臺灣博碩士論文加值系統

目前在組織工程的發展方面，仍以人工皮膚的研發最為純熟，本
研究欲建立一個皮膚替代物的立體模型來取代一般的體外細胞毒性試驗，以減少實驗動物的犧牲與龐大經濟成本的支出。本實驗主要分為四個階段:第一階段，萃取來自豬皮的第一型膠原蛋白。第二階段，將膠原蛋白溶液加入幾丁聚醣和硫酸軟骨素，以冷凍乾燥的方式製備出多孔性海綿狀基質。第三階段主要是材料性質分析，以熱穩定分析儀（示差掃描熱卡計）、傅立葉轉換紅外光分光光譜儀以及動態分析儀對基質的基本性質進行分析，並配合掃描式電子顯微鏡的結果作一初步探討。第四階段進行細胞培養，以初級培養的人類纖維母細胞和角質細胞分別於基質中進行培養，將培養兩個星期的基質做H & E染色(Hematoxylin and eosin stain；H & E stain)切片並配合電子顯微鏡觀察細胞於基質中生長的情形。

So far, the most established system in tissue engineering is the development of the artificial skin. In order to reduce the number of animals used in trials, an in vitro model would be helpful. In this study, the idea is to create a skin substitute that could mimic the real skin by first develop a composite matrix for cell and tissue culture. There are four parts in this plan. First, obtain the type I collagen from porcine skin. Second, prepare a 3-D, sponge-liked (porous) collagen based matrix with chitosan and chondroitin sulfate. Third, investigate the physicochemical properties of the prepared material by Differential Scanning Calorimetry (DSC), Fourier-Transform Infrared (FTIR) spectroscopic studies, Dynamic Mechanical Analysis (DMA) and Scanning Electron Microscopic (SEM) studies. Fourth, culture the human dermal fibroblasts and epidermal keratinocytes into the matrix and to observe the cell growth and proliferation by SEM and H & E stain.

目錄
目錄……………………………………………………………………………… I
附表目錄 ………………………………………………………………………… III
附圖目錄 ………………………………………………………………………… IV
中文摘要 ………………………………………………………………………… V
英文摘要 ………………………………………………………………………… VI
第一章 緒論 …………………………………………………………………… 1
1.1 皮膚的構造與功能……………………………………………………… 1
1.1.1 表皮層…………………………………………………………… 1
1.1.2 真皮層…………………………………………………………… 2
1.2 研究動機 ………………………………………………………………… 3
1.3 膠原蛋白………………………………………………………………… 3
1.3.1 膠原蛋白的結構………………………………………………… 3
1.3.2 膠原蛋白之來源與應用………………………………………… 4
1.4 添加多醣類的目的……………………………………………………… 4
1.4.1 幾丁聚醣 & 硫酸軟骨素的使用………………………………… 4
1.5 未來應用與發展………………………………………………………… 5
第二章 人工皮膚基質之製備………………………………………………… 14
2.1 實驗目的………………………………………………………………… 14
2.2 實驗材料………………………………………………………………… 14
2.3 實驗方法………………………………………………………………… 15
2.3.1 膠原蛋白萃取…………………………………………………… 15
2.3.2 膠原蛋白定性分析……………………………………………… 16
2.3.3 基質之製備……………………………………………………… 17
2.4 結果與討論……………………………………………………………… 18
第三章 人工皮膚基質之物性評估……………………………………………… 25
3.1 實驗目的………………………………………………………………… 25
3.2 實驗方法………………………………………………………………… 25
3.2.1 熱性質分析……………………………………………………… 25
3.2.2 紅外光傅立業轉換光譜分析…………………………………… 26
3.2.3 拉張力測試……………………………………………………… 27
3.2.4 基質形態觀察…………………………………………………… 28
