Temporal-controlled Dexamethasone Releasing Chitosan Nanoparticle System Enhances Odontogenic Differentiation of Stem Cells from Apical Papilla

doi:10.1016/j.joen.2015.03.024

Journal of Endodontics

Volume 41, Issue 8, August 2015, Pages 1253-1258

https://doi.org/10.1016/j.joen.2015.03.024 Get rights and content

Highlights

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This study highlights development and characterization of 2 novel temporal-controlled dexamethasone releasing nanoparticle systems.
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Chitosan-based nanoparticles are non-cytotoxic to SCAP, and temporal-controlled release of Dex enhanced the odontogenic differentiation potential.
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The proposed morphogen delivering method can be used for the spatial and temporal control in regenerative endodontic procedures.

Abstract

Introduction

The spatial and temporal control of stem cell differentiation into odontoblast-like cells remains one of the major challenges in regenerative endodontic procedures. The current study aims to synthesize and compare the effect of dexamethasone (Dex) release from 2 variants of Dex-loaded chitosan nanoparticles (CSnp) on the odontogenic differentiation of stem cells from apical papilla (SCAP).

Methods

Two variants of Dex-loaded CSnp were synthesized by encapsulation (Dex-CSnpI) and adsorption (Dex-CSnpII) methods. The physicochemical characterization of Dex-CSnpI and Dex-CSnpII was assessed by transmission electron microscopy, Zetasizer, and Fourier transform infrared spectroscopy, whereas the Dex release kinetics was assessed by spectrophotometry. A previously characterized SCAP cell line was cultured onto CSnp, Dex-CSnpI, or Dex-CSnpII. The biomineralization potential was determined by alizarin red staining. Alkaline phosphatase, dentin sialophosphoprotein, and dentin matrix protein-1 gene expressions were analyzed by real-time reverse-transcription polymerase chain reaction.

Results

Dex-CSnpI resulted in slower release of Dex compared with Dex-CSnpII, but both demonstrated sustained release of Dex for 4 weeks. Biomineralization of SCAP was significantly higher (P < .05) in presence of Dex-CSnpII compared with that in Dex-CSnpI at 3 weeks. Alkaline phosphatase gene expression was significantly higher in the presence of Dex-CSnpII compared with Dex-CSnpI, with peak expression seen at 2 weeks (P < .05). The expression of odontogenic specific marker dentin matrix protein-1 was significantly higher in presence of Dex-CSnpII compared with Dex-CSnpI at 3 weeks (P < .05).

Conclusions

Collectively, these data suggest that sustained release of Dex results in enhanced odontogenic differentiation of SCAP. These findings highlight the potential of temporal-controlled delivery of bioactive molecules to direct the spatial- and temporal-controlled odontogenic differentiation of dental stem cells.

Section snippets

Materials and Methods

CS, sodium tripolyphosphate, Dex, and α-minimum essential medium were purchased from Sigma-Aldrich Inc (St Louis, MO). Fetal bovine serum, L-glutamine solution, and antibiotic:antimycotic solution were obtained from Gibco (Carlsbad, CA). All other chemicals were of analytical grade (purity ≥ 95%).

Synthesis and Characterization of Nanoparticles

The physicochemical properties of CSnp, Dex-CSnpI, and Dex-CSnpII are presented in Table 2. The nanoparticles appeared spherical in shape, with a particle size of 94–200 nm. The size of CSnp was 93.9 nm, and an increase in size was observed after Dex incorporation. The sizes of Dex-CSnpI and Dex-CSnpII were 179.1 nm and 112 nm, respectively. The charge of CSnp, Dex-CSnpI, and Dex-CSnpII was +33.9, +18.6, and +20.3 mV, respectively. FTIR spectra analysis of CSnp, Dex-CSnpI, and Dex-CSnpII

Discussion

The concept of temporal-controlled release is the process of presenting specific bioactive molecules to the stem cells at a predetermined rate to maintain their optimum bioavailability at the required time as well as to protect their biological activity through the desired period of interaction. Precise control of bioactive molecules on a temporal basis is of utmost importance for stem cells, while differentiating into specialized cell types of the tissue and organ (24). Dex at a concentration

Acknowledgments

This study was supported by the University of Toronto Start-up Fund, Ontario, Canada and a research grant from the American Association of Endodontists (AAE) Foundation (2014).

The authors deny any conflicts of interest related to this study.

References (35)

V.E. Arana-Chavez et al.
Odontoblasts: the cells forming and maintaining dentine
Int J Biochem Cell Biol
(2004)
D. Tziafas et al.
Designing new treatment strategies in vital pulp therapy
J Dent
(2000)
A. Bakopoulou et al.
Comparative analysis of in vitro osteo/odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP)
Arch Oral Biol
(2011)
G.T. Huang et al.
Formation of odontoblast-like cells from cultured human dental pulp cells on dentin in vitro
J Endod
(2006)
K. Näsström et al.
Narrowing of the dental pulp chamber in patients with renal diseases
Oral Surg Oral Med Oral Pathol
(1985)
S. Zhang et al.
One-step preparation of chitosan solid nanoparticles by electrospray deposition
Int J Pharm
(2010)
S.A. Agnihotri et al.
Recent advances on chitosan-based micro- and nanoparticles in drug delivery
J Control Release
(2004)
A. Kishen et al.
An investigation on the antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal disinfection
J Endod
(2008)
Z. Shi et al.
Antibacterial and mechanical properties of bone cement impregnated with chitosan nanoparticles
Biomaterials
(2006)
S. Shrestha et al.
Temporal-controlled release of bovine serum albumin from chitosan nanoparticles: effect on the regulation of alkaline phosphatase activity in stem cells from apical papilla
J Endod
(2014)

N.B. Ruparel et al.

Characterization of a stem cell of apical papilla cell line: effect of passage on cellular phenotype

J Endod

(2013)

T. Mosmann

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

J Immunol Methods

(1983)

Q. Yuan et al.

Controlled and extended drug release behavior of chitosan-based nanoparticle carrier

Acta Biomater

(2010)

J. Meng et al.

Engineering tenofovir loaded chitosan nanoparticles to maximize microbicide mucoadhesion

Eur J Pharm Sci

(2011)

S.E. Bae et al.

Fabrication of covered porous PLGA microspheres using hydrogen peroxide for controlled drug delivery and regenerative medicine

J Control Release

(2009)

C. Gómez-Gaete et al.

Encapsulation of dexamethasone into biodegradable polymeric nanoparticles

Int J Pharm

(2007)

P.C. Tobias Duarte et al.

Histopathological condition of the remaining tissues after endodontic infection of rat immature teeth

J Endod

(2014)

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Basic ResearchTemporal-controlled Dexamethasone Releasing Chitosan Nanoparticle System Enhances Odontogenic Differentiation of Stem Cells from Apical Papilla

Highlights

Abstract

Introduction

Methods

Results

Conclusions

Section snippets

Materials and Methods

Synthesis and Characterization of Nanoparticles

Discussion

Acknowledgments

Int J Biochem Cell Biol

J Dent

Arch Oral Biol

J Endod

Oral Surg Oral Med Oral Pathol

Int J Pharm

J Control Release

J Endod

Biomaterials

J Endod

J Endod

J Immunol Methods

Acta Biomater

Eur J Pharm Sci

J Control Release

Int J Pharm

J Endod

Basic Research
Temporal-controlled Dexamethasone Releasing Chitosan Nanoparticle System Enhances Odontogenic Differentiation of Stem Cells from Apical Papilla