Faculty of Dentistry, University of Granada Colegio Máximo de Cartuja s/n, Granada 18071, Spain.
Department of Dermatology, Stomatology, Radiology and Physical Medicine, Morales Meseguer Hospital, Biomedical Research Institute (IMIB), Regional Campus of International Excellence "Campus Mare Nostrum", Faculty of Medicine, University of Murcia, Murcia 30008, Spain.
Dent Mater. 2024 Oct;40(10):1591-1601. doi: 10.1016/j.dental.2024.07.012. Epub 2024 Jul 26.
Drug-loaded non-resorbable polymeric nanoparticles (NPs) are proposed as an adjunctive treatment for pulp regenerative strategies. The present in vitro investigation aimed to evaluate the effectiveness of tideglusib-doped nanoparticles (TDg-NPs) in mitigating the adverse effects of bacterial lipopolysaccharide endotoxin (LPS) on the viability, morphology, migration, differentiation and mineralization potential of human dental pulp stem cells (hDPSCs).
Cell viability, proliferation, and differentiation were assessed using a MTT assay, cell migration evaluation, cell cytoskeleton staining analysis, Alizarin Red S staining and expression of the odontogenic related genes by a real-time quantitative polymerase chain reaction (RT-qPCR) were also performed. Cells were tested both with and without stimulation with LPS at various time points. One-way ANOVA and Tukey's test were employed for statistical analysis (p < 0.05).
Adequate cell viability was encountered in all groups and at every tested time point (24, 48, 72 and 168 h), without differences among the groups (p > 0.05). The analysis of cell cytoskeleton showed nuclear alteration in cultures with undoped NPs after LPS stimulation. These cells exhibited an in blue diffuse and multifocal appearance. Some nuclei looked fragmented and condensed. hDPSCs after LPS stimulation but in the presence of TDg-NPs exhibited less nuclei changes. LPS induced down-regulation of Alkaline phosphatase, Osteonectin and Collagen1 gene markers, after 21d. LPS half-reduced the cells production of calcium deposits in all groups (p < 0.05), except in the group with TDg-NPs (decrease about 10 %).
LPS induced lower mineral deposition and cytoskeletal disorganization in hDPSCs. These effects were counteracted by TDg-NPs, enhancing osteogenic differentiation and mineralization.
载药不可吸收聚合物纳米颗粒（NPs）被提议作为牙髓再生策略的辅助治疗。本体外研究旨在评估他克莫司掺杂纳米颗粒（TDg-NPs）对减轻细菌脂多糖内毒素（LPS）对人牙髓干细胞（hDPSCs）活力、形态、迁移、分化和矿化潜能的不良影响的有效性。
通过 MTT 检测评估细胞活力、增殖和分化，通过细胞迁移评估、细胞骨架染色分析、茜素红 S 染色和实时定量聚合酶链反应（RT-qPCR）检测牙源性相关基因的表达来评估细胞分化和矿化潜能。还在不同时间点用 LPS 刺激和不刺激细胞来测试这些细胞。采用单因素方差分析和 Tukey 检验进行统计学分析（p<0.05）。
所有组在所有测试时间点（24、48、72 和 168 小时）都有足够的细胞活力，且组间无差异（p>0.05）。细胞骨架分析显示，LPS 刺激后未掺杂 NPs 的培养物中细胞核发生改变。这些细胞呈现出蓝色弥散和多灶性外观。一些核看起来碎片化和浓缩。LPS 刺激后但存在 TDg-NPs 的 hDPSCs 显示出较少的核变化。LPS 诱导碱性磷酸酶、骨粘连蛋白和 Collagen1 基因标志物在 21d 后下调。LPS 使所有组的细胞钙沉积产量减少一半（p<0.05），但 TDg-NPs 组除外（减少约 10%）。
LPS 诱导 hDPSCs 中矿化减少和细胞骨架紊乱。TDg-NPs 可拮抗这些作用，增强成骨分化和矿化。
