Anisotropy of collagen fiber orientation in sheep tendon by 1H double-quantum-filtered NMR signals (original) (raw)
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Study of order and dynamic processes in tendon by NMR and MRI
Journal of Magnetic Resonance Imaging, 2007
The anisotropy of the angular distribution of collagen fibrils in a sheep tendon was investigated by 1 H double-quantum (DQ) filtered NMR signals. Double-quantum build-up curves generated by the five-pulse sequence were measured for different angles between the direction of the static magnetic field and the axis of the tendon plug. Proton residual dipolar couplings determined from the DQ build-up curves in the initial excitation/reconversion time regime which mainly represent the bound water are interpreted in terms of a model of spin-1/2 pairs with their internuclear axes oriented on average along the fibril direction in the presence of proton exchange. The angular distribution of collagen fibrils around the symmetry axis of the tendon measured by the anisotropy of the residual dipolar couplings was described by a Gaussian function with a standard deviation of 12°AE 1°and with the center of the distribution at 4°AE 1°. The existence of this distribution is directly reflected in the finite value of the residual dipolar couplings at the magic angle, the value of the angular contrast, and the oscillatory behavior of the DQ build-up curves. The 1 H residual dipolar couplings were also measured from the doublets recorded by the DQ-filtered signals. From the angular dependence of the normalized splitting the angular distribution of the collagen fibrils was evaluated using a Gaussian function with a standard deviation of 19°AE 1°and with the center of distribution at 2°AE 1°. The advantages and disadvantages of these approaches are discussed.
Multiple Quantum Filtered NMR Studies of the Interaction between Collagen and Water in the Tendon
Journal of the American Chemical Society, 2002
We studied the physical processes and the chemical reactions involved in magnetization transfer between water and large proteins, such as collagen, in bovine Achilles tendon. Since the NMR spectrum for such proteins is broadened by very large dipolar interactions, the NMR peaks of the various functional groups on the protein cannot be separated from one another on the basis of their different chemical shifts. A further complication in observing the protein spectrum is the intense narrow peak of the abundant water. Thus, magnetization transfer (MT) within the protein or between water and the protein cannot rely on differences in the chemical shifts, as is commonly possible in liquids. We present a method that separates the protein spectrum from that of the water spectrum on the basis of their different intramolecular dipolar interactions, enabling exclusive excitation of either the protein or water. As a result, the protein spectrum as well as the effect of spin diffusion within the protein can be measured. In addition, the MT rates from the protein to water and vice versa can be measured. Two types of mechanisms were considered for the MT: chemical exchange-and dipolar interaction-related processes (such as NOE). They were distinguished by examining the effects of the following experimental conditions: (a) temperature; (b) pH; (c) ratio of D 2O to H2O in the bathing liquid; (d) interaction of the protein with small molecules other than water, such as DMSO and methanol. Our results lead us to the conclusion that the MT is dominated below the freezing point by the dipolar interaction between the protein and water, while an exchange of protons between the protein and the water molecules is the most significant process above the freezing point. On the basis of the fact that the spin temperature is established for the protein on a time scale much shorter than that of the MT, we could measure protein spectra that are distinguished by the contributions made to them by the various functional groups; i.e., contributions of methylenes were distinguished from those of methyls.
Structure and dynamics of water in tendon from NMR relaxation measurements
Biophysical Journal, 1990
Nuclear magnetic relaxation times were measured in collagen tissue when varying the orientation of the fiber with respect to the static field. T, was found to be only slightly dependent on 6, the fiber-to-field angle, but T2 was very sensitive to the orientation, with a maximum value at the magic angle. The transverse decay curves were multiexponential. Their deconvolution displayed four components; the ones that decayed most slowly were almost independent of 0, but the two fastest ones showed a strong angular dependence that was interpreted with a cross-relaxation model. Quadrupolar dips were visible in the 1 / T1 dispersion curves. These dips were independent of 0, so that the magnetization transfer could also be assumed to be independent of the fiber orientation. Finally, each component was assigned to a fraction of protons localized in the macromolecular structure and characterized by particular dynamics. The model of Woessner was applied to the water molecules tightly bound into the macromolecules, which resulted in a dynamical description of this water fraction. This description is compatible with the two-sites model of Ramachandran based on x-ray diffraction and with the extensive studies of Berendsen. However, the important indications obtained from the deconvolution lead to a less static representation of the tissue.
