纳米Ag/TiO_2涂层托槽的研制及其抗菌性能研究
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摘要
在口腔正畸临床领域,固定矫治器作为一种高效能矫治器得到广泛应用,并取得良好疗效。但其主体成分托槽结构复杂,导致菌斑附着增加并影响口腔微环境,从而引起牙釉质脱矿及牙周损害。预防并控制菌斑附着是预防龋病及牙周炎症的关键,如何对托槽材料进行改进,抑制口腔致病菌的黏附与增生是目前口腔正畸学领域研究的方向之一。随着口腔材料学的发展,纳米抗菌材料的出现为开发新型托槽材料提供了新选择。
绝大多数纳米抗菌材料使用的是单一纳米抗菌剂,如纳米TiO2、纳米银等,这些材料自身具有一定的缺陷,限制了它们的应用。以纳米TiO2为载体的复合银系无机抗菌剂成为新型抗菌材料研究的一个重要方向,具有独特的力学性能、抗菌性能及生物学相容性,应用前景非常广阔。
目前国内外尚无将纳米Ag/TiO2材料与技术应用于口腔正畸托槽的报导,本研究拟在普通金属托槽表面制备纳米Ag/TiO2涂层,研制具有美观、高强、耐磨及可见光催化甚至在黑暗环境中仍具有抑菌性能的纳米Ag/TiO2涂层托槽,通过理化性能、抗菌性能和生物相容性检测,证实托槽具有纳米材料独特的力学及生物学性能,为解决托槽周边牙釉质龋和正畸治疗引起的牙周损伤等正畸临床常见棘手问题提供有效途径,并推进纳米技术在口腔正畸材料领域的广泛应用。本研究包括以下三章内容：
第一章纳米Ag/TiO2涂层托槽的制备及理化性能检测
目的：
制备纳米Ag/TiO2涂层托槽,并对其理化性能进行检测。
方法：
本研究选择临床上常用的普通金属托槽,抛光、超声清洗、冲洗干燥后备用。使用溶胶-凝胶法制备TiO2凝胶,用旋转涂敷法在普通金属托槽表面涂敷TiO2薄膜,然后浸渍在AgNO3溶液中,获得Ag/TiO2薄膜,经过120℃热处理后分别在120、200、300℃下退火,冷却,制备出不同退火温度下纳米Ag/TiO2涂层托槽A、B、C组。
在扫描电镜下对普通金属托槽、纳米TiO2涂层托槽和纳米Ag/TiO2涂层托槽的表面形貌、微观结构及A组纳米Ag/TiO2涂层托槽的断面形貌进行观察。
测量普通金属托槽、纳米TiO2涂层托槽及A、B、C组纳米Ag/TiO2涂层托槽的表面粗糙度。
采用划痕实验法检测纳米TiO2涂层和A、B、C组纳米Ag/TiO2涂层与基体托槽的结合强度。
用X射线衍射仪分析纳米TiO2涂层托槽及A、B、C组纳米Ag/TiO2涂层托槽表面涂层的晶体结构和物相。
对纳米TiO2涂层托槽及A组纳米Ag/TiO2涂层托槽进行X射线光电子能谱分析。
采用紫外—可见分光光度计测量石英玻璃片上纳米TiO2涂层及A、B、C组纳米Ag/TiO2涂层的透光率。
结果：
扫描电镜图片显示金属托槽表面虽经抛光,但仍较粗糙,存在较大的沟壑状和坑状凹陷。纳米TiO2涂层是由许多球形微小颗粒组成的颗粒膜,组成薄膜的Ti02颗粒粒径为10-30nm。纳米Ag/TiO2涂层也是由许多球形微小颗粒组成的颗粒膜,具有严整的组织结构；组成薄膜的Ti02颗粒粒径均匀；Ag颗粒均匀分布在薄膜上,粒径约为50-100nnm,很少出现团聚现象。A、B、C组托槽中纳米Ag颗粒的直径随着退火温度的升高逐渐增大且分布更密集。
对纳米Ag/TiO2涂层托槽的断面形貌观察显示纳米Ag/TiO2涂层托槽表面涂层的厚度约为120nm,厚度均匀,表面平整、光滑,表面光洁度高,并可见Ag颗粒沉积在涂层上。
粗糙度测试结果显示纳米TiO2涂层托槽和纳米Ag/TiO2涂层托槽与普通商业用金属托槽表面粗糙度差异无统计学意义。纳米TiO2涂层与基体的结合强度为1.18kg, A、B、C组纳米Ag/TiO2涂层的结合强度分别为1.16、1.12、1.26kg。
XRD谱图显示纳米TiO2和Ag/TiO2涂层的XRD曲线上均出现锐钛矿型TiO2的衍射峰；纳米Ag/TiO2涂层的XRD曲线上均出现Ag0的衍射峰,且随着退火温度的升高,Ag衍射峰的强度也增大。
XPS全谱图显示纳米TiO2和Ag/TiO2涂层的主要成分分别为：Ti、O、C和Ag、 Ti、O、C; TiO2中的Ti2p能级分解为2个能级：Ti2p1/2和Ti2p3/2,中心峰值分别为464.6ev和458.9ev；2者峰值差为5.7ev；加入Ag后,Ti2p的结合能峰值向较高能值漂移；Ag/TiO2中的Ag3d能级分解为2个能级：Ag3d5/2和Ag3d3/2,其中心峰值分别为368.1ev和374.1ev；2者峰值差为6ev。
纳米TiO2涂层及A、B、C组纳米Ag/TiO2涂层的透光率谱图显示TiO2涂层在波长<350nm的紫外光区有完全吸收；Ag沉积后,吸收边发生红移,透光率下降,且随着退火温度的降低,透光率也逐渐下降。
结论：
1.本研究采用溶胶-凝胶法在普通金属托槽表面成功地制备了纳米Ag/TiO2薄膜涂层。
2.该纳米Ag/TiO2涂层分布均匀,厚度和纳米Ag、TiO2颗粒均为纳米尺度,保证了纳米材料的特性；且表面光洁度高,具有足够的涂层基体结合强度,可以满足口腔正畸临床的需要。
3.纳米Ag/TiO2涂层中TiO2为锐钛矿型,Ag以单质形式存在,随着退火温度的升高,Ag颗粒的结晶度提高且颗粒长大。
4.Ag的沉积增加了TiO2的光催化活性,并使其具有可见光催化性能。
5.随着Ag沉积颗粒粒径的减小,TiO2在可见光激发条件下的光催化活性增强。
6.退火温度120℃较为合适,制成的纳米Ag/TiO2涂层中Ag和TiO2颗粒粒径较小,既具备足够的结合强度又有较强的可见光催化活性。
第二章纳米Ag/TiO2涂层托槽的抗菌性能研究
目的：
研究纳米Ag/TiO2涂层托槽的抗菌性能,初步探讨抗菌机制,为临床应用提供理论依据。
方法：
制备A组纳米Ag/TiO2涂层托槽,对照组为普通金属托槽和同条件下纳米TiO2涂层托槽,消毒后备用。使用同样方法在不锈钢17-4表面涂敷纳米TiO2薄膜涂层和Ag/TiO2薄膜涂层。复苏变形链球菌、血链球菌、伴放线放线杆菌、具核梭杆菌、牙龈卟啉单胞菌和中间型普氏菌,培养并制备成菌悬液。使用贴膜法检测纳米Ag/TiO2涂层对上述口腔常见菌的抗菌活性,计算抗菌率并在不同时间点观察灭菌效果。
将普通金属托槽、纳米TiO2涂层托槽和纳米Ag/TiO2涂层托槽浸泡于混合菌液中,培养48h后在扫描电镜下观察抗细菌黏附效果。
将变形链球菌与纳米Ag/TiO2涂层托槽共同培养6h、18h、24h、48h,提取GTF粗酶,采用Somogyi-Nelson法测定上清液中还原糖含量,计算GTF活性单位,分析不同时间点纳米Ag/TiO2涂层托槽对变形链球菌GTF活性的影响。
向纳米Ag/TiO2涂层上滴加牙龈卟啉单胞菌菌悬液,分不同抗菌时间测量胞内外牙龈素的活性单位与蛋白含量,计算牙龈素比活,分析纳米Ag/TiO2涂层托槽对胞内外牙龈素比活的影响。
结果：
抗菌实验结果显示纳米Ag/TiO2涂层在黑暗环境下对所有实验菌均有抗菌活性,20min后抗菌率达到了80.2%以上。黑暗环境下纳米Ag/TiO2涂层作用30-60min后即可完全杀死菌液中的口腔常见细菌,而纳米TiO2涂层作用240min后也未显示出抗菌活性。纳米Ag/TiO2涂层对不同菌种的杀菌时间存在差异,对变形链球菌、血链球菌及牙龈卟啉单胞菌的杀灭时间最短,为20-30min；对伴放线放线杆菌、具核梭杆菌及中间型普氏菌的杀灭时间延长为30-60min。
扫描电镜照片显示纳米Ag/TiO2涂层托槽表面黏附的细菌数量明显少于普通金属托槽及纳米TiO2涂层托槽。普通金属托槽表面有大量的细菌黏附,呈致密片状分布,形成多层菌膜；纳米TiO2涂层托槽表面也见大量的细菌黏附,呈颗粒状,但未见多层菌膜形成；而纳米Ag/TiO2涂层托槽表面黏附的细菌明显少于前2者,呈散在分布。
在各时间点,普通托槽组、纳米TiO2涂层托槽组和纳米Ag/TiO2涂层托槽组GTF活性依次下降；纳米Ag/TiO2涂层托槽组GTF活性比其余3组明显降低；除48h时间点外,TiO2托槽组GTF活性比空白对照组下降,差异有统计学意义；普通托槽组GTF活性与空白对照组差异无统计学意义。Ag/TiO2托槽对GTF活性的抑制作用强弱顺序为：18h>24h>6h>48h,除18与24h时间点外,其余时间点两两比较Ag/TiO2托槽对GTF活性的抑制作用差异均有统计学意义。
