As shown in Fig. 1, three-dimensional structural analyses were performed by the SkyScan software for the following regions: (1) 0.5-mm-long sections at proximal (25% of the bones’ length from their proximal ends), proximal/middle (37%), middle (50%) and distal (75%) sites in cortical bone of the tibiae; The parameters
evaluated included periosteally enclosed volume, bone volume and medullary volume in the regions of cortical bone and percent bone volume (bone volume/tissue volume), trabecular number and trabecular thickness in the trabecular regions. After scanning by μCT, the bones were dehydrated, cleared and embedded in methyl methacrylate as previously described [33]. Transverse segments were Ku-0059436 manufacturer obtained by cutting with an annular diamond saw. Images of calcein and alizarin-labelled
bone sections were visualized using the argon 488-nm laser and the HeNe 543-nm laser, respectively, of a confocal laser scanning microscope (LSM 510; Carl Zeiss MicroImaging GmbH, Jena, Germany) at similar regions as the μCT analysis. In the cortical regions, periosteal and endosteal labels and inter-label bone areas were measured as newly formed bone area at each region and normalized by total cortical bone area using ImageJ software (version 1.42; http://rsbweb.nih.gov/ij/) [30]. All data are shown as mean ± SE. Body weight was compared by one-way ANOVA. In the analysis of bones, the left and right sides in each group were compared by paired t-test, and then those in all three groups by one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test. Statistical find more analysis was performed using SPSS for Windows (version
17.0; SPSS Inc., Chicago, IL), and p < 0.05 was considered as significant. As shown in Table 1 and Table 2, there were no statistically significant differences in body weight or longitudinal lengths of the tibiae, fibulae, femora, ulnae and radii. Analysis by μCT showed that in the cortical regions of the tibiae in the DYNAMIC + STATIC group, fantofarone periosteally enclosed and cortical bone volumes in the right loaded side were markedly higher than those of the contra-lateral non-loaded side at the proximal (+15.5 ± 1.0% and +35.9 ± 3.2%, respectively; p < 0.01), proximal/middle (+18.8 ± 0.6% and +32.7 ± 1.6%, respectively; p < 0.01) and middle (+13.3 ± 2.2% and +24.0 ± 2.2%, respectively; p < 0.01) sites ( Table 3; Fig. 2A). There were no significant differences at the distal site. Medullary volume in the cortical region of the right loaded tibiae was smaller compared to that of the left tibiae at the proximal site (− 10.2 ± 2.8%; p < 0.01). In contrast to these differences between loaded and non-loaded bones in the DYNAMIC + STATIC group, there were no significant differences in the periosteally enclosed bone volume, cortical bone volume or medullary volume between the left and right tibiae in the STATIC or NOLOAD group.