Cytotoxicity of Chelating Agents Used In Endodontics and Their Influence on MMPs of Cell Membranes

Abstract This study evaluated the cytotoxic effect and the ability to inhibit matrix metalloproteinases (MMP-2 and MMP-9) of 0.2% chitosan (CH) and 1% acetic acid (AA) compared with 17% ethylenediaminetetraacetic acid (EDTA). Cell viability assay was performed according to ISO 10993-5 with mouse fibroblasts (L929). The culture was exposed to 0.2% CH, 1% AA, and 17% EDTA. The chelating agents were evaluated immediately after contact with the cells and after 6 h, 12 h, and 24 h of incubation. Cell viability was analyzed using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Inhibition of the gelatinolytic activity of MMP-2 and MMP-9 was evaluated by gelatin zymography. Different concentrations of CH were evaluated: 50 mM, 5 mM, 0.5 mM, and 0.05 mM. EDTA (0.5 mM) was used as a positive control. The results demonstrated that CH and AA had an initial cytotoxic effect, which decreased after 6 h, 12 h, and 24 h, being statistically similar to EDTA (P > 0.05). Additionally, CH at concentrations of 50 mM, 5 mM, and 0.5 mM had an inhibitory effect on MMP-2 and MMP-9, similar to that of the control with EDTA. The chelating agents had no cytotoxic effects after 24 h. MMP-2 and MMP-9 were inhibited by the experimental solutions.

摘要 本研究以17%乙二胺四乙酸（ethylenediaminetetraacetic acid，EDTA）为对照，评估了0.2%壳聚糖（chitosan，CH）与1%乙酸（acetic acid，AA）的细胞毒性作用，以及其对基质金属蛋白酶（matrix metalloproteinases，MMP-2、MMP-9）的抑制活性。本实验参照ISO 10993-5标准，以小鼠成纤维细胞（L929）为模型开展细胞活力检测。将细胞分别暴露于0.2% CH、1% AA与17% EDTA中，分别于接触即刻、孵育6h、12h及24h后评估螯合剂的细胞毒性。采用噻唑蓝（3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide，MTT）法检测细胞活力。通过明胶酶谱法评估MMP-2与MMP-9的明胶水解活性抑制效果，同时设置不同浓度梯度的CH实验组：50 mM、5 mM、0.5 mM及0.05 mM，并以0.5 mM EDTA作为阳性对照。实验结果显示，CH与AA在接触即刻呈现细胞毒性，该毒性在孵育6h、12h及24h后显著降低，且与EDTA组无统计学差异（P>0.05）。此外，浓度为50 mM、5 mM及0.5 mM的CH对MMP-2与MMP-9均表现出与EDTA阳性对照相当的抑制活性。24h后，各螯合剂均未表现出细胞毒性。受试溶液均可抑制MMP-2与MMP-9的活性。