*2.1. Animals*

Animal procedures were approved by the appropriate institutional animal care and use committee (protocol 65 DEF\_IGSSA\_SP). Interventions were performed at an accredited animal facility. Male Sprague Dawley rats (Charles River, France) were housed individually in cages with controlled temperature and lighting conditions, and food and water supplied ad libitum. The rats were eight weeks old (mean weight of 200 g) when they underwent the

first surgical procedure. In cases of postoperative complications, such as deep infection or bone fixation failure, the animals were excluded from the study and euthanized. Animals were killed by the intraperitoneal injection of sodium pentobarbital (150 mg/kg) at the age of 12 weeks (for IM analysis) or 22 weeks (for bone repair assessment).

### *2.2. Surgical Procedures*

IMT surgery (steps 1 and 2) was performed as previously described [15]. The first stage of surgery was performed under general anesthesia induced by the intraperitoneal administration of a ketamine/medetomidine mixture (60 and 0.42 mg/kg, respectively). Rats were placed in the prone position and an incision was made through the skin and muscle to expose the right femur. A mini external fixator (RatExFix RISystem, Davos, Switzerland) was screwed onto the anterolateral surface of the femur shaft, and a Gigli wire saw was used to create a 6 mm segmental defect. The bone defect was filled with either hand-made PMMA or metakaolin spacers (*n* = 5/group). Four weeks later, the animals underwent the second stage of graft surgery or were killed for stage 1 membrane studies. This time point was chosen based on the results of our previous model validation study [15]. For stage 2 of the IMT surgery, rats (*n* = 5/group) were anesthetized with isoflurane (1.5 to 2% isoflurane in 1 to 1.5 L of O2/min). An incision was carefully made in the membrane for spacer removal. The defect was then filled with a morselized corticocancellous allograft harvested from the distal femur of littermates killed on the same day. On three consecutive days after each surgical procedure, the animals received subcutaneous injections of a cephalosporin antibiotic (10 mg/kg enrofloxacin) and an opioid painkiller (0.05 mg/kg buprenorphine, twice daily). Unprotected weight-bearing activity was allowed immediately after surgery. The animals were weighed daily, and animal facility staff also evaluated their behavior, pain, normal movements and the appearance of the wound every day. Radiographic follow-up evaluations were performed every two weeks to check for incorrect spacer positioning and implantation failure. The animals were killed after 10 weeks for bone-healing assessment.