Journal of Magnetic Resonance, 1999
and the proton exchange rate at a temperature of 24°C and above, where no dipolar splitting is evident. The values obtained for these parameters at 24°C were 300 and 50 Hz and 3000 s ؊1 , respectively. The results for the residual dipolar interactions were verified by repeating the above measurements at a temperature of 1.5°C, where the spectra of the H 2 O molecules were well resolved, so that the 1 H-1 H dipolar interaction could be determined directly from the observed splitting. Analysis of the MQF experiments at 1.5°C, where the proton exchange was in the intermediate regime for the 1 H-2 H dipolar interaction, confirmed the result obtained at 24°C for this interaction. A strong dependence of the intensities of the MQF signals on the proton exchange rate, in the intermediate and the fast exchange regimes, was observed and theoretically interpreted. This leads to the conclusion that the MQF techniques are mostly useful for tissues where the residual dipolar interaction is not significantly smaller than the proton exchange rate. Dependence of the relaxation times and signal intensities of the MQF experiments on the orientation of the tendon with respect to the magnetic field was observed and analyzed. One of the results of the theoretical analysis is that, in the fast exchange regime, the signal decay rates in the MQF experiments as well as in the spin echo or CPMG pulse sequences (T 2 ) depend on the orientation as the square of the second-rank Legendre polynomial. © 1999 Academic Press Key Words: dipolar interaction; proton-proton dipolar interaction; proton-deuteron dipolar interaction; deuteron-deuteron dipolar interaction; heteronuclear triple-quantum filter; homonuclear triple-quantum filter; proton-deuterium spin correlation.
Anisotropy of spin relaxation of water protons in cartilage and tendon
NMR in Biomedicine, 2000
Transverse spin relaxation rates of water protons in articular cartilage and tendon depend on the orientation of the tissue relative to the applied static magnetic field. This complicates the interpretation of magnetic resonance images of these tissues. At the same time, relaxation data can provide information about their organisation and microstructure. We present a theoretical analysis of the anisotropy of spin relaxation of water protons observed in fully hydrated cartilage. We demonstrate that the anisotropy of transverse relaxation is due almost entirely to intramolecular dipolar coupling modulated by a specific mode of slow molecular motion: the diffusion of water molecules in the hydration shell of a collagen fibre around the fibre, such that the molecular director remains perpendicular to the fibre. The theoretical anisotropy arising from this mechanism follows the 'magic-angle' dependence observed in magnetic-resonance measurements of cartilage and tendon and is in good agreement with the available experimental results. We discuss the implications of the theoretical findings for MRI of ordered collagenous tissues.
Anisotropy in Tendon Investigated in Vivo by a Portable NMR Scanner, the NMR-MOUSE
Journal of Magnetic Resonance, 2000
Ordered tissue like tendon is known to exhibit the magic-angle phenomenon in magnetic resonance investigations. Due to the anisotropic structure the transverse relaxation time T 2 depends on the orientation of the tendon in the magnetic field. In medical imaging, relaxation measurements of tendon orientation are restricted by the size of the object and the space available in the magnet. For humans, tendon orientation can only be varied within small limits. As a consequence, the magic-angle phenomenon may lead to a misjudgement of tendon condition. It is demonstrated that the NMR-MOUSE (mobile universal surface explorer), a hand-held NMR sensor, can be employed to investigate the anisotropy of T 2 in Achilles tendon in vivo. The NMR-MOUSE provides a convenient tool for analyzing the correlation of T 2 and the physical condition of tendon.
The Magic Angle Effect in NMR and MRI of Cartilage
This chapter reviews the molecular basis of “magic angle” effect in cartilage beginning from fundamental concepts of physics and physical chemistry. MA effect is due to the unique oriented structure of collagen fibrils that occur in cartilage, tendon, ligaments and other connective tissues. Water is bound to the backbone of the collagen molecule in a repetitive manner that forms a constant time average proton-proton vector P-P parallel coaxial with fibril orientation. Constant P-P causes induction of frequency shifts 1000 Hz due to fixed orientation of an exchangeable proton relative to the fixed proton magnetic field B ~ ±10 Gauss of the neighboring immobilized bridge proton on the same water molecule. T2 relaxation times vary with orientation of collagen fibrils with the MRI magnetic field Bo as the effective local field in the collagen fibril is Be = Bo +B() and B() varies as a function of the angle between the vectors Bo and P-P. The Stoichiometric Hydration Model provides tools to relate changes in T1, T2 and T1 with orientation to specific changes in collagen structure. It is hoped that further study will relate measurable molecular shifts to disease progression in osteoarthritis and other injuries to cartilage.
