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研究生:黃彥富
論文名稱:鯊魚軟骨素與物理刺激對於體外軟骨再生組織的影響
論文名稱(外文):The effect of physical stimulations and chondroitin sulfate modification on the neocartilage formation
指導教授:徐善慧徐善慧引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:90
中文關鍵詞:軟骨細胞鯊魚軟骨素超音波生物反應器
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本研究要以體外測試探討以新改質物質鯊魚軟骨素應用在軟骨組織工程上的可能性,及豬關節軟骨細胞植入支架在不同的轉速的生物反應器內的生長情況。
在新改質物質上的研究,chondroitin sulfate (CS)在促進軟骨細胞的增生及基質分泌上沒有明顯效應,但是卻有促進外圍的軟骨細胞向支架內生長的現象。而在經過超音波刺激的軟骨細胞的確能增加細胞在支架上的貼附狀況。
利用小豬關節軟骨細胞在植入支架後經由靜置培養及不同轉速的rotator型生物反應器培養四週後的研究中,發現15∼18 rpm的生物反應器所培養出的細胞支架組織,其內部的細胞活性都比靜態的良好,且均沿著支架材料穩固的貼附及生長,細胞也有較聚集且均勻分佈在支架內部。因此15∼18 rpm的rotator型生物反應器培養系統較能有效提供養份及廢物的質傳與適當的機械應力刺激,進而促進體外關節軟骨的再生及維持細胞生長及型態。
In this study, polyester scaffolds modified by type collagen and chondroitin sulfate (CS) were evaluated as tissue engineering cartilage scaffolds. The 2D and 3D culture of chondrocytes harvested form porcine knee articular cartilage revealed that there were no major differences in the cell number and biochemical contents between CS modified scaffolds and control. However, CS appeared to promote cellular ingrowth and cartilaginous tissue formation, based on the histologic studies. Chondrocytes pre-exposed with ultrasound attached better on scaffolds.
In vitro chondrogenesis was also studied under controlled dynamic conditions. The porcine chondrocytes were seeded into the scaffolds and cultivated in either static condition or rotating-type bioreactor with different rotating speeds. It was demonstrated that the rotating-type bioreactor (15~18 rpm) could promote the growth of chondrocytes, help to maintain their phenotype and increase the production of extracellular matrix. Cells proliferated into the scaffolds and became more uniformly distributed after 4 weeks. In conclusion, CS, ultrasound and rotating-type bioreactor in 15~18 rpm could have the potential to improve the quality of engineered cartilage.
目 錄
符號縮寫 ………………………………………………………… I
中文摘要 ………………………………………………………… III
英文摘要 ………………………………………………………… IV
目錄 ………………………………………………………… V
圖目錄 ………………………………………………………… X
表目錄 ………………………………………………………… XII
研究目的 ………………………………………………………… XIII
第一章 文獻回顧…………………………………………… 1
1.1 軟骨組織生理學……………………………………… 1
1.1.1 軟骨組織……………………………………………… 1
1.1.2 軟骨組織的分類……………………………………… 1
1.1.3 軟骨細胞……………………………………………… 2
1.1.4 軟骨基質……………………………………………… 2
1.1.4.1 軟骨基質-蛋白質多醣……………………………… 4
1.1.4.2 軟骨基質-膠原蛋白(collagen)……………………… 5
1.2 組織工程(Tissue Engineering)……………………… 6
1.2.1 組織工程三元件之一:支架(scaffold)……………… 6
1.2.2 組織工程三元件之二:細胞(cell)…………………… 7
1.2.3 組織工程三元件之三:細胞訊息因子……………… 8
