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The serum samples were immediately separated from the blood by centrifugation at 1,000 �� g for 10 min, and the serum was frozen and stored at ?80��C until used for analysis. Femurs were isolated for evaluation, and the soft tissue was removed. Femurs were wrapped in saline-soaked gauze and stored at ?80��C until used for analysis. Bone morphometry and density The right femur was fixed in 10% neutral buffered formalin, degreased in 100% ethanol, re-fixed in cyanuric chloride Anticancer Compound Library price (Wako Pure Chemical Industries, Osaka, Japan) and decalcified with formic acid. Thin sections of the right femur were made with a sliding microtome (Leica SM-2000R; Leica Biosystems, Nussloch, Germany). The sections were stained with hematoxylin and eosin (H&E) stain. The amount and width of each trabecular bone, correlated with bone strength, was traced distal to epiphyseal line using a bio-imaging navigator (Biorevo BZ-9000; Keyence, Osaka, Japan). The range of calcified bone was extracted. The area of the calcified Ceramidase bone in the traced range was calculated using analytical software (Hybrid Cell Count Software; Keyence). Bone strength The bending strength of the left femur was measured with a three-point bending test using a mechanical testing machine (AG-X; Shimadzu, Kyoto, Japan). The specimen was placed horizontally in the loading section of the machine. The center of the diaphysis was pressed with 50 kgf; the distance from the support point was 15 mm. The breaking strength in Newtons (N) was used as a measure of bone strength. Serum biochemistry Biochemical measurements, including serum calcium, phosphorus, total protein and albumin were measured with an auto-analyzer (Hitachi 7180; Hitachi High-Technologies, Tokyo, Japan). Bone metabolism Serum tartrate-resistant acid phosphatase 5b (TRACP-5b), a bone resorption marker, and bone-specific alkaline phosphatase (BAP), a bone formation marker, were used as biochemical markers of turnover and, measured using an enzyme immunoassay Selleckchem AZD8055 (Rat TRACP-5b ELISA; Cusabio Biotech Co., Ltd., Wuhan, China). Statistical analysis Continuous variables are expressed as mean �� standard deviation. Comparisons between groups were made using the Mann-Whitney U test. P