Simvastatin Promotes Odontoblastic Differentiation and Expression of Angiogenic Factors via Heme Oxygenase-1 in Primary Cultured Human Dental Pulp Cells

doi:10.1016/j.joen.2009.11.021

Journal of Endodontics

Volume 36, Issue 3, March 2010, Pages 447-452

https://doi.org/10.1016/j.joen.2009.11.021 Get rights and content

Abstract

Introduction

Although simvastatin has multiple demonstrable effects, its function in dentinogenesis remains unclear. In this study, we tested the hypothesis that the addition of simvastatin to human dental pulp cells (HDPCs) stimulates odontogenesis both by promoting odontoblastic differentiation and by favoring the release of angiogenic factors. In addition, the role of heme oxygenase-1 (HO-1) in these effects was investigated.

Methods

The expression of markers for odontoblastic differentiation and angiogenesis was analyzed by means of alkaline phosphatase (ALP) activity, alizarin red staining, and Western blotting.

Results

Simvastatin enhanced the differentiation of HDPCs by up-regulating mineralization nodules and odontogenic markers as well as angiogenic markers. These phenomena were then correlated with the induction of HO-1 protein levels. The inducing effect of simvastatin on odontoblastic differentiation and angiogenesis was nullified by an HO-1 inhibitor and a carbon monoxide (CO) scavenger.

Conclusions

These results suggested that simvastatin exerts its odontoblastic differentiation and angiogenesis-inducing effects in HDPCs through a mechanism that involves the action of HO-1 and its product CO.

Section snippets

Primary Culture of HDPCs

The human dental pulp tissues obtained from sectioned teeth were removed aseptically, rinsed with Hanks' buffered saline solution, and placed in a 60-mm Petri dish. The dental pulp tissues were then minced with a blade into small fragments and cultured in Dulbecco modified Eagle medium (Biofluids Inc, Rockville, MD) containing 10% fetal bovine serum (Gibco, Carlsbad, CA) along with 100 U/mL penicillin and 100 U/mL streptomycin (Life Technologies, Rockville, MD). Cultures were maintained at 37°C

Effects of Simvastatin on ALP Activity and Mineralization Behavior

In this study, ALP activity was measured as an early marker for differentiation of HDPCs treated with or without simvastatin. We noted increased ALP activity during the 14-day culture period in a dose-dependent manner (Fig. 1A).

Next we examined the mineralization of the extracellular matrix in vitro when HDPCs were cultured in the presence of 1 μmol/L of simvastatin. After 7 days, the amount of dye bound to the cells with 1 μmol/L statin was similar to that of the control. However, after 14

Discussion

Statin is a potent bone formation stimulator that activates the promotion of the osteoblastic growth factor gene BMP-2. In vivo, the subcutaneous administration of statin in mice has been noted to stimulate bone formation (13). Further studies have shown that statin promotes osteoblastic differentiation, enhances ALP production and bone mineralization (14), and up-regulates the expression of bone anabolic factors such as VEGF (19). However, the mechanism of odontoblastic differentiation and

Acknowledgments

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2008-013-E00056).

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Citation Excerpt :
Simvastatin is a reductase inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), used for inhibiting cholesterol synthesis in hyperlipidemia therapy [25]. Min et al. demonstrated that simvastatin stimulated odontogenesis and angiogenesis by upregulating odontoblastic differentiation and heme oxygenase-1 (HO-1) expression in hDPSCs [26]. In addition, Jia et al. clarified that simvastatin (Sim) effectively promoted hDPSC mineralization and pulp and dentin regeneration after pulpotomy [27].
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The results revealed that 70 wt% CPC (CPC7) with 30 wt% CSH (CSH3) exhibited optimal setting time and porous structure for clinical use. The cell viability and cytotoxicity analysis demonstrated that CPC7–CSH3 with or without simvastatin or collagenase did not injure hDPSCs. In vivo, the CPC7–CSH3–Sim–Col induced dentin bridge formation.
CPC7–CSH3–Sim–Col in this study has great potential as a VPT biomaterial to enhance the dentin bridge formation.
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The improvement of biomaterials capable of driving the regeneration of the pulp-dentin complex mediated by resident cells is the goal of regenerative dentistry. In the present investigation, a chitosan scaffold (CHSC) that released bioactive concentrations of simvastatin (SIM) was tested, aimed at the development of a cell-free tissue engineering system.
First, we performed a dose-response assay to select the bioactive dose of SIM capable of inducing an odontoblastic phenotype in dental pulp cells (DPCs); after which we evaluated the synergistic effect of this dosage with the CHSC/DPC construct. SIM at 1.0 μmol/L (CHSC-SIM1.0) and 0.5 μmol/L were incorporated into the CHSC, and cell viability, adhesion, and calcium deposition were evaluated. Finally, we assessed the biomaterials in an artificial pulp chamber/3-dimensional culture model to simulate the cell-free approach in vitro.
SIM at 0.1 μmol/L was selected as the bioactive dose. This drug was capable of strongly inducing an odontoblastic phenotype on the DPC/CHSC construct. The incorporation of SIM into CHSC had no deleterious effect on cell viability and adhesion to the scaffold structure. CHSC-SIM1.0 led to significantly higher calcium-rich matrix deposition on scaffold/dentin disc assay compared with the control (CHSC). This biomaterial induced the migration of DPCs from a 3-dimensional culture to its surface as well as stimulated significantly higher expressions of alkaline phosphatase, collagen type 1 alpha 1, dentin matrix acidic phosphoprotein 1, and dentin sialophosphoprotein on 3-dimensional–cultured DPCs than on those in contact with CHSC.
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^‡: Drs Min and Lee contributed equally to this article.

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Basic ResearchSimvastatin Promotes Odontoblastic Differentiation and Expression of Angiogenic Factors via Heme Oxygenase-1 in Primary Cultured Human Dental Pulp Cells

Abstract

Introduction

Methods

Results

Conclusions

Section snippets

Primary Culture of HDPCs

Effects of Simvastatin on ALP Activity and Mineralization Behavior

Discussion

Acknowledgments

J Endod

Arch Oral Biol

J.Endod

J Endod

Trends Pharmacol Sci

Biochem Biophys Res Commun

Biochem Biophys Res Commun

J Endod

J Endod

Trends Immunol

J Endod

J Endod

Cell Biol Int

Free Radic Biol Med

Cell Signal

J Endod

J Biol Chem

Effects of calcium hydroxide-containing pulp-capping agents on pulp cell migration, proliferation, and differentiation

J Dent Res

Basic Research
Simvastatin Promotes Odontoblastic Differentiation and Expression of Angiogenic Factors via Heme Oxygenase-1 in Primary Cultured Human Dental Pulp Cells