Sigmascan pro

The calvarial samples were subjected to dehydration and embedding. The segments were embedded to show the sagittal sections in paraffin blocks. The paraffin blocks were sliced (5 μm) and stained with hematoxylin and eosin. The detailed staining procedure followed the standard method in the manufacturer’s manual. The selected sections were photographed with a digital camera (DP-73; Olympus, Tokyo, Japan). The images were analyzed with Sigma Scan Pro (SPSS, Chicago, IL). The ratio of the remaining graft was calculated based on the ratio of the remaining graft to the original size of the graft.

The samples were harvested, decalcified in 5 % nitric acid for 5 days, and dehydrated in ethyl alcohol and xylene. After separation of the calvarial bones, the samples were embedded in paraffin blocks. The paraffin blocks were sliced into sections that were then stained with hematoxylin and eosin. The section with the occlusal plane area was selected.
The staining procedure for hematoxylin and eosin staining was as follows. First, de-wax and hydrate paraffin sections. The slide was stained in hematoxylin for 5 min. Overstained sections can easily be differentiated by agitating for a second in acid-alcohol then washing in tap water for 5 min. The slides were immersed in eosin for 30 s and then washed in running tap water for 1 min. The slides were dehydrated and cleared in xylene.
Digital images of the selected sections were captured with a digital camera (DP-73; Olympus, Tokyo, Japan). The images were analyzed by Sigma Scan pro (SPSS, Chicago, IL). The thickness of the masseter muscle was measured from the perpendicular line to the mandibular ramus.

The calvarial samples were harvested, decalcified in 5 % nitric acid for 5 days, and dehydrated in ethyl alcohol and xylene. After separation of the parietal bones through the midline sagittal suture, the calvarial samples were embedded in paraffin blocks. The paraffin blocks were sliced into sections that were then stained with hematoxylin and eosin. The section with the largest defect area was selected, along with sections 50 μm proximal and distal to the largest defect section.
The staining procedure for hematoxylin and eosin staining was as follows. First, de-wax and hydrate paraffin sections. The slide was stained in hematoxylin for 5 min. Overstained sections can easily be differentiated by agitating for a second in acid-alcohol, then washing in tap water for 5 min. The slides were immersed in eosin for 30 s and then wash them in running tap water for 1 min. The slides were dehydrated and clear in xylene.
Digital images of the selected sections were captured with a digital camera (DP-73; Olympus, Tokyo, Japan). The images were analyzed by Sigma Scan pro (SPSS, Chicago, IL). The new bone formation was calculated as the percentage of newly formed bone in the calvarial defect area.

After the μ-CT analysis, the samples were decalcified in 5% nitric acid for 2 weeks and dehydrated in ethyl alcohol and xylene. The samples were separated through the midline sagittal suture and embedded in paraffin blocks. The paraffin blocks were sliced into sections that were then stained with hematoxylin and eosin. The sections that showed the sagittal image of the parietal bone and bone grafted area were selected for histologic analysis. Digital images of the selected sections were taken with a digital camera (DP-73; Olympus, Tokyo, Japan). The images were analyzed by Sigma Scan pro (SPSS, Chicago, IL, USA). The amount of newly formed bone was calculated as the percentage of the total region of the bone graft area.