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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊台鴻 | zh_TW |
dc.contributor.advisor | Tai-Horng Young | en |
dc.contributor.author | 曾子豪 | zh_TW |
dc.contributor.author | Tzu-Hao Tseng | en |
dc.date.accessioned | 2024-02-22T16:14:40Z | - |
dc.date.available | 2024-02-23 | - |
dc.date.copyright | 2024-02-22 | - |
dc.date.issued | 2024 | - |
dc.date.submitted | 2024-01-29 | - |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/91688 | - |
dc.description.abstract | 在治療骨科感染時,常常使用含抗生素的骨水泥珠子(beads)或填充器(spacer)。然而,這些材料中抗生素釋放不足可能無法消除微生物病原體,導致治療失敗。各種骨水泥配方展現出不同的藥物溶出特性,其中Simplex P骨水泥雖然因易於獲得而廣泛使用,但其抗生素釋放量最低。以往增強這些骨水泥中抗生素釋放的嘗試並未有效轉化為臨床應用。我們的研究著重於利用臨床容易獲得的材料來提高PMMA中抗生素的釋放效果,這一策略使臨床醫生能在手術中應用改進後的方法,有望提高治療成果。
在這項研究中,我們使用的是Stryker Surgical的Simplex P骨水泥(Stryker, Kalamazoo, MI),這是一種全球廣泛使用的商業骨水泥,但其抗生素釋放效果較差。我們的研究選擇了Vancomycin作為抗生素,因為它在臨床上常用,並對抗甲氧西林耐藥金黃色葡萄球菌(MRSA)有效,MRSA是骨髓炎和植入物相關感染的主要原因之一。 在研究的第一部分中,我們創新地使用了Vicryl Rapide縫合線來創造持續釋放的骨水泥珠子。這是通過將縫合線多次穿過珠子和/或將縫合線段混入骨水泥中來達成。這種技術顯著提高了抗生素的釋放效果,且可持續七周。具體而言,第一天的釋放增加了9.0%,從第二天到第四十九天的釋放量增加了118.0%。然而,僅添加縫合線段並沒有增加釋放量。以肉眼及電子顯微鏡觀察,Vicryl Rapide縫合線在周邊完全降解,中心部分部分降解。這種降解促進了抗生素顆粒在縫合線區域的釋放,與抗生素在對照組中有大量抗生素被包覆且無法釋放的情況形成對比。重要的是,加入縫合線的組別展現出明顯更強的抗菌效果。 N-乙酰半胱氨酸(NAC)在臨床上廣泛認可為有效的化痰劑和對抗acetaminophen毒性的解毒劑。有一些研究發現NAC可中和聚甲基丙烯酸甲酯(PMMA)的細胞毒性潛力,儘管NAC本身不是抗生素,許多研究也展現了潛在的抗菌特性。在我們研究的第二部分中,我們將NAC和/或vancomycin混合進PMMA骨水泥中以探討可能的效果。實驗分為六個不同組別,根據其特定組成劃分:(1)2g vancomycin,(2)4g vancomycin,(3)2g vancomycin + 2g NAC,(4)2g vancomycin + 4g NAC,(5)2g vancomycin + 6g NAC,和(6)6g NAC。我們的結果顯示,加入NAC可以延長vancomycin的釋放效率。其中較具臨床意義的是,在有2g vancomycin時,再加入2g、4g或6g NAC的骨水泥配方,都可在七天後顯示出比僅含4g vancomycin的配方更多的vancomycin釋放。此外,添加2g和4g NAC被觀察到能改善細胞活性,但添加過多(6g) NAC會顯著降低細胞活性。在抗菌效果方面,含有NAC和2g vancomycin的PMMA骨水泥在七天後對抗甲氧西林敏感金黃色葡萄球菌(MSSA)和MRSA的活性優於僅含2g或4g vancomycin的骨水泥。這種抗菌功效隨著時間的延長而增加。然而,僅含NAC的骨水泥配方沒有顯示出任何顯著的抗菌特性。 在我們骨水泥研究的剩餘時間裡,我們還探討了IL-6對前十字韌帶(ACL)的影響,作為我們研究的第三部分。這旨在瞭解ACL斷裂後或手術後關節腔內IL-6的增加是否可能是創傷後骨關節炎的成因之一。我們的發現如下: IL-6濃度增加會增強ACL纖維細胞中的periostin(POSTN)基因表達。同樣,西方墨點法分析也證明POSTN蛋白的產生會受到被IL-6的濃度和時間影響。急性或慢性ACL傷害不會對POSTN的產生造成影響,我們觀察到,無論是在傷害後3個月內還是至少6個月後獲得的ACL組織,其POSTN蛋白的產生相似。使用PI3K/Akt阻斷劑可以緩解IL-6對ACL組織的影響。相反,Ras/MAPK和JAK/STAT途徑並未減少POSTN的產生。此外,ACL和IL-6的共同存在導致軟骨細胞增加MMP-13和ADAMTS-4的產生。因此,我們的研究顯示,ACL斷裂後或手術後,IL-6與ACL組織的相互作用可能是軟骨退化的一個重要因素。 | zh_TW |
dc.description.abstract | Antibiotic-loaded bone cement beads and spacers are commonly employed in treating orthopaedic infections. However, inadequate antibiotic release from these materials can fail to eliminate microbial pathogens, potentially resulting in unsuccessful treatments. Various cement formulations exhibit distinct elution characteristics, with Simplex P cement showing the lowest antibiotic release despite its widespread use due to easy accessibility. Prior attempts to enhance antibiotic elution from these cements have not effectively transitioned into clinical application. Our approach focuses on utilizing readily accessible materials to enhance the release profiles of Vancocycin from bone cement. This strategy enables orthopaedic surgeons to apply the improved method during surgery, potentially enhancing treatment outcomes.