在纳米Ag/TiO2涂层作用下,牙龈卟啉单胞菌胞内外牙龈素比活随抗菌时间推移均呈下降趋势,10min内细菌胞内牙龈素比活明显大于胞外,10min后两者无明显差异；胞外牙龈素比活水平在20min内基本持平,之后随时间延长呈缓慢下降趋势；胞内牙龈素比活20min内出现明显下降,之后下降趋势减慢。
结论：
1.纳米Ag/TiO2涂层托槽在黑暗环境下对口腔常见细菌有较强的抗菌作用。
2.纳米Ag/TiO2涂层托槽对口腔常见细菌有较好的抗黏附作用。
3.纳米Ag/TiO2涂层托槽能有效抑制变形链球菌葡糖基转移酶的活性,从而对变形链球菌有良好抗黏附作用。
4.纳米Ag/TiO2涂层托槽在杀死牙龈卟啉单胞菌的同时还可降解细菌胞内外的牙龈素,显著抑制其活性。
第三章纳米Ag/TiO2涂层托槽的生物相容性评价
目的：
对纳米Ag/TiO2涂层托槽进行生物相容性测试,为其在临床的安全使用提供实验依据。
方法：
在不锈钢17-4圆片表面涂敷纳米TiO2薄膜涂层和Ag/TiO2薄膜涂层,加入DMEM培养液制取浸提液,取复苏后的L929细胞与其共同培养,在倒置相差显微镜下观察细胞形态；使用MTT法计算细胞增殖度并进行材料毒性程度分级；用碘化丙啶染色细胞后荧光显微镜下观测细胞死亡情况；用流式细胞仪检测分析细胞周期,进行细胞毒性试验。
将纳米TiO2薄膜涂层和Ag/TiO2薄膜涂层试样用生理盐水浸泡提取浸提液,加入新鲜稀释抗凝兔血,测量吸光度值,计算材料溶血率,进行溶血试验。
将纳米TiO2薄膜涂层和Ag/TiO2薄膜涂层试样用生理盐水浸泡提取浸提液,对小鼠灌胃,观察动物行为是否有毒性体征出现；对试验前后体重进行测量,观察体重变化；处死动物,进行病理解剖,肉眼观察动物重要脏器有无病理变化；在显微镜下观察小鼠心、肺、脾、肺、肾有无病理改变,进行短期全身毒性试验。
取涂敷纳米Ag/TiO2薄膜涂层的不锈钢17-4圆片和同样规格的口腔用牙胶圆片,缝合固定到金黄色仓地鼠的颊囊黏膜上,观察动物的活动情况,有无全身不良反应；观察试件与口腔内黏膜接触处的局部黏膜反应；处死动物,对颊囊部全层组织进行病理组织学观察,并进行病理反应分级,进行口腔黏膜刺激试验。
结果：
细胞毒性试验形态学观察结果显示试验组细胞形态数量与对照组无明显区别,可见大量分裂细胞；荧光显微镜下见只有极少量细胞被PI染成红色,可能为正常凋亡细胞,各组未见明显差异；MTT试验结果显示各组细胞增殖度较为接近,毒性等级均为0-1级；细胞周期分析未见异常,各组之间无明显差异。
溶血试验中试验组的吸光度值明显低于阳性对照组,TiO2组和Ag/TiO2组的溶血率分别为0.255%、0.637%,均小于5%,即均不会引起急性溶血。
短期全身毒性试验中试验组和对照组60只小鼠无一例死亡,临床毒性体征分级都是无症状；试验组小鼠体重增长与对照组比较差异均无统计学意义；病理解剖肉眼观察动物重要脏器未见充血、出血、水肿等明显病理变化；显微镜下观察小鼠心、肺、脾、肺、肾未出现变性、萎缩、坏死等病理变化。
口腔黏膜刺激试验中动物活动正常,饮食正常；与试验材料和对照材料接触的颊囊黏膜表面及其周围未见充血、水肿、糜烂及溃疡等反应；试验组与阴性对照组接触处颊囊黏膜组织切片基本相同,未见异常变化,病理反应评分除1侧为轻度外,其余均为0级。
结论：
1.纳米Ag/TiO2涂层托槽对细胞不产生明显毒性。
2.纳米Ag/TiO2涂层托槽不会引起急性溶血反应。3.纳米Ag/TiO2涂层托槽浸提液对小鼠产生的影响与生理盐水无差异,没有短期全身毒性。
4.纳米Ag/TiO2涂层托槽对仓地鼠口腔黏膜没有刺激性,具有良好的生物安全性。
综上所述,本研究成功研制出纳米Ag/TiO2涂层托槽,具有良好的机械性能；对口腔常见细菌具有优异的抗菌性能和抗黏附作用；对机体无毒性,生物相容性良好,可以满足正畸临床需要,但还需进一步深入研究之后才能应用于临床。
In orthodontic clinic, fixed appliance has been widely used for its efficiency and gained fine therapeutic effect. But the structure of its main composition-brackets is complex, leading to dental plaque increasing and having impact on micro-circumstance in oral cavity, so enamel decalcification, gingivitis and periodontitis were caused. Prevention and control of dental plaque adhesion is the key of dental caries and periodontitis prevention. Improve the material of brackets so to inhibit adhension and growth of pathogenic bacterium in oral cavity is one of the investigative directions in orthodontics sphere. Along with the development of the science of dental materials, the appearance of nanometer antibiosis materials provids new choice for new bracket materials exploitation.
Most nanometer antibiosis materials utilize single nanometer antibacterial agent such as nanometer titanium dioxide, nanometer sliver and so on. These materials have defect of their own, so the application are constrainted. Silver-loaded titanium dioxide becomes an important direction of new antibiosis materials investigation. It has specific mechanical property, antibiosis function and biocompatibility, so it has broad prospect of application.
There is no report of using nanometer Ag/TiO2materials and technique in orthodontics brackets at home and abroad. This study plans to make nanometer Ag/TiO2coating on ordinary metal brackets so to develop nanometer Ag/TiO2coating on brackets that has fine appearance, high intensity, visible photocatalytic activity and bacteriostasis effect even in dark circumstances. Detect the efficiency of physics, chemistry, antibiosis and biocompatibility of the brackets so as to confirm the specific mechanical and biological properties of the brackets. Our aim is to provide effective method for solving adamantine caries and periodontal inflammation, and to promote the use of nanometer technology in orthodontic materials area. This study consists of three chapters below:
