Graphene quantum dots enhance the osteogenic differentiation of PDLSCs in the inflammatory microenvironment

Abstract

Background and objective: Graphene quantum dots (GQDs), a type of carbon-based nanomaterial, have remarkable biological, physical, and chemical properties. This study investigated the biological mechanisms of the proliferation and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) induced by GQDs in an inflammatory microenvironment.

Materials and methods: PDLSCs were cultured in osteogenic-induced medium with various concentrations of GQDs in standard medium or medium mimicking a proinflammatory environment. The effects of GQDs on the proliferation and osteogenic differentiation activity of PDLSCs were tested by CCK-8 assay, Alizarin Red S staining, and qRT‒PCR. In addition, Wnt/β-catenin signalling pathway-related gene expression was measured by qRT‒PCR.

Results: Compared with the control group, the mRNA expression levels of ALP, RUNX2, and OCN and the number of mineralized nodules were all increased in PDLSCs after treatment with GQDs. Moreover, during the osteogenic differentiation of PDLSCs, the expression levels of LRP6 and β-catenin, which are Wnt/β-catenin signalling pathway-related genes, were upregulated.

Conclusion: In the inflammatory microenvironment, GQDs might promote the osteogenic differentiation ability of PDLSCs by activating the Wnt/β-catenin signalling pathway.

Keywords: Graphene quantum dots; Osteogenic differentiation; Periodontal ligament stem cells; Wnt/β-catenin signalling pathway.

Fig. 1

Morphological and molecular characteristics of isolated PDLSCs. (a-b) microphotography of alive primary periodontal ligament cells (a) magnification 40×; (b) magnification 100×. (c) colony culture of PDLSCs and single-cell colony culture (inset panel). (d) microphotography of PDLSCs colony stained with Crystal Violet. (e) microphotography of representative data for Alizarin red-S staining of PDLSCs differentiates into osteoblasts. (f) microphotography of representative data for Oil Red O staining of PDLSCs differentiates into adipocyte. (g-j) flow cytometry was used to analyse the surface markers of PDLSCs; Positive rate: (g) CD90-100%, (h) CD146- 90.4%, (i) STRO-1-82.7%, (j) CD34-0.5%.

Fig. 2

Effects of different concentrations (0–30 µg/mL) of GQDs on the proliferation of PDLSCs under the media mimicking proinflammatory environment and the standard media as the control group. (SM-standard media, IM-inflammatory microenvironment)

Fig. 3

Effect of GQDs on the osteogenic differentiation of PDLSCs. (a-d) the expression of ALP, RUNX2, OCN, and COL-1 were detected by qRT-PCR at Day 7. Data are expressed as mean ± SD from three independent experiments performed in triplicate.*p<0.05, **p<0.01, ns: No statistical difference. (e) Alizarin S Red staining was performed to detect the mineralized nodules on Day 7 and Day 14

Fig. 4

(a)Schematic demonstrated that GQDs promote osteogenic differentiation of PDLSCs through the Wnt/β-catenin signalling pathway. (b) LRP6 expression (c) β-catenin and (d) LEF1 were assessed by qRT-PCR on Day 7. *p<0.05, **p<0.01, ns: No statistical difference