Supplement of nitric oxide through calcium carbonate-based nanoparticles contributes osteogenic differentiation of mouse embryonic stem cells

Abstract

This study investigated the delivery of S-nitrosothiol (GSNO) as a nitric oxide (NO) donor loaded into calcium carbonate-based mineralized nanoparticles (GSNO-MNPs) to regulate cell signaling pathways for the osteogenic differentiation of mouse embryonic stem cells (ESCs). GSNO-MNPs were prepared by an anionic block copolymer template-mediated calcium carbonate (CaCO3) mineralization process in the presence of GSNO. GSNO-MNPs were spherical and had a narrow size distribution. GSNO was stably loaded within the MNPs without denaturation. TEM analysis also demonstrated the localization of GSNO-MNPs within membrane-bound structures in the cell, indicating the successful introduction of GSNO-MNPs into the cytosol of ESCs. Intracellular levels of NO and cGMP were significantly increased upon treatment with GSNO-MNPs, compared with the control group. When cells were exposed to GSNO-MNPs, the effects of nanoparticles on cell viability were not statistically significant. GSNO-MNPs treatment increased ALP activity assay and intracellular calcium levels. Real-time RT-PCR also revealed highly increased expression levels of the osteogenic target genes ALP, osteocalcin (OCN), and osterix (OSX) in GSNO-MNP-treated ESCs. The protein levels of OSX and Runt-related transcription factor 2 (RUNX2) showed similar patterns of expression based on real-time RT-PCR. These results indicate that GSNO-MNPs influenced the osteogenic differentiation of ESCs. Transcriptome profiling identified several significantly enriched and involved biological networks, such as RAP1, RAS, PI3K-AKT, and MAPK signaling pathways. These findings suggest that GSNO-MNPs can modulate osteogenic differentiation in ESCs via complex molecular pathways.