,与之相反,喷砂+酸蚀+微弧氧化组BIC明显升高,为77.49±4.96%。结论1.喷砂+酸蚀+微弧氧化处理的纯钛表面比其他三组具有最大的粗糙度,最高的膜层结合强度和相对最好的浸润性,推测其具有最高的生物活性。2.喷砂+酸蚀+微弧氧化处理的纯钛表面的生物相容性明显优于其他三组。3.喷砂+酸蚀+微弧氧化处理的纯钛表面种植体的骨结合速度及成骨能力明显优于其他三组。更多还原

【Abstract】 The biological material is a multidisciplinary approach research field and is also a key technology among the national economic and social development,and has already become a hot topic of research at home and abroad. As a typical representative of the biological materials, dental implant materials have become the clinical and scientific research hotspot at present. Implant surface treatment methods plays a vital role in improving the success rate of implanting. At present, there are so many methods of the implant surface treatment among kinds of products in the market, different processing methods have their respective advantages and disadvantages, but their common goal is to form a rough surface, making it a better bone combination ability and biological activity.The implant surface feature is one of the main factors which can directly affect the rate of bone integration process, and plays a significance clinical value in improving the success rate of dental implanting.Scholars have been trying to find the best way of the implant surface treatment for many years in order to achieving the best implant-bone interface.In the early time, the mechanical surface treatment of titanium implants has many shortcomings, such as:hard to form the chemical combination with the tissue in the early time, slow to achieve the bone combination, long healing period and so on. The hotspot of dental implant surface treatment method is to use different process to change the physical and chemical properties of the implant surface, promoting the early implant-bone combination, so as to ensuring the long-term function of implants.Pure titanium implant surface composite sandblasting acid corrosion of micro-arc oxidation is to realizing the best biological effect of the coating surface by changing the process condition and the design, adjusting the structure of the oxide film, element composition and the biological characteristics. The technology also has the characteristics of simple process, ecologically,and is worth for further demonstration and usage in clinical.This paper intends to conduct the research in the following respects: Materials and methods:PART I:The preparation and characterization of MAO combined with sand-blasting acid-etching surface treatment pure Ti implant.Sand-blasting:The samples used a certain granularity Al2O3grinding grain and jet to the prepared TA2pure Ti specimen surface with a high speed. The main process parameters that can affect the sand-blasting pure Ti substrate surface property:Particle size is200microns, gas pressure is0.9bar, gas flow velocity of210m/min, jet angle is70°, nozzle distance of15cm.Acid-etching:With a mixture of HF and HCI hydrochloride acid solution to corrode TA2pure Ti substrate surface, the main process parameters that can affect the acid-etchingpure Ti substrate surface property:Hydrofluoric acid concentration is40%, hydrochloric acid concentration was38%, the mixture of HCl and HF ratio of6:4, corrosion solution volume of40ml, pickling temperature is25℃, acid etching time was100min.MAO:MAO is a method that changing the working area by introducing the ordinary Faraday area of anodic oxidation process into the high pressure discharge area and creating oxide ceramic membrane in aluminum,magnesium and titanium metal surface with the use of the high working voltage.The electrode voltage is500V, electrode frequency is550HZ, oxidation temperature is less than40℃, oxidation time is18min,(CH3COO)2Ca·H2O concentration is30mmol/L, C3H7Na2O6P·5H2O is10mmol/L, NaOH is8mmo/L.Due to the formation of MAO is a complex process, influenced by various factors and regulations,this early experiments have already optimized a set of process parameters, combined sand-blasting and acid-etching on the basis of this process parameters.Ⅱ. Samples were divided into4groups according to the surface treatment: group A:MAO treatment; group B:MAO sand-blasting treatment; group C:MAO acid-etching treatment; group D:MAO sand-blasting and acid-etching treatment, characterized and analized each group separately from the surface morphology, surface energy spectrum analysis, membrane thickness, surface roughness, film bonding strength and so on.PART II The effects of MAO sand-blasting acid-etching corrosion of pureTi surface treatment on osteoblast biology behaviorMachining the TA2pure Ti cylindrical specimens with CNC wire-cutting machine.Processing of height3.0mm, base diameter of8.0mm. Processing of height3.0mm, base diameter of8.0mm. Burnishing the sample step by step with200-1000#metallographic sand paper, and then placing it into the60～80℃oxide sodium solution to deal with the oil after polishing, the soak time is5min. Firstly we cleaning the sample with the deionized water and soaking it into hydrochloric acid and nitric acid solution for10-15s, adequately removed the old oxidation layer, and then dry it using deionized water at the room temperature. Samples were divided into4groups according to the surface treatment:group A:MAO treatment; group B:MAO sand-blasting treatment; group C:MAO acid-etching treatment; group D:MAO sand-blasting and acid-etching treatment.The four groups cultivated with ST2cell lined respectively, and then analysised the cell proliferation, adhesion, differentiation through SEM observation, cell toxicity detection, ALP activity, osteocalcin content detection,comparative analysis the biological compatibility of four groups of different surface treatment pure Ti implant.Part Ⅲ The rabbit femoral implant experiment of MAO sand-blasting acid-etching pureTiMachining the TA2pure Ti cylindrical specimens with CNC wire-cutting machine.Processing of height3.0mm, base diameter of8.0mm. Burnishing the sample step by with200～1000#metallographic sand paper, Burnishing the sample