The aggregation of pig articular chondrocyte and synthesis of extracellular matrix by a lactose-modified chitosan.

A reductive amination reaction (N-alkylation) obtained exploiting the aldheyde group of lactose and the amino group of the glucosamine residues of chitosan (d.a. 89%) afforded a highly soluble engineered polysaccharide (chitlac) for a potential application in the repair of the articular cartilage. Chitosan derivatives with 9% and 64% of side chain groups introduced have been prepared and characterized by means of potentiometric titration, (1)H-NMR and intrinsic viscosity. Both polymers, with respect to the unmodified chitosan, induce cell aggregation when in contact with a primary culture of pig chondrocytes, leading to the formation of nodules of considerable dimensions (up to 0.5-1 mm in diameter). The nodules obtained from chondrocytes treated with chitlac with the higher degree of substitution have been studied by means of optical and electron microscopy (SEM, TEM) and the production of glycosaminoglycans (GAGs) and collagen has been measured by means of colorimetric assays. The chondro-specificity of GAG and collagen was determined by RT-PCR. The results show that the lactose-modified chitosan is non-toxic and stimulates the production of aggrecan and type II collagen.

Transverse relaxation mechanisms in articular cartilage.

Relaxation rates in the rotating frame (R1rho) and spin-spin relaxation rates (R2) were measured in articular cartilage at various orientations of cartilage layer to the static magnetic field (B0), at various spin locking field strengths and at two different static magnetic field strengths. It was found that R1rho in the deep radial zone depended on the orientation of specimens in the magnet and decreased with increasing the spin locking field strength. In contrast, R1rho values in the transitional zone were nearly independent of the specimen orientation and the spin locking field strength. Measurements of the same specimens at 2.95 and 7.05 T showed an increase of R1rho and most R2 values with increasing B0. The inverse B0 dependence of some R2 values was probably due to a multicomponent character of the transverse magnetization decay. The experiments revealed that the dominant T1rho and T2 relaxation mechanism at B0 < or = 3 T is a dipolar interaction due to slow anisotropic motion of water molecules in the collagen matrix. On average, the contribution of scalar relaxation due to rapid proton exchange in femoral head cartilage at 2.95 T is about 6% or less of the total R1rho at the spin locking field of 1000 Hz.

Numerical simulation of trabecular bone magnetic resonance imaging.

A numerical simulation of trabecular bone structure MR imaging is described. The input of the model is derived from synchrotron 3D muCT trabecular bone images with a resolution of 14mummu14mumx14mum. The static magnetic field perturbation in the bone sample induced by the differences in magnetic susceptibility values between mineralized bone and bone marrow is computed and the MRI experiment for a selected imaging sequence is modeled.

Short-TE projection reconstruction NMR microscopy of trabecular bone.

The aim of this study was to assess the potential of projection reconstruction (PR) NMR microscopy in the quantitative evaluation of trabecular bone architecture. Short-TE PR spin-echo microimages were acquired at 7.05 T on normal bone explants. The main structural parameters such as bone volume fraction (BVF), trabecular thickness (Tb.Th.) and trabecular separation (Tb.Sp.) were obtained from the 3D microimages using the method of directed secants. Quantitative structural data were then compared with those derived from conventional spin-echo microimages. Our study indicates that projection reconstruction NMR microscopy promises to be more accurate than the conventional FTI method in the analysis of trabecular bone.

Proteoglycan depletion and magnetic resonance parameters of articular cartilage.

Calcium ions and various amounts of proteoglycans were removed from porcine articular cartilage explants using ethylenediaminetetraacetic acid or guanidinium chloride solutions. The water proton magnetic parameters such as T(1) and T(2) relaxation times, diffusion (D), and magnetization transfer (M(S)/M(0)) were then measured by 1D MR microscopy on native specimens, after incubation in the extracting solutions and after final reconditioning in a physiological saline. While the replacement of the interstitial fluid by the treating solutions strongly affected the various MR parameters, calcium depletion did not show any influence on the MRI appearance of the chondral tissue. Interestingly, only the longitudinal relaxation time T(1) and the diffusion coefficient D were seen to be sensitive to an extensive proteoglycan depletion of the tissue. Our results indicate that a modest proteoglycan depletion, as it occurs in the early stage of a pathological cartilage degradation, has little relevance to the above MR parameters. Further MRI studies on the macromolecular components of cartilage are, therefore, necessary for a better understanding of the interaction mechanisms between water and extracellular matrix that might lead to the early diagnosis of the cartilage damage.

