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系統識別號 U0026-1508201111474000
論文名稱(中文) 發展具光交聯及生物可相容性的三維細胞支架作為細胞之應用
論文名稱(英文) Development of Photo-Crosslinking Biocompatible Three Dimensional Pattern Scaffold for Cell Application
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 99
學期 2
出版年 100
研究生(中文) 王鈺富
研究生(英文) Yu-Fu Wang
電子信箱 novajason@hotmail.com
學號 P86981164
學位類別 碩士
語文別 英文
論文頁數 46頁
口試委員 指導教授-葉明龍
口試委員-陳家進
口試委員-林睿哲
口試委員-粘譽薰
中文關鍵字 光交聯  烯基化聚乙二醇  水膠  雙光子雷射掃瞄顯微鏡 
英文關鍵字 Photon Cross-linking  Poly (ethylene) glycol-diacrylate (PEG-DA)  Hydrogel  Two Photon Laser Scanning Microscopy 
學科別分類
中文摘要 在三維的支架中可以提供細胞一個更接近真實以及讓細胞生長的環境,同時也提供細胞在多維度下的訊息傳遞。目前,越來越多得文獻回顧也提及在三維空間的細胞培養,相較於二維空間的細胞培養有個明顯的差異性。雙光子雷射掃瞄系統是一個可以深入活體組織,並減少侵入性的雷射掃瞄系統。此系統不僅僅可以作為掃瞄系統,也同時可以作為光交聯聚合物材料之光源,利用此雷射系統可以在基材內準確的製造出三維之光交聯圖形。因此,結合三維的支架細胞培養,以及雙光子雷射掃瞄系統之加工過程,我們希望可以發展出一套引導細胞生長及移動之網路。
此篇研究目的為找出利用烯基化聚乙二醇作為材料之支架,並配合雙光子雷射掃瞄系統之應用範疇內,最適當之加工參數。結果顯示,利用自製的烯基化聚乙二醇做為支架,其適當的光交聯濃度為百分之十五。同時雙光子雷射掃瞄系統也可適當的作為支架之加工工具。當支架表面含有吸引細胞貼附之胜肽時,可以增加其細胞之貼附狀況。最後,也發現利用纖維蛋白膠混入細胞可以作為支架之中心結構。再建構了整個基本系統後,此系統是具有潛力進一步發展成利用三維、可生物降解支架,來引導細胞之生長和移動。
英文摘要 The three dimensional (3D) cultures can provide a more realistic environment for cell growth with its resemblance in cell-to-cell interaction in all dimensions. More studies have shown that cells cultured in 3D environment respond differently to those in two dimensions. Two photon laser scanning microscopy can image the living tissue at higher depth and less invasive. The two photon laser can also work as light source for photo-crosslinking polymers to create precise 3D crosslinking pattern. Therefore, by combining 3D cell culture scaffolds with two photon laser microprocessing, the cell guiding networks can be achieved.
The purpose of this study was to develop the foundation of the poly (ethylene) glycol-diacrylate (PEG-DA) hydrogel with the two photon laser scanning system. Our results showed that the most appropriate concentration of self-made PEG-DA for gelation was 15 % ( w/v). The two photon laser scanning system could be used as a processing tool to the PRG-DA hydrogel. With the cell adhesive ligand, GRGDK, at the surface of the scaffold, it could increase the condition of the cell attachment. Finally, we also developed the core structure with the fibrin gel. With the basic condition established, the further step could be developed. With cells inside this kind of 3D patterned and biodegradable scaffold, cell migration and proliferation could be guided. The cell manipulation and signal recording in the 3D environment could also potentially be achieved.
