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系統識別號 U0026-2007201014254400
論文名稱(中文) 聚丙烯腈奈米纖維補片於心肌組織工程上之應用
論文名稱(英文) Development of a Novel Myocardial Thinfilm Using Electrospun Polyacrylonitrile Nanofibers
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 98
學期 2
出版年 99
研究生(中文) 吳淑婷
研究生(英文) Su-Ting Wu
學號 n3697402
學位類別 碩士
語文別 英文
論文頁數 38頁
口試委員 指導教授-吳文騰
共同指導教授-謝清河
口試委員-王盈錦
口試委員-張德生
口試委員-陳美瑾
中文關鍵字 心肌梗塞  電紡織法  心肌補片 
英文關鍵字 Myocardial infarction  electrospinning  myocardial thinfilm 
學科別分類
中文摘要 心臟衰竭是國人最主要的死因之一,其病因大多是由於心臟的功能受損會伴隨著有害於心臟的心肌重塑(remodeling)。目前心肌組織工程被視為可用於補強受損心肌、增進心臟功能的技術。本研究中,我們將利用電紡織法開發出一個具生物相容性,可用於修補受損心肌,並有結合細胞治療潛力進而長期改善心臟功能的心肌補片。
研究指出心肌細胞間的規則排列有助於化學信息的交流與傳遞;因此,我們利用電紡織法製備出順向性的聚丙烯腈(ES-PAN)奈米纖維補片,以作為細胞生長與排列的支架,並以非順向性做為對照組。接著將內皮細胞培養於ES-PAN補片上,五天後以細胞存活率分析(MTT)及細胞週期分析法(PI-staining)進行ES-PAN補片生物毒性測試。結果顯示培養於ES-PAN補片上無明顯毒性,證明ES-PAN補片具有生物相容性。此外,經由細胞免液染色法分析後,相較於非順向性ES-PAN補片,培養於順向性ES-PAN補片上之內皮細胞與心肌細胞上皆擁有較好的順向性排列。
在活體測試中,利用心肌梗塞老鼠模式,植入ES-PAN補片於心臟受損表面,經兩個月觀察,超音波結果顯示植入ES-PAN補片之心臟功能明顯比未做任何修補的組別好。
總結以上研究,我們證實順向性ES-PAN補片可成功誘導細胞生長方向,並有助於提升受損後之心臟功能。相信結合兩者好處,亦即將培養內皮與心肌細胞之順向性ES-PAN補片,用於修復受損心肌會有更好的效果。因此,未來將著重於結合上述兩者,應用於心臟組織修復,使其成為心肌組織工程中一個具有潛力的治療方式。
英文摘要 Heart failure is associated with a significant impact on morbidity and mortality attributable largely to myocardial dysfunction accompanied by progressive harmful cardiac remodeling. The use of cardiac grafts has been considered for replacing damaged myocardium to enhance cardiac function. In this study, we tried to design and develop a biocompatible myocardial thinfilm by electrospinning and combining with cell therapy to patch infarcted myocardium and improve long-term heart function.
Studies have verified that a compact, end-to-end cardiac myocyte arrangement enhances electrophysiological connection and function. To achieve this characteristic, we have developed well-aligned electrospun polyacrylonitrile (ES-PAN) nanofiber thinfilms and unaligned as a control. Then, we examined the cytotoxicity of ES-PAN thinfilms by MTT and PI-staining. The percentage of endothelial cell apoptosis was lower when cultured on tissue-culture polystyrene (TCPS) than on ES-PAN thinfilm, with no significant difference demonstrating the biocompatibility of PAN thinfilm. In addition, endothelial cells and cardiomyocytes were cultured on ES-PAN thinfilms and showed better alignment when cultured on patterned ES-PAN thinfilms than on unaligned thinfilms. For in vivo testing, we implanted an empty ES-PAN thinfilm onto the infarcted left ventricular wall surface of a rat model. Results of echocardiography revealed that implantation of an ES-PAN thinfilm onto a myocardial infarction improved contractile function compared with infarct group after 8 weeks (P < 0.05).
These results demonstrated that physical patterning of ES-PAN thinfilms affects cell organization and implantation of the ES-PAN thinfilms preserves cardiac functions after myocardial infarction (MI).
Aligned endothelial cells and cardiomyocytes integrated with implantation of an ES-PAN thinfilm into the infarct myocardium may have synergistic therapeutic effect in myocardial infarction treatments. Future works will focus on the combination of cell therapy and biomaterial implantation to achieve higher recovery rates of the myocardial infarct heart.
論文目次 TABLE OF CONTENTS
Abstract in English I
Abstract in Chinese III
Acknowledgement V
Table of contents VI
Index of figures VIII
Index of tables IX

