臺灣博碩士論文加值系統

心血管疾病造成的死亡率歷年來一直居高不下，而繞道手術是臨床常用的一種治療心血管疾病方式。到目前為止，仍未發現適合用於動脈繞道手術的材料。因此本研究以靜電紡絲的方法將蠶絲中的絲素蛋白與人類頭髮中的角蛋白製備成多孔隙纖維管，以利血管組織工程移植物的發展。
首先將絲素蛋白(F)與角蛋白(K)分別以9:1 (FK91)、8:2 (FK82)、7:3 (FK73)的重量比混合均勻後，以靜電紡絲噴成纖維膜。藉由掃描式電子顯微鏡可得知所得之纖維膜具有多孔且互相連通的結構，且F、FK91、FK82、FK73纖維膜的平均直徑隨著角蛋白的含量增加而降低。接著進一步以拉伸試驗機測得F、FK91、FK82、FK73纖維膜的最大拉伸強度亦隨蛋白的含量增加而降低。藉由親/疏水性測試可得知F、FK91、FK82、FK73纖維膜的潤濕性因角蛋白的含量增加而提升。
由體外試驗結果得知纖維膜的細胞貼附能力與其潤濕性一致，且其對於人類臍帶靜脈內皮細胞不具有毒性。免疫螢光染色與即時聚合酶鏈鎖反應分析亦證實了纖維膜並不影響細胞的表型。根據纖維膜的最大拉伸強度、細胞貼附能力與微觀結構，本研究認為FK82與血管相似，可用於後續動物植入實驗。藉由微電腦斷層掃瞄、組織學染色分析得知將大鼠主動脈內皮細胞種在FK82之多孔纖維管(FK82C)，並移植至大鼠腹主動脈一個月後，大鼠並未發生再狹窄與鈣化的情況，綜合以上結果顯示其具有血管組織工程的材料的潛力。

Cardiovascular diseases (CVD) are one of the leading causes of death and bypass surgery is one of the common treatment options for the critical CVD patients. There is no ideal material available for arterial bypass surgery.
Herein, fibroin and keratin porous fibrotic tube was prepared by electro-spinning and proposed for tissue-engineered vascular graft (TEVG). The purified fibroin (F) and keratin (K) was mixed in different weight ratios of 9:1 (FK91), 8:2 (FK82), and 7:3 (FK73) to obtain membrane. The SEM images revealed that the electro-spinned membranes have fibrous interconnected porous structure. The average diameter and the ultimate tensile strength (UTS) of the developed membrane decreased with the increased keratin content. The wettability of the developed membrane increased with keratin content. The wettability results were in agreement with that of the cell adhesion to the electro-spinning membranes. The attached HUVECs on the developed membranes showed no cytotoxicity. The immunocytochemistry staining and qPCR analysis showed that, the phenotype of keratin-fibroin membrane was not altered. The results of ultimate tensile strength, cell adhesion and microstructure revealed that FK82 is similar to that of native vessels and could be used in vivo test. The rat aorta endothelial cells were seeded in FK82 porous fibrous tube (FK82C) and then implanted to the rat abdominal aorta for one month. FK82C vascular graft could not be restenosis and calcification. Therefore, it could be considered as a potential material for TEVG.

