The spin-lattice relaxation time T1
H of rat bone matrix, 1
H of polymer blend, and 31
P of rat bone mineral, HA, KPF6
) was measured and is listed in . The results show that the proton T1
of the polymer blend was very similar to that of solid bone matrix. Based on the T1
values and excitation angles, a TR
of 0.15 s was chosen in the following WASPI measurements of bone matrix. The correction factors F
under these experimental conditions were calculated using equation 2
. The F
values of the bone specimen and polymer phantom was very close, with the difference being about 4% (). Therefore, no proton T1
correction was needed. The 31
of the bone specimens were much longer than that of KPF6
, which serves as calibration reference. The correction factors F
were calculated for each group with TR
= 1 s and flip angle β
(). The long 31
of bone mineral necessitated correction for T1
effects using the correction factor F
described in the Materials and methods section.
Table 1 Spin-lattice relaxation times T1 of the protons in polymer blend and rat bone specimens, and 31P spins in hydroxyapatite (HA), KPF6 and rat bone specimens at 4.7 T. Excitation pulse β was obtained from the fitting of equation 1.
Non-suppression MRI and WASPI experiments were performed on each bone specimen along with the polymer calibration phantom and the marrow tube. shows typical non-suppression MRI and WASPI images of the calibration phantoms, and the trabecular and cortical bone specimens. In non-suppression MRI (), the liquid-state proton signal (water and fat) from bone marrow dominates the image and both the marrow tube and bone specimens are very bright. In the WASPI images, the marrow tube is invisible and the water and fat signals from the bone marrow are suppressed to less than 4% in WASPI (). Due to the magnetic susceptibility discontinuity at the fluid-air interface, there is a substantial field gradient present, and a thin superficial layer of marrow signal is not suppressed (). This susceptibility artifact is greatly alleviated in vivo where the bone is surrounded by soft tissue. The WASPI signal of the rat bone () thus originates from solid bone matrix (collagen, tightly bound water, and other immobile molecules).
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 (more ...)
In order to evaluate the accuracy of the bone tissue volume TV obtained by counting the total voxel number in non-suppression MR images, three tubes of water with known volumes of 0.112, 0.056, and 0.015 cm3 measured by weighing the water was imaged by non-suppression MRI (). The total number of voxels above a threshold intensity were obtained for each water tube. These numbers were converted to the volume in cubic centimeter by multiplying by a factor (FOV/64)3. The volumes obtained were plotted against their known volumes and displayed in . A strong positive correlation (r2=0.9997) and a slope close to 1 indicate that the volumes measured by non-suppression MRI are very close to the real volumes. The errors of the volumes measured by non-suppression MRI are less than 5%.
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 (more ...)
The calibration curve that linked the polymer phantom image density obtained from the WASPI image to the actual polymer mass density was obtained for each imaging experiment. WASPI derived bone matrix image density values were converted to polymer equivalent mass density (polymer g.cm−3) according to these regression relationships (). The true matrix densities of the bone tissue were obtained as described in the Materials and methods section.
Fig. 3 Process of obtaining bone matrix densities from WASPI experiments. Calibrations were created by linear regression of the physical densities (Dcpi) of the three polymer calibration pellets to their WASPI image densities (Dcwi). The arrows denote the process (more ...)
To test the quantitative accuracy of the 31P measurements, five HA phantoms with known HA weights were measured using 31P spectroscopy. The results are shown in . The 31P T1 of the reference KPF6 was 3 times longer than that of HA, resulting in a KPF6 correction factor F 39% larger than that of HA (). Without T1 correction, significant errors were observed; however, after T1 correction, the 31P NMR measurements proved to be very accurate with errors within 5% ().
Accuracy of 31P NMR spectroscopy measurement of mineral content using five HA phantoms with known mass.
shows the bone mineral densities measured by 31P NMR spectroscopy and gravimetric analysis for the CON, OVX, and NFR groups. The mineral density values obtained by 31P NMR were close to those obtained by gravimetric analysis, which proves the accuracy of these 31P NMR measurements. For cortical bone specimens, both 31P NMR measurements and gravimetric analysis showed that the BMD of the OVX group was not different from that of the CON group (p = 0.98 and 0.97 respectively), yet the BMD of the NFR was 22.1% (by 31P NMR, p < 0.001) and 17.5% (by gravimetric analysis, p = 0.004) lower than that of the CON group. The BMD of trabecular bone tissue was found to be about 40.5% lower in the OVX group relative to the CON group (p < 0.001) by 31P NMR, and 24.6% lower (p = 0.04) by gravimetric analysis. Decreased BMD was also found in the trabecular NFR group by both 31P NMR (26.8% lower) and gravimetric analysis (21.5% lower) relative to the CON group; the differences were significant (p < 0.001 by 31P NMR and p = 0.02 by gravimetric analysis).
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.
shows the bone matrix densities measured by WASPI and gravimetric analysis for the CON, OVX and NFR groups. No significant changes of cortical bone matrix density in the OVX group were observed by either WASPI or gravimetric analysis relative to the CON group, yet the cortical bone matrix densities in the NFR group were 13.9% lower than that of the CON group by gravimetric analysis (p = 0.01) and 10.3% lower by WASPI (p = 0.06). On the contrary, although the trabecular bone matrix density in the NFR group was 3.7% lower by WASPI and 12.5% lower by gravimetric analysis relative to the CON group, the decreases are not significant (p = 0.70 by WASPI and p = 0.07 by gravimetric analysis), yet the trabecular bone matrix densities in the OVX group were 30.8% lower than that of the CON group by gravimetric analysis (p=0.002) and 38.0% lower by WASPI (p = 0.001).
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.
The extents of bone mineralization (EBMs) were calculated as the ratio of BMD to bone matrix density from the above measurements (). Although the EBM values of the OVX specimens are somewhat higher than those of the CON group, they are not significantly different. On the contrary, EBMs of the cortical and trabecular specimens were found to be about 12.4% and 26.3% lower in the NFR group relative to the CON group (p = 0.06 and p = 0.02) by MR, and 4.0% and 11.9% lower (p = 0.02 and p = 0.17) by gravimetric analysis. Except for the last measurement, these differences are all statistically or marginally significant; the p = 0.17 comparison is due to the large standard deviation in the trabecular NFR gravimetry measurement.
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.
Histopathology studies were performed on representative samples from each group after WASPI. Microscopic evaluation revealed morphologic differences in the rat femoral diaphysis bone specimens examined. The control specimen showed normal cortical and cancellous bone with evidence of previous endochondral ossification. The ovariectomized specimen showed a normal cortex with evidence of previous endochondral ossification. With microCT studies on the OVX group, evidence of osteoporosis was found (published elsewhere) [45
]. The partially nephrectomized specimen showed evidence of marked osteoclastic activity with cortical cutting cones and significantly resorbed trabeculae. Also present was abundant woven bone formation deposited on the surfaces of the trabeculae and endosteum of the cortex and in the cutting cones. Also noted are several calcospherules within fibrous tissue filling some of the spaces where bone was previously resorbed. These morphologic findings are consistent with severe secondary hyperparathyroidism ().
Histology study of the rat bone samples from CON, OVX, and NFR groups.