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姓名 徐藝庭(Yi-ting Hsu) 查詢紙本館藏 畢業系所 化學工程與材料工程學系 論文名稱 利用導電高分子聚吡咯為基材以電刺激促進幹細胞分化
(Electrical Stimulation to Promote Stem cell Differentiation using Conducting Polypyrrole Films)相關論文 檔案 [Endnote RIS 格式]
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摘要(中) 聚吡咯(Polypyrrole, PPy)是一種具導電性的生醫材料。在本研究中，我們將PPy膜作為培養間葉幹細胞(mescnchymal stem cells, MSCs)的基材，觀察PPy對於細胞分化成骨的影響。
利用化學氧化的方式，以過硫酸銨(ammonium persulfate, APS)作為起始劑可以成功製備出PPy膜，藉由改變單體濃度及與起始劑的莫爾比，可控制所製備的PPy薄膜具有不同導電度。首先以傅立葉紅外光譜(Fourier transform infrared spectroscopy, FTIR)進行確認以PPy膜成功地沉積在基材上，在材料的物性分析中，我們使用四點探針來量測材料片電阻，並以掃描電子顯微鏡(scanning electron microscopy, SEM)和原子力顯微鏡(atomic force microscopy, AFM)觀察薄膜厚度及表面形貌。另外，藉由電子能譜儀(X-ray spectroscopy, XPS)探討薄膜表面元素組成，解析C1s的吸收峰後發現PPy薄膜的導電性質與高分子的分枝率成正比，推測合成高交聯度的聚吡咯可以提升高分子網絡結構的完整性。
最後我們將PPy膜用於培養大鼠的骨髓間葉幹細胞(RM1)， MTT分析結果表明PPy膜是生物相容性材料，並且以骨分化培養液進行細胞培養，將RM1細胞誘導分化為成骨細胞。根據鹼性磷酸酶(alkaline phosphatase activity assay, ALP)的活性檢測顯示，PPy膜應具有提早分化成骨的效果。以茜素紅染色(Alizarin red stain, ARS) 及Calcium-o- cresolphthalein complexone的分析結果表明，聚吡咯薄膜能促進骨的礦化，以增加細胞外基質中的鈣沉積。最後，藉由PPy膜上施加恆定電場，探討電刺激成骨作用，結果顯示經過電刺激培養的細胞比起未經處理的PPy膜，其鈣沉積明顯增加，說明電刺激可能是能夠增進細胞骨化的效果。這些結果表明，聚吡咯具有優越的生物傳導性，適合作為骨組織修復用的生醫材料。
摘要(英) Polypyrrole (PPy) is a conductive biomaterial. In this study we would like to apply PPy films as substrates to culture mesenchymal stem cells (MSCs) to evaluate if PPy may promote osteogenesis. Chemical oxidation polymerization using ammonium persulfate (APS) as initiator was utilized to polymerize PPy films. By changing monomer concentrations and initiator ratio, films with different conductivity were fabricated. Fourier transform infrared spectroscopy (FTIR) was performed to confirm that PPy films were successfully deposited. Physical properties of PPy films were characterized using four point probe, scanning electron microscopy (SEM) and atomic force microscopy (AFM) to demonstrate sheet resistance, thickness, and surface morphology of PPy films, respectively. In addition, X-ray spectroscopy (XPS) was applied to investigate film composition. By diagnosing C1s peaks, PPy films with higher conductivities always had higher branch ratio, which should be due to that complete network would be formed by highly-crosslinked PPy chains.
These films were then applied for culturing rat bone marrow-derived mesenchymal stem cells (RM1). MTT assay suggested that PPy films were biocompatible materials. By using osteogenic medium, RM1 cells were differentiated into osteoblasts. The alkaline phosphatase (ALP) activity assay revealed that PPy films may accelerate osteogenesis. Alizarin red stain (ARS) and calcium-o-cresolphthalein complexone assays suggested that PPy film promoted mineralization to increase calcium deposition in extracellular matrix.
Finally, PPy films were subjected in a constant electric field to elucidate the effect of electrical stimulation on osteogenesis. Cells cultured on PPy films with electrical stimulation deposited more calcium than that cultured on untreated PPy films, suggesting that electrical stimulation may be able to improve cell differentiation. These results indicated that PPy should be an appropriate biomaterial with superior bioconductivity, which should be capable
III
of facilitating bone regeneration in tissue engineering application.
