臺灣博碩士論文加值系統

近年來研究指出，褪黑激素 (Melatonin, Mel) 結合骨替代材料，能賦予材料有骨傳導與骨誘導活性，但褪黑激素半衰期短，使用上受到限制；氫氧基磷灰石 (Hydroxyapatite) 為常見的骨替代材料，但藥物攜帶量不佳。故本研究透過 pH 介導沉澱法 (pH-mediated precipitation method) 結合幾丁聚醣 (Chitosan, CS) 提升磷灰石攜帶藥物能力，形成攜帶褪黑激素之幾丁聚醣/氫氧基磷灰石複合物 (Mel-loaded CS/HA) 與幾丁聚醣/缺鈣型氫氧基磷灰石複合物 (Mel-loaded CS/CDHA)，探討其藥物攜帶與釋放情形及對小鼠成骨細胞 (MC3T3E1) 分化之影響。實驗結果顯示 : 利用 pH 值介導沉澱法可將褪黑激素與幾丁聚醣共沉澱於磷灰石表面，而幾丁聚醣/氫氧基磷灰石複合物與幾丁聚醣/缺鈣型氫氧基磷灰石對褪黑激素之攜帶率分別為 56%、59%；能持續釋放褪黑激素長達九天，釋放率為 26% 與 77%。由複合材料釋放的褪黑激素促進小鼠前驅成骨細胞增生。提高小鼠前驅成骨細胞鹼性磷酸酶活性，促進礦化作用的進行，縮短礦化作用時間至 11 天，並能提高 Runx-2、ALP、Osteocalcin 的基因表現。

Recently, researches about melatonin combined with bone implants afford materials to bone osteoconduction and osteoinduction, but the effects of melatonin is limited due to its short half-life. Hydroxyapatite is a common bone implant, but the ability of drug loading is poor. In this study, we combine chitosan with hydroxyapatite by pH-mediated precipitation method to promote the ability of drug loading. Melatonin-loaded chitosan/hydroxyapatite and melatonin-loaded chitosan/ calcium-deficient-hydroxyapatite are fabricated to investigate the drug release and its effects on murine osteoblast-like cell line (MC3T3-E1) differentiation. The results display that apatite is able to combine with chitosan and melatonin by pH-mediated precipitation method. The loading efficiency of chitosan/hydroxyapatite and chitosan/ calcium-deficient-hydroxyapatite is 56% and 59%, respectively. The percentage of accumulation release is 26% and 77%, respectively. The released melatonin promoted MC3T3-E1 cell proliferation, alkaline phosphatase activity and mineralization, and shorten the time of mineralization to 11 days. Moreover, the released of melatonin increased gene expression of Run-x, alp and osteocalcin.

目錄
摘要 I
Abstract II
目錄 III
第一章、 前言 1
第二章、 文獻回顧 3
2.1 牙周組織介紹 3
2.1-1 齒槽骨 3
2.1-2 牙周韌帶 3
2.1-3 牙周病 3
2.2 骨頭 3
2.2-1 骨組織組成 3
2.2-2 骨重塑作用 4
2.3 牙周組織再生工程 4
2.3-1牙周組織再生 4
2.3-2 骨組織修復 5
2.3-3骨材料種類 5
2.4氫氧基磷灰石 (Hydroxyapatites) 6
2.4-1 氫氧基磷灰石的合成 6
2.4-2 逆微乳化技術 (Reverse-Micro-emulsion) 合成磷灰石 7
2.4-3 缺鈣型氫氧基磷灰石 (Calcium-deficient-Hydroxyapatite, CDHA) 7
2.5幾丁聚醣 7
2.5-1 幾丁聚醣結構 7
2.5-1 幾丁聚醣於骨組織工程之應用 8
2.6 褪黑激素 (Melatonin) 8
2.6-1 褪黑激素於骨組織再生之影響 9
2.6-2 褪黑激素控制釋放 9
第三章、 實驗動機與目的 10
第四章、 實驗架構 11
第五章、 實驗流程 12
第六章、 實驗材料及方法 13
6.1 實驗儀器 13
6.2 實驗藥品 14
6.3 實驗溶液配製 16
6.4 實驗方法 17
6.4-1逆微乳化法合成氫氧基磷灰石與缺鈣型氫氧基磷灰石 17
6.4-2 幾丁聚醣/磷灰石複合物製備與鑑定 17
6.4-3幾丁聚醣/磷灰石/褪黑激素複合物製備 17
6.4-4 幾丁聚醣/褪黑激素微粒物化特性分析 17
6.5 體外細胞實驗 18
6.5-1 細胞株培養 18
6.5-2 細胞相容性測試 18
6.5-3 鹼性磷酸酶活性 (ALP activity) 18
6.5-4 礦化測試 (Mineralization) 19
6.5-5 mRNA表現 19
6.6 統計分析 20
第七章、 結果與討論 21
7.1 氫氧基磷灰石與缺鈣型氫氧基磷灰石鑑定 21
7.1-1 XRD晶體結構分析 21
7.1-2 FT-IR 成份分析 21
7.1-3物理特性分析 21
7.2 幾丁聚醣/磷灰石複合物鑑定 22
7.2-1 幾丁聚醣/磷灰石複合物的自發螢光 22
7.2-2 幾丁聚醣/磷灰石複合物的FTIR成分分析 22
7.3 幾丁聚醣/磷灰石顆粒攜帶褪黑激素特性分析 23
7.3-1 幾丁聚醣/磷灰石顆粒攜帶褪黑激素之鑑定 23
7.3-2 褪黑激素包覆率測定 24
7.4 幾丁聚醣/磷灰石顆粒攜帶褪黑激素對細胞之影響 24
7.4-1 幾丁聚醣/磷灰石顆粒對成骨前驅細胞毒性試驗 24
7.4-2 褪黑激素控制釋放 25
7.4-3 褪黑激素釋放對於成骨前驅細胞增生情形 25
7.4-4 成骨前驅細胞鹼性磷酸酶活性之影響 25
7.4-5 成骨前驅細胞礦化程度 26
7.4-5 mRNA 基因表現 26
第八章、 結論 28
第九章、 參考文獻 29
第十章、 圖表 36

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