Collagen fibrils are differently organized in weight-bearing and not-weight-bearing regions of pig articular cartilage.

The magnetic resonance (MR) appearance of the weight-bearing ("loaded") and not-weight-bearing ("unloaded") regions in T(2)-weighted images of pig articular cartilage is different. On the hypothesis that this difference may be ascribed, at least in part, to a different collagen fibre organization in the two regions, this organization was studied using biochemical, histological, and X-ray diffraction methods. While the mean concentrations of collagen and of its cross-links were the same in the two regions, a regular small angle X-ray diffraction pattern was observed only for the habitually "loaded" tissue. It was also seen by light microscopy that the four typical functional zones were well displayed in the "loaded" cartilage whereas they were not clearly depicted in the "unloaded" tissue. Collagen presented a high concentration of fibrils forming an intricate and dense meshwork at the surface of both "loaded" and "unloaded" cartilage. A second zone of high collagen concentration was present at the upper layer of the deep zone of "loaded" cartilage. By contrast, this lamina of highly concentrated fibrils was lacking in "unloaded" cartilage and collagen fibrils appear thinner. Our study proves that the organization of collagen fibres is different for the "loaded" and "unloaded" regions of articular cartilage. It also suggests that this different organization may influence the MR appearance of the tissue. J. Exp. Zool. 287:346-352, 2000.

Short-TE projection reconstruction MR microscopy in the evaluation of articular cartilage thickness.

The aim of this study was to assess the potential of projection-reconstruction (PR) MR microscopy in the accurate measurement of cartilage thickness. Short-TE PR microimages were acquired at 7.05 T on bone-cartilage cylindrical plugs excised from four regions of two disarticulated femoral heads (i. e. superior, inferior, posterior and anterior), using an NMR instrument equipped with a microimaging accessory. The PR microimages were then correlated with conventional spin-echo (SE) microimages and with histology. On PR microimages, acquired with an echo time of 3.2 ms, the cartilage signal was increased, allowing an accurate delineation of the cartilage from the tidemark/cortical bone region. As a consequence, by the PR method a more precise measurement of cartilage thickness compared with that performed by the conventional SE approach was feasible. An excellent correlation between PR microimages and histology was also obtained (r = 0.90). By the proposed method it is possible to accurately determine the cartilage thickness better than with the conventional SE sequences.

Intercellular Ca2+ waves in mechanically stimulated articular chondrocytes.

Articular cartilage is a tissue designed to withstand compression during joint movement and, in vivo, is subjected to a wide range of mechanical loading forces. Mechanosensitivity has been demonstrated to influence chondrocyte metabolism and cartilage homeostasis, but the mechanisms underlying mechanotransduction in these cells are poorly understood. In many cell types mechanical stimulation induces increases of the cytosolic Ca2+ concentration that propagates from cell to cell as an intercellular Ca2+ wave. Cell-to-cell communication through gap junctions underlies tissue co-ordination of metabolism and sensitivity to extracellular stimuli: gap junctional permeability to intracellular second messengers allows signal transduction pathways to be shared among several cells, ultimately resulting in co-ordinated tissue responses. Mechanically-induced Ca2+ signalling was investigated with digital fluorescence video imaging in primary cultures of rabbit articular chondrocytes. Mechanical stimulation of a single cell, obtained by briefly distorting the plasmamembrane with a micropipette, induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18 alpha-glycyrrhetinic acid, suggesting the involvement of gap junctions in signal propagation. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in microinjection experiments while the expression of connexin 43 could be detected in Western blots. A series of pharmacological tools known to interfere with the cell calcium handling capacity were employed to investigate the mechanism of mechanically-induced Ca2+ signalling. In the absence of extracellular Ca2+ mechanical stimulation induced communicated Ca2+ waves similar to controls. Mechanical stress induced Ca2+ influx both in the stimulated chondrocyte but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Cells treatment with thapsigargin and with the phospholipase C inhibitor U73122 blocked mechanically-induced signal propagation. These results provide evidence that in chondrocytes mechanical stimulation activates phospholipase C, thus leading to an increase of intracellular inositol 1,4,5-trisphosphate. The second messenger, by permeating gap junctions, stimulates intracellular Ca2+ release in neighbouring cells. Intercellular Ca2+ waves may provide a mechanism to co-ordinate tissue responses in cartilage physiology.

Articular cartilage repair in rabbits by using suspensions of allogenic chondrocytes in alginate.

