臺灣博碩士論文加值系統

活髓治療為面對牙根尚未完全發育完成及開放性牙根尖之理想的治療方式，成功的覆髓治療可以保存牙髓的活性，維持其正常生理功能。Mineral trioxideaggregate (MTA) 為一具有良好封閉能力與優異生物相容性之材料，目前實驗已證實MTA 對牙骨質與牙本質均有誘導再生之能力，應用於根尖逆充填、根管穿孔修補、及根尖成形術與活髓治療方面均有不錯的效果，但是操作材料需高度技巧與熟練度，且硬化時間太長為其缺點。目前有兩種不同組成的MTA，新一代MTA 調整成分組成之後不含鐵元素，因而呈現白色外觀解決美觀上的需求。之前研究顯示材料之優異性質均為灰色MTA 的表現，雖然這灰色與白色MTA主要組成成份均為卜特蘭水泥（Portland Cement），但組成成分的差異是否影響材料的基本性質與生物相容性？這方面的研究並不多，且說法迥異。
本研究第一部份目的為探討不同組成比例的MTA 材料性質及水合反應的差異。藉由掃瞄式電子電子顯微鏡（SEM）、X 光晶體繞射分析（XRD）、能量散射光譜分析儀(Energy dispersive spectrometer. EDS)及微硬度測試的研究方法，分析不同組成之MTA 在不同時間點所表現表面形態、結晶性及晶體結構與組成差
異，以瞭解MTA 水合反應使否因組成成分改變而有所差異，同時以全自動生化測定機及酸鹼值測量儀分析七天之內兩種材料所釋放的鈣離子與酸鹼值。SEM表面觀察與XRD 分析結果顯示白色MTA 水合反應較快，在第三天其晶體形態與結晶性均趨穩定，灰色MTA 需到第七天方能呈現水合反應穩定之晶相。而EDX 能譜分析顯示兩種MTA 的材料表面晶體組成相似，晶體部分組成元素為鈣與氧，應為氫氧化鈣之結晶，而基質部分含有鈣、矽及氧，為C-S-H。微硬度分
析結果顯示初期灰色MTA 的硬度較高，但是七天之後兩者的硬度並無統計上之差異。在鈣離子釋放量上，七天之內灰色MTA 所釋放的鈣離子濃度均大於白色MTA。而酸鹼值方面兩種材料均維持一定的高鹼性，初始酸鹼值為11.35 至11.38，3 小時之後兩者酸鹼值可以維持在11.88 至12.1 之間，此酸鹼值可以穩定維持72 小時。
本實驗第二部分之目的為探討兩種不同組成成分之MTA 的生物相容性與材料對牙髓纖維母細胞的影響，並分析成分C3S/C2S、C3A、C4AF 的細胞毒性，藉由個別成分之比較瞭解MTA 的生物相容性。
以ISO 測試細胞毒性標準流程進行實驗，結果顯示C3A 的細胞毒性略高於其他組成成分，兩種MTA 的細胞毒性均很溫和，為生物相容性佳之材料，但白色MTA 有略高細胞毒性的趨勢。
進一步以組織細胞功能性之表現釐清材料對細胞的影響之差異，在牙髓纖維母細胞的ALP 染色分析部分結果顯示灰色MTA 對細胞表現ALP 活性的影響與控制組相近，而白色 MTA 的ALP 活性表現較少，C3S/C2S 組牙髓纖維母細胞仍維持部分ALP 活性，但 C3A、C4AF 幾乎沒有ALP 之表現，表示細胞的鈣化能力受到影響。細胞衰老實驗以β-galactocidase 為指標，結果顯示之兩種MTA均不會引發細胞衰老之表現。
綜合實驗結果所得結論為：MTA 主成分中以C3S/C2S 的生物性質較理想，C3A 細胞毒性略大、C4AF 雖無細胞毒性但會影響細胞顯型（phenotype）的表現。不同組成成分比例MTA 中以灰色MTA 對細胞生物相容性較理想，而白色MTA材料硬化較快，增加操作上的方便，但是生物相容性不如灰色MTA 好。本實驗發現MTA 生物相容性與材料性質的確因成分比例不同而有所改變，此部分的研究之結果可為日後研發改良新的活髓治療生醫材料之參考，保留生物相容性佳的成分，調整材料操作性質，應可有更為理想之覆髓生醫材料的出
現。

Mineral Trioxide Aggregate (MTA) has been developed at Loma Linda University to seal off the pathways of communication between the root canal system and the external surface of the tooth. A series of experiments have been conducted to assess the suitability of MTA, these experiments began with physical properties of the material and biocompatibility studies, followed by clinical studies. The results showed MTA has good sealing ability and excellent biocompatibility. MTA has been applied on vital pulp therapy because of these properties. The results favored the usage of MTA than calcium hydroxide and others capping agents.
