Both human core bone samples and mouse hind limbs were excised, cleaned of soft tissue, and fixed in 3.7% formaldehyde for 72 hours. Isolated bone tissue were dehydrated in graded alcohols (70 to 100%), cleared in xylene and embedded in methyl methacrylate. Plastic tissue blocks were cut into 5µm sections using a Polycut-S motorized microtome(Reichert-Jung, Nossloch, Germany).
After the mouse bone sections were used to measure the fluorochrome labeled surface and interlabel width, they were deplasticized in xylene and then stained with Goldner’s Trichrome.
Randomly selected regions of interest (ROIs) within three sections per limb were visualized for fluorochrome labeling using a Nikon Eclipse 90i microscope and Nikon Plan Fluor 10X objective. ROIs from the same sections were visualized using a Nikon Eclipse 90i microscope and 4X and 20X objectives for Goldner’s Trichrome staining. Image capture was performed using NIS Elements Imaging Software 3.10 Sp2 and a Photometrics Coolsnap EZ camera. The Bioquant Osteo II digitizing system (R&M Biometrics, Nashville, TN) according to the manufacturer’s instructions, or sequentially Adobe Photoshop® and Image J software, were used for image analysis. The following primary measurements for dynamic parameters of bone formation were collected from the trabecular surface in defined ROIs (100 µm distal to the growth plate and 50 µm in from the endosteal cortical bone) at 100X magnification: single-label perimeter (sL.PM), double-labeled perimeter measured along the first label (dL.Pm) and interlabel distance. The same sections were then evaluated under brightfield microscopy after Goldner’s Trichrome staining to determine static parameters of bone formation including: tissue volume (TV), bone volume (BV) and osteoid volume (OV).