臺灣博碩士論文加值系統

覆髓治療為牙齒深度窩洞之微創治療術式，為能防止牙髓受到外界的刺激而受到損傷，其上的復形物常常會用的光聚合複合樹脂，使缺損的牙冠恢復其型態及維持牙齒功能。然而複合樹脂黏著系統，含有一些能對牙髓細胞造成損害的單體，如TEGDMA、HEMA、BisGMA等，也有機會在治療後通過牙本質小管進入牙髓腔，造成牙髓組織發炎壞死的情形。本研究計畫目的為探討含奈米鈣介孔矽質泡材(CMCF)作為窩洞基底覆髓生醫材料，能在牙本質小管中形成仿生結晶，封閉牙本質小管，達到保護牙髓組織的功能，且期望此材料也能進一步誘導受損部位牙本質母細胞分化為牙本質組織，進一步增加覆髓治療的成功率。
本研究分為三部分，第一部分為材料性質與牙本質小管內結晶形成探討，透過牙本質模型以掃描式電子顯微鏡，分析小管結晶/牙本質黏著劑/生醫材料之交互作用機轉，並以紫外光-可見光光譜分析儀，及高效液相層析儀分析牙本質黏著劑與其中單體對於受含奈米鈣介孔矽質泡材處理後的牙本質穿透能力的影響。第二部分為體外細胞相容性測試以及體外深度窩洞模型，首先確認含奈米鈣介孔矽質泡材做為生醫材料應具備良好之細胞相容性，而後以牙本質薄片模擬窩洞覆髓治療時，透過本材料形成仿生結晶封閉牙本質小管，保護牙髓細胞免受複合樹脂及牙本質黏著劑內的單體所損傷。第三部分為材料對牙髓細胞再礦化能力的影響。
實驗結果顯示，生醫材料含奈米鈣介孔矽質泡材，與30%磷酸作用後，透過掃描式電子顯微鏡觀察，能在牙本質小管內形成與牙齒結構類似之磷酸二氫鈣結晶。在生物相容性測試方面，顯示對細胞無明顯毒性，且低於牙本質黏著劑對細胞造成的毒性；透過牙本質薄片模擬臨床深度窩洞覆髓治療，含奈米鈣介孔矽質泡材可藉由形成仿生結晶阻擋複合樹脂及牙本質黏著劑中毒性單體影響牙髓細胞。為了進一步了解含奈米鈣介孔矽質泡材誘導細胞再礦化能力，透過鹼性磷酸酶定性、定量試驗以及DSPP、DMP-1相關蛋白表現量分析，也發現本材料能誘導細胞分泌較多鹼性磷酸酶以及牙本質分化與礦化相關蛋白，推斷其具誘導細胞再礦化之能力。
綜合以上結論，顯示本次實驗研發之含奈米鈣介孔矽質泡材作為生醫材料，用於臨床深度窩洞間接覆髓治療時，具備良好生物相容性，形成仿生結晶保護層，減少牙本質黏著劑滲透，保護牙髓組織以及促進牙髓組織再礦化，在臨床應用上有相當潛力。

Pulp capping is applied to a deep tooth cavity for keeping the pulp vitality. The following light cured composite resin is always applied to recovery the original morphology and chewing function. However, it containing toxic monomers may permeate through dentinal tubules to cause pulp damage. The purpose of this research project is to develop nano-calcium encapsulated mesocellular siliceous foams (denoted as nCa-MCFs) as a capping biomaterial that can form biomimetic crystallization in dentinal tubule when nCa-MCFs works with phosphoric acid (denoted as nCa-MCFs-HP). The biomimetic crystal layer would serve as a protective barrier protect pulp tissues from the toxic monomer of dentin bonding agents, and further induces the differentiation of odontoblast.
This study carries out three parts—Part Ι: To investigate the properties of nCa-MCFs-HP material and the formation of biomimetic crystallization in dentin tubule. We would analyze the interaction of tubule crystallization and biomaterials by using scanning electron microscope. The elution of dentin bonding agents and monomers through 0.2 mm dentin disc after nCa-MCFs-HP treatment would analyzed by UV-Vis spectroscopy and HPLC. Part ΙΙ: To evaluate the biocompatibility of nCa-MCFs-HP biomaterials, and to establish the in vitro deep dentin disc model. Part ΙΙΙ: To evaluate the pulp cell mineralization after nCa-MCFs-HP treatment.
The results revealed that the nCa-MCFs-HP can form calcium phosphates crystallization in tubule. The biocompatibility of nCa-MCFs-HP was also confirmed by WST-1 and LDH test, which indicates no significant cytotoxicity, and lower cytotoxicity than the group of dentin bonding agents. The simulated deep cavity treating with nCa-MCFs-HP material can form a biomimetic crystalline barrier which may protect pulp cell from toxicity monomers released from composite resins and dentin bonding agents. ALP staining assay, ALP quantitative assay, and mineralization-related protein – DMP-1, DSPP expression further indicated the nCa-MCFs-HP treated dentin has the great help of the induction of pulp cell mineralization.
Based on the given situation, we concluded that the nCa-MCFs-HP biomaterial has good biocompatibility, and the forming biomimetic crystallization protect layer could reduce the toxic monomers permeation, protect pulp tissues, and promote mineralization as a pulp capping material.

