Evaluation of surface bioactivity and biocompability of sol-gel calcium silicate cements

以鈣矽酸鹽為主成分的三氧礦化聚合物（Mineral trioxide aggregate, MTA）因具有良好的封閉能力、表面生物活性與生物相容性，且在臨床應用可達到成功治療的結果，所以被廣泛應用於牙髓病的修補治療。不過MTA卻有硬化時間較長、於液態環境中會釋放重金屬物質、操作性質較差且價格昂貴等缺點。研究指出MTA的物理化學及生物性質，由主成分矽酸三鈣（tricalcium silicate, C3S）所主導，因此本研究團隊藉由改變反應r值（r = [H2O] / Si[OR]4)與催化劑用量，利用溶膠-凝膠法（so-gel technique）研發出具孔洞性之高純度矽酸三鈣，命名為so400與sn200。同時研究也證實以溶膠-凝膠法所備製之矽酸三鈣具有較短的硬化時間，以及與市售MTA材料相當的物理和化學性質，但其在表面生物活性和生物相容性部分則仍待評估。因此本研究主要目的是要評估以溶膠-凝膠法製成之矽酸三鈣（so400和sn200）與傳統高溫融熔淬火法備製之矽酸三鈣（melt-quench method C3S, mC3S）和市售產品灰色MTA（GMTA）與白色MTA（WMTA）於表面生物活性與生物相容性的表現。在表面生物活性部分，將測試材料浸泡於模擬體液（simulated body fluid, SBF）中，且以浸泡於水中的組別做對照，於不同時段下以SEM-EDS觀察其水合的表面結構形態與組成元素，並以XRD與SS-NMR進行水合產物晶相分析與分子結構鑑定，然後使用ICP-MS分析材料與環境離子交換的行為並紀錄pH值的改變。於生物相容性部分，使用大鼠的牙髓細胞株（mineralizing rat pulpal cell line, MRPC-1）做測試細胞，以直接接觸與間接接觸模型測試材料的生物相容性。SEM-EDS分析結果顯示，與在水中硬化之組別樣本相比較，三種鈣矽酸鹽與兩種市售MTA在SBF中硬化之水合產物表面會有一層含有鈣、磷元素且表面呈細針狀次級結構之球狀沉澱物的產生。XRD分析顯示，鈣矽酸鹽材料於SBF中硬化之水合產物與在水中硬化的組別相同，都可觀察到氫氧化鈣相關繞射峰出現，並且隨著反應時間增加，氫氧化鈣繞射峰的強度會逐漸減弱甚至消失。此外，當在SBF水合反應時間增長至28天時，可觀察到2θ=29.4⁰和2θ=25.9⁰、31.8⁰的繞射峰出現的位置與強度有所變化，推測可能與碳酸鈣與氫氧機磷灰石的產生有關。在材料與環境交互作用方面，無論在水中或是SBF的組別，鈣離子自材料釋放的行為大致相同，但SBF中各組別所測得之鈣離子濃度皆約為水中的1.5~2倍。這可能是因為水中硬化組別pH值於反應初期會急遽升高至pH11-12，不利於鈣離子釋放；而SBF的組別則因為其緩衝效應造成pH值隨反應時間增加而上升的效應較緩慢（pH 8.5~10），而較有利於鈣離子的釋放。不同於水中硬化組別於反應一天後均未檢出矽離子釋放的結果，在SBF中硬化的組別皆可觀察到有矽離子的釋放，於反應1天時濃度升高之後逐漸下降，不過在WMTA組別則是於第1天上升後則未再下降。此外，在SBF中硬化的組別，均可觀察到溶液中之磷離子在反應1-4天內即大幅降低至無法檢測的程度，推測材料表面生物活性層的生成在材料水合初期即開始進行。而以SS-NMR檢測SBF中硬化水合產物之分子鑑定與結構分析結果發現， 31P MAS光譜於2.6ppm出現代表氫氧機磷灰石（hydroxyapatite, HAP）的訊號，證實各組鈣矽酸鹽於SBF中生成之表面沉澱物為氫氧基磷灰石。而在生物相容性方面，mC3S與so400雖然在4小時細胞貼附表現稍差，但24小時後所有材料均呈現良好的細胞貼附結果；同時以材料與細胞共養之間接接觸模型進行之細胞存活率測試結果顯示，除WMTA組別有顯著略低於控制組的情形，其他組別的細胞存活率與控制組沒有顯著差異。總結來說，以溶膠-凝膠法製備的矽酸三鈣（so400, sn200）與市售材料MTA相同，在模擬體液環境中可於材料表面生成氫氧基磷灰石生物活性層，具有表面生物活性特質，同時亦具備良好的生物相容性表現。若進一步考量其較短的硬化時間以及與MTA相當的物理性質，以溶膠-凝膠法製備的sn200相較於MTA於牙髓病修補治療的應用上，為一極具潛力的材料。Mineral trioxide aggregate (MTA), a type of calcium silicate cement, is widely used in endodontic repair therapies due to its good sealing ability, surface bioactivity and biocompability. However, several disadvantages of MTA have been reported such as long setting time, difficulty in handling, expensive and the releasing of heavy metal ions. Tricalcium silicate (C3S), the main constituent of MTA, has been proved to govern the properties of MTA including the physicochemical, biocompatible and bioactive properties. Therefore, our research team developed new tricalcium silicate cements with high purity, porous structure, and short setting time, named sn200 and so400, via sol-gel process by modification of mixing order (r ratio) and concentration of catalyst. Moreover, their physicochemical properties were tested and confirmed to be comparable with MTA. However, their surface bioactivity and biocompability were unknown and needed further evaluation. The purpose of this study is to evaluate the surface bioactivity and biocompability of sol-gel tricalcium silicate cements (so400 and sn200) in comparison with melt-quench method C3S (mC3S), commercial gray MTA (GMTA) and