All samples underwent dehydration using increasing grades of alcohol and embeded in light-curing resin (Technovit 7200 VLC + BPO, Kulzer and Co., Wehrheim, Germany). Samples were further handled using a cutting and grinding equipment (Exakt Apparatebau, Norderstedt, Germany). Thin- ground sections of around 100 µm were prepared in a plane with the sagittal suture and through the center of the defect, before being stained with Levai-Laczko dye, a variant of the Giemsa dye that allows to distinguish reliably between old bone and new bone [26 (link)].
The slices were scanned using an Olympus BX61VS digital virtual microscopy system (DotSlide 2.4, Olympus, Tokyo, Japan) with a 20× objective resulting in a resolution of 0.32 µm per pixel and then were segmented manually using Photoshop CS 4 (Adobe Systems Inc., San Jose, CA, USA). The segmented images were then measured in Fiji using the Bone J plugin based on the color-coded thresholding that was obtained from the correspondent histograms [27 (link)]. The histomorphometric analysis was performed at three regions of interest (ROIs) representing the central compartment within the defect margins, the adjacent ectocranial compartments, and the outer compartment on the surface of the host’s cortical bone.
The slices were scanned using an Olympus BX61VS digital virtual microscopy system (DotSlide 2.4, Olympus, Tokyo, Japan) with a 20× objective resulting in a resolution of 0.32 µm per pixel and then were segmented manually using Photoshop CS 4 (Adobe Systems Inc., San Jose, CA, USA). The segmented images were then measured in Fiji using the Bone J plugin based on the color-coded thresholding that was obtained from the correspondent histograms [27 (link)]. The histomorphometric analysis was performed at three regions of interest (ROIs) representing the central compartment within the defect margins, the adjacent ectocranial compartments, and the outer compartment on the surface of the host’s cortical bone.
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