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研究生:李靜雯
研究生(外文):Ching-Wen LI
論文名稱:奈米結構與微水平震動平台對細胞生長之影響研究
論文名稱(外文):Investigations of the effects of nanostructure and micro horizontal vibration on cell proliferation
指導教授:王國禎
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生醫工程研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:60
中文關鍵詞:真空抽氣法奈米結構微水平震動牛頸動脈內皮細胞
外文關鍵詞:vacuum air-extraction processnanostructuredPLGA scaffoldmicro horizontal vibrationsBEC
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細胞與支架材料之交互作用乃是組織工程重要之研究主題之ㄧ,本研究以具生物相容性與具生物可降解特性之PLGA高分子材料做為支架,分別探討內皮細胞於具不同奈米結構之PLGA支架以及對PLGA支架施以微水平震動刺激後其生長情形。
PLGA為廣泛應用之生物可降解材料之一,而具奈米結構之PLGA更是在組織工程上有極大之應用潛能。本研究利用陽極氧化鋁模(AAO)結合真空抽氣法之物理方式翻模,製作不同奈米結構之PLGA支架,以牛頸動脈內皮細胞之生長比較不同奈米結構對於細胞生長與貼附的影響。將PLGA溶液澆鑄於陽極氧化鋁模(AAO)上,經過1-2小時使得表面凝固;再放入真空烘箱經由抽氣使得PLGA半凝固態之PLGA材料能擠壓進入奈米孔洞中,形成柱狀奈米結構,奈米結構之尺寸可由抽氣時間加以控制;接著,將抽氣後的PLGA試片浸泡於磷酸溶液中去除氧化鋁,或直接脫膜,即可得到奈米結構PLGA支架,細胞貼附測試結果,研究結果顯示表面粗糙度為25奈米之柱狀奈米結構PLGA支架,較利於內皮細胞之生長。
微水平震動部份,本研究以微水平震動平台對內皮細胞施加頻率為0.5Hz、1Hz、2Hz,震幅為20μm之微水平震動刺激。研究結果發現,具奈米結構PLGA支架較不適合施加微水平震動刺激,其原因乃是材料表面奈米化,使材料表面較為疏水,故細胞於粗糙度越小之PLGA支架生長情形越差。若對無奈米結構之PLGA支架,施以1Hz之微水平震動,細胞之增生率甚至較在24-well中為佳。
本研究發現,適當之奈米結構以及適當頻率之微水平震動皆可有效刺激內皮細胞增生。然而,兩種刺激方式並無法同時使用,主要原因乃是奈米結構會降低材料表面之疏水性且減少細胞與材料之接觸面積,在微水平震動時,培養液較不易保留且細胞貼附面之剪應力增加,反而不利於細胞之增生。
Interactions between cell and scaffold material is one of the main interesting topics in tissue engineering. In this research, influences of nanostructured PLGA scaffolds and micro horizontal vibrations on bovine endothelial cells (BEC) were investigated, respectively.
In the study of the interactions between BEC and nanostructured PLGA scaffolds, two simple fabrication methods to fabricate orderly nanostructured PLGA scaffolds using anodic aluminum oxide (AAO) template were conducted. In the vacuum air-extraction approach, the PLGA solution was cast on an AAO template first. The vacuum air-extraction process was then applied to suck the semi-congealed PLGA into the nanopores of the AAO template to form a bamboo sprouts array of PLGA. The surface roughness of the nanostructured scaffolds, ranging from 20 nm to 68 nm, can be controlled by the sucking time of the vacuum air-extraction process. In the replica molding approach, the PLGA solution was cast on the orderly scraggy barrier-layer surface of an AAO membrane to fabricate a PLGA scaffold of concave nanostructure. Cell culture experiments using BEC demonstrated that the nanostructured PLGA membrane can increase the cell growing rate, especially for the bamboo sprouts array scaffolds with smaller surface roughness.
