*2.6. Implant Surgery*

The in vivo study was performed at the facility of the Department of Emergencies and Organ Transplantation of the University of Bari. After the approval of the Italian Minister of Health (Approval number 654/2020 PR), 16 sheep underwent the implantation, in a blind matter, of the two kinds of prothesis. Sheep were divided in two groups: the first one that was sacrificed after 6 months from surgery and the second one after 3 months. As far as the surgery is concerned, an incision was performed at the level of the parietal bone on the skin and subcutaneous tissue (Figure 11a), the custom-made mask was positioned on the bone using screws (Figure 11b) and the ostectomy was performed via piezosurgery (Mectron, Italy) [21] (Figure 11c); the bone gusset and the mask were then removed and the former was replaced with the device that was subsequently anchored using the same holes created for the mask positioning (Figure 11d).

**Figure 11.** Surgical procedure for the in vivo studies: (**a**) exteriorizing of parietal bone; (**b**) guiding mask fixed to the bone with screws; (**c**) bone gusset removal through piezosurgery osteotomy; (**d**) device implantation and fixation with screws.

‐ ‐ The above tissues were than sutured and the animals were awakened from anaesthesia. After the surgery, depending of the group they belonged to, animals underwent the administration of different bone markers (Tetracycline, Xilenol Orange and Calcein Blue were used for the three months group, whereas Calcein Blue, Tetracyclin Xilenol and Orange and Alizarin Red were used for the six months group). Moreover, the absence of alisteresis and screws and prosthesis mobility, as well as any periprosthetic inflammatory reaction, infections, sub cutaneal exudation and soft tissue scarring times were checked. The sheep's temperature was also regularly monitored to exclude fever.

‐ After 24 h from the last administration, the animals were euthanized using a propofol overdose, then the heads were removed and were subjected to a CT scan to evaluate the

effects of the implant on the bone. The devices were removed using the piezosurgery making a gusset of bone 1 cm from the device on all the sides, then put in formaline for seven days, washed under running water for 24 h and put in 70◦ ethanol to undergo histological evaluations.
