Effects of applied load on bone ultrastructure explored by Raman microspectroscopy and imaging.

Abstract

The mechanical properties of bone are of interest to researchers in many fields. However, the mechanical properties of bone ultrastructure---the collagenous organic matrix and apatitic mineral---have not been explored, owing to the difficulty in observing mechanical effects at this structural level using standard techniques. Elucidation of the effects of mechanical load on bone ultrastructure is vital to the understanding of this tissue's macroscopic mechanical properties. Raman spectroscopy is particularly well-suited to the study of bone mechanical properties, since it gives simultaneous information about the organic and mineral components of the tissue, and is non-destructive to the specimen. In addition, Raman spectroscopy has excellent spatial resolution (on the order of single microns), and is known to be a useful tool in the study of protein conformation and mineral composition. By extending Raman spectroscopy into an imaging mode, spatially-relevant compositional information can be obtained. To reduce the vast amount of spectral data in an image to usable size, multivariate and univariate statistics are employed. The research reported here describes several experiments. Initial experiments were performed on macroscopically-fractured fresh and fixed mouse femora. In these experiments, new mineral species were observed in the area of fracture, and there was evidence that the collagenous organic matrix was undergoing crosslink rupture. Subsequent experiments using a more controlled form of deformation, cylindrical indentation, showed that new mineral species were also observed within the indent area, while crosslink rupture was clearly observed at the edge of indents, where shear forces were strongest. This work is the first indication that bone mineral may undergo a phase transformation as a response to fracture. Preliminary results in using Raman as a real-time method for measuring the effects of deformation on bone tissue were also obtained. Deformation of the organic matrix with increased strain is clearly visible spectroscopically. Finally, a murine model for the genetic disease osteogenesis imperfecta was investigated. Spectroscopic differences in the carbonate content as well as the mineral to matrix ratio were observed in adolescent mice. These observations may explain the increase in bone strength observed in adolescent mice and humans afflicted with this disease.