Raman spectroscopy demonstrates prolonged alteration of bone chemical composition following extremity localized irradiation.

introductionRadiotherapy to the appendicular skeleton can cause an increased risk of developing catastrophic fractures with delayed bone healing or non-union, and may subsequently require multiple procedures and amputation. Biomechanical studies suggest that irradiated bone is more brittle, but the cause is unclear and cannot be explained by changes to bone structure or quantity, suggesting that there are crucial changes in irradiated bone material properties. Raman spectroscopy provides a means to assess the chemical properties of the mineral and matrix constituents of bone, which could help explain post-radiation embrittlement. In this study we use a murine tibial model with focal irradiation and perform Raman spectroscopy to test the hypothesis that changes in bone chemistry following irradiation is consistent with reduced bone quality and persists in the long term after irradiation.

methodsFemale BALB/F mice aged 12weeks were subjected to unilateral, localized hindlimb irradiation in 4 daily 5Gy fractions (4×5Gy) totaling 20Gy, and were euthanized at 1, 4, 8, 12, and 26weeks post-irradiation (n=6/group). The irradiated (right) and non-irradiated contralateral control (left) tibiae were explanted and assessed by non-polarized and polarized Raman spectroscopy over the proximal cortical bone surface. Raman parameters used included the mineral/matrix ratio, mineral crystallinity, carbonate/phosphate ratio, collagen cross-link ratio, and depolarization ratio.

resultsSignificantly increased collagen cross-link ratio and decreased depolarization ratio of matrix were evident at 1week after irradiation and this persisted through 26weeks. A similar significant decrease was observed for depolarization ratio of mineral at all time points except 8 and 26weeks. At 4weeks after irradiation there was a significantly increased mineral/matrix ratio, increased mineral crystallinity, and decreased carbonate/phosphate ratio compared to controls. However, at 12weeks after irradiation these parameters had moved in the opposite direction, resulting in a significantly decreased mineral/matrix ratio, decreased crystallinity and increased carbonate/phosphate ratio compared to controls. At 26weeks, mineral/matrix, crystallinity and carbonate/phosphate ratios had returned to normal. DISCUSSION: In this mouse model, Raman spectroscopy reports both bone mineral and collagen cross-link radiation-induced abnormalities that are evident as early as one week after irradiation and persists for 26weeks. The picture is one of extensive damage, after which there is an attempt at remodeling. We hypothesize that pathological cross-links formed by radiation damage to collagen are poorly resorbed during the altered remodeling process, so that new tissue is formed on a defective scaffold, resulting in increased bone brittleness.