white MTA (WMTA). To study the surface bioactivity, the test materials were immersed in simulated body fluid (SBF) and distilled water, respectively and the behaviors of bioactive layer formation of test materials were analyzed via SEM-EDS, XRD and SS-NMR. The ions exchange between material and environment were measured by ICP-MS and the environmental pH was recorded. To study the biocompability, mineralizing rat pulpal cell line (MRPC-1) was used to evaluate the cell adhesion and the cytotoxicity of tested materials using the direct and indirect contact models, respectively. Utilizing SEM-EDS, we found the calcium-phosphate contenting precipitates in granular appearance with acicular surface formed on the surface of all test materials of SBF group, in comparison to those samples of distilled water group. After hydration, the peaks corresponding to Ca(OH)2 were identified by XRD either in SBF or distilled water groups and those peaks decreased in intensity or even disappeared as immersion time increased. When hydrated in SBF for 28 days, some small and broad peaks at 2θ=29.4⁰ and 2θ=25.9⁰, 31.8⁰ were observed in XRD pattern, which may corresponding to CaCO3 and poor crystallized hydroxyapatite (HAP). Different to the environmental pH increased rapidly to 11~12 in the first day of hydration in distilled water, lower pH value (8.5~10) was found in SBF group, which is favorable for the calcium ions releasing into environment. Consistently, the Ca+2 concentration of SBF group was 1.5~2 times of distilled water group although both groups demonstrated the similar trend of the change of Ca+2 ions as the time increased. In addition, we found the silicon ions increased rapidly in the first day of hydration and then decreased gradually as the time increased in all materials of SBF group, except WMTA. Meanwhile, no silicon ions were detected in distilled water group. For all of test materials, the phosphate ions of SBF decreased rapidly and became undetectable within 1~4 days, which may imply the formation of bioactive layer starts in the early stage of hydration. Besides, the signal at 2.6ppm was obtained in all test materials hydrated in SBF using 31P MAS SS-NMR analysis, which confirmed the HAP formed on the surface. Furthermore, good cell adhered to all test materials was observed in 24-hour culture, although the scores for cell adhered to mC3S and so400 were slightly lower in 4-hour culture. Using Transwell indirect contact model, we found there were no significant difference of cell viabilities among the test materials, but WMTA demonstrated the significant lower cell viability than that of control group without material treatment. Based on these findings, we proved the sol-gel tricalcium silicate cements (so400, sn200) have surface bioactive property with a HAP layer formed on their surface and the good biocompatibility in vitro. Moreover, take the advantages of its short setting time and the comparable physical properties of MTA, the sol-gel C3S of sn200 is considered as a potential material in endodontic applications