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研究生:王銘嘉
研究生(外文):Ming-Jia Wang
論文名稱:負載抗生素之多孔性氫氧基磷灰石對金黃色葡萄球菌抑制效應之研究
論文名稱(外文):Inhibiting Effects of Antibiotics Loaded Porous Hydroxyapatite Microspheres on Staphylococcus Aureus
指導教授:顔秀崗
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生醫工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
畢業學年度:97
語文別:英文
論文頁數:55
中文關鍵詞:氫氧基磷灰石微米球抑菌細胞培養
外文關鍵詞:hydroxyapatitemicrosphereantibacterialcell culture
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本研究以濕性化學法為合成方法,利用具有生物活性之無機陶瓷(氫氧基磷灰石)與生物可降解性之高分子製備出均勻且可控制粒徑大小之複合微米球。各種檢測技術譬如X光繞射(XRD)、傅立葉轉換紅外線光譜(FTIR)、電子顯微鏡觀察(SEM/TEM)、感應耦合電漿質譜儀分析(ICP-MS)與熱分析(TGA/DSC)等被用來鑑定氫氧基磷灰石-生物高分子複合微米球之結晶相、成分組成、表面形貌、化學結構及熱穩定性,分析結果發現此微米球呈現多孔狀且擁有大量的微孔與介孔,因而展現出高比表面積的特色,藉由此優點將微米球作為藥物載體應用於骨組織感染治療可大幅提高載藥量,因此本研究以微米球負載gentamicin、vancomycin、teicoplanin和zyvox四種不同的抗生素,探討其釋放行為及其對金黃色葡萄球菌(staphylococcus aureus)之抑菌效應。釋放曲線顯示出在初期12小時內,四種抗生素於50 ml 之磷酸鹽緩衝溶液中有爆發性釋放,其濃度針對金黃色葡萄球菌皆能高於最低抑菌濃度(MIC),隨後則維持著緩慢釋放,而由實驗結果亦可得知化學結構中具有羧基之抗生素如vancomycin和teicoplanin與氫氧基磷灰石之間有較強之鍵結,因而導致有較長期緩慢且持續性的釋放行為。利用從藥物釋放試驗所獲得之釋放溶液,以瓊脂擴散法進行抑菌試驗,不同時間點所獲得之釋放液皆有顯著的抑菌圈,且抑菌圈直徑大小和各時間點所測得之抗生素濃度成正相關,此現象證實抗生素被負載於複合微米球再經過釋放之後,仍保有化學活性與穩定性。在細胞實驗中,因為本研究所合成的微米球含有生物高分子的成分,擁有細胞增殖所需要的胺基酸,加上氫氧基磷灰石具有生物活性及導骨性,因此以微米球進行體外細胞培養結果指出此微米球能增加類骨母細胞的增殖與分化,且以微米球之萃取液進行細胞毒性測試亦顯示出其對細胞不具毒性。整體而言,本研究成功的製備出具有高抗生素負載量及可注射式之氫氧基磷灰石-生物高分子複合微米球,並且達到有效延長藥物釋放、抗菌目的及提升細胞生長,此將有助於解決骨組織疾病所引發之棘手問題,並發揮微小傷口及骨整合等優點。
In this study, composite microspheres composed of HA and biopolymer with uniform morphology and controllable size were synthesized from a mixed solution of calcium phosphate and biodegradable polymer by wet-chemical method. Techniques such as X-ray diffraction (XRD), Fourier transform infrared spectrograph (FTIR), electron microscopy examination (SEM/TEM), inductively coupled plasma-mass spectroscopy (ICP-MS) and thermal analysis (TGA/DSC) were used to characterize HA-biopolymer microspheres by considering their crystalline phase, composition, morphology, chemical bonding and thermal stability. The results indicated that the obtained microspheres were porous in nature and revealing remarkable micropore and mesopore volume, and high specific surface area which are suitable for the drug carriers. Taking advantage of the high drug loading ability, different antibiotics were loaded into microspheres and their release and efficacy of against bacteria were investigated for the treatment of bone infection. Gentamicin, vancomycin, teicoplanin, and zyvox were the antibiotics and Staphylococcus aureus was the bacteria used in this research. The release profiles revealed the concentration of burst release within 1 h in 50 mL PBS were higher than minimum inhibitory concentration (MIC) of all antibiotics for Staphylococcus aureus and then followed by a long-term sustaining release. The data also suggested that the chemical bonding of antibiotic containing carboxylic groups such as vancomycin and teicoplanin were able to bind with HA strongly, resulting in a slower release rate over a prolonged period. The antibacterial activity of the elution fluid containing antibiotics obtained from the release test was checked using the agar diffusion method. The elution fluid revealed a distinct bacterial inhibitory zone which was related to the measured concentration at each immersion time. This demonstrated that antibiotics loaded into the composite microspheres remained chemically stable and active after release. Because of the peptide of biopolymer and the bioactivity and osteoconductivity of HA, the in vitro cellular attachment with microspheres, the composite microspheres could enhance the proliferation and differentiation of osteoblast-like cells. The results of cytotoxic test with extracts of microspheres also showed that the microspheres prepared in this research were non-toxic. In general, the high antibiotics-loaded and injectable microspheres have been prepared successfully to achieve drug sustaining release for antibacterial purpose and promote osteoblast growth. This achievement should be very helpful to resolve the tough problems of bone disease because of the advantages of micro-invasion and osseointegration.
摘要……………………………………………………………………………………i
Abstract………………………………………………………………………………iii
Contents………………………………………………………………………………v
Table captions…………………………………………………………………………vi
Figure captions………………………………………………………………………vii
Chapter 1 Introduction…………………………………………………………………1
Chapter 2 Materials and methods………………………………………………………6
2-1 Materials preparation and characterization……………………………………6
2-2 Drug loading and release kinetic………………………………………………8
2-3 Antibacterial assessment………………………………………………………9
2-4 Cell experiment………………………………………………………………10
Chapter 3 Results and discussion………………………………………………………13
3-1 Materials characterization……………………………………………………13
3-2 Drug release……………………………………………………………………31
3-3 Antibacterial assessment………………………………………………………38
3-4 Cell experiment………………………………………………………………42
Chapter 4 Conclusions…………………………………………………………………48
Chapter 5 References…………………………………………………………………50
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