The aim of this study was to investigate molecular interactions between a bisphosphonate (BP), zoledronic acid, and bone tissue by the use of Raman microspectroscopy. In this way, samples of hydroxyapatite (HA), as a bone model, and Wistar rat femurs were soaking in zoledronic acid solutions. Sample surfaces were studied by Environmental Scanning Electron Microscopy and Raman spectroscopy. The amount of zoledronic acid incorporated onto the samples and the inorganic phosphate released in solution were determined by (31)P NMR spectroscopy. Total carbonate content in solution was evaluated by inorganic carbon analyser. After impregnation new Raman bands with frequencies close to characteristic peaks of zoledronic acid (in particular phosphate moieties and imidazole ring of the R2 side-chain) were observed on both types of samples. Physico-chemical parameters of the bone were also significantly modified (P<0.0001). The mineral to organic ratio and the carbonate to phosphate ratio decreased and the crystallinity increased. Released inorganic phosphate and carbonate were detected in the solutions. The Raman shift of the bands corresponding to the phosphate groups and the imidazole ring of the BP highlight their implication in the binding to the mineral. The detection of released inorganic phosphate and carbonate in solution, the modifications of the mineral to phosphate ratio and the carbonate to phosphate ratio reveal that BP decrease the amount of inorganic phosphate and limit the dissolution of bone mineral. The increase of the crystallinity after BP binding shows a re-organisation of the lattice with a higher symmetry. Thus, it seems that zoledronic acid has an important contribution on the increase of crystallinity. The use of Raman spectrometry brings new and complementary information on the impact of zoledronic acid on bone composition at molecular level. Raman spectrometry could help to understand by which way BPs improve bone strength and decrease fracture risk.
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