Aluminum conductive tape

The residual methylmethacrylate embedded tibia sample blocks from bone histomorphometry were used for the osteocyte analysis. Blocks were trimmed and the bone surface was sequentially ground with silicon carbide sandpaper of increasing grid number (Scientific Instrument Services Inc., NJ, USA). The sample surface was then carbon coated by vacuum evaporation (Auto 306 Vacuum Coater, Boc Edwards, UK) followed by fixation on the specimen mount with aluminum conductive tape (Ted Pella Inc., CA, USA). A digital scanning electron microscope (SEM, Supra 55 VP, Zeiss, Oberkochen, Germany, Center for Nanoscale Systems in Harvard University, Cambridge, MA) was employed with an accelerating voltage of 20kV, a working distance of 10mm and 500× magnification for taking backscattered electron images of a standardized tibial midshaft area located 4.5mm distal from the tibia-fibula junction. Images were analyzed with the Image J software (NIH, MD) for measuring osteocyte lacunae area and density (Qing et al., 2012 ). The total osteocyte lacunae area was measured in the number of pixels and then converted to the metric system. The total area was divided by the number of osteocytes to get the lacunae area. The number of osteocyte was divided by mineralized bone area to get the lacunae density.