臺灣博碩士論文加值系統

明膠為生物可分解高分子，因具有良好生物相容性及可塑性等優點而被廣泛應用於生醫材料領域。然而明膠材料之機械性質與生物活性不佳之缺點嚴重抑制其應用面。因此本研究中添加入具高硬度與良好生物活性誘導能力之二氧化矽作為明膠材料之補強材料，並添加低毒性的天然交聯劑綠槴子素(genipin)交聯明膠，使其形成較穩固的交聯結構後，利用冷凍乾燥法製備出多孔性的綠槴子素交聯明膠/二氧化矽複合支架。
FT-IR結果顯示成功地以溶膠-凝膠(sol-gel)法製備出二氧化矽奈米顆粒，SEM觀察二氧化矽粒徑大小為70nm其大小尺寸均一。另外以UV-Vis和DSC觀察明膠複合支架之交聯指數與熱性質，結果指出添加至1-5 wt%二氧化矽及0.17-1.0 wt%綠槴子素交聯處理之明膠複合支架其交聯程度約為42-89%，而變性溫度(TD)約為85-91℃，相較於純明膠上升2-8℃。此外，測量明膠複合支架之膨潤度約為511-279%。物理性質檢測顯示明膠複合支架孔洞間具有良好連通性，支架內部孔徑大小分布為200-700μm的三度空間立體結構，其孔徑大小變化隨著綠槴子素的添加量增加具有先上升而後下降的趨勢，且其孔隙率多介於80-90%。機械性質檢測結果顯示明膠中添加0.67 wt%綠槴子素交聯劑與5 wt%二氧化矽所製備支架支彈性模數相較於純明膠支架有約40%支提升。此外，生物可降解性實驗顯示綠槴子素的添加具有延緩明膠複合支架降解速率之效果，然而二氧化矽的添加會加速明膠支架的降解速率。
支架表面生長氫氧基磷灰石(HA)之結果顯示氫氧基磷灰石生長的數量隨著明膠支架浸泡於模擬體液(SBF)中的天數增長而增多，當支架浸泡於模擬體液中3天後，XRD與SEM結果證實氫氧基磷灰石已成功披覆於支架表面，且二氧化矽的添加確實能誘發磷酸鈣晶體的成核成長，生長的數量隨著二氧化矽的添加量增加而增加，以添加5 wt%二氧化矽具有較佳誘發氫氧基磷灰石生長的能力。
生物毒性測試顯示添加0.67 wt%綠槴子素交聯明膠支架的降解物質並不會對細胞生長造成毒害；添加二氧化矽之明膠支架的降解物質可促進細胞生長速度。此外，生物相容性結果顯示明膠複合支架支架表面披覆一層膠原蛋白能誘導細胞往支架的方向生長，且當細胞接觸到支架表面後會逐漸往支架內部生長，而無明顯的排斥現象。
綜合以上結果，明膠與5 wt%二氧化矽掺混再添加入0.67 wt%綠槴子素進行交聯處理之明膠複合支架具有最佳的孔洞性質、膨潤度、穩固的交聯結構、良好熱穩定性，以及適當的降解速率，且與純明膠支架相比具有良好的生物活性誘導能力，能促進細胞生長的速度以及良好的生物相容性，可作為理想的生醫植入材。

High biodegradability, biocompatibility and plasticity of gelatin can be used as biomaterial for biomedical application. However, there is some limitation due to its inappropriate mechnical properties and poor bioactive ability. Therefore the addition of bioactive silica and low cytotoxic nature of crosslinking agent such as, genipin, into gelatin was expected to improve the bioactivity and mechanical stability of gelatin. In this study, the high-porosity gelatin/SiO2 composite scaffolds with presence of genipin were sucessfully prepared by freeze-drying technique.
