In vitro study on flavonoid NO donor nanoparticles promoting osteogenic differentiation of PDLSCs by regulating macrophage polarization
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 lipopo-lysaccharide,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 miner-alization 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 en-hance the osteogenic differentiation of PDLSCs.
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