論文目次 摘要 II
ABSTRACT III
致謝 IV
TABLE OF CONTENTS V
LIST OF FIGURES VII
LIST OF TABLES IX
CHAPTER 1 INTRODUCTION 1
1.1 BACKGROUND 1
1.2 CHOICE OF POLYMER 2
1.3 PHOTO-POLYMERIZATION 3
1.4 DEGRADATION OF SCAFFOLD 3
1.5 TWO PHOTON LASER SCANNING MICROSCOPY 4
1.6 PURPOSE AND SPECIFIC AIMS 6
CHAPTER 2. MATERIALS AND METHODS 7
2.1 FLOW CHART OF THE EXPERIMENT 7
2.2 MATERIALS AND INSTRUMENTS 8
2.3 CELL CULTURE 9
2.4 CHARACTERISTIC OF PEG-DA GEL 10
2.4.1 PEG-DA Synthesis 10
2.4.2 UV Effect to the Cell 11
2.4.3 Swelling Ratio 11
2.4.4 Cell compatibility with PEG-DA Gel 12
2.5 2D HYDROGEL SCAFFOLD 12
2.5.1 PEG-DA gel with R6G 13
2.5.2 PEG-DA gel with 5-FAM 13
2.5.3 PEG-DA gel with 5-FAM-GRGDK 14
2.5.4 PEG-DA gel with 5-FAM-GRGDK and cell 15
2.6 3D HYDROGEL SCAFFOLD 16
2.6.1 3D scaffold with designed pattern 16
2.6.2 Core structure of the 3D hydrogel scaffold 17
2.7 STATISTICAL ANALYSIS 18
CHAPTER 3. RESULTS 19
3.1 PEG-DA SYNTHESIS 19
3.2 UV EFFECT TO THE CELLS 20
3.3 SWELLING RATIO 21
3.4 CELL COMPATIBILITY WITH PEG-DA GEL 22
3.5 PEG-DA GEL WITH R6G 23
3.6 PEG-DA GEL WITH 5-FAM 24
3.7 PEG-DA GEL WITH 5-FAM-GRGDK 25
3.8 PEG-DA GEL WITH 5-FAM-GRGDK AND CELLS 27
3.9 3D SCAFFOLD WITH DESIGNED PATTERN 29
3.10 CORE STRUCTURE OF THE 3D HYDROGEL SCAFFOLD 30
CHAPTER 4. DISCUSSION 32
4.1 CHARACTERISTIC OF PEG-DA GEL 32
4.2 2D HYDROGEL SCAFFOLD 34
4.3 3D HYDROGEL SCAFFOLD 37
4.4 EXPERIMENT LIMITATION 37
4.5 FUTURE PROSPECTION 38
CHAPTER 5. CONCLUSION 39
REFERENCES 40
CURRICULUM VITAE 45

參考文獻 1. Kloxin, A.M., M.W. Tibbitt, A.M. Kasko, J.A. Fairbairn, and K.S. Anseth, Tunable Hydrogels for External Manipulation of Cellular Microenvironments through Controlled Photodegradation. Advanced Materials, 2010. 22(1): p. 61-66.
2. Li, G.N., L.L. Livi, C.M. Gourd, E.S. Deweerd, and D. Hoffman-Kim, Genomic and morphological changes of neuroblastoma cells in response to three-dimensional matrices. Tissue Engineering, 2007. 13(5): p. 1035-1047.
3. Tayalia, P., C.R. Mendonca, T. Baldacchini, D.J. Mooney, and E. Mazur, 3D Cell-Migration Studies using Two-Photon Engineered Polymer Scaffolds. Advanced Materials, 2008. 20(23): p. 4494-4498.
4. Shoichet, M.S., Polymer Scaffolds for Biomaterials Applications. Macromolecules, 2010. 43(2): p. 581-591.
5. Nicodemus, G.D., I. Villanueva, and S.J. Bryant, Mechanical stimulation of TMJ condylar chondrocytes encapsulated in PEG hydrogels. Journal of Biomedical Materials Research Part A, 2007. 83A(2): p. 323-331.
6. Cruise, G.M., D.S. Scharp, and J.A. Hubbell, Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels. Biomaterials, 1998. 19(14): p. 1287-1294.
7. Hahn, M.S., J.S. Miller, and J.L. West, Laser scanning lithography for surface micropatterning on hydrogels. Advanced Materials, 2005. 17(24): p. 2939-2942.
