牙科臨床上使用Mineral Trioxide Aggregate (MTA)材料的目的，主要是用於根尖之填充，封閉牙根尖端與牙周組織間通道，並促進組織癒合。過去對這種材料的研究顯示，大多數是有關於細胞毒性測試之探討和其他常用填充材料的比較等文獻報告，但是對於MTA材料所引起的細胞毒性進而誘發細胞變化的報告卻很少。 研究動機：MTA經實驗證實具有較佳的封閉能力和生物相容性，但長達三到四小時的硬化時間是潛在的缺點，而體外實驗是想要瞭解MTA在口腔環境中硬化的這段期間，MTA對細胞生物學上的影響以及變化。 研究目的：探討MTA材料作用於U2OS骨細胞後的生物學上的反應，於細胞外觀的變化、細胞毒性分析、DNA的裂解，再進一步探討細胞內之蛋白質訊息傳遞的路徑。 材料與方法：以浸泡在培養液中3、6、9天之MTA柱狀材料萃取液，利用MTT分析細胞活性方法，檢測其細胞的存活率；應用酵素連結免疫吸附定量法(enzyme linked-immunosorbent assay，ELISA)測定細胞的存活，並決定細胞半致死濃度(ID50)，並依據此濃度，以西方點墨法分析apoptosis的路徑，以DNA fragment assay判斷DNA複製是否受損傷及基因毒性。 結果：實驗中發現細胞毒性隨添加劑量的不同濃度(0、2.5、5、10、15、20 mg/ml)增加而增加，顯示MTA具有細胞毒性，在光學顯微鏡下可以發現有細胞凋零體(apoptosis body)的出現。依據此ID50濃度，用西方點墨法(SDS-PAGE)分析體內蛋白質，結果發現p53蛋白質增加，Erk蛋白質減少的現象，且bax蛋白質無明顯變化並推論為經由此路徑導致發生細胞凋零的結果。 結語： 本研究證明高濃度之MTA會對骨細胞具有毒性，其毒性之結果會造成細胞凋零，這一表現在細胞凋零的過程中，由可觀察到的p53蛋白質明顯的增加，而被證實。 This study investigated the biocompatibility of mineral trioxide aggregate (MTA), by culturing U2OS human osteosarcoma cells in the presence of materials. Observing cytomorphology, cytotoxicity assay and DNA fragmentation which was induced by MTA extracted liquid. The extraction were added to the human osteosarcoma cell line (U2OS) in which the survival rate was evaluated by MTT assay. The cell inhibition dose (ID50) was calculated. The mechanism of the cell death induced by MTA extracted liquid was analyzed by western blotting. The one way ANOVA analysis of variance statistic method was performed by Jump software. The results showed that MTA material was revealed degree toxicity to U2OS (P<0.05). Under microscope observation (100X) U2OS cell line had chromatin aggregate. The apoptotic bodies were found. The pathway of signal transduction were detected by western blotting and the p53 protein was expressed when cell excited. Conclusion: From the present study, cell apoptosis was observed in U2OS cells treated by MTA extract. The mechanism of cell death was through p53 protein which was mediated by ERK protein gene activation.
