Biomedical subjects

C Picart

Publications and source records attributed to C Picart.

12 recordsLinked to original sources

PubMed2003

Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification.

Endothelial cell seeding constitutes an appreciated method to improve blood compatibility of small-diameter vascular grafts. In this study, we report the development of a simple innovative technique based on multilayered polyelectrolyte films as cell adhesive substrates. Polyelectrolyte multilayered films ending by poly(sodium-4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) or poly(L-glutamic acid)/poly(D-lysine) (PGA/PDL) could enhance cell adhesion by modification of the physico-chemical properties of the surface. The biological responses of human umbilical vein endothelial cells seeded on the polyelectrolyte multilayer films, on PDL or PAH monolayers, and on control surfaces, were evaluated in terms of initial attachment, growth, cellular metabolic activity, endothelial phenotype, and adhesion. The results showed that polyelectrolyte multilayers neither induce cytotoxic effects nor alter the phenotype of the endothelial cells. The polyelectrolyte multilayered films enhanced initial cell attachment as compared to the polyelectrolyte monolayer. Cell growth observed on the films was similar to that on TCPS. Among the different coating tested, the film ending by PSS/PAH exhibited an excellent cellular biocompatibility and appeared to be the most interesting surface in terms of cellular adhesion and growth. Such films could be used to cover hydrophobic (cell resistant) substrates in order to promote cell colonization, thereby constituting an excellent material for endothelial cell seeding.

Biocompatible Materials↗

PubMed2003

Modeling of the detachment of a molecule from a surface: illustration of the "Bell-Evans effect".

This article deals with the modeling of the detachment of a molecule initially adsorbed on a surface and submitted to an external force whose strength increases with time. By means of an atomic force microscope (AFM), it is possible to measure the force when the molecule separates from the substrate. However, it is known that this force depends to a large extend on the rate at which the pulling force is applied ("Bell-Evans effect"). Two models are described to illustrate this behavior. First, a random walk approach is suggested to reveal the fundamental principle of the escape over a time-dependent energy barrier. Second, a multi bead-and-spring model is proposed to mimic the AFM experiment and numerical simulations, based on Brownian dynamics, are performed.

Binding Sites↗

PubMed2002

Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers.

The structure of poly(l-lysine) (PLL)/hyaluronan (HA) polyelectrolyte multilayers formed by electrostatic self-assembly is studied by using confocal laser scanning microscopy, quartz crystal microbalance, and optical waveguide lightmode spectroscopy. These films exhibit an exponential growth regime where the thickness increases exponentially with the number of deposited layers, leading to micrometer thick films. Previously such a growth regime was suggested to result from an "in" and "out" diffusion of the PLL chains through the film during buildup, but direct evidence was lacking. The use of dye-conjugated polyelectrolytes now allows a direct three-dimensional visualization of the film construction by introducing fluorescent polyelectrolytes at different steps during the film buildup. We find that, as postulated, PLL diffuses throughout the film down into the substrate after each new PLL injection and out of the film after each PLL rinsing and further after each HA injection. As PLL reaches the outer layer of the film it interacts with the incoming HA, forming the new HA/PLL layer. The thickness of this new layer is thus proportional to the amount of PLL that diffuses out of the film during the buildup step, which explains the exponential growth regime. HA layers are also visualized but no diffusion is observed, leading to a stratified film structure. We believe that such a diffusion-based buildup mechanism explains most of the exponential-like growth processes of polyelectrolyte multilayers reported in the literature.

Chemical Phenomena↗

PubMed2000

Actin protofilament orientation in deformation of the erythrocyte membrane skeleton.

The red cell's spectrin-actin network is known to sustain local states of shear, dilation, and condensation, and yet the short actin filaments are found to maintain membrane-tangent and near-random azimuthal orientations. When calibrated with polarization results for single actin filaments, imaging of micropipette-deformed red cell ghosts has allowed an assessment of actin orientations and possible reorientations in the network. At the hemispherical cap of the aspirated projection, where the network can be dilated severalfold, filaments have the same membrane-tangent orientation as on a relatively unstrained portion of membrane. Likewise, over the length of the network projection pulled into the micropipette, where the network is strongly sheared in axial extension and circumferential contraction, actin maintains its tangent orientation and is only very weakly aligned with network extension. Similar results are found for the integral membrane protein Band 3. Allowing for thermal fluctuations, we deduce a bound for the effective coupling constant, alpha, between network shear and azimuthal orientation of the protofilament. The finding that alpha must be about an order of magnitude or more below its tight-coupling value illustrates how nanostructural kinematics can decouple from more macroscopic responses. Monte Carlo simulations of spectrin-actin networks at approximately 10-nm resolution further support this conclusion and substantiate an image of protofilaments as elements of a high-temperature spin glass.

Actins↗

PubMed2000

[Morphometric analysis of erythrocyte aggregates in vitro: pharmacological application with buflomedil].

