Molecular spectroscopic identification of the water compartments in bone

Matrix bound water is a correlate of bone's fracture resistance and assessment of bound water is emerging as a novel measure of bone's mechanical integrity. Raman spectroscopy is one of the few nondestructive modalities to assess the hydration status in bone; however, it has not been used to study the OH-band in bone. A sequential dehydration protocol was developed to replace unbound (heat drying) and bound (ethanol or deuterium) water in bone. Raman spectra were collected serially to track the OH-band during dehydration. Spectra of synthetic hydroxyapatite, demineralized bone and bulk water were collected to identify mineral and collagen contributions to the OH-band. Band assignments were supported by computational simulations of the molecular vibrations of Gly-Pro-Hyp amino acid sequence. Experimentally and theoretically obtained spectra were interpreted for band-assignments. Water loss was measured gravimetrically and correlated to Raman intensities. Four peaks were identified to be sensitive to dehydration: 3220cm(-1) (water), 3325cm(-1) (NH and water), 3453cm(-1) (hydroxyproline and water), and 3584cm(-1) (mineral and water). These peaks were differentially sensitive to deuterium treatment such that some water peaks were replaced with deuterium oxide faster than the rest. Specifically, the peaks at 3325 and 3584cm(-1) were more tightly bound to the matrix than the remaining bands. Comparison of dehydration in mineralized and demineralized bone revealed a volume of water that may be locked in the matrix by mineral crystals. The OH-range of bone was dominated by collagen and the water since the spectral profile of dehydrated demineralized bone was similar to that of the mineralized bone. Furthermore, water associates to bone mainly by collagen as findings of experimentally and theoretically spectra. The current work is among the first thorough analysis of the Raman OH stretch band in bone and such spectral information may be used to understand the involvement of water in the fragility of aging and in diseased bone.