3.2.5 基質孔洞分析…………………………………………………… 29
3.3 結果與討論……………………………………………………………… 30
第四章 人工皮膚基質與皮膚細胞相容性試驗………………………………… 50
4.1 實驗目的………………………………………………………………… 50
4.2 實驗材料………………………………………………………………… 50
4.3 實驗方法………………………………………………………………… 51
4.3.1 纖維母細胞與角質細胞的初級培養…………………………… 51
4.3.2 細胞生長於基質內之觀察……………………………………… 52
4.3.3 細胞形態之觀察………………………………………………… 53
4.4 結果與討論……………………………………………………………… 53
第五章 結論…………………………………………………………………… 60
附表目錄
表1-1 膠原蛋白種類、基因位置及分布組織………………………………… 7
表1-2 各形式的膠原蛋白於生醫材料上之應用……………………………… 11
表1-3 GAGs的種類與其本身的細胞訊息傳導接受器……………………… 12
表2-1 電泳用膠體配製法……………………………………………………… 20
表2-2 電泳緩衝液配製法……………………………………………………… 20
表2-3 兩倍樣品緩衝液配製法………………………………………………… 21
表3-1 具代表性之官能基IR圖譜…………………………………………… 40
附圖目錄
圖1-1 皮膚結構圖……………………………………………………………… 6
圖1-2 膠原蛋白分子的交聯反應……………………………………………… 9
圖1-3 第一型膠原蛋白之結構圖……………………………………………… 10
圖1-4 各式醫療用之膠原蛋白產品…………………………………………… 11
圖1-5 幾丁質與幾丁聚醣之結構式…………………………………………… 13
圖1-6 硫酸軟骨素之結構式…………………………………………………… 13
圖2-1 SDS-PAGE 電泳分析結果……………………………………………… 22
圖2-2 各種基質之成品………………………………………………………… 23
圖2-3 不均質之基質…………………………………………………………… 24
圖3-1 DSC裝置簡圖…………………………………………………………… 33
圖3-2 FTIR之外觀與固定樣品之裝置………………………………………… 34
圖3-3 拉張力測試向量圖……………………………………………………… 35
圖3-4 電子尺之外觀…………………………………………………………… 36
圖3-5 DMA之拉張力測試夾具………………………………………………… 37
圖3-6 電子束照射對樣品所產生之效應……………………………………… 38
圖3-7 DSC 之結果……………………………………………………………… 39
圖3-8 FTIR圖譜………………………………………………………………… 41
圖3-9 拉張力示意圖…………………………………………………………… 43
圖3-10 各基質之拉張力圖……………………………………………………… 45
圖3-11 各基質之斷裂模數……………………………………………………… 46
圖3-12 基質水相耐受性之表現………………………………………………… 47
圖3-13 基質之孔洞分析………………………………………………………… 49
圖4-1 皮膚細胞初級培養流程圖……………………………………………… 55
圖4-2 人類角質細胞與纖維母細胞之外觀(×200)…………………………… 56
圖4-3 纖維母細胞於基質內培養兩週之H&E染色切片結果………………… 57
圖4-4 SEM下不同基質與纖維母細胞之形態………………………………… 58
圖4-5 SEM下角質細胞與纖維母細胞之形態………………………………… 59

1. H.Beele. Artificial skin: past, present and future.The International Journal of Artificial Organs 25: 163-173, 2002.
2. AR Haake, K Holbrook. The structure and development of skin. In Freedberg, et
al,ed.Fitzpatrick’s Dermatology in General Medicine,5th edn. McGraw Hill, 1999.
3. K Nocka, S Majumder, B Chabot. Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice-evidence for an impaired c-kit kinase in mutant mice. Gene Dev 3: 816-26, 1989.
4. 劉克(責任編輯)。北京青年報2003年4月14日。
5. K. Gelse, E. Po¨schl, T. Aigner. Collagens─structure, function, and biosynthesis. Advanced Drug Delivery Reviews 55: 1531—1546, 2003.
6. Huang-Lee, L.L.H., Wu, J.H. and M.E Nimni. Effects of hyaluronan on collagen fibrillar matrix contraction by fibroblasts. J.Biomed. Mater.Res.28: 123-132, 1994.
