(Margate, UK) and group-housed in sterilized polypropylene cages with free access to water and a maintenance diet containing 0.73% calcium, 0.52% phosphorus, and 3.5 IU/g vitamin D (RM1; Special Diet Services Ltd., Witham, UK) in a 12-h light/dark cycle, with room temperature at 21°C ± 2°C. The mice were used for experiments when almost skeletally mature at 19 weeks of age. 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). In vivo external mechanical loading The apparatus and protocol for axial loading of the mouse tibia have been reported previously [24–26]. Non-invasive, dynamic loads [0.1 s trapezoidal-shaped pulse (period 0.025 s loading, 0.05 s hold, and 0.025 s unloading); GDC-0994 order 10 s rest time between each pulse; 40 cycles/day] were applied between the right flexed knee and ankle under isoflurane-induced anesthesia (approximately 7 min/day). This rest time enhances the osteogenic potential of loading [27]. The flexed joints are positioned in concave cups; the upper cup, into which the knee is positioned, is attached to the actuator arm of a servo-hydraulic loading machine (Model HC10; Zwick Testing Machines Ltd., Leominster, UK) and the lower cup to a dynamic load

cell. The servo-hydraulic mechanism of the loading machine operates to apply controlled dynamic compressive loads axially to the tibia. The left non-loaded tibia MI-503 was used as an internal control, as has previously Resveratrol been validated in the present model [25] and confirmed by others in the rat ulna axial loading model [28]. Normal activity within the cages was allowed between loading periods. In the present study, a peak load of 13.5 N was CYT387 clinical trial selected since this has previously been shown to induce significant bone gain through an increase in bone formation at both cortical and trabecular sites [7, 25]. Assessment of loading-induced strain Single element strain gauges were attached ex vivo, in a longitudinal orientation, to the proximal

lateral tibial shaft of similar 19-week-old female C57BL/6 mice. These showed that a peak load of 13.5 N engendered a peak longitudinal strain of approximately 1,800 με in that region. Since the mouse tibia is not large enough to permit attachment of multiple gauges, the predictions of the normal strain distribution throughout the bone induced by loading were extended to full bone normal strain characterizations using finite element (FE) analysis. A voxel-based FE model (voxel size, 40 μm) was constructed by processing the micro-computed tomography (μCT) images using a computer program developed in house in the Department of Orthopaedics and Sports Medicine, University of Washington [29]. The bone material properties were assumed to be homogeneous, linear, and isotropic (Young’s modulus, 17 GPa; Poisson’s ration, 0.3) in order to approximately match the above strain gauge reading.