臺灣博碩士論文加值系統

本研究針對第一型膠原蛋白重組纖維結構吸附於固體載體上的微結構變化進行探討。本研究成功使用原子力顯微鏡觀察膠原蛋白纖維的微結構。藉由原子力顯微鏡觀察比較膠原蛋白吸附於雲母片、聚甲基丙烯酸甲酯及左旋聚乳酸高分子薄膜基材上的纖維形態，以及，改變溶液的濃度、酸鹼值及製備樣品的方式，來研究重組膠原蛋白纖維微結構所受的影響。結果顯示，重組的膠原蛋白纖維有許多種形態，特別是載體基材性質的影響極為顯著。當膠原蛋白溶液的濃度改變時，會對於先重組再吸附於基材上的膠原蛋白纖維之尺寸有所影響。但是，若先將溶液吸附於基材上再進行重組，則濃度對重組纖維尺寸的影響就不明顯。當膠原蛋白溶液的酸鹼值超過其等電點時，除雲母片外，其他載體上皆無法觀察到纖維結構的存在。膠原蛋白經尿素處理後，纖維結構會受到破壞，而呈現放射狀纖維結構。

The microstructure of reconstructed type I collagen adsorbed on solid substrates has been investigated by atomic force microscopy. In this work, we utilized different substrates such as mica, PMMA, and PLLA as the supporting surface that the self-assembly of collagen molecules adsorption onto this surface were characterized. We also exploited the morphology of the adsorption collagen varied by the pH values, concentrations, and adsorption procedures of collagen solutions. These results indicated that collagen has different adsorption patterns according to its concentration, variable heterogeneous surfaces of substrates, adjusting the pH of buffer solutions and detergents. We have observed the fibril microstructure of type I collagen by AFM successfully. Further, we will investigate how the chemical structure of the supporting polymer surfaces and the protein-surface interactions will affect protein adsorption processes.

目錄 頁碼
致謝-----------------------------------------------------------------I
中文摘要------------------------------------------------------------II
英文摘要-----------------------------------------------------------III
目錄----------------------------------------------------------------IV
圖目錄--------------------------------------------------------------VI
表目錄------------------------------------------------------------VIII

第一章 緒論---------------------------------------------------------1

第一節 膠原蛋白之簡介------------------------------------------------2
第二節 膠原蛋白之分類------------------------------------------------5
第三節 膠原蛋白的生合成------------------------------------------------7
第四節 膠原蛋白之生物功能及應用----------------------------------------10
第五節 膠原蛋白之微結構-----------------------------------------------12
第六節 實驗動機及目的-------------------------------------------------17

第二章 實驗儀器----------------------------------------------------18

第一節 原子力顯微鏡原理介紹-------------------------------------------18

第三章 材料方法----------------------------------------------------21

第一節 實驗藥品及設備-----------------------------------------------24
第二節 實驗方法------------------------------------------------------24
1. 載體表面微結構檢測-------------------------------------------------24
2. 膠原蛋白之重組纖維微結構觀察----------------------------------------25
(一) 膠原蛋白溶液配置-------------------------------------------------25
(二) 膠原纖維微結構觀察-----------------------------------------------26

第四章 結果及討論---------------------------------------------------28

第一節 載體表面微結結構檢測-------------------------------------------28
第二節 膠原蛋白吸附於載體表面重組之微結構觀察------------------31
(1) 膠原蛋白吸附於不同特性基材對其微結構之影響-------------------31
(2) 膠原蛋白溶液之酸鹼值對重組微結構之影響-------------------------34
(3) 不同膠原蛋白濃度吸附於高分子基材對纖維結構之影響----------38
(4)變性劑對膠原蛋白重組微結構之影響----------------------------------40
第三節 膠原蛋白重組後吸附於載體表面之微結構觀察---------------42

