Ovariectomy and Femur Osteotomy in Mice

OVX and femur osteotomy were performed under general anesthesia using 2% isoflurane (Florene, Abbott, Wiesbaden, Germany). For analgesia, mice received 25 mg/ml tramadol hydrochloride (Tramal®, Gruenenthal, Aachen, Germany) in the drinking water, starting one day preoperatively until three days postoperatively. Before surgery, mice received one subcutaneous injection of the antibiotics clindamycin-2-dihydrogenphosphate (45 mg/kg, Ratiopharm, Ulm, Germany). Mice were bilaterally ovariectomized by ligation of the oviduct and removal of the ovary. Femur osteotomy was performed as described previously^{23 (link)}. Briefly, at the midshaft of the right femur an osteotomy gap (0.4 mm) was created and stabilized using an external fixator (axial stiffness 3 N/mm, RISystems, Davos, Switzerland). The mice were injected after 10 days with calcein green (0.03 g/ml, Sigma-Aldrich, St. Louis, USA) and after 15 days with alizarin red (0.045 g/ml, Sigma-Aldrich) for dynamic bone histomorphometry.