Comparison of wide-field/deconvolution and confocal microscopy in bioengineering. Interest of multi-photon microscopy in the study of articular cartilage.

The increase in lateral and spatial resolutions is one of the major targets of research and development in the field of optical microscopies applied to living tissue. The optical geometry of Confocal Laser Scanning Microscopy (CLSM) demonstrates its undeniable advantage on conventional fluorescence microscopy by segregating the planes outside the focussing plane. The methodological and technological advances of the last five years have been fast evolving, especially with regard to the optimisation of CLSM and deconvolution process. The limited analysis in thick tissue have given rise to the development of other techniques, multi-photon excitation microscopy in particular. In this paper, we have applied these techniques on major biological applications in bioengineering (endothelial cell, chondrocyte in 3D-culture, human cartilage) and discussed the technical limitations and perspectives.

Rheologic characterization of vegetal lectins by dissociation of induced erythrocyte agglutinates.

Energy evolved from hemagglutination reaction or spent in dissociating erythrocyte agglutinates has been proved to be an excellent parameter for analyzing cell-cell interactions mediated by bridging molecules such as antibodies or lectins. We developed a new rheo-optical method to estimate the energy of dissociation of red blood cell agglutinates. In a Couette shear field agglutinates can be dissociated until a suspension of monodispersed cells is obtained. Intensity of light backscattered by suspended agglutinates increases during their mechanical dissociation. Variation of backscattered light intensity correlates with the energy spent in the process. The adhesive energy of erythrocyte agglutination induced by lectins has been estimated by applying this method. Two specific lectins (Dolichus Biflorus agglutinin and Ulex Europaeus agglutinin) and a new lectin obtained from Amarantus Cruentus seeds which specificity is unknown were studied. Results obtained in this work for Dolichus Biflorus lectin are comparable with values published by other authors. An asymptotic decrease of adhesive energy was observed when the mechanical dissociation was applied several times on the same sample. Our results suggest that the cell detachment is accompanied by the extraction of membrane receptors. This finding is consistent with results obtained by other authors.

Effects of an angelica extract on human erythrocyte aggregation, deformation and osmotic fragility.

In Chinese traditional medicine, angelica is widely used for its known clinical effects of ameliorating blood microcirculation. But the mechanism of these beneficial effects still remains unclear. In this work the rheological behaviour of human erythrocytes treated by angelica was studied in vitro. Normal RBCs incubated with an angelica extract at different concentrations (5, 10 or 20 mg/ml) for 60 min at 37 degrees C and then their aggregation, deformation and osmotic fragility were measured with different recently developed optical techniques, namely Erythroaggregometer (Regulest, Florange, France), LORCA (Mechatronics, Amsterdam) and Fragilimeter (Regulest, Florange, France). Experimental results show that angelica (20 mg/ml) significantly decreased normal RBCs' aggregation speed (p<0.01) and could inhibit the hyperaggregability caused by dextran 500. However, the strength of normal RBCs aggregates were not influenced by angelica. When a calcium ionophore A23187 (1.9 microM) was used to harden cell membrane, angelica (20 mg/ml) could significantly (p<0.01) protect erythrocytes against the loss of their deformability even it had no effects on normal RBCs deformation. Finally angelica (5 and 10 mg/ml) decreased significantly (p<0.01) normal RBCs osmotic fragility. In conclusion angelica plays a rheologically active role on human erythrocytes, and this study suggests a possible mechanism for angelica's positive effects against certain cardiovascular diseases.

Validation of a test of the red cell membrane osmotic resistance.

The aim of the present work was to validate a new technique for the measurement of resistance of the red blood cell membrane using an automated apparatus called a Fragilimeter. Its principle lies in the measurement of the extinction of a laser beam projected through a red blood cell suspension subjected, by diffusion, to a variation of salinity from an isotonic equilibrium (154 mM NaCl) to, a hypotonic one, 25 mM NaCl. The variation of osmotic pressure induces on the cells a progressive lysis and a modification of the extinction of the transmitted light. The validation of the method was based on the comparison between results obtained with the Fragilimeter and those obtained using the reference DACIE technique. Analyses were based on blood samples from healthy donors. The determination of the initial, the 50% and the full haemolysis thresholds allowed observation of the fragility of the cell, through its membrane resistance. The physical phenomenon measured in these cells when subjected to various ionic strengths is discussed on the basis of observations realised by means of an optical microscope.

Influence of a calcium antagonist on blood rheology and arterial compliance in hypertension: comparison with a thiazide diuretic.