第五章 結論---------------------------------------------------------45

參考文獻------------------------------------------------------69










圖目錄 頁碼
圖1-1.1已知的膠原蛋白其超分子自我重組圖示---------------------------4
圖1-2.1第一型膠原蛋白分子之圖示----------------------------------------6
圖1-3.1 膠原蛋白之合成示意圖及所參與之酵素---------------------------9
圖2-1.1 原子力顯微鏡原理示意圖-------------------------------------20
圖3-2-1.1 聚甲基丙烯酸甲酯之化學結構式---------------------------------24
圖3-2-1.2 左旋聚乳酸之化學結構式--------------------------------------25
圖4.1. Mica基材以原子力顯微鏡觀察之表面微結構--------------------46
圖4.2. 聚甲基丙烯酸甲酯基材以原子力顯微鏡觀察之微結構---------47
圖4.3. 左旋聚乳酸基材以原子力顯微鏡觀察之微結構------------------48
圖4.4. 左旋聚乳酸孔洞之直徑及高度分布圖------------------------------49
圖4.5. 0.1N 醋酸滴置於左旋聚乳酸基材後之微結構--------------------50
圖4.6. 0.2 mg/ml(pH2.75)膠原蛋白吸附於不同特性基材之微結構----51
圖4.7. 膠原蛋白吸附於不同特性高分子基材對纖維尺寸之分析圖--52
圖4.8. 不同酸鹼值之0.2mg/ml膠原蛋白溶液吸附於不同特性之材之微結構--------54
圖4.9. 不同酸鹼值之0.2mg/ml collagen 吸附於不同特性基材其重組纖維尺寸分析圖-----------------------------------------------------------55
圖4.10. 膠原蛋白吸附於 PMMA基材後，纖維重組之纖維形態-------------57
圖4.11. 膠原蛋白吸附於 PMMA基材後，纖維重組之纖維形態------------58
圖4.12. 不同濃度膠原蛋白吸附於 PMMA基材後，纖維重組之尺寸分布圖-----------59
圖4.13. 膠原蛋白吸附於PMMA基材後，經纖維重組所形成之網狀纖維其纖維間距分析圖----------------------------------------------------------------60
圖4.14. 含8M 尿素之膠原蛋白溶液吸附於PMMA基材之微結構--------61
圖4.15. 含8M 尿素之膠原蛋白溶液吸附於PMMA基材，其纖維尺寸分析統計圖-------62
圖4.16. 含8M 尿素之0.2mg/ml膠原蛋白溶液吸附於PMMA基材所形成之顆粒性纖維與分支纖維尺寸分析-------------------------------------------63
圖4.17. 含8M 尿素之0.2mg/ml膠原蛋白溶液吸附於PMMA基材所形成之大區塊纖維尺寸分析---------------------------------------------------------64
圖4.18. 不同濃度之膠原蛋白溶液經兩小時纖維重組後吸附於 PMMA 之微結構-----65
圖4.19. 0.2 mg / ml 膠原蛋白溶液經兩小時纖維重組後吸附於 PMMA 所觀察到的膠原分子結構及其尺寸-----------------------------------------66
圖4.20. 不同膠原蛋白濃度經纖維重組後吸附於 PMMA所觀察之纖維結構寬度及高度分布--------------------------------------------------------------67









表目錄 頁碼
表1-2.1 膠原蛋白的分類及特性-----------------------------------------------------6
表1-4.1 將膠原蛋白製成不同型式及其應用-------------------------------------12
表3-2-1.1 高分子之物理特性-------------------------------------------------------25
表4.1. 左旋聚乳酸孔洞之直徑及高度統計表-----------------------------------49
表4.2. 膠原蛋白吸附於不同特性高分子基材其纖維尺寸之統計表--------52
表4.3. 膠原蛋白溶液之酸鹼值對吸附於不同特性基材其重組纖維尺寸分析表----------------------------------------------------------------------------------------56
表4.4. 不同濃度膠原蛋白吸附於 PMMA 基材後，纖維重組之尺寸統計表--------------------------------------------------------------------------------------------59
表4.5. 膠原蛋白吸附於PMMA基材後，經纖維重組所形成之網狀纖維其纖維間距分析表---------------------------------------------------------------------------60
表4.6. 含8M 尿素之膠原蛋白溶液吸附於PMMA基材其纖維尺寸分析表-62
表4.7. 不同膠原蛋白濃度經重組後吸附於 PMMA所觀察之纖維結構寬度及高度統計-------------------------------------------------------------------------------68

