All mice were allowed normal cage activity in between loading sessions. At 19 weeks of age, the mice were euthanized and their tibias dissected free of soft tissue and stored in 70% ethanol. At 14 weeks of age, female and male Lrp5HBM+, Lrp5−/−, WTHBM− and WT+/+ mice (n = 6 to 9) underwent unilateral sciatic neurectomy to remove functional load

bearing of the right tibia [26]. The mice were anaesthetised using halothane and oxygen, the sciatic nerve approached from its dorsal surface and a 3 mm section excised. Forskolin The wound was sutured and the mice recovered in a heated cage. The left tibia served as a control. Three weeks after neurectomy the mice were euthanized and the right and left tibia were extracted and stored in 70% ethanol until they were scanned using microCT. The entire tibias from loaded and sciatic neurectomised groups were scanned ex-vivo at a resolution of 4.9 μm × 4.9 μm

using micro computed tomography (Skyscan 1172, Belgium). Analysis of cortical bone was performed using a 0.49 mm long segment (or 100 tomograms) at Selleckchem Capmatinib 37% of the tibias’ length from their proximal ends. This was the site where the strain gauges were attached and where previous experiments had established a substantial osteogenic response to loading [23]. For analysis of the cortical bone compartment, 2D computation was used and parameters determined for each of the 100 tomograms

which were then averaged. The parameters chosen for cortical bone were: total (periosteally enclosed) area, medullary (endosteally enclosed) area and cortical bone area (total–medullary). For trabecular bone, we analysed a region of secondary spongiosa located distal to the growth plate in the proximal metaphysis and extending 0.98 mm (or 200 tomograms) distally. Woven bone was detected in less than 10% of all loaded mice. Histomorphometric analysis in 2- and 3-dimensions (2D, 3D) was performed by Skyscan software (CT-Analyser v.1.5.1.3). For analysis of cancellous bone the cortical shell was excluded by operator-drawn Dimethyl sulfoxide regions of interest and 3D algorithms used to determine: bone volume percentage (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular spacing (Tb.Sp). Coefficients of variation (CVs) were determined by repeating full scan (including repositioning) reconstruction and analysing the same sample 4 times. The CV of each parameter was determined as the ratio between the standard deviation and the mean. The CVs for relevant parameters are the following: BV/TV: 1.57% and Tb.Th: 1.61% and cortical area: 0.11%.