誌謝………………………………………………………………..…………………..i
摘要………………………………………………………………..………………….ii
ABSTRACT………………………………………………………………………….iii
LIST OF FIGURES…………………………………………………………………vi
LIST OF TABLES……………………………………………………………….…viii
LIST OF ABBREVIATION…………………………………………………….…ix
Chapter 1 Introduction…………………………………………………………….. 1
1.1 Cardiovascular diseases………………………………………………...……1
1.2 Vascular bypass grafting………………………………………………..……4
1.2.1 Autograft/Allograft…………………………………………………...……4
1.2.2 Synthetic graft………………………………………………………...……4
1.2.3 Biological graft……………………………………………………….……5
1.3 Object of study………………………………………………………….……5
Chapter 2 Theoretical basis…………………………………………………………7
2.1 Silk fibroin……………………………………………………………...……7
2.2 Human hair keratin……………………………………………….…………10
2.3 Electro-spinning………………………………………………………….…12
2.4 Vascular endothelial cells…………………………………………..………14
Chapter 3 Materials and methods…………………………………………………16
3.1 Fibroin-Keratin porous membrane……………………………….…………17
3.1.1 Isolation and purification of fibroin from cocoon silk…………..……17
3.1.2 Isolation and purification of keratin from human hair……………..…19
3.1.3 Electro-spinning of keratin/fibroin membrane……………………..…21
3.1.4 Microstructure and diameter of electro-spinning fibers………………21
3.1.5 Tensile strength measurement…………………………………………21
3.1.6 Water contact angle measurement…………………………….………22
3.1.7 Cell culture………………………………………….…………………22
3.1.8 Cell viability………………………………………………………...…22
3.1.9 Cytotoxicity……………………………………………………………23
3.1.10 Cell adhesion test………………………………………….…………23
3.1.11 Immunocytochemistry staining………………………………………24
3.1.12 Gene expression by quantitative PCR (qPCR)…...…………………24
3.1.13 Statistical analysis……………………………………………………25
3.2 Vascular graft…………………………………….…………………………25
3.2.1 Electro-spinning of keratin/fibroin tube……….……………...………25
3.2.2 Cell seeding method…………………………………………...………25
3.2.3 Graft implantation………………………………………………..……26
3.2.4 Imaging of the grafts in vivo………………………………..…………28
3.2.5 Histology, immunohistochemistry………………………….…………28
Chapter 4 Results……………………………………………………………….…30
4.1 Morphology of fibrous membrane and cell…………………………………30
4.2 Diameter analysis of reconstructed fibers……………………..……………32
4.3 Tensile strength…………………………………………………………..…34
4.4 Wettability………………………………………..…………………………36
4.5 Cell viability and cytotoxicity………………………………………………38
4.6 Cell adhesion study…………………………………………………………39
4.7 HUVECs gene expression analysis…………………………………………40
4.8 Operative results and patency evaluation………………………..…………46
4.9 Histology and immunohistochemistry after implantation…………..………49
Chapter 5 Discussion………………………………………….. …………………53
Chapter 6 Conclusion…………………………………………………………..…56
REFERENCE…………………………………………………………………….…57

1. Roger VL, Go AS, Lloyd-Jones DM, Adams RJ, Berry JD, Brown TM, Carnethon MR, Dai S, de Simone G, Ford ES, Fox CS, Fullerton HJ, Gillespie C, Greenlund KJ, Hailpern SM, Heit JA, Ho PM, Howard VJ, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Makuc DM, Marcus GM, Marelli A, Matchar DB, McDermott MM, Meigs JB, Moy CS, Mozaffarian D, Mussolino ME, Nichol G, Paynter NP, Rosamond WD, Sorlie PD, Stafford RS, Turan TN, Turner MB, Wong ND, Wylie-Rosett J, American heart association statistics committee and stroke statistics subcommittee, Circulation 2011; 123(4): 18-209.
2. Diagram showing a heart attack, NIH: National Heart, Lung and Blood Institute, Retrieved July 26, 2017, from http://www.nhlbi.nih.gov/health/health-topics/topics/heartattack
3. Coronary artery bypass grafting, NIH: National Heart, Lung and Blood Institute, Retrieved July 26, 2017, from https://commons.wikimedia.org/wiki/File:Coronary_artery_bypass_grafting.jpg
4. Cooper GJ, Underwood MJ, Deverall PB, Arterial and venous conduits for coronary artery bypass, European Journal of Cardio-Thoracic Surgery: Official Journal of the European Association for Cardio-Thoracic Surgery 1996; 10(2): 129-140.
5. Nwasokwa ON, Coronary artery bypass graft disease, Annals of Internal Medicine 1995; 123(7): 528-545.
6. Motwani JG, Topol EJ. Aortocoronary saphenous vein graft disease. Circulation 1998; 97: 916-931.
7. Wu HC, Wang TW, Kang PL, Tsuang YH, Sun JS, Lin FH, Coculture of endothelial and smooth muscle cells on a collagen membrane in the development of a small-diameter vascular graft, Biomaterials 2007; 28(7): 1385-1392.
8. Foster ED, Kranc MA, Alternative conduits for aortocoronary bypass grafting Circulation 1989; 79: 34–39.
9. Conte MS, The ideal small arterial substitute: a search for the Holy Grail? , FASEB J 1998; 12(1): 43-45.
10. Hu J, Sun X, Ma HY, Xie CQ, Chen E, Ma PX, Porous nanofibrous PLLA scaffolds for vascular tissue engineering, Biomaterials 2010; 31: 7971-7977.
