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研究生:史漢欽
研究生(外文):Han-Chin Shih
論文名稱:戊二醛/1-(3-二甲基胺丙基)-3-乙基碳二亞胺交聯的明膠支架於腸平滑肌細胞組織工程之研究
論文名稱(外文):Glutaraldehyde/1-(3- Dimethylaminopropyl)-3-Ethylcarbodiimide Crosslinked Gelatin Scaffold for Tissue Engineering of Intestinal Smooth Muscle Cells
指導教授:謝明發謝明發引用關係
指導教授(外文):Ming-Fa Hsieh
學位類別:碩士
校院名稱:中原大學
系所名稱:醫學工程研究所
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:98
中文關鍵詞:戊二醛明膠1-(3-二甲基胺丙基)-3-乙基碳二亞胺小腸組織工程
外文關鍵詞:glutaraldehydegelatin1-(3-dimethylaminopropyl)-3-ethylcarbodiimideintestine tissue engineering
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短腸症患者由於腸道被切除,會有營養、吸收不良的症狀。目前的醫學解決方式多為使用全靜脈營養治療、藥物治療以及外科手術治療,但均帶有副作用,所以利用組織工程的技術幫助小腸再生為目前亟欲發展的治療方式。本研究以天然高分子明膠為基底材料,準備10、15、20 wt%三種不同濃度的明膠溶液,利用兩種交聯劑戊二醛與1-(3-二甲基胺丙基)-3-乙基碳二亞胺進行化學交聯,再使用冷凍乾燥法製備出外徑0.8 cm、內徑0.3 cm、長度3 cm可應用於小腸組織工程的管狀生物材料支架。本研究對支架進行物理/化學性質鑑定、靜態培養平滑肌細胞於支架並評估支架的體外/體內生物相容性。以掃描式電子顯微鏡觀察GA交聯明膠支架孔洞大小約為100-300 μm,利用茚三酮試劑測得支架的交聯率為30 %,支架在去離子水中24小時後支架含水率為600 %,以阿基米得原理測得支架孔洞率24-40 %,管狀支架的楊氏係數64-102 MPa,在降解率測試的結果發現,GA交聯的明膠支架在磷酸鹽緩衝溶液中24小時後失去支架結構,以ISO10993-5體外生物相容性評估顯示GA交聯的明膠支架細胞毒性高,在材料正下方纖維母細胞死亡率達70 %,皮下植入結果發現,在植入1週後有大量免疫細胞出現在支架周圍。以相同的鑑定方法得到結果顯示,EDC交聯明膠支架的孔洞大小為100-300 μm,交聯率為80 %,含水率為320-500 %,孔洞率為40-64 %,楊氏係數為52-59 MPa,在磷酸鹽緩衝溶液中可維持支架形狀達14天,添加5 U/mL的膠原蛋白酶後支架形狀可維持7天,體外生物相容性結果顯示細胞毒性較低,在材料正下方纖維母細胞死亡率為40 %,皮下植入結果發現,與GA交聯明膠支架比較,在植入1週後並沒有出現大量免疫細胞,表示使用EDC交聯的支架具有較佳的生物相容性。利用離心法在EDC交聯的管狀明膠支架種植平滑肌細胞種植率達到71 %,靜態培養1及7天後分別以掃描式電子顯微鏡觀察在管狀明膠支架上的平滑肌細胞,使用SMA與α-smooth muscle-cy3標定平滑肌細胞上的肌動蛋白後,觀察在管狀支架上的平滑肌細胞生長情形,與第1天相比在第7天時細胞數量明顯增多。在未來將把此材料移植入生物體內評估對小腸再生的影響,預期對於小腸再生會有良好的助益。
Short bowel syndrome (SBS) patients is massive resection of the small intestine. The patients require total parenteral nutrition (TPN), medication, and surgical operation to different degrees, but these therapies have side effects. This study aims to crosslink gelatin with glutaraldehyde (GA) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) to fabricate a tubular scaffold for small intestine tissue engineering. This study aims to crosslink 10, 15, 20 wt% difference concentration gelatin with glutaraldehyde (GA) and 1-ethyl-3-3-dimethylaminopropyl carbodiimide hydrochloride (EDC) than use freeze dryer to fabricate a outside 0.3 cm, inside 0.8 cm higher 3 cm tubular scaffold for small intestine tissue engineering. The scaffold’s physical and chemical properties were characterized. Its biocompatibility was evaluated by observing smooth muscle cell (SMC) seeding on the scaffold and in vivo, in vitro study. Use Scanning Electron Microscope observe the glutaraldehyde crosslinked scaffold had a pore size range of 100-300 μm, Ninhydrin test get 30 % crosslinking index, Use ddH2O make Swelling tset get 600 % swelling ratio, Archimedes test get porosity of 24-40 % and the machine test get Young’s modulus of 64-102 MPa. The results obtained from in vitro degradation study showed that the GA groups degrade before 24 hours. The biocompatibility results also show GA groups were cytotoxic to L929 cells, under the scaffold’s cell cytotoxic ratio is 70%. In vivo study observed many immunization cell around scaffold after implantation 1 week. Meanwhile, the EDC crosslinked scaffolds resulted to a pore size range of 100-300 μm, 80 % crosslinking index, 320-500 % swelling ratio, porosity of 40-64 % and a Young’s modulus of 55 MPa, scaffold can maintain their structure in PBS more than 14 days, scaffold can maintain there structure in collagenase PBS 7 days. In vivo study observed few immunization cell around scaffold after implantation 1 week. Cells were seeded on the EDC crosslinked scaffolds using centrifugal cell immobilization (CCI) method. The seeding efficiency was 71 %. The cell’s morphology and growth on the scaffold were observed with the use of SEM, immunofluorescence (IF) and Immunohistochemistry (IHC) after static growth. At future will implantation in biological’s body to evalulate for intenstine’s regeneration.
