Multi-modality study of the compositional and mechanical implications of hypomineralization in a rabbit model of osteomalacia

Abstract

Osteomalacia is characterized by hypomineralization of the bone associated with increased water content. In this work we evaluate the hypotheses that 1) 3D solid-state magnetic resonance imaging (MRI) of (31)P (SSI-PH) and (1)H (SSI-WATER) of cortical bone can quantify the key characteristics of osteomalacia induced by low-phosphate diet; and 2) return to normophosphatemic diet (NO) results in recovery of these indices to normal levels. Twenty female five-week old rabbits were divided into four groups. Five animals were fed a normal diet for 8 weeks (NOI); five a hypophosphatemic diet (0.09%) for the same period to induce osteomalacia (HYI). To examine the effect of recovery from hypophosphatemia an additional five animals received a hypophosphatemic diet for 8 weeks, after which they were returned to a normal diet for 6 weeks (HYII). Finally, five animals received a normal diet for the entire 14 weeks (NOII). The NOI and HYI animals were sacrificed after 8 weeks, the NOII and HYII groups after 14 weeks. Cortical bone was extracted from the left and right tibiae of all the animals. Water content was measured by SSI-WATER and by a previously reported spectroscopic proton-deuteron nuclear magnetic resonance (NMR) exchange technique (NMR-WATER), phosphorus content by SSI-PH. All MRI and NMR experiments were performed on a 9.4 T spectroscopy/micro-imaging system. Degree of mineralization of bone (DMB) was measured by micro-CT and elastic modulus and ultimate strength by 3-point bending. The following parameters were lower in the hypophosphatemic group: phosphorus content measured by SSI-PH (9.5+/-0.4 versus 11.1+/-0.3 wt.%, p<0.0001), ash content (63.9+/-1.7 versus 65.4+/-1.1 wt.%, p=0.05), ultimate strength, (96.3+/-16.0 versus 130.7+/-6.4 N/mm(2), p=0.001), and DMB (1115+/-28 versus 1176+/-24 mg/cm(3), p=0.003); SSI-WATER: 16.1+/-1.5 versus 14.4+/-1.1 wt.%, p=0.04; NMR-WATER: 19.0+/-0.6 versus 17.4+/-1.2 wt.%, p=0.01. Return to a normophosphatemic diet reduced or eliminated these differences (SSI-PH: 9.5+/-0.9 versus 10.6+/-0.8 wt.%, p=0.04; DMB: 1124+/-31 versus 1137+/-10 mg/cm(3), p=0.2; US: 95.6+/-18.6 versus 103.9+/-7.5 N/mm(2), p=0.2; SSI-WATER: 12.4+/-0.6 versus 12.2+/-0.3 wt.%, p=0.3) indicating recovery of the mineral density close to normal levels. Phosphorus content measured by SSI-PH was significantly correlated with DMB measured by micro-CT (r(2)=0.47, p=0.001) as well as with ultimate strength (r(2)=0.54, p=0.0004). The results show that the methods presented have potential for in situ assessment of mineralization and water, both critical to the bone's mechanical behavior.

Figure 1

Timeline showing the preparation and execution of the different phases in the study. HYI and NOI groups were sacrificed after 8 weeks. HYII was switched to normal diet after 8 weeks and both HYII and the control group NOII) were sacrificed after 14 weeks.

Figure 2

3D radial projection imaging sequence using ramp sampling for phosphorus and proton imaging designed for uniform mapping of k-space. The initial preparatory pulse, P₁ (60°), played out only once, was used for phosphorus with long T₁ in order to prepare the magnetization into the steady state.

Figure 3

Central 20 slices from the data set of one of the bone specimens, with reference capillaries (in endosteal cavity) used for quantification: a) ³¹P (SSI-PH); b) water (SSI-WATER). Voxel size: 277×277×277 μm³ (a) and 183×183×183 μm³ (b). Volume rendered images of the same specimen obtained by SSI-PH (c) and SSI-WATER d).

Figure 3

Central 20 slices from the data set of one of the bone specimens, with reference capillaries (in endosteal cavity) used for quantification: a) ³¹P (SSI-PH); b) water (SSI-WATER). Voxel size: 277×277×277 μm³ (a) and 183×183×183 μm³ (b). Volume rendered images of the same specimen obtained by SSI-PH (c) and SSI-WATER d).

Figure 3

Central 20 slices from the data set of one of the bone specimens, with reference capillaries (in endosteal cavity) used for quantification: a) ³¹P (SSI-PH); b) water (SSI-WATER). Voxel size: 277×277×277 μm³ (a) and 183×183×183 μm³ (b). Volume rendered images of the same specimen obtained by SSI-PH (c) and SSI-WATER d).

Figure 3

Central 20 slices from the data set of one of the bone specimens, with reference capillaries (in endosteal cavity) used for quantification: a) ³¹P (SSI-PH); b) water (SSI-WATER). Voxel size: 277×277×277 μm³ (a) and 183×183×183 μm³ (b). Volume rendered images of the same specimen obtained by SSI-PH (c) and SSI-WATER d).

Figure 4 a

Group differences showing lower measures of bone phosphorus quantified by solid state ³¹P MRI (SSI-PH) (wt%), ash (wt%), Ultimate strength (US) in N/mm² and degree of mineralization of bone (DMB) in mg/cm³ in the hypophosphatemic group (HYI) as compared to the same age control group (NOI) in phase I. Bars indicate mean±SD and p represents the statistical significance.

Figure 4 b

Same measures as in Figure 4a comparing hypophosphatemic (HYII) and control group (NOII) in phase II indicating partial recovery of HYII animals upon return to normophosphatemic diet.

Figure 5 a

Group differences showing higher water content quantified by solid state ¹H MRI (SSI-WATER) (wt%), exchange NMR (NMR-WATER) (wt%) and gravimetry (Drying-WATER) (wt%) in the hypophosphatemic group (HYI) as compared to the same age control group (NOI) in phase I. Bars indicate mean±SD and p represents the statistical significance.

Figure 5 b

Same measures as in Figure 5a comparing hypophosphatemic (HYII) and control group (NOII) in phase II indicating partial recovery of HYII animals upon return to normophosphatemic diet.