中孔洞鈣氧化矽材料應用於治療牙齒敏感症之機制及細胞相容性

Abstract

牙本質敏感症的治療截至目前，仍沒有一個令人滿意的臨床結果，本團隊之前的研究顯示以明膠為模板的含碳酸鈣中孔洞二氧化矽複合材料GCMS (gelatin-templated calcium mesoporous silicate)簡稱中孔洞鈣氧化矽材料與30 %磷酸調拌，可以在10分鐘內，有效地阻塞牙本質小管達30 μm，並且不對牙齒造成酸蝕破壞，具有治療牙本質敏感症極大的潛力。然而做為一個理想的牙科臨床治療材料，除了有效性以外，生物相容性更是不可忽視的問題，並且本團隊希望進一步了解其沉澱結晶的機制，因此，本研究的目標為 (1) 評估GCMS的生物相容性，以了解其應用於臨床治療的可能性；(2) 評估GCMS/磷酸製劑及氫氧化鈣/去離子水製劑經由牙齒滲透所析出的離子濃度，以分析GCMS/磷酸製劑攜帶鈣離子及磷酸根離子穿透牙齒的情形；(3) 並使用不同的含鹼土金屬中孔洞二氧化矽複合材料，找出其中與GCMS/磷酸製劑結晶沉澱型態相似的材料，作元素分析，了解結晶沉澱的來源是否主要來自於所塗抹的材料，以期進一步了解GCMS用於治療牙本質敏感症形成結晶沉澱的機制。 第一部分，以二氧化矽孔洞材料作為擔體，加入碳酸鈣，在400℃下5小時，經由簡便之含浸法及鍛燒過程製造出GCMS，利用WST-1、LDH、ALP，分別測試GCMS、GCMS/磷酸製劑 (pH 4.5-5.0) 之材料萃取液，以及模擬臨床材料經由牙齒滲透接觸細胞的生物相容性，並以氫氧化鈣以及Seal & Protect做為其對照組。第二部分，以Transwell dentin disc model及離子層析儀確認GCMS/磷酸製劑、氫氧化鈣/去離子水製劑經由0.2 mm牙本質試片滲透所釋放的鈣離子及磷酸根離子濃度。第三部分，本團隊使用前述的含浸法製造出中孔洞鎂氧化矽材料、中孔洞鍶氧化矽材料，並且將GCMS、中孔洞鎂氧化矽材料、中孔洞鍶氧化矽材料及氫氧化鈣，與不同比例磷酸調拌，調整至相近的pH值塗抹於牙本質試片，利用電子顯微鏡觀察牙齒表面的顯微結構，並以EDS做元素分析。 細胞實驗的結果為GCMS的材料萃取液有好的生物相容性，而GCMS/磷酸製劑材料萃取液、氫氧化鈣材料萃取液、Seal & Protect材料萃取液則有較高的細胞毒性；GCMS/磷酸製劑若經由牙本質試片滲透則能得到好的生物相容性，對照組經過牙本質試片亦有好的生物相容性表現。Transwell dentin disc model及離子層析儀的結果顯示，氫氧化鈣/去離子水製劑不會經由0.2 mm的牙本質試片析出鈣離子，反觀GCMS/磷酸製劑經由0.2 mm的牙本質試片，能在5分鐘開始測得磷酸根離子，而後於10分鐘開始測得鈣離子，顯示鈣離子本身不易穿透牙本質屏障，酸性環境的磷酸根離子可能扮演前導的角色，磷酸根離子濃度提高而影響電位，對於鈣離子產生引力，使鈣離子進入牙本質小管，而後由於牙齒有緩衝的作用，pH值升高形成沉澱結晶。不同製劑塗抹於牙本質試片的SEM結果則顯示，中孔洞鎂氧化矽材料/磷酸製劑形成的結晶沉澱較不連續；中孔洞鍶氧化矽材料/磷酸製劑與中孔洞鈣氧化矽材料/磷酸製劑所形成的沉澱結晶外型則較為相似，將中孔洞鍶氧化矽材料/磷酸製劑的牙本質試片做EDS元素分析，結果顯示結晶沉澱的區域含有大量的鍶，顯示沉澱結晶的來源大部分來自於塗抹於牙齒的材料。 本研究證實GCMS材料萃取液及GCMS/磷酸製劑穿過牙本質屏障後可以得到相當好的生物相容性。酸性環境下，磷酸根離子扮演前導的角色，攜帶鈣離子穿透牙本質屏障，GCMS由於其中孔洞二氧化矽材料具有高表面積、釋放快、作用迅速等特性，快速大量釋放鈣離子，使得結晶沉澱大部分來自於所塗抹的材料，對牙齒酸蝕破壞較少，有成為牙科臨床治療牙本質敏感症的潛力。但是否只有酸性環境的磷酸根離子，或是其他帶負電離子亦能使得GCMS/磷酸製劑進入牙本質小管，這部分仍待進一步釐清。