8. DeLong, S.A., A.S. Gobin, and J.L. West, Covalent immobilization of RGDS on hydrogel surfaces to direct cell alignment and migration. Journal of Controlled Release, 2005. 109(1-3): p. 139-148.
9. Watanabe, T., M. Akiyama, K. Totani, S.M. Kuebler, F. Stellacci, W. Wenseleers, K. Braun, S.R. Marder, and J.W. Perry, Photoresponsive hydrogel microstructure fabricated by two-photon initiated polymerization. Advanced Functional Materials, 2002. 12(9): p. 611-614.
10. Fedorovich, N.E., M.H. Oudshoorn, D. van Geemen, W.E. Hennink, J. Alblas, and W.J.A. Dhert, The effect of photopolymerization on stem cells embedded in hydrogels. Biomaterials, 2009. 30(3): p. 344-353.
11. Fairbanks, B.D., M.P. Schwartz, C.N. Bowman, and K.S. Anseth, Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility. Biomaterials, 2009. 30(35): p. 6702-6707.
12. Nguyen, K.T. and J.L. West, Photopolymerizable hydrogels for tissue engineering applications. Biomaterials, 2002. 23(22): p. 4307-4314.
13. West, J.L. and J.A. Hubbell, Polymeric biomaterials with degradation sites for proteases involved in cell migration. Macromolecules, 1999. 32(1): p. 241-244.
14. Kloxin, A.M., M.W. Tibbitt, and K.S. Anseth, Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms. Nature Protocols, 2010. 5(12): p. 1867-1887.
15. Kloxin, A.M., A.M. Kasko, C.N. Salinas, and K.S. Anseth, Photodegradable Hydrogels for Dynamic Tuning of Physical and Chemical Properties. Science, 2009. 324(5923): p. 59-63.
16. Thornton, P.D., R.J. Mart, S.J. Webb, and R.V. Ulijn, Enzyme-responsive hydrogel particles for the controlled release of proteins: designing peptide actuators to match payload. Soft Matter, 2008. 4(4): p. 821-827.
17. Lee, S.H., J.J. Moon, and J.L. West, Three-dimensional micropatterning of bioactive hydrogels via two-photon laser scanning photolithography for guided 3D cell migration. Biomaterials, 2008. 29(20): p. 2962-2968.
18. Lee, S.H., J.J. Moon, J.S. Miller, and J.L. West, Poly(ethylene glycol) hydrogels conjugated with a collagenase-sensitive fluorogenic substrate to visualize collagenase activity during three-dimensional cell migration. Biomaterials, 2007. 28(20): p. 3163-3170.
19. Hahn, M.S., J.S. Miller, and J.L. West, Three-dimensional biochemical and biomechanical patterning of hydrogels for guiding cell behavior. Advanced Materials, 2006. 18(20): p. 2679-2684.
20. Wylie, R.G. and M.S. Shoichet, Two-photon micropatterning of amines within an agarose hydrogel. Journal of Materials Chemistry, 2008. 18(23): p. 2716-2721.
21. Patrick, A.G. and R.V. Ulijn, Hydrogels for the Detection and Management of Protease Levels. Macromolecular Bioscience, 2010. 10(10): p. 1184-1193.
22. Nagy, L.J., L. Ding, L.S. Xu, W.H. Knox, and K.R. Huxlin, Potentiation of Femtosecond Laser Intratissue Refractive Index Shaping (IRIS) in the Living Cornea with Sodium Fluorescein. Investigative Ophthalmology & Visual Science, 2010. 51(2): p. 850-856.
23. Wong, J.Y., A. Velasco, P. Rajagopalan, and Q. Pham, Directed movement of vascular smooth muscle cells on gradient-compliant hydrogels. Langmuir, 2003. 19(5): p. 1908-1913.
24. Salinas, C.N. and K.S. Anseth, The influence of the RGD peptide motif and its contextual presentation in PEG gels on human mesenchymal stem cell viability. Journal of Tissue Engineering and Regenerative Medicine, 2008. 2(5): p. 296-304.
