中文摘要
本研究主要目的為探討以生物相容性較高、毒性較低之交聯劑(Genipin、
Chitosan、Ribose)來交聯不同分子量大小與分布範圍之台灣鯛魚鱗膠原蛋白。
將各個不同比例之交聯劑與膠原蛋白進行交聯反應後分別凍乾成膜，再利用電
子掃描顯微鏡(SEM)、膨潤度(Swelling ratio)、交聯指數(Ninhydrin assay)、熱變
性溫度(Td)等進行物性分析測試來探討出最佳之交聯條件。
由結果我們可以發現，其最佳交聯條件為: 1%幾丁聚醣+ 1%梔子素+ 0.6g
膠原蛋白，且使用分子量較大、分布平均之膠原蛋白時，其交聯膜呈現最佳之
交聯效果。由SEM放大兩千倍加以觀察，發現其表面幾乎沒有孔洞與皺褶，呈
現極佳之交聯狀態。在DSC分析中也可觀察到，其熱變性溫度由未交聯前之魚
鱗膠原蛋白的20~25℃上升到最高可達131.6℃。由IR光譜可發現，醯胺鍵的
吸收訊號在交聯前與交聯後產生些微位移，但大部分膠原蛋白的吸收訊號仍然
存在且維持不變，由此可加以推測交聯發生在膠原蛋白案及結構中之醯胺
(amide)位置，且主要結構不變。此外，在實驗過程中發現梔子素之比例上升
時，其交聯膜之藍色也會相對增加，推測是因其分子內共振增加使其顏色更加
明顯且交聯程度越好。
魚鱗膠原蛋白之醫用敷料在市場上有極大的發展性，本研究雖探討出有最
佳交聯效果之交聯劑與膠原蛋白比例，但仍須配合後人的生物實驗來加以探討
出何種孔徑大小與交聯程度最適合細胞的貼附與增生，來增加其實用性且期望
在醫用敷料的應用上有良好的潛力。

關鍵字: 膠原蛋白、幾丁聚醣、梔子素、核醣、交聯

SUMMARY
In this study, the main purpose is to discuss the feasibility of preparing the biomaterial film from the Taiwan Tilapia scale collagen which have difference molecular weight by cross-linkes with genipin, chitosan, and/or ribose. We used HPLC to compare the difference of molecular weight between the different extraction times. It is very different from previous studies that only focused on the crosslinking agents. The physical properties are measured by scanning electron microscopy (SEM), cross-linked index (Ninhydrin assay), degree of swelling (Swelling ratio) and thermal denaturation temperature (Td). From the results, we can find out that the best cross-linking conditions are: 1% chitosan + 1% genipin + 0.6g Collagen. In addition, when using the collagen with larger molecular weight, the cross-linked membranes show the best interation effect. Besides, during the experiment we found out that when the content of genipin increased, not only the cross-linking degree increased, but the blue color became darker.

Key words: fish scale collagen、Chitosan、Genipin、Ribose、Cross-linking

第六章 參考文獻
1. Ahmad, M.; Nirmal, N. P.; Chuprom, J., Molecular characteristics of collagen extracted from the starry triggerfish skin and its potential in the development of biodegradable packaging film. RSC advances 2016, 6 (40), 33868-33879.
2. Fathke, C.; Wilson, L.; Hutter, J.; Kapoor, V.; Smith, A.; Hocking, A.; Isik, F., Contribution of bone marrow–derived cells to skin: collagen deposition and wound repair. Stem cells 2004, 22 (5), 812-822.
3. 林建智, 台灣鯛魚鱗膠原蛋白核糖交聯之研究. 成功大學化學系學位論文 2010, 1-88.
4. 赵玉榕, 台湾渔业的困境与出路. 两岸关系 2006, (10), 15-17.
5. 王品捷, 酵素水解之台灣鯛魚鱗膠原蛋白生物活性之研究. 成功大學化學系學位論文 2013, 1-72.
