Transverse sections (40 μm thick) of tibial cortex were cut at tibia–fibula junction using a diamond wire saw (Well 3241, Norcross, GA, USA). The sections were cover-slipped with Eukitt (Calibrated Instruments, Hawthorne, NY, USA) and mounted unstained for visualization under fluorescent microscopy (Eclipse E400; Nikon, Japan) for quantitative morphometry using image analysis software (Bioquant Image Analysis Corporation, Nashville, TN, USA). Endocortical and periosteal measurements included single- and double-labeled perimeter and interlabel width, which were used to calculate the mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR) at both the endocortical and periosteal

bone surfaces according to the standard guidelines BI 2536 purchase previously published for bone histomorphometry [31]. For

those samples not displaying a double label, a minimum MAR was assigned (0.5 μm/day) and was used to calculate BFR. Quantification of advanced glycation end-product accumulation A fluorometric assay was performed in order to evaluate the extent of AGEs in HFD and LFD bone. The tibial mid-shafts were demineralized using EDTA and confirmed using contact radiographs. The demineralized bone samples C646 research buy were then hydrolyzed using 6 N HCl (24 h, 110°C). AGE content was determined using fluorescence readings taken using a microplate reader at the excitation wavelength of 370 nm and emission wavelength of 440 nm. These readings were standardized to a quinine-sulfate standard and then normalized to the amount of collagen present in each bone sample. The amount of collagen for each sample was determined based on the amount of hydroxyproline, the latter being determined Suplatast tosilate using a chloramine-T colorimetric

assay that recorded the absorbance of the digested samples against a hydroxyproline standard at the wavelength of 585 nm [32]. Mechanical testing Size-dependent measures such as failure load and energy absorption do not account for changes in the bone cross-section area, thereby confounding the effects of bone quality and quantity. To understand the mechanical integrity of the bone and its resistance to fracture, size-independent mechanical properties (yield and maximum stresses, stiffness, and fracture toughness1) also need to be measured [19, 33] as part of a larger plan of study which includes bone distribution and bone quantity measures. Prior to testing, the femora were thawed in room-temperature HBSS, and the size and geometry of all samples were measured with calipers. The left femora were tested in unnotched three-point bending to evaluate bending strength and stiffness. The right femora were tested in notched three-point bending to assess the fracture toughness. For toughness testing, the femoral shaft was sharply notched in the mid-diaphyseal region through the posterior wall using the method described by Ritchie et al. [33].