關鍵字(中) ★ 成骨
★ 間質幹細胞
★ 電刺激
★ 聚吡咯膜
★ 化學氧化聚合關鍵字(英) ★ Osteogenesis
★ Mesenchymal stem cells
★ Electrical stimulation
★ Polypyrrole
★ Chemical oxidative polymerization論文目次 摘要 .................................................................................................................................................. I
Abstract ...........................................................................................................................................II
目錄 ............................................................................................................................................... IV
表目錄 ........................................................................................................................................... VI
圖目錄 ..........................................................................................................................................VII
第一章序論 .................................................................................................................................. 1
1-1 前言 ......................................................................................................................... 1
第二章文獻回顧與理論基礎 ...................................................................................................... 3
2-1 組織工程 ................................................................................................................. 3
2-2 幹細胞 ..................................................................................................................... 4
2-2-1 間葉幹細胞(Mesenchymal stem cell, MSC) ............................................ 5
2-3 骨組織學 ................................................................................................................. 8
2-4 生醫材料 ............................................................................................................... 11
2-5 導電高分子 ........................................................................................................... 12
2-5-1 聚吡咯Polypyrrole .................................................................................. 13
2-5-2 聚吡咯的導電機制 ................................................................................... 14
2-5-3 導電高分子對細胞之文獻回顧 .............................................................. 16
2-5-4 電刺激對細胞分化之文獻回顧 .............................................................. 19
第三章實驗方法與設備 ............................................................................................................ 23
3-1 實驗藥品 ............................................................................................................... 23
3-2 實驗儀器 ............................................................................................................... 25
3-3 試藥製備與實驗方法 .......................................................................................... 25
3-3-1 Polypyrrole 製備 ......................................................................................... 25
V
3-3-2 細胞培養 ................................................................................................... 26
3-3-3 MTT 分析 .................................................................................................. 28
3-3-4 鹼性磷酸酶(Alkaline phosphatase assay, ALP) ..................................... 28
3-3-5 茜素紅染色(Alizarin red S，ARS) ......................................................... 29
3-3-6 Calcium-O-Cresolphthalein complexone ................................................. 29
3-3-7 乳酸脫氫酶(LDH)-CytoTox96R Non-Radioative cytotoxicity Assay . 30
3-4 實驗設計與架構 ................................................................................................... 31
3-4-1 合成導電高分子聚吡咯(Polypyrrole, PPy) ........................................... 31
3-4-2 導電高分子材料對於骨分化的影響 ..................................................... 31
3-4-3 電刺激對骨分化的影響 .......................................................................... 32
3-5 物性分析與結構鑑定 .......................................................................................... 33
3-5-1 結構之鑑定 ............................................................................................... 33
3-5-2 電性質分析 ............................................................................................... 34
3-5-3物理性質分析 ............................................................................................ 35
3-6 生物性質分析 ....................................................................................................... 35
3-6-1 MTT細胞活性測試 ................................................................................... 35
3-6-2 鹼性磷酸酶(Alkaline phosphatase assay, ALP) ..................................... 36
3-6-3 茜素紅染色(ARS) .................................................................................... 37
3-6-4 Calcium-O-Cresolphthalein complexone ................................................. 37
3-6-5 乳酸脫氫酶(lactate dehydrogenase , LDH)細胞毒性分析 ................... 37
3-6-6 骨分化之基因表現 ................................................................................... 38
第四章結果與討論 ..................................................................................................................... 40
4-1 導電性質(Conductivity) ....................................................................................... 40
4-2 掃描式電子顯微鏡(Scanning electron microscopy, SEM) ............................... 42
4-3 原子力顯微鏡(Atomic force microscopy, AFM) ............................................... 44
4-4 紅外線光譜分析(FTIR spectroscopy) ................................................................ 46
4-4 電子能譜儀(X-Ray spectroscopy , XPS) ............................................................ 48
4-5 生物相容性(Biocompatibility) .......................................................................... 57
4-6 導電性薄膜對於骨分化的影響 .......................................................................... 59
4-6-1 鹼性磷酸酶分析(Alkaline phosphatase assay, ALP) ............................. 59
4-6-2鈣沉積分析 ................................................................................................ 60
4-6-2-1茜素紅S染色分析 ......................................................................... 60
4-6-2-2 Ca離子定量分析(Calcium-O-Cresolphthalein complexone) . 61
4-6-3 骨分化之基因表現 ................................................................................... 65
4-7 電刺激促進細胞分化 .......................................................................................... 67
第五章 結論 ................................................................................................................................ 70
第六章、參考文獻 ....................................................................................................................... 71
