Edta 2na

The rabbits were anesthetized using intravenous administration with 3% sodium pentobarbital (30 mg/kg) in an ear vein and the animals were subsequently sacrificed via air embolism. The left femoral head was divided into two parts along the coronal plane of the central hole and fixed in 10% formaldehyde solution (Sangon Biotech Co., Ltd., Shanghai, China) at 4°C for 48 h, decalcified for 3 months with 0.27 mol/l EDTA-2Na (Sangon Biotech Co., Ltd.) at 25°C with pH 7.4. H&E and Sudan III (performed on 5 µm and 20 µm sections obtained from the same part of the femoral head, respectively, at room temperature for 15 min), and PATH staining (performed on 7 µm sections at room temperature for 24 h; all Sigma-Aldrich; Merck KGaA) was performed to observe histopathological changes using a light microscope (Olympus Corporation, Tokyo, Japan). Following H&E staining, the number of empty bone cells were counted at a magnification, ×400 to calculate the empty bone trap rate. Following Sudan III staining, the fat embolism number (number of blood vessels with fat embolization in the femoral head cartilage) was counted by eye using a microscope at magnification, ×200. Following PATH staining, the thrombosis number was observed and the thrombosis rate was calculated (thrombosis number/total blood vessels).

After μCT scanning, the radius specimens were decalcified in 12% ethylenediaminetetraacetic acid disodium salt (EDTA-2Na; Sangon Biotech, China) for 4–5 weeks, dehydrated in a graded series of alcohol, and embedded in paraffin for histological sectioning. Sections with a thickness of 6 μm were cut and stained with hematoxylin and eosin (HE). The stained sections were observed microscopically, and newly formed bone was evaluated with Image Pro Plus 6.0 software (Media Cybernetics, USA).