Histomorphometry.

The right tibiae were fixed in 4% PFA overnight, washed under tap water, and then decalcified in 0.5 M EDTA (pH 7.4) for 3 wk before embedding in paraffin. Six-micrometer-thick sections were cut for H&E staining. The left tibiae were processed for embedding in methylmethacrylate. Six-micrometer-thick longitudinal sections were cut with a Polycut-S motorized microtome (Reichert) and stained with Goldner’s trichrome solutions. Images corresponding to the secondary spongiosa region were acquired via a Nikon Eclipse 90i and quantified using Bioquant Osteo Software (Bioquant Image Analysis). Osteoblast number per bone surface and osteoclast number per bone surface were calculated as described earlier (11). The thicknesses of the proliferative zone and hypertrophic zone of the growth plate were expressed as the average of 12 measurements that were taken at sites that were evenly distributed across the entire growth plate in the paraffin sections as described previously (55). The proliferative zone is defined as the area that contains flattened cells in a longitudinal and columnar arrangement. The hypertrophic zone is defined as the area at which the cell size starts to increase to the area where cartilage becomes bone.