Gsk219

RAW 264.7 cells were cultured with high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, South Logan, UT, USA) containing 10% FBS (Hyclone) and 100 U/mL of penicillin/streptomycin (Thermo Fisher Scientific). A total of 5 × 10³/well RAW264.7 cells at passages 3 to 5 were seeded into 24-well plates and induced by M-CSF (Peprotech, Cranbury, NJ, USA) and sRANKL (R&D Systems) at a concentration of 20 ng/mL for osteoclast differentiation. Human DPSC-derived exosomes were administrated with a final concentration of 5 × 10⁷/mL.
To investigate the influence of TRPV4, the RAW 264.7 cells induced by M-CSF and sRANKL were treated with GSK219 (10 μM; Selleck, Houston, TX, USA), a specific TRPV4 inhibitor [46 (link)]. TRPV4 agonist GSK101 (Selleck Selleck, Houston, TX, USA) was delivered at a concentration of 100 nM [37 (link)]. DMSO was administrated as a control.

Human PDL progenitor cells were isolated as previously described and were identified following previous protocols,^{34 (link)} which used at passage 4. Compressive force loading was provided by glass layers and 50 mL plastic tube caps containing weighed metal balls as previously described.^{35 (link),36 (link)} 1.5 g/cm² compressive force was applied to PDL progenitor cells for different time points (3–24 h), and different compressive force (0.5–2.0 g/cm²) was applied to PDL progenitor cells for 6 h. In addition, after being subjected to 1.0 g/cm² and 1.5 g/cm² compressive force for 6 h, PDL progenitor cells were collected for further experiments of optical microscope (OM, Olympus, Japan), scanning electron microscope (SEM) and transmission electron microscope (TEM).
To confirm the influence of pyroptosis under mechanical stimuli, pyroptosis activator PPVI (4 μmol/L), pyroptosis inhibitor MCC950 (10 μmol/L) and Caspase-1 inhibitor Belnacasan (VX765, 20 μM, S2228, Selleck) were added to PDL progenitor cells for 18 h in advance, then 1.5 g/cm² force was applied to PDL progenitor cells for 6 h.^{31 ,37} In addition, TRPV4 inhibitor GSK219 (10 mmol/L, Selleck) were applied to PDL progenitor cells for 1 h and then stimulated with force loading (1.5 g/cm², 6 h).¹⁷ PDL progenitor cells without force-loaded and drug treatment served as controls.