Hybrid chitosan/gelatin/nanohydroxyapatite scaffolds promote odontogenic differentiation of dental pulp stem cells and in vitro biomineralization

Georgia Vagropoulou; Maria Trentsiou; Anthie Georgopoulou; Eleni Papachristou; Oleg Prymak; Aristeidis Kritis; Matthias Epple; Maria Chatzinikolaidou; Athina Bakopoulou; Petros Koidis

doi:10.1016/j.dental.2020.09.021

Hybrid chitosan/gelatin/nanohydroxyapatite scaffolds promote odontogenic differentiation of dental pulp stem cells and in vitro biomineralization

Dent Mater. 2021 Jan;37(1):e23-e36. doi: 10.1016/j.dental.2020.09.021. Epub 2020 Nov 15.

Affiliations

¹ Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Greece.
² Department of Materials Science and Technology, University of Crete, Greece.
³ Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Essen, Germany.
⁴ Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Greece.
⁵ Department of Materials Science and Technology, University of Crete, Greece; Foundation for Research and Technology Hellas-Institute of Electronic Structure and Laser FORTH-IESL, Heraklion, Greece. Electronic address: mchatzin@materials.uoc.gr.
⁶ Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Greece. Electronic address: abakopoulou@dent.auth.gr.
⁷ Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Greece. Electronic address: pkoidis@dent.auth.gr.

PMID: 33208264
DOI: 10.1016/j.dental.2020.09.021

Abstract

Objective: Hybrid chitosan/gelatin/nanohydroxyapatite (CS/Gel/nHA) scaffolds have attracted considerable interest in tissue engineering (TE) of mineralized tissues. The present study aimed to investigate the potential of CS/Gel/nHA scaffolds loaded with dental pulp stem cells (DPSCs) to induce odontogenic differentiation and in vitro biomineralization.

Methods: CS/Gel/nHA scaffolds were synthesized by freeze-drying, seeded with DPSCs, and characterized with flow cytometry. Scanning Electron Microscopy (SEM), live/dead staining, and MTT assays were used to evaluate cell morphology and viability; real-time PCR for odontogenesis-related gene expression analysis; SEM-EDS (Energy Dispersive X-ray spectroscopy), and X-ray Diffraction analysis (XRD) for structural and chemical characterization of the mineralized constructs, respectively.

Results: CS/Gel/nHA scaffolds supported viability and proliferation of DPSCs over 14 days in culture. Gene expression patterns indicated pronounced odontogenic shift of DPSCs, evidenced by upregulation of DSPP, BMP-2, ALP, and the transcription factors RunX2 and Osterix. SEM-EDS showed the production of a nanocrystalline mineralized matrix inside the cell-based and - to a lesser extent - the cell-free constructs, with a time-dependent production of net-like nanocrystals (appr. 25-30nm in diameter). XRD analysis gave the crystallite size (D=50nm) but could not distinguish between the initially incorporated and the biologically produced nHA.

Significance: This is the first study validating the potential of CS/Gel/nHA scaffolds to support viability and proliferation of DPSCs, and to provide a biomimetic microenvironment favoring odontogenic differentiation and in vitro biomineralization without the addition of any inductive factors, including dexamethasone and/or growth/morphogenetic factors. These results reveal a promising strategy towards TE of mineralized dental tissues.

Keywords: Chitosan; Dentin; Electron microscopy; Gelatin; Hydroxyapatite; Scaffolds; Stem cells; Tissue engineering.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biomineralization
Cell Differentiation
Cell Proliferation
Cells, Cultured
Chitosan*
Dental Pulp
Gelatin*
Odontogenesis
Stem Cells
Tissue Scaffolds

Substances

Gelatin
Chitosan