本文選題：抗菌　切入點：生物膜　出處：《武漢大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：目的：(1)評價納米銀(silver nanoparticles, AgNPs)溶液沖洗對牙本質(zhì)糞腸球菌(Enter ococcusfaecalis, E. faecalis)生物膜的抗菌效果；(2)評價AgNPs凝膠封藥對牙本質(zhì)E. faecalis生物膜的抗菌效果；(3)評價AgNPs凝膠處理的牙本質(zhì)抵抗E. faecalis的附著和生物膜形成的能力；(4)制備并表征介孔生物活性玻璃(mesoporous bioactive glasses, MBGs)和載銀介孔生物活性玻璃(Ag-loaded mesoporous bioactive glasses, Ag-MBGs),初步評價Ag-MBGs對根管E. faecalis生物膜的抗菌效果。 方法：實驗一：在牙本質(zhì)片上構(gòu)建4周E. faecalis生物膜,然后將96個牙本質(zhì)片隨機(jī)均分為4組(n=24),分別以0.1%AgNPs溶液、2%次氯酸鈉(sodium hypochlorite, NaOC1)溶液和生理鹽水為沖洗液,使用注射器針頭沖洗牙本質(zhì)E.faecalis生物膜2分鐘,未處理標(biāo)本作為對照組。利用掃描電鏡(scanning electron microscopy, SEM)觀察每組12個標(biāo)本E.faecalis生物膜的結(jié)構(gòu),每組另外12個標(biāo)本用激光共聚焦掃描顯微鏡(confocal laser scanning microscopy, CLSM)結(jié)合LIVE/DEAD熒光染色法分析E.faecalis生物膜內(nèi)活菌比,評價0.1%AgNPs溶液沖洗對牙本質(zhì)E.faecalis生物膜的抗菌效果。實驗二：在牙本質(zhì)片上和牙根錐塊內(nèi)構(gòu)建4周E.faecalis生物膜,然后將80個牙本質(zhì)片和40個牙根錐塊隨機(jī)均分為4組(每組20個牙本質(zhì)片和10個牙根錐塊),分別用0.02%AgNPs凝膠、0.01%AgNPs凝膠、氫氧化鈣(calcium hydroxide, Ca(OH)2)糊劑和生理鹽水封藥7天。SEM觀察每組10個牙本質(zhì)片上E. faecalis生物膜的結(jié)構(gòu),CLSM結(jié)合LIVE/DEAD熒光染色法分析每組另外10牙本質(zhì)片上殘留E. faecalis生物膜內(nèi)活菌比。獲取牙根錐塊內(nèi)0～100μm和100～200μm深度的牙本質(zhì)碎屑,分散、梯度稀釋后涂布BHI瓊脂培養(yǎng)板,培養(yǎng)24小時后計數(shù)菌落形成單位(colony forming units, CPUs),評價藥物對牙本質(zhì)小管內(nèi)E. faecalis的抗菌效果。實驗三：將200個牙本質(zhì)片平均分為5組：生理鹽水組、Ca(OH)2糊劑組、2%洗必泰(Chlorhexidine, CHX)凝膠組、0.01%和0.02%AgNPs凝膠組。標(biāo)本經(jīng)上述藥物處理7天后接種E. faecalis懸液,37℃厭氧培養(yǎng)1天或7天。SEM定性評價E. faecalis對牙本質(zhì)的附著和生物膜形成情況,CLSM結(jié)合LIVE/DEAD熒光染色法定量分析牙本質(zhì)表面附著的E. faecalis總量和活菌比。制備牙本質(zhì)片20個,分別用0.01%和0.02%AgNPs凝膠封藥7天。將牙本質(zhì)片放入無菌24孔板中,每孔加入1mL ddH2O。1天和7天時,分別吸取每組5個孔內(nèi)的液體,電感耦合等離子原子發(fā)射光譜儀測量液體中Ag+濃度。實驗四：采用溶劑揮發(fā)誘導(dǎo)自組裝結(jié)合凝膠-溶膠技術(shù)合成MBGs,采用離子吸附法制備Ag-MBGs,表征MBGs和Ag-MBGs的理化性能,評價MBGs和Ag-MBGs在Tris-HCl緩沖液中的離子釋放特征,以及浸入模擬體液(simulated body fluids, SBF)后的pH值變化情況。在12個根管預(yù)備后的單根下頜前磨牙根管內(nèi)構(gòu)建4周E. faecalis生物膜,分別用Ca(OH)2糊劑、MBGs和Ag-MBGs封藥7天。利用SEM、CLSM結(jié)合LIVE/DEAD熒光染色法觀察藥物處理后根管內(nèi)的E. faecalis生物膜結(jié)構(gòu),初步評價Ag-MBGs對根管E. faecalis生物膜的抗菌效果。 