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研究生:周欣誼
研究生(外文):Hsin-Yi Chou
論文名稱:人類間葉幹細胞於多胜肽多層交聯薄膜上之成骨分化
論文名稱(外文):Osteogenic Differentiation of Human Mesenchymal Stem Cells on Crosslinked Polypeptide Multilayer Films
指導教授:羅俊民
指導教授(外文):Chun-Min, Lo
學位類別:碩士
校院名稱:國立陽明交通大學
系所名稱:生物醫學工程學系
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:英文
論文頁數:60
中文關鍵詞:人類間葉幹細胞成骨分化多胜肽多層薄膜交聯組織工程
外文關鍵詞:human mesenchymal stem cells (hMSCs)osteogenic differentiationpolypeptide multilayer filmscrosslinkingtissue engineering
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多胜肽多層薄膜的一大特點是能夠經由少量表面改質吸附在所有材料上,目前已經發展為各種生醫材料。本研究使用帶正電的聚左旋離胺酸(poly-L-lysine)和帶負電的聚左旋麩胺酸(poly-L-glutamic acid)以1-乙基-(3-二甲基氨基丙基)碳醯二亞胺/N-羥基硫代琥珀醯亞胺(EDC/Sulfo-NHS)作為交聯劑,以層層堆疊法(layer-by-layer technique)製備多胜肽多層交聯薄膜(六層PLL/PLGA交聯薄膜),並在其上吸附(coating)一型膠原蛋白(collagen type I, COL I),試驗其對人類間葉幹細胞(human mesenchymal stem cell; hMSC)進行成骨分化(osteogenic differentiation)的影響。在成骨分化期間,使用顯微鏡觀察hMSC的貼附、細胞型態、排列和增長的同時,也運用細胞-基質電阻抗儀(electric cell-substrate impedance sensing, ECIS)以非侵入、即時的方式監控細胞行為,並透過茜素紅染色(Alizarin Red S staining)和定量反轉錄聚合酶鏈鎖反應(quantitative reverse transcription polymerase chain reaction)來量化其效果。結果顯示hMSC的成骨分化效果於皆吸附COL I的玻片和六層PLL/PLGA交聯薄膜上並無顯著差異,但於六層PLL/PLGA交聯薄膜的鈣質沉積比在玻片要均勻。總結而言,六層PLL/PLGA交聯薄膜雖未提升hMSC的成骨分化效果,但仍能讓hMSC進行成骨分化,有做為吸附材料的潛力,例如可用做其他生醫材料的表面改質材料等。最後,此研究展示了細胞-基質電阻抗儀能夠在體外即時監測hMSC的成骨分化,並能用數學模型及量化的參數,來解釋成骨分化過程中的細胞行為。
Polypeptide multilayer films have been developed as a versatile biomaterial and they can be adsorbed on nearly every material with few or no modification. In this study, alternatively deposited six layers of cationic poly-L-lysine (PLL) and anionic poly-L-glutamic acid (PLGA) films were fabricated by the layer-by-layer (LbL) technique, crosslinked with EDC/Sulfo-NHS, and coated with collagen type I (COL I). The six-layer crosslinked PLL/PLGA films were compared to the cover glass, both coated with COL I, to examine effects of the films on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). During the differentiation process, cell attachment and spreading, morphology, alignment, and proliferation were observed by microscopic images and the electric cell-substrate impedance sensing (ECIS) system. Osteogenic differentiation was evaluated by Alizarin Red S (ARS) staining and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results showed that the hMSCs on both substrates coated with COL I underwent osteogenic differentiation, and no significant differences were spotted except for a more evenly distributed calcium deposition on the six-layer crosslinked PLL/PLGA films. For conclusion, the six-layer crosslinked PLL/PLGA films did not demonstrate significantly improved osteogenic differentiation of the hMSCs, but could still serve as a potential coating material for other biomedical substrates as hMSCs were proved to differentiate into osteoblastic cells on the films. Lastly, the ECIS system can be a practical way for real-time monitoring the osteogenic differentiation process of the hMSCs in vitro and the model fitting could describe cell behaviors in a quantitative way.
