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研究生: 林新蓉
swary - fransiska delimartin
論文名稱: 表面微結構對骨類細胞行為之效應
The Effects of Surface Micro-Patterns on Osteoblastic cells' Behaviors
指導教授: 何明樺
Ming Hua, Ho
口試委員: 王孟菊
Meng Jiy, Wang
蔡偉博
Wei Bor, Tsai
李伯訓
Bor Sun, Lee
蘇忠傑
Jung chieh, Su
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2008
畢業學年度: 96
語文別: 英文
論文頁數: 169
中文關鍵詞: 微米溝槽大鼠骨瘤細胞接觸引導現象骨礦化
外文關鍵詞: microgrooved, osteoblast cell, contact guidance, bone mineralization
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在本研究中,吾人使用微影技術(Photolithography)於矽晶片上製備同心圓狀微米級溝槽(microgrooves),其溝槽寬度由圓心(約20贡um)往邊緣(約1贡um)遞減,其溝槽深度則維持在轨贡1 um左右。於此一矽晶片上進行大鼠骨瘤細胞(ROS)的培養,接觸引導(Contact guidance)的現象清晰可見,且細胞之貼附數目將隨溝槽寬度的增加而下降,意即最高之細胞貼附密度發生在具有最小溝槽寬度的位置,此外,較窄的溝槽同樣有效地促進了細胞的增生。

吾人接著將微米溝槽由矽晶圓複製至PDMS基材上,並以氬氣電漿調整其親疏水性。實驗結果指出在親水性的PDMS基材上,細胞排列之方向性、細胞貼附與細胞增生的情形皆較於疏水性PDMS基材上之細胞為佳,這主要是由於表面親水性的增加提高了基材本身對細胞的親和性,也因如此,細胞能偵測到表面微結構的差異,故細胞的排列、貼附與增生皆會隨著溝槽寬度的減少而提升。相反地,在水性的PDMS基材上,由於其與細胞親和性差,故無法發現溝槽的結構或存在對細胞的數量與方向性產生顯著的影響。在細胞分化方面,吾人亦觀察到在親水性PDMS基材上的細胞更傾向於分化成骨組織,此點可由其上之骨細胞表現出較明顯的骨分化指標而證明。此外,降低溝槽寬度亦能有效促進骨蛋白與細胞鈣化的表現,以上的現象可能與蛋白質在表面上的沉積狀況有關。

最後,吾人使用臭氧活化方式將RGD胜肽片段接枝於有溝槽結構的PDMS基材上,與未改質之樣品相較,雖然兩者之親水性相近,但RGD片段確實促進了細胞的貼附,且所培養之細胞也能偵測到基材表面之結構狀態,也就是說,溝槽寬度之降低能成功提昇細胞的貼附、增生、排列與骨分化。由以上之結果可發現,藉由結合材料表面的物理結構與化學性質,骨類細胞的生長與行為可被成功控制。


Photolithography methods are used in this thesis to create microgroove pattern on Si wafer. The Si wafer has characteristics of continuous groove width varied from 1 to 20μm, while the depth was kept as 1μm. ROS cells were cultured onto Si wafer surfaces. “Contact guidance” phenomena were found on the microgrooved substrates. Increasing the groove width would result in the lower cell attachment and cell density. Microgroove pattern was successfully replicated from Si wafer to PDMS substrates. The groove depth was decreased to 0.8 μm. Ar plasma treatment was used to create hydrophilic PDMS. The ROS cell observation was conducted both on hydrophobic and hydrophilic PDMS. The cell alignment on the hydrophilic PDMS was significantly higher than on hydrophobic samples. Hydrophilic PDMS could enhance the cells attachment and proliferation, due to its high affinity to the cells, and the cells were able to sense the topographical cues and affect their proliferation. Decreasing the groove width would result in a higher cell proliferation and bone mineralization, with the highest expression on the smallest groove width. On hydrophobic PDMS, the cell affinity was poor, and the cells could not recognize the topographical cues, hence the variation in groove width was not efficient on the cell proliferation. RGD peptide was also grafted on PDMS surfaces via ozone-induced grafting process. Even though the RGD-modified PDMS was hydrophobic, however, the topographical cues still could be sense by the cells. The combination of topographical and chemical cues could enhance the cell attachment and determined the cell behavior and final cellular mineralization as well.

