Bones were decalcified in Cal-Ex II Fixative/Decalcifier (Fisher Scientific, Pittsburgh, PA, USA) for 4 to 6 days or in 14.5% EDTA (pH 7.2) for 4 weeks. Samples then were embedded in paraffin, and 5-μm-thick coronal sections at the interproximal area between the first and second maxillary molars were made. Thus each section included a complete cross section through the entire maxilla, which allowed a side-by-side comparison of the bone, teeth, and soft tissues from the ligature (right) and nonligature (left) sites.
To quantify the area of osteonecrosis and periosteal thickness, hematoxylin and eosin (H&E)–stained slides were digitally scanned using the Aperio XT automated slide scanner and the Aperio ImageScope Version 10 software (Aperio Technologies, Inc., Vista, CA, USA). One author (SD) marked the area(s) of osteonecrosis, defined as loss of more than five contiguous osteocytes with confluent areas of empty lacunae, and the total area was calculated by the ImageScope software. The ruler tool in ImageScope was used to measure the greatest area of buccal periosteal thickness on both the ligature and nonligature sides. Numbers of empty and total osteocytic lacunae were counted manually on the digital whole-slide image over a 1-mm-long and 0.25-mm-wide area of bone (length and width measured with the ImageScope ruler tool) at the buccal alveolus adjacent to the D1 root.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining using the DeadEnd Colorimetric TUNEL System Kit (Promega, San Luis Obispo, CA, USA) was performed on the section adjacent to that used for the H&E stain. For TUNEL quantification, TUNEL+ osteocytes and total osteocytes were counted manually within 1 mm adjacent to osteonecrotic foci or (if osteonecrosis was not present) within a 1 mm along the buccal alveolar bone.
Using a standard brightfield microscope, areas of mucosa overlying the alveolar crest with the greatest inflammation were identified. The high-power (×40) field with the greatest numbers of inflammatory cells was selected, and the numbers of polymorphonuclear cells and lymphocytes were counted manually.
All histology and digital imaging were performed at the Translational Pathology Core Laboratory (TPCL) at UCLA.
To quantify the area of osteonecrosis and periosteal thickness, hematoxylin and eosin (H&E)–stained slides were digitally scanned using the Aperio XT automated slide scanner and the Aperio ImageScope Version 10 software (Aperio Technologies, Inc., Vista, CA, USA). One author (SD) marked the area(s) of osteonecrosis, defined as loss of more than five contiguous osteocytes with confluent areas of empty lacunae, and the total area was calculated by the ImageScope software. The ruler tool in ImageScope was used to measure the greatest area of buccal periosteal thickness on both the ligature and nonligature sides. Numbers of empty and total osteocytic lacunae were counted manually on the digital whole-slide image over a 1-mm-long and 0.25-mm-wide area of bone (length and width measured with the ImageScope ruler tool) at the buccal alveolus adjacent to the D1 root.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining using the DeadEnd Colorimetric TUNEL System Kit (Promega, San Luis Obispo, CA, USA) was performed on the section adjacent to that used for the H&E stain. For TUNEL quantification, TUNEL+ osteocytes and total osteocytes were counted manually within 1 mm adjacent to osteonecrotic foci or (if osteonecrosis was not present) within a 1 mm along the buccal alveolar bone.
Using a standard brightfield microscope, areas of mucosa overlying the alveolar crest with the greatest inflammation were identified. The high-power (×40) field with the greatest numbers of inflammatory cells was selected, and the numbers of polymorphonuclear cells and lymphocytes were counted manually.
All histology and digital imaging were performed at the Translational Pathology Core Laboratory (TPCL) at UCLA.
