本文關鍵詞：骨粉內納米銀緩釋及抗菌性能研究，由筆耕文化傳播整理發(fā)布。

        背景：口腔種植中，多種原因（拔牙后牙槽骨萎縮、創(chuàng)傷、感染、腫瘤、先天畸形等）可引起牙種植體植入區(qū)骨量不足從而影響種植體的植入及遠期效果。骨組織替代材料的植入成為解決這一問題的必要手段，而如何控制術后感染成為種植治療中的難點及熱點。目前廣泛研究的抗生素如萬古霉素、四環(huán)素、左氧氟沙星等負載到骨粉上技術因細菌耐藥性、抗生素釋放持續(xù)時間短暫等，而未能達到理想的控制骨粉感染的目的。銀、鋅、銅等無機抗菌劑摻雜入骨粉材料內可有效抑制細菌感染，但其對哺乳動物細胞的毒性成為應用中的隱憂，且如何使其持續(xù)有效的發(fā)揮作用還未很好解決。此外，如何有效利用納米技術來延緩抗菌劑的釋放還需進一步研討。研究目的：本研究嘗試用環(huán)式糊精（CD）及殼聚糖（CS）作為配體合成納米銀，再將不同配體的納米銀（NP）負載到人工骨粉內，結合乳化技術，外包裹聚乳酸羥基乙酸（PLGA），制成能延緩釋放無機抗菌劑納米銀的骨粉生物材料。本實驗的特點是利用環(huán)糊精、殼聚糖對納米銀的覆蓋并不同程度增強其抗菌性，及負載納米銀的羥基磷灰石外包裹聚乳酸羥基乙酸，制成骨粉材料控制其感染，延緩納米銀的釋放。通過本實驗，希望獲得能持續(xù)有效釋放納米銀，直到自體骨形成，達到理想的控制骨粉感染的目的。方法與結果：1、環(huán)式糊精配體納米銀的制備及性能借鑒文獻[1]中納米銀的合成方法合成環(huán)式糊精配體納米銀，方法是：不同濃度的β-CD水溶液作為配體，硼氫化鈉溶液還原硝酸銀水溶液制得CD-NP。通過掃描電鏡、透射電鏡觀察制得的CD-NP的表面形貌、粒子直徑大小及粒子的聚集情況，觀察其穩(wěn)定性，檢測不同濃度納米銀的抗菌性。紫外分光光度計觀測CD-NP的吸收峰值。結果顯示：據文獻[1]報道,隨環(huán)式糊精濃度增加，制得納米銀抗菌性增強，因此選用20mM的環(huán)式糊精作為配體，與2mM的硝酸銀溶液持續(xù)攪拌20分鐘后，加入20mM的硼氫化鈉水溶液，變黑30s后形成黃褐色溶液，攪拌24小時后可制得穩(wěn)定性良好的納米銀。電鏡結果顯示：納米銀顆粒成圓形，粒徑在24-33nm之間，分布較均勻，無明顯聚集。紫外吸收光譜顯示：CD-NP的吸收峰值在410nm。細菌實驗表明：殼聚糖配體納米銀有明顯抗菌性，且隨納米銀的濃度增高，抗菌性增強。2、殼聚糖配體納米銀的制備及性能借鑒文獻[2]中納米銀的合成方法合成殼聚糖配體納米銀，方法是利用殼聚糖作為配體和還原劑，氫氧化鈉和乙酸調節(jié)PH值，制得CS-NP。將CS-NP溶于0.1%乙酸溶液中通過掃描電鏡、透射電鏡觀察制得的CD-NP的表面形貌、粒子直徑大小及粒子的聚集情況，觀察其穩(wěn)定性，檢測不同濃度納米銀的抗菌性。紫外分光光度計觀測CS-NP的吸收峰值。結果顯示：脫乙酰度>75%的殼聚糖溶于1%的乙酸溶液中，制得1mg/ml的殼聚糖溶液，加熱至45℃并穩(wěn)定攪拌至殼聚糖完全溶解形成均質溶液，24ml的殼聚糖均質液與1ml100mM的硝酸銀溶液混合后，將0.1M的氫氧化鈉溶液75ml緩慢加入上述混合液中，得黃褐色溶液。電鏡結果顯示：CS-NP成大小不等的卵圓形，粒徑在5-25nm之間，有聚集現(xiàn)象，粒子大小分布不太均勻。紫外吸收光譜顯示：CS-NP的吸收峰值在400nm。細菌實驗表明：殼聚糖配體納米銀有明顯抗菌性，且隨納米銀的濃度增高，抗菌性增強。3、羥基磷灰石負載納米銀的制備及性能方法：利用凍干法將不同濃度的環(huán)式糊精和殼聚糖配體納米銀負載到羥基磷灰石里，通過掃描電鏡、透射電鏡觀察制得的CD-NP-HA/CS-NP-HA的表面形貌、粒子直徑大小及粒子的聚集情況，觀察其穩(wěn)定性，并測試負載不同配體不同濃度納米銀的羥基磷灰石的抗菌性。將納米銀羥基磷灰石浸泡于模擬體液中，檢測其體外緩釋性能。結果顯示：將0.1umol、0.2umol、0.5umol、1.0umol、2.0umol、4.0umol、10.0umol的CD-NP加入到100mg羥基磷灰石水溶液中（10mg/ml）攪拌4小時后凍干。將0.1umol、0.2umol、0.5umol、1.0umol、2.0umol、4.0umol、10.0umol的CS-NP加入到100mg羥基磷灰石水溶液中（10mg/ml）攪拌4小時后凍干。凍干后的負載納米銀的羥基磷灰石呈白色粉末狀。電鏡結果顯示：CD-NP-HA/CS-NP-HA成大小不等的顆粒狀，，粒徑在9nm--60nm，顆粒聚集，穩(wěn)定性好。細菌實驗結果表明：負載0.1umol及以上濃度的CD-NP-HA/CS-NP-HA均有抗菌性。緩釋實驗結果表明：在第14天檢測時釋放達高峰。4、包裹聚乳酸羥基乙酸的羥基磷灰石制備及其性能方法：將聚乳酸羥基乙酸（PLGA）溶于氯仿中至完全溶解，負載納米銀的羥基磷灰石凍干粉溶于上述溶液中，劇烈攪拌至凍干粉溶解，將上述溶液迅速倒入含0.4%甲基纖維素的蒸餾水中，劇烈攪拌直至氯仿完全揮發(fā)至盡。離心、水洗后，將得到的固體凍干。通過掃描電鏡、透射電鏡觀察制得的CD-NP-HA-PLGA/CS-NP-HAPLGA的表面形貌、粒子直徑大小及粒子的聚集情況，觀察其穩(wěn)定性，并測試負載不同配體不同濃度納米銀的羥基磷灰石的抗菌性。將包裹PLGA的納米銀羥基磷灰石浸泡于模擬體液中，檢測其體外緩釋性能。結果：將200mg的PLGA溶于20ml氯仿，50mg負載不同濃度納米銀的羥基磷灰石溶于上述溶液中，溶解后倒入200ml含0.4%甲基纖維素的蒸餾水中，得到粉末狀的緩釋納米銀的骨粉材料。電鏡結果顯示：凍干粉成大小不等的球狀，直徑相差叫懸殊。最大直徑可達100多nm，最小可至10nm以下。細菌實驗結果顯示：含0.1umolCD-NP和0.1umolCS-NP及以上濃度的緩釋微球均具有明顯抗菌性。緩釋實驗結果顯示：至第42天時，其釋放納米銀仍呈上升趨勢，未見明顯下降。結論：1、通過不同方法合成環(huán)式糊精與殼聚糖配體的納米銀，再通過凍干法將納米銀負載到羥基磷灰石里，再將其表面包裹聚乳酸聚羥基乙酸，凍干后得目標骨粉。含0.1umol納米銀的羥基磷灰石及含0.1umol納米銀的羥基磷灰石再包裹聚乳酸聚羥基乙酸均有明顯的抗菌性，體外緩釋實驗結果顯示，納米銀羥基磷灰石在第14天時釋放納米銀達高峰，包裹聚乳酸聚羥基乙酸后，在第42天時釋放納米銀仍呈上升趨勢，未見明顯下降。2、還需細胞相關實驗檢測負載不同濃度納米銀的羥基磷灰石及包裹聚乳酸聚羥基乙酸的羥基磷灰石對細胞的影響，及動物實驗檢測其在種植體周圍炎模型中是否可以正常成骨且可以抗感染，有望成為好的緩釋載體材料。

