Discovery v8

After specimens were tested, fracture type was evaluated with a stereomicroscope (1.5× magnifications) (Discovery V8, Zeiss, Oberkochen, Germany) and classified as adhesive fracture, cohesive fracture, and mixed fracture. A cohesive failure occurs when the fracture occurs completely within the dentin structure or within the restorative material; an adhesive failure occurs when the bond fails at the interface of the adhesive and the restorative material; and it is considered mixed when a combination of adhesive and cohesive fractures occurs [4 (link)].

Dye penetration extent was evaluated with a stereomicroscope (1.0× magnifications) (Discovery V8, Zeiss, Germany) and scored between 0 and 3 for each side of every tooth. Score 0 was attributed to the absence of dye penetration; score 1 was attributed to stain at the interface; score 2 was attributed to stain across the interface < 1 mm; and score 3 was attributed to stain across the interface ≥ 1 mm. The highest score for each tooth was the one used (Figure 3).

Hamsters were nasally anesthetized with 4% sevoflurane in O₂. The right eye was used as healthy control, and the left eye was used for induction of A. griffini keratitis, except for the sham group, who were inoculated with saline solution at 0.9% only. A total of 50,000 trophozoites in a volume of 5 μl resuspended in saline solution at 0.9% were inoculated into each cornea using a 34 G needle and a stereoscope (ZEISS Discovery.V8).

The functionality of the osteoclasts was analyzed by using two different resorption assays: Osteo-assay Plates (#3987, Corning, Corning, NY, USA) and ivory slices. The Osteo-assay Plates are coated with inorganic three-dimensional crystalline material (bone biomimetic synthetic surface). Ivory slices were cut with a low speed water-cooled diamond saw. The slices had a diameter of 7 mm and a thickness of 0.5 mm. Osteoclasts were generated from hPBMCs in monoulture and co-cultures with cementoblasts as described above. After 21 days of culture, the cells were lysed by 2.5% chlorinated soda solution for 5 min at room temperature and washed by distilled water. Ivory slices were stained with a solution of 1% toluidine blue (#89640, Sigma-Aldrich, St Louis, MO, USA) in 1% sodium borate (#S9640, Sigma-Aldrich, St Louis, MO, USA) for 4 min. Resorption pits were observed and quantified using photomicrographs from five randomly chosen fields by inverted phase-contrast microscopy for Osteo-assay Plates and stereomicroscopy Discovery V8 (Zeiss, Oberkochen, Germany) for the ivory slices.

Whole-mount Oil Red O stain was performed as described (Kim et al., 2015 (link)). For histologic analysis, larvae were fixed in 10% formalin and embedded in paraffin. 6 μm sections were stained with hematoxylin and eosin (H&E). Images were obtained on a Carl Zeiss Discovery V8 stereomicroscope using a Zeiss Axioncam MRc5 camera (Carl Zeiss, Jena, Germany).