纳米银对牙本质粪肠球菌生物膜的抗菌效果研究

发布时间：2018-03-20 16:27

本文选题：抗菌　切入点：生物膜　出处：《武汉大学》2014年博士论文　论文类型：学位论文

【摘要】：目的：(1)评价纳米银(silver nanoparticles, AgNPs)溶液冲洗对牙本质粪肠球菌(Enter ococcusfaecalis, E. faecalis)生物膜的抗菌效果；(2)评价AgNPs凝胶封药对牙本质E. faecalis生物膜的抗菌效果；(3)评价AgNPs凝胶处理的牙本质抵抗E. faecalis的附着和生物膜形成的能力；(4)制备并表征介孔生物活性玻璃(mesoporous bioactive glasses, MBGs)和载银介孔生物活性玻璃(Ag-loaded mesoporous bioactive glasses, Ag-MBGs),初步评价Ag-MBGs对根管E. faecalis生物膜的抗菌效果。方法：实验一：在牙本质片上构建4周E. faecalis生物膜,然后将96个牙本质片随机均分为4组(n=24),分别以0.1%AgNPs溶液、2%次氯酸钠(sodium hypochlorite, NaOC1)溶液和生理盐水为冲洗液,使用注射器针头冲洗牙本质E.faecalis生物膜2分钟,未处理标本作为对照组。利用扫描电镜(scanning electron microscopy, SEM)观察每组12个标本E.faecalis生物膜的结构,每组另外12个标本用激光共聚焦扫描显微镜(confocal laser scanning microscopy, CLSM)结合LIVE/DEAD荧光染色法分析E.faecalis生物膜内活菌比,评价0.1%AgNPs溶液冲洗对牙本质E.faecalis生物膜的抗菌效果。实验二：在牙本质片上和牙根锥块内构建4周E.faecalis生物膜,然后将80个牙本质片和40个牙根锥块随机均分为4组(每组20个牙本质片和10个牙根锥块),分别用0.02%AgNPs凝胶、0.01%AgNPs凝胶、氢氧化钙(calcium hydroxide, Ca(OH)2)糊剂和生理盐水封药7天。SEM观察每组10个牙本质片上E. faecalis生物膜的结构,CLSM结合LIVE/DEAD荧光染色法分析每组另外10牙本质片上残留E. faecalis生物膜内活菌比。获取牙根锥块内0～100μm和100～200μm深度的牙本质碎屑,分散、梯度稀释后涂布BHI琼脂培养板,培养24小时后计数菌落形成单位(colony forming units, CPUs),评价药物对牙本质小管内E. faecalis的抗菌效果。实验三：将200个牙本质片平均分为5组：生理盐水组、Ca(OH)2糊剂组、2%洗必泰(Chlorhexidine, CHX)凝胶组、0.01%和0.02%AgNPs凝胶组。标本经上述药物处理7天后接种E. faecalis悬液,37℃厌氧培养1天或7天。SEM定性评价E. faecalis对牙本质的附着和生物膜形成情况,CLSM结合LIVE/DEAD荧光染色法定量分析牙本质表面附着的E. faecalis总量和活菌比。制备牙本质片20个,分别用0.01%和0.02%AgNPs凝胶封药7天。将牙本质片放入无菌24孔板中,每孔加入1mL ddH2O。1天和7天时,分别吸取每组5个孔内的液体,电感耦合等离子原子发射光谱仪测量液体中Ag+浓度。实验四：采用溶剂挥发诱导自组装结合凝胶-溶胶技术合成MBGs,采用离子吸附法制备Ag-MBGs,表征MBGs和Ag-MBGs的理化性能,评价MBGs和Ag-MBGs在Tris-HCl缓冲液中的离子释放特征,以及浸入模拟体液(simulated body fluids, SBF)后的pH值变化情况。在12个根管预备后的单根下颌前磨牙根管内构建4周E. faecalis生物膜,分别用Ca(OH)2糊剂、MBGs和Ag-MBGs封药7天。利用SEM、CLSM结合LIVE/DEAD荧光染色法观察药物处理后根管内的E. faecalis生物膜结构,初步评价Ag-MBGs对根管E. faecalis生物膜的抗菌效果。结果：实验一：经2%NaOC1溶液冲洗的牙本质E. faecalis生物膜结构明显破坏,仅少量的生物膜残留在牙本质表面；0.1%AgNPs溶液冲洗并不能破坏牙本质E.faecalis生物膜结构完整性,生物膜内活菌比低于未处理组(P0.05),但与生理盐水冲洗组无统计学差异(P0.05)。实验二：AgNPs凝胶显著破坏牙本质E. faecalis生物膜的结构并杀灭生物膜内细菌。