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

• Published in: Dental materials 2021-01, Vol.37 (1), p.e23-e36
• Main Authors: Vagropoulou, Georgia, Trentsiou, Maria, Georgopoulou, Anthie, Papachristou, Eleni, Prymak, Oleg, Kritis, Aristeidis, Epple, Matthias, Chatzinikolaidou, Maria, Bakopoulou, Athina, Koidis, Petros
• Format: Article
• Language: English
• Subjects: Biomimetics; Biomineralization; Cbfa-1 protein; Cell culture; Cell Differentiation; Cell morphology; Cell Proliferation; Cells, Cultured; Chitosan; Crystallites; Crystals; Cytology; Dental Pulp; Dental restorative materials; Dentin; Dentistry; Dexamethasone; Differentiation; Electron microscopy; Flow cytometry; Freeze drying; Gelatin; Gene expression; Hydroxyapatite; In vitro methods and tests; Microenvironments; Mineralization; Morphology; Nanocrystals; Odontogenesis; Scaffolds; Scanning electron microscopy; Stem Cells; Structural analysis; Time dependence; Tissue engineering; Tissue Scaffolds; Transcription factors; Viability; X-ray diffraction; X-ray spectroscopy
• ISSN: 0109-5641; 1879-0097
• DOI: 10.1016/j.dental.2020.09.021

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. 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. 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. 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.