In a pig model ePTFE grafts will sustain for 6 weeks a confluent endothelial cell layer formed in vitro under shear stress conditions.

OBJECTIVE: to investigate in a pig model whether small diameter ePTFE grafts will sustain a confluent endothelial cell layer formed in vitro under shear stress conditions. MATERIALS AND METHODS: thirteen ePTFE (4 mm) grafts were implanted end to end in the right femoral artery of; 8 grafts had been endothelialized in vitro. Grafts were left in situ for 6 weeks then evaluated with ultrasound and histology. RESULTS: seven endothelialized graft were patent with confluent endothelial cell lining. None of the control grafts were patent or showed evidence of an endothelial lining. CONCLUSION: in this pig model ePTFE grafts sustained for 6 weeks a confluent endothelial cell layer formed in vitro under shear stress.

Cryopreservation of artificial cartilage: viability and functional examination after thawing.

In biomedical research and in reconstructive surgery, preservation of intact tissue has been an unsolved problem. In this study, we investigated the viability of cryopreserved artificial cartilage and its synthetic activity of cartilage-specific matrix proteins after thawing for in vitro use. A polymer fleece cylinder (diameter = 3 mm; height = 3 mm) was loaded with a suspension of bovine chondrocytes (25 x 10(6)/ml) and encapsulated with fibrin glue. After a culture period of 1 week, the artificial cartilage units were frozen in a cryoprotection solution containing 10% basal medium (RPMI 1640), 10% DMSO and 80% FCS. The freezing procedure consisted of three steps: a 30-min period at +4 degrees C followed by a 24-hour storage at -80 degrees C. After that, the tissue units were transferred into liquid nitrogen (-196 degrees C) for final storage. Using histochemical staining techniques of cryogenic slices, we investigated the ability of cryopreserved artificial cartilage to produce its specific matrix after thawing. A modified MTT assay was used to determine the viability of frozen tissue units in comparison with unpreserved samples at different moments after thawing. Depending on the chondrocytes used for the formation of artificial cartilage, the viability of cryopreserved tissue varied between 65 and 85%. Both the intensity of alcian blue staining for proteoglycans and the azan staining for collagens increased proportionally with incubation time after thawing. These findings indicate that cryopreservation of small artificial cartilage units is possible with a minor loss of cell viability. Secondly, its synthetic activity of cartilage-specific matrix did not decline after the freezing process.

Endothelialization of PTFE vascular grafts under flow induces significant cell changes.

BACKGROUND: To minimize thrombogeneity of small diameter PTFE grafts they are usually coated in vitro with endothelial cells under static culture conditions. The disadvantage of this technique is that a cell layer is formed that fails to withstand shear stress typical in normal blood flow. METHOD: Since the in vivo functional and structural status of endothelial cells correlates with the applied shear stress, we developed a computer-controlled perfusion system to seed and culture cells on PTFE-grafts up to a confluent monolayer under the influence of increasing shear stress. The confluence of endothelial coating was defined by immunohistological staining of cross sections, and by upper light microscopy of flattened graft samples. In addition, the expression of fibronectin as an important adhesion molecule was estimated. RESULTS AND DISCUSSION: The application of pulsatile shear stress (6.6 dyn/cm2, 5 min) to grafts endothelialized under perfusion (n = 7) did not lead to a disruption of the confluent cell layer. In contrast, a 5 min long shear stress of 3 dyn/cm2 was sufficient to wash more than 50% of cells off the PTFE-graft cultured under static conditions (n = 6). The perfusion cultures showed a significantly higher proliferation rate in comparison with static cultures. This effect was reproducibile in both serum-containing and serum-free culture media. The expression of fibronectin by endothelial cells was significantly higher in the perfused graft compared to the static one. These results suggest the practicability of endothelialized PTFE vascular grafts, preconditioned to shear rates similar to the in vivo situation, as an alternative bypass material in cardiac surgery.

Long-term effects of rhEPO therapy on erythrocyte rheology in dialysis patients with different target hematocrits.