In the study of the influences of micro horizontal vibrations on BEC proliferations, different frequencies 0.5 Hz, 1 Hz, and 2 Hz respectively with a 20 micro-meter amplitude, using a piezoelectric micro-positioning stage, were applied to nanostructured and flat PLGA scaffolds during cell cultivation. It was found that the proliferation of BEC on nanostructured PLGA scaffold under micro horizontal vibrations was suppressed. It is presumed that the nanostructures made the surface of the scaffold more hydrophobic, hence degraded the adhesions between BECs and the scaffold. On the contrary, the proliferations of BECs on a flat PLGA scaffold were enhanced under vibrations of 1 Hz frequency and 20 micro-meter amplitude. The 1 Hz frequency is close to the pulse rate of mature cattle (60-80 Hz).
In this research, both the nanostructured PLGA scaffolds and the 1 Hz micro horizontal vibrations on flat PLGA scaffolds were discovered to be able to enhance the proliferation of BECs. However, the micro horizontal vibrations using a piezoelectric micro-positioning stage is much easier to be implemented.
目錄
致謝.............................................................................................................I
中文摘要...................................................................................................II
英文摘要..................................................................................................IV
目錄..........................................................................................................VI
表目錄.......................................................................................................X
圖目錄......................................................................................................XI
第一章 緒論
1-1前言......................................................................................................1
1-2研究動機與目的..................................................................................3
第二章 文獻回顧
2-1生醫材料回顧......................................................................................6
2-1-1生物可降解之高分子材料............................................................7
2-2奈米材料............................................................................................10
2-3奈米製程............................................................................................11
2-4奈米表面結構對細胞貼附/生長的影響...........................................15
2-5壓電致動器(PZT).............................................................................18
2-6微震動對細胞之影響........................................................................20
2-7水平震動應力(Vibration stress)推導...............................................23
第三章 奈米結構對細胞生長之影響
3-1實驗藥品............................................................................................26
3-1-1具奈米結構PLGA支架製備.......................................................26
3-1-2牛頸動脈內皮細胞培養..............................................................26
3-2實驗器材............................................................................................28
3-2-1具奈米結構PLGA支架製備.......................................................28
3-2-2牛頸動脈內皮細胞培養..............................................................28
3-3藥品配置............................................................................................29
3-3-1具奈米結構PLGA支架製備.......................................................29
3-3-2牛頸動脈細胞培養......................................................................29
3-4實驗方法............................................................................................30
3-4-1陽極氧化鋁膜製備......................................................................30
3-4-2無奈米結構PLGA支架製備.......................................................32
3-4-3具奈米結構PLGA支架製備.......................................................32
3-4-3牛頸動脈細胞培養......................................................................34
3-5實驗結果............................................................................................35
3-5-1陽極氧化鋁膜製備......................................................................35
3-5-2具奈米結構PLGA支架製備.......................................................37
3-5-3內皮細胞植覆PLGA支架與培養...............................................39
第四章 微水平震動對細胞生長之影響
4-1實驗器材............................................................................................42
4-1-4親水性量測..................................................................................42
4-1-2微水平震動..................................................................................42
4-2微水平震動平台簡介........................................................................42
4-2-1微水平震動平台運動方式..........................................................42
4-2-2鐵氟龍固定夾具..........................................................................43
4-3實驗方法............................................................................................44
