Fig. 2

Force-Exos block NLRP3-mediated PDLC pyroptosis to reduce M1 polarization and osteoclast formation. (A) Scheme of co-culture system. Exosomes were isolated from DFSCs with or without mechanical stimulation and co-cultured with PDLCs. (B) Intracellular localization of exosomes in PDLCs. Exosomes were labeled with PKH26 (red). PDLC cytoskeleton was stained with Phalloidin (green) and nuclei were counterstained with DAPI (blue). Subsequent to co-culturing exosomes with PDLCs, the fluorescence assays indicated the endocytosis of PDLCs on exosomes. Scale bar: 50 μm. (C) Hoechst 33,342 and propidium iodide (PI) fluorescence staining revealed that force upregulated the number of PI-positive cells (pyroptotic cells), which was downregulated by exosomes, especially Force-Exos. Scale bar: 50 μm. n = 5. (D) Flow cytometry showed the number of Annexin V⁺PI⁺ cells (pyroptotic cells) apparently decreased in Force + Force-Exo group, compared with Force + PBS group and Force + Ctrl-Exo group. Scale bar: 50 μm. n = 5. (E) Enzyme-linked immunosorbent assay showed IL-1β levels increased under mechanical stimulation, which was significantly suppressed after the exosome treatment and Force-Exos promoted the effect. n = 4. (F) The Western blot analysis indicated exosomes reduced the protein expression levels of NLRP3, pro-caspase-1, caspase-1, GSDMD, GSDMD-N, pro-IL-1β, IL-1β and IL-18. Force-Exos significantly enhanced the effect. Full‑length blots are presented in Fig. S6: Fig. 2F. (G) Ultrastructural comparison by transmission electron microscopy among Force + PBS and Force + Force-Exo groups. In contrast to the disappeared chromatin and non-viable mitochondria in Force + PBS group (red arrow), cell membrane in Force + Force-Exo group was relatively intact and chromatin condensed (blue arrow). Scale bar: 2 μm. (H) The qRT-PCR demonstrated exosomes significantly decreased the mRNA level of M1 markers including iNOS, TNF-α, IL-1β, whereas the mRNA level of M2 markers including CD163, Arg-1 and IL-4R was increased. Moreover, Force-Exos pronounced the impact, which was consistent with the efficacy of MCC950. n = 5. (I) Flow cytometry showed that the ratio of CD11b⁺ CD86⁺ cells (M1 macrophages) to CD11b⁺ CD206⁺ cells (M2 macrophages) was reduced in the Force + Force-Exo group and Force + MCC950 group, compared with other groups. n = 5. (J) The Western blot analysis revealed that the utilization of Force-Exos markedly diminished the protein expression levels of osteoclast markers (NFAT2, MMP9, CTSK), analogous to the efficacy of MCC950. Full‑length blots are presented in Fig. S6: Fig. 2J. (K) TRAP staining revealed the number of TRAP-positive osteoclasts decreased after treatment with exosomes and MCC950, and Force-Exos promoted the reduction. Scale bar: 200 μm. n = 5. *P < 0.05, **P < 0.01, ***P < 0.001