1 3 4 6 5 7 Many cross-sectional studies have determined the effects of bisphosphonates in rats either as a preventive treatment, before development of OVX-induced bone loss, or as a recovering treatment after development of bone loss. Although the results are difficult to compare directly due to differences in experimental design, preventive treatment in animals may lead to the same amount of final bone mass as recovering treatment. The associated microstructure in terms of number and thickness of trabeculae, however, can be different. While the influence of the time point of treatment is highly relevant clinically, no studies were found that directly compared the effects of a preventive and a recovering treatment on final bone mass and associated microstructure. In addition, the fact that only results of cross-sectional studies have been reported complicates comparison of the final and original bone structure and provides little or no information on the changes in structure over time. Here also we expect that longitudinal in vivo micro-CT imaging will potentially provide more information about the specific changes in the bone, both spatially and temporally. 8 9 Methods Animals n n n n Micro-CT Scanning 10 10 11 Mechanical Testing After sacrifice, all left tibiae were dissected and used for two different mechanical tests. First, a standard three-point bending test was applied. The tibiae were placed on the lateral surface on two rounded supporting bars with a distance of 2.4 mm. A preload of 1 N was applied (Z020; Zwick, Ulm, Germany) at the medial surface of the diaphysis by lowering a third rounded bar. A constant displacement rate of 6 mm/minute was applied until failure. Displacement was measured from the actuator displacement transducer of the testing machine. Stiffness, ultimate force, and ultimate displacement were calculated for each sample. 12 Histomorphometry 13 Statistics t t t P Results Ovariectomy 1 1 2 t Fig. 1 Average percentage change in structural parameters in the metaphyseal proximal tibia and upper standard deviation for all groups at all time points. Brackets indicate P < 0.05 at week 16  Fig. 2 a b c d e f Green line  1 Table 1 Mean values and standard deviation of all structural parameters of all groups at the start of the experiment  Group BV/TV (1)  Conn.D (1/mm) SMI (1) Tb.N (1/mm)  Tb.Th (μm)  Tb.Sp (μm)  Control 0.17 (0.056) 41 (47) 1.52 (0.53) 1.89 (1.38) 105 (49) 613 (368) OVX 0.26 (0.055) 141 (76) 1.22 (0.39) 3.77 (1.66) 84 (10) 335 (174) OVX + late ZOL 0.23 (0.062) 86 (66) 1.23 (0.34) 2.77 (1.55) 89 (13) 482 (227) OVX + early ZOL 0.17 (0.056) 38 (21) 1.70 (0.39) 1.86 (0.80) 95 (7) 647 (207) ZOL Treatment The early ZOL treatment and control groups were not significantly different at all time points for all structural parameters. 2 Table 2 P t Structural parameter P P BV/TV * * Conn.D 0.249 0.181 SMI * * Tb.N * * Tb.Th * * Tb.Sp 0.344 * * P 3 Table 3 P Group  ΔBV/TV ΔConn.D ΔSMI 2 4 8 12 16 2 4 8 12 16 2 4 8 12 16 1 vs. 2 * * * * * * * * * * * * * * * 1 vs. 3 * * * * * 0.341 * * * * 0.929 0.473 0.141 0.305 0.24 1 vs. 4 0.999 0.999 0.999 0.999 0.322 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 2 vs. 3 0.999 0.999 0.579 * * 0.999 0.906 0.811 * * 0.598 0.443 0.667 0.148 0.37 2 vs. 4 * * * * * 0.079 * * * * * * * * * 3 vs. 4 * * * * * 0.9 * * * * 0.421 0.29 0.104 0.331 0.206 Group  ΔTb.N ΔTb.Th ΔTb.Sp 2 4 8 12 16 2 4 8 12 16 2 4 8 12 16 1 vs. 2 0.088 * * * * * * 0.673 0.999 * 0.21 * * * * 1 vs. 3 0.108 * * * * * * 0.325 0.999 0.999 0.099 0.072 0.2 0.802 0.999 1 vs. 4 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 2 vs. 3 0.999 0.999 0.353 * * 0.999 0.999 0.999 0.999 0.152 0.999 0.999 0.128 * * 2 vs. 4 0.45 * * * * * * 0.265 0.999 * 0.492 * * * * 3 vs. 4 0.623 * * * * * * 0.109 0.999 0.999 0.279 0.084 0.187 0.667 0.999 Groups: 1, control; 2, OVX; 3, late ZOL; 4, early ZOL * P CT-derived mineralization values in all groups did not change over time and did not differ between groups at all time points. Aging An ANOVA with repeated measures on all structural parameters of the control group was performed to determine if there was a significant effect of aging. BV/TV and Tb.N were found to significantly decrease and Tb.Sp was found to significantly increase due to aging. All other structural parameters did not change significantly in 16 weeks. An ANOVA with repeated measures was performed on the percentage change in structural parameters comparing the control and early treatment groups to determine any significant differences. BV/TV and Tb.N showed a significant effect of age, treatment, and age * treatment and Tb.Sp showed a significant effect of age and age * treatment. While BV/TV decreased significantly over time in both the early treatment and control groups, it was found to decrease at a higher rate in the latter. Tb.N significantly decreased over time in the control group; however, it did not significantly change in the early treatment group as a net result of OVX, aging, and ZOL treatment. Tb.Sp significantly increased in the control group due to aging, while it did not in the early treatment group. Cortical Thickness 3 Fig. 3 Average percentage change in cortical thickness in the metaphyseal proximal tibia and upper standard deviation for all groups at all time points. At week 4 the OVX and the late ZOL groups were significantly lower than the early ZOL group, and at week 16 the OVX group was significantly higher than the control group  Mechanical Testing 4 Fig. 4 a b P  4 Histomorphometry 5 Fig. 5 a b c d P  Discussion In this study we assessed the effects of OVX and of early and late ZOL treatments after OVX on the microstructure of metaphyseal bone in the proximal tibia of aged, female Wistar rats over a 16-week time course, using in vivo high-resolution micro-CT. 2 14 16 5 7 7 8 17 18 19 8 17 20 21 23 20 24 25 26 27 28 29 34 35 9 36 37 38 20 39 P Another limitation to this study was the fact that on average, at 8 weeks in the study, the OVX group was in a more deteriorated condition than the late treatment group, while there was no demonstrable explanation for this. Although the ANOVA showed no significant differences between the groups, the treatment group still had a more favorable starting point when ZOL was administered. However, because after ZOL injection significant improvement took place while the OVX group still deteriorated, it is shown that this will not have affected the final conclusions. The method of in vivo CT scans combined with image registration software presented here has enabled us to study our goals in a more efficient way in terms of number of animals needed in the experiment. Follow-up micro-CT scans were made of the same animals, enabling us to use paired statistics and relative percentage changes in structural parameters. By registering all CT scans, we were able to select the same part of the metaphyseal trabecular bone for every measurement, thereby decreasing the variation in structural parameters due to a varying selected region of interest. In conclusion, OVX induced rapid changes in trabecular bone mass and structure. A single injection of ZOL at OVX completely inhibited these changes in bone mass and structure seen in osteoporotic rats. A single ZOL injection after OVX-induced bone loss improved bone mass and structure, per group and compared to the control group. BV/TV and the associated bone microstructure at the end point of this study were, however, significantly less favorable than in the early treatment group.