Chapter 1. Introduction 1
1.1 Congestive heart failure 1
1.2 Current therapeutic strategies for heart failure 1
1.3 Tissue engineering 2
1.4 Tissue engineering strategies to regenerate heart function 4
1.5 Challenge of cardiac patch engineering 5
1.6 Electrospinning 6
1.6.1 Technique improvement on electrospinning: Fiber alignment 7
1.6.2 Electrospinning apply to tissue engineering 9
Chapter 2. Materials and methods 10
2.1 Electrospun-PAN thinfilm fabrication 10
2.2 Mechanical testing 11
2.3 Analyses of fiber and cell orientation 11
2.4 Cell isolation 11
2.5 Cell culture 12
2.6 MTT assay 12
2.7 Flow cytometry 12
2.8 Immunocytochemistry 13
2.9 Rat myocardial infarction model and PAN thinfilm implantation 13
2.10 Echocardiography 14
2.11 Masson's trichrome staining 14
2.12 Statistical analysis 15
Chapter3. Results 16
3.1 Fabrication and characterization of the ES-PAN thinfilms 16
3.2 ES-PAN thinfilms cytotoxicity testing 16
3.3 Endothelial cells culturing and response to ES-PAN thinfilms 17
3.4 Cardiomyocytes culturing and response to ES-PAN thinfilms 17
3.5 Endothelial cells and cardiomyocytes co-culturing and response to ES-PAN thinfilms 18
3.6 Implantation an ES-PAN thinfilm to repair myocardial defect in a rat model 18
3.6.1 ES-PAN thinfilm implantation improves cardiac functions after MI 18
3.6.2 ES-PAN thinfilm implantation reduces infarct size after MI 19
Chapter 4. Discussion 32
4.1 Non-biodegradable materials provide adequate strength for long-term in vivo mechanical support 32
4.2 Blood vessel formation contributed to improved cardiac function 33
4.3 Conclusion and future work 33
References 35

Index of figures
Figure 1. Key factors of tissue engineering. 3
Figure 2. Schematic diagram of the electrospinning. 7
Figure 3. Electrospnning setup used to obtain aligned nanofibers. 10
Figure 4. Characterization of ES-PAN thinfilms. 20
Figure 5. Stress–strain curves. 21
Figure 6. Endothelial cells viability on ES-PAN thinfilms measured by MTT assay. 22
Figure 7. Endothelial cells align unidirectionally when cultured on aligned ES-PAN thinfilms.. 23
Figure 8. Cardiomyocytes align when cultured on aligned ES-PAN thinfilms. 24
Figure 9. Endothelial cells and cardiomyocytes align when cultured on aligned ES-PAN thinfilms. 25
Figure 10. Aligned ES-PAN thinfilms improve cardiomyocytes outgrowth at 5 days. 26
Figure 11. ES-PAN thinfilm implantation improves cardiac functions after MI for 2 months. 27
Figure 12. Representative images of ES-PAN at 8 weeks after implantation and histological sections. 29
Figure 13. ES-PAN thinfilm implantation reduces infarct size after MI. 31

Index of tables
Table 1. Polymers used in tissue engineering 4
Table 2. Schematic diagram of various electrospinning set-ups to obtain aligned nanofibers. 8
Table 3. Mechanical properties of ES- PAN thinfilms 21
Table 4. Endothelial cells apoptosis when cultured on ES-PAN thinfilms. 22
Table 5. Endothelial cells proliferation when cultured on ES-PAN thinfilms. 22
Table 6. Statistical analysis: correlation coefficient 23
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