Micro-CT scanning Vertebrae were thawed to room temperature and scanned with a desktop micro-CT system (microCT40, Scanco Medical AG, Bruettisellen, Switzerland) at an isotropic resolution of 16 μm (55 kV, 145 μA, 500 projections per 180°, 200 ms integration time). After scanning, samples were frozen again until mechanical testing. Images were Gaussian filtered (sigma = 0.8, support = 1 voxel) and binarized to separate bone from background using a global thresholding procedure [35]. From the CT scans, the trabecular region was manually selected starting ten slices below the cranial growth plate and ending

ten slices above the caudal growth plate, resulting in a trabecular region of approximately 5 mm in axial direction. From this region, six bone structural parameters (bone volume fraction APR-246 (BV/TV), connectivity density (Conn.D), structure model index (SMI), trabecular number (Tb.N), trabecular thickness (Tb.Th), and separation (Tb.Sp)) were automatically determined. Cortical bone was semi-automatically delineated from the CT scans by drawing contour lines, using the same set of slices as used for trabecular bone measurements. Specimen preparation To achieve plano-parallel ends, vertebrae were fixed

in a custom-made jig. A double-blade, wafering, low-speed diamond saw (Isomet, Buehler, Lake Bluff, IL, USA) was used under constant saline irrigation to remove cranial and caudal ends including the growth plate. After sawing, the exact HKI-272 mouse vertebral height was measured using a caliper and Sorafenib molecular weight found to be 4.06 ± 0.09 mm (mean ± SD). An example of a processed vertebra can be seen in Fig. 1. A single-blade, wafering, low-speed diamond saw was used under constant saline irrigation to remove all posterior pedicles and processes. Anterior elements were clipped off using a rounger, resulting in a separated vertebral body. CT scans taken for pilot samples had shown no splintering Parvulin resulted from sawing and clipping. Vertebrae were kept frozen in a

0.9% saline solution until fatigue testing. Fig. 1 Schematic of fatigue loading test. The lower platen, designed as a cup, contained the vertebra. The top platen, smaller in diameter than the cup, was lowered onto the vertebra to a compressive preload of 5 N, at which point the displacement was set at zero. A 0.9% saline solution containing protease inhibitors was added to the cup to prevent the vertebra from dehydrating and to inhibit microorganism growth Fatigue compression tests Vertebrae were thawed to room temperature prior to mechanical testing. In total, all samples were frozen and thawed for three cycles. Previously, five cycles of freezing and thawing has been found not to affect mechanical properties determined in a static, compression [36], and indentation test [37]. Therefore, we assumed that fatigue properties determined in our study were not affected by the freezing and thawing.