In this study, we focused on Stryker Surgical Simplex P [Stryker, Kalamazoo, MI], a globally utilized commercial bone cement, known for its less effective antibiotic elution properties. Our research targeted Vancomycin as the antibiotic of choice due to its prevalent use in clinical settings and effectiveness against methicillin-resistant Staphylococcus aureus (MRSA), a leading cause of osteomyelitis and implant-related infections. In the first part of our research, we innovatively employed Vicryl Rapide sutures to create sustained-release cement beads. This was achieved by threading the sutures multiple times through the beads and/or incorporating suture pieces into the cement powder. This technique notably enhanced the antibiotic elution profiles over a seven-week period. Specifically, there was a 9.0% increase in release on the first day and a substantial 118.0% rise from the second to the forty-ninth day. However, the incorporation of suture pieces alone could not amplify the release quantities. Notably, the sutures underwent complete peripheral degradation and partial central degradation. This degradation facilitated the release of antibiotic particles around the suture area, in contrast to the control group where antibiotics remained densely trapped. The groups with suture incorporation demonstrated markedly stronger antimicrobial effects. N-Acetylcysteine (NAC) is widely recognized in clinical settings as an effective mucolytic agent and as an antidote for acetaminophen toxicity. Emerging research highlights NAC's potential in neutralizing the cytotoxicity of PMMA (Polymethyl methacrylate), and despite not being an antibiotic, NAC demonstrates inherent antimicrobial properties. In the second part of our reasearch, we integrated NAC and/or vancomycin into PMMA bone cement to explore these effects. The experiment was structured into six distinct groups, differentiated by their specific compositions: (1) 2g vancomycin, (2) 4g vancomycin, (3) 2g vancomycin + 2g NAC, (4) 2g vancomycin + 4g NAC, (5) 2g vancomycin + 6g NAC, and (6) 6g NAC. This was achieved by adding precise quantities of vancomycin powder (Sigma, USA) and/or NAC powder (Sigma) to 40g of PMMA powder for each respective group. Our findings reveal that incorporating NAC enhances the release rate of vancomycin. Notably, the bone cement formulations containing 2g vancomycin with incremental additions of 2g, 4g, and 6g NAC showed a more substantial release of vancomycin after seven days compared to formulations with 4g vancomycin alone. Moreover, the addition of 2g and 4g NAC was observed to improve cell viability, whereas a 6g NAC inclusion initially reduced cell viability significantly. In terms of antibacterial effectiveness, PMMA bone cement infused with both NAC and 2g vancomycin exhibited superior activity against MSSA (Methicillin-Sensitive Staphylococcus Aureus) and MRSA after seven days, outperforming bone cement containing either 2g or 4g vancomycin alone. This antibacterial efficacy was found to increase over time. However, bone cement compositions containing only NAC showed no significant antibacterial properties. During the remaining duration of our bone cement research, we also explored the impact of IL-6 on the Anterior Cruciate Ligament (ACL) as the third part of our research. This was aimed at understanding whether the increased intra-articular IL-6 levels following ACL rupture or post-surgical intervention could be a contributing factor to post-traumatic osteoarthritis (OA). Our findings are as follows: Quantitative real-time PCR analysis indicated that IL-6 enhanced POSTN gene expression in ACL fibroblasts in a dose-dependent manner. Similarly, Western blot analysis demonstrated that POSTN protein production was