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研究生:林燕霜
研究生(外文):Yen-Shuang Lin
論文名稱:動態式淨壓對軟骨細胞代謝之影響
論文名稱(外文):The Effect of Dynamic Compressive Pressure on Chondrocytes Metabolism
指導教授:鄭誠功鄭誠功引用關係孫瑞昇孫瑞昇引用關係
指導教授(外文):Cheng-Kung ChengJui-Sheng Sun
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:80
中文關鍵詞:軟骨細胞動態式淨壓組織工程生物反應器物理性刺激
外文關鍵詞:chondrocytesdynamic compressive pressuretissue engineeringbioreactorphysical stimulation
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退化性關節炎為老年人口生活品質下滑之主要原因之ㄧ,近年來,由於組織工程技術之蓬勃發展,應用在關節軟骨組織之修復亦逐漸受到重視。然而在體外進行軟骨培養時,由於與體內環境上的差異,軟骨組織面臨著去分化 (de-
differentiation) 的問題,而造成所培養出來的組織與體內組織在型態及功能上之歧異。關節軟骨在體內實際的環境,是不斷的受到循環負載而發生週期性的形變和回復,循環負載及養分提供是軟骨細胞執行正常功能和維持細胞外基質正常表現的重要因素。
本研究以體外三度空間培養方式,將軟骨細胞包埋於瓊脂膠 (agarose gel) 內,結合壓縮刺激的生物反應器 (bioreactor) 上,給予細胞物理性刺激,進而來探討不同頻率 (0.5Hz、1Hz、2Hz、3Hz) 及不同單軸壓縮變形量 (5%、10%、15%) 的刺激條件下,細胞增生 (cell proliferation)、蛋白多醣 (glucosaminoglycans
,GAGs)、一氧化氮 (nitric oxide) 及總膠原蛋白 (total collagen) 合成分泌之關係。實驗結果發現,軟骨細胞處於低頻率 (0.5Hz) 及低變形量 (5%) 條件下,會降低細胞增生和抑制GAGs合成而增加NO的含量,對軟骨細胞可能造成傷害,變形量 10% 的刺激下可以使軟骨細胞有較好的增生情況,而頻率 1Hz 對於 GAGs 分泌則有良好的結果,膠原蛋白則受動態式淨壓影響較小。刺激後的靜置培養對軟骨細胞的增生和代謝結果隨著天數的增加而越好,生物反應器結合適當的力學刺激可以增加軟骨細胞增生及代謝,希望對於軟骨組織工程 (Cartilage Tissue Engineering) 關節修復方面有良好的應用。
Osteoarthritis (OA) is a major cause of deterioration of the quality of life in the aged population. With the development of tissue engineering, particular attention has been paid to the application of tissue engineering in cartilage repair. However, in vitro method for expanding chondrocytes in two-dimension culture will cause the original characteristics to be lost due to the change of their morphology and function in their production from type Ⅱ collagen to typeⅠ (de-differentiation). In actual vivo environment, articular cartilage is unceasing to receive the circulation loading to have the periodic distortion and the recovery. The circulation loading and the nutrient provides are the important factor that the chondrocytes can carry out the normal function and maintain the expression of extracellular matrix (ECM).
The present study embeds the chondrocytes into the agarose gel by three- dimension culture methods and apply compressive stress to condrocytes at the same time, after that to discuss the different degree of frequency (0.5Hz、1Hz、2Hz、3Hz) and axial deformation (5%、10%、15%) stimulation, and the relationship of chondrocytes metabolism by analysis of cell proliferation、glucoseaminoglycans (GAGs)、nitric oxide (NO) and total collagen. As a result, low frequency (0.5Hz) and low amplitude (5%) could inhibit cell proliferation and GAGs synthesis and stimulate NO production that may cause cell damage. 10% deformation could make good cell proliferation and 1Hz could stimulate GAGs synthesis well, but dynamic compression has less influence in collagen synthesis. Culture periods after dynamic compression make better cell proliferation and metabolism by increasing days. Combination of physical stimulation with bioreactor could increase cell proliferation and metabolism and have great promise in application of Cartilage Tissue Engineering in repair of joint injury.
