Imagej 1.52a software

The bone resorption assay kit (CSR-BRA-48KIT; COSMOBIO, Tokyo, Japan) was used to check resorption activity of osteoclast cells according to the manufacturer’s protocol. Briefly, Raw264.7 cells (5 × 10³ cells/well) were seeded into calcium phosphate (CaP)-coated 48-well culture plate. Additional collagen type I coating was performed using 50 μg/mL collagen type I (3447-020-01; R&D System, Minneapolis, MN, USA) to mimic a bone biomimetic surface. Raw264.7 cells were cultured at 37 °C and 5% CO₂ in DMEM containing 10% FBS. After 24 h, the medium was changed to complete α-MEM medium without phenol-red and then pretreated with a vehicle (DMSO) and citreoviridin (0.5 and 1μg/mL) for 1 h. Subsequently, RANKL (100 ng/mL) was added in the medium to induce an osteoclast differentiation. RANKL and citreoviridin were re-treated every 2 days for 6 days. On day 6, the cells were washed with PBS and treated with 5% sodium hypochlorite for 5 min to remove the cells. Then, the plate was washed with D.W. and dried. Osteoclast resorbing areas were captured using a digital camera (Olympus) attached to microscope (Olympus) and the pit areas obtained from 20 different regions by 4 independent experiments were measured by NIH ImageJ 1.52a software.

To study the effect of A‐PRF‐XBSM mixture exudates on cell migration, a scratch wound‐healing assay mimics cell migration during wound healing in vitro. hPDLSCs were seeded into six‐well plates (3 × 10⁵ cells/well; Nunc, Denmark) and cultured until reaching 80% confluence. A scratch was introduced into the single‐layer on each dish using a pipette tip (100–1000 µl tip; Thermo Scientific, USA). Non‐adherent cells were extracted by washing once with 1× phosphate‐buffered saline (PBS) (Gibco, USA). The cells were then serum‐starved overnight. The A‐PRF‐XBSM mixture exudates were added to each well. Complete medium and DMEM/F12 without FBS were used as positive and negative controls, respectively. The images of cell migration into the scratch area at 0 and 24 h were taken using an Olympus CKX‐RCD microscope (Olympus, Japan) set up with a DP2‐BSW microscope camera and then analyzed using ImageJ 1.52a software (USA).