Evaluation methods

Plane radiographs
The experimental animals were sacrificed and plane radiographs of the full length of the tibia were obtained at 45 kV for 12 ms. The implants were evaluated for loosening, pullout, or differences in the surrounding osseous tissue.

Micro-CT evaluation
The experimental animals were sacrificed and micro-CT was performed on all specimens by harvesting samples that included the implanted part of the tibia. Micro-CT (SkyScan 1173, Zwijnaarde, Belgium) was performed using an aluminum filter in a mid-resolution of 20 μm at 30 kV, 60 μA. The newly formed bone area ratio (the ratio of the area of new bone formation at the space between two threads in the pitch) and the bone-to-implant contact ratio (the ratio of bone directly attached to the thread of the screw) were evaluated. The quality of new bone was evaluated based on a total of 42 pictures analyzed by CTAn, a micro-CT analysis program. The bony tissue of the new bone was analyzed with an ROI (region of interest) at a width of 0.99 mm and a height of 2.48 mm. The bone fraction directly attached to the screw thread was analyzed with an ROI at a width of 0.97 mm and a height of 2.48 mm. For quantification of the quantity and quality of the new bone, the following items were evaluated: percent bone volume (bone volume/trabecular volume), specific surface (bone surface/volume ratio), trabecular bone pattern factor (the parameter of the connectedness of these bone patterns), trabecular thickness (the thickness of the trabeculae), trabecular number (the number of trabeculae), and trabecular separation (the distance between the trabeculae).

Histologic evaluation
The full length of the tibia specimen was fixed in formalin for 5 days and the tibiae containing each specimen were divided into two parts. Gross section tissue was put into a cassette and washed for 6 h and then dehydrated in 100 % alcohol. The tissue was put into methacrylate-based chemical curing resin and stirred for 2 days, and then it was stirred and embedded by dissolving in benzoyl peroxide. The block was trimmed and sectioned along the longitudinal axis of the dental implant fixture using an EXAKT cutting instrument (BS-3000 N). Moreover, a 4 μm section was made at the right center of the implant along the sagittal plane and included the surrounding tibia. Grinding was carried out using an EXAKT grinding machine (4,110), and an acrylic slide attachment was performed. The slide was stained with hematoxylin and eosin (H&E) staining and viewed with a light microscope to determine the bond between the bone and the implant and the new bone formation around the implant. Histomorphometric evaluations of the dental implant fixtures were carried out after a scaled calibration using a morphometry program (LEICA IM50 Image Manager, version 4.0). The bone-to-implant contact ratio was measured in the marrow space of the tibia. All measurements were performed using a (X12.5) magnification objective.