Formation of spheroids by dental pulp 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 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 KruskalWallis 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), 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 increased the pro-angiogenic capacity of spheroids whilst not interfering with their formation. Preclinical studies will reveal whether pre-conditioning of spheroids with hypoxia and HMA can effectively improve the efficiency of cell transplantation approaches for regenerative endodontics.

The response of gingiva monolayer, spheroid, and ex vivo tissue cultures to collagen membranes and bone substitute

“…GC spheroids were produced using 3D Petri Dishes® (Microtissues, Inc., Providence, RI, USA), following the instructions of the manufacturer (Janjić, Lilaj, Moritz, & Agis, 2018). Three GC spheroids with a total cell number of 50,000 and an average diameter of 356 μm/spheroid were seeded onto 1 cm 2 of a collagen membrane (Geistlich Bio‐Gide®; Geistlich Pharma AG) or bone substitute (Geistlich Bio‐Oss®; Geistlich Pharma AG) covering 1 cm 2 of plastic.…”

Contraction dynamics of dental pulp cell rod microtissues

“…Isolation and culture of dental pulp cells Human DPC were harvested in a sterile environment from extracted third molars without pulpitis, with a prior informed consent [16]. Pulp tissue underwent explant cultures to isolate DPC through outgrowth.…”

“…In transplanted tissues, mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT cellsignaling pathways play a vital role [1,13]. Their properties and broad range of interaction with the microenvironment in both dental embryogenesis [14] and in vitro pulp regeneration significantly regulate the self-assembly of cells [15,16]. Recent studies have shown that PI3K has a role in the leading edge of migrating fibroblasts via F-actin filament branching [17].…”

The Influence of Pro-Inflammatory Factors on Sclerostin and Dickkopf-1 Production in Human Dental Pulp Cells Under Hypoxic Conditions

“…Hypoxia-based strategies aim to improve angiogenesis in regenerative approaches. One approach is based on the idea to pre-condition cells with hypoxic conditions or hypoxia mimetic agents "to train" them for the hypoxic region of a defect where they are supposed to support regeneration (Janjić et al, 2018b). Diverse effects of hypoxia on Sost and Dkk-1 were discussed (Genetos et al, 2010;Chen et al, 2013;Guo et al, 2014).…”