UNLABELLED: Successful treatment of renal anemia with recombinant erythropoietin (rhEPO) raises the question of whether the renal anemia symptom complex requires complete correction. Current arguments against increasing hemoglobin (Hb) levels above 10-11 g/dl are impaired hemodynamics, increased risk of vascular access occlusion, unmanageable hypertension and dialysis complications. The aim of the study was to determine whether sustained Hb normalization using long-term rhEPO causes hemorheological changes with a potentially negative hemodynamic impact. The study was conducted in 42 rhEPO-treated dialysis patients with stable Hb > 11.0 g/dl for at least 20 weeks. The mean Hb of the total study group was 12.8 1.1 g/dl. To study the effect of Hb as a risk indicator in greater detail, the patients were divided into two groups, with hematocrits above and below 0.40. Hemorheology (erythrocyte deformability and aggregation, plasma viscosity) showed no significant changes, including vs a healthy control group. Throughout the period of increased rhEPO administration, no increase was observed in the incidence of hypertension or vascular thrombosis. CONCLUSION: the marked additional quality-of-life benefit achieved by complete correction of renal anemia harbors no substantial increase in treatment risk.

Complete correction of renal anemia by recombinant human erythropoietin.

The target-hematocrit (Hct) for the correction of renal anemia by recombinant human erythropoeitin (rhEPO) therapy is discussed controversially. A normalization of the Hct that could lead to a further improvement of the patients status, is often rejected, because of possible side effects as a result of an increase in blood viscosity. Hemodialysis (HD) induces an acute hemoconcentration due to ultrafiltration that might influence these risk factors negatively and therefore conflict with the normalization of Hct. The aim of this study was to investigate the changes in rheological and biochemical parameters in chronic HD patients with a normal initial Hct before hemodialysis. Results in 39 patients are given as mean +/- SD before/after HD: Hct 0.42 +/- 0.05/0.45 +/- 0.05 (p < 0.001), hemoglobin (g/dI) 13.3 +/- 1.0/14.4 +/- 1.3 (p < 0.001), MCV (fl) 99.3 +/- 5.7/99.1 +/- 5.5, MCHC (mM/l) 19.9 +/- 0.6/20.1 +/- 0.6 (p < 0.01), red blood cell (RBC) elongation (%) 60.97 +/- 3.67/60.99 +/- 3.73, RBC aggregation index AI0 0.52 +/- 0.12/0.50 +/- 0.12, AI4 0.52 +/- 0.14/0.51 +/- 0.12, plasma viscosity 1.74 +/- 0.14/1.92 +/- 0.20 (p < 0.001), whole blood viscosity (WBV), etaabs.100(mPas) 5.91 +/- 0.78/6.80 +/- 1.2 (p < 0.001), etaabs.0.01(mPas) 75.81 +/- 35.48/167.656 +/- 98.686 (p < 0.05), ultrafiltration (FM) 2.1 +/- 1.1. The biochemical parameters protein, albumin, IgG, IgA, IgM, cholesterol, transferrin and fibrinogen are significantly increased after HD. The hemoconcentration during HD is associated with a significant increase in WBV, mainly associated with the increase in Hct (r = 0.83), but not exceeding the normal range compared to healthy controls. The increase in plasma viscosity is correlated mainly with an increase in protein (r = 0.80), albumin (r = 0.74), and fibrinogen (r = 0.54). No significant changes in RBC aggregation and deformability were observed during the HD session. In conclusion, from the rheological point of view it is unlikely that the normalization of the Hct will contribute to an increased risk in access thrombosis or thromboembolic events in HD patients.

A novel perfusion system for the endothelialisation of PTFE grafts under defined flow.