表目錄
表一 導電高分子在生物方面的應用 ....................................................................................... 17
表二電刺激對細胞分化之文獻回顧 ....................................................................................... 22
表三配製不同濃度聚吡咯所需的單體用量 ........................................................................... 26
表四配製不同濃度聚吡咯所需的起始劑用量 ....................................................................... 26
表五beta Actin primer序列 ........................................................................................................ 38
表六Core binding factor 1 (Cbfa 1) primer序列 ...................................................................... 39
表七Ostocalcin (OC) primer序列 .............................................................................................. 39
圖目錄
圖2-1 骨組織工程之四大要素 .................................................................................................... 4
圖2-2 間葉幹細胞的分化可塑性示意圖 ................................................................................... 6
圖2-3 骨母細胞表型的發展 ........................................................................................................ 7
圖2-4 骨修復過程中新生骨組織的階段性替換情形 .............................................................. 7
圖2-5 Wolff理論下骨折癒合過程 ............................................................................................ 9
圖2-6 Wolff理論骨順應最大應力方向成長 ......................................................................... 9
圖2-7 將機械應力轉變為電流的壓電晶體與骨骼的壓電現象 ........................................... 10
圖2-8 模擬骨的壓電現象(磷灰石-膠原接面) ......................................................................... 10
圖2-9 芳香雜環類的導電高分子 ............................................................................................. 12
圖2-10 聚吡咯之反應機構 ........................................................................................................ 15
圖2-11 內皮細胞培養於聚吡咯基材上之型態(A)氧化態之聚吡咯(B) 經-0.5V 4小時還原之聚吡咯 .................................................................................................................................. 18
圖2-12 細胞培養於不同材料之型態(A)TCPS (B) 純材料PPy .......................................... 20
圖2-13 軸突長度統計圖(A)於PPy膜上通電刺激(B) 純材料PPy (C) 於溶液中通電刺激(D) TCPS ..................................................................................................................................... 20
圖2-14 外部刺激對細胞影響 .................................................................................................... 21
圖3-1 材料性質檢定之實驗設計圖 ......................................................................................... 32
圖3-2 電場與材料對細胞分化影響之實驗設計圖 ................................................................ 32
圖3-3 實驗裝置圖 ...................................................................................................................... 33
圖3-4 XPS原理示意圖 ................................................................................................................ 34
圖3-5 四點探針原理示意圖 ...................................................................................................... 34
圖3-6 MTT酵素反應式 ............................................................................................................ 36
圖3-7 pNPP酵素反應式 ........................................................................................................... 36
圖3-8 茜素紅分子式 .................................................................................................................. 37
圖4-1 四點探針量測不同條件之聚吡咯的表面片電阻 ........................................................ 41
圖4-2 不同條件之聚吡咯的表面片電導值 ............................................................................. 41
圖4-3 不同反應速率下聚合狀況(a) 正常速率；(b) 反應速率較快，增加聚合物之分枝 ....................................................................................................................................................... 42
圖4-4 掃描式電子顯微鏡觀察各種聚吡咯之表面結構 ........................................................ 43
圖4-5 掃描式電子顯微鏡觀察各種聚吡咯之截面厚度 ........................................................ 43
圖4-6 原子力顯微鏡觀察各種聚吡咯之表面粗糙度 ............................................................ 45
圖4-7 聚吡咯之IR圖譜 .............................................................................................................. 46
圖4-8 過氧硫酸銨之標準圖譜 .................................................................................................. 47
圖4-9 FTIR光譜測定不同條件下聚吡咯之結構 ..................................................................... 47
圖4-10聚吡咯鍍膜之XPS全光譜圖表面元素檢測 ................................................................ 49
圖4-11 (a)α-α’與α-β鍵結；(b) 聚合過程中，兩分枝高分子鍵結成網狀高分子 ............ 50
圖4-12 聚吡咯中的C1s之XPS光譜圖 ................................................................................... 51
圖4-13不同條件下的聚吡咯之C1s -XPS光譜圖 ................................................................... 54
圖4-14不同合成條件之聚吡咯分枝率隨著(a)單體濃度(b)起始劑比例改變的影響 ........ 55
圖4-15 聚吡咯高分子的分枝率與片電導之關係圖 .............................................................. 56
圖4-16電子在分子內與分子間的傳導 .................................................................................... 56
圖4-17 倒立式電子顯微鏡觀察老鼠間葉幹細胞之貼附行為 ............................................. 57
圖4-18 MTT分析各種材料之生物相容性 ............................................................................... 58
圖4-19 0~8天的鹼性磷酸酶分析結果 ..................................................................................... 60
圖4-20 茜素紅染色(細胞培養第11天) .................................................................................. 63
圖4-21 茜素紅染色(細胞培養第14天) .................................................................................. 63
圖4-22 Calcium-O-Cresolphthalein complexone分析7~28天之鈣沉積 ............................... 64
圖4-23 Calcium-O-Cresolphthalein complexone分析7~28天的鈣沉積(以細胞數進行正規化處理) ......................................................................................................................................... 64
圖4-24材料片電導與第14天時以細胞數正規化處理之鈣沉積關係圖 ............................ 65
圖4-25 第7、11天的Cbfa1基因表現量 ................................................................................ 66
圖4-26 第11天的Osteocalcin基因表現量 .............................................................................. 66
圖4-27 0.5M Py APS/Py 0.2經電刺激後培養第11天之相對鈣沉積量 ........................... 68
(b) .................................................................................................................................................. 69
圖4-28 RM1培養於三種聚吡咯表面，並經電刺激(1V, 4hr)後培養第11天之相對鈣沉積量(a)相對於TCPS控制組；(b) 相對於各個material控制組 .................................................. 69
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指導教授 胡威文(Wei-wen Hu) 審核日期 2012-8-23 推文 plurk
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