結(jié)果：實驗一：經(jīng)2%NaOC1溶液沖洗的牙本質(zhì)E. faecalis生物膜結(jié)構(gòu)明顯破壞,僅少量的生物膜殘留在牙本質(zhì)表面；0.1%AgNPs溶液沖洗并不能破壞牙本質(zhì)E.faecalis生物膜結(jié)構(gòu)完整性,生物膜內(nèi)活菌比低于未處理組(P0.05),但與生理鹽水沖洗組無統(tǒng)計學(xué)差異(P0.05)。實驗二：AgNPs凝膠顯著破壞牙本質(zhì)E. faecalis生物膜的結(jié)構(gòu)并殺滅生物膜內(nèi)細(xì)菌。其中0.02%AgNPs凝膠組牙本質(zhì)E. faecalis生物膜內(nèi)活菌比、牙本質(zhì)小管內(nèi)0～100μm和100～200μm深度的CFUs少于0.01%AgNPs組和Ca(OH)2組(P0.05),后兩組又顯著少于生理鹽水組(P0.05)。實驗三：生理鹽水和Ca(OH)2處理的牙本質(zhì)表面聚集大量E. faecalis,隨著培養(yǎng)時間延長逐漸形成稠密的E. faecalis生物膜；AgNPs凝膠處理的牙本質(zhì)表面附著的細(xì)菌較少,形成的E. faecalis生物膜稀疏；而2%CHX處理的牙本質(zhì)表面附著的細(xì)菌最少。2%CHX組平均細(xì)菌總量和活菌比顯著低于AgNPs凝膠組(P0.05),后者又顯著低于生理鹽水組和Ca(OH)2組(P0.05)；培養(yǎng)1或7天,0.01%和0.02%AgNPs凝膠組的平均細(xì)菌總量和活菌比均無明顯差異(P0.05)。 AgNPs凝膠處理后的牙本質(zhì)浸泡在ddH2O中1天或7天,0.02%AgNPs凝膠組牙本質(zhì)釋放的Ag+量均高于0.01%AgNPs凝膠組(P0.05)。0.02%或0.01%AgNPs凝膠組牙本質(zhì)浸泡在ddH2O中7天釋放的Ag+量高于1天,但差異無統(tǒng)計學(xué)意義(P0.05)。實驗四：合成的MBGs為典型的介孔材料：表面光滑均勻平整,內(nèi)部有規(guī)則的介孔通道,Ⅵ型氮氣吸附-脫附等溫線和H1型滯后環(huán),平均孔徑為4.7nm,對應(yīng)的XRD小衍射角出現(xiàn)三個衍射峰。Ag-MBGs內(nèi)部保持介孔結(jié)構(gòu),但介孔通道邊緣變得不規(guī)整,表面和內(nèi)部有大量納米顆粒,EDS能譜證實為Ag元素。MBGs和Ag-MBGs浸泡在SBF中,SBF的pH值輕度上升,并保持穩(wěn)定；在Tris-HCl中各元素呈緩釋狀態(tài),釋放速率隨時間延長而降低。經(jīng)Ag-MBGs處理的根管E. faecalis生物膜結(jié)構(gòu)破壞明顯,僅殘留少量細(xì)菌在牙本質(zhì)小管口內(nèi)；而經(jīng)MBGs處理的E. faecalis生物膜結(jié)構(gòu)完整,殘留大量活菌。有少量Ag-MBGs和MBGs顆粒殘留在根管壁上。 結(jié)論：1.AgNPs對牙本質(zhì)E. faecalis生物膜的抗菌效果與其應(yīng)用方式有關(guān)。AgNPs溶液沖洗不能破壞牙本質(zhì)表面E. faecalis生物膜結(jié)構(gòu),但AgNPs凝膠封藥能破壞牙本質(zhì)表面的E. faecalis生物膜結(jié)構(gòu),并有效殺滅牙本質(zhì)表面和牙本質(zhì)小管內(nèi)的E. faecalis.2.2%CHX處理的牙本質(zhì)能產(chǎn)生明顯的持續(xù)抗菌效果。AgNPs凝膠處理的牙本質(zhì)浸泡在液體中迅速釋放Ag+,不能形成緩釋；AgNPs凝膠處理的牙本質(zhì)與E. faecalis接觸早期可導(dǎo)致E. faecalis死亡,但7天后出現(xiàn)E. faecalis對牙本質(zhì)的附著和生物膜的形成。Ca(OH)2處理的牙本質(zhì)無持續(xù)抗菌效果。3.Ag-MBGs形成Ag+緩釋系統(tǒng),對根管內(nèi)E. faecalis生物膜具有較強(qiáng)的抗菌效果,抗菌效果與其釋放的Ag+有關(guān)。
[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.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R781.3

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10 谷海晶;凌均h，

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