參考文獻
Chen, Y., Mak, A. F. T., Wang, M., Li, J. & Wong, M. S. PLLA scaffolds with biomimetic apatite coating and biomimetic apatite/collagen composite coating to enhance osteoblast-like cells attachment and activity. Surface and Coatings Technology, Epud ahead of print (2005).
Chernoff, E. A. G. C., D.A. Atomic force microscope images of collagen fibers. J. Vac. Sci. Technol. A 10, 596-599 (1992).
Chevallay, B. & Herbage, D. Collagen-based biomaterials as 3D scaffold for cell cultures: applications for tissue engineering and gene therapy. Med. Biol. Eng. Compu. 38, 211-8 (2000).
Christiansen, D. L., Huang, E. K. & Silver, F. H. Assembly of type I collagen: fusion of fibril subunits and the influence of fibril diameter on mechanical properties. Matrix Biol. 19, 409-20 (2000).
Chvapil, M., Kronenthal, L. & Van Winkle, W., Jr. Medical and surgical applications of collagen. Int. Rev. Connect Tissue Res. 6, 1-61 (1973).
Cisneros, D. A., Hung, C., Franz, C. M. & Muller, D. J. Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy. J. Struct. Biol. 154, 232-45 (2006).
Dupont-Gillain, C. C., Nysten, B., & Rouxhet, P. G. Collagen adsorption on poly(methyl methacrylate): net-like structure formation upon drying. Polymer international 48, 271-276 (1999).
Dupont-Gillain, C. C., Jacquemart, I. & Rouxhet, P. G. Influence of the aggregation state in solution on the supramolecular organization of adsorbed type I collagen layers. Colloids Surf. B Biointerfaces 43, 179-86 (2005).
Elliott, J. T. et al. Vascular smooth muscle cell response on thin films of collagen. Matrix Biol. 24, 489-502 (2005).
Flaumenhaft, R. & Rifkin, D. B. The extracellular regulation of growth factor action. Mol. Biol. Cell 3, 1057-65 (1992).
Gale, M., Pollanen, M. S., Markiewicz, P. & Goh, M. C. Sequential assembly of collagen revealed by atomic force microscopy. Biophys. J. 68, 2124-8 (1995).
Gelman, R. A., Poppke, D. C. & Piez, K. A. Collagen fibril formation in vitro. The role of the nonhelical terminal regions. J. Biol. Chem. 254, 11741-5 (1979).
Gelman, R. A., Williams, B. R. & Piez, K. A. Collagen fibril formation. Evidence for a multistep process. J. Biol. Chem. 254, 180-6 (1979).
Gelse, K., Poschl, E. & Aigner, T. Collagens--structure, function, and biosynthesis. Adv. Drug Deliv. Rev. 55, 1531-46 (2003).
Ginger A. Abraham, J. M., Kristen Billiar, Susan J. Sullivan. Evaluation of the porcine intestinal collagen layer as a biomaterial. J. Biomed. Mater. Res. 51, 442-452 (2000).
Grinnell, F. Fibroblast biology in three-dimensional collagen matrices. Trends Cell Biol. 13, 264-9 (2003).
Hafemann, B. et al. Use of a collagen/elastin-membrane for the tissue engineering of dermis. Burns 25, 373-84 (1999).
Hansma, H. G. et al. Reproducible imaging and dissection of plasmid DNA under liquid with the atomic force microscope. Science 256, 1180-4 (1992).
Hashimoto, M. et al. SEM and TEM analysis of water degradation of human dentinal collagen. J. Biomed. Mater Res. B Appl. Biomater. 66, 287-98 (2003).
Hattori, S. et al. Alkali-treated collagen retained the triple helical conformation and the ligand activity for the cell adhesion via alpha2beta1 integrin. J. Biochem. (Tokyo) 125, 676-84 (1999).
He, W., Ma, Z., Yong, T., Teo, W. E. & Ramakrishna, S. Fabrication of collagen-coated biodegradable polymer nanofiber mesh and its potential for endothelial cells growth. Biomaterials 26, 7606-15 (2005).
Hodge, A. J. In Treatise on Collagen I, Academic Press, New York. 185, 205. (1967).
