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1H nuclei compartmentalization, exchange and self-diffusion in cortical bone by one- and two-dimensions NMR in homogeneous and inhomogeneous fields
Leonardo Brizi1, Marco Barbieri1, Claudia Testa1, and Paola Fantazzini1

1Physics and Astronomy, University of Bologna, Bologna, Italy

Synopsis

There is increasing interest in the study of water content, compartmentalization, exchange and its interaction with collagen in cortical bone for the evaluation of bone fracture risk. Here, we present the NMR characterization of 1H nuclei signals of the cortical bone. Different components (collagen, lipid, water) and different water compartments are identified measuring NMR properties and self-diffusion coefficients. The exchange between collagen and water protons is observed and an average residence time in the collagen is estimated. The results can contribute to optimize MRI protocols specifically for bone imaging and to characterize the role of water in this tissue.

INTRODUCTION

Reduced bone strength is associated to a loss of bone mass, usually evaluated by the estimation of the Bone Mineral Density (BMD) with Dual energy X-ray absorptiometry technique. BMD cannot fully explain the mechanical properties for this tissue as well, since other determinants are involved, and there is evidence of the significant role of water in its mechanical properties (1, 2). There is increasing interest in the study of water content, compartmentalization, exchange and its interaction with collagen in cortical bone for the evaluation of bone fracture risk (3). Recently, NMR/MRI studies (1, 4-5) have enabled clinical imaging of cortical bone, giving a new perspective to the diagnosis of bone diseases. Moreover, there is increasing awareness that also water content have a role in determining the so- called “quality” of the bone. Here, we present the NMR characterization of 1H nuclei signals, including water content, compartmentalization, exchange and diffusion, whose knowledge may help to optimize MRI protocols, and to characterize to role of water in the cortical bone system by low field- low cost portable devices (6).

METHODS

One and two dimensional 1H-NMR analyses were performed on cylindrical pig cortical bone samples (diameter~ 1 cm, height~ 1.5 cm) cored from the pig shoulder as shown in Figure 1. NMR measurements were executed by both a laboratory device (JEOL C60 - homogeneous magnetic field B0=0.473 T) and a low field portable, NMR single-sided device (MOUSE PM10, Magritek - inhomogeneous magnetic field with an average B0= 0.327 T). In the homogeneous field, a one pulse sequence was acquired to determine the FID behavior and a IR-FID sequence to study the longitudinal relaxation time. By the single-sided device, the STE-CPMG sequence was performed to estimate D-T2 correlation function. Data inversions were performed using UPEN for 1D Inverse Laplace Transform (ILT) and I2DUPEN for the 2D ILT (7, 8).

RESULTS AND DISCUSSION

We characterized 1H spin pools as low and high mobility protons and water compartments (bound water and pore water) by means of the different combinations of the measured parameters. FID analysis identified a fast quasi-Gaussian (solid-like) component due to low-mobility nuclei (almost all collagen protons) with a relaxation time Tgauss in the range 10 μs – 20 μs and a slower exponential decay component due to the high-mobility 1H nuclei (tentatively water and lipid) as shown in Figure 2. The analysis of IR-FID data indicated that all the FIDs, obtained at the different inversion times, revealed the presence of two 1H spin groups. The filtering technique (9-10) allowed us to separate the signal of the solid-like and the liquid-like components for which the quasi-continuous T1 distributions were computed. Because a cross-relaxation effect was expected, we performed the inversion without imposing the usual non-negative amplitude constraint. The results, shown in Figure 3, indicated magnetization and/or proton exchange between the two spin pools. Moreover, the exchange of magnetization between low and high mobility protons was detected by a spin-group analysis. The exchange between collagen and water protons has been demonstrated with an average residence time in the collagen of ~220 µs (Figure 4). The results of the 2D inversion of the D-T2 raw data, obtained by the single-sided device, are depicted in Figure 5. The diffusion coefficient of liquid-like components in cortical bone had D values in the range (1.2 – 0.03) µm2/ms. Three main compartments were found: pore water, lipids and bound water; the latter was presumably constituted by a slower component, (loosely-bound water) and a faster one (bound water).

