Acta Orthopaedica 2011; 82 (5): 628–632 g were used. To unload the bone, 24 animals were injected with Botox (Allergan, Irvine, CA) in the extensor muscles of the right hind leg 3 days before surgery. All animals had a stainless steel screw inserted unilaterally in the right proximal tibia (Agholme et al. 2010). After surgery, the rats were ran- domly divided into 4 groups of 12 animals. One Botox-treated (unloaded) group and one untreated (loaded) group received subcutaneous injections of 25 mg/kg Scl-Ab twice weekly for 4 weeks, with injections starting 3 days after surgery. The other 2 groups were injected with saline solution at the same time points. The rats were killed 4 weeks after surgery. Implants Stainless steel (316L) screws (thread M 1.7) were used. The threaded part of the screw is 2.8 mm long. The screws were custom-made and fitted with a head that enabled it to be mounted in a materials testing machine. The head has a 3.3-mm long portion that protrudes into the subcutaneous space. This type of screw has been used in this model previ- ously (Agholme et al. 2010). Antibody An anti-sclerostin monoclonal antibody (Scl-AbVI) specifi- cally designed for rat studies was provided by Amgen Inc. (Thousand Oaks, CA). Botox injection In animals allocated to Botox treatment, the right hind limb was shaved and cleaned with chlorhexidine alcohol. The rats were anesthetized with isoflurane and using an insulin syringe, they were given 5 × 1 U of Botox intramuscularly in the calf and quadriceps femoris muscles. 2 days after injection, none of the injected animals were using the injected limb. After 2 weeks, the injection procedure was repeated to ensure that the effect of the Botox did not recede. Surgical procedure The rats were anesthetized with isoflurane and operated under sterile conditions. Each rat received 7 mg oxytetracycline and 0.015 mg buprenorphine at the time of surgery. The right hind limb was shaved and cleaned with chlorhexidine alcohol. The rat was placed in a sterile surgical glove and the shaved leg was pulled out through a hole in the glove. Sterile tape was wrapped around the paw, and the leg was cleaned once more with chlorhexidine alcohol. The medial proximal metaphysis was exposed with a longitudinal incision. The periosteum was reflected proximally up to the epiphysis. An insertion hole was hand-drilled in the cancellous bone, approximately 3 mm distal to the physis, using a regular 1.2-mm injection needle. A screw was inserted in the hole and screwed in place. The skin was sutured using a 4/0-monofilament nylon suture. The animals were allowed to bear full weight immediately after waking from anesthesia. They received 0.007 mg buprenor- phine as postoperative analgesic every 12 h for 48 h. The rats 629 Figure 1. Region of interest used for µCT analysis. Frontal and trans- verse sections of the proximal tibia (epiphysis removed) from saline- treated (A) and Scl-Ab-treated (B) weight-bearing animals. A 1.5-mm long region in the metaphysis was chosen (dashed lines). In this region, only the trabecular bone was evaluated (white trace). This region was situated away from the screw (*) so that it would not be influenced by any bone formation around the screw. were given free access to food and water during the experi- ment, and were housed 3 per cage at 21°C in a room with a cycle of 12 h light and 12 h dark. The study was approved by the Regional Ethics Committee for Animal Experiments and we followed institutional guidelines for care and treatment of laboratory animals. Mechanical evaluation The rats were killed using carbon dioxide at the designated time point. Harvested bones were kept moist by saline irriga- tion and all bones were tested within 1 h of harvesting. All screws were tested for pull-out strength in a computerized materials testing machine (100 R; DDL Inc.), at a cross-head speed of 0.1 mm/s. The machine recorded the maximum force and the energy uptake until the force had dropped to 90% of maximum. The stiffness was then decided by the operator as the slope of the linear portion of the force/distance curve. The peak pull-out force was considered the primary variable. All analyses were performed while being blinded for treatment. 3 animals were excluded, 2 due to surgical mistakes and 1 because the tibia fractured at the screw during harvesting. All exclusions were done under blind conditions. The 3 animals that were excluded belonged to Botox-treated groups. Micro-CT (µCT) After pull-out testing, the right proximal tibia of all animals was scanned using μCT (Skyscan 1172 v. 1.5; Skyscan, Aar - teselar, Belgium). Figure 1 shows the region of interest ana- lyzed. The μCT scanner acquired topographic images of the bone with an isotropic voxel size of 15 μm at energy settings of 100 kV and 100 μA, using aluminum filter of 0.5 mm and 10 repeated scans. The images were reconstructed using NRecon