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研究生:張嘉珮
研究生(外文):Chang chia pei
論文名稱:軟骨組織工程中細胞與培養環境之最適化應用於氣管補綴片的評估
論文名稱(外文):Optimization of the cell sources and culture conditions in cartilage tissue engineering for application in tracheal patches
指導教授:徐善慧徐善慧引用關係
指導教授(外文):Hsu shan hui
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:101
中文關鍵詞:軟骨細胞氣管補綴片動態培養生物反應器
外文關鍵詞:chondrocytestracheal patchesdynamic culturebioreactor
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本研究即以組織工程的原理,自小豬取出不同部位的軟骨細胞(耳朵、肋骨、膝關節),分別植入幾丁聚醣及動物明膠混摻的天然材料支架(CG)及利用第二型膠原蛋白改質PLLA /PLGA人工合成材料支架(BCII)中,接著分別置於靜態及動態兩種培養環境下進行培養。培養28天後,分別進行生化分析、機械性質分析、組織切片染色及SEM等一系列的分析及評估。實驗結果發現,細胞在兩種支架中皆可大量生長並分泌基質成分,另外,在組織切片圖中亦可見支架周圍有類軟骨組織的形成。SEM圖的觀察,發現細胞會呈現圓形的型態。最後經由綜合評述比較,發現耳朵軟骨細胞在靜態培養環境下的生長情況最好,而BCII支架可提供較佳的機械強度。因此,針對氣管修復上氣管補綴片的需求,採用BCII支架並植入耳朵軟骨細胞,在靜態條件下進行培養,將是促進軟骨組織生成的最佳組合。
Based on the principle of tissue engineering in this, the chondrocytes harvested from baby porcine ear, costal and knee articular cartilage were seeded into chitosan-gelatin complexes (CG) and the synthetic PLLA/PLGA blends modified by the type II collagen (BCII), and then cultivated in vitro under static and dynamic conditions. After 28d of culture, matrices were evaluated by biochemical analyses, mechanical tests, histochemical staining and scanning electron microscopy (SEM). It was found that these two scaffolds could promote the growth of chondrocytes and enhanced production of extracellular matrix (ECM). In addition, the histological sections showed the generation of cartilage-like tissues in the outer layer of the scaffolds. Scanning electron micrographs showed that the chondrocytes had a round morphology. In conclusion, the static condition was optimal for the baby porcine ear chondrocytes, and BCII scaffolds had better mechanical properties. It would be optimal for tracheal patches application to seed baby porcine ear chondrocytes into BCII scaffolds and cultivate in vitro under static condition.
目 錄
符號縮寫 ………………………………………………………… I
中文摘要 ………………………………………………………… II
英文摘要 ………………………………………………………… III
目錄 ………………………………………………………… IV
圖目錄 ………………………………………………………… VIII
表目錄 ………………………………………………………… X
研究目的 ………………………………………………………… XI
第一章 文獻回顧…………………………………… 1
1.1 軟骨組織學………………………………………… 1
1.1.1 軟骨的分類………………………………………… 1
1.1.2 軟骨細胞…………………………………………… 3
1.1.3 軟骨基質…………………………………………… 4
1.1.3.1 蛋白質多醣…………………………………………. 4
1.1.3.2 膠原蛋白…………………………………………... 5
1.2 氣管軟骨學………………………………………… 6
1.2.1 氣管結構…………………………………………… 6
1.3 關節軟骨的缺損…………………………………… 7
1.3.1 軟骨的鈣化………………………………………… 7
1.3.2 氣管切開術………………………………………… 7
1.4 氣管重建…………………………………………… 8
1.5 組織工程…………………………………………… 9
1.5.1 組織工程的創立…………………………………… 9
1.5.2 組織工程三大元件………………………………… 9
1.6 組織工程的研究發展………………………………. 11
1.6.1 支架的材料研究…………………………………….. 11
1.6.1.1 人工合成材料……………………………………….. 11
1.6.1.2 天然材料…………………………………………….. 12
1.7 組織工程生物反應器……………………………….. 13
1.8 總結………………………………………………….. 15
第二章 實驗儀器、器材與藥品…………………… 16
