臺灣博碩士論文加值系統

注射型水凝膠可完全填補不規則形狀的受損部位並減少外科手術放入植入物所引起的風險，同時，可藉由簡單的混合過程攜帶藥物，經由溶膠-凝膠態轉換特性進而局部釋放治療藥物，有助於減少外科手術風險及增進受損組織的修復之用，成為相當具有潛力的一種新型藥物載體策略。本研究將採用幾丁聚醣（Chitosan, CS）之水溶性衍生物羧甲基幾丁聚醣（N, O carboxymethyl chitosan, NOCC）與透明質酸（hyaluronic acid, HA）之衍生物氧化透明質酸（Aldehyded Hyaluronic Acid, AHA），經希夫鹼反應（Schiff base）的方式，製備出具酸鹼敏感性的水凝膠。
於傅立葉紅外線光譜儀（Fourier transform infrared spectrometer ,FTIR）結果中顯示，能成功於幾丁聚醣之葡萄胺單元之amino及primary hydroxyl端改質成具有羧甲基之官能基，並增加於中性環境下之水溶性。於TNBSA的分析結果顯示，透明質酸之氧化程度為44％與羧甲基幾丁聚醣改質程度為70％。由流變儀分析評估不同濃度NOCC/ AHA水凝膠得知，隨著NOCC和AHA濃度上升，凝膠強度隨之提升（G’由0.489到7.08）和凝膠點（gelling point）時間隨之縮短（NOCC濃度由2%的11分鐘到5%的25秒）。並由SEM觀察結果得知，隨著NOCC的濃度提高，較多的胺基（NH2）與AHA的醛基（CHO）交聯（Crosslink），會得到較緻密、較小的孔徑且互相連接的孔洞。於藥物釋放研究中，隨NOCC濃度提高時，鞣花酸（Ellagic acid , EA）釋放速率隨之減緩。不同酸鹼環境下時，pH 4.5有較佳緩效藥物釋放的能力，而於6小時內，pH 7.4具有較佳的累積釋放量。於Live/Dead Viability結果顯示，細胞封裝在添加藥物鞣花酸的水凝膠中，具有作為細胞載體的發展潛力。本研究成功開發以NOCC/ AHA注射型酸鹼應答性水凝膠系統，可原位成型作為局部藥物控制釋放的新型載體應用於生物醫學領域。

Injectable hydrogels do not require a surgical procedure for implantation, and various therapeutic drugs can be incorporated through simple mixing, due the sol - gel state conversion characteristics. Preparation of pH-sensitive hygrogel in this study, the use of chitosan modified, prepared as a N,O carboxymethyl chitosan（NOCC）and hyaluronic acid （HA） derivative aldehyded hyaluronic acid （AHA）, via schiff base reaction. FTIR analysis, observation chitosan successfully modified N,O carboxymethyl chitosan（NOCC）. TNBSA analysis, determination of the degree of the oxide hyaluronic acid and modified degree of N,O carboxymethyl chitosan（NOCC）. Rheological Behavior analysis, rate hydrogel gelling point and intensity of hydrogel. Situ forming hydrogel NOCC / AHA from the form of a liquid into a gel state about within 25 seconds -11 minutes, with NOCC concentration increases gelling time shorter, due NOCC concentration improve hove more amine group （NH2） with AHA have aldehyde group （CHO） crosslink. SEM analysis, observed NOCC concentration increases have smaller pores. Drug release study, pH 4.5 lower drug release capability with pH 7.4 higher drug release capability, but NOCC concentration improve have a lower drug release capability, due NOCC concentration have impact the pores of the hydrogel. In brief, an injectable NOCC/AHA pH-sensitive hydrogel system was successfully developed for in situ local controlled release as novel drug carrier for biomedical applications.