1.3 組織工程的研究發展………………………………… 9
1.3.1 支架的材料研究……………………………………… 9
1.3.1.1 天然高分子材料……………………………………… 9
1.3.1.2 合成高分子材料……………………………………… 10
1.3.1.3 支架的改質研究……………………………………… 11
1.4 超音波物理刺激效應(Ultrasound)………………… 13
1.5 組織工程生物反應器(Bioreactor)………………… 14
1.6 總結…………………………………………………… 16
第二章 超音波刺激系統架設與生物反應器設計……… 17
2.1 超音波刺激系統架設………………………………… 17
2.1.1 超音波刺激器………………………………………… 17
2.1.2 架設設計圖…………………………………………… 17
2.2 自行設計的生物反應器設計圖……………………… 20
第三章 實驗儀器、器材與藥品…………………………… 21
3.1 實驗儀器……………………………………………… 21
3.2 實驗器材……………………………………………… 22
3.3 實驗藥品……………………………………………… 22
3.3.1 支架製作……………………………………………… 22
3.3.2 膠原蛋白萃取………………………………………… 23
3.3.3 表面改質……………………………………………… 23
3.3.4 體外細胞相容性測試………………………………… 24
3.3.4.1 動物軟骨細胞取得…………………………………… 24
3.3.4.2 細胞培養……………………………………………… 24
3.3.4.3 DNA定量分析……………………………………… 24
3.3.4.4 GAG定量分析……………………………………… 25
3.3.4.5 Collagen定量分析…………………………………… 25
3.3.4.6 電子顯微鏡觀察……………………………………… 26
3.3.4.7 組織切片分析………………………………………… 26
第四章 實驗方法…………………………………………… 27
4.1 鯊魚軟骨素對於體外軟骨再生影響之實驗設計…… 27
4.2 評估物理刺激應用於體外軟骨再生之實驗設計…… 28
4.3 軟骨細胞的取得……………………………………… 29
4.3.1 成豬與小豬膝關節軟骨細胞的取得………………… 29
4.4 薄膜製作……………………………………………… 30
4.4.1 天然材料薄膜製作…………………………………… 30
4.5 薄膜材料的細胞相容性測試………………………… 31
4.6 支架製作……………………………………………… 32
4.7 膠原蛋白的萃取與分析……………………………… 32
4.7.1 膠原蛋白的萃取……………………………………… 32
4.7.2 膠原蛋白電泳分析…………………………………… 33
4.8 支架改質……………………………………………… 34
4.8.1 第二型膠原蛋白改質………………………………… 34
4.8.2 第二型膠原蛋白+鯊魚軟骨素改質………………… 35
4.9 支架體外培養………………………………………… 36
4.9.1 PC細胞的植入支架方法…………………………… 36
4.10 支架的生化分析……………………………………… 37
4.10.1 細胞數分析(DNA分析)……………………………… 37
4.10.2 細胞基質-葡萄糖胺聚合醣分析(GAG分析)……… 38
4.10.3 細胞基質--膠原蛋白分析(Collagen分析)…………… 38
4.11 掃描式電子顯微鏡觀察……………………………… 40
4.12 組織切片分析………………………………………… 40
4.13 機械性質分析………………………………………… 42
第五章 實驗結果…………………………………………… 44
5.1 新材料PLGA的評估………………………………… 44
5.1.1 新材料PLGA的PC細胞相容性比較……………… 44
5.1.2 Blend-N的3D多孔支架製作與孔洞分析…………… 45
5.2 自行萃取的成豬type collagen純度測定………… 45
5.2.1 type collagen純度測定(SDS-PAGE)……………… 45
5.3 以鯊魚軟骨素改質type collagen的研究………… 46
5.3.1 C薄膜經CS改質後對PC貼附及生長之影響…… 46
5.3.2 超音波前處理對PC在以CS改質的C薄膜上PC貼附及生長…………………………………………… 47
5.4 成豬膝關節軟骨細胞植入支架實驗………………… 49
5.4.1 支架表面及孔洞觀察………………………………… 49
5.4.2 生化分析……………………………………………… 49
5.4.3 支架外觀型態觀察…………………………………… 52
5.4.4 支架組織切片分析…………………………………… 52
5.4.5 SEM圖分析………………………………………… 53
5.4.6 動態機械性質分析…………………………………… 53
5.4.7 成豬膝關節軟骨細胞植入支架實驗總結…………… 54
5.5 初代小豬關節軟骨細胞植入支架在不同動態環境下培養評估實驗………………………………………… 55
5.5.1 支架的SEM圖……………………………………… 56
5.5.2 生化分析……………………………………………… 56
5.5.3 動態機械性質分析…………………………………… 57
5.5.4 支架組織切片分析…………………………………… 59
5.5.5 初代小豬關節軟骨細胞植入支架在不同動態環境下培養評估實驗總結…………………………………… 60
第六章 結論…………………………………………………… 62
參考文獻 ………………………………………………………… 61
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