The Fourier Transform Infrared (FTIR) spectrum of SiO 2 show typical Si-O-Si stretching vibration peaks at 470 cm-1, 790 cm-1 and 1060~1233 cm-1. This result indicated SiO2 nanoparticles were successfully synthesized by using sol-gel peocessing. The Field-Emission Scanning Electron Microscope (FE-SEM) image of SiO2 show spherical particle with average particle size at about 70 nm. The crosslinking index and thermal stability of gelatin/silica composite scaffolds with presence of ginipin were characterized by UV-Vis and DSC. The crosslinking index increased to 42-89% and the denature temperature was about 85-91℃ with increasing amounts of SiO2 and genipin up to 5wt% and 1.0 wt%, respetively. Compared to pure gelatin, the denature temperature increased 2-6℃. However, the swelling ratio of composite scaffolds about 511-279%. The porosity of the composite scaffolds with well-developed and open-channel micropores was about 80-90%. The pore size roughly about 200-700μm in diameter decreased with increasing genipin amount. The mechanical properties of 5 wt% SiO2 and 0.67 wt% composite scaffolds were enhanced about 40% compared to pure organic matrix.
For biodegradable test, the degradation rate of composite scaffolds decreased as the concentration of genipin increased, but it increased with increasing SiO2 amount. The scaffolds were then soked in a simulated body fluid (SBF) up to 3 days to evaluate its in vitro bioactivity. The SiO2-containing scaffolds show excellent bioactivity as their biomimetically deposited apatite, identified by SEM and XRD. The formation of the apatite was induced by silanol group in the hydrated silica gel formed on the suface. The cytotoxicity test was noted that residual genipin concentration in the 0.67 wt% geipin composite scaffold didn’t affect the cell growth. Fourthemore, the biocompatible test showed the composite scaffold surface coated with collagen can induce cells to migrate into composite scaffold.
High porosity of 5 wt% gelatin/SiO2 composite scaffolds with 0.67 wt% crosslinking agent genipin showed excellent swelling properties and thermal stability with suitably degradable rate. The cells attached to the nanocomposite scaffold are higher than that to the pure gelatin scaffold. These findings suggest that the 5wt% gelatin/SiO2 composite scaffold with 0.67 wt% genipin can be considered a very good alternative material for biomedical applications.

中文摘要.............................................. I
ABSTRACT .............................................II
總目錄 .............................................IV
圖目錄 .............................................VI
表目錄 .............................................X
第一章 緒論...........................................1
1-1前言 ..............................................1
1-2研究動機............................................3
1-3研究方向............................................3
第二章 文獻回顧........................................4
2-1骨組織工程..........................................4
2-1-1金屬材料..........................................5
2-1-2陶瓷類材料........................................5
2-1-3高分子材料........................................5
2-2人工支架............................................6
2-2-1冷凍乾燥法........................................6
2-3天然可降解性材料....................................8
2-3-1膠原蛋白..........................................8
2-3-2明膠 .............................................11
2-4交聯劑.............................................11
2-4-1 綠槴子素........................................12
2-4-2綠槴子素和明膠的交聯反應機制.....................12
2-4-3綠槴子素交聯生物組織.............................16
2-5利用溶膠-凝膠法(SOL-GEL)製備二氧化矽...............21
2-5-1溶膠-凝膠法原理及反應機制........................22
2-5-2製備參數對溶膠-凝膠法製備SiO2之影響..............24
2-6有機高分子-無機材料複合材料........................26
2-7仿生礦化及生物適應性評估 .......................... 30
第三章 實驗方法與步驟.................................35
3-1 實驗材料..........................................35
3-2 實驗儀器..........................................37
3-3 實驗流程圖........................................38
3-4實驗步驟...........................................39
3-4-1不同粒徑單分散二氧化矽之製備.....................39
3-4-2綠槴子素交聯明膠/奈米二氧化矽複合支架之製備......40
3-4-3綠槴子素交聯明膠/奈米二氧化矽複合支架之交聯指數分析
.................................................42
3-4-4綠槴子素交聯明膠/奈米二氧化矽複合支架之生物降解..43
3-4-5綠槴子素交聯明膠/奈米二氧化矽複合支架表面披覆HA..45
3-4-6綠槴子素交聯明膠/奈米二氧化矽複合支架毒性測試....47
3-5支架材料性質分析...................................47
3-6 實驗樣品及其代碼..................................49
第四章 結果與討論.....................................50
4-1奈米二氧化矽粒子之製備與結構分析...................50
4-2綠槴子素交聯明膠/奈米二氧化矽複合支架材料性質分析..53
4-2-1交聯程度測試.....................................53
4-2-2熱性質測試.......................................56
4-2-3 膨潤度測試......................................59
4-2-4 明膠支架之巨觀型態..............................61
4-2-5 明膠複合支架之微觀孔洞形貌......................62
4-2-6孔隙率測試.......................................68
4-3 綠槴子素交聯明膠/奈米二氧化矽複合支架之機械性質測試
..................................................69
4-4 綠槴子素交聯明膠/奈米二氧化矽複合支架之生物降解測試
..................................................71
4-5 綠槴子素交聯明膠/奈米二氧化矽複合支架表面IN VITRO生長氫氧基磷灰石(HA)........................................76
4-6綠槴子素交聯明膠/奈米二氧化矽複合支架毒性測試......88
4-6-1明膠複合支架降解物質之毒性測試...................88
4-6-2明膠複合支架與肺腺癌細胞共培養結果...............91
第五章 結論...........................................94
參考文獻..............................................96

[1]Langer R, Vacanti J.P, Tissue engineering, Science 260 (1993) 920-26.
