More than 100 indents were made in the selected region of size varying from 300 to 500 μm. selleckchem A maximum load of 5000 μN was used. Anatomical areas were selected based on qBEI images, and results were normalized for calcium content. The elastic modulus was calculated using the method of Oliver and Pharr [29], by fitting the unloading curve with a second order

polynomial, differentiating and therefore evaluating the elastic recovery at maximum load to determine the contact depth. The parameters measured during the experiment were peak load (Pmax), peak displacement hmax, contact area Ac, and stiffness S. The stiffness was calculated from the slope of the initial unloading curve; the region between 20 and 95% of the maximum load was used to determine the slope of the unloading curve. The hardness H and reduced modulus Er are calculated from unloading contact stiffness, S, and the indenter contact area Ac: H=Pmax/AcH=Pmax/Ac Er=π1/2S/2Ac1/2Er=π1/2S/2Ac1/2 Thin sections (~ 4 μm) were cut from the L5 vertebrae, and spectral images acquired in the area of trabecular bone using a Bruker Equinox 55 (Bruker Optics) spectrometer interfaced to a Mercury Cadmium Telluride (MCT) focal plane array detector (64 × 64 array) imaged onto the focal plane of an IR microscope (Bruker Hyperion 3000; Bruker Optics). Each area imaged was 400 × 400 μm, corresponding to an optimal spatial resolution of ~ 6.3 × 6.3 μm.

Alectinib chemical structure Spectral resolution was 4 cm− 1. Background spectral images were collected under identical conditions from the same BaF2 windows at the beginning and end of each experiment to ensure instrument stability. Both instruments were continuously powered to minimize warm-up instabilities and purged with dry-air (Bruker Optics) to minimize the water vapor and CO2 interference. Following this, individual spectra were extracted

from trabecular surfaces that were exhibiting either primary mineralization packets, or resorption pits, based on the previously acquired qBEI images (six different trabecular surfaces per animal were analyzed). BCKDHB The individual spectra were processed as published elsewhere to derive the following spectroscopic parameters: (i) Mineral/matrix ratio (integrated areas under the phosphate (mineral) 900–1200 cm− 1 and amide I 1592–1728 cm− 1 (matrix; mainly collagen) absorbance peaks, respectively; corresponds to ash weight measurements) [30], (ii) mineral maturity/crystallinity (through curve-fitting of the phosphate (mineral) 900–1200 cm− 1 peak and the calculation of the 1030 to 1020 cm− 1 sub-band peak area) [31] and [32], and (iii) the ratio of PYD/divalent collagen cross-links (through curve-fitting of the Amide I and II peaks and the calculation of the 1660 to 1690 cm− 1 sub-band peak area) [33]. For each animal, the values of each parameter at a particular anatomical site (forming or resorbing) were averaged and the resultant value was treated as a single statistical unit.