11. Nerem RM, Seliktar D, Vascular tissue engineering, Annu Rev Biomed Eng 2001; 3: 225-243.
12. Kuo SM, Chang SJ, Yao CH, Manousakas I, A perspective view on the preparation of micro- and nanoparticulates of biomaterials from electrostatic and ultrasonic methods, Biomed Eng Appl Basis Commun 2009; 21: 343-353.
13. Rao JW, Ouyang LQ, Jia XL, Quan DP, Ouyang LQ, Rao JW. The fabrication and characterization of 3D porous sericin/fibroin blended scaffolds. Biomed Eng Appl Basis Commun 2012; 23: 1-12.
14. Sierpinski P, Garrett J, Ma J, Apel P, Klorig D, Smith T, Koman LA, Atala A, Van Dyke M, The use of keratin biomaterials derived from human hair for the promotion of rapid regeneration of peripheral nerves, Biomaterials 2008; 29: 118-128.
15. Rouse JG, Van Dyke ME, A review of keratin-based biomaterials for biomedical applications, Materials 2010; 3: 999-1014.
16. Li M, Tao W, Lu S, Kuga S, Compliant film of regenerated Antheraea pernyi silk fibroin by chemical crosslinking, Int J Biol Macromol 2003; 32: 159-163.
17. Aluigi A, Zoccola M, Vineis C, Tonin C, Ferrero F, Canetti M, Study on the structure and properties of wool keratin regenerated from formic acid, Int J Biol Macromol 2007; 41: 266-273.
18. Vasconcelos A, Freddi G, Cavaco-Paulo A, Biodegradable materials based on silk fibroin and keratin, Biomacromolecules 2008; 9: 1299-305.
19. Liu H, Li X, Zhou G, Electrospun sulfated silk fibroin nanofibrous scaffolds for vascular tissue engineering, Biomaterials 2011; 32: 3784-3793.
20. Wang SD, Zhang YZ, Yin GB, Wang HW, Dong ZH, Fabrication of a composite vascular scaffold using electrospinning technology, Mater Sci Eng C 2010; 30: 670-676.
21. Chew SY, Mi R, Hoke A, Leong KW, The effect of the alignment of electrospun fibrous scaffolds on Schwann cell maturation, Biomaterials 2008; 29: 653-661.
22. Hakimi O, Knight DP, Vollrath F, Vadgama P, Spider and mulberry silkworm silks as compatible biomaterials, Composites Part B 2007; 38: 324-337.
23. Dyakonov T, Yang CH, Bush D, Gosangari S, Majuru S, Fatmi A, Design and characterization of a silk-fibroin-based drug delivery platform using naproxen as a model drug, J Drug Deliv 2012; 2012: 490514.
24. Allardyce BJ, Rajkhowa R, Dilley RJ, Atlas MD, Kaur J, Wang X, The impact of degumming conditions on the properties of silk films for biomedical applications, Text Res J 2016; 86: 275-287.
25. Zhang X, Tsukada M, Morikawa H, Aojima K, Zhang G, Miura M, Production of silk sericin/silk fibroin blend nanofibers, Nanoscale Res Lett 2011; 6: 510.
26. Silk beta-sheets, Biofoundations, Retrieved July 26, 2017, from http://biofoundations.org/brain-factor7-bf-7-from-silkworms-enhancing-brain-health
27. Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen J, Lu H, Richmond J, Kaplan DL, Silk-based biomaterials, Biomaterials 2003; 24: 401-416.
28. Wang B, Yang W, McKittrick J, Meyers MA, Keratin: Structure, mechanical properties, occurrence in biological organisms, and efforts at bioinspiration, Prog Mater Sci 2016; 76: 229-318.
29. Dowling LM, Crewther WG, Inglis AS, The primary structure of component 8c-1, a subunit protein of intermediate filaments in wool keratin. Relationships with proteins from other intermediate filaments, Biochem J 1986; 236: 695-703.
30. Sierpinski P, Garrett J, Ma J, Apel P, Klorig D, Smith T, Koman LA, Atala A, Van Dyke M, The use of keratin biomaterials derived from human hair for the promotion of rapid regeneration of peripheral nerves, Biomaterials 2008; 29: 118-128.
31. Ki CS, Baek DH, Gang KD, Lee KH, Um IC, Park YH, Characterization of gelatin nanofiber prepared from gelatin–formic acid solution , Polymer 2005; 46: 5094-5102.