Hohenester, E. & Engel, J. Domain structure and organisation in extracellular matrix proteins. Matrix Biol. 21, 115-28 (2002).
Holmes, D. F., Chapman, J. A., Prockop, D. J. & Kadler, K. E. Growing tips of type I collagen fibrils formed in vitro are near-paraboloidal in shape, implying a reciprocal relationship between accretion and diameter. Proc. Natl. Acad. Sci. U S A 89, 9855-9 (1992).
Holmes, D. F., Graham, H. K., Trotter, J. A. & Kadler, K. E. STEM/TEM studies of collagen fibril assembly. Micron 32, 273-85 (2001).
Hong, Y., Gao, C., Xie, Y., Gong, Y. & Shen, J. Collagen-coated polylactide microspheres as chondrocyte microcarriers. Biomaterials 26, 6305-13 (2005).
Hulmes, D. J. Building collagen molecules, fibrils, and suprafibrillar structures. J. Struct. Biol .137, 2-10 (2002).
Jacquemart, I., Pamula, E., De Cupere, V. M., Rouxhet, P. G. & Dupont-Gillain Ch, C. Nanostructured collagen layers obtained by adsorption and drying. J. Colloid Interface Sci. 278, 63-70 (2004).
Jiang, F., Horber, H., Howard, J. & Muller, D. J. Assembly of collagen into microribbons: effects of pH and electrolytes. J. Struct. Biol. 148, 268-78 (2004).
K.A.Piez. Molecular and aggregate structures of the collagens, in:K.A.Piez. A.H. Reddi(Eds), Extracellualr Matrix Biochemistry, Elsevier, New York, 1-40 (1984).
Kadler, K. E., Holmes, D. F., Trotter, J. A. & Chapman, J. A. Collagen fibril formation. Biochem. J. 316 (Pt 1), 1-11 (1996).
Keresztes, Z., Rouxhet, P. G., Remacle, C. & Dupont-Gillain, C. Supramolecular assemblies of adsorbed collagen affect the adhesion of endothelial cells. J. Biomed. Mater. Res. A 76, 223-33 (2006).
Kim, B. S., Baez, C. E. & Atala, A. Biomaterials for tissue engineering. World J. Urol. 18, 2-9 (2000).
Kuivaniemi, H., Tromp, G. & Prockop, D. J. Mutations in fibrillar collagens (types I, II, III, and XI), fibril-associated collagen (type IX), and network-forming collagen (type X) cause a spectrum of diseases of bone, cartilage, and blood vessels. Hum. Mutat. 9, 300-15 (1997).
Kuzuya, M. & Kinsella, J. L. Induction of endothelial cell differentiation in vitro by fibroblast-derived soluble factors. Exp. Cell Res. 215, 310-8 (1994).
Liu, X., Won, Y. & Ma, P. X. Porogen-induced surface modification of nano-fibrous poly(L-lactic acid) scaffolds for tissue engineering. Biomaterials 27, 3980-7 (2006).
M. Mertig, U. T., J. Bradt, G. Leibiger, W. Pompe and H. Wendrock. Scanning force microscopy and geometric analysis of two-dimensional collagen network formation. Surface and interface and analysis 25, 514-521 (1997).
Miller, J. M., Zoll, D. R. & Brown, E. O. Clinical observations on use of an extruded collagen suture. Arch. Surg. 88, 167-74 (1964).
Miyata, T., Taira, T. & Noishiki, Y. Collagen engineering for biomaterial use. Clin. Mater. 9, 139-48 (1992).
Murata, M., Maki, F., Sato, D., Shibata, T. & Arisue, M. Bone augmentation by onlay implant using recombinant human BMP-2 and collagen on adult rat skull without periosteum. Clin. Oral Implants Res. 11, 289-95 (2000).
Myllyharju, J. & Kivirikko, K. I. Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet. 20, 33-43 (2004).
Nagata, K. Hsp47: a collagen-specific molecular chaperone. Trends Biochem. Sci. 21, 22-6 (1996).
Nimni, M. E. Fibrillar Collagen: Their Biosynthesis, Molecular Structure, and Mode of Assembly. In Mark A.Zern and Lola M.Reid (Eds.). Extracellular Matrix: Chemistry, Biology, and Pathobiology with Emphasis on the Liver, 121-148 (1993).