Part One Development of nanometer Ag/TiO2coated brackets and physicochemical properties detection
Objective
To develop nanometer Ag/TiO2coated bracket and detect its physicochemical properties.
Methods
In this study, we chose ordinary metal brackets widely used in clinic, polish, ultrasonic clean, flush and dry. TiO2gel was produced by sol-gel method. TiO2film was coated on brackets surface, and then soaked in AgNO3solution. Ag/TiO2film was heat treated under120degree, then annealing under120,200,300degree respectively, then nanometer Ag/TiO2coated bracket A,B,C were produced.
Scanning electron microscope (SEM) was used to observe the micromorphology of surface of ordinary metal brackets, nanometer TiO2coated bracket and nanometer Ag/TiO2coated bracket. Cross-section topography of nanometer Ag/TiO2coated bracket group A was also observed.
The surface roughness of metal bracket, nanometer TiO2coated bracket and nanometer Ag/TiO2coated bracket were measured.
The combination intensity between smear layer of nanometer TiO2and nanometer Ag/TiO2with basis material were analyzed by scratch experimentation.
The crystal structure and phase of nanometer TiO2coated bracket and nanometer Ag/TiO2coated bracket were analyzed by XRD.
The chemical components of nanometer TiO2coated bracket and nanometer Ag/TiO2coated bracket were characterized using XPS.
The transmittance of nanometer TiO2coating and nanometer Ag/TiO2coating on quartz glass were mearsured by UV-VIS.
Results
SEM results showed that the surface of ordinary metal bracket was rough and existenced major ravine andpitlike depression. Nanometer TiO2coating film was constructed by many sphericity micro-particles, the diameter of which was10-30nm. Nanometer Ag/TiO2film was also constructed by many sphericity micro-particles, with orderly structure, the diameter of TiO2particles were uniformity, Ag particles were distributed on the film, the diameter of which was50-100nm. Aggregation phenomenon was seen seldomly. The diameter of Ag particles became bigger and distributed more intensively with the annealing temperature arising.
The section micromorphology showed that the thickness of nanometer Ag/TiO2film was about120nm, the film was well-distributed and had smooth surface, Ag particles could be seen deposited on the film.
There were no statistically differences among the surface roughness of five groups of brackets. The combination intensity of the nanometer TiO2smear layer with basis material was1.18kg, the numerical value of A,B,C groups of nanometer Ag/TiO2were1.16,1.12,1.26kg respectively.
XRD showed that there were anatase TiO2diffraction peak in both curves of TiO2and Ag/TiO2films. Ag0diffraction peak appeared in the curve of Ag/TiO2, and the intensity became larger as the temperature arising.
XPS showed main components of TiO2and Ag/TiO2were Ti, O,C and Ag,Ti,O,C respectively. The Ti2p level in TiO2was divided into2levels:Ti2p1/2and Ti2p3/2, the central peak were464.6ev and458.9ev, the difference was5.7ev. The peak value of binding energy of Ti2p drifted to high energy after Ag deposoted. The Ag3d level in Ag/TiO2was divided into2levels:Ag3d5/2and Ag3d3/2, the central peak were368.1ev and374.1ev, the difference was6ev.
The TiO2transmittancy spectrogram showed ultraviolet light<350nm could be completely absorbted, red shift of the absorption happened and transmittancy decreased after Ag deposited. The transmittancy decreased as the annealing temperature decreasing.
