Longitudinal Effects of Single Hindlimb Radiation Therapy on Bone Strength and Morphology at Local and Contralateral Sites

Abstract

Radiation therapy (RTx) is associated with increased risk for late-onset fragility fractures in bone tissue underlying the radiation field. Bone tissue outside the RTx field is often selected as a "normal" comparator tissue in clinical assessment of fragility fracture risk, but the robustness of this comparison is limited by an incomplete understanding of the systemic effects of local radiotherapy. In this study, a mouse model of limited field irradiation was used to quantify longitudinal changes in local (irradiated) and systemic (non-irradiated) femurs with respect to bone density, morphology, and strength. BALB/cJ mice aged 12 weeks underwent unilateral hindlimb irradiation (4 × 5 Gy) or a sham procedure. Femurs were collected at endpoints of 4 days before treatment and at 0, 1, 2, 4, 8, 12, and 26 weeks post-treatment. Irradiated (RTx), Contralateral (non-RTx), and Sham (non-RTx) femurs were imaged by micro-computed tomography and mechanically tested in three-point bending. In both the RTx and Contralateral non-RTx groups, the longer-term (12- to 26-week) outcomes included trabecular resorption, loss of diaphyseal cortical bone, and decreased bending strength. Contralateral femurs generally followed an intermediate response compared with RTx femurs. Change also varied by anatomic compartment; post-RTx loss of trabecular bone was more profound in the metaphyseal than the epiphyseal compartment, and cortical bone thickness decreased at the mid-diaphysis but increased at the metaphysis. These data demonstrate that changes in bone quantity, density, and architecture occur both locally and systemically after limited field irradiation and vary by anatomic compartment. Furthermore, the severity and persistence of systemic bone damage after limited field irradiation suggest selection of control tissues for assessment of fracture risk or changes in bone density after radiotherapy may be challenging. © 2017 American Society for Bone and Mineral Research.

Keywords: BIOMECHANICS; BONE µ-CT; FRACTURE RISK ASSESSMENT; PRECLINICAL STUDIES; RADIATION-INDUCED BONE DISEASE.

Figure 1

A) Experimental design: female BALB/cJ mice aged 12 weeks were exposed to four consecutive daily unilateral hindlimb irradiation exposures of 5 Gy each. The contralateral hindlimb and body were shielded with lead. These animals yielded the RTx and Contra samples. A separate group of age-matched animals were anesthetized but not irradiated, yielding the Sham samples. At end points of 4 days prior to treatment and 0, 1, 2, 4, 8, 12, and 26 weeks after treatment, animals were euthanized and femurs collected. B) Average mouse body mass for each treatment group (arithmetic mean ± standard error) over the course of the study. There were no significant differences in body mass at any time point between the sham and irradiated mice (contralateral femurs are derived from the non-irradiated limb of RTx group mice). C) Schematic of the μCT volumes of interest. D) Graphical description of the femur strength and cortical bone strength outcome measures.

Figure 2

Representative μCT-derived cross-sections of bones at 0, 4, 8, 12, and 26 weeks after treatment for each group. For each treatment group, the upper row presents a sagittal section of the distal femur, and the lower row presents a transverse metaphyseal cross-section.

Figure 3

Mid-diaphyseal (cortical) bone geometric parameters of femora as a function following 4x5 Gy hindlimb irradiation for Sham, Contralateral, and RTx groups. A) Cortical area (Ct.Ar); B) mean cortical thickness (Ct.Th); C) endosteal area (Es.Ar), and D) total cross sectional area (Tt.Ar) results are shown with arithmetic mean ± standard error bars. Statistically significant differences between groups are denoted for p < 0.05.

Figure 4

Metaphyseal cortical bone of the distal femur as a function of time following 4x5 Gy hindlimb irradiation for Sham, Contralateral, and RTx groups. A) Cortical area (Ct.Ar) and B) mean cortical thickness (Ct.Th) results are shown with arithmetic mean ± standard error bars. Statistically significant differences between groups are denoted for p < 0.05.

Figure 5

Metaphyseal trabecular bone of the femur as a function of time following 4x5 Gy hindlimb irradiation for Sham, Contralateral, and RTx groups. A) Bone volume fraction (BV/TV); B) trabecular number (Tb.N); C) connectivity density (Conn.D); and D) trabecular thickness (Tb.Th) results are presented as arithmetic mean ± standard error bars. Statistically significant differences between groups are denoted for p < 0.05.

Figure 6

Epiphyseal trabecular bone of the femur as a function of time following 4x5 Gy hindlimb irradiation for Sham, Contralateral, and RTx groups. A) Bone volume fraction (BV/TV); B) trabecular number (Tb.N); C) connectivity density (Conn.D); and D) trabecular thickness (Tb.Th) results are presented as arithmetic mean ± standard error bars. Statistically significant differences between groups are denoted for p < 0.05.

Figure 7

Tissue mineral density (TMD) of the femur as a function of time following 4x5 Gy hindlimb irradiation for Sham, Contralateral, and RTx groups. Volumes of interest included A) Diaphyseal cortical (Dp TMD); B) metaphyseal trabecular (Mp TMD); and C) epiphyseal trabecular (Ep TMD). Results are presented as arithmetic mean ± standard error bars. Statistically significant differences between groups are denoted for p < 0.05.

Figure 8

Mechanical strength of the femurs in bending as a function of time following 4x5 Gy hindlimb irradiation for Sham, Contralateral, and RTx groups. A) Bending strength of the femur B) bending stiffness of the femur; C) bending strength normalized to individual mouse body weight; D) energy to break; and E) post-yield displacement. Data presented as arithmetic mean ± standard error, with significance between groups denoted at p < 0.05.

Figure 9

Mechanical properties of the mid-diaphyseal cortical bone in bending as a function of time following 4x5 Gy hindlimb irradiation for Sham, Contralateral, and RTx groups. A) Flexural strength and B) flexural yield strength of the bone material; and C) flexural modulus of the cortical bone. Data presented as arithmetic mean ± standard error, with significance between groups denoted at p < 0.05.

Figure 10

Summary of post-irradiation changes in bone morphology, density, mechanics, and biochemistry as a function of time after treatment in this murine model of limited field radiotherapy. Biochemical and histomorphologic data derived from: A) Oest et al. J Orthop Res 2015 33(3):334-42 (ref #10); B) Gong et al. Bone 2013 57(1):252-8 (ref #16), C) Oest et al. Bone 2016 86:91-7 (ref #17), D) Oest and Damron Radiat Res 2014 181(4):439-43 (ref #18). Abbreviations: Tt.Ar: total area, Ct.Th: cortical thickness, Ct.TMD: cortical tissue mineral density, MAR: mineral apposition rate, Tb. BV/TV: trabecular bone volume fraction, Tb.TMD: trabecular tissue mineral density, AGEs: advanced glycation end products. All studies reflected in this figure were conduced on BALB/c mice, used in several studies over many years. Mice from the study detailed in this manuscript were not the same as the mice from which osteoclast, MAR, and biochemical data were obtained.