630 Acta Orthopaedica 2011; 82 (5): 628–632 Figure 2. Effect of treatment with anti-sclerostin antibody (Scl-Ab) on screw fixation. A. Peak pull-out force. B. Pull-out energy. C. Stiffness. Compared to controls, Scl-Ab treatment significantly increased peak force (a: p = 0.01; b: p < 0.001) and energy (c: p = 0.02; d: p < 0.001) both with and without loading. Stiffness was significantly increased in an unloaded setting (e: p = 0.03). Unloading had a large effect on screw fixation, causing a large reduction in peak force (p < 0.001). (Skyscan v. 1.5.1.4; Aarteselar), by correcting for ring arti- facts and beam hardening. Analysis of bone volume fraction (BV/TV, when measured by μCT) and trabecular thickness, separation, and number (Tb.Th, Tb.Sp, Tb.N) were performed in CTAn (Skyscan v. 1.9.1.0; Aarteselar). BV/TV was con- sidered the primary variable. All analyses were performed in a blinded manner. 4 additional samples had to be excluded since the μCT measurement region (specified in Figure 1) was too damaged to be properly evaluated. These exclusions were done while blinded. All animals that were excluded belonged to the Botox-treated groups. Statistics Results are presented as mean (SD). Comparisons for group mean differences were calculated for treatment effects on log- transformed data, if variances were unequal—as decided by Levene’s test. Differences were tested using 2-way ANOVA with Scl-Ab and Botox treatment as fixed factors, and t-test for post hoc comparisons. A result was considered to be sta- tistically significant at p-values < 0.05. All statistical analyses were performed using SPSS v. 18.0. Results The mean body weight of all groups increased during the experiment, but Botox-injected rats grew less. They weighted 22 (6) g less than uninjected rats at the time of surgery, and 75 (11) g less at the end of the experiment. The latter weight dif- ference cannot be explained only by a reduced muscle mass in the treated hind limbs (data not shown). Implant fixation 2-way ANOVA confirmed an effect of both Scl-Ab and unloading on pull-out force (p < 0.001 for both), but we did not find any statistically significant interaction between them. Thus, the response to the antibody appeared to be similar in loaded and unloaded bone. Unloading reduced the pull-out force by 56% compared to the controls with weight bearing (p < 0.001) (Figure 2). In unloaded bone, Scl-Ab treatment increased the pull-out force by 62% (p = 0.01) with corresponding increases in energy and stiffness (Figure 2). Antibody treatment did not restore the pull-out force (p = 0.05) or energy (p < 0.001) to values corresponding to those for weight-bearing controls. In weight- bearing bone, Scl-Ab treatment almost doubled the pull-out force (p = 0.003) and increased pull-out energy and stiffness (Figure 2). µCT analysis 2-way ANOVA confirmed an effect of both Scl-Ab (p = 0.029) and unloading (p < 0.001) on BV/TV (Figure 3). Both fac- tors also influenced trabecular thickness (p < 0.001 for both). Unloading, but not antibody treatment, influenced trabecular number and trabecular spacing (p < 0.001 for both). How- ever, there was no statistically significant interaction between unloading and antibody treatment for BV/TV or other mor- phometric parameters. In unloaded controls, there was a 90% decrease in cancel- lous BV/TV compared to weight-bearing controls (p < 0.001). This decrease in BV/TV was primarily due to a profound decrease in trabecular number (p < 0.001). Trabecular thick- ness also decreased (p < 0.001), while trabecular spacing increased (p < 0.001).