其中0.02%AgNPs凝胶组牙本质E. faecalis生物膜内活菌比、牙本质小管内0～100μm和100～200μm深度的CFUs少于0.01%AgNPs组和Ca(OH)2组(P0.05),后两组又显著少于生理盐水组(P0.05)。实验三：生理盐水和Ca(OH)2处理的牙本质表面聚集大量E. faecalis,随着培养时间延长逐渐形成稠密的E. faecalis生物膜；AgNPs凝胶处理的牙本质表面附着的细菌较少,形成的E. faecalis生物膜稀疏；而2%CHX处理的牙本质表面附着的细菌最少。2%CHX组平均细菌总量和活菌比显著低于AgNPs凝胶组(P0.05),后者又显著低于生理盐水组和Ca(OH)2组(P0.05)；培养1或7天,0.01%和0.02%AgNPs凝胶组的平均细菌总量和活菌比均无明显差异(P0.05)。 AgNPs凝胶处理后的牙本质浸泡在ddH2O中1天或7天,0.02%AgNPs凝胶组牙本质释放的Ag+量均高于0.01%AgNPs凝胶组(P0.05)。0.02%或0.01%AgNPs凝胶组牙本质浸泡在ddH2O中7天释放的Ag+量高于1天,但差异无统计学意义(P0.05)。实验四：合成的MBGs为典型的介孔材料：表面光滑均匀平整,内部有规则的介孔通道,Ⅵ型氮气吸附-脱附等温线和H1型滞后环,平均孔径为4.7nm,对应的XRD小衍射角出现三个衍射峰。Ag-MBGs内部保持介孔结构,但介孔通道边缘变得不规整,表面和内部有大量纳米颗粒,EDS能谱证实为Ag元素。MBGs和Ag-MBGs浸泡在SBF中,SBF的pH值轻度上升,并保持稳定；在Tris-HCl中各元素呈缓释状态,释放速率随时间延长而降低。经Ag-MBGs处理的根管E. faecalis生物膜结构破坏明显,仅残留少量细菌在牙本质小管口内；而经MBGs处理的E. faecalis生物膜结构完整,残留大量活菌。有少量Ag-MBGs和MBGs颗粒残留在根管壁上。结论：1.AgNPs对牙本质E. faecalis生物膜的抗菌效果与其应用方式有关。AgNPs溶液冲洗不能破坏牙本质表面E. faecalis生物膜结构,但AgNPs凝胶封药能破坏牙本质表面的E. faecalis生物膜结构,并有效杀灭牙本质表面和牙本质小管内的E. faecalis.2.2%CHX处理的牙本质能产生明显的持续抗菌效果。AgNPs凝胶处理的牙本质浸泡在液体中迅速释放Ag+,不能形成缓释；AgNPs凝胶处理的牙本质与E. faecalis接触早期可导致E. faecalis死亡,但7天后出现E. faecalis对牙本质的附着和生物膜的形成。Ca(OH)2处理的牙本质无持续抗菌效果。3.Ag-MBGs形成Ag+缓释系统,对根管内E. faecalis生物膜具有较强的抗菌效果,抗菌效果与其释放的Ag+有关。
[Abstract]:Objective: (1) evaluation of silver nanoparticles (silver nanoparticles AgNPs) solution on dentin of Enterococcus faecalis (Enter ococcusfaecalis, E. faecalis) the antibacterial effect of biofilm; (2) to evaluate the effect of antibacterial drugs on AgNPs gel sealing dentin E. faecalis membrane; (3) to evaluate the ability of dentin AgNPs gel processing E. faecalis resistance attachment and biofilm formation; (4) preparation and characterization of mesoporous bioactive glass (mesoporous bioactive, glasses, MBGs) and silver loaded mesoporous bioactive glass (Ag-loaded mesoporous bioactive glasses, Ag-MBGs), a preliminary evaluation of the antibacterial effect of Ag-MBGs on root canal E. faecalis biofilm.
