臺灣博碩士論文加值系統

目 錄
誌　　謝 I
摘 要 II
ABSTRACTS III
第一章 緒論 1
第二章 文獻回顧 3
2-1 組織工程 3
2-2 生物支架 5
2-3 澱粉(Starch) 7
2-4 海藻酸鈉(Sodium Alginate) 9
2-5 明膠(Gelatin) 10
2-6 介孔生醫活性玻璃(Mesoporous Bio-glasses, MBG) 11
2-7 交聯劑 12
2-7-1 碳酸鈣(Calcium carbonate)與氯化鈣(Calcium chloride) 12
2-7-2 1-乙基-(3-二甲基氨基丙基)碳醯二亞胺 (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide，EDC) 14
2-8 冷凍乾燥原理 15
2-9 前驅實驗 16
2-10 實驗目的 20
第三章 實驗材料與方法 21
3-1實驗材料與儀器 21
3-2 實驗流程 23
3-2-1 實驗流程圖 23
3-2-2 實驗樣本製備 23
3-2-2-1 MBG粉末製備 23
3-2-2-2 生物支架製備 24
3-2-3 實驗組別 25
3-3 生物支架之型態觀察 25
3-3-1 光學顯微鏡觀察(Optical microscope, OM) 25
3-3-2 掃描式電子顯微鏡觀察(Scanning Electron Microscope, SEM)、能量色散X射線譜(Energy dispersive spectroscopy, EDS)及孔徑分析 25
3-3-3 孔隙率測試 26
3-4 生物支架之物化性質分析 27
3-4-1 傅立葉紅外線光譜儀(Fourier Transform Infrared Spectroscopy, FTIR) 27
3-4-2 交聯度測試 28
3-4-3 吸水量測試 28
3-4-4 降解速率測試 29
3-4-5 抗壓強度測試 30
3-5 生物支架之生物相容性測試 31
3-5-1 滅菌處理 31
3-5-2 細胞培養 31
3-5-3 萃取液的製備 32
3-5-4 細胞毒性測試 32
3-5-5 細胞貼附測試 33
3-5-6 細胞增生測試 33
3-5-7 鹼性磷酸酶(Alkaline phosphatase, ALP)半定量與染色測試 33
3-6 統計方法 34
第四章 結果與討論 35
4-1 MBG之孔徑分析 35
4-2 生物支架光學影像及光學顯微鏡觀察 37
4-3 SEM觀察、EDS元素分析及支架孔徑分析 39
4-4 孔隙率測試 43
4-5 FTIR-ATR測試 44
4-6 交聯度測試 46
4-7 吸水量測試 47
4-8 降解速率測試 49
4-9 抗壓強度試驗 52
4-10 細胞毒性測試 55
4-11 D1細胞貼附測試 57
4-14 D1細胞增生測試 61
4-15 鹼性磷酸酶(ALP)半定量測試 63
4-16 鹼性磷酸酶(ALP)染色分析 67
第五章 結論 69
參考文獻 70


圖目錄
Fig. 1.組織工程示意圖 5
Fig. 2.澱粉結構圖 (a)直鏈澱粉 (b)支鏈澱粉 8
Fig. 3.澱粉顆粒形成凝膠過程 8
Fig. 4.海藻酸鈉結構圖 9
Fig. 5.明膠結構圖 10
Fig. 6.MBG形成介孔示意圖 12
Fig. 7. 鈣離子(Ca2+)取代海藻酸鈉的鈉離子(Na+)示意圖 13
Fig. 8. EDC交聯機制示意圖 15
Fig. 9.前驅實驗流程圖 16
Fig. 10. Alginate/Starch及Gelatin/Starch分別浸泡兩種不同交聯劑24小時後樣本實照圖 18
Fig. 11.Alginate/Starch與Gelatin /Starch支架吸水量曲線圖 19
Fig. 12.本研究實驗示意圖 20
Fig. 13.實驗流程圖 23
Fig. 14.MBG的(a)氮氣吸/脫附曲線圖(b)孔徑大小分布圖 36
Fig. 15. MBG之雷射粒徑分析測試結果 36
Fig. 16. MBG的表面型態及微相結構分析(a)SEM；5000倍 (b)TEM；40000倍 37
Fig. 17.生物支架光學影像及光學顯微鏡影像圖 38
Fig. 18.生物支架之SEM圖(100X) 40
Fig. 19.生物支架之EDS分析Si及P元素 (1000X) 41
Fig. 20.生物支架之EDS分析Si及Ca元素 (1000X) 41
Fig. 21.生物支架之平均孔徑尺寸分析 42
Fig. 22.生物支架之孔隙率測試 43
Fig. 23.生物支架之FT-IR/ATR測試 45
Fig. 24.生物支架之交聯度測試 46
Fig. 25.生物支架之吸水量曲線圖 48
Fig. 26.生物支架浸泡至去離子水中之降解速率測試 50
Fig. 27.生物支架浸泡至Hank’s solution之降解速率測試 51
Fig. 28.生物支架之應力-應變曲線圖 (a)乾樣本 (b)浸泡在去離子水10分鐘後的濕樣本 53
Fig. 29.生物支架在應變50%時之抗壓強度 (a)乾樣本 (b)浸泡在去離子水10分鐘後的濕樣本 54
Fig. 30.生物支架與L929細胞培養1天後之細胞毒性定量結果 55
Fig. 31.生物支架與L929細胞培養1天後之細胞型態定性圖 56
Fig. 32.生物支架與D1細胞接觸培養1小時後之SEM型態觀察圖 58
Fig. 33.生物支架與D1細胞接觸培養1天後之SEM型態觀察圖 59
Fig. 34.生物支架與D1細胞接觸培養2天後之SEM型態觀察圖 60
Fig. 35.生物支架與D1細胞接觸培養，培養時間之細胞增生 62
Fig. 36.生物支架與D1細胞接觸培養，培養時間之吸光值(OD 405nm) 65
Fig. 37.生物支架與D1細胞接觸培養，培養時間之ALP半定量 66
Fig. 38.生物支架與D1細胞接觸培養，培養不同時間之ALP染色定性圖 68

 
表目錄
表1、實驗材料 21
表2、實驗儀器 22
表3、實驗組別 25
表4、實驗組別 36
表5、不同交聯製程支架的孔隙率比較 44

Badhe, R. V., Bijukumar, D., Chejara, D. R., Mabrouk, M., Choonara, Y. E., Kumar, P., . . . Pillay, V. (2017). A composite chitosan-gelatin bi-layered, biomimetic macroporous scaffold for blood vessel tissue engineering. Carbohydrate polymers, 157, 1215-1225.
