Kouqiang yixue (Nov 2024)
In vitro study on flavonoid NO donor nanoparticles promoting osteogenic differentiation of PDLSCs by regulating macrophage polarization
Abstract
Objective As a signaling molecule, NO regulates key physiological processes and is closely related to periodontitis. To investigate the effect of flavonoid NO donor composite nanoparticles (G10@HAP/MSN@ZnO@COS) on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) by regulating macrophage polarization. Methods The novel NO donor drug G10 was loaded on hydroxyapatite/mesoporous silicanant particles (HAP/MSN), filled with zinc oxide (ZnO), and then coated with chitosan (COS) to prepare composite nanoparticles (G10@HAP/MSN@ZnO@COS). The best concentration of G10@HAP/MSN@ZnO@COS was screened to promote cell proliferation by CCK-8 cell experiment. After the mouse mononuclear macrophages were stimulated by lipopolysaccharide, the mice were divided into four groups: Control group, G10 group, HAP/MSN@ZnO@COS group and G10@HAP/MSN@ZnO@COS group. Each group was cultured with fresh medium, 5 μg/mL G10, 5 μg/ mL HAP/MSN@ZnO@COS and 5 μg/mL G10@HAP/MSN@ZnO@COS for 72 h respectively. ELISA and RT-qPCR were used to detect the expression of cytokines (TNF-α, IL-6, IL-1β, iNOS, IL-10) and mRNA expression in each group, and the phenotypic changes of M1/M2 were evaluated. The supernatant of each culture medium was used as conditioned medium to culture PDLSCs, and the osteogenic ability and cell mineralization were evaluated by alkaline phosphatase activity test and alizarin red staining. Results CCK-8 experiment showed that G10@HAP/MSN@ZnO@COS of 5 μg/mL could significantly promote the proliferation of PDLSCs. The results of ELISA showed that compared with Control group, the expression of M1 type marker IL-1β, IL-6, TNF-α and iNOS in G10@HAP/MSN@ZnO@COS group was significantly decreased (P<0.000 1), while the expression of M2 type marker IL-10 was significantly increased (P<0.000 1). The results of RT-qPCR were consistent with those of ELISA, which showed that the expression of M1-related genes in G10@HAP/MSN@ZnO@COS group decreased significantly (P<0.01). The results of alizarin red staining and alkaline phosphatase activity test showed that the number of mineralized nodules and alkaline phosphatase activity in G10@HAP/MSN@ZnO@COS-CM group were significantly higher than those in other groups (P<0.000 1). Conclusion Composite nanoparticles (G10@HAP/MSN@ZnO@COS) can effectively inhibit the polarization of macrophages to M1 phenotype and promote it to M2 phenotypic polarization. The anti-inflammatory microenvironment regulated by G10@HAP/MSN@ZnO@COS can enhance the osteogenic differentiation of PDLSCs.
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