step by step with200～1000#metallographic sand paper, and then placing it in the60～80℃oxide sodium solution to deal with the oilthe samples after polishing, the soak time is5min.Firstly we cleaning the sample with the deionized water and soaking it into hydrochloric acid and nitric acid solution for1015s, adequately removed the old oxidation layer, and then dry it using deionized water at the room temperature.Samples were divided into4groups according to the surface treatment: group A:MAO treatment; group B:MAO sand-blasting treatment; group C:MAO acid-etching treatment; group D:MAO sand-blasting and acid-etching treatment.We implanted the pure Ti specimen into the rabbit femoral respectively, removed the specimens at the time of healing4w and8w.Then calculated the bone mass and quality around the implant and evaluated implant-bone bonding strength after MAO sand-blasting acid-etching treatment by push-out test, bone and implant contact rate, researched bone features from the organizations form metrology.Results:Part Ⅰ:The Surface Roughness:pure Ti substrate surface roughness Ra is0.2μm. And after the processing surface of MAO process, the Ra into0.8μm.The increase of surface roughness after MAO treatment is mainly for the appearance of the micro-arc oxide film holes.The roughness Ra of MAO sand-blasting pure Ti substrate micro-arc oxidation film is1.4μm, slightly larger than the sample MAO only. And the roughness Ra of MAOacid-etching pure Ti substrate micro-arc oxidation film is1.4μm, better than MAO sample.But it reached2.2μm after MAO treatment combined the sand-blasting and acid-etching.It shows that the MAO film roughness of sand-blasting and acid-ecthing has significantly increased, which are apparently manifested on the emergence of more uniform holes in oxide film.Membrane Layer Thickness:MAO film thickness is37.2μm;MAO sand-blasting film is28.3μm; MAO acid-ecthing film thickness can reach40.2μm; MAO sand-blasting acid-etching thickness is43.1μm,the thickest of all. It made difference of the thickness of the oxide film because of the different pretreatment.Bonding Strength:MAO film load is20.7N;MAO sand-blasting film is23.2N; MAO acid-ecthing film thickness can reach27.9N; MAO sand-blasting acid-etching thickness is31.2N. It made difference of the thickness of the oxide film because of the different pretreatment.Contact Angle:The test result is as follows:MAO contact angle is82.9°;MAO sand-blasting contact angle is76.7°; MAO acid-ecthing is69.3°; And MAO sand-blasting acid-etching is63.2°.Part II:Cytotoxicity test:According to the OD determined by MTT calculating the relative generation rate (RGR),RGR%=Dexperimental group/ODnegative control group X100%.We classificated the cytotoxicity according to the different period RGR of each group(CTS).The experiment result showed that the implant leaching solution of modified four groups have no significant difference when compared with negative control group(P>0.05).The CTS classification are level O.It is showed that the pure Ti implant of this experiment is non-toxic, and is conformed to the 《Requirements Of The Medical Equipment Biological Assement National Standard GB/T16886.1-2001》to the cytotoxicity of medical equipment.The results of cell adhesion:SEM (2500x):MAO group ST2cells are long fusiform or polygon, the pseudopodia spreaded to the porous titanium implant surface; Cells in MAO acid-etching group presented a long spindle appearence, the cyton lies in the primary hollow of acid corrosion,and the pseudopodia attached to the micro hole in planus; In MAO sand-blasting group,cells lied in the rough surface in irregular polygon shape, pseudopodia and antenna attached to the secondary micro hole. MAO sand-blasting acid-etching group:Cells showed irregular shape, the cyton embedded into the primary hollow with good extensibility.Multiple celluar processes swelled into the bone trabecular structure with strong adhesion.We can know the effects samples made to the cell proliferation function according to the cells quantity change at1,3,5, and7days which inoculation to the sample surface. Results:As time went by, OD increased in the whole four groups;At the same time, OD in the four groups MAO、MAO sand-blasting、MAO acid-etching and MAO sand-blasting acid-etching increased gradually.Except for Group B and C, there shows significant difference between groups(P<0.05).Cell differentiation results:After three days, the difference of ALP activity in Group A、B、C is not abviously(P>0.05),but it shows significant difference between groups(P<0.05) because the ALP activity in Group D increased than before.The tendency was same as before after five days and seven days. Part Ⅲ:Iconography examination:Imaging examination:X-ray examination results showed that the implantation direction was perpendicular to the surface of the femur, and the implant impaled both side of the bone wall.And there is no abnormal phenomenon such as fracture in the area.Put out test:In4weeks and8weeks,the bone-implant interface shear strengthen showed group D> group B> group C> group A, group D is the highest and otherwise group D is the lowest, both showed statistical significance than other groups(B and C)(P<0.05).Histological examination:The hard tissue biopsies found that the bone around the implant have the similar situation in group B and C, BIC were58.74±6.57%、51.09±6.39%.On the contrary, group A declined and group D increased obviously:30.13±6.14%and77.49±4.96%.Conclusion:l.The surface of MAO sand-blasting acid-etching pure Ti have the largest roughness, the highest film bond strength and the relatively best invasive than the other three groups, so we speculates that it has the highest biological activity.2.The biocompatibility of the surface of MAO sand-blasting acid-etching pure Ti is superior to the other three groups.3.The bone combineation speed and osteogenesis ability of MAO sand-blasting acid-etching pure Ti implant is better than the other three groups.更多还原