    【Background】In implantation,a variety of reasons (alveolar bone atrophy after exodontia,trauma, infection, cancer, birth defects, etc.) can cause lack of bone mass in dentalimplants implanted area,which will affect the implant and long-term effect.Implanted bone substitute materials become the essential means to solve this problem,but how to control for postoperative infection is a growing difficulty andhotspot in the treatment of implantation. Currently,widely research of antibioticssuch as vancomycin, tetracycline, levofloxacin due to load onto the bone technologiessuch as bacterial drug resistance and antibiotic release duration is short, but failed toachieve the goal of the ideal control bone infection. Silver, zinc, copper and otherinorganic antibacterial agent doping into bone powder materials can effectively inhibitbacterial infections, but its toxicity to mammalian cells become concerns in theapplication, and how to make its sustained and effective work is not very good solve.In addition, how to effectively use nanotechnology to delay the release ofantimicrobial agent still need further discussion.【Aim】This study try to use beta-cyclodextrin (β-CD) and chitosan (CS) as ligandsynthesis of silver nanoparticles, then different ligands of silver nanoparticles (NP)load in the artificial bone, combined the technology of emulsification, Polylacticacid hydroxy acetic acid(PLGA), PLGA outside the package made antibacterial bonebiomaterial to delay the release of inorganic nano silver. This experiment use thecharacteristics of cyclodextrin, chitosan to cover silver nanoparticles and enhance itsantimicrobial properties in different levels, and nano silver hydroxyapatite wrappedby Polylactic acid hydroxy acetic acid(PLGA),made into bone powder materialcontrol the infection, and slow release of silver nanoparticles. Through theexperiment, hopely can get sustained release of nano silver, until autologous boneformation, achieve the objective of the ideal control bone infection.【Methods and results】1.Preparation and properties of CD-NP Reference literature[1]in the synthesis of silver nanoparticles synthesis nano silverof β-CD ligand, the method is: different concentrations of beta-CD aqueoussolution as a ligand, sodium borohydride as reducts, reduct silver nitrate aqueoussolution to CD-NP. By scanning electron microscopy (sem), transmission electronmicroscopy (sem) observate CD-NPs’ surface morphology, particle diameter andparticle size of aggregate, the stability of the observation period.Spectrophotometerobservate CD-NPs’ absorption peak.Results：As reported in literature[1], with cyclodextrin concentration increased, theobtained nano-silver antibacterial properties enhanced.so that20mM β-CD as ligands,and2mM silver nitrate solution, stirring after20minutes, add sodium borohydridesolution of20mM, get dark solution，30s later, brown solution was formed，after24hs’ stiring, can obtain good stable nano-silver. Electron microscopy (sem)results showed that nano-silver particles into a round shape, particle size, between24to33nm are uniformly distributed and no obvious aggregation and test antimicrobialproperties of different concentrations of silver nanoparticles.. Ultraviolet absorptionspectra showed that CD-NP absorption peak at410nm. Bacteria experiment showedthat chitosan ligands nano-silver have obvious antimicrobial properties, and