[2]Mooney D.J, Mikos A.G, Growing new organs, Sci Am 280 (1999) P 60-65.
[3]Mikos A.G, Netherlands Organisation for Scientific Research, 2003
[4]Pieper J.S, Hafman T, Veekamp J.H, Kuppevelt T.H, Development of tailor-made collagen glycosaninoglycan matrices: EDC/NHS crosslinking and ultrastructural aspects, Biomaterials 21 (2000) P581-593
[5]Tabata Y, Tissue regeneration based on growth factor release, Tissue Eng 9 (2003) P5-15
[6]Biomet 2005 annual report
[7]Ravaglioli A, Krajewski A, Bioceramics Materials. Properties, Applications. Chapman and Hall (1992) P1-5
[8] Shapiro J. In: Acrylic cement in orthopadeic surgery. Personal Communication to J Charneley, E & S Livingstone, Edinburgh and London 127.
[9]Mittelmeier H, Katthagen B.D, Clinical experience with the implantation of collagen-apatite for local bone regeneration, Zeitschrift fur Orthop und Ihre Grenzgebiete 121 (1983) P115-123
[10] Kang H.W, Tabata Y, Ikada Y, Fabrication of porous gelatin scaffolds for tissue engineering, Biomaterials 20 (1999) 1339-1344
[11]湯加潤 組織工程中的膠原蛋白 科學發展 308期2004年8月p20-23
[12]Becker W.M, Kleinsmith L.J, Hardin J, The world of the cell 4th ed San Francisco: Addison Wesley Longman, Inc.( 2000) 301-303.
[13]Tu R, Lu C.L, Thyagarajan K, Wang E, Nguyen H, Shen S, Hata C, Quijano R C, Kinetic study of collagen with polyepoxy ffixatives, J Biomed Mater Res 27(1993) 3-9
[14]Zeeman R, Dijkstra P.J, Wachem P.B.Van, Luynb M.J.A.Van, Hendriksc M, Cahalanc P.T, Feijena J, Successive epoxy and carbodiimide ceoss-linking of dermal sheep collagen, Biomaterials 20 (1999) 921-931
[15]Tu R, Lu C.L, Thyagarajan K, Wang E, Nguyen H, Shen S, Hata C and Quijano R.C, Kinetic study of collagen with polyepoxy fixatives, J. Biomed Mater. Res. 27 (1993) 3-9
[16]Nimni M.E,Cheung D, Strates B, Odama M.K, Sheikh K, Nimni M.Eed Collagen, Biotechnology. Florida, Bioprosthesis derived from cross-linked and chemically modified collagenous tissues, Nimni M E.ed Collagen, Biotechnology Florida, CRC Press Inc, 3 (1988) 1-37

[17]Sung H.W, Huang D M, Chang W H, Huang R N, Hsu J C, Evaluation of gelatin hydrogel crosslinked with various crosslinling agents as bioadhesives: In vitro study, J biomed Mater Res (1999) 520-530
[18]Tsai C.C, Huang R.N, Sung H.W, Liang H.C., In vitro evaluation of the genotoxicity of a naturally occurring crosslinking agent (genipin) for biologic tissue fixation, J Biomed Mater Res 52 (2000) 58-65
[19]Sung H.W, Huang R.N, Huang L.L.H, Tsai C.C, Chiu C.T, Biocompatibility study of a biological tissue fixed with a naturally occurring crosslinking reagent, J Biomed Mater Res 42 (1998) 560-567
[20]Zeeman R, Dijkstra P. J, Wachem P. B.van, Luyn M. J. A.van, Hendriks M, Cahalan P. T, Feijen J, Successive epoxy and carbodiimide cross-linking of dermal sheep collagen, Biomaterials 20 (1999) 921-931
[21]馬純媛，以天然交聯劑(genipin)交聯處理的生物材料表面性質的探討，碩士論文，國立中央大學，(2000) 27-30
[22]Liang H.C , Chang W.H, Liang H.F, Lee M.H, Sung M.H, Crosslinking structures of gelatin hydrogels crosslinked with genipin or a water-soluble carbodiimide, J Appl Polym Sci 91 (2004) 4017–4026,