2.1 實驗儀器…………………………………………… 16
2.2 實驗器材…………………………………………… 17
2.3 實驗藥品…………………………………………… 20
2.3.1 支架製作…………………………………………… 20
2.3.2 膠原蛋白萃取……………………………………….. 20
2.3.3 表面改質……………………………………………... 20
2.3.4 體外細胞培養………………………………………. 21
2.3.4.1 動物軟骨細胞的取得……………………………… 21
2.3.4.2 細胞培養…………………………………………… 21
2.3.4.3 細胞量定量分析……………………………………. 22
2.3.4.4 葡萄糖胺聚合糖定量分析………………………… 22
2.3.4.5 膠原蛋白定量分析………………………………… 22
2.3.4.6 電子顯微鏡觀察…………………………………… 23
2.3.4.7 組織切片分析……………………………………… 23
第三章 實驗方法
3.1 軟骨細胞的取得…………………………………… 25
3.1.1 小豬軟骨細胞的取得……………………………… 25
3.2 支架製作…………………………………………… 26
3.3 膠原蛋白的萃取…………………………………… 27
3.4 支架改質…………………………………………… 28
3.4.1 膠原蛋白改質………………………………............. 28
3.5 支架體外培養……………………………………… 28
3.5.1 小豬軟骨細胞的植入支架方法…………………… 29
3.6 支架的生化分析…………………………………… 29
3.6.1 細胞數分析………………………………………… 29
3.6.2 葡萄糖胺聚合糖分析………………………………. 30
3.6.3 膠原蛋白分析……………………………………….. 31
3.7 掃描式電子顯微鏡觀察…………………………….. 32
3.8 組織切片…………………………………………… 32
3.8.1 基質染色分析………………………………………. 32
3.8.2 H-E stain…………………………………………….. 33
3.8.3 Masson’s Trichrome Stain…………………………… 35
3.8.4 E.V.G Stain…………………………………………… 37
3.9 機械性質分析………………………………………. 39
3.10 生物反應器…………………………………………. 41
3.10.1 自行設計的生物反應器設計圖…………………… 41
第四章 實驗結果…………………………………… 43
4.1 小豬軟骨細胞植入天然材料支架實驗…………… 43
4.1.1 生化分析…………………………………………… 43
4.1.2 動態機械性質分析………………………………… 45
4.1.3 支架形態觀察……………………………………… 46
4.1.4 支架組織切片分析………………………………… 46
4.1.5 SEM圖分析………………………………………… 48
4.2 小豬軟骨細胞植入人工合成改質支架實驗 49
4.2.1 生化分析…………………………………………… 49
4.2.2 動態機械性質分析………………………………… 51
4.2.3 支架形態觀察………………………………………. 52
4.2.4 支架組織切片分析………………………………… 52
4.2.5 SEM圖分析………………………………………… 54
第五章 討論………………………………………… 55
第六章 結論……………………………………………… 59
參考文獻 ……………………………………………………… 61
圖 目 錄
圖 1 CG支架植入小豬軟骨細胞體外培養DNA分析... 66
圖 2 CG支架植入小豬軟骨細胞體外培養GAG分析… 67
圖 3 CG支架植入小豬軟骨細胞體外培養collagen分析…………………………………………………….
68
圖 4 細胞植入CG支架作DMA壓縮模式測試………… 70
圖 5 細胞植入CG支架作DMA拉伸模式測試……… 71
圖 6 體外培養14天後的支架外貌……………………. 72
圖 7 體外培養28天後的支架外貌……………………. 73
圖 8 培養24小時後的支架組織切片圖(H&E stain)….. 74
圖 9 培養14天後的支架組織切片圖(H&E stain)……… 75
圖 10 培養28天後的支架組織切片圖(H&E stain)……… 76
圖 11 小豬軟骨細胞植入支架培養28天後SEM圖…… 77
圖 12 BCⅡ支架植入小豬軟骨細胞體外培養DNA分析 79
圖 13 BCⅡ支架植入小豬軟骨細胞體外培養GAG分析 80
圖 14 BCⅡ支架植入小豬軟骨細胞體外培養collagen分析……………………………………………………
81
圖 15 細胞植入BCⅡ支架作DMA壓縮模式測試……. 83
圖 16 細胞植入BCⅡ支架作DMA拉伸模式測試……. 84
圖 17 體外培養14天後的支架外貌…………………… 85
圖 18 體外培養28天後的支架外貌…………………… 86
圖 19 培養24小時後的支架組織切片圖(H&E stain)…. 87
圖 20 培養14天後的支架組織切片圖(H&E stain)……… 88
圖 21 培養28天後的支架組織切片圖(H&E stain)……… 89
圖 22 小豬軟骨細胞植入支架培養28天後SEM圖…… 90
表 目 錄
表 1 CG支架植入小豬軟骨細胞體外培養DNA分析結果…………………………………………………….
65
表 2 CG支架植入小豬軟骨細胞體外培養GAG分析結果……………………………………………………
65
表 3 CG支架植入小豬軟骨細胞體外培養collagen分析結果………………………………………………….
65
表 4 細胞植入CG支架作DMA壓縮模式測試……….. 69
表 5 細胞植入CG支架作DMA拉伸模式測試……….. 69
表 6 BCII支架植入小豬軟骨細胞體外培養DNA分析結果………………………………………………….
78
表 7 BCII支架植入小豬軟骨細胞體外培養GAG分析結果………………………………………………….
78
表 8 BCII支架植入小豬軟骨細胞體外培養collagen分析結果……………………………………………….
78
表 9 細胞植入BCⅡ支架作DMA壓縮模式測試……… 82
表10 細胞植入BCⅡ支架作DMA拉伸模式測試……… 82
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