O'Cearbhaill E, D., Barron, V. & McHugh, P. E. Characterisation of a collagen membrane for its potential use in cardiovascular tissue engineering applications. J. Mater Sci. Mater Med. 17, 195-201 (2006).
Okada, H. & Toguchi, H. Biodegradable microspheres in drug delivery. Crit. Rev. Ther Drug Carrier Syst. 12, 1-99 (1995).
Ottani, V., Martini, D., Franchi, M., Ruggeri, A. & Raspanti, M. Hierarchical structures in fibrillar collagens. Micron 33, 587-96 (2002).
Ottani, V., Raspanti, M. & Ruggeri, A. Collagen structure and functional implications. Micron 32, 251-60 (2001).
Paige, M. F., Rainey, J. K. & Goh, M. C. A study of fibrous long spacing collagen ultrastructure and assembly by atomic force microscopy. Micron 32, 341-53 (2001).
Pamula, E., De Cupere, V., Dufrene, Y. F. & Rouxhet, P. G. Nanoscale organization of adsorbed collagen: influence of substrate hydrophobicity and adsorption time. J. Colloid Interface Sci. 271, 80-91 (2004).
Reddi, A. H. Morphogenesis and tissue engineering of bone and cartilage: inductive signals, stem cells, and biomimetic biomaterials. Tissue Eng. 6, 351-9 (2000).
Revenko, I., Sommer, F., Minh, D. T., Garrone, R. & Franc, J. M. Atomic force microscopy study of the collagen fibre structure. Biol. Cell 80, 67-9 (1994).
Roskelley, C. D., Srebrow, A. & Bissell, M. J. A hierarchy of ECM-mediated signalling regulates tissue-specific gene expression. Curr. Opin. Cell Biol. 7, 736-47 (1995).
Sano, A., Hojo, T., Maeda, M. & Fujioka, K. Protein release from collagen matrices. Adv. Drug Deliv. Rev. 31, 247-266 (1998).
Sano, A., Maeda, M., Nagahara, S., Ochiya, T., Honma, K., Itoh, H., Miyata, T. & Fujioka, K. Atelocollagen for protein and gene delivery. Adv. Drug Deliv. Rev. 55, 1651-77 (2003).
Sekine, T., Nakamura, T., Shimizu, Y., Ueda, H., Matsumoto, K., Takimoto, Y. & Kiyotani, T. A new type of surgical adhesive made from porcine collagen and polyglutamic acid. J. Biomed. Mater. Res. 54, 305-10 (2001).
Sheu, M. T., Huang, J. C., Yeh, G. C. & Ho, H. O. Characterization of collagen gel solutions and collagen matrices for cell culture. Biomaterials 22, 1713-9 (2001).
Silver, F. H., Freeman, J. W. & Seehra, G. P. Collagen self-assembly and the development of tendon mechanical properties. J. Biomech. 36, 1529-53 (2003).
Smith, L. A. & Ma, P. X. Nano-fibrous scaffolds for tissue engineering. Colloids Surf. B Biointerfaces 39, 125-31 (2004).
Sripriya, R., Kumar, M. S. & Sehgal, P. K. Improved collagen bilayer dressing for the controlled release of drugs. J. Biomed. Mater. Res. B Appl. Biomater. 70, 389-96 (2004).
Taatjes, D. J., Quinn, A. S. & Bovill, E. G. Imaging of collagen type III in fluid by atomic force microscopy. Microsc. Res. Tech. 44, 347-52 (1999).
Tan, E. C., Lin, R. & Wang, C. H. Fabrication of double-walled microspheres for the sustained release of doxorubicin. J. Colloid Interface Sci. 291, 135-43 (2005).
Tatiana Yu. Latychevskaia, K. K. L., Michitoshi Hayashi, Chung-Hung Chang, & Alois Renn, U. P. W., Jui-Hung Hsu, Ta-Chau Chang, Sheng Hsien Lin. Single molecule spectroscopy. J. Chin. Chem. Soc. 50, 477-516 (2003).
Thomas, A. C., Campbell, G. R. & Campbell, J. H. Advances in vascular tissue engineering. Cardiovasc Pathol. 12, 271-6 (2003).
Trelstad, R. L., Hayashi, K. & Gross, J. Collagen fibrillogenesis: intermediate aggregates and suprafibrillar order. Proc. Natl. Acad. Sci. U S A 73, 4027-31 (1976).
Veis, A. Collagen fibrillogenesis. Connect Tissue Res. 10, 11-24 (1982).
White, J. F. et al. Collagen fibril formation in a wound healing model. J. Struct. Biol. 137, 23-30 (2002).
Woodcock, S. E., Johnson, W. C. & Chen, Z. Collagen adsorption and structure on polymer surfaces observed by atomic force microscopy. J. Colloid Interface Sci. 292, 99-107 (2005).
Yamada.N., U., E., & Kuroyanagi, Y. Clinical evaluation of an allogeneic cultured dermal substitute composed of fibroblasts within a spongy collagen matrix. Scand. J. Plast. Reconstr. Surg. Hand Surg. 33, 147-154 (1999).
Yang, B.; Adelung, R.; Ludwig, K.; Bossmann, K.; Pashley, D. H. & Kern, M. Effect of structural change of collagen fibrils on the durability of dentin bonding. Biomaterials 26, 5021-31 (2005).
Yang, C., Hillas, P. J.,Baez, J. A., Nokelainen, M., Balan, J., Tang, J., Spiro, R. & Polarek, J. W. The application of recombinant human collagen in tissue engineering. BioDrugs 18, 103-19 (2004).
Yang, J., Tamm, L. K., Somlyo, A. P. & Shao, Z. Promises and problems of biological atomic force microscopy. J. Microsc. 171 (Pt 3), 183-98 (1993).
Zou, Q., Leang, K., Sadoun, E., Reed, M. & Devasia, S. Control Issues in High-speed AFM for Biological Applications: Collagen Imaging Example. Asian J. Control 6, 164-178 (2004).
王盈錦. 生物醫學材料. 國立編譯館, 115~144 (2002).