Keren Keinan-Adamsky

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.

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 to the Disordered Phase and Apatite Crystallite Morphology during Mineralization by an Acidic Mineral Binding Peptide from Osteonectin

Noncollagenous proteins regulate the formation of the mineral constituent in hard tissue. The mineral formed contains apatite crystals coated by a functional disordered calcium phosphate phase. Although the crystalline phase of bone mineral was extensively investigated, little is known about the disordered layers composition and structure, and less is known regarding the function of noncollagenous proteins in the context of this layer. In the current study, apatite was prepared with an acidic peptide (ON29) derived from the bone/dentin protein osteonectin. The mineral formed comprises needle-shaped hydroxyapatite crystals like in dentin and a stable disordered phase coating the apatitic crystals as shown using X-ray diffraction, transmission electron microscopy, and solid-state NMR techniques. The peptide, embedded between the mineral particles, reduces the overall phosphate content in the mineral formed as inferred from inductively coupled plasma and elemental analysis results. Magnetization transfers between disordered phase species and apatitic phase species are observed for the first time using 2D (1)H-(31)P heteronuclear correlation NMR measurements. The dynamics of phosphate magnetization transfers reveal that ON29 decreases significantly the amount of water molecules in the disordered phase and increases slightly their content at the ordered-disordered interface. The peptide decreases hydroxyl to disordered phosphate transfers within the surface layer but does not influence transfer within the bulk crystalline mineral. Overall, these results indicate that control of crystallite morphology and properties of the inorganic component in hard tissue by biomolecules is more involved than just direct interaction between protein functional groups and mineral crystal faces. Subtler mechanisms such as modulation of the disordered phase composition and structural changes at the ordered-disordered interface may be involved.

23Na and 2H magnetic resonance studies of osteoarthritic and osteoporotic articular cartilage

In this study, the short component of the 23Na T2 (T2f) and the 23Na and 2H quadrupolar interactions (νQ) were measured in bone‐cartilage samples of osteoarthritic (OA) and osteoporotic (OP) patients. 23Na νQ was found to increase in osteoarthritic articular cartilage relative to controls. Similar results were found in bovine cartilage following proteoglycan (PG) depletion, a condition that prevails in osteoarthritis. 23Na νQ and 1/T2f for articular cartilage obtained from osteoporotic patients were significantly larger than for control and osteoarthritic cartilage. Decalcification of both human and bovine articular cartilage resulted in an increase of 23Na νQ and 1/T2f, showing the same trend as the osteoporotic samples. Differences in the ratio of the intensity of the large 2H splitting to that of the small one in the calcified zone were also observed. In osteoporosis, this ratio was twice as large as that obtained for both control and osteoarthritic samples. The 2H and 23Na results can be interpreted as due to sodium ions and water molecules filling the void created by the calcium depletion and to calcium ions being located in close association with the collagen fibers. To the best of our knowledge, this is the first study reporting differences of NMR parameters in cartilage of osteoporotic patients. Magn Reson Med, 2010.

The effect of decalcification on the microstructure of articular cartilage assessed by 2H double quantum filtered spectroscopic MRI

Abstract2H quadrupolar splitting of deuterated water molecules is a sensitive measure of the order and density of the collagen fibers in articular cartilage. In the calcified zone, near the bone, two pairs of quadrupolar split satellites were previously observed. To examine whether the large splitting observed originates from the presence of calcium ions and hydroxyapatite, one-dimensional 2H single and double quantum filtered spectroscopic imaging were performed on articular cartilage-bone plugs before and after decalcification. After decalcification, the magnitude splitting of the two pairs of satellites did not change and orientation dependency was kept. However, the intensity of the large splitting was greatly enhanced. According to these results the two pairs of satellites do not stem from the presence of calcium ions and hydroxyapatite but originate from the presence of two groups of collagen fibers with different degrees of hydration. The enhanced intensity of the large splitting is attributed to an increased amount of water molecules that fill the void, resulting from the removal of hydroxyapatite, which resides near the fibers responsible for the large splitting. The quadrupolar splitting observed in the trabecular bone was not orientation-dependent, indicating a random orientation of the collagen fibers in that tissue.

