Fixative solution

Osteogenic induction was applied to detect the osteogenic differentiation capacity of PDLCs induced by H₂O₂ in the presence or absence of LIPUS pretreatment. Cells were seeded at a density of 1 × 10⁵ cells per well in a 6-well culture plate and incubated. When the cells reached 80% confluency, the medium was replaced with stem osteogenic differentiation medium [a-MEM supplemented with 10% FBS, 50 µg/mL of ascorbic acid (Sigma), 5 mM L-glycerophosphate (Sigma), and 100 nM dexamethasone (Sigma) for 7-21 days depending on the experiment.
Alkaline phosphatase (ALP) staining was determined using NBT/BCIP (Beyotime) according to the manufacturer's protocol after 7 days of osteogenic induction. ALP activity was evaluated using an AKP analysis kit (Nanjing Jiancheng Bioengineering Institute, China) after 7 days, according to the manufacturer's instructions.
Calcium accumulation was detected by fixing with 4% fixative solution (Solarbio Co., Ltd., Beijing, China) and staining with 0.2% Alizarin Red (Solarbio) after 21 days of osteogenic induction. To quantify mineralization, cells stained with Alizarin Red were destained with 10% cetylpyridinium chloride monohydrate (Solarbio), following which the extracted stain was transferred to a 96-well plate, and the absorbance at 405 nm was measured using a microplate reader (Perkin Elmer, USA).

SA-β-gal activity was assayed as previously reported [28 (link)]. Briefly, adipose tissue chunks were collected in PBS, fixed with fixative solution (Solarbio) for 15 min. Then, the adipose tissue chunks were washed 3 times in PBS and placed in SA-β-gal activity solution (Solarbio). After 10–14 h of incubation at 37 °C CO₂-free incubator, the adipose tissue chunks were rinsed with PBS. Micrographs were captured using an Axio Zoom V16 (ZEISS) microscope and processed in ZEN software (ZEISS).