Dataviewer program

The remaining 30 implants with PEO-treated surfaces (15 PEO-Ti and 15 PEO-Ca) were installed into the sheep mandibular body by the extra-oral access following the standard surgical protocol. Investigations on animals were carried out at Stavropol State Medical University (Russia) and were approved by the local ethical committee (extract from protocol No.98 of May 20, 2021).
After 2, 4, and 8 weeks, the material was collected (3 DI from each sheep) and tested by means of microcomputed tomography. The Skyscan 1176 micro-CT scanner (Bruker microCT, Belgium) was used to examine the bone structure. The scanning parameters in the Skyscan 1176 program, v. 10.0.0.0, are as follows: radiograph voltage of 90 kV; radiograph current of 270 μA; 0.1-mm filter diameter; image pixel size of 17.74 μm; tomographer rotation of 360º; rotation pitch of 0.2; frame averaging of 4. The scanned objects were reconstructed in the Nrecon, v. 1.7.4.2 program (Bruker microCT, Belgium) using the following main parameters: smoothing of 2, circle reduction of 20, X-ray hardness of 41, contrast range of all images of 0.015–0.11. Spatial orientation (x, y, z) and selection of separate regions of the reconstructed material were performed using DataViewer program, v. 1.5.6.2 (Bruker microCT, Belgium).

The extracted skulls were studied using a μCT machine (SKYSCAN1173, Ver. 1.6, BRUKER-CT, KONTICH, Belgium) for imaging. The pre-set imaging conditions were 60 μA tube current, 130 kVp tube voltage, 500 ms exposure time, 1 mm aluminum filter, and 0.3° rotation angle. The pixel size was 13.85 μm, and the number of pixels was 2240 × 2240. With the help of a μCT scan, a total of 800 raw high-resolution images were obtained. The NRecon program (Ver 1.7.0.5, BRUKER-CT, KONTICH, Belgium) was used for the cross-sectional reconfiguration. The Dataviewer program (Ver. 1.5.1.3, BRUKER-CT, KONTICH, Belgium) and the Ct-VOX program (Ver. 1.14.4.2, BRUKER-CT, KONTICH, Belgium) were used for 3D reconstruction. The volume of newly formed bone inside the scaffolds was calculated using the difference in grayscale level. In the program, since the HU (Housefield unit) of the scaffold was 400, the HU of the cortical bone was 600 or more, and that of the soft tissue was 100 or less, the grayscale threshold of the new bone was set to 150–350 HU. Newly formed bone volume in the scaffolds was calculated using these programs, according to the following calculation: $$\text{Percent}\text{bone}\text{volume}\left(\%\right)=\text{New}\text{bone}\text{volume}/\text{Total}\text{volume}\text{in}\text{scaffold}\times 100$$

The root surfaces of all crown-bonded specimens were painted with two layers of nail varnish. Specimens were immersed in 50% silver nitrate (AgNO3) in the dark for 12 h followed by washing (water). Specimens were further placed in a photo-developing solution under a fluorescent light for 12 h. For the assessment of microleakage, bonded specimens were securely placed in a micro-CT chamber (Bruker SkyScan 1173 Kontich, Belgium). Scanning configuration included 86 kV voltage, 93 μA anode current, 620 ms exposure time, isotropic resolution of 16 μm image pixel size, brass filter, 0.25 rotation step for 360° angle, frame averaging of four for improved signal to noise ratio, and random movement of eight to minimize ring artifacts. The projected images were reconstructed using N-Recon^® program version 1.6.1.3 (Bruker Skyscan, Kontich, Belgium) to produce cross-sections of images. Images were saved in a 16 bit TIF file format. Reconstructed images were loaded in the Dataviewer^® program (Bruker Skyscan, Kontich, Belgium) software to determine accurate positioning and visual inspection. The volume of silver nitrate penetration was quantified with the CTAn^® program (Bruker Skyscan, Kontich, Belgium) by selecting a binarized threshold value corresponding to the amount (silver nitrate).