8 March 2013 Polarization Raman spectroscopy to explain rodent models of brittle bone
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Activation Transcription Factor 4 (Atf-4) is essential for osteoblast maturation and proper collagen synthesis. We recently found that these bones demonstrate a rare brittleness phenotype, which is independent of bone strength. We utilized a confocal Renishaw Raman microscope (50x objective; NA=.75) to evaluate embedded, polished cross-sections of mouse tibia from both wild-type and knockout mice at 8 weeks of age (24 mice, n<=8 per group). Analysis of peak ratios indicated statistically significant changes in both mineral and collagen; however, compositional changes did not fully encompass biomechanical differences. To investigate the impact of material organization, we acquired colocalized spectra aligning the polarization angle parallel and perpendicular to the long bone axis from wet intact femurs. To validate our results, we used MMP9-/- mice, which have a brittleness phenotype that is not explained by compositional Raman measures. Polarization angle difference spectra show marked significant changes in orientation of these compositional differences when comparing wild type to knockout bones. Relative to wild-type, Atf4 -/- and MMP9 -/- bones show significant differences (t-test; p<0.05) in prominent collagen peaks. Further investigation of known peak ratios illustrates that this physical anisotropy of molecular organization is tightly clustered in brittle knockout bones. Such findings could have alternate interpretations about net collagen orientation or the angular distribution of collagen molecules. Use of polarization specific Raman measurements has implicated a structural profile that furthers our understanding of models of bone brittleness. Polarization content of Raman spectra may prove significant in future studies of brittle fracture and human fracture risk.
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Alexander J. Makowski, Alexander J. Makowski, Jeffry S. Nyman, Jeffry S. Nyman, Anita Mahadevan-Jansen, Anita Mahadevan-Jansen, "Polarization Raman spectroscopy to explain rodent models of brittle bone ", Proc. SPIE 8565, Photonic Therapeutics and Diagnostics IX, 85656L (8 March 2013); doi: 10.1117/12.2006487; https://doi.org/10.1117/12.2006487

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