Cellular behaviours of bone marrow-derived mesenchymal stem cells towards pristine graphene oxide nanosheets

Abstract

Objectives: Graphene oxide (GO), the derivative of graphene with unique properties, has attracted much attention for applications in dental implants. The aim of this study was, by two biomimetic cell culture methods, to investigate the quantitative relationship between the concentration of pristine GO nanosheets and their cellular behaviours towards bone marrow-derived mesenchymal stem cells (BMSCs).

Materials and methods: The cells were firstly characterized according to their morphology, self-renewal capabilities and multipotency. Subsequently, adhesion density, proliferation, alkaline phosphatase activity and mineralization of BMSCs treated with various concentrations of GO were analysed. In addition, osteogenic-related proteins were measured for further verification of the GO-induced osteogenic differentiation.

Results: Pristine GO nanosheets inhibited the proliferation of BMSCs at a high concentration of 10 μg/mL during the first 3 days with two seeding methods and facilitated proliferation of BMSCs at a low concentration of 0.1 μg/mL after 5 days with a sequential-seeding method compared to a co-seeding method. Analogously, osteogenic differentiation was promoted when BMSCs were treated with 0.1 μg/mL of GO. Both the proliferation and differentiation showed concentration-dependent behaviour. Interestingly, Wnt/β-catenin signalling pathway appeared to be involved in osteogenic differentiation induced by pristine GO nanosheets.

Conclusions: Pristine GO nanosheets at a concentration of 0.1 μg/mL provide benefits to promote BMSCs proliferation and osteogenesis under a sequential-seeding method, contributing to the use of GO for dental implantation.

Figure 1

Characterization of pristine graphene oxide (GO) nanosheets and GO suspension. (A) Morphology of pristine GO nanosheets with TEM. (B) GO suspension with concentrations of 0.02 and 0.1 mg/mL, recorded by means of a digital camera

Figure 2

Characterization of bone marrow‐derived mesenchymal stem cells (BMSCs) (P3 and P8) morphology after 5 days of culture. (a) Low and (b) high magnification, as visualized by (A) inverted microscopy. (B) Microscopic images of BMSCs when stained with (a) alizarin red and (b) Oil Red O for the presence of calcium deposits and lipid droplets by successful differentiation into osteoblasts and adipocytes, respectively, in in vitro culture

Figure 3

Adhesion density of bone marrow‐derived mesenchymal stem cells (BMSCs) that were sequentially seeded and co‐seeded into GO/DMEM at concentrations of 0.01~10 μg/mL and morphology of BMSCs treated with/without GO. (A) Confocal laser scanning images of BMSCs at low magnification show adhesion density after 72 h of incubation. The control group had no pristine GO nanosheets. Sample cells with abnormal morphology are circled with a dashed yellow line. (B) The high‐magnification images show the morphology of BMSCs after 72 h of incubation with and without pristine GO nanosheets. Blue stains nuclei and green stains cytoskeleton

Figure 4

The proliferation of bone marrow‐derived mesenchymal stem cells treated with different final concentrations (0, 0.01, 0.1, 1 and 10 μg/mL) of GO/DMEM with sequential‐seeding (A, B) and co‐seeding methods (C, D) after 1, 3, 5 and 7 days. *P<.05, **P<.01, ***P<.001 and ****P<.0001 vs the control group (n=5)

Figure 5

The effect of GO on ALP activity after 3 and 7 days' differentiation. Post‐cultured GO/DMEM was collected for the detection of ALP activity secreted by BMSCs with (A) sequential seeding and (B) co‐seeding methods. (C) The ALP activity within BMSCs treated with GO/OIM was measured. ***P<.001 and ****P<.0001 vs the control group

Figure 6

Mineralization of BMSCs treated with GO/DMEM and GO/OIM. (A) Alizarin red staining after culture with GO/DMEM for 21 days. (B) Optical photos and microscopic images showed the formation of calcium deposits after culture with GO/OIM for 21 days. (C) 10% cetylpyridinium chloride solution was used for the quantification of alizarin red staining. ****P<.0001

Figure 7

Expression profiles of osteogenic‐related proteins for differentiation of BMSCs in GO/OIM after 14 days. (A) Representative Western blot scans of each protein. (B, C) ImageJ software was used for semiquantitative analyses of Runx2 and β‐catenin expression. *P<.05, **P<.01, ***P<.001 and ****P<.0001