32. Li D, Xia Y, Electrospinning of nanofibers: Reinventing the wheel? , Adv Mater 2004; 16: 1151-1170.
33. Rodriguez-Morata A, Garzon I, Alaminos M, Garcia-Honduvilla N, Sanchez-Quevedo MC, Bujan J, Campos A, Cell viability and prostacyclin release in cultured human umbilical vein endothelial cells, Ann Vasc Surg 2008; 22: 440-448.
34. Sumpio BE, Riley JT, Dardik A, Cells in focus: endothelial cell, Int J Biochem Cell Biol 2002; 34: 1508-1512.
35. Yang Y, Chen X, Ding F, Zhang P, Liu J, Gu X, Biocompatibility evaluation of silk fibroin with peripheral nerve tissues and cells in vitro, Biomaterials 2007; 28: 1643-1652.
36. Marelli B, Alessandrino A, Fare S, Freddi G, Mantovani D, Tanzi MC, Compliant electrospun silk fibroin tubes for small vessel bypass grafting, Acta Biomater 2010; 6: 4019-4026.
37. Amiraliyan N, Nouri M, Kish MH, Effects of some electrospinning parameters on morphology of natural silk-based nanofibers, J Appl Polym Sci 2009; 113: 226-234.
38. Reichl S, Films based on human hair keratin as substrates for cell culture and tissue engineering, Biomaterials 2009; 30: 6854-6866.
39. Alemdar A, Iridag Y, Kazanci M, Flow behavior of regenerated wool-keratin proteins in different mediums, Int J Biol Macromol 2005; 35: 151-153.
40. Rockwood DN, Preda RC, Yucel T, Wang X, Lovett ML, Kaplan DL, Materials fabrication from Bombyx mori silk fibroin, Nat Protocols 2011; 6: 1612-1631.
41. Corey JM, Lin DY, Mycek KB, Chen Q, Samuel S, Feldman EL, Martin DC, Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth, J Biomed Mater Res A 2007; 83: 636-645.
42. Zhang T, Fan HT, Xu XJ, Liu H, Uniaxially aligned In2O3 nanofibers based sensors with fast response to ethanol, Biomed Eng Appl Basis Commun 2012; 24: 105-109.
43. Young TH, Chung YC, Chen MH, Yu BY, Chen RS, The behavior of rat tooth germ cells on 3-hydroxyl-butyrate-co-3-hydroxy-hexanoate (PHBHHx) membranes, Biomed Eng Appl Basis Commun 2007; 19: 279-288.
44. Wang MH, Hsu CK, Soung HS, Tang TP, Chang KC, Methyl cellulose enhance gelatin membrane as guidance channels for peripheral nerve regeneration, Biomed Eng Appl Basis Commun 2012; 24: 85-98.
45. Tsai HT, Liao SC, Wu HM, Chou CY, Chen KS, Temperature sensitivity of composite hydrogel prepared by surface graft polymer of nipaam and bamboo charcoal powder, Biomed Eng Appl Basis Commun 2012; 24: 171-177.
46. Huang KK, Chao YC, Shih YC, Shih MK, Inhibitory activity of natural active compounds against human papilloma virus pseudovirus and novel dosage form for use therewith, Biomed Eng Appl Basis Commun 2013; 25: 1350036.
47. Lin FH, Wu HC, Evaluation of magnetic-hydroxyapatite nanoparticles for gene delivery carrier, Biomed Eng Appl Basis Commun 2010; 22: 33-39.
48. Wang KP, Hsu SH, Huang TB, Low-intensity ultrasound increases the endothelial nitric oxide synthase (eNOS) expression of endothelial cells possibly via the phosphatidylinositol 3-kinase/akt/protein kinase a signaling pathway, Biomed Eng Appl Basis Commun 2010; 22: 367-376.
49. Dang JM, Sun DD, Shin-Ya Y, Sieber AN, Kostuik JP, Leong KW, Temperature-responsive hydroxybutyl chitosan for the culture of mesenchymal stem cells and intervertebral disk cells, Biomaterials 2006; 27: 406-418.
50. Cheng YH, Yang SH, Liu CC, Gefen A, Lin FH, Thermosensitive hydrogel made of ferulic acid-gelatin and chitosan glycerophosphate, Carbohydr Polym 2013; 92: 1512-1519.