摘要 i
Abstract iii
目錄 v
第一章 文獻回顧 1
1.1保存牙髓之重要性 1
1.1.1牙髓組織之發育與構造 1
1.1.2 保留牙髓於臨床治療的意義 2
1.2 覆髓治療 2
1.3現今常用覆髓治療材料 3
1.3.1氫氧化鈣 (Calcium hydroxide) 3
1.3.2三氧化礦物(Mineral Trioxide Aggregate ,MTA) 4
1.3.3 Biodentine TM 4
1.4牙本質黏著劑之退變與生物毒性 5
1.5牙本質小管 6
1.6中孔洞矽質生醫材料與仿生結晶 7
1.6.1中孔洞材料的發展 7
1.6.2本實驗室研究中孔洞材料用於牙本質疾病之沿革 7
1.7牙髓組織再礦化 8
1.7.1牙本質母細胞分化 8
1.7.2Dentin sialophosphoprotein (DSPP) 8
1.7.3 Dentin Matrix Protein 1 (DMP1) 9
第二章 實驗動機與目的 10
第三章 材料與方法 11
3.1 實驗材料之製備與性質分析 11
3.1.1含奈米鈣介孔矽質泡材(CMCF)之合成與性質最佳化 11
3.1.2 X射線繞射分析(X-Ray Diffractometer, XRD) 12
3.2 材料在牙本質小管結晶與牙本質黏著劑交互作用探討 12
3.2.1 牙本質試片製作 12
3.2.2掃描式電子顯微鏡觀察 13
3.2.3 穿透式電子顯微鏡觀察 13
3.2.4 牙本質黏著劑內單體之釋放 14
3.2.4.1 紫外光-可見光分光光度分析儀 (UV-visible Spectroscopy )檢測 牙本質黏著劑釋出 14
3.2.4.2 高效液相層析儀(HPLC)檢測HEMA之釋放 15
3.3 體外生物相容性測試暨模擬深度窩洞之牙本質模型探討 15
3.3.1 材料萃取液製備 15
3.3.2 牙本質黏著劑萃取液製備 16
3.3.3人類牙髓細胞之初級培養 16
3.3.4 WST-1 細胞存活率測試 (WST-1 cell viability assay) 17
3.3.5 LDH細胞毒性測試 (Lactate Dehydrogenase Cytotoxicity Assay) 18
3.3.6牙本質薄片模型之細胞毒性檢測 19
3.4 體外細胞礦化行為分析 21
3.4.1鹼性磷酸酶定性染色分析 (ALP Staining Assay) 21
3.4.2 鹼性磷酸酶定量 (ALP Activity Quantitative Assay) 22
3.4.3 礦化相關蛋白表現量分析 24
第四章 實驗結果 29
4.1 材料性質分析 29
4.1.1 實驗材料之X射線繞射分析 29
4.2 材料在牙本質小管結晶與牙本質黏著劑交互作用探討 29
4.2.1 掃描式電子顯微鏡觀察 29
4.2.2 穿透式電子顯微鏡觀察 30
4.3 牙本質黏著劑內單體之釋放 30
4.3.1紫外光-可見光分光光度分析儀 (UV-visible Spectroscopy )檢測牙本質黏著劑釋出 30
4.3.2高效液相層析儀(HPLC)檢測HEMA之釋放 31
4.4 體外生物相容性測試暨深度窩洞牙本質模型探討 31
4.4.1材料萃取液與牙本質黏著劑萃取液之 WST-1 細胞存活率測試 31
4.4.2材料萃取液與牙本質黏著劑萃取液之LDH細胞毒性測試 31
4.4.3牙本質薄片模型之LDH細胞毒性檢測 32
4.5體外細胞礦化行為分析 32
4.5.1 材料萃取液與牙本質黏著劑萃取液之鹼性磷酸酶定性染色分析 32
4.5.2 材料萃取液與牙本質黏著劑萃取液之鹼性磷酸酶定量 33
4.5.3 材料萃取液對牙髓細胞礦化相關蛋白表現量分析 33
4.5.4 牙本質薄片模型之鹼性磷酸酶定量 33
4.5.5 牙本質薄片模型對牙髓細胞礦化相關蛋白表現量分析 33
4.6統計方法 34
第五章 討論 35
5.1 CMCF-HP鈣磷莫耳比1:1.5的選擇 35
5.2 濃度30%磷酸製劑選擇 35
5.3 CMCF-HP在牙本質小管內形成結晶之討論 35
5.4 UV-Vis與HPLC檢測CMCF對於減少牙本質黏著劑與其中HEMA滲透之討論 36
5.5牙本質黏著劑萃取液與CMCF-HP材料萃取液之生物相容性探討 36
5.6 牙本質薄片模型之生物相容性的意義 37
5.7牙本質黏著劑FL-Bond ΙΙ 之選擇與細胞毒性 37
5.8 CMCF-HP材料對細胞礦化行為的影響 38
第六章 結論 40
第七章 未來研究方向 41
參考資料 42

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第四章
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表4-3………………………………………………………………………………….67

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