Hematoxylin-eosin-stained section images were digitized and analyzed in order to recognize the presence of native bone tissue by the presence of osteocyte lacunae-containing cells and the newly formed bone tissue recognized by the absence of lacunae. Also, the characteristics of peri-implant bone, presence or absence of bone fractures, and the shape and contour of bone grooving resultant of the trephine action were analyzed. Histological analysis was performed in images of the semi-serial slices of each peri-implant bone. They were captured by a digital camera (Samsung, South Korea) coupled to a light microscope (Zeiss, Germany) with original ×200 magnification and resolution of 600 dpi. Images around 116–80 cm were captured of each third of the peri-implant bone. Then, a digital framework of entire peri-implant bone was built by the combining three images.

Immunohistochemical analysis also was performed on three thirds on each sample with collagen I and osteocalcin. The same image capture and construction were made, but they were measured, and the value was defined by the positive-staining samples and was used to automatically analyze images of all samples that were stained under identical conditions for both proteins and implant removals.

In the analysis of both mandible sides, the images were acquired at ×200 magnification using a Nikon E600 microscope (Nikon Instruments Inc, Melville, USA). The integral optical density (IOD) of target protein was measured with Image-Pro Plus 5.0 (Media Cybernetics, Rockville, MD, USA). In the process of measurement, the values were defined firstly by determining the positive staining of control sections and were used to automatically analyze images of all samples that were under identical conditions (u/pixel) [25].

Statistical analyses were performed with SPSS software (SPSS, Chicago, Ill). Student’s t test was used to determine statistical differences in the values between the 9M and IR samples. Data were presented as means with standard deviations. The level of significance was set at 5% (P < 0.05).