Magnesium-containing nanostructured hybrid scaffolds for enhanced dentin regeneration

Abstract

Dental caries is one of the most prevalent chronic diseases in the United States, affecting 92% of adults aged 20-64 years. Scaffold-based tissue engineering represents a promising strategy to replace damaged dental structures and restore their biological functions. Current single-component scaffolding materials used for dental tissue regeneration, however, cannot provide the proper microenvironment for dental stem/progenitor cell adhesion, proliferation, and differentiation; new biomimetic hybrid scaffolds are needed to promote better dental tissue formation. In this work, we developed a biomimetic approach to prepare three-dimensional (3D) nanofibrous gelatin/magnesium phosphate (NF-gelatin/MgP) hybrid scaffolds. These scaffolds not only mimic the nanostructured architecture and the chemical composition of natural dentin matrices but also constantly present favorable chemical signals (Mg ions) to dental pulp stem cells (DPSCs), thus providing a desirable microenvironment to facilitate DPSC proliferation, differentiation, and biomineralization. Synthesized hybrid NF-gelatin/MgP possesses natural extracellular matrix (ECM)-like architecture, high porosity, high pore interconnectivity, well-defined pore size, and controlled Mg ion release from the scaffold. Adding MgP into NF-gelatin also increased the mechanical strength of the hybrid scaffold. The sustained release of Mg ions from the NF-gelatin/MgP (MgP=10% wt/wt) scaffold significantly enhanced the proliferation, differentiation, and biomineralization of human DPSCs in vitro. The alkaline phosphatase (ALP) activity and the gene expressions for odontogenic differentiation (collagen I [Col I], ALP, osteocalcin [OCN], dentin sialophosphoprotein [DSPP], and dentin matrix protein 1 [DMP1]) were all significantly higher (p<0.05) in the NF-gelatin/MgP group than in the NF-gelatin group. Those results were further confirmed by hematoxylin and eosin (H&E) and von Kossa staining, as shown by greater ECM secretion and mineral deposition in the hybrid scaffold. Consistent with the in vitro study, the DPSCs/NF-gelatin/MgP constructs produced greater ECM deposition, hard tissue formation, and expression of marker proteins (DSPP, DMP1, Col I) for odontogenic differentiation than did the DPSCs/NF-gelatin after 5 weeks of ectopic implantation in rude mice. The controlled release of metallic ions from biomimetic nanostructured hybrid scaffolds, therefore, is a promising approach to enhancing the biological capability of the scaffolds for dental tissue regeneration.

FIG. 1.

Effects of adding Mg ions in the culture medium of 2D culture plates on the proliferation, differentiation, and biomineralization of dental pulp stem cells (DPSCs). (A) MTT assay showed that adding 100 ppm Mg ions increased DPSC proliferation compared to the control group from 1 to 7 days. However, the DPSCs had lower proliferation rates when high concentrations of Mg ions (500 and 1000 ppm) were added to the culture medium. (B) Relative gene expression (collagen type I [Col I], osteocalcin [OCN], dentin matrix protein 1 [DMP1], alkaline phosphatase (ALP), and dentin sialophosphoprotein [DSPP]) after human DPSCs were cultured on a 12-well plate for 3 weeks (*p<0.05 between the Mg ions treated and the control groups). (C) Alizarin red staining revealed that the DPSCs treated by 100, 500, and 1000 ppm Mg ions conditioned media displayed more mineralized nodules than the control group after 4 weeks of culturing. Color images available online at www.liebertpub.com/tea

FIG. 2.

Scanning electron microscope (SEM) micrographs of nanofibrous (NF)-gelatin and NF-gelatin/magnesium phosphate (MgP) hybrid scaffolds. (A) NF-gelatin scaffold, overview. (B) High magnification of (A) showing pore wall morphology of the NF-gelatin scaffold. (C) High magnification of (B) showing gelatin nanofibers of the NF-gelatin scaffold. (D) NF-gelatin/MgP (MgP=10% wt/wt) scaffold overview. (E) High magnification of (D) showing pore wall morphology of the NF-gelatin/MgP scaffold. (F) High magnification of (E) showing gelatin nanofibers of the NF-gelatin/MgP scaffold.

FIG. 3.

(A) Attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectra of NF-gelatin/MgP hybrid scaffolds with different amounts of MgP in the scaffolds. Dashed lines I and II assigned to gelatin, and dash line III assigned to Mg ions. (B) The energy dispersive X-ray spectroscopy (EDS) spectra of the NF-gelatin/MgP (MgP=10% wt/wt) hybrid scaffold. (C) The EDS mappings of Mg element within the NF-gelatin/MgP (MgP=10% wt/wt) hybrid scaffold. Color images available online at www.liebertpub.com/tea

FIG. 4.

