Elsevier

Bone

Volume 46, Issue 6, June 2010, Pages 1582-1590
Bone

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

https://doi.org/10.1016/j.bone.2010.02.020Get rights and content

Abstract

In this study, bone mineral density (BMD) of normal (CON), ovariectomized (OVX), and partially nephrectomized (NFR) rats was measured by 31P NMR spectroscopy; bone matrix density was measured by 1H 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 31P NMR) and 17.5% (by gravimetry) lower than CON. For trabecular bone specimens, BMD of the OVX group was 40.5% (by 31P NMR) and 24.6% (by gravimetry) lower than CON; BMD of the NFR group was 26.8% (by 31P 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 31P 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 31P NMR/WASPI method is capable of discerning the difference in EBM between animals with osteoporosis and those with impaired bone mineralization.

Introduction

According to the US Surgeon General's report on “Bone Health and Osteoporosis” released in 2004, there are roughly 10 million Americans over the age of 50 with osteoporosis. Additionally, another 34 million have low bone mass or “osteopenia” of the hip, which puts them at risk for osteoporosis, fracture, and the associated complications later in life [1].

Osteopenia is defined by the World Health Organization as having a bone mineral density (BMD) score between 1.0 and 2.5 standard deviations below the BMD of a normal young adult, and osteoporosis is defined as having a BMD lower than 2.5 standard deviations. In practice, BMD is measured by X-ray-based bone densitometry methods, such as Dual Energy X-ray Absorptiometry (DXA) and Quantitative and Peripheral X-ray Computed Tomography (QCT and pQCT) [2], [3], [4], [5]. However, in addition to osteoporosis, other metabolic bone diseases can also result in low BMD [6], [7], [8], [9].

In the case of osteoporosis, bone formation is not able to compensate for bone resorption, leading to thinning and loss of connectivity in trabecular bone and deep resorption cavities in cortical bone [10]. Studies and guidelines for diagnosis and management of osteoporosis have shown that even though the amount of mineral per unit volume of bone substance (mineral plus matrix) might not differ substantially from normal, the amount of mineral per unit volume of bone tissue (bone substance, bone cells, marrow, blood vessels, and other soft tissues) is decreased. In other words, while the extent of mineralization (EBM) of osteoporotic bone may be close to normal, BMD of osteoporotic bone tissue is definitely low [9], [11].

In the case of impaired bone mineralization, the osteoid thickness is increased and mineralization lag time is prolonged. Hence, the amount of mineral per unit volume of bone substance is lower, and the amount of mineral per unit volume of bone tissue also is decreased, that is, both the EBM and BMD are lower. The causes of impaired bone mineralization can be inadequate nutritional supply of vitamin D, calcium, or phosphorus; renal failure; or interference of certain drugs with calcium and vitamin D metabolism [12].

The critical point is that measurement of BMD alone does not permit the physician to distinguish osteoporosis from impaired bone mineralization, which could result in treatment for the wrong condition.

At present, the best direct measurements of EBM are histomorphometric indices, which can only be obtained invasively by bone biopsy [13]. Although serum vitamin D measurements can indicate that a mineralization defect is present, routine vitamin D blood testing frequently fails to detect the condition and cannot serve as a quantitative measure of EBM. There is an urgent need to develop new noninvasive methods that can measure not only BMD but bone matrix as well and provide physicians with information on bone mineralization.

The broad resonance lines and very short spin–spin relaxation times (T2) of the magnetic resonance signals of bone substance (31P signal from mineral and 1H signal from organic bone matrix) are the major obstacles for traditional MRI to image bone substance. New methods to overcome these obstacles have been developed recently. Ultrashort echo time (UTE) MRI has been developed by Robson et al., Reichert et al., and Du et al. to image solid components of human bone [14], [15], [16]. More recently, a radial projection pulse sequence with long-T2 soft tissue suppression has been developed by Techawiboonwong and co-workers to image the solid-like components of bone matrix [17]. Another approach to visualize short-T2 hard tissues, sweep imaging with Fourier transformation (SWIFT), has been developed by Idiyatullin and co-workers, in which magnetic resonance time-domain signals are acquired during a swept RF excitation in a time-shared manner [18]. Anumula et al. first investigated hypomineralization by 31P solid-state MRI [19], and later by 1H and 31P solid-state MRI [20].

Our group has been engaged in developing 31P solid-state magnetic resonance imaging (SMRI) to visualize bone mineral [21], [22], [23] and quantitative water- and fat-suppressed proton projection MRI (WASPI) to visualize solid bone matrix [24], [25], [26]. With these new methods, MRI measurements of bone mineral and matrix density have become available. These two methods emphasize a pair of key features that ensure the quantitative accuracy of the intensity of the solid-state signals: (a) the use of very short duration (∼ 10 μs) hard RF excitation pulses that uniformly excite large spectral bandwidths and (b) minimal delay (also ∼ 10 μs) between the end of the RF pulse and the beginning of data acquisition. These constraints require that no magnetic field gradient is switched in the interval from the start of the RF pulse to the end of data acquisition. Therefore, when imaging is performed, the excitation pulse is applied during a constant amplitude gradient pulse, and the image is reconstructed from free induction decays (FIDs) [22].

The purpose of this study is to assess the validity of 31P measurement of bone mineral content and 1H measurement of bone matrix content in rat models of reduced total bone density and reduced mineralization by comparison with gravimetric analysis. Because the size of rat bone specimens is small enough to be placed well within the effective volume of the MRI coil, single-pulse 31P spectroscopy was utilized to measure the total mineral content. WASPI 1H solid-state MRI was used to measure the matrix content. Reduced total bone was obtained with the ovariectomized (OVX) rat model, which has been widely used to study the effects of menopause on bone mass, trabecular microstructure, and fracture risk and to model human osteoporosis [27], [28], [29], [30], [31], [32], [33], [34], [35]. Reduced mineralization is achieved with the partially nephrectomized (NFR) rat model, which has been used to study the effects of renal osteodystrophy on bone metabolism [36], [37], [38], [39], [40], [41], [42].

Section snippets

Animal model and specimen preparation

Thirty mature female Sprague–Dawley (SD) rats (250–275 g, ∼ 15 weeks old) were obtained from Charles River Laboratories (Charles River, Charlestown, MA, USA) and were divided into three equally sized groups: the animals in the control group (CON) were not subjected to any surgical or dietary interventions. The OVX group underwent bilateral ovariectomy at the animal supplier's facility 1 week prior to arrival in the laboratory to induce a state of low bone mass and micro-architectural

Results

The spin–lattice relaxation time T1 (1H of rat bone matrix, 1H of polymer blend, and 31P of rat bone mineral, HA, KPF6) was measured and is listed in Table 1. 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 Eq. (2). The F values of the

Discussion

The OVX rat model has been widely used to induce osteoporosis in rat bone. In this study, 31P NMR spectroscopy showed significant decrease of both BMD and bone matrix density in OVX trabecular bone, while the ratio of these two measurements remained similar to that of the CON group, which strongly indicated the presence of osteoporosis: reduced bone mass while the extent of bone mineralization is normal.

The NFR model that has been reported in the literature would create renal insufficiency,

Acknowledgments

This work was supported by a grant from National Institutes of Health (EB004012). MR facilities were supported in part by National Institutes of Health grant P41RR14075 from the National Center for Research Resources, the MIND Institute, and the Athinoula A. Martinos Center for Biomedical Imaging. We thank Dr. Robert M. Neer of Massachusetts General Hospital for helpful discussions and Dr. Melvin J. Glimcher of Children's Hospital Boston for inspiration and guidance during the research and

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