臺灣博碩士論文加值系統

牙周炎是一種慢性發炎疾病，常造成齒槽骨破壞，嚴重時甚至造成牙齒脫落。由於口腔環境多細菌的特殊性，以及牙周骨缺損形狀不一，牙根分叉區域骨移植材料不易充填等問題，造成許多治療上的困難與不良的治療結果。近年來，原位形成凝膠系統被廣泛討論，注射型水凝膠，可攜帶藥物、生長因子及細胞，進入細微孔洞中膠凝並固定於患處協助修復，在組織工程領域極具發展潛力。第一型膠原蛋白是細胞外基質的主要成分，近年來膠原蛋白材料是體外培養細胞最常用的材料之一，但其機械性能較脆弱使其在組織工程發展上受到限制。許多研究發現，添加幾丁聚醣可提高膠原蛋白水凝膠的機械穩定性和抗壓強度。
本研究採用第一型膠原蛋白為水凝膠主材料，並於水凝膠中加入幾丁聚醣，用以提高水凝膠的機械穩定性和抗壓強度。本研究設計不同比例的膠原蛋白和幾丁聚醣組成的水凝膠，觀察其機械強度、抗酵素分解能力、吸水性與抗菌能力，並研究前骨母細胞培養於水凝膠表面其貼附、增生、分化及礦化情形，以得到最佳的水凝膠比例，開發出可注射型溫敏性膠原蛋白-幾丁聚醣複合水凝膠，評估作為骨組織再生材料的潛力。
本研究將膠原蛋白和幾丁聚醣以不同比例混合，比例分別為10：0（Col），9：1（Ch-1），8：2（Ch-2）和5：5（Ch-5）。並以FTIR分析水凝膠組成，發現膠原蛋白與幾丁聚醣混合後，其化學結構特徵相較於混合前，沒有顯著的變化。利用掃描式電子顯微鏡觀察水凝膠表面與縱切面形貌，觀察到添加幾丁聚醣會使膠原蛋白纖維聚集，在Ch-5組別中更是出現塊狀的複合結構出現。進一步測試水凝膠之各種物理特性如抗壓強度，抗酵素分解能力與吸水性，結果顯示幾丁聚醣的添加，可增加水凝膠之抗酵素分解能力與吸水性。接著將變形鏈球菌(Streptococcus mutans, SM, strain UA159)培養於水凝膠表面，偵測綠色螢光蛋白(green fluorescent protein)，分析細菌貼附於水凝膠表面情形，結果顯示Ch-5組別具有最好的抗細菌貼附能力，掃描式電子顯微鏡也觀察到類似的結果。參照ISO 10993-5規範，分析各組水凝膠之細胞毒性，發現本研究之各組水凝膠對於前骨母細胞無毒性。進一步使用alamarblue試劑，檢測前骨母細胞培養於水凝膠上之增生情形，實驗結果顯示前骨母細胞在Col和Ch-1組別中增生情形較佳。掃描式電子顯微鏡觀察細胞貼附型態，發現細胞於各組水凝膠上培養一天，細胞型態沒有顯著差異；而培養七天後，前骨母細胞在Col與Ch-1組別，形成較明顯的纖維狀型態。西方墨點法分析前骨母細胞貼附在不同水凝膠上，其FAK與p-ERK的表現，發現細胞在Ch-1組中FAK的表現有增加的趨勢，而在Col組中則有較強的磷酸化ERK1/2表現。同時也利用西方墨點法分析水凝膠對前骨母細胞表現Runx2與OCN的影響，結果顯示前骨母細胞培養在水凝膠上，其Runx2的表現有增加的趨勢，其中在Col組中，Runx2的表現顯著高於對照組。與對照組相比，水凝膠組別的OCN表現同樣有上升的趨勢，其中Ch-1組的OCN有較高的表現。接著利用Von Kossa染色法觀察水凝膠對前骨母細胞礦化的影響，結果顯示細胞在Col組中，礦物質沉積的情形較明顯。本研究透過添加幾丁聚醣改善了膠原蛋白水凝膠的物理性質與抗菌能力，在前骨母細胞分化與礦化效果上，則需要進一步的研究與探討。

