Altered mechanical behavior of demineralized bone following therapeutic radiation

Abstract

Post-radiotherapy (RTx) bone fragility fractures are a late-onset complication occurring in bone within or underlying the radiation field. These fractures are difficult to predict, as patients do not present with local osteopenia. Using a murine hindlimb RTx model, we previously documented decreased mineralized bone strength and fracture toughness, but alterations in material properties of the organic bone matrix are largely unknown. In this study, 4 days of fractionated hindlimb irradiation (4 × 5 Gy) or Sham irradiation was administered in a mouse model (BALB/cJ, end points: 0, 4, 8, and 12 weeks, n = 15/group/end point). Following demineralization, the viscoelastic stress relaxation, and monotonic tensile mechanical properties of tibiae were determined. Irradiated tibiae demonstrated an immediate (day after last radiation fraction) and sustained (4, 8, 12 weeks) increase in stress relaxation compared to the Sham group, with a 4.4% decrease in equilibrium stress (p < .017). While tensile strength was not different between groups, irradiated tibiae had a lower elastic modulus (-5%, p = .027) and energy to failure (-12.2%, p = .012) with monotonic loading. Gel electrophoresis showed that therapeutic irradiation (4 × 5 Gy) does not result in collagen fragmentation, while irradiation at a common sterilization dose (25 kGy) extensively fragmented collagen. These results suggest that altered collagen mechanical behavior has a role in postirradiation bone fragility, but this can occur without detectable collagen fragmentation. Statement of Clinical Significance: Therapeutic irradiation alters bone organic matrix mechanics and which contribute to diminished fatigue strength, but this does not occur via collagen fragmentation.

Keywords: bone biomechanics; demineralized bone; radiation therapy.

FIGURE 1

A bilateral hindlimb murine model (A) was used to deliver four fractionated doses of 5 Gy over a 4-day period. Following demineralization, tibiae were prepared for uniaxial tensile loading (B). Mechanical loading consisted of a stress relaxation experiment (C) with a five-parameter Maxwell–Wiechert model used to characterize the stress relaxation process. This was followed by a monotonic tensile test to failure (D).

FIGURE 2

Cross-sectional area of the mouse tibia (A) as a function of axial position determined from micro-CT scans. A solid model of the demineralized tibia (B) was created from thresholded micro-CT scans and converted to a voxel finite element mesh (C). Longitudinal stresses due to an axial tensile load are shown on the tibial surface (C) and cross-sections (D).

FIGURE 3

The five parameters of the Maxwell–Wiechert phenomenological model are plotted as a function of time in vivo for Sham or RTx treatment groups (mean ± SE). Regression parameter estimates are shown for the independent variables of treatment and time (weeks). Stress (σ) and time constant (τ) components are indicated. RTx, radiation therapy

FIGURE 4

Stress relaxation for Sham and RTx groups based on mean values from the five-parameter Maxwell–Wiechert model. Short (2 τ₁) and long time constant (2 τ₂) values are shown representing 87% of exponential decay. Results illustrate a similar relaxation response for short time period (<30 s) with more relaxation for the RTx group for long time periods. RTx, radiation therapy

FIGURE 5

Monotonic tensile loading results for tensile strength, elastic modulus, and energy to failure are plotted as a function of time in vivo for Sham or RTx treatment groups (mean ± SE). Regression parameter estimates are shown for the independent variables of treatment and time (weeks). RTx, radiation therapy

FIGURE 6

Collagen fragmentation was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Intact collagen is observed in the form of α1 and α2 bands (~115–130 kDa) and β11 and β12 bands (~250 kDa) for the 0 Gy, and 20 Gy ex vivo irradiation groups, as well as the 4 × 5 Gy and 0 Gy in vivo groups obtained from animals 12 weeks after treatment. In the 25 kGy ex vivo irradiation group, the α and β bands are diminished, and visible smears indicate collagen fragments less than 100 kDa in size. L, ladder; RTC, rat tail collagen control

FIGURE 7

Instability fracture toughness of mineralized cortical bone (as we previously reported) increased with greater demineralized bone energy to failure. Mean and SE of mean shown with all-time points grouped together