The feasibility of allogenic implants of chondrocytes in alginate gels was tested for the reconstruction in vivo of artificially full-thickness-damaged articular rabbit cartilage. The suspensions of chondrocytes in alginate were gelled by the addition of calcium chloride solution directly into the defects giving in situ a construct perfectly inserted and adherent to the subchondral bone and to the walls of intact cartilage. The tissue repair was controlled at 1, 2, 4 and 6 months after the implant by NMR microscopy, synchrotron radiation induced X-ray emission to map the sulfur of glycosaminoglycans and by histochemistry. Practically a complete repair of the defect was observed 4-6 months from the implant of the chondrocytes with the recovery of a normal tissue structure. Controls in which Ca-alginate alone was implanted developed only a fibrous cartilage.

Inhomogeneous alginate gel spheres: an assessment of the polymer gradients by synchrotron radiation-induced X-ray emission, magnetic resonance microimaging, and mathematical modeling.

It has been previously demonstrated that calcium alginate gels prepared by dialysis often exhibit a concentration inhomogeneity being the polymer concentration considerably lower in the center of the gel than at the edges. Inhomogeneity may be a preferred structure in microcapsules due to low porosity and higher stability so that it is interesting to evaluate the polymer gradient in spherically symmetrical small alginate beads (1.0-0.7 mm diameter) obtained in different conditions. In this paper, two complementary techniques have been used to investigate this aspect. The concentration gradient of alginate has been analyzed by measuring both the spatial distribution of calcium ions in sections of alginate gel spheres, by means of x-ray fluorescence spectroscopy, and the T2 relaxation behavior on intact gel beads using magnetic resonance microimaging. The experimentally determined gradients from three-dimensional gels provide data to reevaluate the parameter estimates in the recently reported mathematical model for alginate gel formation (A. Mikkaelsen and A. Elgsaeter, Biopolymers, 1995, Vol. 36, pp. 17-41). The model may account for the gels being less inhomogeneous when nongelling sodium or magnesium ions are added during gelation.

Zinc mapping in bone tissues by histochemistry and synchrotron radiation-induced X-ray emission: correlation with the distribution of alkaline phosphatase.

Zinc distribution in osteons was mapped by synchrotron radiation-induced X-ray emission analysis in both human and porcine adult bone, as well as in porcine bone by histochemistry using Timm's method. Both procedures showed that zinc is not uniformly distributed, being in its highest concentration on haversian bone surfaces. When Timm's method was applied in conjunction with a procedure leading to partial zinc extraction, three zinc pools were specifically detected: a loose one, found in the mineralizable osteoid; a mineral one, bound to the bone mineral; and a tenacious one, firmly bound to an organic component located in the osteoid and mineralizing organic matrix. The alkaline phosphatase distribution was also mapped in porcine adult bone by histochemistry and immunohistochemistry and it was found codistributed with tenacious zinc mainly at the calcification front. The data suggest that alkaline phosphatase is buried as a bone matrix protein during initial mineralization.

Sensitivity of chondrocytes of growing cartilage to reactive oxygen species.

Vascular invasion of calcified cartilage, during endochondral ossification, is initiated and sustained by invasive cells (endothelial cells and macrophages) which degrade the tissue by releasing lytic enzymes. Concurrently, reactive oxygen species (ROS) are also released by these cells and we hypothesize that ROS also contribute to the degradation of the tissue. As a preliminary approach to this problem, the antioxidant activities and the effect of ROS on hypertrophic cartilage and chondrocytes (HCs) were investigated. Compared to resting or articular chondrocytes, HCs exhibited higher catalase but lower SOD specific activities and lower PHGPx concentration, thus revealing a defence activity specific against H2O2. Moreover, dose-dependent depletion of ATP occurred after few minutes of exposure to ROS, and a long-term treatment (16 h incubation with ROS) promoted the release of LDH activity and a significant variation of the poly- to mono-unsaturated fatty acid ratio. Finally, the incubation of HCs with low ROS doses induced the release of sedimentable alkaline phosphatase activity (matrix vesicles). How the obtained results fit the in vivo occurring events is discussed.

Magnetic resonance imaging of articular cartilage: ex vivo study on normal cartilage correlated with magnetic resonance microscopy.

