Actin tracker green

Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and then permeabilized with 0.1% Triton X-100 for 10 min. Cells were incubated with Actin-Tracker Green (Beyotime, Shanghai, China) at room temperature for 1 hour. Then, the nuclei of cells were stained with Hoechst33342 and cells were subsequently visualized under a fluorescence microscope (Olympus BX51, USA). Adobe Photoshop CS6 (Adobe Systems, USA) was used to prepare merged images.

M2‐exos were labeled with fluorescent 3,3′‐dioctadecyloxacarbocyanine perchlorate (DiD; Invitrogen) according to the manufacturer's recommendations. Briefly, purified M2‐exos were incubated in 5 × 10⁻⁶m DiD for 15 min at 37 °C and were then ultracentrifuged at 100 000 g for 90 min to remove unbound dye. After being washed twice in PBS with centrifugation at 100 000 g, the labeled exosomes were resuspended in PBS before use. hPDLCs seeded on 24‐well plates were incubated at 37 °C with DiD‐labeled M2‐exos. Uptake was stopped after 4 h by washing. Then, the cells were fixed in 4% paraformaldehyde (PFA) for 30 min and permeabilized in 0.5% Triton‐X (Beyotime Biotechnology) in PBS for 15 min. Samples were stained sequentially with Actin‐Tracker Green (Beyotime Biotechnology) for 30 min and 4′,6‐diamidino‐2‐phenylindole (Beyotime Biotechnology) for 5 min at room temperature.

EGCG (≥98% purity), 3-MA and 4′,6-diamidino-2-phenylindole (DAPI), MDC, and MTT were provided by Sigma (St. Louis, MO, USA). EGCG was dissolved in deionized water to prepare 10 mM stock solutions at −20 °C. Dulbecco’s modified Eagle’s medium (DMEM), OPTI-MEM I medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). AFP and LC3 mouse monoclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin was obtained from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies were purchased from Wuhan Boster Biological Engineering Co. (Wuhan, China). Cy3-labeled anti-mouse antibody, Actin-Tracker Green, Mitochondrial Membrane Potential Assay Kit with JC-1 and the BCA Protein Assay Kit were purchased from Beyotime Institute of Biotechnology (Beijing, China).

Recombinant RANKL and M-CSF were purchased from PeproTech (Princeton, NJ, United States). Icariside I (ICS; C₂₇H₃₀O₁₁; MW: 530.53), baohuoside Ⅰ (BS; C₂₇H₃₀O₁₀; MW: 512.52), icariin (ICA; C₃₃H₄₀O₁₅; MW: 676.68), icaritin (ICT; C₂₁H₂₀O₆; MW: 368.38), and a Cell Counting Kit-8 (CCK-8) were purchased from Target Molecule Corp. (Boston, MA, United States). Trypsin-EDTA (0.05%), PBS, FBS, and MEM-Alpha basic were purchased from Gibco. Antibodies against p38 (#8690), p-p38 (#4511), ERK (#9102), p-ERK (#4370), JNK (#9252), p-JNK (#4668), p65 (#8242), p-p65 (#3033), IκBα (#4812), p-IκBα (#2859), and RANK (#4845) were purchased from Cell Signaling Technology (Boston, MA, United States). Antibodies against TRAP (ab52750) and cathepsin K (ab19027) were purchased from Abcam (Cambridge, MA, United States). Antibodies against NFATc1 (66963-1-Ig), uPAR (10286-1-AP), and β-actin (66009-1-Ig) were purchased from Proteintech Group Inc. (Wuhan, China). DAPI and Actin-Tracker Green were obtained from Beyotime (Shanghai, China). Cy3-labelled goat anti-rabbit antibody was purchased from Boster (Wuhan, China). Unless noted otherwise, other reagents were of the highest purity available and were obtained from Sigma-Aldrich (St. Louis, MO, United States).

There are six groups: GO (GO hydrogel), GO + US (GO hydrogel and ultrasound cavitation), 0.1KBGO (0.1KBGO hydrogel), 0.1KBGO + US (0.1KBGO hydrogel and ultrasound cavitation), 0.5KBGO (0.5KBGO hydrogel), and 0.5KBGO + US (0.5KBGO hydrogel and ultrasound cavitation). The GO, 0.1KBGO, and 0.5KBGO hydrogels were added to 48-well plates. Then BMSCs were seeded in each well at a density of 2 × 10⁴. After that, the US groups received ultrasonic loading with a sound intensity of 1.5 W/cm² for 5 min per day, while other groups were left untreated. After 48 h, a Calcein-AM/PI Live/Dead assay kit (BB-4126, Bestbio, China) was used to monitor the viability of the seeded BMSCs under a CLSM. After 1, 4, and 7 days, a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was applied to evaluate the proliferation of BMSCs on different hydrogels, and the optical density (OD) was detected by a microplate spectrophotometer (Epoch2, BioTek, USA) at 450 nm. And the cell nuclei and cytoskeleton of BMSCs were stained by DAPI staining solution (C1006, Beyotime, China) and actin-tracker green (C2201S, Beyotime, China) and then visualized by a CLSM to evaluate the cell spreading and adhesion after being cultured for 48 h.