Elsevier

Journal of Endodontics

Volume 41, Issue 11, November 2015, Pages 1852-1861
Journal of Endodontics

Basic Research
Effects of Prolyl Hydroxylase Inhibitor L-mimosine on Dental Pulp in the Presence of Advanced Glycation End Products

https://doi.org/10.1016/j.joen.2015.08.002Get rights and content

Highlights

  • We evaluated the response of monolayer cultures of dental pulp–derived cells and tooth slice organ cultures to the prolyl hydroxylase inhibitor L-mimosine in the presence of advanced glycation end products.

  • In the presence of advanced glycation end products, L-mimosine increases vascular endothelial growth factor and interleukin-8 but not interleukin-6 in monolayer cultures of dental pulp–derived cells.

  • In the presence of interleukin-1β and advanced glycation end products, L-mimosine increases vascular endothelial growth factor but not interleukin-6 and interleukin-8 in monolayer cultures of dental pulp–derived cells.

  • L-mimosine decreases alkaline phosphatase and matrix mineralization under the influence of advanced glycation end products in monolayer cultures of dental pulp–derived cells.

  • In the presence of advanced glycation end products, L-mimosine increases vascular endothelial growth factor but not interleukin-6and interleukin-8 in tooth slice organ cultures.

  • Our data suggest that prolyl hydroxylase inhibitors can increase the production of angiogenic growth factors in the dental pulp in the presence of advanced glycation end products.

Abstract

Introduction

Proangiogenic prolyl hydroxylase (PHD) inhibitors represent a novel approach to stimulate tissue regeneration. Diabetes mellitus involves the accumulation of advanced glycation end products (AGEs). Here we evaluated the impact of AGEs on the response of human pulp tissue to the PHD inhibitor L-mimosine (L-MIM) in monolayer cultures of dental pulp–derived cells (DPCs) and tooth slice organ cultures.

Methods

In monolayer cultures, DPCs were incubated with L-MIM and AGEs. Viability was assessed based on formazan formation, live-dead staining, annexin V/propidium iodide, and trypan blue exclusion assay. Vascular endothelial growth factor (VEGF), interleukin (IL)-6, and IL-8 production was evaluated by quantitative polymerase chain reaction and immunoassays. Furthermore, expression levels of odontoblast markers were assessed, and alizarin red staining was performed. Tooth slice organ cultures were performed, and VEGF, IL-6, and IL8 levels in their supernatants were measured by immunoassays. Pulp tissue vitality and morphology were assessed by MTT assay and histology.

Results

In monolayer cultures of DPCs, L-MIM at nontoxic concentrations increased the production of VEGF and IL-8 in the presence of AGEs. Stimulation with L-MIM decreased alkaline phosphatase levels and matrix mineralization also in the presence of AGEs, whereas no significant changes in dentin matrix protein 1 and dentin sialophosphoprotein expression were observed. In tooth slice organ cultures, L-MIM increased VEGF but not IL-6 and IL-8 production in the presence of AGEs. The pulp tissue was vital, and no signs of apoptosis or necrosis were observed.

Conclusions

Overall, in the presence of AGEs, L-MIM increases the proangiogenic capacity, but decreases alkaline phosphatase expression and matrix mineralization.

Section snippets

Monolayer Culture of DPCs

Extracted third molars from nondiabetic patients without any sign of inflammation were used to harvest human DPCs after informed consent was obtained (Ethics Committee of the Medical University of Vienna, Vienna, Austria). Teeth were opened under sterile conditions, and pulp tissue was collected. DPCs were cultured in alpha-minimal essential medium (Invitrogen Corporation, Carlsbad, CA) supplemented with 10% fetal calf serum (PAA Laboratories, Linz, Upper Austria, Austria) and penicillin G at

DPCs Remain Vital When Incubated with AGEs in the Presence of L-MIM in Monolayer Cell Cultures

DPCs stimulated with AGE at 1, 10, and 100 μg/mL in the presence of L-MIM at 1 mmol/L showed no pronounced changes in formazan formation. No significant difference was observed when AGE-treated cells were compared with BSA-treated cells (Fig. 1A). Also, the addition of 10 ng/mL IL-1β made DPCs more susceptible to AGEs with regard to formazan formation (Fig. 1B). Staurosporine, which served as the positive control, reduced formazan formation at 30 and 100 nmol/L in the MTT assay (Fig. 1C).

Discussion

The dental pulp, as part of the dentin-pulp complex, is a highly vascularized tissue in which chronically dysbalanced blood glucose levels lead to the accumulation of AGEs in dentin (6). AGEs have been proposed to lead to compromised oral tissue regeneration 15, 16. Therefore, it is of relevance to assess the impact of AGEs on regenerative approaches. A novel experimental approach proposed for regenerative endodontics is the application of PHD inhibitors to stimulate revascularization and

Acknowledgments

The authors thank M. Pensch (Department of Oral Surgery, Medical University of Vienna, Vienna, Austria) for skillful technical assistance. The authors acknowledge S. Tangl and his team (Karl Donath Laboratory for Hard Tissue and Biomaterial Research, Department of Oral Surgery, Medical University of Vienna, Vienna, Austria) and D. Teplanova and R. Szoeloesi (Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria) for

References (46)

  • H.D. Muller et al.

    Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor in dental pulp-derived cells

    J Endod

    (2012)
  • T. Mosmann

    Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays

    J Immunol Methods

    (1983)
  • J.F. Telfer et al.

    Placental alkaline phosphatase activity is inversely related to cell growth rate in HeLaS3 cervical cancer cells

    FEBS Lett

    (1993)
  • L. Jiang et al.

    Effects of deferoxamine on the repair ability of dental pulp cells in vitro

    J Endod

    (2014)
  • L. Li et al.

    Hypoxia promotes mineralization of human dental pulp cells

    J Endod

    (2011)
  • S. Hama et al.

    Involvement of inducible nitric oxide synthase and receptor for advanced glycation end products in periapical granulomas

    J Endod

    (2007)
  • M.F. Leite et al.

    Diabetes induces metabolic alterations in dental pulp

    J Endod

    (2008)
  • S. Yamagishi et al.

    Advanced glycation end products (AGEs) and diabetic vascular complications

    Curr Diabetes Rev

    (2005)
  • M.S. Devi et al.

    Differential modulation of angiogenesis by advanced glycation end products

    Exp Biol Med (Maywood)

    (2011)
  • Y. Abiko et al.

    The mechanism of protracted wound healing on oral mucosa in diabetes. Review

    Bosn J Basic Med Sci

    (2010)
  • Y. Nakajima et al.

    Advanced glycation end products increase expression of S100A8 and A9 via RAGE-MAPK in rat dental pulp cells

    Oral Dis

    (2015)
  • K. Bala et al.

    Interaction of glycated protein and DFO mimicked hypoxia in cellular responses of HUVECs

    Mol Biosyst

    (2012)
  • P.C. Chang et al.

    Soft-tissue wound healing by anti-advanced glycation end-products agents

    J Dent Res

    (2014)
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