Characterization of the effects of x-ray irradiation on the hierarchical structure and mechanical properties of human cortical bone

Abstract

Bone comprises a complex structure of primarily collagen, hydroxyapatite and water, where each hierarchical structural level contributes to its strength, ductility and toughness. These properties, however, are degraded by irradiation, arising from medical therapy or bone-allograft sterilization. We provide here a mechanistic framework for how irradiation affects the nature and properties of human cortical bone over a range of characteristic (nano to macro) length-scales, following x-ray exposures up to 630 kGy. Macroscopically, bone strength, ductility and fracture resistance are seen to be progressively degraded with increasing irradiation levels. At the micron-scale, fracture properties, evaluated using insitu scanning electron microscopy and synchrotron x-ray computed micro-tomography, provide mechanistic information on how cracks interact with the bone-matrix structure. At sub-micron scales, strength properties are evaluated with insitu tensile tests in the synchrotron using small-/wide-angle x-ray scattering/diffraction, where strains are simultaneously measured in the macroscopic tissue, collagen fibrils and mineral. Compared to healthy bone, results show that the fibrillar strain is decreased by ∼40% following 70 kGy exposures, consistent with significant stiffening and degradation of the collagen. We attribute the irradiation-induced deterioration in mechanical properties to mechanisms at multiple length-scales, including changes in crack paths at micron-scales, loss of plasticity from suppressed fibrillar sliding at sub-micron scales, and the loss and damage of collagen at the nano-scales, the latter being assessed using Raman and Fourier Transform Infrared spectroscopy and a fluorometric assay.

Fig. 1

The structural hierarchy of bone. At the smallest level, at the scale of the tropocollagen molecules and mineralized collagen fibrils, (intrinsic) toughening is achieved through plasticity, principally via mechanisms of molecular uncoiling and intermolecular sliding of molecules and mineralized collagen fibrils. Cross-links form at these length-scales between the collagen molecules and between the fibrils [40]. At micrometer dimensions, the breaking of sacrificial bonds at the interfaces of fibril arrays contributes to increased energy dissipation, together with crack bridging of microcracks by collagen fibrils. At the largest length-scales in the range of 10 s to 100 s μm, the primary sources of toughening are extrinsic and result from extensive crack deflection and crack bridging/twisting by uncracked ligaments, both mechanisms that are motivated by the occurrence of microcracking [5,6].

Fig. 2

Schematic showing the beamline setup for in situ tensile testing of bone with real time small-angle x-ray scattering and wide-angle x-ray diffraction (SAXS/WAXD) imaging. The 10 keV x-ray beam penetrates the longitudinally oriented human cortical bone sample perpendicular to the tensile set up. The SAXS detector is positioned to record the meridional D-spacing in the staggered array of mineralized collagen fibrils. The WAXD detector is positioned to record patterns from the crystallites with c-axis along the tensile direction. Tissue strain is determined by the marker lines on the sample (camera not shown in schematic). Images (a) and (b) show WAXD pattern and the SAXS pattern of bone respectively, with the region for azimuthal integration shown in the pie-shaped sector outlined in both. Figures (c) and (d) show the integrated intensity variation over this region. Graph (c) shows a pronounced (0002) diffraction peak for the hydroxyapatite, and graph (d) shows a pronounced first order diffraction peak due to the fibrillar D-spacing (67 nm).

Fig. 3

Mechanical properties of hydrated human cortical bone (transverse orientation) subjected to varying degrees of x-ray irradiation. (a) Stress-strain curves from three-point bending tests (offset for clarity) and (b) fracture toughness R-curves showing resistance to fracture in terms of the stress intensity, K_J, as a function of crack extension, Δa, both as a function of prior x-irradiation dosage. K_J fracture toughness values were back-calculated from J measurements using the J-K equivalence for mode I fracture. The end of the curves indicates the critical toughness values, K_Jc, at which complete failure occurred.