ABSTRACT i ACKNOWLEDGMENT v CONTENTS vii FIGURE LIST xi TABLE LIST xxiii Chapter I Introduction 1 Chapter II Literature Review 5 II.1 Tissue Engineering 5 II.2 Cells and Extracellular Matrix (ECM) 7 II.2.1 Characteristics of ECM 7 II.2.2 Interactions between cells and ECM 10 II.3 Biomaterial Surface Modification 13 II.3.1 Surface topographical modification 15 II.3.1.1 Photolithography 16 II.3.1.2 Soft lithography 18 II.3.2 Surface chemistry modification 20 II.3.2.1 Plasma surface treatment 21 II.3.2.2 Ozone induced grafting 23 II.4 Biological Response to Pattern Surface 24 II.5 Polydimethylsiloxane (PDMS) 28 II.6 Rat Osteosarcoma Cells (ROS 17/2.8 cells) 31 II.7 Bone Marker of Osteoconductivity 31 Chapter III Materials and Experimental Procedure 35 III.1 Chemicals 35 III.2 Experimental Apparatus 37 III.3 Experimental Procedure 38 III.3.1 Silicon wafer cleaning procedure 38 III.3.2 Preparation of PDMS membrane 39 III.3.3 Plasma surface treatment 41 III.3.4 Ozone treatment and RGD grafting 42 III.3.5 Characterization of substrates 43 III.3.5.1 FTIR-ATR spectroscopy 43 III.3.5.2 AFM measurement 43 III.3.5.3 SEM measurement 44 III.3.5.4 Water contact angle measurement 44 III.3.5.5 Electron spectroscopy for chemical analysis 45 III.3.5.6 Amino acid analysis 45 III.3.6 Cell culture on Si wafer and PDMS membranes 45 III.3.7 SEM analysis for cell-cultured samples 46 III.3.8 In vitro mineralization 46 III.3.9 Alkaline phosphatase (ALP-ase) quantification assay 47 III.3.10 Bicinchoninic acid (BCA) protein assay 48 III.3.11 Alkaline phosphatase staining 49 III.3.12 Osteopontin and bone sialoprotein staining 49 III.3.13 Von kossa staining 51 III.3.14 Protein deposition assay 52 Chapter IV Results and Discussions 53 IV.1 Silicon Wafer Characterization 53 IV.2 Cell Culture on Si Wafer 58 IV.3 Mineralization of ROS cells on Si wafer. 68 IV.4 Polydimethylsiloxane (PDMS) Characterization 70 IV.5 Cell Culture in Pristine and Plasma-treated PDMS Sample. 80 IV.5.1 Cell culture on pristine PDMS 81 IV.5.2 Cell culture on plasma-treated PDMS 89 IV.6 Bone Marker Staining of ROS Cells Cultured on PDMS 102 Substrates IV.7 Mineralization of ROS Cells on PDMS Substrates 108 IV.8 Ozone Surface Treatment and RGD-grafting on PDMS 118 Substrates IV.8.1 RGD peptide grafting 125 IV.8.2 Cell culture on RGD-grafted PDMS 128 IV.8.3 Histochemistry stainings of osteoblast cells cultured 130 on RGD-grafted PDMS CHAPTER IV CONCLUSIONS AND SUGGESTIONS 143 IV.1 Conclusions 143 IV.2 Suggestions 146 REFERENCES 147 APPENDIX CELL CHARACTERISTIC CALCULATION 167

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