[23]Bigi A, Cojazzi G, Panzavolta S, Roveri N, Rubini K, Stabilization of gelatin films by crosslinking with genipin, Biomaterials 23 (2002) 4827– 4832
[24]Yao C.H, Liu B.S, Chang C.J, Hsu S.H, Chen Y.S, Preparation of networks of gelatin and genipin as degradable biomaterials, Materials Chemistry and Physics 83 (2004) 204–208
[25]Yao C.H, Liu B.S, Hsu S.H, Chen Y.S, Tsai C.C, Biocompatibility and biodegradation of a bone composite containing tricalcium phosphate and genipin crosslinked gelatin, Journal of Biomedical Materials Research Part A 69A (4) ( 2004) 709-717
[26]Ebelmwn H.W, Ann. Chimie. Phys. 16 (1846) 129
[27]Graham T, J. Chem. Soc 17 (1864) 318
[28]Coradin T, Bah S, Livage J, Gelatin/silicate interactions: from nanoparticles to composite gels, Colloids and Surfaces B: Biointerfaces 35 (2004) 53–58
[29]梁義林，以天然交聯劑(Genipin)交聯生物組織材料最適化條件及其穩定性的探討，碩士論文，國立中央大學，(1999) 28-46
[30]Jirgensons B and Straumanis M.E, Coloid Chemistry, MvMillan Co., New York (1962)
[31]Schaefer D.W et al., AIP Con. Proc.,63 (1987) 154
[32]Sarjeant P.T, Roy R., J. American Ceramic Society 52(1) (1969) 57-58
[33]Satoh T, Akitaya M, Konno M, Saito S, Particle Size Distributions Produced by Hydrolysis and Condensation of Tetraethylorthosilicate, Journal of Chemical Engineering of Japan 30 (1997) 759-762
[34]Tamon H, Kitamura T, Okazaki M, Heterogeneous Nucleation of Water Vapor on Submicrometer Particles of SiC, SiO2, and Naphthalene, J. Colloid and Interface Science, 197 (1998) 353-359
[35]Iler R.K, The chemistry of silica, Wiley-Inter Science (1979)
[36]Kim K.D, Kim H.T, Formation of Silica Nanoparticles by Hydrolysis of TEOS Using a Mixed Semi-Batch/Batch Method, Journal of Sol-Gel Science and Technology 25 (2002) 183-189
[37]許麗文，SiO2單分散體之合成及其性質之研究，化學研究所碩士論文,國立成功大學 (1999)
[38] Butler M.F, Ng Y.F, Pudney P.A, Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin, J Polym Sci Part A: Polym Chem 41 (2003) 3941–3953,
[39]Hench L.L, Wilson J, World Scientific (1993) 1-24
[40]El-Ghaannam A, Ducheyne P, Shapiro IM, Bioactive material template for in vitro, synthesis of bone, J Biomed Mater Res 29 (1995) 359-370
[41]Kokubo T, Kushitani H, Sakka S, Kitsugi T, Yamamuro T, Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W3, J Biomed Mater Res 24 (1990) 721-734
[42]Ohtsuki C, Kokubo T, Yamamuro T, Mechanism of apatite formation on CaO SiO2 P2O5 glasses in a simulated body fluid, J Noncryst Solids 143 (1992) 84-92
[43]Li P, Ohtsuki C, Kokubo T, Nakanishi K, Soga N, Nakamura T, Yamamuro T, Process of formation of bone-like apatite layer on silica gel, J Mater Sci: Mater Med 4 (1993) 127-131
[44]Fuentes S, Retuert J, Gozalez G, Reiz-Hitzky E, Chitosan Based Films. Synthesis and Crystalline Properties of Nanocomposites with Amine Propyl Siloxane, Int. J. Polym. Mater 35 (1997) 61
[45]Retuert J, Nunez A, Yazdani-Pedram M, Martinez F, Synthesis of polymeric organic-inorganic hybrid materials. Partially deacetylated chitin-silica hybrid, Macromol. Rapid Commun., 18 (1997) 163
[46]Madihally S.V, Matthew H.W.T, Porous chitosan scaffold for tissue engineering, Biomaterials 20 (1999) 1133-1142