Periodontitis causes irregular alveolar bony defects that are difficult to compact and stabilize particulate graft materials, resulting in prognosis of poor treatment. In recent years, an increasing number of in situ-polymerized gel systems have been applied to various biomedical engineering. These systems are injectable fluids that can fill a cavity or a defect prior to solidifying or gelling within the desired site. Type I collagen is one of the most commonly used materials of hydrogel in tissue engineering. However, its poor mechanical properties are fragile to limit its application. Previous studies have revealed that the addition of chitosan could improve the mechanical stability of collagen-base hydrogel.
In this study, the type I collagen was used as the backbone of the hydrogel. Different proportions of chitosan were added into the collagen to obtain the optimum conditions of the injectable thermosensitive hydrogel for the osteoblastic adhesion, proliferation as well as bone tissue regeneration.
Collagen and chitosan were mixed in different ratios that collagen to chitosan were 10:0(Col), 9:1(Ch-1), 8:2(Ch-2) and 5:5(Ch-5). The results of Fourier transform infrared spectroscopy (FTIR) showed no significant difference of collagen-chitosan mixed hydrogels compared to the pure ingredients. The results of mechanical properties showed that the hydrogel containing chitosan revealed higher young’s modulus than the Col group. In enzymatic degradation, Ch-5 showed higher enzymatic resistance than the other counter parts. Swelling ratio results showed that the scaffolds could bind more water as the chitosan composition increased. Antimicrobial properties of hydrogels were conducted by using Streptococus mutains with green fluorescent protein. The results of antimicrobial properties showed Ch-5 exhibited highest antibacterial property. The cytotoxicity was analyzed according to the ISO10993-5. It was found that all the hydrogels were non-toxic to cells. Furthermore, the results of proliferation detected by alamarBlue reagent showed more cells on the Col and Ch-1 groups than the other ones. In addition, the cellular morphology observed under SEM showed that good cellular spreading on Col and Ch-1 groups. Western blot analysis showed that cells on Col hydrogel revealed higher ERK phosphorylation, and higher FAK expression on Ch-1. The bone differentiation marker Runx2 and OCN were increased on Col group and Ch-1 group respectively. The Von Kossa staining results showed that the mineralization of preosteoblasts on Col group was more obvious than the other groups. In this study, the addition of chitosan improved the enzymatic resistance and swelling property. Ch-5 exhibited highest antibacterial ability. Col and Ch-1 hydrogels showed higher osteoblastic proliferation. The expression of Runx2 was higher in Col group than the control. Col revealed favorable mineralization accumulation.