6. Rubin, A. L.; Miyata, T.; Stenzel, K. H., Collagen: medical and surgical applications. Journal of Macromolecular Science—Chemistry 1969, 3 (1), 113-118.
7. Raiskup-Wolf, F.; Hoyer, A.; Spoerl, E.; Pillunat, L. E., Collagen crosslinking with riboflavin and ultraviolet-A light in keratoconus: long-term results. Journal of Cataract & Refractive Surgery 2008, 34 (5), 796-801.
8. Jorge-Herrero, E.; Fernandez, P.; Turnay, J.; Olmo, N.; Calero, P.; Garcı́a, R.; Freile, I.; Castillo-Olivares, J., Influence of different chemical cross-linking treatments on the properties of bovine pericardium and collagen. Biomaterials 1999, 20 (6), 539-545.
9. 劉媛婷, 台灣鯛魚鱗膠原蛋白-明膠-幾丁聚醣交聯創傷敷料之製備與特性研究. 2015.
10. Guang, X. Z.; Moon, C.-M.; Kwak, J.-S.; Oh, K.-W.; Bae, H.-I.; Han, J.-Y., Biomedical application of PHBV/collagen (PHCP) nanofiber as the wound dressing. Journal of Biomedical Research 2011, 12 (3), 147-155.
11. 高煒堯, 利用魚鱗萃取膠原蛋白製備傷口敷料之性質與改良. 中興大學化學工程學系所學位論文 2017, 1-62.
12. Muralidharan, N.; Shakila, R. J.; Sukumar, D.; Jeyasekaran, G., Skin, bone and muscle collagen extraction from the trash fish, leather jacket (Odonus niger) and their characterization. Journal of Food Science and Technology 2013, 50 (6), 1106-1113.
13. Shoulders, M. D.; Raines, R. T., Collagen structure and stability. Annual review of biochemistry 2009, 78, 929-958.
14. Mays, P.; McANULTY, R. J.; Campa, J. S.; Laurent, G. J., Age-related changes in collagen synthesis and degradation in rat tissues. Importance of degradation of newly synthesized collagen in regulating collagen production. Biochemical Journal 1991, 276 (2), 307-313.
15. 陳詩燕, 以幾丁聚醣, 核醣, 梔子素交聯台灣鯛膠原蛋白膜之特性研究. 成功大學化學系學位論文 2011, 1-93.
16. Bailey, A.; Bazin, S.; Sims, T.; Le Lous, M.; Nicoletis, C.; Delaunay, A., Characterization of the collagen of human hypertrophic and normal scars. Biochimica et Biophysica Acta (BBA)-Protein Structure 1975, 405 (2), 412-421.
17. Muzzarelli, R. A.; El Mehtedi, M.; Bottegoni, C.; Aquili, A.; Gigante, A., Genipin-crosslinked chitosan gels and scaffolds for tissue engineering and regeneration of cartilage and bone. Marine drugs 2015, 13 (12), 7314-7338.
18. Bornstein, P.; Piez, K. A., The nature of the intramolecular cross-links in collagen. The separation and characterization of peptides from the cross-link region of rat skin collagen. Biochemistry 1966, 5 (11), 3460-3473.
19. Bailey, A.; Robins, S.; Balian, G., Biological significance of the intermolecular crosslinks of collagen. Nature 1974, 251 (5471), 105-109.
20. Bailey, A., Intermediate labile intermolecular crosslinks in collagen fibres. Biochimica et Biophysica Acta (BBA)-Protein Structure 1968, 160 (3), 447-453.
21. 楊人龍, 台灣鯛魚皮膠原蛋白, 明膠和膠原蛋白水解液之特性研究與比較. 2008.
22. Kittiphattanabawon, P.; Benjakul, S.; Visessanguan, W.; Nagai, T.; Tanaka, M., Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus). Food chemistry 2005, 89 (3), 363-372.