Erythrocyte aggregation is usually evaluated through indirect measurements such as the sedimentation rate and rheometric measurements of aggregation and disaggregation thresholds. The aim of this study was a preliminary evaluation of a morphometric approach of aggregates in vitro. A sample of blood (on EDTA) was examined under the microscope, and a picture with magnification X 640 was obtained after stabilization of the cellular structure. The digital image was analyzed with a home made dedicated software that allows the measurement of the cellular density and the evaluation of several shape parameters. Ten samples obtained from different donors were analysed within one hour after blood withdrawal and 24 hours later (preservation temperature: 4 degrees C), with and without adjunction of Buflomedil (4 microg/ml). Results showed an important anti-aggregant effect of Buflomedil administrated in these conditions, both on fresh and preserved blood (p<10(-3)). These results illustrate the potential interest of a morphometric approach to erythrocyte aggregation. They show new pharmacological properties of Buflomedil administered in vitro, which have to be confirmed with a therapeutic administration of the compound.

Densitometry↗

PubMed1999

Actin protofilament orientation at the erythrocyte membrane.

The short actin filaments in the erythrocyte's membrane skeleton are shown to be largely oriented tangent to the lipid bilayer. Actin "proto"-filaments have previously been described as junctional centers intertriangulated by spectrin; however, the protofilaments may simultaneously serve as pinning centers between the network and the overlying bilayer. The latter function now seems of particular importance because near-normal network assembly has been reported with transgenic mouse sphero-erythrocytes that lack the primary linkage protein Band 3. To assess possible physical constraints on actin protofilaments in intact membranes, fluorescence polarization microscopy (FPM) has been used to study rhodamine phalloidin-labeled red cell ghosts. A basis for interpreting FPM images of cells is provided by FPM applied to isolated actin filaments. These are labeled with the same rhodamine probes and imaged at various orientations with respect to the polarizers, including filament orientations perpendicular to the image plane. High aperture and fluorophore conjugation effects are found to be minimal, enabling development of a simple, semi-empirical model which indicates that protofilaments are generally within approximately 20 degrees of the membrane tangent plane.

Actins↗

PubMed1999

Blood yield stress in systemic sclerosis.

Blood is a weak percolating physical gel at low shear rates, in which clusters of aggregates can be reversibly disaggregated or formed again. This phenomenon is of potential importance in the microvascular pathophysiology of ischemic and vasospastic disorders such as systemic sclerosis. The aim of this work was to determine blood yield stress using low-shear-rate rheometry with a homemade roughened Couette device in 10 patients with systemic sclerosis compared with 10 healthy controls. Biochemical plasmatic parameters were assessed independently. Results showed a significantly increased stress (+56%, P < 0.05 at 60% hematocrit) for scleroderma patients. The best biochemical predictor for yield stress was the ratio of albumin to globulins; 69% of its variance was explained by plasmatic factors (albumin, fibrinogen, and globulins) in scleroderma patients and 23.4% in healthy controls. Additional microscopic observations showed different microstructures. These results support the hypothesis of an abnormal red blood cell organization process in scleroderma patients that could be partly responsible for the severity of ischemic complications of the disease.

Adult↗

PubMed1999

Muscle cell peeling from micropatterned collagen: direct probing of focal and molecular properties of matrix adhesion.

To quantitatively elucidate attributes of myocyte-matrix adhesion, muscle cells were controllably peeled from narrow strips of collagen-coated glass. Initial growth of primary quail myoblasts on collagen strips was followed by cell alignment, elongation and end-on fusion between neighbors. This geometric influence on differentiation minimized lateral cell contact and cell branching, enabling detailed study of myocyte-matrix adhesion. A micropipette was used to pull back one end of a quasi-cylindrical cell while observing in detail the non-equilibrium detachment process. Peeling velocities fluctuated as focal roughness, microm in scale, was encountered along the detachment front. Nonetheless, mean peeling velocity ( microm/second) generally increased with detachment force (nN), consistent with forced disruption of adhesion bonds. Immunofluorescence of beta1-integrins correlated with the focal roughness and appeared to be clustered in axially extended focal contacts. In addition, the peeling forces and rates were found to be moderately well described by a dynamical peeling model for receptor-based adhesion (Dembo, M., Torney, D. C., Saxman, K. and Hammer, D. (1988). Proc. R. Soc. Lond. B 234, 55-83). Estimates were thereby obtained for the spontaneous, molecular off-rate (kooff, (less than or equal to)10/seconds) and the receptor complex stiffness (kappa, approx. 10(-5)-10(-6) N/m) of adherent myocytes. Interestingly, the local stiffness is within the range of flexible proteins of the spectrin superfamily. The overall approach lends itself to elucidating the developing function of other structural and adhesive components of cells, particularly skeletal muscle cells with specialized components, such as the spectrin-homolog dystrophin and its membrane-linked receptor dystroglycan.

Animals↗

PubMed1998

Threshold of shear stress in human blood for healthy and sick subjects.

