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. 2018 Nov 25;23(12):3081.
doi: 10.3390/molecules23123081.

Raman Spectroscopic Analysis to Detect Reduced Bone Quality after Sciatic Neurectomy in Mice

Yasumitsu Ishimaru  1 , Yusuke Oshima  2   3   4 , Yuuki Imai  5 , Tadahiro Iimura  6   7 , Sota Takanezawa  8 , Kazunori Hino  9 , Hiromasa Miura  10
Affiliations

Raman Spectroscopic Analysis to Detect Reduced Bone Quality after Sciatic Neurectomy in Mice

Yasumitsu Ishimaru et al. Molecules. .

Abstract

Bone mineral density (BMD) is a commonly used diagnostic indicator for bone fracture risk in osteoporosis. Along with low BMD, bone fragility accounts for reduced bone quality in addition to low BMD, but there is no diagnostic method to directly assess the bone quality. In this study, we investigated changes in bone quality using the Raman spectroscopic technique. Sciatic neurectomy (NX) was performed in male C57/BL6J mice (NX group) as a model of disuse osteoporosis, and sham surgery was used as an experimental control (Sham group). Eight months after surgery, we acquired Raman spectral data from the anterior cortical surface of the proximal tibia. We also performed a BMD measurement and micro-CT measurement to investigate the pathogenesis of osteoporosis. Quantitative analysis based on the Raman peak intensities showed that the carbonate/phosphate ratio and the mineral/matrix ratio were significantly higher in the NX group than in the Sham group. There was direct evidence of alterations in the mineral content associated with mechanical properties of bone. To fully understand the spectral changes, we performed principal component analysis of the spectral dataset, focusing on the matrix content. In conclusion, Raman spectroscopy provides reliable information on chemical changes in both mineral and matrix contents, and it also identifies possible mechanisms of disuse osteoporosis.

Keywords: Raman spectroscopy; bone matrix; bone quality; collagen crosslink; hydroxyapatite; mineral maturity; osteoporosis; sciatic neurectomy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The average Raman spectra obtained from neurectomized (NX group) mice and Sham operation mice (Sham group). Previous studies can be used to assign apparent peaks in Raman spectra originating from the bone mineral and matrix components (Table 1). Raman peaks originating from the mineral components are as follows: ν2 PO43−, 430–450 cm−1; ν4 PO43−, 584–610 cm−1; ν1 PO43−, 960 cm−1; and ν1 CO32− ν3 PO43−, 1035–1076 cm−1. Those originating from the matrix components are as follows: Proline, 855 cm−1 and 922 cm−1; hydroxyproline, 876 cm−1; phenylalanine, 1002 cm−1; amide III, 1243–1320 cm−1; CH2, 1448 cm−1; and amide I, 1664 cm−1.
Figure 2
Figure 2
Raman peak intensity ratios in the NX and Sham groups. (a) Carbonate/phosphate ratio (1069 cm-1/960 cm−1), (b) mineral/phenylalanine ratio (960 cm−1/1002 cm−1), and (c) mineral/proline+hydroxyproline ratio (960 cm−1/922 cm−1 + 855 cm−1 + 876 cm-1) were significantly higher in the NX group than in the Sham group. (d) Mineral/CH2 ratio (960 cm−1/1448 cm−1) and (e) mineral/amide I ratio (960 cm−1/1664 cm−1) were also higher in the NX group, and (f) amide I/CH2 ratio (1664 cm−1/1448 cm−1) was lower in the NX group, although there were no significant differences. ** p < 0.01.
Figure 3
Figure 3
Score plots of the Principal Component Analysis (PCA) analysis of Raman spectra. The X and Y axes include any principal components (PCs) that differed between the NX and Sham groups, with the distance on the axis indicating the degree of difference. PCA plots were built with (a) all spectral regions, (b) a partially extracted spectral region for the mineral component, and (c) a partially extracted spectral region for the matrix component.
Figure 4
Figure 4
Mean bone mineral density (BMD) of the (a) femurs and (b) tibiae. The BMD of both the femurs and tibiae was significantly decreased in the NX group compared to the Sham group. * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
Volume rendering images constructed from X-ray micro-CT data of the proximal tibiae of (a) Sham and (b) NX mice. In the NX mouse, the thickness of the cortical bone is obviously decreased, and the microstructure of the cancellous bone is altered.
Figure 6
Figure 6
Micro-CT parameters. Relative to the Sham group, the NX group demonstrated an altered (a) bone volume (BV/TV), (b) connectivity density, (c) structural model index (SMI), (d) trabecular number, (e) trabecular thickness, (f) trabecular separation, (g) cancellous BMD, (h) cortical thickness, and (i) cortical BMD. * p < 0.05, ** p < 0.01.
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