Conclusion
1. The results confirmed that nanometer Ag/TiO2coated brackets were developed successfully by sol-gel method.
2. This nanometer Ag/TiO2coating was well-distributed, the thickness and diameter of particles were all in nanometer scale, so that the characteristics of nanometer materials were guaranteed. It had smooth surface and enough combination intensity with basis material, which could meet the clinical demand of orthodontics.
3. TiO2in the film was anatase TiO2. Ag existed by the form of simple substance. Ag particles grew up as the annealing temperature increasing.
4. The photochemical catalytic activity of TiO2increased by the deposition of Ag, and made it owned visible light photoresponse.
5. The visible light photoresponse increased by the decrease of diameter of Ag.
6. The result indicated that choose120degree as annealing temperature is suitable, for the diameter of Ag and TiO2were small, which owned enough combination intensity and strong visible light photoresponse.
Part Two
Research on bactericidal ability of nanometer Ag/TiO2coated brackets
Objective
To study the bactericidal ability of nanometer Ag/TiO2coated bracket and investigate the mechanism initially, in order to provide theoretical evidence.
Methods
A group nanometer Ag/TiO2bracket was prouduced and TiO2and Ag/TiO2coatings were coated on stainless steel17-4with the same method. S.mutans, S.sangui, A.actinomycetemcomitans, F.nucleatum, P.gingivalis and P.intermedia were anabiosised and cultured into bacteria liquid. Film applicator coating method was used to test the antibacterial efficacy of Ag/TiO2coating.
3groups of brackets were soaked in the mixed bacteria liquid. After48h, SEM was used to observe the anti-adhesive efficacy of the coating.
Cocultured S.mutans with Ag/TiO2coated bracket for6,18,24,48h, then extracted coarse GTF. The content of reducing sugar in supernatant was tested by Somogyi-Nelson method, and activity of GTF was calculated in order to analyze the influence of Ag/TiO2coating on GTF in different times.
The activity of gingpains ecto-cellular and intra-cellular and protein level were measured and specific activity of gingpains was calculated in order to analyze the effect of Ag/TiO2coating on gingpains.
Results
The result showed more than80.2%of bacteria were killed within20minutes in dark circumstance which confirm fine antibacterial effect of Ag/TiO2coating. The coating could kill all the bacteria after30-60min, while TiO2coating did not exhibit antibacterial effect after240min. There were differences among the time of killing different strains.
SEM showed the bacterial number adhered on the surface of nanometer Ag/TiO2coated bracket was obviously fewer than on the surface of metal bracket and nanometer TiO2coated bracket. There were considerable bacteria adhere to the surface of metal bracket, distributed compactly and formed multilayer pellicle. There were also massive bacteria adhere to the surface of nanometer TiO2coated bracket, in grainy structure, no multilayer pellicle was seen. There were obvious slight bacteria adhere to the surface of nanometer Ag/TiO2coated bracket, scattered distributed.
In different times, GTF activity of metal bracket group, nanometer TiO2coated bracket group and nanometer Ag/TiO2coated bracket group descended by turns, GTF activity in nanometer Ag/TiO2coated bracket group was obviously lower than3orther groups, GTF activity in nanometer TiO2coated bracket group was lower than blank control group besides at48h, there were statistically differences among these groups. There were no statistically differences between GTF activity of metal bracket group and nanometer TiO2coated bracket group. The sequence of inhibition of nanometer Ag/TiO2coated bracket on GTF activity were:18h>24h>6h>48h. There were statistically differences between the inhibition in different times besides18h and24h.
The specific activity of gingpains ecto-cellular and intra-cellular both had downtrend as the time lasting. The specific activity of gingpains intra-cellular was obviously bigger than ecto-cellular within10min, the difference became small after10min. The specific activity of gingpains ecto-cellular did not change much within20min, then assumed slow downtrend. The specific activity of gingpains intra-cellular descented obviously within20min, then steped down downtrend.
Conclusion
1. The results confirmed the outstanding antibacterial property of nanometer Ag/TiO2coated bracket in dark circumstance.
2. Nanometer Ag/TiO2coated bracket had fine adherence resistance to common bacteria in oral cavity.
3. The bracket could effectively restrain the activity of GTF so that it has excellent adherence resistance to S.mutans.
4. The bracket could lead to gingpains degradation ecto-cellular and intra-cellular while killing P.gingivalis.
Part Three
Biocompatibility evaluation of nanometer Ag/TiO2coated bracket
Objective
Biocompatibility of nanometer Ag/TiO2coated bracket was examined in order to provide experimental evidence for its use in clinic.
Methods
TiO2and Ag/TiO2coatings were spreaded on the surface of stainless steel wafer. Add DEME to the wafers, and cocultured with L929cell. The morphous of cell were observed under inverted phase contrast microscope. Relative growth rate was calculated by MTT method and carried out toxity degree grading. The death of cell was observed under fluorescene microscope. Cell cycle was detected by flow cytometry.
Leaching liquor was extracted from TiO2and Ag/TiO2coatings and added to fresh anticoagulation cony blood, optical density was measured and hemolysis rate was calculated.
Lavaging mouse with leaching liquor extracted from TiO2and Ag/TiO2coatings, observing toxicity physical sign, measuring body weigh change before and after experiment, and then observe pathological change of organs heart, hepar, lien, lung and kidney.
Sutured the wafer to the cheek pouch of inaurate hamster, observed the activity of animals and mucous membrane reaction in the contact region, then observe the full-thickness of cheek pouch under microscope, and carry out pathology reaction grading.
Results
There were no differences among3groups of cells under inverted phase contrast microscope and fluorescene microscope.Toxicity levels of3groups were0-1. There were no abnormality seen in cell cycle.
Optical density in test group was obviously lower than positive control group. The hemolysis rate of TiO2group and Ag/TiO2group were0.255%and0.637%. They were both less than5%, which meaned no acute haemolysis was induced.
There was no death in all the animals, clinical toxicity physical sign were all asymptomatic, there was no difference between test group and control group in body weigh increasing, the organs were observed under microscope and no pathological change was found.
The animals acted and took food normally, no pathological change was observed in the contact mucous membrane of cheek pouch both in macroscopic and microscope observation.
Conclusion
1. Nanometer Ag/TiO2coated bracket had no toxicity for cell.
2. No acute haemolysis was induced by nanometer Ag/TiO2coated bracket.
3. The influence of leaching liquor extracted from Ag/TiO2coating on mouse had no difference with sodium chloride and no short-term systemic toxicity was observed.