Monitoring of the effect of intervertebral disc nucleus pulposus ablation by MRI

In order to investigate intervertebral disc (IVD) degeneration and repair, a quantitative non‐invasive tool is needed. Various MRI methods including qCPMG, which yields dipolar echo relaxation time (TDE), magnetization transfer contrast (MTC), and 1H and 2H double quantum filtered (DQF) MRI were used in the present work to monitor changes in rat IVD after ablation of the nucleus pulposus (NP), serving as a model of severe IVD degeneration. In the intact IVD, a clear distinction between the annulus fibrosus (AF) and the NP is obtained on T2 and TDE weighted images as well as on MTC maps, reflecting the high concentration of ordered collagen fibers in the AF. After ablation of the NP, the distinction between the compartments is lost. T2 and TDE relaxation times are short throughout the disc and MTC is high. 1H and 2H DQF signal, which in intact discs is obtained only for the AF, is now observable throughout the tissue. These results indicate that after ablation, there is an ingression of collagen fibers from the AF into the area that was previously occupied by the NP, as was confirmed by histology. Copyright

1,1‐Diamino‐2,2‐dinitroethylenes are always zwitterions

The nitration of tetraiodoethylene (7) yields 1,1‐diiodo‐2,2‐dinitroethylene (8). The latter reacts with alkylamines 9 or alkyldiamines 11 to give the corresponding acyclic 1,1‐diamino‐2,2‐dinitroethylenes 10 or their cyclic analogs 12, respectively. On the basis of liquid and solid‐state 13C and 15N NMR data, x‐ray analysis and ab initio calculations, we suggest that the title compounds are always zwitterionic and that the CA–CN bond is not a true double bond. Copyright

Ubiquitin immobilized on mesoporous MCM41 silica surfaces – Analysis by solid-state NMR with biophysical and surface characterization

Deriving the conformation of adsorbed proteins is important in the assessment of their functional activity when immobilized. This has particularly important bearings on the design of contemporary and new encapsulated enzyme-based drugs, biosensors, and other bioanalytical devices. Solid-state nuclear magnetic resonance (NMR) measurements can expand our molecular view of proteins in this state and of the molecular interactions governing protein immobilization on popular biocompatible surfaces such as silica. Here, the authors study the immobilization of ubiquitin on the mesoporous silica MCM41 by NMR and other techniques. Protein molecules are shown to bind efficiently at pH 5 through electrostatic interactions to individual MCM41 particles, causing their agglutination. The strong attraction of ubiquitin to MCM41 surface is given molecular context through evidence of proximity of basic, carbonyl and polar groups on the protein to groups on the silica surface using NMR measurements. The immobilized protein exhibits broad peaks in two-dimensional 13C dipolar-assisted rotational resonance spectra, an indication of structural multiplicity. At the same time, cross-peaks related to Tyr and Phe sidechains are missing due to motional averaging. Overall, the favorable adsorption of ubiquitin to MCM41 is accompanied by conformational heterogeneity and by a major loss of motional degrees of freedom as inferred from the marked entropy decrease. Nevertheless, local motions of the aromatic rings are retained in the immobilized state.

Dynamic Behavior of Supramolecular Comb Polymers Consisting of Poly(2-Vinyl Pyridine) and Palladium-Pincer Surfactants in the Solid State

When poly(2-vinyl pyridine) is combined with Pd-pincer-based organometallic surfactants, a mesomorphic structure forms due to weak stacking interactions between the pyridine units and the Pd-pincer headgroups. The weak binding between the surfactant and the polymer competes with the tendency of the aliphatic tails of the surfactant to crystallize. Here, we demonstrate that over extended periods of incubation, the crystallization tendency of the surfactant tails causes the surfactant molecules to detach from the polymer and gives rise to additional packing modes of the alkyl tails featuring higher crystalline order. The dynamic behavior of these aged structures was investigated by variable-temperature small-angle X-ray scattering (SAXS) and solid-state (13)C NMR, and revealed the influence of thermal changes on the molecular level, and how these changes propagate to the mesoscale structure.