2.5.5. Micro Computed Tomography (μCT)

The collected samples were fixed in phosphate-buffered formaldehyde (pH 7.4, 0.1 M PBS) and dehydratied in ethanol 70%. Three dimensional (3D) μCT images were obtained to determine bone mineral densities and bone volumes surrounding implants in defect areas. Specimens were wrapped using Parafilm M® (Pechiney Plastic Packaging, Chicago, IL, USA) to prevent dry during scanning, and scanned at 130 kV and 60 μA with a resolution of 12μm pixels using a bromine filter (0.25 mm) (Skyscan-1173 Skyscan®, Kontich, Belgium). In addition, calibration rods of standard bone mineral densities were also scanned. Cone-beam reconstruction (version 2.15, Skyscan®, Kontich, Belgium) was performed, and all scan and reconstruction parameters that were applied were identical for all the specimens and calibration rods. The collected data were analyzed by a CT analyser (version 1.4, Skyscan®, Kontich, Belgium). The region of interest (ROI) was defined as annular region of thickness 1 mm surrounding a defect area in the marginal peri-implant from the first microthread to the last microthread. Bone volumes (mm3) were measured in this region (Figure 2) and were expressed as percentages of the total ROI volumes (mm3).

**Figure 2.** Micro-computed tomography (μCT) images in each group. (**a**) Buccolingual section image; (**b**) three-dimensional (3D) image; (**c**) Horizontal section image; and (**d**) Mesiodistal section image. Region of interest (ROI) was defined as an annular area of thickness 1 mm surrounding the defect area (red circle) in the marginal portion of the peri-implant from the first microthread to the last microthread. Bone volumes were measured in this ROI.

#### 2.5.6. Histologic and Histometric Analysis

The harvested specimens were imsersed in neutral buffered formalin (Sigma Aldrich, St Louis, MO, USA), fixed for two weeks, and dehydrated in ascending concentrations of ethanol (70%, 80%, 90%, and 100%), and embedded in Technovit 7200 VLC resin (Heraeus KULZER, South Bend, IN, USA). Embedded specimen blocks were sectioned longitudinally from the center of implant using an diamond cutter (KULZER EXAKT 300, EXAKT, Norderstedt, Germany). The final slides (30 μm) were prepared from initial 400 μm slides by grinding sections using an grinding machine (KULZER EXAKT 400CS, EXAKT, Norderstedt, Germany). Hematoxylin-eosin staining was perfomed, and images were captured by computer connected to light microscope (Olympus BX, Olympus, Tokyo, Japan) attached

to a CCD camera (Polaroid DMC2 digital Microscope camera (Polaroid Corporation, Cambridge, MA, USA). All assessments were made by one skilled investigator using SPOT Software (Ver. 4.0, Diagnostic Instrument, Inc., Sterling Heights, MI, USA).

The following parameters [36] were evaluated:


After measuring the percentage of bone to implant contact (BIC, %), the ratio of bone formation area on intra-threads of implant to overall threads was calculated to determine intra-thread bone density (ITBD, %). Height of newly formed marginal bone by implants was measured. images of specimens were captured at a magnification of ×12.5 and ×40. For the histometric analysis, a magnification of ×40 was used.
