臺灣博碩士論文加值系統

第壹部份
題目 : 以新式溫感型奈米水膠包埋抗生素治療骨髓炎

骨髓炎引起發炎反應通常會造成大量骨缺損及附近軟組織破壞。治療上採用大範圍清創手術及抗生素治療四至六週，為避免長期使用抗生素的併發症產生，使用局部攜帶抗生素系統治療骨髓炎是目前世界上發展的新趨勢。在此我們研發一項新式溫感型奈米水膠(由mPEG 及 PLGA 組成)包埋 Teicoplanin 治療骨髓炎，奈米水膠材料特性將被詳細評估及測試，並且近一步使用兔子骨髓炎實驗模型作為動物實驗體內基礎。結果顯示此新式溫感型奈米水膠具備以下特點 : 1.製程容易。2. 有百分之百的藥物包埋率。3. 接近直線型的藥物釋放曲線。4. 可注射式的設計，適合小傷口使用，增加可應用性。5. 以及高度生物相容性。和傳統使用骨水泥包埋抗生素治療骨髓炎相比較，在組織學、分子生物學、及組織免疫學成效上，兩者治療成效相當類似。因此我們認為新式溫感型奈米水膠，在治療骨髓炎，效果相當顯著。在其他骨科相關疾病上，新式溫感型奈米水膠將有相當廣泛的應用範圍。


第貳部分
題目: 使用蛋白質體研究法分析人工髖關節鬆脫的生物標記

人工髖關節鬆脫是接受關節置換手術後最大的併發症，針對鬆脫機轉，已經有許多理論被提出。人工關節磨耗出的顆粒，引發發炎反應，進一步活化噬骨細胞，造成骨溶解及人工髖關節零件鬆脫，是目前最常被提出的理論。而特殊的細胞激素；如干擾素及介白質等，也會活化噬骨細胞。其他一些轉譯因子如MAP Kinase 、 NFκB ，也會活化噬骨細胞造成人工髖關節鬆脫。
近幾年許多反應氧族(ROS)如(hydrogen peroxide [H2O2], hydroxyl radicals radical [OH*] and superoxide anion [O2-*]).在髖關節零件鬆脫的機轉中扮演的角色越來越被重視。在這篇研究中，我們使用蛋白質體研究法，分析人工髖關節鬆脫患者關節液中的生物標記。成功的發現transthyretin (TTR) 及 peroxiredoxin 2 (PRDX 2) 兩個ROS相關蛋白質，並且進一步分析驗證ROS相關抗氧化媒(Catalase, SOD 1, SOD 2 and SOD 3)在髖關節零件鬆脫中的角色。經由西方點墨法及組織免疫染色法，證實在人工髖關節鬆脫患者的關節液中，TTR 及 SOD 3 相對於人工髖關節未鬆脫及初置換人工髖關節患者而言，表現明顯上升。並且在人工髖關節鬆脫患者的關節囊上，PDRX 2及SOD 2 的表現相對於人工髖關節未鬆脫及初置換人工髖關節患者，也有明顯的上升。以上幾個ROS相關性的蛋白質我們相信是文獻上第一次被證實的人工髖關節的生物標記，對於人工髖關節鬆脫的機轉及探討，將有相當大的助益。

Part I : Treatment of osteomyelitis with teicoplanin-encapsulated biodegradable thermosensitive hydrogel nanoparticles
Osteomyelitis characterized by an inflammatory response often leads to bone loss and the spread of bacterial infection to surrounding soft tissues. To overcome the side effects induced by the systemic antibiotic treatment for osteomyelitis, recent investigations have explored the use of antibiotic-loaded undegradable or biodegradable delivery implants at the infected bone. Here, we show a novel biodegradable thermosensitive implant composed of poly(ethylene glycol) monomethyl ether (mPEG) and poly(lactic-co-glycolic acid) (PLGA) copolymer as a sol-gel drug delivery system for treating bone infection. The physical properties of a series of mPEG-PLGA nanocomposites, including the critical micelle concentration (CMC), particle size, polyindex (PI), sol-gel transition, viscosity and degradation rate, have been characterized in vitro. This sol-to-gel drug delivery system could provide several advantages in treating osteomyelitis, including easy preparation, 100 % encapsulated rate, near-linear sustained release of drugs, injectable design and in situ gelling at the target tissue. Similar to the undegradable teicoplanin-impregnated polymethylmethacylate (PMMA) bone cements, we showed that implantation of the mPEG-PLGA hydrogel containing teicoplanin was effective for treating osteomyelitis in rabbits as detected by the histological staining and immunoblotting analyses. The use of the mPEG-PLGA-based biodegradable hydrogels may hold great promise as a therapeutic strategy for other infected diseases.


