臺灣博碩士論文加值系統

電氣紡絲之系統廣泛被應用於組織工程上，主要之原因為其操作過程簡單並且可有效率的製造出奈米等級之纖維；其纖維之構形提供較大的表面積，以利細胞貼附生長。於電氣紡絲系統中，膠原蛋白/玻尿酸之溶解度及電氣紡絲不同參數(如：電壓、不同溶劑比、不同重量百分比)會對纖維直徑之影響。本實驗以formic acid / HFIP混合溶液作為電氣紡絲之最佳溶劑，由掃描式電子顯微鏡結果圖得知其電壓、不同溶劑比及不同重量百分比之參數對膠原蛋白/玻尿酸奈米複合纖維之直徑大小沒有顯著的差異。而由螢光顯微鏡及FT-IR結果圖可證明膠原蛋白與玻尿酸的確存在於膠原蛋白/玻尿酸共紡之複合奈米纖維內。於細胞的結果圖中，可以得知膠原蛋白/玻尿酸重量百分比為16/3於第二天及第四天時，細胞生長的情形與培養皿有明顯的差異(P＜0.05)，而在第六天時，彼此間沒有明顯的差異(P＞0.05)，且細胞型態以round-like呈現也和培養皿的spindle-like型態有所差異。基因表現的結果圖中，可以發現膠原蛋白/玻尿酸重量百分比為16/3的複合奈米纖維，可使MMP-1/TIMP-1比值增加，由此可推知膠原蛋白/玻尿酸重量百分比為16/3之基材可能會減少疤痕組織之形成。由以上之結果可得知本實驗所開發的膠原蛋白/玻尿酸複合奈米纖維於傷口癒合的應用上，極具有開發之價值。

Electrospinning is a simple and effective method for producing nanofibers. The fibrous matrix had high specific surface area. This property could enhance the cell adhesion and proliferation. We successfully fabricated Type I collagen / hyaluronic acid nanofibrous matrix in HFIP/ formic acid system. Scanning electron microscope revealed that different factors, including voltage, concentration, and solution ratio, were not significantly effected the average diameter of type I collagen / hyaluronic acid nanofiber. On the other hand, we utilized FTIR and fluorescent label method to prove the nanofibers containing hyaluronic acid and collagen. Furthermore, foreskin cells cultured on the various weight ratio of collagen/hyaluronic acid nanofibrous matrix displayed differential cell attachment, proliferation rate, cell morphology and gene expression. Cells expressed higher levels of MMP-1/TIMP-1 ratio on the ratio of collagen to hyaluronic acid was 16/3. The increased ratio of MMP to TIMP expression, which is characteristic of scales wound. Together the data demonstrated that collagen/hyaluronic acid nanofibrous matrix has potential as a wound dressing for skin regeneration.

目錄
中文摘要 I
Abstract II
目錄 III
圖目錄 VI
表目錄 VIII
表目錄 VIII
第一章 前言 1
第二章 研究動機 3
第三章 文獻回顧 5
3.1 皮膚 5
3.2 傷口癒合過程 7
3.3 傷口治療之方式 9
3.4 人工皮膚具備之條件 11
3.5 市售之人工皮膚 13
3.6 基質之製備 16
3.7 電氣紡絲 17
3.8 膠原蛋白 21
第四章 實驗藥品 29
第五章 實驗儀器與耗材 31
第六章 實驗方法 33
6.1 膠原蛋白純化 33
6.2 膠原蛋白之定性及定量 35
6.3 膠原蛋白、玻尿酸及膠原蛋白/玻尿酸複合物之溶解度測試 38
6.4 不同重量百分比之膠原蛋白/玻尿酸混合溶液配置 39
6.5 膠原蛋白/玻尿酸複合奈米纖維製成 40
6.6 膠原蛋白螢光之標定 41
6.7 玻尿酸螢光之標定 42
6.8 不同重量百分比之膠原蛋白/玻尿酸複合奈米纖維之1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)交鏈 43
6.9 MTT assay 46
6.10 不同重量百分比之膠原蛋白/玻尿酸複合奈米纖維基材對纖維母細胞之影響 47
6.11 基因表現 49
第七章 研究結果與討論 53
7.1 膠原蛋白/玻尿酸之溶劑選擇 53
7.2 膠原蛋白/玻尿酸奈米複合纖維之製備 55
7.3 EDC交鏈時間對交鏈膠原蛋白/玻尿酸之影響 61
7.4 Rhodamine-膠原蛋白/FITC-玻尿酸螢光標定之分析 62
