Hadassah Shinar

Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells

Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.

NMR relaxation studies of intracellular Na+ in red blood cells.

The state of intracellular Na+ in human and dog erythrocytes was characterized by 23Na-NMR using dysprosium complexes as shift reagents. Intracellular Na+ concentrations were determined using integration of the inner Na+ NMR signals and measurements of the intracellular volume using 59Co-NMR of extracellular Co(CN)3-6. T2 was found to be significantly shorter than T1, indicating some binding to macromolecules. While the longitudinal magnetization decay follows a single exponential, the transverse magnetization could be fitted with a double-exponential function. It was shown that neither the binding to the inner side of the membrane nor binding to hemoglobin contributes to the relaxation enhancement.

Mapping the fiber orientation in articular cartilage at rest and under pressure studied by 2H double quantum filtered MRI.

The one‐dimensional 2H double quantum filtered (DQF) spectroscopic imaging technique was used to study the orientation of collagen fibers in articular cartilage. The method detects only water molecules in anisotropic environments, which in cartilage is caused by their interaction with the collagen fibers. A large quadrupolar splitting was observed in the calcified zone and a smaller splitting in the radial zone. In the transitional zone the splitting was not resolved and a small splitting was again detected in the superficial zone. From measurements performed at two orientations of the plug relative to the magnetic field it was deduced that in the calcified and radial zones the fibers are oriented perpendicular to the bone, bending at the transitional zone and flattening at the superficial zone. The effect of load applied to the cartilage–bone plug was monitored by the same technique. At low loads there is a small decrease in the quadrupolar splitting in the calcified zone, a marked decrease in the radial zone, and an increase of the splitting accompanied by a thickening of the superficial zone. Under high loads, while the thickening and the splitting of the superficial zone further increase, the splitting in the radial and calcified zones completely collapse. Pressure‐induced changes in the thickness of the surface zone indicate flattening of the collagen fibers near the surface. The marked collapse of the splitting near the bone at high pressures may result from crimping of the collagen fibers. Magn Reson Med 48:322–330, 2002.

Sodium interaction with ordered structures in mammalian red blood cells detected by Na-23 double quantum NMR.

Na-23 double and triple quantum filtered NMR spectra of intact dog and human red blood cells were measured with the pulse sequence 90 degrees-tau/2-180 degrees-tau/2-theta degrees-t1-theta degrees-t2(Acq). For theta = 90 degrees the triple quantum filtered spectra exhibited the typical multiple quantum filtered lineshape, characteristic of isotropic media, while the double quantum filtered ones presented a superposition of two signals, whose proportion depended on the creation time tau. This effect is due to the formation of both second and third rank tensors. The formation of the second rank tensor, T21 results from non-zero residual quadrupolar interaction and is related to the anisotropic motion of sodium ions. Measurements of the double quantum filtered spectra with theta = 54.7 degrees enabled the detection of the contribution of T21 exclusively. No residual quadrupolar interaction was detected for sodium in the cytoplasm, while unsealed ghosts displayed the double quantum filtered spectral pattern, similar to that of intact cells. The anisotropy of motion of the sodium at the plasma membrane of mammalian erythrocytes depended on the integrity of the cytoskeleton network. Theoretical analysis of the double quantum filtered spectra gave a value of residual quadrupolar splitting of approximately 20 Hz for intact unsealed ghosts. The data presented prove that double quantum filtering is a sensitive technique for detection of motional anisotropies in biological systems.

Multinuclear NMR and MRI studies of the maturation of pig articular cartilage

The maturation of pig articular cartilage was followed by 2H in‐phase double quantum filtered (IP‐DQF) spectroscopic MRI, 1H T2 MRI, and 23Na DQF and triple quantum filtered MRS. The results all lead to the conclusion that the order and density of the collagen fibers in articular cartilage increase from birth to maturity. At birth, both 2H IP‐DQF signal and 1H T2 were homogeneous throughout the cartilage and their values independent of the orientation of the plug relative to the magnetic field. At maturation, the 2H IP‐DQF spectrum near the bone is composed of two pairs of quadrupolar split satellites and the 1H T2 relaxation is biexponential, indicating the presence of two groups of collagen fibers. The 2H satellites are orientation dependent, indicating that the two groups of fibers are well ordered at maturation. The fast component of 1H T2 is also orientation dependent and thus we have concluded that this component results from residual dipolar interaction, while the slow T2 component in mature cartilage, as well as the T2 relaxation in immature cartilage, is governed by other mechanisms. Magn Reson Med, 2006.

Changes in axonal morphology in experimental autoimmune neuritis as studied by highb-value q-space1H and2H DQF diffusion magnetic resonance spectroscopy

Experimental autoimmune neuritis (EAN) has been studied in rat sciatic nerves by a combination of high b‐value 1H and 2H double quantum filtered (DQF) diffusion MRS. The signal decays of water in the 1H and 2H DQF diffusion MRS were found to be not monoexponential and were analyzed using the q‐space approach. The q‐space analysis of the 1H diffusion data detected two diffusing components, one having broad and the other having narrow displacement profiles. These components were shown to be very sensitive to the progression of EAN disease. The q‐space parameters were found to be abnormal at day 9 postimmunization before the appearance of clinical signs. The assignment of the component with the narrow displacement profile to axonal water has been corroborated by the 2H DQF diffusion MRS results. The displacement and the relative population of this slow and restricted diffusing component followed the processes of demyelination, axonal loss, and remyelination that occur in EAN. The displacements extracted from the slow‐diffusing component with the narrow displacement correlated well with the average size of the axons as deduced from electron microscopy (EM). The component with the broad displacement showed significant changes which were attributed to the formation of endoneurial edema. This observation was also corroborated by the 2H DQF diffusion MRS experiments. It seems, therefore, that q‐space analysis of high b‐values diffusion MRS is a promising new approach for early detection and better characterization of the different pathologies associated with EAN. This study demonstrates the utility of high‐b‐value q‐space diffusion MRS for studying white matter‐associated disorders in general. Magn Reson Med 48:71–81, 2002.