Methods: experiment one: Construction of 4 weeks E. faecalis biofilm on dentine slices, then 96 dentine slices were randomly divided into 4 groups (n=24, 0.1%AgNPs) respectively with 2% sodium hypochlorite solution (sodium, hypochlorite, NaOC1) and saline solutions for washing liquid, using an injection needle irrigation dentin E.faecalis the 2 minute film, untreated specimens as the control group. Using scanning electron microscopy (scanning electron, microscopy, SEM) structure were observed in 12 specimens of E.faecalis biofilm, each other 12 specimens by confocal laser scanning microscopy (confocal laser scanning microscopy, CLSM E.faecalis) analysis of living bacteria in biofilm than LIVE/DEAD staining combined with fluorescence the evaluation of 0.1%AgNPs, solution washing antibacterial effect on dentin E.faecalis membrane. Experiment two: Construction of 4 weeks E.faecalis biofilm on dentine slices and the root cone block, and then 80 dentin slices and 40 root cone block were randomly divided into 4 groups (each group of 20 dentin slices and 10 root cone block), respectively with 0.02%AgNPs gel, 0.01%AgNPs gel, calcium hydroxide (calcium hydroxide, Ca (OH) 2) and saline sealing paste structure with 10.SEM on the 7 day dentin on-chip E. faecalis membrane, CLSM combined with LIVE/DEAD fluorescence staining analysis of each other 10 dentin slices of residual E. faecalis living bacteria in biofilm. Dentin debris, get the root cone block within 0~100 m and 100~200 m depth dispersion, after gradient dilution coating BHI agar plate culture. 24 hours after counting the colony forming units (colony forming, units, CPUs), to evaluate the effect of antibacterial drugs on dentinal tubules in E. faecalis. Experiment three: 200 dentine plates were divided into 5 groups: saline group, Ca (OH) 2 paste group, 2% chlorhexidine (Chlorhexidine, CHX gel group, 0.01%) and 0.02%AgNPs gel group. After treatment for 7 days, the specimens were inoculated with E. faecalis suspension, 37 C incubated for 1 days or 7 days.SEM qualitative evaluation of E. faecalis on dentin adhesion and biofilm formation, CLSM combined with LIVE/DEAD fluorescence staining quantitative dentin attached on the surface of E. total faecalis and analysis of live bacteria ratio. Preparation of dentin slices of 20, 0.01% and 0.02%AgNPs respectively for 7 days. The gel sealing dentin pieces into a sterile 24 well plates, each hole joined 1mL ddH2O.1 and 7 days, respectively, from each of the 5 hole liquid, the concentration of Ag+ atomic emission spectrometer in liquid inductively coupled plasma. Experiment four: by evaporation induced self-assembly with sol gel synthesis technology of MBGs, Ag-MBGs prepared by ion adsorption method. The physicochemical characterization of MBGs and Ag-MBGs, MBGs and Ag-MBGs in the evaluation of Tris-HCl buffer solution. Ion release characteristics, and immersed in the simulated body fluid (simulated body, fluids, SBF) after the pH value changes. In 12 a single mandibular premolar after root canal preparation of root canal construction 4 weeks E. faecalis biofilm, respectively Ca (OH) 2 paste, 7 days MBGs and Ag-MBGs by SEM medicine. CLSM LIVE/DEAD E. faecalis, combined with the fluorescence of biofilm structure within the root canal treatment method observation after staining, the preliminary evaluation of the antibacterial effect of Ag-MBGs on root canal E. faecalis biofilm.
