Dose- and time-dependent effects of taxifolin on viability and mineralization markers of osteoblast-like cells

Abstract The current study evaluated the effects of taxifolin treatments on the viability of osteoblast-like cells, and on the expression of early mineralization markers, as part of the ongoing search for new endodontic materials able to induce periapical healing without causing cytotoxicity. Saos-2 osteoblast-like cells were exposed to different concentrations of taxifolin (5 and 10 µM), applied as pretreatments either for 24h and 72h, or continuously throughout the experimental protocol. Cell viability using the methylthiazole tetrazolium (MTT) assay, alkaline phosphatase activity using thymolphthalein monophosphate assays, deposition of mineralized nodules using alizarin red staining, and expression of ALP and COL-1 by qPCR were determined after 6 and 13 days of treatment. The data were analyzed statistically (p<0.05). Taxifolin was not cytotoxic in the concentrations tested. Pretreatments with taxifolin for 24h and 72h at 10 µM stimulated ALP activity, and increased mineralized nodule deposition by Saos-2 cells. Continuous treatment with taxifolin was not effective in stimulating ALP activity and mineralization. ALP and COL-1 gene expression increased with taxifolin pretreatments, since the highest mRNA levels were observed after 72h of pretreatment with taxifolin at 10 µM on day 13. In conclusion, taxifolin was cytocompatible, and induced mineralization markers when applied for short periods in osteoblast-like cell culture.

摘要 本研究旨在评估花旗松素（taxifolin）处理对成骨样细胞（osteoblast-like cells）活力及早期矿化标志物表达的影响，作为当前研发可诱导根尖周愈合（periapical healing）且无细胞毒性（cytotoxicity）的新型牙髓治疗材料（endodontic materials）探索工作的一部分。将Saos-2成骨样细胞暴露于5 μM、10 μM两种浓度的花旗松素中，分别设置24 h、72 h预处理组及全程持续给药实验组。分别于处理第6天和第13天，采用四甲基偶氮唑盐（methylthiazole tetrazolium，MTT）法检测细胞活力，麝香草酚酞单磷酸法检测碱性磷酸酶（alkaline phosphatase，ALP）活性，茜素红染色法检测矿化结节沉积情况，并通过实时荧光定量聚合酶链反应（qPCR）检测ALP与I型胶原（Collagen Type I，COL-1）的基因表达水平。所有数据均采用统计学方法分析（p<0.05为差异具有统计学意义）。实验结果表明，受试浓度范围内的花旗松素均未表现出细胞毒性。10 μM浓度下24 h、72 h的花旗松素预处理可显著提升Saos-2细胞的碱性磷酸酶活性，并促进矿化结节沉积；而全程持续给予花旗松素则未对碱性磷酸酶活性及矿化过程产生有效刺激。花旗松素预处理可上调ALP与COL-1的基因表达，其中以10 μM浓度预处理72 h后第13天的mRNA表达水平为最高。综上，花旗松素具备细胞相容性，在成骨样细胞培养体系中短期给药可诱导矿化标志物的表达。