Influences of construct properties on the proliferation and matrix synthesis of dedifferentiated chondrocytes cultured in alginate gel.

To investigate whether the chondrocytes-alginate construct properties, such as cell seeding density and alginate concentration might affect the redifferentiation, dedifferentiated rat articular chondrocytes were encapsulated at low density (LD: 3 x 10(6) cells/ml) or high density (HD: 10 x 10(6) cells/ml) in two different concentrations of alginate gel (1.2% or 2%, w/v) to induce redifferentiation. Cell viability and cell proliferation of LD culture was higher than those of HD culture. The increase in alginate gel concentration did not make an obvious difference in cell viability, but reduced cell proliferation rate accompanied with the decrease of cell population in S phase and G2/M phase. Scan electron microscopy observation revealed that chondrocytes maintained round in shape and several direct cell-cell contacts were noted in HD culture. In addition, more extracellular matrix was observed in the pericellular region of chondrocytes in 2% alginate culture than those in 1.2% alginate culture. The same tendency was found for the synthesis of collagen type II. No noticeable expression of collagen type I was detected in all constructs at the end of 28-day cultures. These results suggested that construct properties play an important role in the process of chondrocytes' redifferentiation and should be considered for creating of an appropriate engineered articular cartilage.

Multidetector computed tomography sizing of aortic annulus prior to transcatheter aortic valve replacement (TAVR): Variability and impact of observer experience.

PURPOSE: To evaluate intra- and inter-observer variability of multidetector computed tomography (MDCT) sizing of the aortic annulus before transcatheter aortic valve replacement (TAVR) and the effect of observer experience, aortic valve calcification and image quality. METHODS: MDCT examinations of 52 consecutive patients with tricuspid aortic valve (30 women, 22 men) with a mean age of 83±7 (SD) years (range: 64-93 years) were evaluated retrospectively. The maximum and minimum diameters, area and circumference of the aortic annulus were measured twice at diastole and systole with a standardized approach by three independent observers with different levels of experience (expert [observer 1]; resident with intensive 6 months practice [observer 2]; trained resident with starting experience [observer 3]). Observers were requested to recommend the valve prosthesis size. Calcification volume of the aortic valve and signal to noise ratio were evaluated. RESULTS: Intra- and inter-observer reproducibility was excellent for all aortic annulus dimensions, with an intraclass correlation coefficient ranging respectively from 0.84 to 0.98 and from 0.82 to 0.97. Agreement for selection of prosthesis size was almost perfect between the two most experienced observers (k=0.82) and substantial with the inexperienced observer (k=0.67). Aortic valve calcification did not influence intra-observer reproducibility. Image quality influenced reproducibility of the inexperienced observer. CONCLUSION: Intra- and inter-observer variability of aortic annulus sizing by MDCT is low. Nevertheless, the less experienced observer showed lower reliability suggesting a learning curve.

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.

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.

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.

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.

Multicenter study of an erythro-aggregometer: quality control and standardization.

There is a need for quality assurance procedures in hemorheology, especially for clinical and pharmacological studies, which require reliable and well-calibrated instruments to be interpretable and comparable. Preliminary investigations allowed preparation of standardized SM (normal NS and hyperaggregable HS), and checking of storage conditions (in accordance with the guidelines of the SSC of ISTH) of RBC in nutritive SAG mannitol for at least 2 or 3 weeks with subsequent washings and resuspension in SM. In our study, we compared erythro-aggregometers of the same brand in 6 laboratories, using the same red blood cells (RBC) and suspending media (SM) for each series of tests. EA was measured by laser backscattering with determination of aggregation time (AT), partial dissociation threshold (PDT) and aggregation index (AI). Prior to the study, devices were set up on the same day with the same standardized blood, and calibration data were then analyzed. Within-assay precision (WAP) was assessed on 3 days for the 2 types of SM (n = 18 x 2). Between-assay precision (BAP) was assessed on 6 occasions, once every month (n = 6 x 2 x 6). Accuracy was studied with 3 series of RBC resuspended in 10 SM of "unknown" aggregability. Good agreement was observed between 5/6 centers for the 3 parameters of EA. WEP was good: CV of AT ranging from 1.4 to 2.5% for the NS and from 1.4 to 2.4% for the HS. In each center, BAP was slightly lower than WEP (CV: 8-11.8% for the NS and 3.8-4.7% for the HS over the 6-month study), with no drift, except for one center. Precision was always better with the HS than with the NS which seemed a better tool to assess it. As to accuracy, non-significant differences were generally found between centers, with similar data to the reference values. This work also stressed the importance of the RBC parameter itself in rheological data. For the first time, a protocol for standardization of EA has been developed and evaluated, permitting the Quality Control of this technique.

[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)

Hydroxyapatite incorporated into collagen gels for mesenchymal stem cell culture.

Collagen gels could be used as carriers in tissue engineering to improve cell retention and distribution in the defect. In other respect hydroxyapatite could be added to gels to improve mechanical properties and regulate gel contraction. The aim of this work was to analyze the feasibility to incorporate hydroxyapatite into collagen gels and culture mesenchymal stem cells inside it. Human bone marrow mesenchymal stem cells (hMSC-BM) were used in this study. Gels were prepared by mixing rat tail type I collagen, hydroxyapatite microparticles and MSCs. After polymerization gels were kept in culture while gel contraction and mechanical properties were studied. In parallel, cell viability and morphology were analyzed. Gels became free-floating gels contracted from day 3, only in the presence of cells. A linear rapid contraction phase was observed until day 7, then a very slow contraction phase took place. The incorporation of hydroxyapatite improved gel stability and mechanical properties. Cells were randomly distributed on the gel and a few dead cells were observed all over the experiment. This study shows the feasibility and biocompatibility of hydroxyapatite supplemented collagen gels for the culture of mesenchymal stem cells that could be used as scaffolds for cell delivery in osteoarticular regenerative medicine.