Tartrate-resistant acid phosphatase (TRAP) staining

Cells were fixed with 4% paraformaldehyde, washed with distilled water, and stained for TRAP in a humid and light-protected incubator for 1 h using the Leukocyte Acid Phosphatase Assay kit (Sigma). TRAP-positive cells containing 2 or more nuclei were regarded as osteoclasts.

A microscope (80i Eclipse, Nikon, Japan) was used to capture images of TRAP-stained pouch sections and observe TRAP-positive osteoclast-like cells (400× magnification). Due to the blurred boundaries of TRAP-positive cells stained dark red in the calvaria sections, analysis software (Image-Pro Plus 6.0) was used to quantify the number of TRAP-positive osteoclast-like cells on the bone-membrane interface by comparing the dark red colored area with the total implanted bone area (400× magnification).