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

<b><i>Background/Aims:</i></b> 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). <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> 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. <b><i>Conclusion:</i></b> These findings suggest that iNOS-MNPs can induce osteogenic differentiation in ESCs by integrating complex signaling pathways.

背景与目的：本研究探讨了负载诱导型一氧化氮合酶的矿化纳米颗粒（inducible nitric oxide synthase-loaded mineralized nanoparticles，iNOS-MNPs）对小鼠胚胎干细胞（embryonic stem cells，ESCs）成骨分化的影响。
方法：本研究在诱导型一氧化氮合酶（iNOS）存在的条件下，采用阴离子嵌段共聚物模板介导的碳酸钙（calcium carbonate，CaCO₃）矿化工艺制备iNOS-MNPs。所制备的iNOS-MNPs呈球形，粒径分布狭窄，且iNOS可稳定负载于纳米颗粒内部且未发生变性。为验证iNOS-MNPs成功导入ESCs胞质，采用荧光分析法检测细胞内一氧化氮（nitric oxide，NO）水平；同时采用竞争性酶联免疫吸附实验定量检测NO效应分子环磷酸鸟苷（cyclic guanosine 3',5'-monophosphate，cGMP）的含量。采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）法检测iNOS-MNPs处理后的细胞活力。通过碱性磷酸酶（alkaline phosphatase，ALP）活性检测、细胞内钙定量实验及茜素红S染色检测基质矿化水平，以探究ESCs的成骨分化情况。此外，采用免疫荧光染色与蛋白质印迹法检测成骨相关因子Runt相关转录因子2（Runt-related transcription factor 2，RUNX2）、骨钙蛋白（osteocalcin，OCN）及osterix（OSX）的蛋白表达水平。通过基于网络的分析方法，评估与iNOS-MNPs诱导ESCs成骨分化相关的复杂信号通路。
结果：与对照组相比，经iNOS-MNPs处理的细胞内NO与cGMP水平显著升高。iNOS-MNPs处理未对细胞活力产生不良影响。重要的是，摄取iNOS-MNPs可促进ESCs的成骨分化。通过转录组分析，我们共筛选得到1836个差异诱导表达基因，并采用ClueGO与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）进行功能富集分析。上述分析鉴定出多个显著富集且相互关联的分子通路，包括蛋白激酶活性、雌激素受体活性、骨形态发生蛋白（bone morphogenetic protein，BMP）受体结合、配体门控离子通道活性及磷脂酰肌醇3-磷酸结合等。
结论：上述研究结果表明，iNOS-MNPs可通过整合复杂的信号通路诱导ESCs发生成骨分化。