The aims of this study were (a) to compare the MR appearance of normal articular cartilage in ex vivo MR imaging (MRI) and MR microscopy (MRM) images of disarticulated human femoral heads, (b) to evaluate by MRM the topographic variations in articular cartilage of disarticulated human femoral heads, and subsequently, (c) to compare MRM images with histology. Ten disarticulated femoral heads were examined. Magnetic resonance images were obtained using spin-echo (SE) and gradient-echo (GE) sequences. Microimages were acquired on cartilage-bone cylindrical plugs excised from four regions (superior, inferior, anterior, posterior) of one femoral head, using a modified SE sequence. Both MRI and MRM images were obtained before and after a 90 degrees rotation of the specimen, around the axis perpendicular to the examined cartilage surface. Finally, MRM images were correlated with histology. A trilaminar appearance of articular cartilage was observed with MRI and with a greater detail with MRM. A good correlation between MRI and MRM features was demonstrated. Both MRI and MRM showed a loss of the trilaminar cartilage appearance after specimen rotation, with greater evidence on MRM images. Cartilage excised from the four regions of the femoral head showed a different thickness, being thickest in the samples excised from the superior site. The MRM technique confirms the trilaminar MRI appearance of human articular cartilage, showing good correlation with histology. The loss of the trilaminar appearance of articular cartilage induced by specimen rotation suggests that this feature is partially related to the collagen-fiber orientation within the different layers. The MRM technique also shows topographic variations in thickness of human articular cartilage.

Correlation between biochemical composition and magnetic resonance appearance of articular cartilage.

OBJECTIVE: The objective of this study was to find a correlation between magnetic resonance (MR) appearance and biochemical composition of the normal articular cartilage by comparing the laminar aspects with the distribution of the two principal matrix components: proteoglycans and collagen. DESIGN: T2-weighted MR microimages of porcine cartilage-bone plugs, excised from both the habitually loaded and habitually unloaded regions of the proximal end of the humerus, were obtained using a spin-echo sequence. Proteoglycans (PGs) were monitored by histology and by measuring the uronate and the sulfur content of the tissue; a histologic method and the chemical determination of hydroxyproline were used for the evaluation of the collagen content. RESULTS: The 'loaded' cartilage exhibited the expected MR laminar appearance whereas the 'unloaded' tissue appeared to be more homogeneous. The PG content in the 'loaded' cartilage, was found to be 2.4 times higher than in the habitually unloaded tissue, exhibiting an increasing trend from the articular surface to the bone. In the 'unloaded' cartilage the uronate distribution was more uniform with a higher concentration in the intermediate zone. The mean collagen content of both cartilage regions was found to be about 39% of the tissue dry weight. Histology and hydroxyproline distribution pattern showed that collagen was particularly concentrated at the surface and in a central zone of the 'loaded' cartilage whereas in the 'unloaded' tissue collagen was evident only at the surface. In accordance with the collagen distribution, transverse relaxation (T2) times in 'loaded' cartilage showed a minimum value at the articular surface and another minimum in a central region. On the contrary, the average T2 value of the 'unloaded' tissue was high at the surface and decreased rapidly in the deeper zones. CONCLUSION: These results demonstrate that the MR appearance of articular cartilage correlates with the collagen content, but not with that of PGs, of the different zones. Other matrix components might, however, influence the MR appearance by contributing to the macromolecular organization of the tissue.

Membrane stretch activates a potassium channel in pig articular chondrocytes.

Activity of stretch-activated potassium channels has been recorded in articular chondrocytes using patch-clamp technique. Pressure dependence is described by a sigmoidal function with a half-maximum effect at -20.5 mbar. Selectivity for potassium is demonstrated by agreement between the reversal potential measured at different [K+]o and the prediction of Nernst equation and by block of these channels by caesium.

Propagation of intercellular Ca2+ waves in mechanically stimulated articular chondrocytes.

Intercellular Ca2+ signalling in primary cultures of articular chondrocytes was investigated with digital fluorescence video imaging. Mechanical stimulation of a single cell induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18alpha-glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. In the absence of extracellular Ca2+ mechanical stimulation failed to induce Ca2+ responses and communicated Ca2+ waves. Under these conditions Ca2+ microinjection induced intercellular waves involving the cells immediately surrounding the stimulated one. Mechanical stress induced Ca2+ influx in the stimulated, but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Cell treatment with thapsigargin failed to block mechanically induced signal propagation, but significantly reduced the number of cells involved in the communicated Ca2+ wave. Similar results were obtained with the phospholipase C inhibitor U73122, which is known to prevent InsP3 generation. These results provide evidence that mechanical stimulation induces a cytosolic Ca2+ increase that may permeate gap junctions, thus acting as an intercellular messenger mediating cell-to-cell communication in articular chondrocytes.

Gap junctions mediate intercellular calcium signalling in cultured articular chondrocytes.