Baniasadi, H., SA, A. R., & Mashayekhan, S. (2015). Fabrication and characterization of conductive chitosan/gelatin-based scaffolds for nerve tissue engineering. International journal of biological macromolecules, 74, 360-366.
Brinker, C. J., Lu, Y., Sellinger, A., & Fan, H. (1999). Evaporation‐induced self‐assembly: nanostructures made easy. Advanced materials, 11(7), 579-585.
Bueno, E. M., & Glowacki, J. (2009). Cell-free and cell-based approaches for bone regeneration. Nature Reviews Rheumatology, 5(12), 685.
Dong, Z., Wang, Q., & Du, Y. (2006). Alginate/gelatin blend films and their properties for drug controlled release. Journal of Membrane Science, 280(1-2), 37-44.
Dvir, T., Timko, B. P., Kohane, D. S., & Langer, R. J. N. n. (2011). Nanotechnological strategies for engineering complex tissues. 6(1), 13.
George, M., & Abraham, T. (2007). pH sensitive alginate–guar gum hydrogel for the controlled delivery of protein drugs. International journal of pharmaceutics, 335(1-2), 123-129.
George, M., & Abraham, T. E. (2006). Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan—a review. Journal of controlled release, 114(1), 1-14.
Gleeson, J., Plunkett, N., & O’Brien, F. (2010). Addition of hydroxyapatite improves stiffness, interconnectivity and osteogenic potential of a highly porous collagen-based scaffold for bone tissue regeneration. Eur Cell Mater, 20(218), 30.
Hamman, J. H. (2010). Chitosan based polyelectrolyte complexes as potential carrier materials in drug delivery systems. Marine drugs, 8(4), 1305-1322.
Hutmacher, D. W. (2000). Scaffolds in tissue engineering bone and cartilage. In The biomaterials: Silver jubilee compendium (pp. 175-189): Elsevier.
Kakkar, P., Verma, S., Manjubala, I., & Madhan, B. (2014). Development of keratin–chitosan–gelatin composite scaffold for soft tissue engineering. Materials Science and Engineering: C, 45, 343-347.
Koski, C., Onuike, B., Bandyopadhyay, A., & Bose, S. (2018). Starch-hydroxyapatite composite bone scaffold fabrication utilizing a slurry extrusion-based solid freeform fabricator. Additive manufacturing, 24, 47-59.
Lai, J.-Y., & Li, Y.-T. (2010). Evaluation of cross-linked gelatin membranes as delivery carriers for retinal sheets. Materials Science and Engineering: C, 30(5), 677-685.
Leong, K., Cheah, C., & Chua, C. (2003). Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs. Biomaterials, 24(13), 2363-2378.
Lien, S.-M., Ko, L.-Y., & Huang, T.-J. (2009). Effect of pore size on ECM secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering. Acta biomaterialia, 5(2), 670-679.
Martins, M., Barros, A. A., Quraishi, S., Gurikov, P., Raman, S., Smirnova, I., . . . Reis, R. L. (2015). Preparation of macroporous alginate-based aerogels for biomedical applications. The Journal of Supercritical Fluids, 106, 152-159.