CONCLUSION

Our results have further clarified compartmentalization, diffusion and exchange of water in cortical bone. Different components (collagen, lipid, water) and different water compartments, can be identified measuring different NMR properties (signal intensity and relaxation times) and self-diffusion coefficients, by diffusion-relaxation measurements. Therefore, our results show the possibility to develop a NMR method, based on low-cost, mobile and non-ionizing single-sided devices for the characterization of bone tissue properties, which can change in pathological conditions.
The longitudinal magnetization transfer and the proton chemical exchange suggest caution in the interpretation of T2 data in cortical bone. These results can contribute to optimize MRI protocols specifically for bone imaging and to characterize the role of water in this tissue.

Acknowledgements

We acknowledge Ms Roberta Fognani, Laboratory of Medical Technology, IRCCS Istituto Ortopedico Rizzoli, Bologna IT, for the preparation of the samples, and Fondazione del Monte di Bologna e Ravenna for the financial support to this project.

References

1. Granke M, et al. The role of Water Compartments in the Material Properties of Cortical Bone. Calcif Tissue Int. 2015;97(3):292-307.
2. Horch RA, et al. Clinically compatible MRI strategies for discriminating bound and pore water in cortical bone. PlosOne. 2011;6(1): e16359.
3. Granke M, et al. Identifying novel clinical surrogates to assess human bone fracture toughness. J Bone Miner Res. 2015;30(7):1119–1347.
4. Horch RA, et al. Characterization of 1H NMR signal in human cortical bone for magnetic resonance imaging. Magn Reson Med. 2010;64(3):680-687.
5. Gul-E-Noor F, et al. The Behavior of Water in Collagen and Hydroxyapatite Sites of Cortical Bone: Fracture, Mechanical Wear, and Load Bearing Studies. 2015;119(37):21528–21537.
6. Blümich B, Perlo J, Casanova F. Mobile single-sided NMR. Progress in Nucl Magn Res Spect. 2008;52(4):197–269.
7. Borgia GC, Brown RJS, Fantazzini P. Uniform-penalty inversion of multiexponential decay data. J Magn Res. 1998;132(1):65-77.
8. Bortolotti V, Brown RJS, Fantazzini P, et al. Uniform Penalty inversion of two-dimensional NMR relaxation data. Inverse Problems 2016:3319 doi:10.1088/1361-6420/33/1/015003.
9. Fantazzini P, et al. Solid-Liquid NMR relaxation and signal amplitude relationships with ranking of seasoned softwoods and hardwoods. J Appl Phys. 2006;100:0749071-7 doi: 10.1063/1.2354322.
10. Fantazzini P, et al. The search for negative amplitude components in quasi-continuous distributions of relaxation times: the example of 1H magnetization exchange in articular cartilage and hydrated collagen. New J Phys. 2011;13:065007.

Figures

Sketch of the transverse section of the pig cortical bone showing the position of the core samples preparation. The inset shows the core side towards the joint.

Example of the FID of a cortical bone sample. Raw data (black dots) and fit (green line). Data show a quasi-gaussian solid-like component due to low-mobility (collagen) protons and an exponential liquid-like component due to high-mobility protons (water and lipid).

The red curve for the solid-like component shows a negative peak and a positive peak. Both the negative and the positive peaks for the solid-like are in the same positions as for the liquid-like (blue curve). The correspondence of the two features for solid-like and liquid-like are a clear fingerprint of magnetization and/or proton exchange between the two spin pools.

Plot of T2FID values of the liquid components versus the ratio 1/α, where α is the ratio between the 1H amount in the solid-like over the liquid-like component. R2 = 0.979 for the fitting of the reported function: T2FID (µs) = 224 (µs) /α + 18 µs. This empirical law can be interpreted by applying the fast exchange theory. Under the assumption of two compartments in exchange at the equilibrium it can be shown that the equation in the inset is satisfied.

Example of a Diffusion-Relaxation map of cortical bone sample. Three main compartments were found: pore water (D = 1.2 µm2/ms, T2 = ~10 ms), lipids (D = 0.1 µm2/ms, T2 = ~2 ms) and bound water; the latter was presumably constituted by a slower component, loosely-bound water (D = 0.4 µm2/ms, T2 = 0.2 ms – 1 ms) and a faster one, bound water (D = (10-1 – 10-2) µm2/ms, T2 = ~10 ms).

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)
1286