Fig. 4

X-ray computed micro-tomography of crack paths in hydrated human cortical bone in the transverse orientation following (a–c) low dose (50 Gy), and (e–g) high dose (~MGy) x-irradiation. Images show crack paths: (a) & (d) x-ray tomographs from side-view perpendicular to the crack plane, (b) & (e) 3-D x-ray tomography images of these paths (3-D crack surface is in purple; Haversian canals are yellow), and (c) & (f) 2-D tomographs of the paths from the back face of the sample. The crack deflects on encountering the osteons; such crack deflection and crack twisting is the prime extrinsic toughening mechanism in bone in the transverse orientation. Note, however, that the frequency of such deflections increases with irradiation while their severity decreases, resulting in less meandering crack paths in the irradiated bone. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Partitioning of strain between the collagen and mineral as a function of the global tissue strain in 70 kGy irradiated vs. unirradiated hydrated human cortical bone. Variation of the strain from SAXS/WAXD measurements in the (a) collagen and (b) mineral as a function of the global applied strain in the bone tissue for unirradiated and 70 kGy irradiated bone. The collagen and mineral strain are binned in regular intervals of tissue strain (N = 20 for each group). Error bars in the graphs are standard deviation of the binned value. Note that for a given strain in the bone tissue the strain in the collagen fibrils is diminished in irradiated vs. unirradiated bone, consistent with the progressive loss in macroscopic plasticity in bone with increasing irradiation.

Fig. 6

Strain in collagen fibril, and mineral in bone as a function of applied strain in the tissue for human cortical bone in the (a) unirradiated and (b) 70 kGy irradiated condition. The upper graphs show the ratios of fibril to tissue strain, ε_F/ε_T, and mineral to tissue strain, ε_M/ε_T, for the (a) unirradiated and (b) 70 kGy irradiated bone, averaged from N = 20 samples for each group. In (a) and (b), the solid lines represent the constant strain ratio expected before yield. In (a), the dotted line represents where the ratio would vary if the fibril and mineral strain remained constant beyond the yield strain. The lower graphs show the corresponding stress-strain curves for the (a) unirradiated and (b) 70 kGy irradiated human cortical bone.

Fig. 7

(a) Schematic illustration of the sample orientation in relation to the bone-matrix microstructure in human cortical bone (top) and schematic model for bone deformation for the various hierarchical length-scales in response to external tensile load. Strain is simultaneously measured at all three levels of the structural hierarchy (tissue, fibril, and mineral nanoparticles). The mineralized fibrils, which are stiffened with collagen cross-links, deform in tension and transfer the stress between neighboring fibrils by shearing in the thin layer of the matrix. Within each mineralized fibril, the stiff mineral platelets deform in tension and transfer stresses between adjacent platelets by shearing in the interparticle collagen matrix. (Red lines demonstrate shearing qualitatively). (Adapted from ref. [10]) (b) At a fixed tissue strain of 0.85%, the individual strain in the fibrils is 42% less in the 70 kGy irradiated bone than in the healthy unirradiated bone. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

UV-Raman spectroscopy of (hydrated) unirradiated and x-irradiated human cortical bone. (a) UV-Raman spectra for five different irradiation groups after doses of 0, 0.05, 70, 210 and 650 kGy, showing specifically the large changes in the relative height of the amide I feature compared to the CH₂ wag peak. The amide I (primarily from C≡O stretch) peak has previously been a good indicator for observing changes in the protein arrangement as the amide is known to play a role in cross-linking and bonding. The amide I peak height of the peak monotonically increases with irradiation, consistent with an increase in cross-linking in the collagen. Some of the other noticeable organic features for the bone-matrix are the CH₂ wag peak (1454–1461 cm⁻¹), amide III (primarily from in-phase combination of NH in-plane bend and CN stretch, 1245–1260 cm⁻¹), and amide II (primarily from out of phase combination of NH in-plane bend and CN stretch). (Additional data from ref. [23]).

Fig. 9

Fourier transform infrared spectroscopy of human cortical bone in the unirradiated and 70 kGy irradiated condition. (a) Shows a comparison of the calculated spectroscopic ratio of the 1660:1690 cm⁻¹ peaks in unirradiated and 70 kGy irradiated samples. The 1660:1690 ratios are calculated through a combination of second derivative spectroscopy to locate the position of the underlying bands within the amide I region and then peak fitting of these subbands to determine the relative percent area of each underlying component. The area 1660:1690 ratio appears to correspond to the ratio of nonreducible/reducible collagen cross-links in bone [39]. Following irradiation of 70 kGy, the 1660:1690 ratio decreases by almost a third. (b) Shows FTIR spectra and the amide I underlying bands for both groups.

Fig. 10

The accumulation of advanced glycation end-products (AGEs) was fluorimetrically quantified in the cortex of the femora in unirradiated and 70 kGy irradiated human cortical bone samples. AGEs increase slightly when bone is irradiated with a dose of 70 kGy which increased the concentration of fluorescent (non-enzymatic) cross-links to ~21% more than in the unirradiated bone.