2.4.2. Mechanical Testing

Four weeks after the insertion of the 3D metallic implants, the rabbits were anesthetized (following the same protocol described above), and euthanized by intracardiac injection, using an overdose of sodium pentobarbital (100 mg/kg).

The anterior incision was resumed and the distal femoral epiphysis, containing the implant after disarticulation and proximal sectioning of the femoral diaphysis (Figure 3), was harvested for evaluation. After careful dissection and cleaning of any adherent soft tissues, the bone tissue–implant block specimens were obtained and immediately fixed in 5% bu ffered formaldehyde solution, prior to being subjected to mechanical tests (within 1 h of euthanasia).

**Figure 3.** (**a**) Photograph of the extracted bone; (**b**) schematic representation of the implant site.

The quantification of the in vivo interfacial extraction force of bone implants represents a biological challenge. One should note that, the analysis of the phenomena that occur at the bone tissue–implant interface is a complex procedure, which requires the use of experimental cutting and grinding techniques, which are quite complicated and expensive. To avoid these shortcomings, the experimental model of in vivo testing of the extraction force of 3D metallic implants was, therefore, applied in this study.

It is important to mention that, the retention between the implant and the bone was evaluated before euthanasia could influence the results (within the first 4 h after euthanasia).

The measurement of the implants extraction force was performed by a tensile traction machine (model WDW, Time Group), which measures both the force and the elongation, and can operate with a maximum force of 5000 N. All tests were performed with a traction speed of 1 mm/min. The implant was positioned into the traction machine support by means of a hexagonal adapter attached with an adhesive on the exterior side of the 3D Ti implant (Figure 4). One should take into account that, when fixing the femur and the implant, the position of the 3D metallic implant should be aligned with

the traction direction. Typical load-displacement curves were recorded (data not shown here), and the failure load was defined as the peak load value of the load-displacement curve.

**Figure 4.** Tensile pull-out measurement: photographs of (**a**) the hexagonal adaptor attached to the 3D Ti implant, and (**b**) both the femur and the adaptor attached to the 3D metallic implant, in the traction machine support.

### 2.4.3. Scanning Electron Microscopy (SEM)

The morphological analysis of the 3D metallic implants' surfaces, following their extraction from the rabbits' femoral condyles, was performed. Therefore, to identify the adherent bone components, surfaces of both control and functionalized 3D Ti implants were examined by scanning electron microscopy (SEM). Using the analysis of backscattering electrons, along with the ImageJ free software (version 1.46r), one could estimate the adherence ratio of the bone tissue remaining on the implant's surface after its extraction from the rabbits' femoral condyle. This parameter is defined as the ratio between the estimated area of the remaining bone tissue and the area of the 3D implant (the area of a circle with a diameter of 3.5 mm).

A SEM SU5000 (Hitachi, Tokyo, Japan), with a resolution of 1.2 nm and an acceleration voltage of 25 kV, was used for these investigations. For comparison reasons only, SEM micrographs of control Ti and Li-C and Li-P structures before surgery are also presented.