[47]Hench L.L, J Am Ceram Soc 74 (1991) 1487-1510
[48]Kokubo T, J Ceram Soc Jpn 99 (1991) 965-973
[49]Ren L, Tsuru K, Hayakawa S, Osaka A, Sol–gel preparation and in vitro deposition of apatite on porous gelatin–siloxane hybrids, Journal of Non-Crystalline Solids 285 (2001) 116-122
[50]Ohtsuki C, Miyazaki T, Tanihara M, Development of bioactive organic-inorganic hybrid for bone substitutes, Master.Sci.Eng. C22 (2002)27
[51]Kim HW, Knowles J.C, Kim H.E, Hydroxyapatite and gelatin composite foams processed via noval freeze-drying and crosslinking for use as temporary hard tissue scaffolds, J Biomed Mater Res 72A (2005) 135-145
[52]Ramachandra R.R, Roopa H.N, Kannan T S, Solid state synthesis and thermal stability of HAP-β-TCP composit ceramic powders, J Biomed Mater Res 8 (1997) 511-518
[53]Cuneyt T.A, Korkusuz F, Timicin M, Akkas N, Investigation of the chemical synthesis and high-temperature sintering behaviour of calcium hydroxyapatite (HA) and tricalcium (TCP) bioceramics, J Mater Sci, Mater Med 8 (1997) 91-96
[54]Layrolle P, Ito A, Tateishi T, Sol-gel synthesis of amorphous calcium phosphate and sintering into microporous hydroxyapatite bioceramics, J. Amer. Cream Soc 81 (1998) 1421-1428
[55]Gorbunoff M.J, The interaction of proteins with hydroxyapatite : I. Role of protein charge and structure, Anal. Biochem 136 (1984) 425-432
[56]Gorbunoff M.J, The interaction of proteins with hydroxyapatite : II. Role of acidic and basic groups, Anal. Biochem 136 (1984) 433-439
[57]Gorbunoff M.J, The interaction of proteins with hydroxyapatite : III. Mechanism, Anal. Biochem 136 (1984) 440-445
[58]Kim H.W, Kim H.E, Salih V, Stimulation of osteoblast responses to biomimetic nanocomposites gelatin-hydroxyapatite for tissue engineering scaffolds, Biomaterials 26 (2005) 5221-5230
[59]Ren L., Tsuru K., Hayakawa S., A. Osaka, Noval approach to fabricate porous gelatin-siloxane hybrids for bone tissue engineering, Biomaterials 23 (2002) 4765–4773
[60]Ryousuke T, Kenichiro I, Yoshio T, Masahiko Y, Studies on the blue pigments produced from genipin and methylamine. Ιstructures of the brownish-red pigments, intermediates leading to the blue pigment, Chem. Pharm. Bull., 24 (1994) 668-673
[61]Prochazkova S, Vårum K.M, Østgaard K, Quantitative determination of chitosans by ninhydrin ,Carbohydrate Polymers 38 (1999) 115-122
[62]Schwarz K, A bound form of Si in glycosaminoglycans and polyuronides, Proc Nat Acad Sci USA 70 (1973) 1608-1612
[63]Carlise E, The nutritional essentiality of silicon, Nutr Rev 40 (1982) 193-8
[64]Lee J.M, Pereira C.A, Kan L.W.K, Effect of molecular structure of poly(glycidyl ether) reagents on crosslinking and mechanical properties of bovine pericardial xenograft materials, J Biomed Mater Res 28 (1994) 981-992
[65]Boyan B.D, Hummert T.W, Dean D.D, Schwartz Z, Role of material surfaces in regulating bone and cartilage cell response, Biomaterials 17 (1996) 137-146
[66]Zigang G, Sophie B, Lee Y.L, Aileen W, Eugene K, Hydroxyapatite–chitin materials as potential tissue engineered bone substitutes, Biomaterials 25 (2004) 1049-1058