4. Nanometer Ag/TiO2coated bracket had no stimulation to oral mucosa of hamster, so it had fine biological properties.
Above all, nanometer Ag/TiO2coated bracket was developed successfully, which could meet the clinical demand for it has excellent mechanical function, owns outstanding bactericidal ability and adhesion resistance, and possesses fine biocompatibility. But futher study should be carried out before it be used in orthodontic clinic.
绝大多数纳米抗菌材料使用的是单一纳米抗菌剂,如纳米TiO2、纳米银等,这些材料自身具有一定的缺陷,限制了它们的应用。以纳米TiO2为载体的复合银系无机抗菌剂成为新型抗菌材料研究的一个重要方向,具有独特的力学性能、抗菌性能及生物学相容性,应用前景非常广阔。
目前国内外尚无将纳米Ag/TiO2材料与技术应用于口腔正畸托槽的报导,本研究拟在普通金属托槽表面制备纳米Ag/TiO2涂层,研制具有美观、高强、耐磨及可见光催化甚至在黑暗环境中仍具有抑菌性能的纳米Ag/TiO2涂层托槽,通过理化性能、抗菌性能和生物相容性检测,证实托槽具有纳米材料独特的力学及生物学性能,为解决托槽周边牙釉质龋和正畸治疗引起的牙周损伤等正畸临床常见棘手问题提供有效途径,并推进纳米技术在口腔正畸材料领域的广泛应用。本研究包括以下三章内容：
第一章纳米Ag/TiO2涂层托槽的制备及理化性能检测
目的：
制备纳米Ag/TiO2涂层托槽,并对其理化性能进行检测。
方法：
本研究选择临床上常用的普通金属托槽,抛光、超声清洗、冲洗干燥后备用。使用溶胶-凝胶法制备TiO2凝胶,用旋转涂敷法在普通金属托槽表面涂敷TiO2薄膜,然后浸渍在AgNO3溶液中,获得Ag/TiO2薄膜,经过120℃热处理后分别在120、200、300℃下退火,冷却,制备出不同退火温度下纳米Ag/TiO2涂层托槽A、B、C组。
在扫描电镜下对普通金属托槽、纳米TiO2涂层托槽和纳米Ag/TiO2涂层托槽的表面形貌、微观结构及A组纳米Ag/TiO2涂层托槽的断面形貌进行观察。
测量普通金属托槽、纳米TiO2涂层托槽及A、B、C组纳米Ag/TiO2涂层托槽的表面粗糙度。
采用划痕实验法检测纳米TiO2涂层和A、B、C组纳米Ag/TiO2涂层与基体托槽的结合强度。
用X射线衍射仪分析纳米TiO2涂层托槽及A、B、C组纳米Ag/TiO2涂层托槽表面涂层的晶体结构和物相。
对纳米TiO2涂层托槽及A组纳米Ag/TiO2涂层托槽进行X射线光电子能谱分析。
采用紫外—可见分光光度计测量石英玻璃片上纳米TiO2涂层及A、B、C组纳米Ag/TiO2涂层的透光率。
结果：
扫描电镜图片显示金属托槽表面虽经抛光,但仍较粗糙,存在较大的沟壑状和坑状凹陷。纳米TiO2涂层是由许多球形微小颗粒组成的颗粒膜,组成薄膜的Ti02颗粒粒径为10-30nm。纳米Ag/TiO2涂层也是由许多球形微小颗粒组成的颗粒膜,具有严整的组织结构；组成薄膜的Ti02颗粒粒径均匀；Ag颗粒均匀分布在薄膜上,粒径约为50-100nnm,很少出现团聚现象。A、B、C组托槽中纳米Ag颗粒的直径随着退火温度的升高逐渐增大且分布更密集。
对纳米Ag/TiO2涂层托槽的断面形貌观察显示纳米Ag/TiO2涂层托槽表面涂层的厚度约为120nm,厚度均匀,表面平整、光滑,表面光洁度高,并可见Ag颗粒沉积在涂层上。
粗糙度测试结果显示纳米TiO2涂层托槽和纳米Ag/TiO2涂层托槽与普通商业用金属托槽表面粗糙度差异无统计学意义。纳米TiO2涂层与基体的结合强度为1.18kg, A、B、C组纳米Ag/TiO2涂层的结合强度分别为1.16、1.12、1.26kg。
XRD谱图显示纳米TiO2和Ag/TiO2涂层的XRD曲线上均出现锐钛矿型TiO2的衍射峰；纳米Ag/TiO2涂层的XRD曲线上均出现Ag0的衍射峰,且随着退火温度的升高,Ag衍射峰的强度也增大。
XPS全谱图显示纳米TiO2和Ag/TiO2涂层的主要成分分别为：Ti、O、C和Ag、 Ti、O、C; TiO2中的Ti2p能级分解为2个能级：Ti2p1/2和Ti2p3/2,中心峰值分别为464.6ev和458.9ev；2者峰值差为5.7ev；加入Ag后,Ti2p的结合能峰值向较高能值漂移；Ag/TiO2中的Ag3d能级分解为2个能级：Ag3d5/2和Ag3d3/2,其中心峰值分别为368.1ev和374.1ev；2者峰值差为6ev。
纳米TiO2涂层及A、B、C组纳米Ag/TiO2涂层的透光率谱图显示TiO2涂层在波长<350nm的紫外光区有完全吸收；Ag沉积后,吸收边发生红移,透光率下降,且随着退火温度的降低,透光率也逐渐下降。
结论：
1.本研究采用溶胶-凝胶法在普通金属托槽表面成功地制备了纳米Ag/TiO2薄膜涂层。
2.该纳米Ag/TiO2涂层分布均匀,厚度和纳米Ag、TiO2颗粒均为纳米尺度,保证了纳米材料的特性；且表面光洁度高,具有足够的涂层基体结合强度,可以满足口腔正畸临床的需要。
3.纳米Ag/TiO2涂层中TiO2为锐钛矿型,Ag以单质形式存在,随着退火温度的升高,Ag颗粒的结晶度提高且颗粒长大。
4.Ag的沉积增加了TiO2的光催化活性,并使其具有可见光催化性能。
5.随着Ag沉积颗粒粒径的减小,TiO2在可见光激发条件下的光催化活性增强。
6.退火温度120℃较为合适,制成的纳米Ag/TiO2涂层中Ag和TiO2颗粒粒径较小,既具备足够的结合强度又有较强的可见光催化活性。
第二章纳米Ag/TiO2涂层托槽的抗菌性能研究
目的：
研究纳米Ag/TiO2涂层托槽的抗菌性能,初步探讨抗菌机制,为临床应用提供理论依据。
方法：
制备A组纳米Ag/TiO2涂层托槽,对照组为普通金属托槽和同条件下纳米TiO2涂层托槽,消毒后备用。使用同样方法在不锈钢17-4表面涂敷纳米TiO2薄膜涂层和Ag/TiO2薄膜涂层。复苏变形链球菌、血链球菌、伴放线放线杆菌、具核梭杆菌、牙龈卟啉单胞菌和中间型普氏菌,培养并制备成菌悬液。使用贴膜法检测纳米Ag/TiO2涂层对上述口腔常见菌的抗菌活性,计算抗菌率并在不同时间点观察灭菌效果。
将普通金属托槽、纳米TiO2涂层托槽和纳米Ag/TiO2涂层托槽浸泡于混合菌液中,培养48h后在扫描电镜下观察抗细菌黏附效果。
将变形链球菌与纳米Ag/TiO2涂层托槽共同培养6h、18h、24h、48h,提取GTF粗酶,采用Somogyi-Nelson法测定上清液中还原糖含量,计算GTF活性单位,分析不同时间点纳米Ag/TiO2涂层托槽对变形链球菌GTF活性的影响。
向纳米Ag/TiO2涂层上滴加牙龈卟啉单胞菌菌悬液,分不同抗菌时间测量胞内外牙龈素的活性单位与蛋白含量,计算牙龈素比活,分析纳米Ag/TiO2涂层托槽对胞内外牙龈素比活的影响。