7.5 膠原蛋白、玻尿酸及膠原蛋白/玻尿酸之FT-IR成分分析 64
7.6 不同重量百分比之膠原蛋白/玻尿酸複合奈米纖維基材對纖維母細胞生長之影響 68
7.7 不同重量百分比之膠原蛋白/玻尿酸複合奈米纖維基材對纖維母細胞型態之影響 71
第八章 結論 76
第九章 參考文獻 77

圖目錄
圖一：皮膚構造 6
圖二：真皮層構造及組成分子 6
圖三：電紡之裝置圖 20
圖四：膠原蛋白D-period之構形 25
圖五: 玻尿酸之結構 28
圖六：EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)之構 44
圖七：膠原蛋白與玻尿酸間之交鏈示意圖 45
圖八：不同重量百分比之膠原蛋白/玻尿酸之混合溶液於電壓23KV、流速為0. 51c.c. /hr、工作距離為10 cm、針頭為18G之電紡條件下，膠原蛋白/玻尿酸之複合奈米纖維於電子掃描顯微鏡圖 57
圖九：不同溶劑比之膠原蛋白/玻尿酸之混合溶液於電壓23KV、流速為0. 51c.c. /hr、工作距離為10 cm、針頭為18G之電紡條件下，膠原蛋白/玻尿酸之複合奈米纖維於電子掃描顯微鏡圖 58
圖十：膠原蛋白/玻尿酸之HFIP/formic acid溶劑比為7/3混合溶液於流速為0. 51c.c./hr、工作距離為10 cm、針頭為18G、不同電壓之電紡條件下，膠原蛋白/玻尿酸之複合奈米纖維於電子掃描顯微鏡圖 59
圖十一：膠原蛋白/玻尿酸奈米複合纖維，利用EDC進行交鏈，於不同時間作用後，在電子掃描顯微鏡下觀察所得之型態 61
圖十二：rhodamine-膠原蛋白/FITC-玻尿酸溶液經電紡所得之奈米複合纖維於螢光顯微鏡下之結果圖 63
圖十三：膠原蛋白、玻尿酸及其混合物於FTIR之結果圖 66
圖十四：纖維母細胞培養於不同基材下，培養二天之MTT assay比較圖 69
圖十五：纖維母細胞培養於不同基材下，培養四天之MTT assay比較圖 69
圖十六：纖維母細胞培養於不同基材下，培養六天之MTT assay比較圖 70
圖十七：纖維母細胞培養在不同基材(培養皿、膠原蛋白纖維及膠原蛋白/玻尿酸複合奈米纖維 72
圖十八：不同重量比之膠原蛋白/玻尿酸奈米纖維對foreskin cell之β-acitin、collagen-1、MMP-1及TIMP-1基因表現的影響 75

表目錄
表1 市售之人工皮膚 15
表2: 組織器官內之膠原蛋白之類型 23
表3: 膠原蛋白應用之優點 24
表4：SDS-PAGE各標準溶液配置 37
表5：β-actin、Collagen、MMP-1、TIMP-1 引子序列之設計 52
表6：不同溶劑對玻尿酸和膠原蛋白及其混合物之溶解度 54
表7：膠原蛋白/玻尿酸物理特性結果表 60
表8：膠原蛋白及玻尿酸於FT-IR之特徵波長 67

Longaker MT, Chiu ES, Harrison MR, Crombleholme TM, Langer JC, Duncan BW, et al. Studies in Fetal Wound-Healing .4. Hyaluronic Acid-Stimulating Activity Distinguishes Fetal Wound Fluid from Adult Wound Fluid. Annals of Surgery 1989 ;210:667-672.
Longaker MT, Chiu ES, Adzick NS, Stern M, Harrison MR, Stern R. Studies in Fetal Wound-Healing .5. a Prolonged Presence of Hyaluronic-Acid Characterizes Fetal Wound Fluid. Annals of Surgery 1991 ;213:292-296.
Peled ZM, Rhee SJ, Hsu M, Chang J, Krummel TM, Longaker MT. The ontogeny of scarless healing II: EGF and PDGF-B gene expression in fetal rat skin and fibroblasts as a function of gestational age. Annals of Plastic Surgery 2001 ;47:417-424.
Hu M, Sabelman EE, Cao Y, Chang J, Hentz VR. Three-dimensional hyaluronic acid grafts promote healing and reduce scar formation in skin incision wounds. Journal of Biomedical Materials Research Part B-Applied Biomaterials 2003 ;67B:586-592.
Elaine N. Marieb, Jon Mallatt, Patricia Brady Wilhelm. Human anatomy;4th edition:P85
MacNeil S. Progress and opportunities for tissue-engineered skin. Nature 2007;445:874-880.
MacKay D. Nutritional support for wound healing. Alternative Medicine Review 2003;8:359-77.
