Development of a new cytotoxicity test method for dental materials

为了建立一种新的细胞毒性试验方法来评估牙科材料可能的毒性作用，本研究建立了对毒物高度敏感的培养细胞系，并建立了细胞毒性试验的定量指标。将SV-40大T抗原导入人牙髓细胞和小鼠颅骨细胞。获得了人牙髓LSC、小鼠颅骨DMCs、LMCs和VMCs 4个永生化细胞系。这些细胞系保留了牙髓或成骨细胞的一些特性。比较四种细胞毒性检测方法的敏感性。四甲基偶氮唑蓝和硝酸甘油比琼脂覆盖法和微孔滤过法显示出更高的敏感性。四种检测方法中，以四甲基偶氮唑蓝比色法重复性最高。Fura 2 AM荧光法测定JTC-12细胞内钙离子浓度([Ca^&lt；2+&gt；]i)。低浓度HgCl2对细胞存活率无明显影响，但可引起细胞内[Ca^&lt；2+&gt；]i快速而持久的升高，[Ca^&lt；2+&gt；]i可作为检测牙科材料对培养细胞初始损伤的有用指标。这些结果表明，新的细胞系和[Ca^&lt；2+]i的变化可能在未来牙科材料毒性测试的研究中有用。

In order to develop a new cytotoxicity test method for assessing possible toxic effects of dental materials, the study was performed to establish new lines of cultured cells with highly susceptible to toxicants and quantitative indexes for cytotoxicity tests.1. The human dental pulp cells and mouse calvaria cells were transfected with the SV-40 large T antigen. Four immortalized cell lines, LSC cells from human pulp and DMCs LMCs and VMCs cells from mouse calvaria were obtained. These cell lines preserved some of the properties of the dental pulp or the osteoblast.2. The susceptibility of four cytotoxicity assays were compared. The MTT and NR assays showed higher susceptibility compared to the agar overlay assay and millipore filter method. The MTT assay showed the highest reproducibility among four tests.3. The intracellular Ca ion concentration ([Ca^<2+>]i) of JTC-12 cells was measured employing Fura 2 AM fluorescence. Exposing cells to low concentrations of HgCl_2, which had no apparent effect on viability, produced a rapid and prolonged increase in [Ca^<2+>]i. The [Ca^<2+>]i was a useful index to detect the initial lesion of cultured cells exposed to dental materials.4. These results suggest that the new cell lines and the [Ca^<2+>]i change may be useful in the future research for testing the toxicity of the dental materials.