Weaners up to 8 weeks of age were fed a standard rodent breeding diet and thereafter a standard rodent maintenance diet (Special Diet Services, South Witham,

UK). All procedures complied with the UK Animals (Scientific Procedures) Act 1986 and were reviewed and approved by the ethics committee of the Royal Veterinary College (London, UK). The magnitude of longitudinal mechanical strain at the tibial midshaft resulting from the loads applied to the tibia was established ex vivo in a sub-sample of male and female Lrp5HBM+ and Lrp5−/− mice and their respective WT littermate controls. In each mouse a single element strain gauge (EA-06-015DJ-120, Vishay Measurement Group, NC) Pexidartinib nmr was bonded with cyanoacrylate adhesive in longitudinal alignment to the medial aspect of the tibia

at 37% of its length from the proximal end. Previous studies have shown that this region corresponds to the site of greatest osteogenic response to similar loading [23]. Strains were measured across a range of peak compressive loads between 8 and 30 N ( Figs. 1 A–B). These peak loads were applied with a ramped trapezoidal waveform using the same servo-hydraulic machine (Dartec HC10, Zwick Roell, Herefordshire, UK) used for in vivo loading. When the Selleck mTOR inhibitor compressive force is applied to the tibia the bone bends in the medial-lateral direction resulting in tension on the medial surface and compression on the lateral surface [24]. From the data in Figs. 1 A–B, three magnitudes of peak load were selected for use in the loading experiment. These were chosen to engender measurable, graded osteogenic responses without causing damage to the bones or joints or the skin through which the loads were applied. Prior to the in vivo loading experiment, strain gauges Bumetanide were used to measure the longitudinal strains applied to the medial surface of the tibia (ex vivo) for each group of mice across a range of compressive forces ( Figs. 1A–B).

The strain engendered at each load (N) was significantly less in the male and female Lrp5HBM+ mice compared with their WTHBM− littermates (p < 0.01) and significantly greater in the male and female Lrp5−/− mice compared with their WT+/+ littermates (p < 0.01). Fig. 1C demonstrates that these strains strongly correlated with the cortical area measured at this site in each group of mice (r2 = 0.83, p < 0.01). Seventeen week old male and female Lrp5HBM+ or Lrp5−/− mice and their respective WT littermates were randomly assigned to one of three loading groups (n = 8/group). While under oxygen and halothane anaesthetic (Merial, Ireland) the right tibia from each mouse was axially loaded on 3 alternate days per week for 2 weeks for 40 cycles/day with a trapezoid waveform, with 14.9 second rest between cycles ( Fig. 1D). The left tibia was used as a non-loaded control to allow side-to-side comparisons for the effects of loading on (re)modelling.