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

Abstract

Aim

To evaluate the impact of hypoxia and hypoxia mimetic agents (HMA) on the formation and activity of spheroids by dental pulp-derived 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 6h and 24h. 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 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), 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).

Conclusions

Pre-conditioning with hypoxia and HMA can increase the pro-angiogenic capacity of spheroids while not interfering with their formation. Preclinical studies will reveal whether pre-conditioning of spheroids with hypoxia and HMA can effectively increase cell transplantation approaches for regenerative endodontics.

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