23. Viguet-Carrin, S.; Garnero, P.; Delmas, P., The role of collagen in bone strength. Osteoporosis international 2006, 17 (3), 319-336.
24. Erwin, R. L., Glucan/collagen therapeutic eye shields. Google Patents: 1990.
25. Miller, B. F.; Olesen, J. L.; Hansen, M.; Døssing, S.; Crameri, R. M.; Welling, R. J.; Langberg, H.; Flyvbjerg, A.; Kjaer, M.; Babraj, J. A., Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise. The Journal of physiology 2005, 567 (3), 1021-1033.
26. Tedder, M. E.; Liao, J.; Weed, B.; Stabler, C.; Zhang, H.; Simionescu, A.; Simionescu, D. T., Stabilized collagen scaffolds for heart valve tissue engineering. Tissue Engineering Part A 2009, 15 (6), 1257-1268.
27. Miller, E. J.; Rhodes, R. K., [2] Preparation and characterization of the different types of collagen. In Methods in enzymology, Elsevier: 1982; Vol. 82, pp 33-64.
28. Ksander, G.; Ogawa, Y., Collagen wound healing matrices and process for their production. Google Patents: 1990.
29. Appell, R. A., Collagen injection therapy for urinary incontinence. The Urologic clinics of North America 1994, 21 (1), 177-182.
30. Okada, T.; Hayashi, T.; Ikada, Y., Degradation of collagen suture in vitro and in vivo. Biomaterials 1992, 13 (7), 448-454.
31. Kakisis, J. D.; Liapis, C. D.; Breuer, C.; Sumpio, B. E., Artificial blood vessel: the Holy Grail of peripheral vascular surgery. Journal of vascular surgery 2005, 41 (2), 349-354.
32. Cullen, B.; Watt, P. W.; Lundqvist, C.; Silcock, D.; Schmidt, R. J.; Bogan, D.; Light, N. D., The role of oxidised regenerated cellulose/collagen in chronic wound repair and its potential mechanism of action. The international journal of biochemistry & cell biology 2002, 34 (12), 1544-1556.
33. 饒梓平, 由魚鱗萃取出膠原蛋白製備人工皮及其在經皮吸收之量化分析. 中興大學化學工程學系所學位論文 2016, 1-61.
34. Schmidt, M.; Dornelles, R.; Mello, R.; Kubota, E.; Mazutti, M.; Kempka, A.; Demiate, I., Collagen extraction process. International Food Research Journal 2016, 23 (3).
35. Longin, R., New method of collagen extraction for radiocarbon dating. Nature 1971, 230 (5291), 241-242.
36. Brown, T. A.; Nelson, D. E.; Vogel, J. S.; Southon, J. R., Improved collagen extraction by modified Longin method. Radiocarbon 1988, 30 (2), 171-177.
37. Kumar, M. N. R., A review of chitin and chitosan applications. Reactive and functional polymers 2000, 46 (1), 1-27.
38. Fei Liu, X.; Lin Guan, Y.; Zhi Yang, D.; Li, Z.; De Yao, K., Antibacterial action of chitosan and carboxymethylated chitosan. Journal of applied polymer science 2001, 79 (7), 1324-1335.
39. Xia, W.; Liu, P.; Zhang, J.; Chen, J., Biological activities of chitosan and chitooligosaccharides. Food hydrocolloids 2011, 25 (2), 170-179.
40. Anjum, S.; Arora, A.; Alam, M.; Gupta, B., Development of antimicrobial and scar preventive chitosan hydrogel wound dressings. International journal of pharmaceutics 2016, 508 (1-2), 92-101.
41. Chen, Z.; Mo, X.; Qing, F., Electrospinning of collagen–chitosan complex. Materials Letters 2007, 61 (16), 3490-3494.