Results: experiment one: after 2%NaOC1 solution washing of dentin E. faecalis biofilm structure was destroyed, only a small amount of biofilm on the surface of residual dentin; 0.1%AgNPs solution washing could not destroy the dentin E.faecalis membrane structure integrity of living bacteria in biofilm is lower than the untreated group (P0.05), but with saline rinse group showed no significant difference (P0.05). Experiment two: bacterial biofilm structure and kill AgNPs gel significantly damage the dentin E. faecalis membrane. The 0.02%AgNPs gel group of dentin E. faecalis living bacteria in biofilm, the dentinal tubules within 0~100 m and 100~200 m depth less than group 0.01%AgNPs and CFUs Ca (OH) 2 group (P0.05), the two group was significantly less than the saline group (P0.05). Experiment three: saline and Ca (OH) 2 dentin surface treatment E. faecalis gathered a lot of training, with the prolongation of time form E. faecalis biofilm dense; AgNPs gel treatment of dentin attached bacteria on the surface of E. less, faecalis biofilm formation and 2%CHX sparse; treatment of dentin attached bacteria on the surface of at least.2%CHX group of average total bacteria and live bacteria ratio was significantly lower than that of AgNPs gel group (P0.05), which was significantly lower than the saline group Ca (OH) and 2 group (P0.05); culture for 1 or 7 days, and 0.01% 0.02%AgNPs gel group average total bacteria and live bacteria ratio had no significant difference (P0.05). After the treatment of dentin AgNPs gel soaked in ddH2O for 1 days or 7 days, the amount of Ag+ 0.02% AgNPs gel group of dentin release the gel was higher than that of 0.01%AgNPs group (P0.05 Ag+).0.02% or 0.01%AgNPs gel group dentin soaked in ddH2O in the 7 day of the release of more than 1 days, but the difference was not statistically significant (P0.05). Experiment four: synthesis of MBGs mesoporous materials: typical surface smooth and flat The mesoporous channel internal rules, type VI N2 adsorption desorption isotherms and H1 hysteresis loop, average pore size is 4.7nm, the corresponding XRD small diffraction angle three diffraction peak of.Ag-MBGs is maintained inside the mesoporous structure, but mesoporous channel edge becomes irregular, and have large internal surface nano particles. EDS spectrum confirmed the Ag elements.MBGs and Ag-MBGs immersion in SBF SBF, the pH value increased slightly, and remained stable in Tris-HCl; the elements in a state of slow release, the release rate decreased with time. After treatment with Ag-MBGs root canal E. faecalis biofilm structure was destroyed, only a small amount of residual bacteria in dentin tubular mouth; and the E. faecalis membrane structure MBGs processing complete, the residual amounts of live bacteria. There is a small amount of Ag-MBGs and MBGs particles remaining in the root canal wall.
Conclusion: 1.AgNPs on dentine E. faecalis biofilm antibacterial effect and its application on.AgNPs solution can destroy the dentin surface E. faecalis biofilm structure, but AgNPs gel drugs can damage E. faecalis biofilm structure on the dentin surface, and effectively kill the dentin surface and dentin tubules in E. treated with faecalis.2.2%CHX dentin can produce obvious sustained antibacterial dentine soaking effect.AgNPs gel processing rapid release of Ag+ in the liquid, can not form a release; dentin and E. faecalis contact AgNPs gel treatment can lead to early death of faecalis E., but 7 days after the formation of.Ca E. faecalis on dentin adhesion and biofilm (OH) 2 no dentin treatment continued antibacterial effect of formation of.3.Ag-MBGs Ag+ drug delivery system, has strong anti-bacterial effect on root canal E. faecalis biofilm, antibacterial effect It is related to the Ag+ that is released.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R781.3

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