also dose-dependently increased by IL-6. When examining the chronicity of ACL injuries, we observed that POSTN protein levels in ACL remnants were similar, regardless of whether the remnants were obtained within 3 months of injury or after a minimum of 6 months. The use of PI3K/Akt blockers was found to mitigate IL-6's effect on ACL fibroblasts. In contrast, Ras/MAPK and JAK/STAT pathways could not reduce POSTN production. Additionally, the presence of both IL-6 and ACL resulted in increased production of ADAMTS-4 and MMP-13 by chondrocytes. Consequently, our study suggests that the interaction between IL-6 and ACL tissue post-rupture or surgery is a significant factor in cartilage degeneration. | en |
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dc.description.tableofcontents | 誌謝 i
中文摘要 ii Abstract iv Chapter 1 1 1.1 Background 1 1.2 Materials and Methods 3 1.2.1 Fabrication of antibiotic-loaded PMMA 3 1.2.2 Antibiotic elution profile 7 1.2.3 In vitro cytotoxicity assay 8 1.2.4 Bioassay of antibiotic activity 9 1.2.5 Gross appearance and microarchitecture of PMMA/sutures 10 1.2.6 Statistical analysis 11 1.3 Results 12 1.3.1 Polymerization temperatures and curing time of cement beads 12 1.3.2 Antibiotic elution profile 14 1.3.3 In vitro cytotoxicity assay 16 1.3.4 Bioassay of antibiotic activity 18 1.3.5 Gross appearance and microarchitecture of PMMA/sutures 20 1.4 Discussion 25 Chapter 2 31 2.1 Background 31 2.2 Matrials and Methods 33 2.2.1 Fabrication of antibiotic-loaded PMMA cement cylinder 33 2.2.2 Vancomycin and NAC elution profile 33 2.2.3 In vitro cytotoxicity assay 35 2.2.4 Bioassay of antibiotic activity 35 2.2.5 Determination of porosity of cement cylinders 36 2.3 Results 37 2.3.1 Vancomycin and NAC elution profile 37 2.3.2 In vitro cytotoxicity assay 38 2.3.3 Bioassay of antibiotic activity 41 2.3.4 Porosity of cement cylinders 44 2.3 Discussion 44 Chapter 3 47 3.1 Background 47 3.2 Materials and Methods 48 3.2.1 Cell culture 48 3.2.2 Immunofluorescence staining 49 3.2.3 Treatment of IL-6 and/or PI3K/Akt, Ras/MAPK, and JAK/STAT blockers 49 3.2.4 Quantitative real-time PCR 50 3.2.5 Western blot 50 3.2.6 Ex vivo culture of ACL explants and immunohistochemical staining 51 3.2.7 Enzyme-linked immunosorbent assay (ELISA) 52 3.2.8 Transwell co-culture system 52 3.2.9 Statistical analysis 54 3.3 Results 54 3.3.1 Immunofluorescence staining 54 3.3.2 IL-6 dose-dependently increased gene expression of POST 55 3.3.3 IL-6 dose-dependently and time-dependently increased protein production of POSTN 56 3.3.4 Regarding the injury chronicity, ACL fibroblasts produces comparable POSTN 57 3.3.5 The effect of IL-6 on POSTN expression was reduced by PI3K/Akt blocker 60 3.3.6 Findings from explant culture and immunohistochemical staining 61 3.3.7 Findings from co-culture system 63 3.4 Discussion 64 Reference 69 | - |
dc.language.iso | en | - |
dc.title | 由易取得的臨床材料製作緩釋抗生素骨水泥的相關研究 | zh_TW |
dc.title | Fabrication of Antibiotic-Loaded Bone Cement with Easily Accessible Clinical Materials for Sustained Release | en |
dc.type | Thesis | - |
dc.date.schoolyear | 112-1 | - |
dc.description.degree | 博士 | - |
dc.contributor.oralexamcommittee | 江鴻生;李亦淇;李亦宸;胡威文 | zh_TW |
dc.contributor.oralexamcommittee | Hongsen Chiang;I-Chi Lee;Yi-Chen Ethan Li;Wei-Wen Hu | en |
dc.subject.keyword | 抗生素,骨水泥,感染,聚甲基丙烯酸甲酯,釋放,萬古黴素,N-乙醯半胱氨酸, | zh_TW |
dc.subject.keyword | Antibiotics,Cement,Infection,PMMA,Release,Vancomycin,NAC, | en |
dc.relation.page | 83 | - |
dc.identifier.doi | 10.6342/NTU202400191 | - |
dc.rights.note | 未授權 | - |
dc.date.accepted | 2024-01-31 | - |
dc.contributor.author-college | 工學院 | - |
dc.contributor.author-dept | 醫學工程學系 | - |
顯示於系所單位: | 醫學工程學研究所 |
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