摘要
Abstract
誌謝
目錄
圖索引
表索引
中英文對照表
第一章 緒論
1-1 研究背景
1-2研究動機及目的
第二章 理論基礎
2-1 小腸生理學
2-1-1 消化系統
2-2-2 小腸生理構造
2-2短腸症
2-3 組織工程簡介
2-3-1 支架
2-3-2 細胞
2-3-3 訊息因子
2-3-4 天然與合成材料於小腸組織工程之文獻回顧
2-4 明膠材料簡介
2-4-1 明膠特性
2-4-4 明膠支架孔洞製作
第三章 研究材料與方法
3-1 研究方法
3-2 儀器設備及藥品
3-3 藥品配置
3-4 材料支架製作方法
3-5 材料分析
3-5-1 掃描式電子顯微鏡
3-5-2 示差掃描卡量計
3-5-3交聯指數
3-5-4 支架含水率
3-5-5 支架孔洞率
3-5-7 支架機械力量測
3-6 生物相容性評估
3-6-1 纖維母細胞解凍
3-6-2 纖維母細胞繼代培養
3-6-3纖維母細胞計數
3-6-4 纖維母細胞冷凍保存
3-6-5 瓊脂擴散間接毒性測試
3-7 種植平滑肌細胞於支架上
3-7-1 平滑肌細胞培養
3-7-2 使用離心方式種植細胞於支架上
3-7-3 細胞支架掃描式電子顯微鏡觀察
3-7-4 免疫螢光染色
3-7-5 免疫化學染色
實驗步驟:
3-8 動物實驗
3-8-1 皮下生物相容性測試
3-9 統計分析方法
第四章 結果與討論
4-1明膠支架製作
4-1-1 使用GA製作明膠支架
4-1-2 使用EDC製作明膠支架
4-2 明膠支架物理/化學分析
4-2-1 支架結構掃描式電子顯微鏡觀察
4-2-2 支架交聯指數測量
4-2-3 支架孔洞率
4-2-4 支架含水率測量
4-2-5 支架示差掃描卡量計分析
4-2-6 支架體外降解測試
4-2-7 支架機械力量測
4-3 體外相容性評估
4-3-1 瓊脂擴散間接毒性測試
4-4 種植平滑肌細胞於支架上
4-4-1 尋找最佳細胞種植方式
4-4-2 細胞於支架上掃描式電子顯微鏡觀察
4-4-3 免疫螢光、免疫化學染色觀察
4-5 動物實驗
4-5-1 皮下生物相容性
第五章 結論
參考文獻
附錄

圖 2-1小腸組織工程概要圖解說明
圖 2-2 明膠利用GA交聯反應機制
圖 2-3 明膠利用EDC及NHS交聯反應機制
圖 3-1實驗架構
圖 3-2 支架示意圖
圖 4-1 比較不同冷凍溫度之GA交聯明膠支架SEM圖
圖 4-2 GA10支架SEM圖
圖 4-3 GA15支架SEM圖
圖 4-4 GA20支架SEM圖
圖 4-5 EDC10支架SEM圖
圖 4-6 EDC15支架SEM圖
圖 4-7 EDC20支架SEM圖
圖 4-8 GA與EDC交聯明膠管狀支架之交聯指數
圖 4-9 GA與EDC交聯明膠管狀支架之支架孔洞率
圖 4-10 明膠管狀支架之支架含水率
圖 4-11 GA與EDC交聯明膠管狀支架之DSC分析
圖 4-12 EDC交聯明膠管狀支架之支架降解率
圖 4-13 使用光學顯微鏡拍攝材料底下纖維母細胞細胞生長情形
圖 4-14使用光學顯微鏡拍攝材料底下纖維母細胞細胞生長情形
圖 4-15 平滑肌細胞生長於EDC交聯明膠管狀支架上1天之SEM圖
圖 4-16 平滑肌細胞生長於EDC交聯明膠管狀支架上7天之SEM圖
圖 4-17 平滑肌細胞生長於EDC交聯明膠管狀支架上1天之免疫螢光染色
圖 4-18 平滑肌細胞生長於EDC交聯明膠管狀支架上7天之免疫螢光染色
圖 4-19 平滑肌細胞生長於EDC交聯明膠管狀支架上1天之免疫化學染色
圖 4-20 GA交聯明膠管狀支架植入小鼠皮下組織觀察發炎情形
圖 4-21 EDC交聯明膠管狀支架植入小鼠皮下組織觀察發炎情形

表 3-1 儀器設備目錄
表 3-2 藥品目錄
表 3-3 Zone index分級
表 4-1 各組支架之玻璃轉移溫度
表 4-2 GA交聯明膠管狀支架之機械性質
表 4-3使用統計方法分析GA各組之差異
表 4-4 EDC交聯明膠管狀支架之機械性質
表 4-5使用統計方法分析EDC各組之差異
表 4-6 GA與EDC交聯的明膠管狀支架與纖維母細胞共培養 24小時後材料下與材料至0.5 cm的細胞存活率
表 4-7比較不同離心力種植細胞於支架上之細胞種植率
表 4-8比較不同離心次數種植細胞於支架上之細胞種植率
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