4-3-1親水性量測..................................................................................44
4-3-2微水平震動細胞培養..................................................................44
4-4實驗結果............................................................................................46
4-4-1親水性量測..................................................................................46
4-4-2具奈米結構PLGA支架微水平震動培養...................................47
4-4-3無奈米結構PLGA支架微水平震動培養...................................47
第五章 結論與未來發展
5-1結論....................................................................................................52
5-1-1 奈米結構對細胞生長之影響.....................................................52
5-1-2微水平震動對細胞生長之影響..................................................52
5-2未來展望............................................................................................52
參考文獻..................................................................................................57

表目錄
表2.1 奈米材料的典型應用與於生醫上的應用...................................11
表2.2常見壓電材料................................................................................20
表2.3各種物理刺激方式........................................................................22
表4.2微水平震動加速度表....................................................................45
表4.3接觸角量測....................................................................................46
圖目錄
圖1.1 組織工程示意圖.............................................................................2
圖1.2兩隻老鼠下腹部的掃描圖,灰色部分為皮下脂肪,紅色為血脂;(a)未震動的老鼠(b)有震動的老鼠......................................................4
圖2.1 PLGA分子式...................................................................................9
圖2.2 不同分子量的聚乙二醇(PEG)接枝在聚胺酯(PU)表面..........12
圖2.3以NaOH對PLGA進行奈米表面處理與表面結構SEM圖........13
圖2.4 電漿蝕刻後之PLGA表面SEM圖..............................................13
圖2.5溶膠噴霧方式製作奈米粒子........................................................14
圖2.6溶膠噴霧方式製作奈米粒子(a)未用(b)用擴散式乾燥器........14
圖2.7電化學沉積奈米金顆粒................................................................15
圖2.8 PDMS微溝槽結構........................................................................16
圖2.9 CTPs細胞於PDMS微溝槽結構上之生長情形..........................17
圖2.10 蛋白質調節細胞貼附材料表面示意圖.....................................17
圖2.11壓電效應示意圖..........................................................................19
圖2.12震動應力之應用(A)細胞培養於24-well底部;以正弦波沿X軸震動(B)假設細胞核相對於黏彈性細胞體是呈現圓球狀............25
圖3.1陽極氧化鋁膜製備流程圖............................................................31
圖3.2陽極處理之設備示意圖................................................................32
圖3.3 柱狀表面結構製程.......................................................................33
圖3.4 碗狀表面結構製程.......................................................................34
圖3.5鋁基材移除前之陽極氧化鋁膜....................................................36
圖3.6 陽極氧化鋁背阻障SEM圖(a)正視圖(b)側視圖......................37
圖3.7阻障層表面處理(a)背阻障層蝕刻120分鐘(b)背阻障層蝕刻40分鐘.................................................................................................37
圖3.8不同奈米結構表面之SEM圖。柱狀結構(a)真空抽氣1小時(b)真空抽氣5小時(c)真空抽氣24小時;(d)利用背阻障翻模之碗狀結構(e)表面無奈米結構之控制組............................................38
圖3.9不同奈米結構表面之AFM圖。柱狀結構(a)真空抽氣1小時(b)真空抽氣5小時(c)真空抽氣24小時;(d)利用背阻障翻模之碗狀結構;(e)表面無奈米結構之控制組..........................................38
圖3.10柱狀表面粗糙度與抽氣時間關係圖..........................................39
圖3.11 牛頸動脈內皮細胞於T-75之生長與光學顯微鏡觀察結果;(a)100倍(b)200倍.......................................................................................40
圖3.12細胞於奈米結構PLGA支架上之貼附染色結果;柱狀表面:(a)真空抽氣1小時(b)真空抽氣5小時(c)真空抽氣24小時;(d)利用背阻障翻模之碗狀結構;(e)表面無奈米結構之控制組..............40
圖3.13 細胞生長計數圖.........................................................................41
圖4.1微水平震動平台運動方式............................................................43
圖4.2鐵氟龍夾具外觀(a)上視圖(b)側視圖........................................43
圖4.3 微水平震動裝置...........................................................................46
圖4.4接觸角量測圖(a)24-Well(b)PLGA.............................................46
圖4.5施加微水平震動於奈米結構PLGA之架之內皮細胞生長計數圖..............................................................................................................47
圖4.6不同微水平震動後牛頸動脈內皮細胞於T-75之型態圖(a)0.5Hz(b)1Hz(c)2Hz(d)靜態培養.................................................................48
圖4.7內皮細胞於平板PLGA支架之震動與靜止生長計數圖(48小時培養)........................................................................................................49
圖4.8內皮細胞於24-Well之震動與靜止生長計數圖(48小時培養)............................................................................................................50
圖4.9內皮細胞於平板PLGA及24-Well之震動培養生長計數圖.......50
圖4.10內皮細胞於PLGA支架之不同培養方式之計數圖...................51
圖5.1不同培養方式細胞增生比較圖....................................................53
圖5.2材料表面之粗糙度影響內皮細胞之生長示意圖........................54
圖5.3 可降解性藥物制放系統製作流程...............................................55
圖5.4 藥物釋放機制...............................................................................55
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