7. I. Karbe, and T. Nishida. Effect of alcohols on the collagen-phosphatidylcholine interaction. Biochemica et Biophysica Acta 581: 106-113, 1979.
8. N. Okumura., T. Nakamura., T. Natsume, Tomihata,Y. Ikada and Y. Shimizu. Experimental study on a new tracheal prosthesis made from collagen-conjugated mesh. J. Thorac.Cardiovasc. Surg.108: 337-345, 1994.
9. Karube and S.Suzuki. Electrochemical aggregation of tropocollagen. Biochem Biophys. Res. Commun.48: 320-325, 1972.
10. T. Nagai, N. Suzuki. Isolation of collagen from fish waste material-skin, bone
and fins. Food chemistry 68: 277-281, 2000.
11. G.Chandrakasan, D.A.Torchia and K.A.Piez. Preparation of intact monomeric
collagen from rat tail tendon and skin and the structure of the nonhelical ends in
solution. J.biol.Chem.251: 6062-6067, 1976.
12. A.Veis, A.Miller, J.Leibovich and W.Traub. The limiting collagen microfibril. The minimum structure demonstrating native axial periodicity. Biochemica et Biophysica Acta 576: 88-98, 1979.
13. I.V.Yannas. Models of organ regeneration processes induced by templates. Eds :
ALES PROKOP DAVID HUNKELER ALAN D. CHERRINGTON. Annals of
the New York academy of science vol. 831 bioartificial organs science, medicine,
and technology. 281-291, 1997.
14. J. F.Burke, I. V. Yannas, W.C. Quinby, JR., C. C. Bondoc & W.K. Jung. Successful use of a physiologically acceptable artificial skin in the treatment of extensive skin injury. Ann.Surg. 194: 413-428, 1981.
15. http://www.bodyfixer.com/images/skin.jpg
16. 王盈錦（編者）。生物醫學材料。合記圖書出版社。56-78，2002。
17. http://www.collagenmatrix.com
18. http://www.gak.co.jp/FCCA/glycoword/PGA12/PGA12E.html
19. K. Tomihata and Y. Ikada.In vitro and in vivo degradation of films of chitin and its
deacetylated derivatives. Biomaterials 18: 567-575, 1997.
20. Jen-Ting Chang, Meng-Shiue Lin, Yu-Chi Chou, Mei-Ju Yang, Shih-Rong Wang.
No evidence of porcine endogenous retrovirus infection in bioartificial skin
composed of porcine dermal matrix. J Artif Organs 5: 265-269, 2002.
21. J. T. Schulz, R. G. Tompkins, and J.F. Burke. Artificial skin. Annu. Rev. Med. 51:
231-244, 2000.
22. E. Khor. Chapter 7: The chemistry of chitin as is known today. Chitin: fulfilling a
biomaterials promise, Elsevier, 2001.
23. 林正雯。第二部分 膠原蛋白膜的特性解析研究。台北醫學院藥學研究所碩　　　　　
士論文，中華民國八十九年。
24. 黃如君。可供細胞生長的膠原蛋白間質之特性研究。台北醫學院藥學研究所
碩士論文，中華民國八十八年。
25. Hsiu-O Ho, Lun-Huei Lin, Ming-Thau Sheu. Characterization of collagen release
isolation and application of collagen gel as a drug carrier. Journal of controlled
44: 103-112, 1997.
26. 陳柏仰。膠原蛋白基質之製備研究。國立臺灣大學化學工程學研究所碩士論
文，中華民國八十九年。
27. 賀孝雍(譯者)原著 Skoog、 West。儀器分析。曉園出版社。263-267， 1980。
28. J. D. Ingle, Jr. S. R.Crouch.Chap.14 Infrared reflection and emission.
Spectrochemical Analysis. 1988.
29. D. Q. M. Craig, A. Fiona. Johnson. Pharmaceutical applications of dynamic
mechanical thermal analysis. Thermochimica Acta 248: 97-115, 1995.
