Osteogenic Effect of Inducible Nitric Oxide Synthase (iNOS)-Loaded Mineralized Nanoparticles on Embryonic Stem Cells

Abstract

Background/Aims: This study investigated the effect of inducible nitric oxide synthase-loaded mineralized nanoparticles (iNOS-MNPs) on the osteogenic differentiation of mouse embryonic stem cells (ESCs). Methods: We prepared iNOS-MNPs using an anionic block copolymer template-mediated calcium carbonate (CaCO₃) mineralization process in the presence of iNOS. iNOS-MNPs were spherical and had a narrow size distribution. iNOS was stably loaded within MNPs without denaturation. In order to confirm the successful introduction of iNOS-MNPs into the cytosol of ESCs, intracellular levels of nitric oxide (NO) was determined with a fluorometric analysis. A NO effector molecule, cyclic guanosine 3’,5’ monophosphate (cGMP) was also quantified with a competitive enzyme immunoassay. Cell viability in response to iNOS-MNP treatment was determined using the cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity assay, intracellular calcium quantification assay, and Alizarin red S staining for matrix mineralization were performed to investigate osteogenic differentiation of ESCs. The protein levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) as osteogenic-related factors were also assessed by immunofluorescence staining and Western blot analysis. The complex pathways associated with iNOS-MNP-derived osteogenic differentiation of ESCs were evaluated by network-based analysis. Results: Cells with iNOS-MNPs displayed a significant increase in NO and cGMP concentration compared with the control group. When cells were exposed to iNOS-MNPs, there were no adverse effects on cell viability. Importantly, iNOS-MNP uptake promoted the osteogenic differentiation of ESCs. Using transcriptome profiling, we obtained 1,836 differentially-induced genes and performed functional enrichment analysis with ClueGO and KEGG. These analyses identified significantly enriched and interconnected molecular pathways such as protein kinase activity, estrogen receptor activity, bone morphogenetic protein (BMP) receptor binding, ligand-gated ion channel activity, and phosphatidylinositol 3-phosphate binding. Conclusion: These findings suggest that iNOS-MNPs can induce osteogenic differentiation in ESCs by integrating complex signaling pathways.

Effect of locally delivered protein complex‐loaded nanoparticles on bone remodelling of atrophic alveolar ridge in beagles

“…The ability of the CaCO 3 ‐mineralized nanoparticle for the delivery of loaded cargos has been verified in various in vitro and in vivo models 15,16 . In particular, we have demonstrated that this CaCO 3 ‐based mineralized hybrid nanoparticle has exhibited a great potential for protein delivery 22‐24 . Based on the verification on the characteristics of P‐NPs, we herein have simply provided the important properties of P‐NPs, such as mean hydrodynamic diameter, size distribution, polydispersity of nanoparticles, zeta potential and the loading efficiencies of DMP‐1 and MMP‐13 in the nanoparticles.…”

“…15,16 In particular, we have demonstrated that this CaCO 3 -based mineralized hybrid nanoparticle has exhibited a great potential for protein delivery. [22][23][24] Based on the verification on the characteristics of P-NPs, we herein have simply provided the important properties of P-NPs, such as mean hydrodynamic diameter, size distribution, polydispersity of nanoparticles, zeta potential and the loading efficiencies of DMP-1 and MMP-13 in the nanoparticles. Further study is anticipated to find out a real in vivo half-life of P-NPs to see whether the number of injection could be minimized during orthodontic treatment in the clinically acceptable level.…”

The Role of Nitric Oxide in Stem Cell Biology

“…Thus, sGC is a potential therapeutic target for osteoporosis [28][29][30]. In this context, it has been reported an increase in mouse ESCs (mESCs) differentiation after treatment with iNOS-loaded mineralized nanoparticles that release NO and increase cGMP intracellular concentration, enhancing osteogenesis-related protein levels [31]. Moreover, this pathway is thought to play a crucial role in mediating vasoconstriction, oxidative stress, and fibrosis in cardiomyocytes [32,33].…”