Raman microspectroscopic studies of normal and pathological musculoskeletal tissues

Diseased states of bone tissue and cartilage were investigated by Raman spectroscopy. Composition standards for the quantitative analysis of bone composition were prepared and evaluated by Raman spectroscopy. These standards included L-proline, trans-4-hydroxy-L-proline and a 6% 13-type carbonated hydroxyapatite. Methods for working with delicate, mineralized tissue specimens, both archived and living were also developed. Raman imaging demonstrated a compatibility with standard histological embedding and staining techniques. The results demonstrated the ability to obtain previously unobtainable chemical information from archived tissue specimens. Raman spectroscopic images of normal and FGF2-induced craniosynostotic fetal day 18.5 mice showed increased mineralization and higher disorder in the mineral deposited in the craniosynostotic tissue. These results agree with previous genetic studies of the same craniosynostosis model. The craniosynostosis study was then expanded to include additional time points prior to and after the 48 hour mark. To accomplish this, a Raman tissue culture cell was developed that would be capable of maintaining a sterile environment and immobilizing the specimen, that allowed for same site observations to be made. Previously hypothesized unstable phases in the mineral composition of the craniosynostotic tissue were observed, phases that occur as early steps in mineral deposition. Mineralization in the craniosynostotic tissue was found to be somewhat different from the normal tissue. This result was not observed in earlier studies in which tissue development was stopped at least several hours before spectroscopic observation. In addition, mineral development was only observed after the first 48 hours of FGF2-induced craniosynostosis. Finally, the study was expanded to include cartilage. A genetic mouse model of osteoarthritis, the Dell (+/-) mouse line, was studied. In the osteoarthritic mice, age-dependent changes in the quality of the collagen and mineral were observed. These changes were observed in four different band area ratios in the Raman spectra of the mouse femurs.