MTA without FeO is made of modification of MTA, a fluxing agent is used to remove the ferrite for production of the tooth-colored powders. Although the major components are similar, there were no sufficient studies supported that MTA without FeO offers the same properties of MTA.
In part 1, we compared the difference of material properties between Gray and White MTA. MTA were analyzed by SEM、XRD、 EDX and Microhardness test in various hydration period. Concentrations of released calcium ion and pH value were also determined every 24 hours for 7 days. In part II, biocompatibility of these two materials were analyzed by MTT assay and Agarose overlay test, these tests were followed the standard procedures in ISO 10993 & 7405. We also analyzed the cytotoxicity of major components in MTA: C3S/C2S、C3A、C4AF. Phenotype of Pulp fibroblast was determined by ALP staining assay and concentration of β-galactocidase.
Results of part 1 showed hydration of White MTA could be stable in 3 days, and Gray MTA took 7 days. This referred White MTA set faster than Gray MTA. White MTA showed higher hardness in first 7 days but after one week there were no difference between both materials. Gray MTA released more calcium ion and both materials could keep in high pH. Biocompatibility of Gray MTA was superior to White MTA. And C3A showed moderate cytotoxicity. Although C3A、C4AF is mild cytotoxicity but both components can’t keep normal phenotype of pulp fibroblast. Only C3S/C2S is mild cytotoxicity, maintained ALP activity of cell and keep cell in normal cell cycle.
Conclusion: Different constitution of MTA results in differences of material properties and biocompatibility. Although White MTA offered faster setting time, better biocompatibility of Gray MTA seems to be more important.

謝誌 i
中文摘要 ii
英文摘要 Abstract iv
目錄 vi
第一章 前言 1
第二章 文獻回顧 3
2.1 活髓治療與根尖成形術之臨床重要性 3
2.2 活髓治療之沿革與材料之需求 4
2.2.1 活髓治療 4
2.2.2 應用於活髓治療之研究 5
2.3 Mineral Trioxide Aggregate（MTA）材料背景 8
2.4 MTA 生物相容性與活髓治療之研究 10
2.5 評估牙科材料生物相容性 11
MTT assay 13
ALP staining assay 14
β-galactocidase assay 15
第三章 動機與目的 16
第四章 材料與方法 18
4.1.1 掃瞄式電子顯微鏡觀察實驗（SEM） 18
4.1.2 X 光晶體繞射分析實驗（XRD） 19
4.1.3 能量散射光譜分析儀實驗（EDX） 20
4.1.4 微硬度測試實驗（Vicker''s hardness test) 21
