Fig 1.
Overview of experimental design and outcome measures.
(A) Animal positioning with lead shielding during irradiation and femur collection at five end points, n = 15 mice/group/end point. (B) Three-point bending configuration showing the notch created on the anterior surface of the femur; crack extension (a) is monitored from the medial side of the femur. (C) Idealized load-displacement plot for a notched femur in three-point bending showing the crack initiation, peak load, and instability points. Inset cartoons show propagation of the fracture surface (crack) corresponding to each of the toughness parameters. The instantaneous half crack angle θinst was determined at each of the three loading points to determine initiation (Kinit), peak load (Kpl), instability (Kinst) fracture toughness. (D) Fracture angle (α) was used as a descriptor femoral fracture pattern.
Fig 2.
Imaging of crack tip propagation.
Crack tip position (black arrows) was documented using reflected white light imaging from the notch tip (NT). The central load pin (LP) was positioned directly above the notch tip. Scale bar represents 0.2 mm.
Fig 3.
Fracture mechanics results for mid-diaphyseal cortical bone are presented as a function of time after treatment for RTX and Sham groups. (A) Initiation fracture toughness (Kinit); (B) peak load fracture toughness (Kpl); (C) instability fracture toughness (Kinst); and (D) fracture angle (α) are all decreased in RTx groups. Data are presented as mean ± standard deviation, n = 15 femurs/group/end point. Asterisks (*) denote p < 0.05 for Sham vs. RTx at each end point via an unpaired Student’s t-test.
Table 1.
Analysis of covariance (ANCOVA) results.
Fig 4.
Mid-diaphyseal femur morphology.
Mid-diaphyseal femur morphology is presented as a function of time after treatment for RTx and Sham groups. (A) Diaphyseal cortical cross-sectional area; (B) diaphyseal cortical thickness; (C) diaphyseal total cross-sectional area; (D) diaphyseal endosteal (marrow) cross-sectional area; (E) diaphyseal minimum moment of inertia (Imin); and (F) maximum moment of inertia (Imax). By 8–12 weeks RTx femurs lose cortical mass through resorption at the endosteal surface. Data are presented as mean ± standard deviation, n = 15 femurs/group/end point. Asterisks (*) denote p < 0.05 for Sham vs. RTx at each end point via an unpaired Student’s t-test.
Fig 5.
Biochemical composition of diaphyseal cortical bone is presented as a function of time after treatment for RTx and Sham groups. (A) Tissue mineral density; (B) mineral to matrix ratio; (C) pentosidine content normalized to collagen mass; (D) non-specific advanced glycation end products normalized to collagen mass; (E) collagen content; and (F) bone mineral content. Density, mineral:matrix ratio, collagen content, and bone mineral content did not differ between treatment groups, and AGE content for RTx femurs is increased only at 4 weeks in this data set. Data are presented as mean ± standard deviation, n = 15 femurs/group/end point. Asterisks (*) denote p < 0.05 for Sham vs. RTx at each end point via an unpaired Student’s t-test.
Fig 6.
Fracture toughness of bone irradiated ex vivo.
Mouse cadaver femurs were devitalized and exposed to 0 Gy or 20 Gy X-irradiation. Fracture mechanics outcomes—including initiation (Kinit), peak load (Kpl), and instability (Kinst) toughness measures—are presented alongside data from the in vivo groups (Sham, RTx) at 0 weeks. Data is presented as mean ± standard deviation, n = 15 femurs/group. Lower case letters denote statistically significant comparisons (p < 0.05) between treatment groups via one-way ANOVA with Tukey’s post-hoc test. Exact p-values are listed in the accompanying table.
Fig 7.
Ex vivo glycation of bone and associated changes in fracture toughness.
To explore the role of AGEs in fracture toughness, mouse femurs were incubated in a ribose solution for 0–14 days ex vivo to induce formation of AGEs. (A) Pentosidine content normalized to collagen mass; (B) non-specific AGEs content normalized to collagen mass; (C) fracture toughness outcomes. Kinst was negatively correlated with ribosylation, but at 3–14 days in ribose solution, the femur AGE content supraphysiologic. Data are presented as mean ± standard deviation, n = 15 femurs/group/end point. Simple linear regression was used to determine correlations between toughness or composition and ribosylation time.
Fig 8.
Known effects of limited field irradiation on murine bone.
In this summary figure, longitudinal changes in bone fracture toughness, morphology, whole bone mechanics, and tissue composition are presented as effect size (Cohen’s d method). Data are derived from the study presented in this manuscript, as well as historical data: aOest ME, JBMR 2017 [11]; bOest ME, Bone 2016 [27]; cOest ME, Radiat Res 2014 [26].