Table 1.
Dosing regimen settings of this study.
Fig 1.
The time schedule of the experiments and sampling, and the pharmacokinetic profiles of the four distinct dosing regimens.
(a) The time schedule of the administration of TPTD, calcein labelling and urine and blood sampling are shown. (b) Six-month-old female rabbits were subjected to one of four TPTD dosing regimens. TPTD (20 μg/kg/day once-daily, 40 μg/kg/day once-daily, 140 μg/kg/week once-weekly or 280 μg/kg/week once-weekly) was administered subcutaneously and the time-course changes in the plasma levels of TPTD were monitored. The data are shown as the mean ± SD (n = 3–4).
Table 2.
Pharmacokinetic parameters of plasma TPTD.
Table 3.
Histopathological examination.
Fig 2.
Temporal changes in the markers of bone metabolism.
(a) The temporal changes in the serum levels of osteocalcin (OC) after the administration of TPTD on days 1 and 22 are shown. In the daily regimen groups (D20 and D40), the OC levels at 0 (pre-dosing), 6 and 24 h after the administration of TPTD were plotted. In the weekly regimen groups (W140 and W280) and the vehicle control (C) group, the OC levels at 0 (pre-dosing), 6 and 24 h, and 3 and 7 days after the administration of TPTD were plotted. (b) The changes in the urinary deoxypyridinoline (DPD) level after the administration of TPTD on days 1 and 29 are shown. The urine samples were collected for 12 hours, from 0 to 12 hours and from 12 to 24 hours after the administration on each day, and the rates of change in the urine DPD levels in comparison to pre-dosing (before the initial administration) were determined. (a) and (b): The data are shown as the mean ± SD (n = 3–4), except for urine DPD in D20 at 0–12 h on day 29, and D40 at all time points, because the urine samples were not obtained. *Indicates p < 0.05 vs. vehicle for each administration frequency (a two-way ANOVA with a post-hoc Dunnett’s test). C, vehicle control; D20, daily 20 μg/kg administration; D40, daily 40 μg/kg administration; W140, weekly 140 μg/kg administration; W280, weekly 280 μg/kg administration.
Fig 3.
Micro- Computed Tomography (CT)-based analyses of the cortical bone of rabbit tibiae.
(a) Three-dimensional reconstructed CT images of the bone structure of rabbit tibiae. The region of cortical porosity is highlighted in light blue. (b) The morphometric values of the cortical bone ratio (Cv /Tv, %), cortical bone thickness (Ct.Th, μm), cortical porosity (Ct.Po, %), perioteal perimeter (Ps.Pm, mm), endosteal perimeter (Es.Pm, mm) and the moment of inertia (mm-5) were measured and compared. The data are shown as the mean ± SD (n = 3–4). *Indicates p < 0.05 vs. vehicle control (ANOVA with post-hoc Dunnett’s test). C, vehicle control; D20, daily 20 μg/kg administration; D40, daily 40 μg/kg administration; W140, weekly 140 μg/kg administration; W280, weekly 280 μg/kg administration.
Fig 4.
Bright and fluorescence images of the transverse sections of rabbit tibiae.
Comprehensive tiling views of the bright field and fluorescence views were obtained from representative specimens in the vehicle control (DV), daily 20 μg/kg/day (D20), 40 μg/kg/day (D40), 140 μg/kg/week (W140), and 280 μg/kg/week (W280) groups (A, B, C, D, E, respectively). The green fluorescence signal from calcein labeling demarcates the sites of active bone formation, while the auto-fluorescence signal derived from the soft tissue provides morphological information. Bright field images were acquired with differential interference contrast (DIC). In the fluorescence view, calcein labelling- and soft-tissue-derived auto-fluorescence is shown in green and red, respectively. Wide-field fluorescence images were processed using the Deconvolution process. The white boxes and white dotted boxes indicate areas shown in Figs 5 and 6, respectively. Scale bars, 1000 μm.
Fig 5.
Magnified images of the endosteum region of the tibiae shown in Fig 4.
Zoom-in views of the medial endosteum region from large views (the white boxed area in each panel) shown in Fig 4. The yellow arrowheads indicate the fibroblastic cell-enriched tissue layer covering the bone surface. These tissue layers in D20 and D40 exhibit the typical appearance of bone marrow fibrosis. Scale bars, 200 μm.
Fig 6.
Magnified images of the cortical region of the tibiae shown in Fig 4.
Zoom-in views of the anteromedial region of cortical bone from large views of the C, D40 and W280 groups (the dotted white boxed area in each panel) shown in Fig 4. The red arrows indicate the porotic cavities. The yellow and green arrows indicate the blood vessel and the calcein-labeled region in the porotic cavity, respectively. Scale bars, 200 μm.
Fig 7.
The histomorphometric analysis of the cortical bone parameters.
The periosteal perimeter, endosteal perimeter and intracortical void were evaluated and compared. mL.Pm, multiple labeled perimeter; dL.Pm, double labeled perimeter; sL.Pm, single labeled perimeter; E.Pm, eroded perimeter; Q.Pm, quiescent perimeter. The data are shown as the mean (n = 3–4).
Fig 8.
Three Dimensional Second Harmonic generation (3D-SHG) imaging detecting bone collagen pattern.
SHG imaging of representative bone sections in the C, D20, D40, W140, and W280 groups and the trabecular bone of the iliac crest from the vehicle control (C iliac) group were acquired to detect bone collagen by focusing on the endosteal region. Three-dimensional projection images of 20 optical slices are shown. Bone collagen-derived SHG signaling, calcein labelling and soft tissue-derived auto-fluorescence are shown in blue, green and red, respectively. The white arrows indicate well-extended collagen fibers. Scale bars, 200 μm.