alongwith the increased concentration of silver nanoparticles, antimicrobial propertiesenhanced.2.Preparation and properties of CS-NPReference literature[2],chitosan as ligand in the synthesis of silver nanoparticles.The method is using chitosan as a ligand and reductant, Sodium hydroxide andacetic acid as adjusts to adjust PH value, obtained CS-NP. CS-NP soluble in0.1%acetic acid solution by scanning electron microscopy (sem) and transmission electronmicroscopy (sem) observate of CS-NP surface morphology, particle diameter andparticle size of aggregate, the stability of CS-NP and test antimicrobial propertiesof different concentrations of silver nanoparticles. Spectrophotometer observateCS-NPs’ absorption peak.Results showed that chitosan(degree>75%) is soluble in1%acetic acidsolution, the chitosan solution was1mg/ml, heating up to45℃and stir until completely dissolved to form chitosan homogeneous solution,24ml chitosanhomogeneous fluid mixed with1ml100mM silver nitrate solution,and add75ml of0.1M sodium hydroxide solution slowly to the mixture, obtaining brown solution.Electron microscopy (sem) results show that CS-NP size ovoid, particle size between5to25nm, have gathered phenomenon, particle size distribution is not uniform.Ultraviolet absorption spectra showed that CS-NP absorption peak at400nm.Bacteria experiment showed that chitosan ligands nano-silver have obviousantimicrobial properties, and along with the increased concentration of silvernanoparticles, antimicrobial properties enhanced.3.Preparation and properties of CD-NP-HA/CS-NP-HAMethods:Different concentration of CD-NP/CS-NP load into the hydroxyapatite bylyophilization.By scanning electron microscopy (sem) and transmission electronmicroscopy (sem) observate CD-NP-HA/CS-NP-HA ‘surface morphology,particle diameter and particle size of aggregate, stability of the observation period,and test antimicrobial properties of hydroxyapatite loading silver nanoparticles indifferent ligand and different concentration.The nano-silver hydroxyapatiteimmersing in simulated body fluid, test slow-release performancein vitro.Results showed that:0.1umol,0.2umol,0.5umol,1.0umol,2.0umol,4.0umol,10.0umol CD-NP to join100mg hydroxyapatite (10mg/ml) in aqueous solution,lyophilization after stirring4hours.0.1umol,0.2umol,0.5umol,1.0umol,2.0umol,4.0umol,10.0umol CS-NP to100mg of hydroxy apatite (10mg/ml) inaqueous solution, lyophilization after stirring4hours. After lyophilizationhydroxyapatite loading nano-silver is into white powder. Electron microscopy(sem) results showed that CD-NP-HA/CS-NP-HA into granular size, particle sizein9nm-60nm, particle aggregation, stability is good. Bacteria experiment resultsshow that the load0.1umol CD-NP-HA/CS-NP-HA have antimicrobial properties.Prolonged-release experimental results show that: on the14th day,releasing is intothe peak.4.Preparation and properties of CD-NP-HA-PLGA/CS-NP-HA-PLGAMethods: Polylactic acid hydroxy acetic acid (PLGA) dissolved in trichloromethane until completely dissolved, CD-NP-HA/CS-NP-HA dissolved in the solution above,violent stir until completely dissolved, above solution