Macrophage polarization effect on BMSCs osteogenesis. (a) A representative illustration of the co-culture system used. (b) ALP staining of BMSCs cultured for 7 days in a co-culture system. Scale bar represents 200 μm. (c) Quantitative analysis of ALP staining (n = 5). (d) ARS staining of BMSCs cultured for 7 days in the co-cultured system. Scale bar represents 200 μm. (e) Quantitative analysis of ARS staining (n = 5). (f) The expression of osteogenesis-related genes, such as Col I, RunX2, OCN, and OPN, was estimated by RT-qPCR after BMSCs co-culture for 7 days (n = 3). (g) RunX2 and OPN immunofluorescent staining of BMSCs was showed 7 days after being cultured in the co-culture system. RunX2 and OPN were stained red and the nuclei was stained blue. Scale bar represents 50 μm. (h) Quantitative analysis of immunofluorescent staining (n = 3). (i) The expression of RunX2 and OPN protein was evaluated by Western blot at 7 days. (j) Quantitative analysis of Western blot (n = 3). ANOVA followed by Bonferroni's multiple comparison test was used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001).