Scanscope cs digital scanner

High-resolution images of 6 μm sections were obtained using either Panoramic MIDI II 2.0.5. scanner or ScanScope CS digital scanner (Aperio Technologies, United States). After calibration, measurements were obtained using Caseviewer (3DHISTECH) and ImageScope (Aperio) softwares. The analysis was carried out in the recipient femoral epiphyses (n = 3 for each timepoint) of the recipient growth plate (native GP), recipient articular cartilage (native AC), growth plate cartilage ectopically placed at the joint surface (GP flip), and articular cartilage ectopically placed inside the epiphysis (AC flip) on Masson’s trichrome stained slides (n = 3, per group and timepoint). All measurements were performed on one slide per sample. For thickness/height measurements of different tissues, 10 individual measurements evenly distributed over the transplanted tissues were obtained and averaged unless stated otherwise.
In order to quantitate the remodeling resulting from hypertrophic inhibitory effect of a putative synovial factor, the distance from the articular surface to the first Col10a1-expressing cell in native AC and GP flip and the distance from the edge of the secondary ossification center to 30 representative Col10a1-expressing cells in native AC and GP flip were assessed (n = 3 per timepoint).

Histological evaluations were performed on Masson's trichrome-stained proximal tibia sections, visualized using a ScanScope CS digital scanner (Aperio Technologies, Inc) under bright-field microscopy. All histological measurements were performed in the central two-third of the proximal tibia growth plate sections. Heights were measured parallel to the chondrocyte columns. Column density was calculated as the number of columns per 500 μm growth plate width. Hypertrophic chondrocytes were operationally defined by a height ≥10 μm (ref. 38 (link)). The terminal hypertrophic chondrocyte was defined as the cell in the last lacuna that was not invaded by metaphyseal blood vessels. For each growth plate section, we performed at least three measurements of PZ height, HZ height and terminal hypertrophic cell height. For terminal hypertrophic cell height, the height of the lacunae, which reflects the actual hypertrophic chondrocytes height before cells condense during tissue fixation and processing, were measured. We also counted the number of proliferative and hypertrophic cells in at least three intact columns from each growth plate section. For each animal, averages were taken on a total of at least 24 different measurements from eight growth plate sections.

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.