Department of Conservative Dentistry and Periodontology, School of Dentistry, Medical University of Vienna, Vienna, Austria.
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
Int Endod J. 2018 Feb;51 Suppl 2:e146-e156. doi: 10.1111/iej.12806. Epub 2017 Aug 12.
To evaluate the impact of hypoxia and hypoxia mimetic agents (HMA) on the formation and activity of spheroids by dental pulp cells (DPC).
DPC on agarose-coated plates were treated with hypoxia and the HMA dimethyloxallyl glycine (DMOG), desferrioxamine (DFO) and L-mimosine (L-MIM). Images of spheroids were taken directly after seeding and at 6 h and 24 h. Spheroid sizes were quantified by area measurement with ImageJ software. Viability was assessed with Live-Dead staining, MTT and resazurin-based toxicity assay. Production of VEGF, IL-8 and SDF-1 was evaluated using immunoassays. Data were analysed using Kruskal-Wallis test and post hoc Mann-Whitney U-test.
DPC formed spheroids in the presence of hypoxia, HMA and combined treatment with hypoxia and HMA. No pronounced difference in spheroid size was found in the groups treated with hypoxia, DMOG, DFO, L-MIM and the combination of hypoxia and the HMA relative to their normoxic controls (P > 0.05). Spheroids appeared vital in Live-Dead and MTT staining and the resazurin-based toxicity assay. Evaluation of protein production with immunoassays revealed significantly enhanced levels of VEGF and IL-8 (P < 0.05), but there was no significant effect on SDF-1 production (P > 0.05). Treatment with a combination of hypoxia and HMA did not further boost VEGF and IL-8 production (P > 0.05).
Pre-conditioning with hypoxia and HMA increased the pro-angiogenic capacity of spheroids whilst not interfering with their formation. Pre-clinical studies will reveal whether pre-conditioning of spheroids with hypoxia and HMA can effectively improve the efficiency of cell transplantation approaches for regenerative endodontics.
评估缺氧和缺氧模拟剂（HMA）对牙髓细胞（DPC）形成和球体活性的影响。
在琼脂糖涂层平板上用缺氧和 HMA 二甲氧乙醯基甘氨酸（DMOG）、去铁胺（DFO）和 L-千里光碱（L-MIM）处理 DPC。直接在接种后和 6 小时和 24 小时拍摄球体的图像。使用 ImageJ 软件通过面积测量定量测量球体大小。用 Live-Dead 染色、MTT 和 resazurin 毒性测定评估活力。用免疫测定法评估 VEGF、IL-8 和 SDF-1 的产生。使用 Kruskal-Wallis 检验和事后 Mann-Whitney U 检验分析数据。
DPC 在缺氧、HMA 以及缺氧和 HMA 的联合处理下形成球体。与常氧对照组相比，缺氧、DMOG、DFO、L-MIM 和缺氧与 HMA 的联合处理组的球体大小没有明显差异（P>0.05）。Live-Dead 和 MTT 染色以及 resazurin 毒性测定显示球体看起来具有活力。免疫测定法评估蛋白质产生显示 VEGF 和 IL-8 水平显著升高（P<0.05），但 SDF-1 产生没有显著影响（P>0.05）。缺氧和 HMA 的联合处理并未进一步增强 VEGF 和 IL-8 的产生（P>0.05）。
缺氧和 HMA 的预处理增加了球体的促血管生成能力，而不干扰其形成。临床前研究将揭示用缺氧和 HMA 预处理球体是否能有效提高细胞移植方法在再生牙髓学中的效率。