25. Salinas, C.N., B.B. Cole, A.M. Kasko, and K.S. Anseth, Chondrogenic differentiation potential of human mesenchymal stem cells photoencapsulated within poly(ethylene glycol)-arginine-glycine-aspartic acid-serine thiol-methacrylate mixed-mode networks. Tissue Engineering, 2007. 13(5): p. 1025-1034.
26. Luo, Y. and M.S. Shoichet, A photolabile hydrogel for guided three-dimensional cell growth and migration. Nature Materials, 2004. 3(4): p. 249-253.
27. Williams, C.G., A.N. Malik, T.K. Kim, P.N. Manson, and J.H. Elisseeff, Variable cytocompatibility of six cell lines with photoinitiators used for polymerizing hydrogels and cell encapsulation. Biomaterials, 2005. 26(11): p. 1211-1218.
28. Bryant, S.J., C.R. Nuttelman, and K.S. Anseth, Cytocompatibility of UV and visible light photoinitiating systems on cultured NIH/3T3 fibroblasts in vitro. Journal of Biomaterials Science-Polymer Edition, 2000. 11(5): p. 439-457.
29. Stephens-Altus, J.S., P. Sundelacruz, M.L. Rowland, and J.L. West, Development of bioactive photocrosslinkable fibrous hydrogels. Journal of BIiomedical Materials Research A, 2011. 00A(00): p. 1-10.
30. West, J.L., M.P. Cuchiara, A.C.B. Allen, T.M. Chen, and J.S. Miller, Multilayer microfluidic PEGDA hydrogels. Biomaterials, 2010. 31(21): p. 5491-5497.
31. Burdick, J.A. and K.S. Anseth, Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering. Biomaterials, 2002. 23(22): p. 4315-4323.
32. West, J.L., S.H. Lee, and J.J. Moon, Three-dimensional micropatterning of bioactive hydrogels via two-photon laser scanning photolithography for guided 3D cell migration. Biomaterials, 2008. 29(20): p. 2962-2968.
33. Thaler, S., C. Haritoglou, T.J. Choragiewicz, A. Messias, A. Baryluk, C.A. May, R. Rejdak, M. Fiedorowicz, E. Zrenner, and F. Schuettauf, In Vivo Toxicity Study of Rhodamine 6G in the Rat Retina.Investigative Ophthalmology & Visual Science, 2008. 49(5): p. 2120-2126.
34. Oefner, P.J., C.G. Huber, F. Umlauft, G.N. Berti, E. Stimpfl, and G.K. Bonn, High-Resolution Liquid-Chromatography of Fluorescent Dye-Labeled Nucleic-Acids. Analytical Biochemistry, 1994. 223(1): p. 39-46.
35. Mann, B.K. and J.L. West, Cell adhesion peptides alter smooth muscle cell adhesion, proliferation, migration, and matrix protein synthesis on modified surfaces and in polymer scaffolds. Journal of Biomedical Materials Research, 2002. 60(1): p. 86-93.
36. Mann, B.K., A.S. Gobin, A.T. Tsai, R.H. Schmedlen, and J.L. West, Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering. Biomaterials, 2001. 22(22): p. 3045-3051.
37. West, J.L., S.H. Lee, J.J. Moon, and J.S. Miller, Poly(ethylene glycol) hydrogels conjugated with a collagenase-sensitive fluorogenic substrate to visualize collagenase activity during three-dimensional cell migration. Biomaterials, 2007. 28(20): p. 3163-3170.
38. Lee, S.H., J.S. Miller, J.J. Moon, and J.L. West, Proteolytically degradable hydrogels with a fluorogenic substrate for studies of cellular proteolytic activity and migration. Biotechnology Progress, 2005. 21(6): p. 1736-1741.
39. West, J.L. and J.C. Hoffmann, Three-dimensional photolithographic patterning of multiple bioactive ligands in poly(ethylene glycol) hydrogels. Soft Matter, 2010. 6(20): p. 5056-5063.
40. Wosnick, J.H. and M.S. Shoichet, Three-dimensional chemical Patterning of transparent hydrogels. Chemistry of Materials, 2008. 20(1): p. 55-60.


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