Gosain A, Luisa MD, DiPietro A. Aging and wound healing. World Journal of Surgery 2004;28:321-326.
Martin P. Wound healing - Aiming for perfect skin regeneration. Science 1997;276:75-81.
Ruszczak Z. Effect of collagen matrices on dermal wound healing. Advanced Drug Delivery Reviews 2003;55:1595-1611.
Mao JS, Zhao LG, de Yao K, Shang QX, Yang GH, Cao YL. Study of novel chitosan-gelatin artificial skin in vitro. Journal of Biomedical Materials Research Part A 2003;64A:301-308.
Boyce ST, Warden GD. Principles and practices for treatment of cutaneous wounds with cultured skin substitutes. American Journal of Surgery 2002 ;183:445-456.
Machens HG, Berger AC, Mailaender P. Bioartificial skin. Cells Tissues Organs 2000;167:88-94.
Balasubramani M, Kumar TR, Babu M. Skin substitutes: a review. Burns 2001;27:534-544.
Park SN, Park JC, Kim HO, Song MJ, Suh H. Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking. Biomaterials 2002;23:1205-1212.
Ma L, Gao CY, Mao ZW, Zhou J, Shen JC, Hu XQ, et al. Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering. Biomaterials 2003;24:4833-4841.
O'Brien FJ, Harley BA, Yannas IV, Gibson L. Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds. Biomaterials 2004;25:1077-1086.
Allemann M, Mizuno S, Eid K, Yates KE, Zaleske D, Glowacki J. Effects of hyaluronan on engineered articular cartilage extracellular matrix gene expression in 3-dimensional collagen scaffolds. Journal of Biomedical Materials Research 2001;55:13-19.
Powell HM, Supp DM, Boyce ST. Influence of electrospun collagen on wound contraction of engineered skin substitutes.Biomaterials.2008;29:834-43
Formhals A 1934 US Patent 1,975,504
Huang ZM, Zhang YZ, Kotaki M, Ramakrishna S. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Composites Science and Technology 2003 ;63:2223-2253.
Demir MM, Yilgor I, Yilgor E, Erman B. Electrospinning of polyurethane fibers. Polymer 2002;43:3303-3309.
Pedicini A, Farris RJ. Mechanical behavior of electrospun polyurethane. Polymer 2003;44:6857-6862.
Casper CL, Stephens JS, Tassi NG, Chase DB, Rabolt JF. Controlling surface morphology of electrospun polystyrene fibers: Effect of humidity and molecular weight in the electrospinning process. Macromolecules 2004;37:573-578
Zong XH, Ran SF, Fang DF, Hsiao BS, Chu B. Control of structure, morphology and property in electrospun poly (glycolide-co-lactide) non-woven membranes via post-draw treatments. Polymer 2003;44:4959-4967.
Megelski S, Stephens JS, Chase DB, Rabolt JF. Micro- and nanostructured surface morphology on electrospun polymer fibers. Macromolecules 2002;35:8456-8466.
Rho KS, Jeong L, Lee G, Seo BM, Park YJ, Hong SD, et al. Electrospinning of collagen nanofibers: Effects on the behavior of normal human keratinocytes and early-stage wound healing. Biomaterials 2006;27:1452-1461.
Noh HK, Lee SW, Kim JM, Oh JE, Kim KH, Chung CP, et al. Electrospinning of chitin nanofibers: Degradation behavior and cellular response to normal human keratinocytes and fibroblasts. Biomaterials 2006;27:3934-3944.
Geng XY, Kwon OH, Jang JH. Electrospinning of chitosan dissolved in concentrated acetic acid solution. Biomaterials 2005;26:5427-5432.
Ji Y, Ghosh K, Shu XZ, Li BQ, Sokolov JC, Prestwich GD, et al. Electrospun three-dimensional hyaluronic acid nanofibrous scaffolds. Biomaterials 2006 ;27:3782-3792.
Zhang YZ, Venugopal J, Huang ZM, Lim CT, Ramakrishna S. Crosslinking of the electrospun gelatin nanofibers. Polymer 2006 ;47:2911-2917.
Zhang YZ, Ouyang HW, Lim CT, Ramakrishna S, Huang ZM. Electrospinning of gelatin fibers and gelatin/PCL composite fibrous scaffolds. Journal of Biomedical Materials Research Part B-Applied Biomaterials 2005 ;72B:156-165.
Li JX, He AH, Zheng JF, Han CC. Gelatin and gelatin-hyaluronic acid nanofibrous membranes produced by electrospinning of their aqueous solutions. Biomacromolecules 2006 ;7:2243-2247.