Mojarradi, H. (2010). Coupling of substances containing a primary amine to hyaluronan via carbodiimide-mediated amidation. In.
Nabavinia, M., Khoshfetrat, A. B., & Naderi-Meshkin, H. (2019). Nano-hydroxyapatite-alginate-gelatin microcapsule as a potential osteogenic building block for modular bone tissue engineering. Materials Science and Engineering: C, 97, 67-77.
Nieto-Suárez, M., López-Quintela, M. A., & Lazzari, M. (2016). Preparation and characterization of crosslinked chitosan/gelatin scaffolds by ice segregation induced self-assembly. Carbohydrate polymers, 141, 175-183.
Pawar, S. N., & Edgar, K. J. (2012). Alginate derivatization: a review of chemistry, properties and applications. Biomaterials, 33(11), 3279-3305.
Sarem, M., Moztarzadeh, F., Mozafari, M., & Shastri, V. P. (2013). Optimization strategies on the structural modeling of gelatin/chitosan scaffolds to mimic human meniscus tissue. Materials Science and Engineering: C, 33(8), 4777-4785.
Stender, E. G., Khan, S., Ipsen, R., Madsen, F., Hägglund, P., Hachem, M. A., . . . Svensson, B. (2018). Effect of alginate size, mannuronic/guluronic acid content and pH on particle size, thermodynamics and composition of complexes with β-lactoglobulin. Food hydrocolloids, 75, 157-163.
Sung, H.-W., Chang, Y., Chiu, Y.-T., Hsu, H.-L., Shih, C.-C., Lu, J.-H., & Yang, P.-C. (1996). Evaluation of an epoxy-fixed biological patch with ionically bound heparin as a pericardial substitute. Biomaterials, 17(17), 1693-1701.
Türe, H. (2019). Characterization of hydroxyapatite-containing alginate–gelatin composite films as a potential wound dressing. International journal of biological macromolecules, 123, 878-888.
Tønnesen, H. H., Karlsen, J. J. D. d., & pharmacy, i. (2002). Alginate in drug delivery systems. 28(6), 621-630.
Takahashi, Y., Yamamoto, M., & Tabata, Y. (2005). Osteogenic differentiation of mesenchymal stem cells in biodegradable sponges composed of gelatin and β-tricalcium phosphate. Biomaterials, 26(17), 3587-3596.
Tkalec, G., Kranvogl, R., Uzunalić, A. P., Knez, Ž., & Novak, Z. (2016). Optimisation of critical parameters during alginate aerogels' production. Journal of Non-Crystalline Solids, 443, 112-117.
Tsigkou, O., Pomerantseva, I., Spencer, J. A., Redondo, P. A., Hart, A. R., O’Doherty, E., . . . Lin, C. P. (2010). Engineered vascularized bone grafts. Proceedings of the National Academy of Sciences, 107(8), 3311-3316.
Wang, C., Jiang, W., Zuo, W., Han, G., & Zhang, Y. (2018). Effect of heat-transfer capability on micropore structure of freeze-drying alginate scaffold. Materials Science and Engineering: C, 93, 944-949.
Wang, C., Liu, H., Gao, Q., Liu, X., & Tong, Z. (2008). Alginate–calcium carbonate porous microparticle hybrid hydrogels with versatile drug loading capabilities and variable mechanical strengths. Carbohydrate polymers, 71(3), 476-480.
Wang, H.-J., Di, L., Ren, Q.-S., & Wang, J.-Y. (2009). Applications and degradation of proteins used as tissue engineering materials. Materials, 2(2), 613-635.
Xie, P., Du, J., Li, Y., Wu, J., He, H., Jiang, X., & Liu, C. (2019). Robust hierarchical porous MBG scaffolds with promoted biomineralization ability. Colloids and Surfaces B: Biointerfaces, 178, 22-31.
Jatnika, R. (2014). 一階段合成銀複合介孔生物活性玻璃 之生物活性與抗菌性探討之研究.
Tian, Y.-J. (2000). 以天然交聯劑 Genipin 交聯幾丁聚醣材料的體外及體內性質評估. National Central University,
李玉寶, 顧寧, & 魏于全. (2006). 奈米生醫材料. 初版, 台北, 五南出版股份有限公司, 162-184.
林哲毅. (2015). 果膠與明膠複合體提升優格發酵劑的抗凍性及儲存存活率之研究. (碩士), 國立中興大學, 台中市.
林裕昇. (2015). 海藻酸鈉與明膠製備球型藥物載體 物化性質與藥物釋放探討. (碩士), 逢甲大學, 台中市.
張文馨. (2011). 以不同交聯劑製備多孔性三維明膠支架及其特性研究. 中興大學材料科學與工程學系所學位論文, 1-93.
陳威志, 胡. (2009). 人工關節是什麼 人工關節的材質與種類.