臺灣博碩士論文加值系統

化學機械法(chemo-mechanical)為一種低侵入性的牙本質齲齒治療方法，但配方中次氯酸鈉之強氧化性對第一型膠原蛋白結構易造成過度破壞，使牙本質中的膠原蛋白雖有殘留，卻未能如預期提升樹脂補綴之接著強度，因此本研究之目的在以氯化鋅溶液取代傳統之次氯酸鈉作為牙本質齲齒移除藥劑，應用化學試劑對膠原蛋白結構之物理性溶解(dissolution)機制，而非化學性之變性(denaturation)或裂解(degradation)機制，使齲齒患部之去礦化牙本質結構因失去膠原蛋白纖維之支撐而軟化，以利後續簡單機械方式移除。實驗採用由牛皮萃取出的膠原蛋白，以1 mg/ml之比例，定量添加含有醋酸、次氯酸鈉、氯化鋅之不同組成分，觀察膠原蛋白在不同的操作環境中的溶解情形，以圓二色光譜(circular dichroism)儀偵測膠原蛋白在不同溶液中二級結構之狀態，及以SDS-PAGE觀察膠原蛋白多胜肽鏈的劣化情形，實驗結果顯示在溶解試驗中以次氯酸鈉的效果最好，但經手搖晃或合併使用超音波後，醋酸及氯化鋅也可達到相同溶解效果，在結構維持方面，在經醋酸及氯化鋅完全溶解後的膠原蛋白溶液中仍可見到其三股螺旋結構與多胜肽鏈的存在，次氯酸鈉則無法見到。本研究並以人為牙本質齲齒樣本進行體外配方效力評估，結合SEM/FESEM之表面形態觀察及FTIR分析膠原蛋白官能基之變化分析，結果顯示0.1～5 wt% 氯化鋅水溶液，可提供良好之膠原蛋白溶解效果，不會影響其二級結構，FT-IR光譜中存在代表膠原蛋白結構之（1662、1637、1553 cm-1）特徵吸收峰，顯示氯化鋅水溶液具有應用於化學機械法之牙本質齲齒移除應用潛力。

Chemo-mechanical removal system is a minimally-invasive treatment for dentin caries. The main component of the system, sodium hypochlorite, however, would destroy the dentin collagen structure to the point that following bonding with resin became poor and unsatisfied. The purpose of this study is to explore the possibility of utilizing zinc chloride, replacing the sodium hypochlorite, not only to dissolve the collagen but also stabilize the protein structure during the dentin caries removal process. Several chemical agents, such as acetic acid, sodium hypochlorite and zinc chloride, were used to realize and compare their influences on type I collagen structure. The results showed sodium hypochlorite dissolved the collagen much more effectively than acetic acid and zinc chloride does in the static environment. However, there was no clear difference among these three agents with the mechanical aid, such as ultrasonic or shacking. After completely dissolution of collagen, its secondary structure and the polypeptide chain were only preserved in acetic acid and zinc chloride solution, observed by circular dichroism and SDS-PAGE. This study also established artificial demineralized dentin to evaluate the removal efficiency of zinc chloride by using SEM/FESEM and FT-IR. The results showed zinc chloride could be used to remove collagen fibers after dentin demineralization and there was no influence on collagen secondary structure, since the major FT-IR functional group absorptions (1662、1637、1553 cm-1) were still be observed. This study recommended the use of 0.1～5 wt% of zinc chloride as an effective ingredient for the chemo-mechanical dentin caries removal system.

目錄
摘要 2
Abstract 4
1-1 研究動機與重要性 11
1-2 研究目的 13
1-3 研究假說 13
第二章 文獻回顧 14
2-1 牙本質齲齒成因與定義 14
2-2 牙本質中膠原蛋白 18
2-2-1 膠原蛋白介紹 18
2-2-2 膠原蛋白的分子結構 18
2-2-3 牙本質中膠原蛋白的特性 21
2-3 目前臨床上治療齲齒的方法的介紹 24
2-4 化學機械齲齒移除方法相關研究 26
2-4-1 Carisolv®介紹與相關研究 27
2-4-2 次氯酸鈉相關研究 30
2-4-2-1 次氯酸在人體內的生理意義與生化反應 30
2-4-2-2 次氯酸對膠原蛋白之影響研究 32
2-4-2-3 次氯酸鈉在牙科上的應用 33
2-5 超音波介紹 33
2-5-1 超音波在牙科上的應用 35
第三章 材料與方法 36
3-1 實驗藥品與儀器 36
3-2 實驗方法 39
3-2-1實驗樣本選擇 39
3-2-1-1牙本質膠原蛋白的萃取 39
3-2-1-2牙本質膠原蛋白的純度分析 39
3-2-2 效力評估 43
3-2-2-1配方篩選之流程圖 43
3-2-2-2 第一型膠原蛋白溶解測試 44
3-2-2-3 第一型膠原蛋白二級結構測試 44
3-2-2-4 第一型膠原蛋白多胜肽鏈裂化觀察 45
3-2-3 In vitro測試 46
3-2-3-1 In vitro測試流程圖 46
3-2-3-2 人為去礦化樣本建立 47
3-2-3-3 實驗配方 in vitro試驗 48
第四章 結果 51
4-1 牙本質膠原蛋白定性分析 51
4-2 第一型膠原蛋白溶解測試 52
4-3 膠原蛋白二級結構觀察 53
4-4 膠原蛋白多胜肽鏈劣化觀察 54
4-5 人工去礦化樣本觀察 56
4-6 不同化學試劑之效力評估 57
第五章 討論 60
5-1 牙本質膠原蛋白定性分析 60
5-2 膠原蛋白溶解測試 60
5-4 膠原蛋白多生肽鏈裂化觀察 63
5-5 人為去礦化樣本觀察 64
5-6 不同化學試劑之效力評估 66
第六章 結論與建議 69


