Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Bone. 2013 Nov;57(1):252-8. doi: 10.1016/j.bone.2013.08.014. Epub 2013 Aug 23.
Radiotherapy 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.
Female 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.
Significantly 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.
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.
四肢骨骼的放射治疗会增加发生灾难性骨折的风险，这些骨折愈合缓慢或不愈合，可能随后需要多次手术和截肢。生物力学研究表明，受照射的骨骼更脆弱，但原因尚不清楚，不能用骨结构或数量的变化来解释，这表明受照射骨骼的材料特性发生了关键性变化。拉曼光谱提供了一种评估骨矿物质和基质成分化学性质的方法，这有助于解释放射后骨的脆性增加。在这项研究中，我们使用一种具有局灶性照射的小鼠胫骨模型，并进行拉曼光谱分析，以检验以下假设：照射后骨化学性质的变化与骨质量降低一致，并在照射后长期持续存在。
12 周龄雌性 BALB/F 小鼠接受单侧、下肢局部照射，每天 4 次，每次 5Gy（4×5Gy），共 20Gy，照射后 1、4、8、12 和 26 周（每组 n=6）处死。取出照射（右侧）和未照射的对侧对照（左侧）胫骨，在近皮质骨表面进行非偏振和偏振拉曼光谱分析。使用的拉曼参数包括矿化/基质比、矿化结晶度、碳酸盐/磷酸盐比、胶原交联比和去偏振比。
照射后 1 周，胶原交联比显著增加，基质去偏振比显著降低，这种情况持续到 26 周。除 8 周和 26 周外，所有时间点的矿化去偏振比均观察到类似的显著降低。照射后 4 周，与对照组相比，矿化/基质比显著增加，矿化结晶度增加，碳酸盐/磷酸盐比降低。然而，在照射后 12 周，这些参数的变化方向相反，导致与对照组相比，矿化/基质比、结晶度和碳酸盐/磷酸盐比显著降低。在 26 周时，矿化/基质、结晶度和碳酸盐/磷酸盐比恢复正常。
在这个小鼠模型中，拉曼光谱报告了骨矿物质和胶原交联的辐射诱导异常，这些异常在照射后 1 周即可检测到，并持续 26 周。其特征是广泛的损伤，随后进行重塑。我们假设，胶原辐射损伤形成的病理性交联在改变的重塑过程中不易被吸收，因此，新组织在有缺陷的支架上形成，导致骨骼脆性增加。