4.1.5 材料釋放鈣離子量之測量 22
4.1.6 材料酸鹼值測量 22
4.2.1 ISO 生物相容性測試 22
4.2.1.1 L929 細胞株培養與培養液之配製 22
4.2.1.2 實驗材料製備 23
4.2.1.3 細胞毒性 - MTT 測試 23
4.2.1.4 細胞毒性 - Agar overlay assay 24
4.2.2 牙髓纖維母細胞細胞細胞顯型與細胞毒性分析 24
4.2.2.1 牙髓纖維母細胞培養 24
4.2.2.2 細胞毒性 - MTT 測試 25
4.2.2.3 ALP staining assay 25
4.2.2.4 β-galactocidase assay 26
第五章 實驗結果 27
5.1 掃瞄式電子顯微鏡觀察實驗（SEM）結果 27
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5.1.1 未水合材料之觀察 27
5.1.2 水合三小時試片 27
5.1.3 水合一天試片 27
5.1.4 水合三天試片 28
5.1.5 水合七天試片 28
5.2 X 光晶體繞射分析試驗結果 28
5.3 微硬度測試實驗（Vicker''s hardness test)結果 29
5.4 材料釋放鈣離子量之測量 30
5.5 材料酸鹼值測量 30
5.6 能量散射光譜分析儀實驗（EDX）結果 30
5.7 ISO 細胞毒性 - MTT 測試結果 30
5.8 ISO 細胞毒性 - Agar overlay assay 結果 31
5.9 ALP staining assay 結果 31
5.10. β-galactocidase assay 32
第六章 討論 33
第七章 結論與未來研究方向 41
圖表
圖4-1 掃瞄式電子顯微鏡：Topcon ABT-60 43
圖4-2 X 光晶體繞射分析儀: Rigaku Geigerflex 43
圖4-3 能量散射光譜分析儀（EDX） 44
圖4-4 Automet® 2 44
圖4-5 a, b 微硬度測試機：SHIMADZU HMV-2 45
圖4-6 TOSHIBA TBA-200FR 全自動生化分析儀 46
圖4-7 電子酸鹼值測量儀：Microcomputer pH/mV/6171 46
圖5-1 a 灰色MTA 粉末材料 SEM 觀察圖片 47
圖5-1 b 白色MTA 粉末材料 SEM 觀察圖片 47
圖5-2 a 灰色MTA 水合三小時試片 48
圖5-2 b 白色MTA 水合三小時試片 48
圖5-2 c 灰色MTA 水合三小時試片 49
圖5-2 d 白色MTA 水合三小時試片 49
圖5-2 e 白色MTA 水合三小時試片（高倍） 50
圖5-3 a 灰色MTA 水合一天試片 51
圖5-4 b 白色MTA 水合一天試片 51
圖5-5 a 灰色MTA 水合三天試片 52
圖5-5 b 白色MTA 水合三天試片 53
圖5-5 a 灰色MTA 水合七天試片 54
圖5-5 b 白色MTA 水合七天試片 55
圖5-6 a 不同時間點灰色MTA 之XRD 圖譜 56
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圖5-6 b 不同時間點白色MTA 之XRD 圖譜 57
圖5-7 細胞型態記錄 (L929) 58
圖5-8 細胞型態記錄 (Pulp fibroblast) 59
圖5-9 Agarose overlay test 60
圖5-9 ALP 定性 62
表2-1 MTA 組成比例 63
表2-2 ISO 10993 64
表2-3 ISO 7405 65
表2-4 標準萃取時間與比例 66
表2-5 Agarose overlay test 標準評估表 67
表4-1 MTT 測試流程表 68
表4-2 ALP 測試流程表 69
表4-3 a β galactosidase staining 標準曲線及回歸程式 70
表4-4 b Total protein stain 標準曲線及回歸程式 70
表5-1 a 不同時間點灰色MTA 硬度測量結果 71
表5-1 b 不同時間點白色MTA 硬度測量結果 72
表5-2 a 灰色MTA 於不同時間點硬度的統計分析結果 73
表5-2 b 白色MTA 於不同時間點硬度的統計分析結果 74
表5-3 不同時間點硬度變化 75
表5-4 每一時間點硬度兩種材料t test result: 75
表5-5 鈣離子釋放量 76
表5-6 pH 值 76
表5-7 a,b EDX 分析材料表面元素 1 天 77
表5-8 a,b EDX 分析材料表面元素 7 天 78
表5-9 MTT 結果 L929 cell line 79
表5-10 MTT 結果 Pulp fibroblast 79
表5-11 Agarose overlay test 結果 80
表5-12 β-galactocidase stain 81
參考書目 82

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