Alp staining kit

To further analyze the catabolic activity in the alveolar bone, mononucleated and multinucleated osteoclasts, as well as polarized resorbing osteoclasts were detected by TRAP staining. The TRAP staining kit (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) was used according to the manufacturer’s protocol. The number of TRAP-positive cells per one section and per mm² of the region of interest were counted by a single examiner and the averages were calculated.
To assess bone formation in the extraction socket, ALP-positive stained area (%) was analyzed using an ALP staining kit (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) at 37 °C for 30 min, according to the manufacturer’s instructions.

The MC3T3-E1 cells were collected and manually counted, and the cell concentration was adjusted to 2 × 10⁴ cells/mL. The suspended cells were added into a 48-well plate (250 μl/well) with α-MEM culture medium and pre-incubated in a humidified incubator (37 °C, 5 % CO₂). Subsequently, upon reaching sub-confluence, switched to osteogenic induction medium containing either basic α-MEM medium or the extract solution of metal materials, to promote osteoblast differentiation. Every two days, the culture medium underwent renewal. After 7 and 14 days, an ALP staining kit (Wako) was applied to evaluate osteoblast differentiation of MC3T3-E1 cells.

PDL cells were seeded into 24 well plates at a density of 5 × 10⁴ cells/well in the osteogenic differentiation medium. ALP activity and mineralization were estimated on days 3, 5, 7, 14, 21, and 28. To assess ALP activity, the cultured cells were washed twice with PBS and fixed in 4% paraformaldehyde solution at room temperature for 15–30 min. Then, the fixed cells were rinsed three times with deionized water and stained using the ALP staining kit (Wako, Osaka, Japan) according to the manufacturer’s protocols. The cells were reacted with 250 μL ALP staining solution for 40 min in the dark and observed by inverted microscopy (CKX41, Olympus, Tokyo, Japan). For the mineralization assay, ARS solution (Sigma-Aldrich, MO, USA) was adjusted with 0.5% NH₄OH to pH 4.1–4.3, followed by filtration through a 0.2 μm filter. The cells were incubated in the solution for 40–60 min, dried at room temperature, washed with deionized water, and observed by inverted microscopy (CKX41, Olympus).