圖目錄 IV
表目錄 VII
第一章 緒論 1
1.1 前言 1
1.2 研究目的 4
第二章 文獻回顧 5
2.1 組織工程 5
2.1.1 細胞 6
2.1.2 支架 6
2.1.3 訊息 8
2.2 水凝膠系統 9
2.2.1 pH敏感性水凝膠 11
2.3 羧甲基幾丁聚醣（N，O-carboxymethyl chitosan:NOCC） 11
2.4 氧化透明質酸（Aldehyded Hyaluronic Acid, AHA） 13
2.5 鞣花酸（ Ellagic acid, EA） 15
第三章 實驗與方法 16
3.1 實驗藥品 16
3.2 實驗流程 17
3.2.1 N，O羧甲基幾丁聚醣合成 18
3.2.2 氧化透明質酸製備 18
3.2.3 水凝膠製備 19
3.2.4 水凝膠搭載藥物 20
3.3 材料分析 21
3.3.1 傅立葉紅外線光譜儀（Fourier transform infrared spectrometer, FT-IR） 21
3.3.2 TNBSA分析 21
3.3.3 澎潤率分析（Swelling Behavior） 23
3.3.4 掃描式電子顯微鏡（Scanning Electron Microscope, SEM） 23
3.3.5 流變儀分析（Rheological Behavior） 24
3.3.6 體外藥物釋放測試 25
3.4 生物相容性測試 26
3.4.1 軟骨細胞培養 26
3.4.2 細胞接種 27
3.4.3 PrestoBlue分析法 28
3.4.4 細胞活性與毒性評估（Live/Dead Viability/Cytotoxicity） 29
第四章 結果與討論 30
4.1 傅立葉紅外線光譜儀（FT-IR） 30
4.2 TNBSA分析 32
4.1 流變儀分析（Rheological Behavior） 34
4.2 掃描式電子顯微鏡（Scanning Electron Microscope, SEM） 40
4.3 澎潤率分析（Swelling Behavior） 42
4.4 體外藥物釋放測試（Drug Release） 43
4.5 PrestoBlue分析法 46
4.6 細胞活性與毒性評估（Live/ Dead Viability &; Cytotoxicity） 48
第五章 結論 52
第六章 參考文獻 54

1.Buckwalter, J.A., Activity vs. rest in the treatment of bone, soft tissue and joint injuries. Iowa Orthop J, 1995. 15: p. 29-42.
2.Park, K.M., et al., Thermosensitive chitosan-Pluronic hydrogel as an injectable cell delivery carrier for cartilage regeneration. Acta Biomater, 2009. 5(6): p. 1956-65.
3.Kolker, S.J., et al., Acid-sensing ion channel 3 expressed in type B synoviocytes and chondrocytes modulates hyaluronan expression and release. Ann Rheum Dis, 2010. 69(5): p. 903-909.
4.Li, F., et al., In-situ forming biodegradable glycol chitosan-based hydrogels: Synthesis, characterization, and chondrocyte culture. Materials Science and Engineering: C, 2012. 32(7): p. 2017-2025.
5.Mano, J.F., et al., Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends. J R Soc Interface, 2007. 4(17): p. 999-1030.
6.葛宇淪，應用於骨組織工程之多孔形HA-PEEK生物複合材料之研究，南台科技大學，化學工程與材枓工程系碩士論文，2008.
7.de la Fuente, M., B. Seijo, and M.J. Alonso, Novel hyaluronan-based nanocarriers for transmucosal delivery of macromolecules. Macromol Biosci, 2008. 8(5): p. 441-50.
8.Duceppe, N. and M. Tabrizian, Factors influencing the transfection efficiency of ultra low molecular weight chitosan/hyaluronic acid nanoparticles. Biomaterials, 2009. 30(13): p. 2625-31.
9.Tang, Y., et al., An improved complex gel of modified gellan gum and carboxymethyl chitosan for chondrocytes encapsulation. Carbohydr Polym, 2012. 88(1): p. 46-53.
10.Li, F., et al., In-situ forming biodegradable glycol chitosan-based hydrogels: Synthesis, characterization, and chondrocyte culture. Materials Science &; Engineering C-Materials for Biological Applications, 2012. 32(7): p. 2017-2025.
11.Nazar, H., et al., Thermosensitive hydrogels for nasal drug delivery: the formulation and characterisation of systems based on N-trimethyl chitosan chloride. Eur J Pharm Biopharm, 2011. 77(2): p. 225-32.
12.Lietman, S.A., et al., Stimulation of proteoglycan synthesis in explants of porcine articular cartilage by recombinant osteogenic protein-1 (bone morphogenetic protein-7). J Bone Joint Surg Am, 1997. 79(8): p. 1132-7.
