Grinding system

The specimens were fixed with 4% paraformaldehyde, dehydrated with a graded series of ethanol solutions (60%, 80%, 90%, and 100%), and embedded in light-curing epoxy resin (Technovit 7200VLC, Hereaus Kulzer, Germany) for 1 week. Then, the specimens were sliced perpendicular to the long axis of the implants and sanding to about 50 µm thickness using a grinding system (Exakt Apparatebau, Germany). Sections were stained with Stevenel’s blue and Van Gieson’s Picrofuchsin stain (20 (link)), and images were taken under a light microscope (OLYMPUS BX43F, Japan). For histomorphometric analysis, BIC and BV/TV were measured using the NIH IMAGE J software (21 (link)). BIC was defined as the line percentage of the implant interface that directly contacts the bone. As for the BV/TV, it indicated the proportion of bone to total tissue volume around the implants.

The abdominal samples were fixed with 10% formalin solution and were decalcified with Calci-Clear Rapid solution (national diagnostics, USA) for 1 week. Then the samples were dehydrated sequentially from 80 to 100% ethyl alcohol and were cleared with xylene and were embedded with paraffin. Then the samples were sectioned with a microtome (Leica RM2255, Leica Biosystems, USA) at a thickness of 4 μm, and were stained with haematoxylin and eosin.
The lumbar spine samples were fixed with 10% formalin and were sequentially dehydrated from 80 to 100% ethyl alcohol. Then the samples were infiltrated and embedded in Technovit 7200 resin (EXAKT, Germany), and the resin was solidified with a polymerization system (EXAKT, Germany). After solidification, the resins were sectioned to 200 μm thick slices with a cutting system (EXAKT, Germany), and the slides were ground to 50 μm thick with a grinding system ((EXAKT, Germany). Then the slices were stained with hematoxylin and eosin.

Two weeks after implant placement, one femur carrying an implant was harvested from each mouse and fixed in 10% buffered formalin for 1 week at 4°C. Specimens were exposed to X-ray (20 kV, 5 seconds), and then dehydrated and embedded in light-curing epoxy resin (TechnoVit 7200VLC, Hereaus Kulzer, Wehrheim, Germany). Embedded specimens were cut perpendicular to the longitudinal axis of the implants at a site 0.5 mm from its apical end. The specimens were then ground to a thickness of about 50 µm with a grinding system (Exakt Apparatebau, Norderstedt, Germany). Sections were stained with Stevenel’s blue and Van Gieson’s picro fuchsin stain, and observed by light microscopy.

After the µCT scanning, the samples were prepared and sectioned as previously described^{8 (link)–10 (link)}. Histological sections were prepared using the Exakt Grinding System (Exakt) and stained with Stevenel’s blue and Alizarin red. The histological description of the tissues was based on light microscopy images obtained using a Leica DMLB light microscope (Leica, Bensheim, Germany).