Formation of spheroids by dental pulp cells in the presence of hypoxia and hypoxia mimetic agents

• Published in: International endodontic journal 2018-02, Vol.51 (S2), p.e146-e156
• Main Authors: Janjić, K., Lilaj, B., Moritz, A., Agis, H.
• Format: Article
• Language: English
• Subjects: 3D culture; Chemokine CXCL12 - metabolism; Deferoxamine - pharmacology; dental pulp; Dental Pulp - cytology; Dental Pulp - drug effects; Dental Pulp - physiology; Enzyme-Linked Immunosorbent Assay; Glycine - analogs & derivatives; Glycine - pharmacology; HIF‐1; Humans; hypoxia; Hypoxia - metabolism; Hypoxia - physiopathology; Interleukin-8 - metabolism; in vitro; microtissues; Mimosine - pharmacology; PHD inhibitors; regenerative endodontics; spheroid; Spheroids, Cellular - cytology; Spheroids, Cellular - drug effects; Spheroids, Cellular - physiology; tissue engineering; Vascular Endothelial Growth Factor A - metabolism
• ISSN: 0143-2885; 1365-2591
• DOI: 10.1111/iej.12806

Aim To evaluate the impact of hypoxia and hypoxia mimetic agents (HMA) on the formation and activity of spheroids by dental pulp cells (DPC). Methodology 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. Results 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). Treatment with a combination of hypoxia and HMA did not further boost VEGF and IL‐8 production (P > 0.05). Conclusions 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.