The mandibles were sectioned into left and right segments, and each peri-implant bone was sectioned again to individualize them. Each peri-implant bone block was fixed in buffered formalin solution, pH 7.0, for 6 days, demineralized in 10% formic acid, and dehydrated through progressing alcohol concentrations and paraffin-embedded. Paraffin blocks were sectioned at 7-μm thick mounted on poly-l-lysine coated glass slides (Sigma–Aldrich, Gillingham, UK) and processed for hematoxylin-eosin staining and for immunohistochemical analysis. Each peri-implant bone paraffin block was longitudinal and colored with hematoxylin-eosin (H&E) to identify sites of new bone destruction or remodeling.

Immunohistochemical analysis was performed on duplicate tissue sections of peri-implant bone from each experimental specimen (9M and IR—randomly chosen). Sections were deparaffinized and rehydrated by rinsing with xylene for 10 min, industrial methylated spirit for 5 min, and more 5 min in tap water. In order put out endogenous peroxidase activity, sections were incubated at room temperature in 3% hydrogen peroxide for 10 min. Two proteins were evaluated due to their sequential expression during bone healing. Collagen type I because it is expressed early in the healing process [24]. Osteocalcin because it is a late marker of bone formation and is expressed during mineralization by osteoblastic cells [24]. To prevent nonspecific protein binding, serum-free blocking agent (DAKO, Hamburg, Germany) was used. The sample was allowed to react for 1 h at room temperature with a primary anti-collagen I antibody (Abcam, Cambridge, UK) and anti-osteocalcin antibody (Takara Biomedicals Europe, France). Immunohistochemical analysis was performed at different thirds of the peri-implant bone (cervical, medium, and apical). Each third was selected at least two times per sample and analyzed. Sample images were captured then observed by means of Leica DM 4000 light microscopy (Leica Cambridge Ltd, Cambridge, UK) incorporating a Leica DFC 320 camera (Leica Cambridge Ltd) for computerized images in histological and immunohistochemistry analysis with a ×40 magnification.