Quantifying Epiphyseal Growth Plate Histology

Masson trichrome–stained proximal tibia epiphyseal sections were used for histological evaluation of the growth plates, which were visualized with a ScanScope CS digital scanner (Aperio Technologies Inc.) under bright-field microscopy. All histological measurements were performed in the central two-thirds of the growth plate sections as previously described (40 (link)). At 2 weeks of age, the beginning of the resting zone was defined as the lower margin of the secondary ossification center. The number of cells in the resting zone was counted per 500 μm growth plate width. For each growth plate section, we performed at least 5 measurements of resting zone height, resting zone cell count, proliferative zone height, hypertrophic zone height, and terminal hypertrophic cell height. For the evaluation of terminal hypertrophic cell height, the height of the lacunae — which reflects the actual hypertrophic chondrocyte height before cells condense during tissue fixation and processing — was measured. For each animal, averages were taken from a minimum of 20 measurements from 4 growth plate sections.