Fig 1.
Rat positioning on the Skyscan 1176 scanner for in vivo scanning.
The rat was placed sideways on the scanning bed while kept anesthetized (anesthesia mask not shown). This configuration was adapted to facilitate the positioning of the irradiated leg (right) into the iso-center of the scanning chamber. The right tibia was secured into a Styrofoam holder (1 cm thick) of cylindrical shape and firmly held with a medical adhesive tape. The non-radiated leg (left) was folded towards the animal’s head and placed alongside the animal with its tail.
Table 1.
Image acquisition and reconstruction parameters of the rat proximal tibiae for the three doses groups.
Fig 2.
Bone growth rates (μm/day) measurements.
(A) 5x magnified microscopic images of the tibial metaphysis labeled twice with calcein for representative irradiated and control tibiae from three doses groups (Ⅰ-ⅤⅠ). Bone growth (ΔX, μm) measured as the mean distance between the two calcein lines, which were modeled as splines and divided by the time interval (3 days) between the two applied injections. (B) Growth rates (μm/day) of rat proximal tibiae for 0.83, 1.65 and 2.47 Gy radiation groups (mean value ± SD). *: a significant difference (p < 0.05) between the control (left) and irradiated (right) tibiae for each radiation dose.
Fig 3.
(A) Growth plate section embedded in MMA and stained with toluidine blue (10x). Evaluation of the hypertrophic and proliferative zonal thicknesses for three doses groups. (B) Growth plate section embedded in MMA and stained with toluidine blue (20x). Evaluation of the hypertrophic cell height and number of proliferative cells per column for three doses groups.
Fig 4.
In vivo scanning of proximal tibia and bone segmentation process.
(a) A representative 3D reconstructed tibia showing the total tibial length (L). (b) Scanned proximal tibial cross-section (10 mm in height) of the rat tibia. This representative image was acquired from a 17.48-μm pixel size scanning at 0.83 Gy radiation dose. VOI consisting trabecular and cortical bone, for morphometric parameters evaluation, beginning at ~1mm distal to the growth plate and extending for 10% of the total tibial length (L). Proximal (f) and distal (c) tibial sections are illustrated. The cortical (d, g) and trabecular (e, h) bone regions were segmented using a semi-automatic bone segmentation algorithm.
Fig 5.
Histomorphometry measurements comparison for control and irradiated tibiae.
(a-d) Growth plate histomorphometry measurements of rat proximal tibiae for 0.83, 1.65 and 2.47 Gy radiation groups (mean value ± SD). *: a significant difference (p < 0.05) between the control (left) and irradiated (right) tibiae for each radiation dose.
Fig 6.
Mean values and standard deviations of the trabecular bone parameters for the left (hatched columns), and right tibia (black columns) at 14th week of age (n = 11/group). *: a significant difference (p < 0.05) between the control (left) and irradiated (right) tibiae for each radiation dose.
Fig 7.
Mean values and standard deviations of the cortical bone parameters for the left (hatched columns), and right tibiae (black columns) at 14th week of age (n = 11/group). *: a significant difference (p < 0.05) between the control (left) and irradiated (right) tibiae for each radiation dose.
Table 2.
Longitudinal assessment of trabecular microarchitecture of the right proximal tibial metaphysis in three doses groups of rats.
Table 3.
Longitudinal assessment of cortical microarchitecture of the right proximal tibial metaphysis in three doses groups of rats.
Table 4.
ANOVA test with Tukey’s multiple comparisons for the trabecular and cortical bone structural properties of the irradiated rat tibiae for three radiation groups on the 14th week.
Fig 8.
Body weight of male Sprague Dawley rats for three doses groups over the adolescent period.
ANOVA test (general linear model) was performed to determine time effects, radiation dose, and their interaction on body weight. N = 11 rats per group (mean value ± SD).
Table 5.
Percentage of unaffected bone marrow cells for 0.83, 1.65 and 2.47 Gy radiation groups extracted from trypan blue test (mean value ± SD).
Fig 9.
Trabecular and cortical bone representation after the 9-weekly in vivo micro-CT scans.
(a—f) Representative 3D micro-CT images of metaphyseal bone structure of the irradiated (right) and non-irradiated control (left) tibiae at 14th week of age after 0.83, 1.65 and 2.47 Gy radiation doses during the rat adolescent period. 3D micro-CT images within each radiation dose portray tibiae from the same rat, randomly selected to be representative of its respective dose group.