*2.5. Immunofluorescence Microscopy (IF) Analysis*

Prior to the sacrifice of the rabbit tibiae, the implants were removed from each tibia and, along with the surrounding bone, the specimens were preserved for 3 h in the refrigerator in the RPMI media, which contained penicillin (50 U/mL) and streptomycin (50 μg/mL). The cells underwent immunostaining and were incubated for 15 min with a protein block (DAKO, Agilent, Santa Clara, CA, USA, X0909) to remove non-specific binding protein. The cells were then incubated for 30 min with a diluted osteocalcin primary antibody (1:100 dilution in 3% bovine serum albumin (BSA), #MA120788, Thermo Fisher Scientific, USA). After being rinsed in PBS, these cells were incubated for 1 h with a diluted secondary antibody (1:200 diluted goat anti-mouse IgG-FITC in 3% BSA, #A10530, Thermo Fisher Scientific, Waltham, MA, USA) in a dark room and washed with PBS. Subsequently, nuclear counterstaining was performed using Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) (1:10,000 dilution) for 5 min. After the counterstaining, the images were obtained by fluorescence microscopy using Axio Observer.A1 (Carl Zeiss, Jena, Germany).

#### *2.6. Transmission Electron Microscopy (TEM) Analysis*

Prior to sacrifice, the implants were removed, and the cells were isolated with a cell scraper and fixed in Karnovsky's solution. After sacrifice, the cells from the bony structures around the area where implants had been placed were collected and fixed in Karnovsky's solution. They were washed in 0.1 M PBS 3 times every 15 min. The specimens were dehydrated through a graded 70–100% ethanol series, exchanged with propylene oxide, and embedded in a mixture of Epon 812 and Araldite. Ultrathin sections (70 nm) were cut using a Leica EM UC6 Ultramicrotome (Leica, Vienna, Austria). A ribbon of

serial ultrathin sections from each bony specimen and implant were collected on copper grids and stained with uranyl acetate and lead citrate. The serial fields were photographed at ×500 magnification using a JEOL 1400-Flash electron microscope (JEOL, Tokyo, Japan) operated at 120 kV.