结果：
抗菌实验结果显示纳米Ag/TiO2涂层在黑暗环境下对所有实验菌均有抗菌活性,20min后抗菌率达到了80.2%以上。黑暗环境下纳米Ag/TiO2涂层作用30-60min后即可完全杀死菌液中的口腔常见细菌,而纳米TiO2涂层作用240min后也未显示出抗菌活性。纳米Ag/TiO2涂层对不同菌种的杀菌时间存在差异,对变形链球菌、血链球菌及牙龈卟啉单胞菌的杀灭时间最短,为20-30min；对伴放线放线杆菌、具核梭杆菌及中间型普氏菌的杀灭时间延长为30-60min。
扫描电镜照片显示纳米Ag/TiO2涂层托槽表面黏附的细菌数量明显少于普通金属托槽及纳米TiO2涂层托槽。普通金属托槽表面有大量的细菌黏附,呈致密片状分布,形成多层菌膜；纳米TiO2涂层托槽表面也见大量的细菌黏附,呈颗粒状,但未见多层菌膜形成；而纳米Ag/TiO2涂层托槽表面黏附的细菌明显少于前2者,呈散在分布。
在各时间点,普通托槽组、纳米TiO2涂层托槽组和纳米Ag/TiO2涂层托槽组GTF活性依次下降；纳米Ag/TiO2涂层托槽组GTF活性比其余3组明显降低；除48h时间点外,TiO2托槽组GTF活性比空白对照组下降,差异有统计学意义；普通托槽组GTF活性与空白对照组差异无统计学意义。Ag/TiO2托槽对GTF活性的抑制作用强弱顺序为：18h>24h>6h>48h,除18与24h时间点外,其余时间点两两比较Ag/TiO2托槽对GTF活性的抑制作用差异均有统计学意义。
在纳米Ag/TiO2涂层作用下,牙龈卟啉单胞菌胞内外牙龈素比活随抗菌时间推移均呈下降趋势,10min内细菌胞内牙龈素比活明显大于胞外,10min后两者无明显差异；胞外牙龈素比活水平在20min内基本持平,之后随时间延长呈缓慢下降趋势；胞内牙龈素比活20min内出现明显下降,之后下降趋势减慢。
结论：
1.纳米Ag/TiO2涂层托槽在黑暗环境下对口腔常见细菌有较强的抗菌作用。
2.纳米Ag/TiO2涂层托槽对口腔常见细菌有较好的抗黏附作用。
3.纳米Ag/TiO2涂层托槽能有效抑制变形链球菌葡糖基转移酶的活性,从而对变形链球菌有良好抗黏附作用。
4.纳米Ag/TiO2涂层托槽在杀死牙龈卟啉单胞菌的同时还可降解细菌胞内外的牙龈素,显著抑制其活性。
第三章纳米Ag/TiO2涂层托槽的生物相容性评价
目的：
对纳米Ag/TiO2涂层托槽进行生物相容性测试,为其在临床的安全使用提供实验依据。
方法：
在不锈钢17-4圆片表面涂敷纳米TiO2薄膜涂层和Ag/TiO2薄膜涂层,加入DMEM培养液制取浸提液,取复苏后的L929细胞与其共同培养,在倒置相差显微镜下观察细胞形态；使用MTT法计算细胞增殖度并进行材料毒性程度分级；用碘化丙啶染色细胞后荧光显微镜下观测细胞死亡情况；用流式细胞仪检测分析细胞周期,进行细胞毒性试验。
将纳米TiO2薄膜涂层和Ag/TiO2薄膜涂层试样用生理盐水浸泡提取浸提液,加入新鲜稀释抗凝兔血,测量吸光度值,计算材料溶血率,进行溶血试验。
将纳米TiO2薄膜涂层和Ag/TiO2薄膜涂层试样用生理盐水浸泡提取浸提液,对小鼠灌胃,观察动物行为是否有毒性体征出现；对试验前后体重进行测量,观察体重变化；处死动物,进行病理解剖,肉眼观察动物重要脏器有无病理变化；在显微镜下观察小鼠心、肺、脾、肺、肾有无病理改变,进行短期全身毒性试验。
取涂敷纳米Ag/TiO2薄膜涂层的不锈钢17-4圆片和同样规格的口腔用牙胶圆片,缝合固定到金黄色仓地鼠的颊囊黏膜上,观察动物的活动情况,有无全身不良反应；观察试件与口腔内黏膜接触处的局部黏膜反应；处死动物,对颊囊部全层组织进行病理组织学观察,并进行病理反应分级,进行口腔黏膜刺激试验。
结果：
细胞毒性试验形态学观察结果显示试验组细胞形态数量与对照组无明显区别,可见大量分裂细胞；荧光显微镜下见只有极少量细胞被PI染成红色,可能为正常凋亡细胞,各组未见明显差异；MTT试验结果显示各组细胞增殖度较为接近,毒性等级均为0-1级；细胞周期分析未见异常,各组之间无明显差异。
溶血试验中试验组的吸光度值明显低于阳性对照组,TiO2组和Ag/TiO2组的溶血率分别为0.255%、0.637%,均小于5%,即均不会引起急性溶血。
短期全身毒性试验中试验组和对照组60只小鼠无一例死亡,临床毒性体征分级都是无症状；试验组小鼠体重增长与对照组比较差异均无统计学意义；病理解剖肉眼观察动物重要脏器未见充血、出血、水肿等明显病理变化；显微镜下观察小鼠心、肺、脾、肺、肾未出现变性、萎缩、坏死等病理变化。
口腔黏膜刺激试验中动物活动正常,饮食正常；与试验材料和对照材料接触的颊囊黏膜表面及其周围未见充血、水肿、糜烂及溃疡等反应；试验组与阴性对照组接触处颊囊黏膜组织切片基本相同,未见异常变化,病理反应评分除1侧为轻度外,其余均为0级。
结论：
1.纳米Ag/TiO2涂层托槽对细胞不产生明显毒性。
2.纳米Ag/TiO2涂层托槽不会引起急性溶血反应。3.纳米Ag/TiO2涂层托槽浸提液对小鼠产生的影响与生理盐水无差异,没有短期全身毒性。
4.纳米Ag/TiO2涂层托槽对仓地鼠口腔黏膜没有刺激性,具有良好的生物安全性。
综上所述,本研究成功研制出纳米Ag/TiO2涂层托槽,具有良好的机械性能；对口腔常见细菌具有优异的抗菌性能和抗黏附作用；对机体无毒性,生物相容性良好,可以满足正畸临床需要,但还需进一步深入研究之后才能应用于临床。
In orthodontic clinic, fixed appliance has been widely used for its efficiency and gained fine therapeutic effect. But the structure of its main composition-brackets is complex, leading to dental plaque increasing and having impact on micro-circumstance in oral cavity, so enamel decalcification, gingivitis and periodontitis were caused. Prevention and control of dental plaque adhesion is the key of dental caries and periodontitis prevention. Improve the material of brackets so to inhibit adhension and growth of pathogenic bacterium in oral cavity is one of the investigative directions in orthodontics sphere. Along with the development of the science of dental materials, the appearance of nanometer antibiosis materials provids new choice for new bracket materials exploitation.