表目錄
表2-1 膠原蛋白的分類………………………………………………23
表2-2 Carisolv®配方組成份 …………………………………………27
表3-1 電泳膠片及相關試劑之配製…………………………………41



圖目錄
圖2.1 牙齒結構剖面圖 15
圖2.2 膠原蛋白纖維組成示意圖 20
圖2.3 膠原蛋白纖維之特殊週期性結構 20
圖2.4 牙本質中膠原蛋白網架 22
圖2.5 以氮為中心的自由基形成示意圖 32
圖2.6 超音波震盪器的構造剖面 35
圖3-1實驗配方篩選之流程圖 43
圖4-2 以CIRCULAR DICHROISM分析不同濃度的次氯酸鈉對膠原蛋白二級結構之影響 79
圖4-3 以CIRCULAR DICHROISM分析不同濃度的醋酸對膠原蛋白二級結構之影響 80
圖4-4 以CIRCULAR DICHROISM分析不同濃度的氯化鋅對膠原蛋白二級結構之影響 81
圖4-5 以CIRCULAR DICHROISM分析添加等量EDTA後不同濃度的氯化鋅對膠原蛋白二級結構之影響 82
圖4-6 利用SDS-PAGE分析不同濃度的次氯酸鈉對膠原蛋白POLYPEPTIDE CHAIN的影響 83
圖4-7 利用SDS-PAGE分析不同濃度的氯化鋅對膠原蛋白POLYPEPTIDE CHAIN的影響 84
圖4-8 利用SDS-PAGE分析添加等量2％ EDTA後不同濃度的氯化鋅對膠原蛋白POLYPEPTIDE CHAIN的影響 85
圖4-9 利用SEM觀察健康的牙本質之表面形態 86
圖4-10 利用SEM觀察健康牙本質內小管間牙本質之形態 86
圖4-11 以FESEM觀察健康牙本質斷面上膠原蛋白纖維形態 87
圖4-12 利用SEM觀察脫鈣4天後的牙本質之表面形態 87
圖4-13 利用FESEM觀察脫鈣後牙本質內膠原蛋白纖維之形態 88
圖4-14 去礦化前後牙本質樣本之FTIR圖譜 90
圖4-15 利用SEM觀察經5WT% ZNCL2處理後的脫鈣樣本之表面形態 91
圖4-16 利用FESEM觀察經5WT% ZNCL2處理後的脫鈣樣本中殘留膠原蛋白纖維之形態 91
圖4-17 利用SEM觀察經CARISOLV®處理後的脫鈣樣本之表面形態 92
圖4-18 利用FESEM觀察經CARISOLV®處理後的脫鈣樣本中殘留膠原蛋白纖維之形態 92
圖4-19 利用SEM觀察經0.95 WT% NAOCL + 0.2 M AMINO ACID混合液處理後的脫鈣樣本之表面形態 93
圖4-20 利用SEM觀察經0.95 WT% NAOCL + PBS混合液處理後的脫鈣樣本之表面形態 93
圖4-21 利用SEM觀察經0.3 WT% ACETIC ACID處理後的脫鈣樣本之表面形態 94
圖4-22 以FTIR分析5 WT% ZNCL2與CARISOLV®對脫鈣後牙本質之表面官能基影響 95
圖4-23 以FTIR分析不同氯化物對脫鈣後牙本質之表面官能基影響 96
圖4-24 以FTIR分析5 WT% ZNCL2與0.03 WT% CH3COOH對脫鈣後牙本質之表面官能基影響 97

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