Min BM, Lee G, Kim SH, Nam YS, Lee TS, Park WH. Electrospinning of silk fibroin nanofibers and its effect on the adhesion and spreading of normal human keratinocytes and fibroblasts in vitro. Biomaterials 2004 ;25:1289-1297.
Liao S, Li BJ, Ma ZW, Wei H, Chan C, Ramakrishna S. Biomimetic electrospun nanofibers for tissue regeneration. Biomedical Materials 2006 ;1:R45-R53.
Fong H, Chun I, Reneker DH. Beaded nanofibers formed during electrospinning. Polymer 1999 ;40:4585-4592.
Huang L, Nagapudi K, Apkarian RP, Chaikof EL. Engineered collagen-PEO nanofibers and fabrics. Journal of Biomaterials Science-Polymer Edition 2001;12:979-993.
Gelse K, Poschl E, Aigner T. Collagens - structure, function, and biosynthesis. Advanced Drug Delivery Reviews 2003 ;55:1531-1546.
Ricard-Blum S, Ruggiero F. The collagen superfamily: from the extracellular matrix to the cell membrane. Pathologie Biologie 2005 ;53:430-442.
Kadler KE, Holmes DF, Trotter JA, Chapman JA. Collagen fibril formation. Biochemical Journal 1996 ;316:1-11.
Matthews JA, Wnek GE, Simpson DG, Bowlin GL. Electrospinning of collagen nanofibers. Biomacromolecules 2002 ;3:232-238.
Park JC, Hwang YS, Lee JE, Park KD, Matsumura K, Hyon SH, et al. Type I atelocollagen grafting onto ozone-treated polyurethane films: Cell attachment, proliferation, and collagen synthesis. Journal of Biomedical Materials Research 2000 ;52:669-677.
Li YH, Huang YD. The study of collagen immobilization on polyurethane by oxygen plasma treatment to enhance cell adhesion and growth. Surface & Coatings Technology 2007 ;201:5124-5127.
Lee CH, Singla A, Lee Y. Biomedical applications of collagen. International Journal of Pharmaceutics 2001 ;221:1-22.
Jiang D, Liang J, Noble PW. Hyaluronan in tissue injury and repair. Annual Review of Cell and Developmental Biology 2007;23:435-461.
Fraser JRE, Laurent TC, Laurent UBG. Hyaluronan: Its nature, distribution, functions and turnover. Journal of Internal Medicine 1997 ;242:27-33.
Torvard C. Ljrent and J. Robert E. Fraser Hyaluronan FASEB J. Laurent and Fraser 6: 2397
Suh H, Lee JE. Behavior of fibroblasts on a porous hyaluronic acid incorporated collagen matrix. Yonsei Medical Journal 2002 ;43:193-202.
Longaker MT, Whitby DJ, Ferguson MWJ, Lorenz HP, Harrison MR, Adzick NS. Adult Skin Wounds in the Fetal Environment Heal with Scar Formation. Annals of Surgery 1994 ;219:65-72.
Lorenz HP, Adzick NS. Scarless Skin Wound Repair in the Fetus. Western Journal of Medicine 1993 ;159:350-355.
Chang CF, Lee MW, Kuo PY, Wang YJ, Tu YH, Hung SC. Three-dimensional collagen fiber remodeling by mesenchymal stem cells requires the integrin-matrix interaction. Journal of Biomedical Materials Research Part A 2007;80A:466-474.
Debelder AN, Wik KO. Preparation and Properties of Fluorescein-Labeled Hyaluronate. Carbohydrate Research 1975;44:251-257.
Tomihata K, Ikada Y. Crosslinking of hyaluronic acid with water-soluble carbodiimide. Journal of Biomedical Materials Research 1997 ;37:243-251.
Kahari VM, Saarialho-Kere U. Matrix metalloproteinases in skin. Eexperimental Dematology Exp Dermatol. 1997 ;6:199-213.
Camacho NP, West P, Torzilli PA, Mendelsohn R. FTIR microscopic imaging of collagen and proteoglycan in bovine cartilage. Biopolymers 2001;62:1-8.
Tomihata K, Ikada Y. Crosslinking of hyaluronic acid with glutaraldehyde. Journal of Polymer Science Part a-Polymer Chemistry 1997 ;35(:3553-3559.
Abatangelo G, Cortivo R, Martelli M, Vecchia P. Cell Detachment Mediated by Hyaluronic-Acid. Experimental Cell Research 1982;137:73-78.
David-Raoudi M, Tranchepain F, Deschrevel B, Vincent JC, Bogdanowicz P, Boumediene K, et al. Differential effects of hyaluronan and its fragments on fibroblasts: Relation to wound healing. Wound Repair and Regeneration 2008;16:274-287.