Copper-induced peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effect of antioxidants and its dependence on the oxidative stress.

In an attempt to deepen our understanding of the mechanisms responsible for lipoprotein peroxidation, we have studied the kinetics of copper-induced peroxidation of the polyunsaturated fatty acid residues in model membranes (small, unilamellar liposomes) composed of palmitoyllinoleoylphosphatidylcholine (PLPC). Liposomes were prepared by sonication and exposed to CuCl(2) in the absence or presence of naturally occurring reductants (ascorbic acid (AA) and/or alpha-tocopherol (Toc)) and/or a Cu(I) chelator (bathocuproinedisulfonic acid (BC) or neocuproine (NC)). The resultant oxidation process was monitored by recording the time-dependence of the absorbance at several wavelengths. The observed results reveal that copper-induced peroxidation of PLPC is very slow even at relatively high copper concentrations, but occurs rapidly in the presence of ascorbate, even at sub-micromolar copper concentrations. When added from an ethanolic solution, tocopherol had similar pro-oxidative effects, whereas when introduced into the liposomes by co-sonication tocopherol exhibited a marked antioxidative effect. Under the latter conditions, ascorbate inhibited peroxidation of the tocopherol-containing bilayers possibly by regeneration of tocopherol. Similarly, both ascorbate and tocopherol exhibit antioxidative potency when the PLPC liposomes are exposed to the high oxidative stress imposed by chelated copper, which is more redox-active than free copper. The biological significance of these results has yet to be evaluated.

The determination of intracellular water space by NMR

A new method for the determination of intracellular water space using NMR spectroscopy is described. The method is based on the measurement of 59Co NMR signal intensity of an inert, stable and membraneimpermeable cobalt(III) compound such as Co(CN)6 3− or Co(imidazole)6 3+ and the 2H or 1H NMR signal intensities of the freely permeable water. As an example of the method, the variation of the intracellular water space of human erythrocytes as a function of osmolality was measured

An observation of 23Na NMR triple-quantum dynamic shift in solution

Mult iplequantum-coherence spectroscopy of spin-: nuclei in solution has become recently a subject of intensive research, particularly in reference to biological systems. It was found, both theoretically and experimentally, that in all cases where the singlequantum-coherence decay is biexponential, higher-order coherences may be observed ( 1, 2). This situation occurs when the nucleus is bound in a slowly tumbling site, i.e., with a reorientation time, rR, of the order of the inverse of the kumor frequency, G’, or larger, or when it exchanges with such a site (3, 4). According to relaxation theory (5) the resonance frequency in such systems is shifted relative to the Larmor frequency. This shift is referred to as a dynamic frequency shift (DFS). The measurement of the DFS for single-quantum (SQ) coherences has been very difficult since the shifts are comparable to the linewidths (6-8). However, it has been found to have a pronounced effect in ESR spectra ( 9). Theory predicts that this shift is smaller than the linewidth for double-quantum (DQ) coherence ( 10) but significantly larger for tr iple-quantum ( TQ ) coherence ( 20, 2 I ) . In the present Communicat ion we report the first experimental observation of a TQ DFS in solution. In order to have a system without complications due to the presence of several species and chemical exchange we have chosen the stable complex of Na+ with 4,7,13,16,21-pentaoxa-l,lO-diazabicyclo[S.8.5] tricosane (Kryptofix 221) ( 12 ) , dissolved in glycerol. In the following we shall denote the complex as Na cryptate. G lycerol was selected as the solvent due to the steep ,temperature dependence of its viscosity, allowing the rotational correlation time to vary over a wide range. The relaxation times of the TQ coherence and the TQ DFS were measured by the pulse sequence (1, 2, 13, 14)

1H double-quantum-filtered MR imaging as a new tool for assessment of healing of the ruptured Achilles tendon

1H double‐quantum–filtered magnetic resonance imaging (DQF MRI) was applied to monitor the healing process of the Achilles tendons in rabbits after tenotomy. DQF MRI provides a new contrast, which is based on the non‐zero average of the dipolar interaction caused by anisotropic motion of water molecules, determined mainly by their interaction with the ordered collagen fibers. Tissues are characterized by the dependence of their DQF signal on the DQ creation time, τ. With the use of DQF MRI, higher tissue contrast is obtained between tendon, bone, skin, and muscle. The tendons, which give weak signals in standard MRI techniques, are highlighted in the 1H DQF image. The image changed dramatically during the healing process of the injured Achilles tendon. These changes matched the phases of the healing process. By using a τ‐weighted contrast, the DQF images indicate the part of tendon that has not completely healed, even after the conventional MRI appeared normal. Magn Reson Med 42:884–889, 1999.