The treatments of dentin hypersensitivity so far do not lead to satisfactory clinical outcome. Our previous study showed that gelatin-templated calcium mesoporous silicate (GCMS) with 30 % H3PO4 could efficiently occlude dentinal tubules by precipitates as deep as 30 μm in 10 minutes without eroding dentin and thus have great potential in treating dentin hypersensitivity. To be an ideal dental material in clinic, both efficiency and biocompatibility are prerequisite as well as the exploration of its mechanism. Thus, the aim of this study was (1) to evaluate the biocompatibility of GCMS for feasibility in treating dentin hypersensitivity clinically, (2) to investigate the release of ion concentration from GCMS/30 % H3PO4 through dentin disc, (3) to evaluate whether the precipitation (crystalline GCSM) is mainly constructed from our materials or incorporated from the dentin substrate using three alkaline-earth metal mesoporous silicates. First, the GCMS was synthesized using mesoporous silica as carrier, impregnated with CaCO3 precursor and calcinated at 400°C for 5 hrs. The biocompatibility was evaluated by both elution and transwell dentin disc model of test materials (GCMS GCMS/30 % H3PO4, Calcium hydroxide and Seal & Protect) using WST-1, LDH and ALP. The cells used for this study were 3T3 fibroblast cells and human dental pulp cells. Second, the release of phosphoric acid and calcium ion of the mixture of GCMS mixing with H3PO4 through dentin disc barrier (0.2 mm in thickness) was determined using ion chromatography. Calcium hydroxide mixing with deionized water served as comparison. Third, three alkaline-earth metal mesoporous silicates with different ration of 30 % H3PO4 by the same pH value was applied to dentin disc samples to examine the crystal penetration by SEM and the elements distribution by EDS. GCMS with 30 % H3PO4, calcium hydroxide and Seal & Protect showed higher cytotoxicity compared to GCMS extraction. However, GCMS with 30 % H3PO4 had great biocompatibility through dentin disc barrier as comparisons. The transwell dentin disc model and ion chromatography revealed that calcium hydroxide with deionized water would not release calcium ion through 0.2 mm dentin disc. However, as to the mixture of GCMS and 30 % H3PO4, PO4 3- could be determined in 5 minutes and then Ca2+ came in 10 minutes. The result demonstrated that calcium ion was not easily penetrate through the dentin barrier without PO4 3-. PO4 3- could be a pilot to usher Ca2+ into dentinal tubules. Thus, we could hypothesize that the concentration of PO4 3- increased to affect the electric property and attracted Ca2+ penetrate into dentinal tubules. Subsequently, the pH value elevated and led to recrystalized penetration. Under EDS analysis, the precipitation in the dentinal tubule presented great amount of strontium ion deposition which revealed mesoporous silicates contributed mostly to the precipitation/ crystallization. The present study demonstrated that the GCMS and GCMS mixing with 30 % H3PO4 through dentin disc barrier have excellent biocompatibility. In acidic condition, phosphoric acid plays a crucial factor to introduce calcium ion to penetrate into dentin disc barrier. GCMS mixing with 30 % H3PO4 could release calcium ion rapidly and massively let penetration mainly comes from our material and show better acidic resistence. GCMS with 30 % H3PO4 has feasibility for treating dentin hypersensitivity clinically.