Beyond their effects on blood pressure, antihypertensive agents may produce additional effects on blood rheology and arterial compliance abnormalities which may play a role in the target organ damage. These effects may depend only on their specific pharmacological properties. We compared the effects of nitrendipine to hydrochlorothiazide in 33 mild to moderate hypertensives in a double blind parallel group trial. Blood rheology and radial artery diameter and compliance were measured at t=0 and t=2 months. Both drugs produced blood pressure lowering. Blood viscosity decreased in the nitrendipine group and increased in the hydrochlorothiazide patients. Red blood cells deformability increased only in the nitrendipine group. Radial artery diameter and compliance were not different between the two groups but there was a trend to an increase of the cross-sectional compliance in hydrochlorothiazide group and to a decrease in nitrendipine group. Our data show that a calcium antagonist (Nitrendipine) could improve rheological parameters.

[Quality control in clinical hemorheology: preliminary study by three centers]. / Contrôle de qualité en hémorhéologie clinique: étude préliminaire sur trois centres.

Clinical or pharmacological trials require reproducible laboratory tests. The aim of this work was to investigate the conditions of quality control of two laboratory tests, blood viscosity and red cell aggregation used for hemorheology trials in three centers. The results showed that viscosity measurements require 2 standards. For red cell aggregation, the control suspension standardized media (normal an pathological aggregation) showed a good intercentre reproducibility of the dissociation thresholds but a wide variability of aggregation times. Complementary studies in 5 or 6 centres are planned in order to define national standards of quality control.

[In vivo effects of plasma substitutes on the rheologic properties of blood]. / Effets in vivo de substituts plasmatiques sur les propriétés rhéologiques du sang.

The objective was to investigate in vivo the rheological effects of plasma substitutes (fluid gelatin, dextran 40, dextran 60, hydroxyethylstarch). The study was performed during a hypervolemic hemodilution in 60 adult volunteers who underwent an odontological surgery. Two control groups were infused with Ringer-Lactate or 4% human albumin. Each patient was infused with 500 ml of plasma substitute or control fluid. Blood samples were obtained at four different times: prior to (t0) and immediately after (t1) the infusion, then 3 hours (t2) and 24 hours (t3) after the hemodilution. At t1 and t2, plasma viscosity did not present important variations, except for the group infused with dextran 60 where a significant increase in plasma viscosity was observed. Blood viscosity and erythrocyte aggregation measurements were performed at hematocrit 40% to determine the intrinsic properties of substitute and eliminate the effects of hemodilution. Blood viscosity at high shear rates underwent minor variations in all groups. In contrast, blood viscosity at low shear rate and aggregation parameters exhibited important variations which markedly depended on the used substitute. The infusion of Ringer-Lactate, albumin and dextran 40 induced a significant decrease in blood viscosity and in erythrocyte aggregation. With dextran 60 and gelatin, blood viscosity and erythrocyte aggregation increased. Finally, the infusion with hydroxyethylstarch did not present important differences as compared to the results recorded at t0. Twenty for hours after the infusion, it should be noted an increase in plasma fibrinogen, probably due to surgery, which caused an increase in erythrocyte aggregation in all groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversing charges or how to improve Wharton's jelly mesenchymal stem cells culture on polyelectrolyte multilayer films.

BACKGROUND: Polyelectrolyte multilayer (PEMs) films made of poly(allylamine hydrochloride) (PAH) as polycation and poly(styrene sulfonate) (PSS) as polyanion, with a PAH ending layer, can be used as a coating in order to improve the anti-thrombogenicity and patency of vascular grafts in vascular engineering field. They induce strong adhesion of mature endothelial cells on glass, expanded polytetrafluoroethylene and cryopreserved arteries. Despite their outstanding effect on mature and progenitor endothelial cells, PEMs ending with PAH showed a poor outcome on Wharton's jelly mesenchymal stem cells (WJ-MSCs) culture. OBJECTIVE: The aim of this work was to examine the influence of the ending charge of PEMs on WJ-MSCs behavior. METHODS: WJ-MSCs amplified until the 3rd passage were seeded and cultured on (PAH-PSS)3-PAH and on (PAH-PSS)4 coated glass for 10 days. Stem cell phenotype was checked by flow cytometry and cell morphology was followed by bright field microscopy. RESULTS: Flow cytometry analysis showed that WJ-MSCs were positive for MSC's markers CD73, CD90 and CD105 and negative for hematopoietic markers CD34 and CD45. Light microscopy showed development of nodule-like structures after 10 days of culture on (PAH-PSS)3-PAH, which resulted in a disturbance of cell monolayer. Whereas WJ-MSCs cultured on (PAH-PSS)4 ending with PSS showed a normal cell growth like on collagen and reached confluence after 10 days. CONCLUSION: The culture surface seems to have a determining role in WJ-MSC's "spatial" behavior, which could be considered in the field of tissue engineering.