51. Unger RE, Peters K, Wolf M, Motta A, Migliaresi C, Kirkpatrick CJ, Endothelialization of a non-woven silk fibroin net for use in tissue engineering: growth and gene regulation of human endothelial cells, Biomaterials 2004; 25: 5137-5146.
52. Hoshi RA, Van Lith R, Jen MC, Allen JB, Lapidos KA, Ameer G, The blood and vascular cell compatibility of heparin-modified ePTFE vascular grafts, Biomaterials 2013; 34: 30-41.
53. Fuchs S, Motta A, Migliaresi C, Kirkpatrick CJ, Outgrowth endothelial cells isolated and expanded from human peripheral blood progenitor cells as a potential source of autologous cells for endothelialization of silk fibroin biomaterials, Biomaterials 2006; 27: 5399-5408.
54. Matthews JA, Wnek GE, Simpson DG, Bowlin GL, Electrospinning of collagen nanofibers, Biomacromolecules 2002; 3: 232-238.
55. Adham M, Gournier JP, Favre JP, De La Roche E, Ducerf C, Baulieux J, Barral X, Pouyet M, Mechanical characteristics of fresh and frozen human descending thoracic aorta, J Surg Res 1996; 64: 32-34.
56. Roeder R, Wolfe J, Lianakis N, Hinson T, Geddes LA, Obermiller J, Compliance, elastic modulus, and burst pressure of small-intestine submucosa (SIS), small-diameter vascular grafts, J Biomed Mater Res 1999; 47: 65-70.
57. Jia L, Prabhakaran M, Qin X, Kai D, Ramakrishna S, Biocompatibility evaluation of protein-incorporated electrospun polyurethane-based scaffolds with smooth muscle cells for vascular tissue engineering, J Mater Sci 2013; 48: 5113-5124.
58. Brewster L, Brey EM, Greisler HP, Chapter Thirty-Nine - Blood vessels. In: Lanza R, Langer R, Vacanti J, editors, Principles of Tissue Engineering (Third Edition). Burlington: Academic Press; 2007, p. 569-584.
59. Tien J, Golden AP, Tang MD. Engineering of blood vessels. In: Shepro D, D''Amore PA, editors. Microvascular Research: Biology and Pathology. San Diego, CA: Academic Press; 2006, p. 1087-1093.
60. Wagenseil JE, Mecham RP, Vascular extracellular matrix and arterial mechanics, Physiol Rev 2009; 89: 957-989.
61. Pedersen EM, Oyre S, Agerbaek M, Kristensen IB, Ringgaard S, Boesiger P, Paaske WP, Distribution of early atherosclerotic lesions in the human abdominal aorta correlates with wall shear stresses measured in vivo, Eur J Vasc Endovasc Surg 1999; 18: 328-333.
62. Tang BT, Cheng CP, Draney MT, Wilson NM, Tsao PS, Herfkens RJ, Taylor CA, Abdominal aortic hemodynamics in young healthy adults at rest and during lower limb exercise: quantification using image-based computer modeling, Am J Physiol Heart Circ Physiol 2006; 291: 668-676.
63. Mowa CN, Jesmin S, Sakuma I, Usip S, Togashi H, Yoshioka M, Hattori Y, Papka R, Characterization of vascular endothelial growth factor (VEGF) in the uterine cervix over pregnancy: effects of denervation and implications for cervical ripening, J Histochem Cytochem 2004; 52: 1665-1674.
64. Kroll J, Waltenberger J, A novel function of VEGF receptor-2 (KDR): rapid release of nitric oxide in response to VEGF-A stimulation in endothelial cells, Biochem Biophys Res Commun 1999; 265: 636-639.
65. Ma Z, Kotaki M, Yong T, He W, Ramakrishna S, Surface engineering of electrospun polyethylene terephthalate (PET) nanofibers towards development of a new material for blood vessel engineering, Biomaterials 2005; 26: 2527-2536.
66. Tara S, Kurobe H, Rocco KA, Maxfield MW, Best CA, Yi T, Naito Y, Breuer CK, Shinoka T, Well-organized neointima of large-pore poly(L-lactic acid) vascular graft coated with poly(L-lactic-co-ε-caprolactone) prevents calcific deposition compared to small-pore electrospun poly(L-lactic acid) graft in a mouse aortic implantation model, Atherosclerosis 2014; 237: 684-691.