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Bone has complex hierarchical structure composed of mineral, collagen, lipids, water and non-collagenous proteins. Being able to probe at the ultrastructural level of bone hierarchy is important to understand age- and disease-related changes in the fracture resistance. Raman spectroscopy (RS) probes molecular vibration and is sensitive to advanced glycation end-products, damage to the bone matrix, thus fracture toughness properties. We studied effect of glycation and mechanical damage on characteristics of Amide I Band. N=20 cadaveric femurs were obtained from Vanderbilt Cadaver Program. We extracted medial and lateral quadrants of cortical bone (~50 mm) along the longitudinal axis of mid-shaft using saw. Dog-bone specimens were created for tensile tests while 2-rectangular beams for glycation incubation. a) For tensile tests, specimens were pulled-to-failure at 5mm/min using 1000 N load cell. b) For glycation, beam-1 (control) was incubated in 50 mM Na3PO4, 0.02% NaN3, protease inhibitor cocktail, and beam-2 (Glucose) with same buffer plus 0.5 M glucose for 5-weeks at 45 °C, pH 7.6. RS acquired before and after both tests indicates that Amide I sub-peak ratio 1670/1640 significantly increased post tensile tests compared to baseline. 1670 and 1640 cm-1 peaks are associated with random coils and alpha helix respectively. Thus, more disorganized structures are formed because of tensile damages. Glycated beams show lower 1670/1640 ratio compared to baseline and control. This in part due to increase in packing density of collagen molecules upon glycation. Overall, our results indicate that RS can assess cortical damage and sugar-mediated changes in the amide I band.
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