Most nanometer antibiosis materials utilize single nanometer antibacterial agent such as nanometer titanium dioxide, nanometer sliver and so on. These materials have defect of their own, so the application are constrainted. Silver-loaded titanium dioxide becomes an important direction of new antibiosis materials investigation. It has specific mechanical property, antibiosis function and biocompatibility, so it has broad prospect of application.
There is no report of using nanometer Ag/TiO2materials and technique in orthodontics brackets at home and abroad. This study plans to make nanometer Ag/TiO2coating on ordinary metal brackets so to develop nanometer Ag/TiO2coating on brackets that has fine appearance, high intensity, visible photocatalytic activity and bacteriostasis effect even in dark circumstances. Detect the efficiency of physics, chemistry, antibiosis and biocompatibility of the brackets so as to confirm the specific mechanical and biological properties of the brackets. Our aim is to provide effective method for solving adamantine caries and periodontal inflammation, and to promote the use of nanometer technology in orthodontic materials area. This study consists of three chapters below:
Part One Development of nanometer Ag/TiO2coated brackets and physicochemical properties detection
Objective
To develop nanometer Ag/TiO2coated bracket and detect its physicochemical properties.
Methods
In this study, we chose ordinary metal brackets widely used in clinic, polish, ultrasonic clean, flush and dry. TiO2gel was produced by sol-gel method. TiO2film was coated on brackets surface, and then soaked in AgNO3solution. Ag/TiO2film was heat treated under120degree, then annealing under120,200,300degree respectively, then nanometer Ag/TiO2coated bracket A,B,C were produced.
Scanning electron microscope (SEM) was used to observe the micromorphology of surface of ordinary metal brackets, nanometer TiO2coated bracket and nanometer Ag/TiO2coated bracket. Cross-section topography of nanometer Ag/TiO2coated bracket group A was also observed.
The surface roughness of metal bracket, nanometer TiO2coated bracket and nanometer Ag/TiO2coated bracket were measured.
The combination intensity between smear layer of nanometer TiO2and nanometer Ag/TiO2with basis material were analyzed by scratch experimentation.
The crystal structure and phase of nanometer TiO2coated bracket and nanometer Ag/TiO2coated bracket were analyzed by XRD.
The chemical components of nanometer TiO2coated bracket and nanometer Ag/TiO2coated bracket were characterized using XPS.
The transmittance of nanometer TiO2coating and nanometer Ag/TiO2coating on quartz glass were mearsured by UV-VIS.
Results
SEM results showed that the surface of ordinary metal bracket was rough and existenced major ravine andpitlike depression. Nanometer TiO2coating film was constructed by many sphericity micro-particles, the diameter of which was10-30nm. Nanometer Ag/TiO2film was also constructed by many sphericity micro-particles, with orderly structure, the diameter of TiO2particles were uniformity, Ag particles were distributed on the film, the diameter of which was50-100nm. Aggregation phenomenon was seen seldomly. The diameter of Ag particles became bigger and distributed more intensively with the annealing temperature arising.
The section micromorphology showed that the thickness of nanometer Ag/TiO2film was about120nm, the film was well-distributed and had smooth surface, Ag particles could be seen deposited on the film.
There were no statistically differences among the surface roughness of five groups of brackets. The combination intensity of the nanometer TiO2smear layer with basis material was1.18kg, the numerical value of A,B,C groups of nanometer Ag/TiO2were1.16,1.12,1.26kg respectively.
XRD showed that there were anatase TiO2diffraction peak in both curves of TiO2and Ag/TiO2films. Ag0diffraction peak appeared in the curve of Ag/TiO2, and the intensity became larger as the temperature arising.
XPS showed main components of TiO2and Ag/TiO2were Ti, O,C and Ag,Ti,O,C respectively. The Ti2p level in TiO2was divided into2levels:Ti2p1/2and Ti2p3/2, the central peak were464.6ev and458.9ev, the difference was5.7ev. The peak value of binding energy of Ti2p drifted to high energy after Ag deposoted. The Ag3d level in Ag/TiO2was divided into2levels:Ag3d5/2and Ag3d3/2, the central peak were368.1ev and374.1ev, the difference was6ev.
The TiO2transmittancy spectrogram showed ultraviolet light<350nm could be completely absorbted, red shift of the absorption happened and transmittancy decreased after Ag deposited. The transmittancy decreased as the annealing temperature decreasing.
Conclusion
1. The results confirmed that nanometer Ag/TiO2coated brackets were developed successfully by sol-gel method.
2. This nanometer Ag/TiO2coating was well-distributed, the thickness and diameter of particles were all in nanometer scale, so that the characteristics of nanometer materials were guaranteed. It had smooth surface and enough combination intensity with basis material, which could meet the clinical demand of orthodontics.
