Fourier transform raman spectroscopy of synthetic and biological calcium phosphates

Abstract

Fourier-transform (FT) Raman spectroscopy was used to characterize the organic and mineral components of biological and synthetic calcium phosphate minerals. Raman spectroscopy provides information on biological minerals that is complimentary to more widely used infrared methodologies as some infrared-inactive vibrational modes are Raman-active. The application of FT-Raman technology has, for the first time, enabled the problems of high sample fluorescence and low signal-to-noise that are inherent in calcified tissues to be overcome. Raman spectra of calcium phosphates are dominated by a very strong band near 960 cm⁻¹ that arises from the symmetric stretching mode $\left( {{\mathbf{\rlap{--} v}}_{\text{1}} } \right)$of the phosphate group. Other Raman-active phosphate vibrational bands are seen at approximately 1075 $\left( {{\mathbf{\rlap{--} v}}_{\text{3}} } \right)$, 590 $\left( {{\mathbf{\rlap{--} v}}_{\text{4}} } \right)$, and 435 cm⁻¹ $\left( {{\mathbf{\rlap{--} v}}_{\text{2}} } \right)$. Minerals containing acidic phosphate groups show additional vibrational modes. The different calcium phosphate mineral phases can be distinguished from one another by the relative positions and shapes of these bands in the Raman spectra. FT-Raman spectra of nascent, nonmineralized matrix vesicles (MV) show a distinct absence of the phosphate ${\mathbf{\rlap{--} v}}_{\text{1}}$band even though these structures are rich in calcium and phosphate. Similar results were seen with milk casein and synthetic Ca-phosphatidyl-serine-PO₄ complexes. Hence, the phosphate and/or acidic phosphate ions in these noncrystalline biological calcium phosphates is in a molecular environment that differs from that in synthetic amorphous calcium phosphate. In MV, the first distinct mineral phase to form contained acidic phosphate bands similar to those seen in octacalcium phosphate. The mineral phase present in fully mineralized MV was much more apatitic, resembling that found in bones and teeth. These findings are consistent with formation of an OCP-like precursor during MV mineral formation that subsequently hydrolyzes to form hydroxyapatite.