It is now well recognized that blood is a yield stress fluid, that is to say that it will not flow below a critical threshold level of stress. This is reflected in vivo with a cessation of flow threshold, of great importance in circulatory physiology and pathophysiology. Values for yield stress obtained by different techniques are scarce. Rheometry at low shear rates is one method of obtaining values. After mitigating the slip effects on the walls of the rheometer, it has been possible to get an approximate value of the yield stress in human blood samples in controls and in ill subjects with different hematocrits. A Low Shear 40 rheometer with a controlled rotation speed was used. Home made geometries were used and the shear rate was varied in the range 10(-3) s-1-100 s-1. Fresh blood samples from healthy donors and patients were anticoagulated with EDTA. Suspensions of red cells in plasma were prepared by removing plasma. The measurements were made at 25 degrees C within four hours following blood sampling. Particular attention was paid to data acquisition at low shear rates (10(-3) s-1, 3.10(-3) s-1, 10(-2) s-1, 3.10(-2) s-1) with a computer linked to the rheometer. Shear stress approached a constant value at low shear rates for geometries with rough surfaces. Shear stress measured at 10(-3) s-1 was taken as an approximation of yield stress. Values were 5 to 6 mPa for hematocrit 60% and 20 to 25 mPa for hematocrit in the 80 to 90% range. For pathological blood samples, accurate measurements were made at shear rate as low as 0.01 s-1. Migrational effects were present at 10(-1) s-1 et 3.10(-3) s-1 but they could be greatly mitigated by the use of the 170 microns surface roughness.

Blood Donors↗

PubMed1998

Systemic sclerosis: blood rheometry and laser Doppler imaging of digital cutaneous microcirculation during local cold exposure.

A combined study of microcirculation hemodynamics in vivo and blood rheometry has been carried out in patients with scleroderma compared to primary Raynaud's phenomenon (PRP) and healthy controls (HC). Laser Doppler perfusion imaging was used to assess superficial skin blood flow in the fingers before, during and after local cold exposure. Blood viscosity was measured at 19 and 37 degrees C. Dynamic and transient flows were also investigated. The scleroderma group showed a significantly lower level of perfusion before the cold test, that was further decreased during and after cold exposure, the difference with PRP being maximal during the rewarming period. It also showed a significantly increased viscosity (p < 0.05) at low shear rates. Hemorheological behavior of the PRP group was similar to the HC group. A significant negative correlation between the LDI perfusion and the apparent blood viscosity at low shear rates was found for the whole population. These findings suggest that rheological factors may be involved in the abnormal cold reactivity of patients with scleroderma.

Blood Flow Velocity↗

PubMedDate not supplied

Blood low shear rate rheometry: influence of fibrinogen level and hematocrit on slip and migrational effects.

Red blood cell (RBC) aggregation is of prime importance in vivo and in vitro for low flow rates. It may be estimated by rheometrical measurements at low shear rates, but these are perturbed by slip and migrational effects which have already been highlighted in the past. These effects lead to a torque decay with time so that the true value of the stress at low shear rates may be greatly underestimated. Elevated aggregation being associated with different diseases, pathological blood samples show more pronounced perturbing effects and a strong time dependency in low shear rate rheometry. To test the dependence of slip and migrational effects on RBC aggregation, and particularly to determine the way in which they depend upon fibrinogen concentration ([Fb]), a home-made measuring system with roughened internal and external walls (170 microns roughness) was used to study low shear rate rheometry for RBC suspensions in PBS buffer containing albumin (at 50 g/l) and fibrinogen at various concentrations. The influences of hematocrit, shear rate, and fibrinogen concentration were investigated. Particular attention was paid to data acquisition at low shear rates (10(-3) s-1 to 3 x 10(-2) s-1). The combined influence of hematocrit and fibrinogen was investigated by adjusting hematocrit to 44 or 57% and fibrinogen concentration ([Fb]) to 3.0-4.5-6.5 g/l. Microscopic observations of the blood samples at rest were performed. They showed that different structures were formed according to fibrinogen concentration. The rheometrical measurements indicated that torque decay with shearing duration was strongly dependent on fibrinogen concentration and on shear rate at fixed hematocrit. Migrational and slip effects were more pronounced as shear rate decreased, fibrinogen concentration was raised, and hematocrit was lowered. The results have been explained on the basis of the expected microstructure of flowing blood in relation to the microscopic observations at rest.

Erythrocyte Aggregation↗

PubMedDate not supplied

Cell interactions with polyelectrolyte multilayer films.

The short-term interactions of chondrosarcoma cells with polyelectrolyte multilayer films built up by the alternate adsorption of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) was studied in the presence and in the absence of serum. The films and their interaction with serum proteins were first characterized by means of optical waveguide lightmode spectroscopy, quartz crystal microbalance, and zeta potential measurements. In a serum-containing medium, the detachment forces measured by the micropipet technique were about eight times smaller on PGA-ending than on PLL-ending films. For these latter ones, the adhesion force decreased when the film thickness increased. In a serum-free medium, the differences between the negative- and positive-ending films were enhanced: adhesion forces on PLL-ending films were 40-100% higher, whereas no cellular adherence was found on PGA-terminating films. PGA-ending films were found to prevent the adsorption of serum proteins, whereas important protein adsorption was always observed on PLL-ending films. These results show how cell interactions with polyelectrolyte films can be tuned by the type of the outermost layer, the presence of proteins, and the number of layers in the film.

Blood Proteins↗