Magnetic Resonance in Chemistry, 2004
We report solid-state NMR investigations of the effect of temperature and hydration on the molecular mobility of collagen isolated from bovine achilles tendon. 13 C cross-polarization magic angle spinning (MAS) experiments were performed on samples at natural abundance, using NMR methods that detect motionally averaged dipolar interactions and chemical shift anisotropies and also slow reorientational processes. Fast motions with correlation times much shorter than 40 µs scale dipolar couplings and chemical shift anisotropies of the carbon sites in collagen. These motionally averaged anisotropic interactions provide a measure of the amplitudes of the segmental motions expressed by a molecular order parameter. The data reveal that increasing hydration has a much stronger effect on the amplitude of the molecular processes than increasing temperature. In particular, the Cg carbons of the hydroxyproline residues exhibit a strong dependence of the amplitude of motion on the hydration level. This could be correlated with the effect of hydration on the hydrogen bonding structure in collagen, for which this residue is known to play a crucial role. The applicability of 1D MAS exchange experiments to investigate motions on the millisecond time-scale is discussed and first results are presented. Slow motions with correlation times of the order of milliseconds have also been detected for hydrated collagen.
Parameter maps of 1H residual dipolar couplings in tendon under mechanical load
Journal of Magnetic Resonance, 2003
Proton multipolar spin states associated with dipolar encoded longitudinal magnetization (DELM) and double-quantum (DQ) coherences of bound water are investigated for bovine and sheep Achilles tendon under mechanical load. DELM decay curves and DQ buildup and decay curves reveal changes of the 1 H residual dipolar couplings for tendon at rest and under local compression forces. The multipolar spin states are used to design dipolar contrast filters for NMR 1 H images of heterogeneous tendon. Heterogeneities in tendon samples were artificially generated by local compression parallel and perpendicular to the tendon plug axis. Quotient images obtained from DQ-filtered images by matched and mismatched excitation/reconversion periods are encoded only by the residual dipolar couplings. Semi-quantitative parameter maps of the residual dipolar couplings of bound water were obtained from these quotient images using a reference elastomer sample. This method can be used to quantify NMR imaging of injured ordered tissues.
Journal of Orthopaedic Research, 2003
It is difficult to monitor the chronic stage of the healing process of ruptured tendons employing the present diagnostic modes. However, the results of this study have shown that 1 H double quantum filtered (DQF) NMR spectroscopy is sensitive to the later stages of the healing process. Regenerated tendons of rabbits were dissected and measured at the end of the acute phase (three weeks), the subacute phase (nine weeks), and the chronic phase (13 and 18 weeks after tenotomy). Four parameters were determined by 1 H DQF NMR spectroscopy: (a) the maximum signal intensity (h max ) relative to the single quantum spectrum, (b) the creation time of the maximum signal intensity (s max ), (c) the decay time from the maximum signal intensity to a value half of that intensity (s 1=2 ) and (d) the residual dipolar splitting of water (d), representing the order of the collagen fibers. The values of h max , s max , s 1=2 , and d of the intact Achilles tendons were 11:3 AE 1:0%, 0:48 AE 0:03 ms, 0:67 AE 0:04 ms and 732 AE 62 Hz (mean AE SEM, n ¼ 6), respectively. In the regenerating tendon, h max increased from 0:41 AE 0:12% at three weeks to 7:07 AE 0:77% at 18 weeks, s max decreased from 1:88 AE 0:31 ms at three weeks to 0:72 AE 0:04 ms at 18 weeks, s 1=2 decreased from 11:6 AE 1:8 ms at 3 weeks to 1:48 AE 0:16 ms at 18 weeks, and d increased from 129 AE 8 Hz at three weeks to 414 AE 29 Hz at 18 weeks. We have concluded that reordering of collagen fibers proceeds continuously even in the chronic stage of healing. Thus, the 1 H DQF NMR spectroscopy is a useful noninvasive technique to evaluate the reconstruction and the order of collagen fibers in regenerating tendon. It is also suggested that s 1=2 and h max are most useful for in vivo DQF NMR spectroscopy and imaging, respectively, in combination with s max .