Gap junction-mediated intercellular communication has been implicated in a variety of cellular functions. Among these, signal transduction can be coordinated among several cells due to gap junctional permeability to intracellular second messengers. Chondrocytes from articular cartilage in primary culture respond to extracellular ATP by rhythmically increasing their cytosolic Ca2+ concentration. Digital imaging fluorescence microscopy of Fura-2 loaded cells was used to monitor Ca2+ in confluent and semi-confluent cell layers. Under these conditions, Ca2+ spikes propagate from cell to cell giving rise to intercellular Ca2+ waves. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in scrape-loading experiments. Intercellular dye transfer was blocked by the gap junction inhibitor 18 alpha-glycyrrhetinic acid. In imaging experiments, the inhibitor caused the loss of synchrony of ATP-induced Ca2+ oscillations, and blocked the intercellular Ca2+ propagation induced by mechanical stimulation of a single cell in a monolayer. It is concluded that gap junctions mediate intercellular signal transduction in cartilage cells and may provide a mechanism for co-ordinating their metabolic activity.

Propofol blocks voltage-gated potassium channels in human T lymphocytes.

The effect of propofol (PR) on voltage-gated potassium channels (KV) in human T lymphocytes (TL) was studied using the patch-clamp technique in the whole-cell configuration. PR was found to reversibly block the KV channels in a dose-dependent manner with a half-blocking concentration of approximately 40 microM. The decrease in the peak current caused by PR was voltage-independent. The activation time constant of the whole-cell potassium currents remained unaffected upon PR treatment, whereas both the rate and extent of the inactivation process were increased, indicating the "open channel block" mechanism. The PR half-blocking concentration was of the same order of magnitude as PR blood concentrations employed in anesthesia. Taking into account the extensive use of PR and the important role of KV channels in human TL, these results suggest a need for investigations into the effect of PR on TL cell-function regulation.

Dual mechanism for cAMP-dependent modulation of Ca2+ signalling in articular chondrocytes.

The ability of cAMP to modulate the actions of Ca(2+)-mobilizing agonists was studied in single Fura-2-loaded pig articular chondrocytes in primary culture. Forskolin and 8-Br-cAMP increased both the frequency and amplitude of Ca2+ oscillations induced by ATP, and, in unstimulated cells, induced single Ca2+ transients or even Ca2+ oscillations. The cAMP-dependent protein kinase inhibitor H89 totally prevented the effect of cAMP-elevating agents on Ca2+ signalling. Forskolin and 8-Br-cAMP promptly increased the rate of Mn2+ quenching, when administered in the presence of ATP, suggesting a potentiation of receptor-mediated Ca2+ influx. In Ca(2+)-free medium, ATP-induced Ca2+ oscillations decreased and stopped after a few cycles: subsequent ATP additions temporarily resumed the activity, an effect that could be mimicked by forskolin. The same agent induced single Ca2+ transients in 42% of the cell population maintained in Ca(2+)-free medium. Thapsigargin prevented Ca2+ responses to both ATP and forskolin. The results indicate a dual mechanism for cAMP-induced potentiation of Ca2+ signalling in articular chondrocytes: an increase of receptor-mediated Ca2+ influx and a positive modulation of intracellular Ca2+ release.

Ca2+ oscillations and intercellular Ca2+ waves in ATP-stimulated articular chondrocytes.

Cytosolic Ca2+ oscillations are known to occur in many cell types stimulated with agonists linked to the phosphoinositide signaling pathway. Trains of repetitive short-lasting Ca2+ spikes could be induced in articular chondrocytes by extracellular ATP, an agonist potently effective in stimulating cartilage resorption. The mechanism of these Ca2+ oscillations was studied by computerized video imaging on primary cultures of articular chondrocytes. Few cycles of oscillatory activity could be evoked in the absence of extracellular Ca2+, while, for oscillations to be sustained, Ca2+ influx was required. Thapsigargin irreversibly blocked Ca2+ oscillations, thus demonstrating the crucial involvement of intracellular stores in triggering the rhythmic activity. Apart from activating intracellular Ca2+ release, extracellular ATP also induced a noncapacitive Ca2+ influx in these cells. This ATP-mediated influx modulates both the oscillation frequency and intracellular stores refilling. In monolayers of confluent cells, Ca2+ oscillations spread from cell to cell in the form of intercellular waves. Propagating waves could also be observed in the absence of extracellular Ca2+, demonstrating that Ca2+ itself is not required for signal coordination. These results demonstrate that complex spatiotemporal pathways of Ca2+ oscillations and intercellular Ca2+ waves could be activated in articular chondrocytes during degenerative diseases.