13.Young, I.S. and J.V. Woodside, Antioxidants in health and disease. J Clin Pathol, 2001. 54(3): p. 176-86.
14.Leung, L.K., et al., Theaflavins in black tea and catechins in green tea are equally effective antioxidants. J Nutr, 2001. 131(9): p. 2248-51.
15.Zern, T.L. and M.L. Fernandez, Cardioprotective effects of dietary polyphenols. J Nutr, 2005. 135(10): p. 2291-4.
16.Mertens-Talcott, S.U., et al., Ellagic acid potentiates the effect of quercetin on p21waf1/cip1, p53, and MAP-kinases without affecting intracellular generation of reactive oxygen species in vitro. J Nutr, 2005. 135(3): p. 609-14.
17.Huang, G., et al., Controlled drug release from hydrogel nanoparticle networks. J Control Release, 2004. 94(2-3): p. 303-11.
18.Drury, J.L. and D.J. Mooney, Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials, 2003. 24(24): p. 4337-51.
19.Gutowska, A., B. Jeong, and M. Jasionowski, Injectable gels for tissue engineering. Anat Rec, 2001. 263(4): p. 342-9.
20.Ruel-Gariepy, E. and J.C. Leroux, In situ-forming hydrogels--review of temperature-sensitive systems. Eur J Pharm Biopharm, 2004. 58(2): p. 409-26.
21.Chenite, A., et al., Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials, 2000. 21(21): p. 2155-61.
22.Petrovic, S.C., W. Zhang, and M. Ciszkowska, Preparation and characterization of thermoresponsive poly(N-isopropylacrylamide-co-acrylic acid) hydrogels: studies with electroactive probes. Anal Chem, 2000. 72(15): p. 3449-54.
23.陳威，利用電漿處理及UV光接枝聚合固定聚丙烯酸藥物釋放水膠並固定幾丁聚醣褐藻酸於316L不鏽鋼表面，大同大學，材料科學與工程學系碩士論文，2010.
24.張永承，聚己內酯混摻幾丁聚醣微粒對複合膜物性影響，東海大學，化學工程與材料工程學系碩士論文，2010.
25.Shu, S., et al., Polyelectrolyte nanoparticles based on water-soluble chitosan-poly(L-aspartic acid)-polyethylene glycol for controlled protein release. Carbohydr Res, 2009. 344(10): p. 1197-204.
26.Weng, L., et al., Non-cytotoxic, in situ gelable hydrogels composed of N-carboxyethyl chitosan and oxidized dextran. Biomaterials, 2008. 29(29): p. 3905-13.
27.Lin, Y.H., et al., Physically crosslinked alginate/N,O-carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs. Biomaterials, 2005. 26(14): p. 2105-13.
28.Chen, S.C., et al., A novel pH-sensitive hydrogel composed of N,O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery. Journal of Controlled Release, 2004. 96(2): p. 285-300.
29.黄强，N, O羧甲基壳聚糖的制备及其理化特性研究，华南理工大学轻工与食品学院，2009年第35卷第7期(总第259期).
30.林倩如，氫氧基磷灰石-膠原蛋白-透明質酸複合微粒對於間葉幹細胞骨分化之影響，國立成功大學，生物科技研究所碩士班，2009.
31.Huang, G.S., et al., Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes. Biomaterials, 2011. 32(29): p. 6929-45.
32.Sharma, G., et al., Biodegradable in situ gelling system for subcutaneous administration of ellagic acid and ellagic acid loaded nanoparticles: evaluation of their antioxidant potential against cyclosporine induced nephrotoxicity in rats. J Control Release, 2007. 118(1): p. 27-37.
33.Scheibmeir, H.D., et al., A review of free radicals and antioxidants for critical care nurses. Intensive Crit Care Nurs, 2005. 21(1): p. 24-8.
34.Hayes ER. N,O-carboxymethyl chitosan and preparative method，US4619995A，1986.
35.Lin, Y.H., et al., Physically crosslinked alginate/N,O-carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs. Biomaterials, 2005. 26(14): p. 2105-2113.
36.Su, W.Y., Y.C. Chen, and F.H. Lin, Injectable oxidized hyaluronic acid/adipic acid dihydrazide hydrogel for nucleus pulposus regeneration. Acta Biomater, 2010. 6(8): p. 3044-3055.