Compressive moduli of the NF-gelatin/Mg hybrid scaffolds with different MgP concentrations. There was a significant difference between the NF-gelatin/MgP (MgP=10% wt/wt) and the NF-gelatin groups (*p<0.05). There was also a significant difference between the NF-gelatin/MgP (MgP=10% wt/wt) and the NF-gelatin/MgP (MgP=20% wt/wt) groups (*p<0.05).

FIG. 5.

Cumulative release of Mg ions from the NF-gelatin/MgP hybrid scaffolds in Tris-HCl buffer at pH 7.4 and 37°C for 3 weeks. Color images available online at www.liebertpub.com/tea

FIG. 6.

The proliferation of human DPSCs cultured on NF-gelatin/MgP and NF-gelatin scaffolds. A total of 5×10⁵ cells was seeded on each scaffold (*p<0.05 between the NF-gelatin/MgP and the NF-gelatin groups). Color images available online at www.liebertpub.com/tea

FIG. 7.

(A) A typical SEM micrograph of human DPSCs cultured on the NF-gelatin scaffold for 3 weeks. (B) A typical SEM micrograph of human DPSCs cultured on the NF-gelatin/MgP scaffold for 3 weeks. More extracellular matrix was deposited on the NF-gelatin/MgP than on the NF-gelatin (red arrows). (C) ALP activities of human DPSCs cultured on the NF-gelatin/MgP and the NF-gelatin scaffolds for 5 weeks (*p<0.05 between the NF-gelatin/MgP and the NF-gelatin groups). Color images available online at www.liebertpub.com/tea

FIG. 8.

Hematoxylin and eosin (H&E) and von Kossa staining images of human DPSCs cultured on NF-gelatin/MgP and NF-gelatin scaffolds for 5 weeks. (A, B) H&E staining of the DPSCs/NF-gelatin construct. (C, D) H&E staining of the DPSCs/NF-gelatin/MgP construct. (E) Quantitative analysis of the newly formed tissue in the DPSCs/NF-gelatin and the DPSCs/NF-gelatin/MgP constructs. (F, G) von Kossa staining of the DPSCs/NF-gelatin construct. (H, I) von Kossa staining of the DPSCs/NF-gelatin/MgP construct. (B, D, G, I) are high-magnification images of (A, C, F, H), respectively. (J) Quantitative analysis of the mineralized area in the DPSCs/NF-gelatin and DPSCs/NF-gelatin/MgP constructs. (*p<0.05 between the NF-gelatin/MgP and the NF-gelatin group). Color images available online at www.liebertpub.com/tea

FIG. 9.

Relative gene expression (Col I, OCN, DMP1, ALP, and DSPP) after human DPSCs were cultured on the NF-gelatin and the NF-gelatin/MgP scaffolds for 5 weeks (*p<0.05 between the NF-gelatin/MgP and the NF-gelatin groups). Color images available online at www.liebertpub.com/tea

FIG. 10.

X-ray and H&E staining images of human DPSCs seeded in the scaffolds with tooth slices and subcutaneously implanted in nude mice for 5 weeks. (A) A representative X-ray image of the NF-gelatin group showing that no minerals were detected. (B) A representative X-ray image of the NF-gelatin/MgP group showing a significant amount of mineral. (C, D) H&E staining images of the DPSCs/NF-gelatin construct. (E, F) H&E staining images of the DPSCs/NF-gelatin/MgP construct. (D, F) are the high-magnification images of (C, E), respectively. (G) Quantitative analysis of the newly formed dental tissue in the DPSCs/NF-gelatin and the DPSCs/NF-gelatin/MgP constructs. (*p<0.05 between the NF-gelatin/MgP and the NF-gelatin group). Color images available online at www.liebertpub.com/tea

FIG. 11.

Immunohistochemical staining imagines of odontogenesis markers for DPSCs/NF-gelatin and the DPSCs/NF-gelatin/MgP constructs after being subcutaneously implanted in nude mice for 5 weeks. (A, B) DSPP expression on the DPSCs/NF-gelatin construct. (C, D) DSPP expression on the DPSCs/NF-gelatin/MgP construct. (E, F) DMP1 expression on the DPSCs/NF-gelatin construct. (G, H) DMP1 expression on the DPSCs/NF-gelatin/MgP construct. (I, J) Col I expression on the DPSCs/NF-gelatin construct. (K, L) Col I expression on the DPSCs/NF-gelatin/MgP construct. (B, D, F, H, J, L) are the high-magnification images of (A, C, E, G, I, K), respectively. Color images available online at www.liebertpub.com/tea