誌謝 I
中文摘要 III
Abstract VI
目錄 VIII
圖目錄 XII
表目錄 XIII
一、緒論 - 1 -
1-1牙周炎與骨缺損 - 1 -
1-2原位形成凝膠系統 - 1 -
1-2膠原蛋白 (collagen) - 2 -
1-3幾丁聚醣 (chitosan) - 4 -
1-4 FAK與ERK1/2在細胞中的角色 - 5 -
1-5 Runx2與OCN在細胞分化中的角色 - 6 -
1-6實驗目的 - 7 -
二、研究方法 - 8 -
2-1純化第一型膠原蛋白(type I collagen) - 8 -
2-2膠原蛋白溶液無菌處理 - 9 -
2-3膠原蛋白溶液濃度測定(Hydroxyproline assay) - 9 -
2-4幾丁聚醣溶液製備 - 10 -
2-5膠原蛋白-幾丁聚醣複合水凝膠製備 - 10 -
2-6 水凝膠之傅立葉轉換紅外線光譜儀分析 - 11 -
2-7觀察水凝膠表面及縱切面之形貌 - 11 -
2-8水凝膠之機械性質測試 - 12 -
2-9水凝膠體外降解測試 - 13 -
2-10水凝膠吸水性測試 - 13 -
2-11培養口腔細菌Streptococcus mutans strain UA159 - 14 -
2-12水凝膠抗菌能力測試 - 14 -
2-13觀察水凝膠表面細菌貼附之形態 - 15 -
2-14細胞培養 - 15 -
2-15水凝膠萃取液之前骨母細胞毒性測試 - 16 -
2-16水凝膠對前骨母細胞增生之分析 - 17 -
2-17觀察水凝膠表面細胞之形態 - 18 -
2-18西方點墨法 - 18 -
2-18-1製備細胞蛋白萃取物 - 18 -
2-18-2蛋白濃度測定 - 19 -
2-18-3西方點墨法 - 20 -
2-19水凝膠影響細胞FAK與ERK1/2表現的影響 - 21 -
2-20水凝膠對細胞OCN及Runx2表現的影響 - 22 -
2-21水凝膠對細胞礦化之分析 - 22 -
2-22統計方法 - 24 -
三、結果 - 25 -
3-1 水凝膠之傅立葉轉換紅外線分析光譜 - 25 -
3-2 水凝膠表陎及縱切陎之形貌 - 26 -
3-3 水凝膠之機械強度 - 27 -
3-4 水凝膠之體外降解比率 - 28 -
3-5 水凝膠支架之吸水性測試 - 28 -
3-6 水凝膠對口腔細菌貼附之影響 - 29 -
3-7 口腔細菌(S. mutans)貼附於水凝膠表陎之形貌 - 29 -
3-8 水凝膠萃取液之細胞毒性測試 - 30 -
3-9 水凝膠對前骨母細胞增生之影響 - 31 -
3-10 水凝膠表陎細胞之形態 - 32 -
3-11 水凝膠對細胞 FAK 及磷酸化 ERK1/2 之影響 - 32 -
3-12 水凝膠對細胞表現 Runx2 與 OCN 之影響 - 33 -
3-13 水凝膠對細胞礦化之影響 - 34 -
四、討論 - 35 -
4-1 水凝膠成分與性質探討 - 35 -
4-2 不同水凝膠之抗菌能力與生物相容性之探討 - 36 -
4-3 不同水凝膠對細胞增生與骨分化影響之探討 - 37 -
4-5 水凝膠培養細胞方式之探討 - 40 -
4-6 總結 - 42 -
五、附圖 - 43 -
六、附表 - 63 -
參考文獻 - 69 -

圖一、水凝膠之傅立葉轉換紅外線光譜儀分析圖譜： - 44 -
圖二、水凝膠之表面形貌： - 46 -
圖三、水凝膠之縱切面形貌： - 48 -
圖四、水凝膠之楊氏係數統計圖： - 49 -
圖五、水凝膠體外降解率統計圖： - 50 -
圖六、水凝膠支架之吸水性統計圖： - 51 -
圖七、水凝膠表面貼附細菌之螢光強度統計圖： - 52 -
圖八、細菌貼附於水凝膠表面之形貌： - 54 -
圖九、水凝膠萃取液之細胞毒性測試： - 55 -
圖十、水凝膠對前骨母細胞增生之影響統計圖： - 56 -
圖十一、水凝膠表面細胞型態： - 58 -
圖十二、水凝膠對細胞表現FAK與p-ERK之影響： - 59 -
圖十三、水凝膠對細胞表現Runx2與OCN之影響： - 60 -
圖十四、水凝膠對細胞礦化之影響： - 62 -

表一、羥脯胺酸檢測體外降解速率相關試劑配方 - 63 -
表二、膠原蛋白與膠原蛋白-幾丁聚醣複合水凝膠配方 - 64 -
表三、細胞固定液 - 64 -
表四、西方點墨法相關試劑： - 65 -
表五、抗體資料： - 68 -

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