摘要………………………………………………………………………i
ABSTRACT…………………………………………………………………ii
目錄……………………………………………………………………iii
圖目錄…………………………………………………………………vii
表目錄……………………………………………………………………ix
第一章 緒論…………………………………………………………1
1.1 前言………………………………………………………………1
1.2 研究目的…………………………………………………………3
第二章 理論基礎……………………………………………………4
2.1 骨骼生長………………………………………………………4
2.1.1 硬骨形成………………………………………………5
2.1.1.1 膜內骨化…………………………………………5
2.1.1.2 軟骨內骨化…………………………………………5
2.1.2 軟骨形成…………………………………………………8
2.2 軟骨組織分類……………………………………………………8
2.3 關節軟骨生理學…………………………………………………9
2.3.1 關節軟骨的結構與功能…………………………………9
2.3.2 關節軟骨組織主要成分…………………………………12
2.3.2.1 軟骨細胞…………………………………………12
2.3.2.2 軟骨基質 – 膠原蛋白…………………………13
2.3.2.3 軟骨基質 – 蛋白多醣…………………………15
2.3.2.4 軟骨基質 – 水及其他小分子…………………17
2.3.2.5 軟骨基質 – 金屬蛋白分解酵素………………18
2.3.3 ㄧ氧化氮對軟骨基質的影響……………………………19
2.3.4 關節軟骨的生物力學及機械功能………………………21
2.3.4.1 黏彈性質…………………………………………21
2.3.4.2 潛變反應…………………………………………22
2.3.4.3 壓力釋放反應……………………………………22
2.3.4.4 軟骨在不同應力下的變形情況…………………23
2.3.5 機械應力對軟骨細胞之影響……………………………25
2.4 關節軟骨發生病變或損傷之原因……………………………25
2.5 退化性關節炎…………………………………………………26
2.6 受傷關節軟骨的治療方法……………………………………28
2.7 組織工程………………………………………………………31
2.7.1 軟骨組織工程…………………………………………32
第三章 生物反應器……………………………………………………33
3.1 生物反應器簡介………………………………………………33
3.2 設計與評估……………………………………………………34
3.2.1 致動器……………………………………………………34
3.2.2 夾具的設計………………………………………………34
3.2.3 荷重元的設計……………………………………………35
3.2.4 防塵套的設計……………………………………………35
3.3. 生物反應器組裝原理…………………………………………36
3.3.1 培養系統…………………………………………………36
3.3.2 壓縮刺激系統……………………………………………36
第四章 材料與方法……………………………………………………38
4.1 材料……………………………………………………………38
4.1.1 實驗儀器…………………………………………………38
4.1.2 實驗試藥與耗材…………………………………………39
4.2 方法……………………………………………………………41
4.2.1 試藥配置…………………………………………………41
4.2.1.1 細胞培養試藥配置………………………………41
4.2.1.2 Agarose Cell Specimens 溶解試藥配置……42
4.2.2 三度空間培養方法……………………………………43
4.2.2.1 軟骨細胞培養方法………………………………43
4.2.2.2 Agarose Cell Specimens 包埋方法…………46
4.2.2.3 Agarose Cell Specimens 溶解方法…………47
4.2.3 培養參數…………………………………………………47
4.2.3.1 動態式淨壓刺激培養……………………………47
4.2.3.2 力學刺激後靜置培養……………………………48
4.2.4 分析與評估………………………………………………48
4.2.4.1 細胞數 (DNA) 分析………………………………48
4.2.4.2 Dimethylmethylene Blue Assay (GAGs)……49
4.2.4.3 Griess Reaction (NO) ………………………50
4.2.4.4 Hydroxyproline Assay (OHP) ………………51
4.2.4.5 Toluidine Blue O (TBO) 染色步驟…………53
第五章 結果與討論……………………………………………………54
5.1 Agarose 三度空間培養………………………………………54
5.1.1軟骨細胞於 agarose 三度空間培養之分佈……………54
5.1.2 Agarose 受動態淨壓之實驗限制……………………56
5.2 細胞數 (DNA) 分析……………………………………………58
5.3 Dimethylmethylene Blue Assay (GAGs) 分析……………60
5.4 Griess Reaction (NO) 分析………………………………62
5.5 Hydroxyproline Assay (OHP) 分析………………………65
5.6 力學刺激後靜置培養…………………………………………67
5.6.1 靜置後細胞數 (DNA) 分析……………………………67
5.6.2 靜置後Dimethylmethylene Blue Assay(GAGs)分析…68
5.6.3 靜置後Griess Reaction (NO) 分析…………………69
5.6.4 靜置後 Hydroxyproline Assay (OHP) 分析…………71
DNA Standard Curve……………………………………………………73
GAGs Standard Curve…………………………………………………73
NO Standard Curve……………………………………………………74
Total Collagen Standard Curve……………………………………74
第六章 結論……………………………………………………………75
參考文獻…………………………………………………………………76
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