30. 陳家全。生物電子顯微鏡學。行政院國科會精密儀器發展中心編印。
111-128，1991。
31. C. J. A. L. Mentink, M. Hendriks, A. A. G.Levels, B. H. R. Wolffenbuttel.
Glucose-mediated cross-linking of collagen in rat tendon and skin. Clinica
Chemica Acta 321:69-76, 2002.
32. J. M. Lee, C. A. Pereira, D. Abdulla, W. A. Naimark and I. Crawford.
A muti-sample denaturation temperature tester for collagenous biomaterials. Med.
Eng. Phys. 17: 115-121, 1995.
33. F. Flandin, C. Buffevant and D. Herbage.A differential scanning calorimetry
analysis of the age-related changes in the thermal stability of rat skin collagen.
Biochemica et Biophysica Acta 791:205-211, 1984.
34. J. S. Pieper, T. Hafmans, J. H. Veerkamp, T. H. van Kuppevelt. Development of
tailor-made collagen-glycosaminoglycan matrices: EDC/NHS crosslinking, and
ultrastructural aspects. Biomaterials 21:581-593, 2000.
35. N. P. Camacho, P. West, P. A. Torzilli, R. Mendelsohn. FTIR Microscopic imaging
of collagen and proteoglycan in bovine cartilage. Biopolymers 62:1-8, 2001.
36. TH. Prasch, G. Knubel, K. schmidt-fonk, S. Ortanderl, S. Nieveler and TH.
Forster. Infrared spectroscopy of the skin : influencing the stratum corneum with
cosmetics products. International Journal of Cosmetics Science 22: 371-383,2000.
37. M.E. Mimni and R.D.Harkness. Collagen Vol I.CRC Press.49-50, 1988.
38. G. D. Pins, F. H. Silver. A self-assembled collagen scaffold suitable for use in soft
and hard tissue replacement.Materials Science and Engineering:C3,101-107, 1995.
39. F. H. Silver, D. L. Christiansen. Mechanical properties of collagenous tissue.
Biomaterials science and biocompatibility. Springer,200-201, 1999.
40. M. N. Taravel and A. Domard. Collagen and its interactions with chitosan.
Biomaterials 17:451-455, 1996.
41. L. G. Wade. Chap.11 Infrared spectroscopy and mass spectroscopy. Organic
Chemistry second ed. Prentice Hall. p466 , 1991.
42. F. W. Billmeyer, JR.Chap.9 Analysis and testing of polymers. Textbook of polymer
science. pp 246-247, 1984.
43. F. Berthod, D. Hayek, O. Damour and C. Collombel. Collagen synthesis by
fibroblasts cultured within a collagen sponge. Biomaterials14: 749-754, 1993.
44. K. Schlotmann, M. Kaeten, A. F. Black, O. Damour, M. Waldmann-Laue and T.
Forster. International Journal of Cosmetics Science 23:309-318, 2001.
45. J. P. Daley and J. M. Donovan. Growth of human keratinocyte in serum-free
medium. Cell Biology vol I. Academic press, 1994.
46. S.T.Boyce and R.G.Ham.Calcium-regulated differentiation of normal human serial
epidermal keratinocyte in chemically defined clonal culture and serum-free
culture. J. Invest. Dermatol 81:33-40, 1983.
47. A. Haake, G.A.Scott and K.A. Holbrook. Chapter 2 :Structure and function of the
skin.The biology of the skin. The Parthenon publishing group, 2001.
48. Yoshimitsu Kuroyanagi, D. Eng, D. rer. nat, D. Med. Sci. Advances in wound
dressings and cultured skin substitutes. J. Artif. Organs 2:97-116, 1999.
49. M. Kremer, E. Lang, A. Berger. Organotypical engineering of differentiated matrix
composite-skin equivalents of human keratinocytes in collagen-GAG (INTEGRA
Artificial Skin) in perfusion culture system. Langenbeck''s Arch Surg 386:357-363
, 2001.