Precision saw

Thin sections were created with a precision saw (EXAKT Advanced Technologies, Norderstedt, Germany) and further reduced to 100–120 µm and polished with a special grinding machine (EXAKT Advanced Technologies, Norderstedt, Germany). With the desktop X-ray unit (Faxitron Cabinet X-ray Systems, Faxitron X-ray, Illinois, IL, USA) at X-ray voltage of 14 kV and 0.25 mA for 2.5 min exposure time, microradiographic images were taken on tooth films of 3.1 x 4.1 cm (Kodak Insight, Rochester, NY, USA). After their development, the microradiographies were digitized with a scanner in grayscale (16 bit, 4800 dpi) (Epson Perfection 4990 Photo, Seiko Epson, Seoul, Korea) and stored as uncompressed files in TIFF format. The images were then analysed with Bioquant OSTEO^® software (BIOQUANT Image Analysis, Nashville, TN, USA). A region of interest (ROI) of 1 × 1 cm was defined for evaluation.

A cone beam computed tomography (KaVo 3D eXam, Kavo Dental, Biberach/Riss, Germany) was used to locate the specimens before further preparation. The specimens were then dissected and fixed by immersion in 1.4% paraformaldehyde at room temperature. Specimens were dehydrated through an ascending alcohol series using a tissue processor (Shandon Citadel 1000, Thermo Shandon, Frankfurt, Germany). Xylene was used as an intermediate fixing agent and Technovit 9100 (Heraeus Kulzer, Wertheim, Germany) for embedding. The polymerization was performed in a cold atmosphere (4 °C) to avoid a negative influence of the polymerization heat. After 20 h, the specimens were completely polymerized and cut through their longitudinal axis in the middle of the graded bone blocks with a precision saw (EXAKT Advanced Technologies, Norderstedt, Germany).