Part II : Identification of ROS-associated Makers in Aseptic Loosened Total Hip Arthroplasty
Aseptic loosening remains the major complication after total hip arthroplasty (THA). Several theories on the cause of aseptic loosening have been proposed. Wear-generated particles are commonly accepted that the initiating cause with an inflammatory response to phagocytosis and resulting in increased proliferation and differentiation of osteoclast precursors into mature osteoclasts. Progressive osteolysis caused by active-osteoclasts can result in prosthesis failure, eventually requiring revision surgery. More recently, it has been established that wear also inhibits the protective actions of antiosteoclastogenic cytokines such as interferon gamma, thus promoting differentiation of macrophages to bone-resorbing osteoclasts. At a molecular level, wear particles activate MAP kinase cascades, NFκB and other transcription factors, and induce expression of suppressors of cytokine signaling. Besides particles, stress shielding, high fluid pressure, genetic variations, and endotoxin were also considered the possible etiologies for loosening. Recently, the role of reactive oxygen species (ROS) (hydrogen peroxide [H2O2], hydroxyl radicals radical [OH*] and superoxide anion [O2-*]) as products of metal corrosion, mostly of titanium, is more often discussed with respect to the overall process of aseptic loosening [8]. In addition, a number of groups studied the influence of H2O2 and ROS from macrophages in process of inflammatory after implantation of prosthesis.
However, aseptic loosening of joint implants is still a major problem and need to identify the possible makers. In present study, a proteomic approach was applied to discover novel loosened THA-specific proteins by comparing the expression profiles of synovial fluid from patients with loosened or non-loosened implant in-vivo. Two ROS-associated proteins, including transthyretin (TTR) and peroxiredoxin 2 (PRDX 2), were identified and verified. We also evaluated the role of ROS-associated enzymes (catalase, SOD 1, SOD 2 and SOD 3) in joint synovial fluid and capsule of loosened THA. Compare to non-loosened THA and primary groups, TTR and SOD 3 in synovial fluid of hip joint shows high expression in loosened THA group (p<0.05). PDRX 2 and SOD 2 in hip capsule demonstrate high expression in loosened THA group than non-loosened and primary THA groups
To our knowledge, these special biomakers were firstly identified in loosen or non-loosened THA in present study. We believe increased knowledge of the biomakers in joint fluid or hip capsule of THA may prove useful for understanding the causes of THA failure.

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誌謝 .................................................v
中文摘要 .............................................vi
第壹部份 .............................................vi
第貳部分 .............................................vii
英文摘要 .............................................viii
目錄 .................................................xi
Directory of Figures ................................xvi
Directory of Tables ................................ xviii
Abstract-part 1 ......................................viii
Abstract-part 2 ......................................ix
Chapter 1 Difficult in Management of osteomyelitis ...x
1.1 Background of osteomyelitis ...............1
1.2 Epidemiology of osteomyelitis ................3
1.3 Classification of osteomyelitis ..................4
1.4 Microbiology of osteomyelitis ....................5
1.5 Pathogenesis of osteomyelitis ....................6
1.6 Diagnosis of Osteomyelitis....................... 8
1.7 Treatment of osteomyelitis ....................... 9
1.7.1 Medicine ..........................................9
1.7.2 Surgery ...........................................10
1.7.3 Adjuvant Therapy ...................................10
1.8 Complications of systemic medical treatment ......11
1.9 Local antibiotic delivery system ..................11
1.10 esearch motive – Develop a novel local delivery ....12
Chapter 2 Study on Novel Biodegradable Thermo-sensitive Hydrogels ...............................................13

2.1 Introduction of biodegradable thermo-sensitive hydrogels ..............................................14
2.2 Advantages of our biodegradable thermo-sensitive hydrogels ...............................................15
2.3 Materials and Methods .....................16
2.3.1. Chemicals .........................................16
2.3.2. Synthesis of mPEG–PLGA diblock copolymers ...16
2.3.3. Proton nuclear magnetic resonance (1H NMR) ....17
2.3.4. Gel permeation chromatography (GPC) ..........17
2.3.5. Critical micelle concentration (CMC) determination 18
2.3.6. Micelle size determination ..........18
2.3.7. Determination of sol–gel phase transition .....18
2.3.8. Viscosity of sol–gel transition .........19
2.4 Results and Discussion ..................19
2.4.1. Characterization of the mPEG–PLGA hydrogel ....19
2.4.2. In vitro drug release .................22
2.5 In vitro preliminary results of novel hydrogels containing teicoplanin demonstrated high potential to
treat osteomyelitis ...................................23

Chapter 3 Treatment of osteomyelitis with teicoplanin-
encapsulated biodegradable thermosensitive hydrogel nanoparticles in a rabbit model .......................24
3.1 In vivo study ...................................25
3.1.1. Production of osteomyelitis in a rabbit model .....25
3.1.2. Histological study ................................26
3.1.3. Western blot analysis .......................26
3.2 Statistic analysis ......................27
3.3 In vivo evaluation ....................28
3.4 Conclusions ........................................31

Part 2 Proteomic Identification of ROS-associated Makers in Aseptic Loosened Total Hip Arthroplasty ...............32
Chapter 4 Proteomic analysis of synovial biomakers in aseptic loosened total hip arthroplasty ......33
4.1 Introduction ...................................34
4.2Mateials and methods .................................35
4.2.1 Participants ......................................35
4.2.2 Radiographic study ................................35
4.2.3 Preparation of synovial fluid of joint .......36
4.2.4 Isoelectric focusing and gel electrophoresis (2-DE) 37
4.2.5 Silver staining ................38
4.2.6 Gel imaging ........................................38
4.2.7 In gel tryptic digestion and protein identification - Matrix-assisted laser desorption ionization time-of-flight mass spectrometry .....................................38
4.2.8 Veridation of identified proteins- Western blot analysis and Immunohistochemistry(IHC) ......39
4.3 Statistical analysis ..........................40
4.4 Results .......................................41
4.4.1 Clinical status of the participants ...........41
4.4.2 2-DE and protein identification ..............41
4.4.3 Varidation of identified protein and associated-ROS enzymes .................................................42
4.5 Discussion ..........................................43
5. Conclusions ..........................................48
Directory of Figures ....................................49
Directory of Tables .....................................71
Directory of Scheme .....................................73
Reference..............................................74
Addendum..............................................84

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