2.5.3 Histological staining

Specimens were decalcified in 10% ethylenediaminetetraacetic acid for 3 weeks at room temperature while being subjected to agitation on a shaker. Specimens were dehydrated in a series of ethanol solutions with concentrations ranging from 75% to 100%, embedded in a paraffin-embedding machine (TKD-BMC, Hubei, China), and sliced into 5-µm-thick posterior sections using a microtome (RM2235, Leica, Germany). Following that, hematoxylin-eosin (HE) staining, Masson’s trichrome staining, saffron-solid green (S&G) staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed separately according to the manufacturer’s instructions. The images were scanned using a NanoZoomer digital pathology scanner (NDP; Hamamatsu, Japan). Three slices were randomly selected based on HE staining, and new bone areas were automatically measured and calculated using Image-Pro Plus 6.0 (IPP). The formula used to calculate new bone area is as follows: New bone area (%) = new bone area/tissue area × 100%.