Quantitative (31)P NMR spectroscopy and (1)H MRI measurements of bone mineral and matrix density differentiate metabolic bone diseases in rat models

Abstract

In this study, bone mineral density (BMD) of normal (CON), ovariectomized (OVX), and partially nephrectomized (NFR) rats was measured by (31)P NMR spectroscopy; bone matrix density was measured by (1)H water- and fat-suppressed projection imaging (WASPI); and the extent of bone mineralization (EBM) was obtained by the ratio of BMD/bone matrix density. The capability of these MR methods to distinguish the bone composition of the CON, OVX, and NFR groups was evaluated against chemical analysis (gravimetry). For cortical bone specimens, BMD of the CON and OVX groups was not significantly different; BMD of the NFR group was 22.1% (by (31)P NMR) and 17.5% (by gravimetry) lower than CON. For trabecular bone specimens, BMD of the OVX group was 40.5% (by (31)P NMR) and 24.6% (by gravimetry) lower than CON; BMD of the NFR group was 26.8% (by (31)P NMR) and 21.5% (by gravimetry) lower than CON. No significant change of cortical bone matrix density between CON and OVX was observed by WASPI or gravimetry; NFR cortical bone matrix density was 10.3% (by WASPI) and 13.9% (by gravimetry) lower than CON. OVX trabecular bone matrix density was 38.0% (by WASPI) and 30.8% (by gravimetry) lower than CON, while no significant change in NFR trabecular bone matrix density was observed by either method. The EBMs of OVX cortical and trabecular specimens were slightly higher than CON but not significantly different from CON. Importantly, EBMs of NFR cortical and trabecular specimens were 12.4% and 26.3% lower than CON by (31)P NMR/WASPI, respectively, and 4.0% and 11.9% lower by gravimetry. Histopathology showed evidence of osteoporosis in the OVX group and severe secondary hyperparathyroidism (renal osteodystrophy) in the NFR group. These results demonstrate that the combined (31)P NMR/WASPI method is capable of discerning the difference in EBM between animals with osteoporosis and those with impaired bone mineralization.

Fig. 1

Typical non-suppression MRI and WASPI images of rat bone specimens along with three polymer phantoms and bone marrow in a glass tube. a, c, e) Non-suppression MR images. A slice showing three polymer phantoms and bone marrow in a glass tube (a); A slice showing a cortical bone specimen (b); A slice showing a trabecular bone specimen (e). b, d, f) WASPI images corresponding to the subjects in a, c and e.

Fig. 2

Volume calibration using three tubes of water with known volumes of 0.112, 0.056, and 0.015 cm³, respectively. a) Non-suppression MR images of the three tubes. b) A volume calibration curve plotted as the volumes of the water tubes obtained from the total voxel numbers of the tubes above a threshold in non-suppression MRI vs. their known volumes.

Fig. 3

Process of obtaining bone matrix densities from WASPI experiments. Calibrations were created by linear regression of the physical densities (D_cpi) of the three polymer calibration pellets to their WASPI image densities (D_cwi). The arrows denote the process of obtaining the polymer equivalent bone matrix densities (D_BmP, expressed in terms of polymer g.cm⁻³) of the rat bone specimens from their WASPI image densities (D_BmW) using the linear regression coefficients. a) Rat normal cortical bone specimen. b) Rat normal trabecular bone specimen. The polymer equivalent bone matrix densities D_BmP were then converted to true bone matrix mass densities D_WmaT (refer to the text).

Fig. 4

Bone mineral densities of the rat bone specimens from CON, OVX, and NFR groups measured by ³¹P NMR spectroscopy and gravimetric analysis. Bars indicate mean ± SD. a) Cortical. b) Trabecular.

Fig. 5

Bone matrix densities of the rat bone specimens from CON, OVX, and NFR groups measured by WASPI and gravimetric analysis. Bars indicate mean ± SD. a) Cortical. b) Trabecular.

Fig. 6

The extents of bone mineralization (EBM), calculated as the ratios of bone mineral density to bone matrix density, of the rat bone specimens from CON, OVX, and NFR groups. Bars indicate mean ± SD. a) Cortical. b) Trabecular.

Fig. 7

Histology study of the rat bone samples from CON, OVX, and NFR groups.