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PRINCIPAL PUBLICATION AND AUTHORS

Primary radiation damage in bone evolves via collagen destruction by photoelectrons and secondary emission self-absorption, K. Sauer (a), I. Zizak (b), J.-B. Forien (c), A. Rack (d), E. Scoppola (e), P. Zaslansky (a), Nat. Commun. 13, 7829 (2022), https:/doi.org/10.1038/s41467-022-34247-z (a) Charité-Universitätsmedizin Berlin, Department for Operative, Preventive and Pediatric Dentistry, Berlin (Germany) (b) Helmholtz-Zentrum Berlin, Department for Structure and Dynamics of Energy Materials (SE-ASD), Berlin (Germany) (c) Lawrence Livermore National Laboratory, Materials Science Division, Livermore, California (USA) (d) ESRF (e) Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Potsdam (Germany)

fibres. These fibres entain strength and toughness to bones and are essential components of the structure.

Measurements of various bony samples, ranging from fish to mammalian bones and teeth, were made using micro-computed tomography imaging (µCT) at beamline ID19 and X-ray diffraction (XRD) mapping at the BESSY II synchrotron in Germany. To image the protein fibril layout directly within each bone type, the samples were examined using second-harmonic generation (SHG) confocal laser scanning microscopy. When the same samples were measured using X-rays, a trail of damage was observed. Regions of damage were clearly visible in sample domains imaged tomographically (Figure 4a), and in points of diffraction irradiation (XRD and XRD-µCT). This was clearly revealed by comparison of SHG images obtained before and after X-ray exposure (Figure 4b and c).

Precise measurements of the XRD beam profile and the dimensions of damage spread revealed that, unexpectedly, collagen is lost even in bone regions that were not directly exposed to the incoming X-ray beam. Measurements of strain relaxation due to damage accumulation, as well as Monte Carlo simulations, revealed the cause of damage: the photons become absorbed, mainly in the mineral, and then, high-energy electrons, emitted from the crystals of bone, scatter in all directions. This means that the nanocrystals become sources of electrons, with high penetration depth in air or vacuum, and they ionise and

destroy the surrounding organic matrix. These can be explained as follows:

1. High-energy photons from incoming diffraction or imaging X-ray beams trigger a cascade of electron excitations (Figure 5a). 2. Electrons ejected from the ionisation of calcium and phosphorus in the mineral scatter and damage the organic collagen framework in bone (Figure 5b). 3. Breakdown of collagen increases with the duration of irradiation but can be identified even with short exposures at high energy and flux.

These new findings support the growing consensus that measurements of bone should be limited to very short exposure times. Caution should be taken in basic medical research to ensure that bone structures of interest are not damaged. X-ray methods are considered non-destructive in materials research, but, at least for research on bone tissues, especially dry samples, this is not true: whereas the mineral is more or less unaffected, the adjacent organic component is severely damaged. These results show the mechanism of damage spread and highlight the vulnerability of the fibrous component of bone. It is thus essential to use the minimum dose necessary to obtain the insights that reflect the bone condition of interest, and to consider that damage to collagen by X-rays is probably unavoidable. Therefore, the results of this work highlight the need to pay close attention to the likely effects of radiation damage.

Fig. 5: Schematic representation of radiation damage expansion in bone. a) A cascade of electron scattering (black circles with minus) and X-ray fluorescence (rings) is created by the incoming X-ray beam (magenta). Calcium (Ca) emits photoelectrons as well as X-ray fluorescence that is absorbed by phosphorous (P) and collagen (cyan). The ejected photoelectrons lead to collagen disintegration. b) The combined effects of lower- energy X-ray fluorescence and secondary photoelectron scattering are the main contributors to collagen breakdown and primary radiation damage in bone.