3. TiO2in the film was anatase TiO2. Ag existed by the form of simple substance. Ag particles grew up as the annealing temperature increasing.
4. The photochemical catalytic activity of TiO2increased by the deposition of Ag, and made it owned visible light photoresponse.
5. The visible light photoresponse increased by the decrease of diameter of Ag.
6. The result indicated that choose120degree as annealing temperature is suitable, for the diameter of Ag and TiO2were small, which owned enough combination intensity and strong visible light photoresponse.
Part Two
Research on bactericidal ability of nanometer Ag/TiO2coated brackets
Objective
To study the bactericidal ability of nanometer Ag/TiO2coated bracket and investigate the mechanism initially, in order to provide theoretical evidence.
Methods
A group nanometer Ag/TiO2bracket was prouduced and TiO2and Ag/TiO2coatings were coated on stainless steel17-4with the same method. S.mutans, S.sangui, A.actinomycetemcomitans, F.nucleatum, P.gingivalis and P.intermedia were anabiosised and cultured into bacteria liquid. Film applicator coating method was used to test the antibacterial efficacy of Ag/TiO2coating.
3groups of brackets were soaked in the mixed bacteria liquid. After48h, SEM was used to observe the anti-adhesive efficacy of the coating.
Cocultured S.mutans with Ag/TiO2coated bracket for6,18,24,48h, then extracted coarse GTF. The content of reducing sugar in supernatant was tested by Somogyi-Nelson method, and activity of GTF was calculated in order to analyze the influence of Ag/TiO2coating on GTF in different times.
The activity of gingpains ecto-cellular and intra-cellular and protein level were measured and specific activity of gingpains was calculated in order to analyze the effect of Ag/TiO2coating on gingpains.
Results
The result showed more than80.2%of bacteria were killed within20minutes in dark circumstance which confirm fine antibacterial effect of Ag/TiO2coating. The coating could kill all the bacteria after30-60min, while TiO2coating did not exhibit antibacterial effect after240min. There were differences among the time of killing different strains.
SEM showed the bacterial number adhered on the surface of nanometer Ag/TiO2coated bracket was obviously fewer than on the surface of metal bracket and nanometer TiO2coated bracket. There were considerable bacteria adhere to the surface of metal bracket, distributed compactly and formed multilayer pellicle. There were also massive bacteria adhere to the surface of nanometer TiO2coated bracket, in grainy structure, no multilayer pellicle was seen. There were obvious slight bacteria adhere to the surface of nanometer Ag/TiO2coated bracket, scattered distributed.
In different times, GTF activity of metal bracket group, nanometer TiO2coated bracket group and nanometer Ag/TiO2coated bracket group descended by turns, GTF activity in nanometer Ag/TiO2coated bracket group was obviously lower than3orther groups, GTF activity in nanometer TiO2coated bracket group was lower than blank control group besides at48h, there were statistically differences among these groups. There were no statistically differences between GTF activity of metal bracket group and nanometer TiO2coated bracket group. The sequence of inhibition of nanometer Ag/TiO2coated bracket on GTF activity were:18h>24h>6h>48h. There were statistically differences between the inhibition in different times besides18h and24h.
The specific activity of gingpains ecto-cellular and intra-cellular both had downtrend as the time lasting. The specific activity of gingpains intra-cellular was obviously bigger than ecto-cellular within10min, the difference became small after10min. The specific activity of gingpains ecto-cellular did not change much within20min, then assumed slow downtrend. The specific activity of gingpains intra-cellular descented obviously within20min, then steped down downtrend.
Conclusion
1. The results confirmed the outstanding antibacterial property of nanometer Ag/TiO2coated bracket in dark circumstance.
2. Nanometer Ag/TiO2coated bracket had fine adherence resistance to common bacteria in oral cavity.
3. The bracket could effectively restrain the activity of GTF so that it has excellent adherence resistance to S.mutans.
4. The bracket could lead to gingpains degradation ecto-cellular and intra-cellular while killing P.gingivalis.
Part Three
Biocompatibility evaluation of nanometer Ag/TiO2coated bracket
Objective
Biocompatibility of nanometer Ag/TiO2coated bracket was examined in order to provide experimental evidence for its use in clinic.
Methods
TiO2and Ag/TiO2coatings were spreaded on the surface of stainless steel wafer. Add DEME to the wafers, and cocultured with L929cell. The morphous of cell were observed under inverted phase contrast microscope. Relative growth rate was calculated by MTT method and carried out toxity degree grading. The death of cell was observed under fluorescene microscope. Cell cycle was detected by flow cytometry.
Leaching liquor was extracted from TiO2and Ag/TiO2coatings and added to fresh anticoagulation cony blood, optical density was measured and hemolysis rate was calculated.
Lavaging mouse with leaching liquor extracted from TiO2and Ag/TiO2coatings, observing toxicity physical sign, measuring body weigh change before and after experiment, and then observe pathological change of organs heart, hepar, lien, lung and kidney.
Sutured the wafer to the cheek pouch of inaurate hamster, observed the activity of animals and mucous membrane reaction in the contact region, then observe the full-thickness of cheek pouch under microscope, and carry out pathology reaction grading.
Results
There were no differences among3groups of cells under inverted phase contrast microscope and fluorescene microscope.Toxicity levels of3groups were0-1. There were no abnormality seen in cell cycle.
Optical density in test group was obviously lower than positive control group. The hemolysis rate of TiO2group and Ag/TiO2group were0.255%and0.637%. They were both less than5%, which meaned no acute haemolysis was induced.
There was no death in all the animals, clinical toxicity physical sign were all asymptomatic, there was no difference between test group and control group in body weigh increasing, the organs were observed under microscope and no pathological change was found.
The animals acted and took food normally, no pathological change was observed in the contact mucous membrane of cheek pouch both in macroscopic and microscope observation.
Conclusion
1. Nanometer Ag/TiO2coated bracket had no toxicity for cell.
2. No acute haemolysis was induced by nanometer Ag/TiO2coated bracket.
3. The influence of leaching liquor extracted from Ag/TiO2coating on mouse had no difference with sodium chloride and no short-term systemic toxicity was observed.
4. Nanometer Ag/TiO2coated bracket had no stimulation to oral mucosa of hamster, so it had fine biological properties.
Above all, nanometer Ag/TiO2coated bracket was developed successfully, which could meet the clinical demand for it has excellent mechanical function, owns outstanding bactericidal ability and adhesion resistance, and possesses fine biocompatibility. But futher study should be carried out before it be used in orthodontic clinic.
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