Root canal dressings for revascularization influence in vitro mineralization of apical papilla cells

Abstract Endodontic revascularization is based on cell recruitment into the necrotic root canal of immature teeth after chemical disinfection. The clinical outcome depends on the ability of surviving cells from the apical tissue to differentiate and promote hard tissue deposition inside the dentinal walls. Objective To investigate the effect of calcium hydroxide (CH) and modified triple antibiotic paste (mTAP – ciprofloxacin, metronidazole and cefaclor) on the viability and mineralization potential of apical papilla cells (APC) in vitro . Material and Methods APC cultures were kept in contact with CH or mTAP (250-1000 µg/mL) for 5 days, after which cell viability was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Next, APCs were subjected to CH or mTAP at 250 µg/mL for 5 days before inducing the differentiation assay. After 14 and 21 days, calcium deposition was assessed by the Alizarin Red S staining method, followed by elution and quantification using spectrophotometry. Data were analyzed using ANOVA followed by Tukey post hoc test. Results CH induced cell proliferation, whereas mTAP showed significant cytotoxicity at all concentrations tested. APC treated with CH demonstrated improved mineralization capacity at 14 days, while, for mTAP, significant reduction on the mineralization rate was observed for both experimental periods (14 and 21 days). Conclusion Our findings showed that CH induces cell proliferation and improves early mineralization, whereas mTAP was found cytotoxic and reduced the mineralization potential in vitro of APCs.

**摘要** 牙髓再生术（Endodontic revascularization）的实施基础为：经化学消毒后，将细胞招募至未成熟恒牙的坏死根管内。其临床预后取决于根尖组织存活细胞的分化能力，以及能否在牙本质管壁内促进硬组织沉积。
**目的** 本研究旨在体外探究氢氧化钙（calcium hydroxide, CH）与改良三联抗生素糊剂（modified triple antibiotic paste, mTAP——环丙沙星、甲硝唑、头孢克洛）对根尖乳头细胞（apical papilla cells, APC）的存活率及矿化潜能的影响。
**材料与方法** 将根尖乳头细胞培养体系分别与浓度为250~1000 μg/mL的CH或mTAP共培养5天，随后采用噻唑蓝（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐，MTT）比色法检测细胞存活率。后续实验中，将根尖乳头细胞以250 μg/mL的CH或mTAP处理5天，随后开展分化检测。分别于诱导分化第14天和第21天，采用茜素红S染色法检测钙沉积情况，随后通过洗脱结合分光光度法进行定量分析。实验数据采用单因素方差分析（ANOVA），并通过Tukey事后检验进行多重比较。
**结果** 氢氧化钙可促进细胞增殖，而改良三联抗生素糊剂在所有受试浓度下均表现出显著的细胞毒性。经氢氧化钙处理的根尖乳头细胞在诱导第14天时矿化能力提升；而经mTAP处理的细胞在两个实验周期（14天、21天）内，矿化速率均出现显著下降。
**结论** 本研究结果表明，氢氧化钙可促进细胞增殖并提升早期矿化能力；而改良三联抗生素糊剂具有细胞毒性，且会在体外环境中降低根尖乳头细胞的矿化潜能。