Fig 2: Osteoclast-secreted miR-324 silences ARHGAP1 during osteogenic differentiation(A) Relative luciferase activity of reporter containing the 3' UTR of ARHGAP1 in MSCs upon culturing with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, and miR-324-sEVs + miR-324 inhibitor; n = 5. (B) Relative mRNA expression levels of Col1a1, Alpl, Sp7, and Runx2 in MSCs cultured with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, miR-324-sEVs + annexin V, miR-324-sEVs + miR-324 inhibitor, and miR-324-sEVs + ARHGAP1; n = 3. (C) Relative expression levels of ARHGAP1 in indicated groups; n = 3. (D) Western blot analysis of ARHGAP1, RhoA, ROCK, p-MYPT1, and ß-actin expression in MSCs cultured with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, miR-324-sEVs + annexin V, miR-324-sEVs + miR-324 inhibitor, and miR-324-sEVs + ARHGAP1; n = 3. (E) Cell viability evaluation of MSCs in indicated groups at 0, 1, 3, 5 and 7 days; n = 3. (F) Representative images of alizarin red staining of MSCs cultured with OC-sEVs, miR-NC-sEVs, miR-324-sEVs, miR-324-sEVs + annexin V, miR-324-sEVs + miR-324 inhibitor, and miR-324-sEVs + ARHGAP1. Scale bar represents 100 µm. (G) Quantification analysis of calcium deposit of MSCs in indicated groups; n = 5. The data in the figures represent the averages ± SD. *p < 0.05, **p < 0.01, for differences between the treatment and control groups.

Fig 3: miR-324 directly binds to ARHGAP1 and induces osteogenic differentiation(A) Relative mRNA expression levels of ARHGAP1 in MSC knockdown or overexpressed ARHGAP1; n = 3. CT represents empty control group without any treatment, and si-CT represents the random non-specific siRNA used for negative control. (B) Relative ALP activity of MSC knockdown or overexpressed ARHGAP1 after osteogenic induction for 14 days; n = 3. (C) Relative mRNA expression levels of Col1a1, Alpl, Sp7, and Runx2 in MSC knockdown or overexpressed ARHGAP1; n = 3. (D) Relative expression levels of miR-324 in MSC knockdown or overexpressed miR-324; n = 3. (E) Relative expression levels of ARHGAP1 in MSC knockdown or overexpressed miR-324; n = 3. Data in the figures represent the averages ± SD. **p < 0.01, for differences between the treatment and control groups.

Fig 4: Osteoclast-derived sEVs promote osteogenic differentiation and mineralization(A) Representative confocal microscopy images show MSCs labeled with CellTracker CM-Dil dye that were cultured with PKH67-labeled sEVs for 24 h. Scale bar represents 10 µm. (B) Relative mRNA expression levels of Col1a1, Alpl, Sp7, and Runx2 in MSCs cultured with OC-sEVs or osteoclast conditioned medium (OC-CM), with or without GW4869 pretreatment; n = 3. (C) Western blot analysis of COL1A1, RUNX2, ALP, and ß-actin in indicated groups. (D) Representative images of alizarin red staining of MSCs cultured with OC-sEVs or OC-CM. Scale bar represents 100 µm. (E) Quantification analysis of calcium deposit of MSCs in indicated groups; n = 5. The data in the figures represent the averages ± SD. **p < 0.01, for differences between the treatment and control groups.

Fig 5: Quantitative analysis of immuno-histochemical staining of RUNX2, VEGF, EphB4, and EphrinB2 proteins 1 and 3 months post operation. (**p < 0.01, *p < 0.05)