Contents
中文摘要 ................................................................................................. i
English Abstract ........................................................................................ii
Contents .............................................................................................. iii
List of Figures ......................................................................................... v
List of Tables ......................................................................................... vi
List of Abbreviations ..................................................................................vii
1. Introduction ......................................................................................... 1
1.1 Stem Cells .......................................................................................... 1
1.2 Mesenchymal Stem Cells .............................................................................. 1
1.3 Osteogenic Differentiation .......................................................................... 4
1.4 Polypeptide Multilayer Films ........................................................................ 5
1.5 Tissue Engineering .................................................................................. 7
1.6 Electric Cell-Substrate Impedance Sensing (ECIS) ................................................... 10
1.7 Objectives ......................................................................................... 12
2. Materials and Methods ............................................................................... 13
2.1 Cell Culture ....................................................................................... 13
2.2 Osteogenic Differentiation ......................................................................... 13
2.3 Using ECIS to observe osteogenic differentiation of hMSCs .......................................... 14
2.4 Film Fabrication ................................................................................... 15
2.5 Experimental Design ................................................................................ 18
2.6 Alizarin Red S Staining Assay ...................................................................... 19
2.7 Quantitative Reverse Transcription Polymerase Chain Reaction........................................ 20
3. Results ............................................................................................. 23
3.1 Osteogenic Differentiation Potential of hMSCs ...................................................... 23
3.2 ECIS Monitoring of hMSCs undergoing osteogenic differentiation ..................................... 25
3.3 Osteogenic Differentiation of hMSCs on PLL/PLGA Films .............................................. 28
3.4 ARS Staining Results ............................................................................... 33
3.5 RT-qPCR Results .................................................................................... 36
4. Discussion .......................................................................................... 37
4.1 Cell Adhesion and Spreading ........................................................................ 37
4.2 Cell Morphology and Alignment ...................................................................... 39
4.3 Proliferation Dynamics ............................................................................. 42
4.4 Calcium Deposition ................................................................................. 44
4.5 RNA expression of the protein markers .............................................................. 46
4.6 Effects of Substrate on the Osteogenic Differentiation of hMSCs .................................... 48
4.7 Future Works and Possible Clinical Applications .................................................... 52
5. Conclusion .......................................................................................... 54
6. References .......................................................................................... 56

List of Figures
Figure 1.1 A TE approach to differentiate hMSCs into osteoblasts. ....................................... 9
Figure 1.2 The basic idea of ECIS. ..................................................................... 10
Figure 1.3 Illustration of ECIS modeling. .............................................................. 11
Figure 2.1 Preparation of the chamber dish. ............................................................ 15
Figure 2.2 LbL fabrication of polypeptide multilayer films. ............................................ 16
Figure 2.3 Experimental scheme.......................................................................... 18
Figure 3.1 Osteogenic differentiation of hMSCs on the first seven days. ................................ 23
Figure 3.2 Osteogenic differentiation of hMSCs for 21 days. ............................................ 24
Figure 3.3 Real-time ECIS of hMSCs in culture and differentiation medium. .............................. 25
Figure 3.4 Normalized parameters of ECIS. .............................................................. 26
Figure 3.5 Images of hMSCs cultured on different substrates and conditions. ............................ 28
Figure 3.6 Box plots of cell area and length/width ratio measured on day 0. ............................ 29
Figure 3.7 Cell orientation angle under different culture conditions. .................................. 31
Figure 3.8 Alizarin Red S staining images. ............................................................. 33
Figure 3.9 The ARS staining images before and after processed. ......................................... 34
Figure 3.10 Bar chart of the ARS-stained area. ......................................................... 35
Figure 3.11 Relative gene expression profiles of Runx2 and OCN. ........................................ 36
Figure 4.1 Timeline of the osteogenic differentiation process. ......................................... 46
Figure 4.2 Multiple factors for stem cell differentiation. ............................................. 48

List of Tables
Table 2.1 Sequences of the primers used in RT-qPCR. .................................................... 22
Table 3.1 ARS staining area and its percentage. ........................................................ 35
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