Macrophage MSR1 exhibits pro-osteogenic differentiation effect of BMSC in a co-culture system. (A) BMSCs were seeded in the lower chamber, and macrophages were cultured in the upper chamber. (B) In a co-culture system for 7 or 14 days, MSR1 KO BMDMs reduced the ability to promote osteogenic differentiation of BMSCs as indicated by AR staining. BMSCs without co-culture were set as the control (Con) group. (C and D) Quantitative evaluation of AR staining results (C) and ALP activities (D) on day 7 and 14 was performed (Values are expressed as mean ± SD, *p < 0.05, **p < 0.01). (E) mRNA expression levels of osteogenic marker genes (Runx2, Ocn, ALP, and Col1) in osteogenic differentiation of BMSCs on day 14 were detected by qPCR in different groups. β-actin was used as an internal control (Values are mean ± SD, *p < 0.05, **p < 0.01). (F) In the co-culture system, MSR1-overexpressing RAW264.7 cells enhanced osteogenic differentiation of BMSCs on day 7 and 14 as revealed by AR staining. BMSCs cultured alone were set as the Con group and RAW264.7 cells without MSR1-plasmid transfection were defined as the blank (BL) group. Vec: vector group, OE: overexpression group. (G and H) Quantitative analyses of AR staining results (G) and ALP activities (H) of osteogenic differentiation of BMSCs on day 7 and 14 were performed. Values are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significance. (I) mRNA expression levels of Col1, ALP, Ocn and Runx2 in osteogenic differentiation of BMSCs on day 14 by qPCR in different groups. β-actin was used as an internal control (Values are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significance).