42. Yan, L. P.; Wang, Y. J.; Ren, L.; Wu, G.; Caridade, S. G.; Fan, J. B.; Wang, L. Y.; Ji, P. H.; Oliveira, J. M.; Oliveira, J. T., Genipin‐cross‐linked collagen/chitosan biomimetic scaffolds for articular cartilage tissue engineering applications. Journal of Biomedical Materials Research Part A 2010, 95 (2), 465-475.
43. Bishop, C.; Cooper, F., Glycosidation of sugars: II. Methanolysis of d-xylose, d-arabinose, d-lyxose, and d-ribose. Canadian Journal of Chemistry 1963, 41 (11), 2743-2758.
44. Bailey, A. J.; Sims, T.; Avery, N. C.; Halligan, E. P., Non-enzymic glycation of fibrous collagen: reaction products of glucose and ribose. Biochemical Journal 1995, 305 (2), 385-390.
45. Liu, T. X.; Wang, Z., Collagen crosslinking of porcine sclera using genipin. Acta ophthalmologica 2013, 91 (4), e253-e257.
46. Pitaru, S.; Noff, M.; Blok, L.; Nir, E.; Pitaru, S.; Goldlust, A.; Savion, N., Long‐Term Efficacy of a Novel Ribose–Cross‐linked Collagen Dermal Filler: A Histologic and Histomorphometric Study in an Animal Model. Dermatologic surgery 2007, 33 (9), 1045-1054.
47. Sarkar, S. D.; Farrugia, B. L.; Dargaville, T. R.; Dhara, S., Chitosan–collagen scaffolds with nano/microfibrous architecture for skin tissue engineering. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials 2013, 101 (12), 3482-3492.
48. Noff, M.; Pitaru, S., Cross-linked collagen matrices and methods for their preparation. Google Patents: 2004.
49. Sundararaghavan, H. G.; Miksan, J. R.; Shreiber, D. In Characterization of genipin-crosslinked collagen gels, 2005 Summer Bioengineering Conference, 2005; pp 1514-1515.
50. 董信光; 李荣玉; 刘志超; 周新刚; 殷炳毅, 生物质与煤混燃的灰分特性分析. 中国电机工程学报 2009, (26), 118-124.
51. López, J.; Imperial, S.; Valderrama, R.; Navarro, S., An improved Bradford protein assay for collagen proteins. Clinica chimica acta 1993, 220 (1), 91-100.
52. Vrijbergen, R.; Soeteman, A.; Smit, J., Calibration of GPC with polydisperse standards. Journal of Applied Polymer Science 1978, 22 (5), 1267-1276.
53. Zhang, X.; Chen, X.; Yang, T.; Zhang, N.; Dong, L.; Ma, S.; Liu, X.; Zhou, M.; Li, B., The effects of different crossing-linking conditions of genipin on type I collagen scaffolds: an in vitro evaluation. Cell and tissue banking 2014, 15 (4), 531-541.
54. Weadock, K.; Olson, R. M.; Silver, F. H., Evaluation of collagen crosslinking techniques. Biomaterials, medical devices, and artificial organs 1983, 11 (4), 293-318.
55. Mentink, C. J.; Hendriks, M.; Levels, A. A.; Wolffenbuttel, B. H., Glucose-mediated cross-linking of collagen in rat tendon and skin. Clinica Chimica Acta 2002, 321 (1-2), 69-76.
56. Barbani, N.; Giusti, P.; Lazzeri, L.; Polacco, G.; Pizzirani, G., Bioartificial materials based on collagen: 1. Collagen cross-linking with gaseous glutaraldehyde. Journal of Biomaterials Science, Polymer Edition 1996, 7 (6), 461-469.
57. Hara, M.; Koshimizu, N.; Yoshida, M.; Haug, I. J.; Ulset, A.-S. T.; Christensen, B. E., Cross-linking and depolymerisation of γ-irradiated fish gelatin and porcine gelatin studied by SEC-MALLS and SDS-PAGE: a comparative study. Journal of Biomaterials Science, Polymer Edition 2010, 21 (6-7), 877-892.