Selenium Nanoparticles by Moderating Oxidative Stress Promote Differentiation of Mesenchymal Stem Cells to Osteoblasts

Abstract

Purpose: Redox homeostasis plays an important role in the osteogenic differentiation of human mesenchymal stem cells (hMSCs) for bone engineering. Oxidative stress (OS) is believed to induce osteoporosis by changing bone homeostasis. Selenium nanoparticles (SeNPs), an antioxidant with pleiotropic pharmacological activity, prevent bone loss. However, the molecular mechanism underlying the osteogenic activity during hMSC-SeNP interaction is unclear.

Methods: This study assessed the effects of different concentrations (25, 50, 100, and 300 ng/mL) of SeNPs on the cell viability and differentiation ability of human embryonic stem cell-derived hMSCs. In addition, we analyzed OS markers and their effect on mitogen-activated protein kinase (MAPK) and Forkhead box O3 (FOXO3) during osteogenesis.

Results: SeNPs increased the cell viability of hMSCs and induced their differentiation toward an osteogenic over an adipogenic lineage by enhancing osteogenic transcription and mineralization, while inhibiting Nile red staining and adipogenic gene expression. By preventing excessive reactive oxygen species accumulation, SeNPs increased antioxidant levels in hMSCs undergoing osteogenesis compared to untreated cells. In addition, SeNPs significantly upregulated the gene and protein expression of phosphorylated c-Jun N-terminal kinase (JNK) and FOXO3a, with no significant change in the expression levels of extracellular signal-related kinase (ERK) and p38 MAPK.

Conclusion: The results approved that low concentrations of SeNPs might enhance the cell viability and osteogenic potential of hMSCs by moderating OS. Increased JNK and FOXO3a expression shows that SeNPs might enhance osteogenesis via activation of the JNK/FOXO3 pathway. In addition, SeNP co-supplementation might prevent bone loss by enhancing osteogenesis and, thus, can be an effective candidate for treating osteoporosis through cell-based therapy.

Keywords: antioxidant; osteogenic differentiation; selenium nanoparticles stem cells.

Figure 1

Characterization of the SeNPs. (A) Transmission electron microscopic image (Scale bar 100 nm); (B) A histogram showing size distribution of the SeNPs; (C) Fourier Transform Infrared spectroscopy (FTIR) of selenium nanoparticles showing the functional characteristics of the nanoparticles.

Figure 2

Fluorescence image of cellular internalization of SeNPs (50 ng/mL) in hMSCs. (A) Control (Scale bar 100µm); (B) Localization of SeNPs () agglomerates in hMSCs (Scale bar 50µm); (C) Aggregated SeNPs within the cytoplasm of the cell (Scale bar 50µm).

Figure 3

Fluorescence intensity of viable hMSCs treated with different SeNP concentrations using alamarBlue assay at 0, 3, 7, and 9 days. Cells treated with lower doses of SeNPs showed significant increase in the number of viable cells from day 0 to day 9 compared to the untreated cells. In contrast, treatment with 300ng/mL SeNPs notably decreased the cell viability on day 9 of the cell culture. Results are mean ±SD of the triplicate experiments, *p<0.05, **p<0.01, ***p<0.001 versus control.

Figure 4

Effect of different SeNP concentrations on osteogenic differentiation of hMSCs. (A) Comparison of the alkaline phosphatase (ALP) activity measured after 7 days of osteogenic induction shows that lower concentrations of SeNPs increased the ALP activity compared to the untreated cells during osteogenesis (B) Quantification of mineralized nodule formation of MSCs visualized by Alizarin red staining after 14 days of osteogenic differentiation shows increased mineralization induced by lower concentration (25, 50, 100ng/mL) of SeNPs. Results are mean ±SD of the triplicate experiments: *p<0.05, **p<0.01, ***p<0.001 versus control.

Figure 5

Effect of different SeNP concentrations on adipogenic differentiation of hMSCs. (A) Quantification of Nile red staining represented as percent fluorescence change relative to control at day 14 of adipogenesis shows that SeNPs significantly suppressed the MSCs differentiation to adipogenic lineage. (B) Nile red staining was used to assess the effect of different concentration of SeNPs on adipogenic differentiation of MSCs. Results are mean ±SD of the triplicate experiments: *p<0.05, **p<0.01, ***p<0.001 versus control.

Figure 6

Effects of different SeNP concentrations on OS parameters measured after 3 days of osteogenic induction. (A) Histogram presentation of flow cytometric analysis of ROS levels in control cells (shaded purple) compared to cells treated with different concentration of SeNPs at 50, 100, 300ng/mL respectively (green lines). (B) Quantitative analysis of DCFA fluorescence intensity with respect to the untreated control shows that lower doses of SeNPs significantly reduced the ROS level whereas, high dose resulted in excessive ROS generation during osteogenesis. (C) Absorbance of the enzymatic antioxidants, SOD and Catalase in control and SeNP treated groups represents that lower doses of SeNPs enhanced the oxidative potential of the cells undergoing osteogenesis. Results are mean ±SD of the triplicate experiments: *p<0.05, **p<0.01, ***p<0.001 versus control.

Figure 7

Relative mRNA expression of osteogenic- and adipogenic-specific gene expression in hMSCs upon SeNP treatment (50 ng/mL). SeNPs upregulated the osteogenic and downregulated the adipogenic transcriptional profile of MSCs undergoing cellular differentiation. Results are mean ±SD of the triplicate experiments: **p<0.01, ***p<0.001 versus control.

Figure 8

Effects of SeNP (50 ng/mL) on protein and gene expression of p38, ERK, JNK, and FOXO3a measured after 3 days of osteogenic induction. (A) SeNPs significantly enhanced the protein expression of p-JNK and p-FOXO3a expression with no notable change in p-p38 and p-ERK expression relative to control; β-actin was used as internal control (B) Density of the protein from Western blot were quantified and expressed as fold change relative to β-actin. (C) Relative fold change in the mRNA expression of MAPK and FOXO3a gene analyzed by RT-PCR. Results are mean ±SD of the triplicate experiments: **p<0.01, ***p<0.001 versus control.