Abstract

BackgroundTo understand the responses of the dental pulp to hypoxia is of high relevance for regenerative endodontics and dental traumatology. Here, we aimed to reveal the effects of hypoxia and the hypoxia mimetic agent L-mimosine (L-MIM) on the production of sclerostin (SOST) and dickkopf-1 (DKK-1) in human dental pulp-derived cells (DPC).MethodsDPC in monolayer, spheroid and tooth slice cultures were treated with L-MIM or hypoxia. Resazurin-based toxicity and MTT assays were performed to determine cell viability. mRNA and protein levels of SOST and DKK-1 were measured with quantitative reverse transcription PCR and ELISA, respectively. To validate the hypoxia-like response, SDF-1, VEGF and IL-8 were assessed. In addition Western blots for HIF-1α, HIF-2α and HIF-3α were done.ResultsCells were vital upon treatment procedures and showed increased levels of HIF-1α, and HIF-2α. In monolayer cultures, mRNA levels of SOST and DKK-1 were downregulated by L-MIM and hypoxia, respectively. A significant downregulation of SOST by hypoxia was found at the protein level compared to untreated cells while the effect on DKK-1 and the impact of L-MIM on SOST and DKK-1 did not reach the level of significance at the protein level. In spheroid cultures, mRNA levels of SOST and DKK-1 were downregulated by L-MIM. A significant downregulation of DKK-1 upon hypoxia treatment was found at the protein level while the impact of hypoxia on SOST and the effect of L-MIM on SOST and DKK-1 did not reach the level of significance. SOST and DKK-1 were also produced in tooth slices, but no pronounced modulation by L-MIM or hypoxia was found. Evaluation of SDF-1, VEGF and IL-8 showed a hypoxia-like response in the culture models.ConclusionsThere is no pronounced influence of hypoxia and L-MIM on DPC viability, SOST and DKK-1 protein production. However, the specific response depends on the culture model and the level of evaluation (mRNA or protein). These results deepen our understanding about the role of hypoxia and the potential impacts of hypoxia-based strategies on dental pulp.

Highlights

  • To understand the responses of the dental pulp to hypoxia is of high relevance for regenerative endodontics and dental traumatology

  • Cell viability of dental pulp-derived cells in monolayer, spheroid and tooth slice culture after treatment with Lmimosine or hypoxia In monolayer cultures (Fig. 1a, d), as well as in spheroid cultures (Fig. 1b, e), Dental pulp-derived cells (DPC) appeared to be viable upon treatment with L-MIM or hypoxia in the resazurin-based toxicity assay

  • Effects of L-mimosine or hypoxia on the production of sclerostin, dickkopf-1, stromal cell-derived factor-1, vascular endothelial growth factor and interleukin-8 in spheroid cultures of dental pulp-derived cells To mimic the 3D situation in the pulp tissue we further evaluated the effect of L-MIM and hypoxia in spheroid cultures of DPC. Messenger ribonucleic acid (mRNA) expression of SOST (0.00001 ± 0.00001), DKK-1 (0.00029 ± 0.00038), Stromal cell-derived factor-1 (SDF-1) (0.00819 ± 0.00931), VEGF (0.01512 ± 0.00940) and IL-8 (0.00029 ± 0.00049) was detected relative to Glycerinaldehyde-3phosphate dehydrogenase (GAPDH) in Quantitative polymerase chain reaction (qPCR) in DPC spheroid cultures under normoxic conditions

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Summary

Introduction

To understand the responses of the dental pulp to hypoxia is of high relevance for regenerative endodontics and dental traumatology. We aimed to reveal the effects of hypoxia and the hypoxia mimetic agent L-mimosine (L-MIM) on the production of sclerostin (SOST) and dickkopf-1 (DKK-1) in human dental pulp-derived cells (DPC) It is of high relevance for regenerative endodontics and dental traumatology to understand the response of the dental pulp to hypoxia. Hypoxia-based strategies such as hypoxia conditioning or the application of hypoxia mimetic agents have been developed as new approaches to stimulate regeneration by inducing hypoxia-responsive factors that support angiogenesis and improve cell survival [3] Hypoxia mimetic agents such as prolyl hydroxylase inhibitors, for example Lmimosine (L-MIM) and deferoxamine, have shown to induce a similar pro-angiogenic response as hypoxia conditioning in dental pulp cells and can induce reparative processes [4,5,6,7,8]. This led researchers to analyse the effects of hypoxia on the dental pulp tissue and its role in regeneration

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