*3.4. In Vivo Implantation*

The Mg-RE sheet with TRC samples was successfully inserted into the subcutaneous tissue of the rat thigh and attached to the femur with an AZ31 screw during surgery, despite the slight mismatches between the Mg-RE sheet and femur owing to manual surgical placement and the fact that there is only one screw to fix the femur and alloy sheet. Slight skin swelling was observed in the hind limbs of the rats during the visual examination of the rats during the study period. The bulge disappeared after a few days.

Figure 10 reveals the surface volume of the degrading Mg-RE alloy sheet and bone growth changes, depicted using MicroCT 3D rendering after various implantation times. As can be seen from Figure 10a,c, two weeks after surgery, no new bone production was found around the Mg-RE alloy sheet and femur, and the screw between them was clearly visible. There are no obvious corrosion pits on the surface of magnesium alloy sheet. Figure 10b,d, six weeks after the surgery, new bone was formed between the Mg-RE alloy sheet and the femur. Figure 10e presents on the cross section of the bone–alloy joint, the new bone is generated and surrounds the original femur. The sheets are attached to the femur by new bone, which appears to be relatively stable in living organisms. In the red area shown in Figure 10, corrosion pits began to appear on the surface of magnesium alloy sheet after six weeks of surgery. This indicates that the films with improved corrosion resistance of Mg-RE alloy sheet began to degrade in vivo after 6 weeks.

Mg-RE sheet degradation and femoral change were assessed by volume quantification in MicroCT 3D rendering separately as shown in Figure 11. Before surgery, the volume of self-made Mg-RE alloy bone sheet was 94.79 mm3. The Mg-RE sheet corroded, resulting in 9.32 ± 0.86 mm<sup>3</sup> volume loss after 6 weeks. With regard to the changes in the femur, the volume of the femur did not change significantly during the two weeks following the surgery. In fact, for some time after the surgery, the rat lost significantly more weight, the reason for this being that it took some time for the body to adapt the a foreign body. After about five days, the rats returned to their normal mental state and began to eat normally. By 6 weeks, as new bone had formed between the sheet and the femur, a noticeable

change in bone volume was observed from 2 to 6 weeks. The rat showed no abnormal physical or physiological responses during subsequent breeding.

Uniquely, magnesium alloy application has the potential to enhance bone formation. Several previous studies have shown that the potential performance, presenting increased bone mass, mineral apposition, and bone mineral density around magnesium alloy implants in bone [36–39]. In our present study, new bone appears in the area where the femur contacts the alloy sheet, which confirmed our findings that the Mg-RE alloy with TRC degradation can lead to the promotion of bone formation. The new bone formation is not common in absorbable polymers or permanent metal devices, thus emphasizing the unique advantages of magnesium fixtures. In this way, the degraded fixture will be gradually replaced in the future.

**Figure 10.** Determination of the volume of the degrading Mg-RE alloy sheet and bone growth changes, depicted in MicroCT 3D rendering after implantation times of 2 weeks (**<sup>a</sup>**,**<sup>c</sup>**) and 6 weeks (**b**,**d**). A cross section slice of femoral-sheet contact area observed after 6 weeks (**e**) shows that the new bone (Nb is represented in green) was formed between the Mg-RE alloy sheet (S) and the original bone (Ob is represented in blue).

**Figure 11.** Mg-RE sheet degradation after 6 weeks was estimated by the volume of quantification. This shows a sheet volume loss of 9.32 ± 0.86 mm<sup>3</sup> after 6 weeks. MicroCT 3D rendering after 2 weeks shows there was no obvious change in bone growth around the degrading Mg-RE sheet. By 6 weeks, as new bone formed between the sheet and the bone, a notable change in bone volume was observed from 2 to 6 weeks.