quickly into the distilledwater containing0.4%methyl cellulose, stir until chloroform volatilize completely todo. After centrifugal, washing, the solid freeze-dried.By scanning electronmicroscopy (sem), transmission electron microscopy (sem) observation of CD-NPHAPLGA/-CS-NP-HA PLGA surface morphology, particle diameter and particlesize of aggregate, observe its stability, and test load different ligand concentration ofsilver nanoparticles hydroxyapatite antimicrobial properties. PLGA nano-silverhydroxyapatite immersing in simulated body fluid, test slow-release performanceinvitro.Results:200mg of PLGA will dissolve in20ml chloroform,50mg hydroxyapatiteloading nano-silver in different concentration，dissolved in above solution，then allpoured into200ml of distilled water containing0.4%methyl cellulose, getting thebone powder that slowly release silver nanoparticles. Electron microscopy (sem)results showed that the product is into the ball size, diameter is disparity. Maximumcan reach more than100nm in diameter, minimum can be below10nm. Bacteriaexperiment results show that:0.1umolCD-NP and0.1umolCS-NPslow-releasemicrospheres have obvious antimicrobial properties. Slow release experiment resultsshow that:To42days, the release of nano-silver is still on the rise, without significantdecline.Conclusion:1.Through different methods for synthesis of CD-NP、 CS-NP bylyophilization,which load into the nano-hydroxyapatite and wrapedd bypoly(lactic-co-glycolic) acid (PLGA) after freeze-dried,getting target bone meal.Hydroxyapatite containing0.1umol NP and NP-HA-PLGA containing0.1umol NPhave obvious antimicrobial properties.Slow-releasing experimental in vitro resultsshow that the nano-silver hydroxyapatite in14days to release up to peak, afterwraped by PLGA，until42days release the nano-silver is still on the rise,withoutsignificant decline.2.It still need to cell-related experiment testing different concentrations of silver nanoparticles hydroxyapatite and NP-HA-PLGA effects on cells, and model of theinflammation around the implant in animal experiments test whether it can benormal osteogenesis and anti-infection,it is expected to become a good slow-releasecarrier material.

骨粉內納米銀緩釋及抗菌性能研究

中文摘要4-8Abstract8-12第1章 緒論16-26    1.1 前言16-17    1.2 相關材料的研究現(xiàn)狀17-26        1.2.1 銀17-19        1.2.2 殼聚糖19-20        1.2.3 環(huán)式糊精20-23        1.2.4 羥基磷灰石23-24        1.2.5 聚乳酸聚羥基乙酸24-26第2章 實驗部分26-52    2.1 材料與設備26-27    2.2 制備方法27-28        2.2.1 環(huán)式糊精、殼聚糖配體納米銀的制備27        2.2.2 負載納米銀的羥基磷灰石（NP-HA）的制備27        2.2.3 聚乳酸聚羥基乙酸包裹負載納米銀的羥基磷灰石（NP-HA- PLGA）的制備27-28    2.3 性質檢測28-30        2.3.1 電鏡觀察28        2.3.2 紫外吸收光譜分析28        2.3.3 傅里葉紅外光譜分析28        2.3.4 抑菌性檢測28-29        2.3.5 緩釋效果檢測29-30        2.3.6 數(shù)據分析30    2.4 結果與分析30-47        2.4.1 電鏡結果30-36        2.4.2 紫外吸收光譜分析結果36-37        2.4.3 紅外吸收光譜分析結果37-40        2.4.4 抑菌性檢測結果40-41        2.4.5 緩釋效果檢測41-47    2.5 結論47-48    2.6 討論48-52        2.6.1 緩釋系統(tǒng)48-49        2.6.2 納米銀的合成49        2.6.3 PLGA 與羥基磷灰石的結合狀態(tài)49-50        2.6.4 銀的有效殺菌濃度50-52參考文獻52-58作者簡介及學期間取得的科研成果58-59致謝59-60

  本文關鍵詞：骨粉內納米銀緩釋及抗菌性能研究，由筆耕文化傳播整理發(fā)布。