Dental pulp stem cells in chitosan/gelatin scaffolds for enhanced orofacial bone regeneration

Athina Bakopoulou; Αnthie Georgopoulou; Ioannis Grivas; Chryssa Bekiari; Oleg Prymak; Κateryna Loza; Matthias Epple; George C Papadopoulos; Petros Koidis; Μaria Chatzinikolaidou

doi:10.1016/j.dental.2018.11.025

Dental pulp stem cells in chitosan/gelatin scaffolds for enhanced orofacial bone regeneration

Dent Mater. 2019 Feb;35(2):310-327. doi: 10.1016/j.dental.2018.11.025. Epub 2018 Dec 7.

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, Heraklion, Greece.
³ Department of Anatomy, Histology & Embryology, School of Veterinary Medicine, Faculty of Health Sciences, A.U.Th, Greece.
⁴ Inorganic Chemistry & Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Germany.
⁵ Department of Materials Science and Technology, University of Crete, Heraklion, Greece; Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH), Heraklion, Greece. Electronic address: mchatzin@materials.uoc.gr.

PMID: 30527589
DOI: 10.1016/j.dental.2018.11.025

Abstract

Objective: Biomimetic chitosan/gelatin (CS/Gel) scaffolds have attracted great interest in tissue engineering of several tissues. However, limited information exists regarding the potential of combining CS/Gel scaffolds with oral cells, such as dental pulp stem cells (DPSCs), to produce customized constructs targeting alveolar/orofacial bone reconstruction, which has been the aim of the present study.

Methods: Two scaffold types, designated as CS/Gel-0.1 and CS/Gel-1, were fabricated using 0.1 and 1% (v/v) respectively of the crosslinker glutaraldehyde (GTA). Scaffolds (n=240) were seeded with DPSCs with/without pre-exposure to recombinant human BMP-2. In vitro assessment included DPSCs characterization (flow cytometry), evaluation of viability/proliferation (live/dead staining, metabolic-based tests), osteo/odontogenic gene expression analysis (qRT-PCR) and structural/chemical characterization (scanning electron microscopy, SEM; energy dispersive X-ray spectroscopy, EDX; X-ray powder diffraction, XRD; thermogravimetry, TG). In vivo assessment included implantation of DPSC-seeded scaffolds in immunocompromised mice, followed by histology and SEM-EDX. Statistical analysis employed one/two-way ANOVA and Tukey's post-hoc tests (significance for p<0.05).

Results: Both scaffolds supported cell viability/proliferation over 14 days in culture, showing extensive formation of a hydroxyapatite-rich nanocrystalline calcium phosphate phase. Differential expression patterns indicated GTA concentration to significantly affect the expression of osteo/odontogenic genes, with CS/Gel-0.1 scaffolds being more effective in upregulating DSPP, IBSP and Osterix. In vivo analysis demonstrated time-dependent production of a nanocrystalline, mineralized matrix at 6, 8 and 10 weeks, being more prominent in constructs bearing rhBMP-2 pre-treated cells. The latter showed higher amounts of osteoid and fully mineralized bone, as well as empty space reduction.

Significance: These results reveal a promising strategy for orofacial bone tissue engineering.

Keywords: Bioinspired chitosan/gelatin scaffolds; Dental pulp stem cells; Orofacial bone regeneration.

Publication types

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

MeSH terms

Animals
Bone Regeneration
Cell Differentiation
Cells, Cultured
Chitosan*
Dental Pulp
Gelatin*
Humans
Mice
Stem Cells
Tissue Engineering
Tissue Scaffolds

Substances

Gelatin
Chitosan