OBJECTIVES: to develop a perfusion system for culturing human endothelial cells on small-diameter PTFE grafts under defined pulsatile shear stress. METHODS: to benefit from a stronger adhesion of endothelial cells to the substrate, we developed a perfusion system which enables culture of endothelial cells on PTFE grafts to confluence under a wide range of shear stress. We also developed an in situ staining method for the determination of the endothelialisation stage by upper light microscopy. RESULTS: the application of pulsatile flow with high shear stress (6.6 dyn/cm2, 5 min) to a graft endothelialised under perfusion did not lead to a disruption of the confluent cell layer. In contrast, a shear stress of 3 dyn/cm2 applied for 5 min was sufficient to wash more than 50% of endothelial cells off the PTFE graft when cultured to confluence under static conditions. CONCLUSIONS: this technique induces a stronger cell adherence of endothelial cells to a PTFE graft in comparison with grafts endothelialised under static conditions. Endothelialised vascular grafts can be pre-conditioned to defined shear stress values.

Perturbation of red blood cell membrane rigidity by extracellular ligands.

It is known that binding of extracellular antibodies against the major sialoglycoprotein, glycophorin A, reduced the deformability of the red blood cell membrane. This has been taken to result from new or altered interactions between the glycophorin A and the membrane skeleton. We have shown by means of the micropipette aspiration technique that antibodies against the preponderant transmembrane protein, band 3, induce similar effects. A definite but much smaller reduction in elasticity of the membrane is engendered by univalent Fab fragments of the anti-band 3 antibodies. By examining cells genetically devoid of glycophorin A or containing a variant of this constituent, truncated at the inner membrane surface, we have shown that the anti-band 3 antibodies do not act through the band 3-associated glycophorin A. We examined the effect of anti-glycophorin A antibodies on homozygous Wr(a+b-) cells, in which an amino acid replacement in band 3 annihilates the Wright b (Wrb) epitope (comprising sequence elements of glycophorin A and band 3) and thus, by implication disrupts or perturbs the band 3-glycophorin A interaction; these cells show a much smaller response to an anti-glycophorin A antibody than do normal controls. We infer that in this case anti-glycophorin A antibodies exert their rigidifying effect through the associated band 3. Another anti-glycophorin A antibody, directed against an epitope remote from the membrane surface, however, increases the rigidity of both Wr(a+b-) and normal cells. This implies that not all antibodies act in the same manner in modifying the membrane mechanical properties. The effect exerted by anti-band 3 antibodies appears not to be transmitted through the band 3-ankyrin-spectrin pathway because the rigidifying effect of the intact antibody persists at alkaline pH, at which there is evidence that the ankyrin-band 3 link is largely dissociated. The large difference between the effects of saturating concentrations of the divalent and univalent anti-band 3 antibodies implies the existence of an overriding effect on rigidity, resulting from the bifunctionality of the intact antigen. Freeze-fracture electron microscopy shows that the anti-band 3 promotes the formation of small clusters of intra-membrane proteins. Extracellular ligands may in general act by promoting strong or transient interactions between integral membrane proteins, thereby impeding local distortion of the membrane skeletal network in response to shear.

Mechanism of regulation of malarial invasion by extraerythrocytic ligands.

Invasion of red cells by Plasmodium falciparum in vitro was inhibited by a range of extracellular ligands, none of which block the major receptors for merozoites. Most effective, in terms of dose response, were two monoclonal antibodies against the Wrb antigen on glycophorin A; wheat germ agglutinin which also binds to glycophorin, and an anti-band 3 monoclonal antibody, caused inhibition of invasion at higher levels of saturation, while concanavalin A, which binds to band 3, was without effect. All the ligands except concanavalin A, increased the rigidity of the host cell membrane. The anti-Wrb antibodies generated the highest dose response effect, but no correlation between invasion and shear elastic modulus of the membrane could be established. All ligands, with the exception of concanavalin A, caused a reduction in the translationally mobile fractions of band 3 and glycophorin, as revealed by fluorescence recovery after photobleaching (FRAP). Invasion diminished with loss of mobile band 3, engendered by bound wheat germ agglutinin or anti-band 3, falling precipitately when the mobile fraction fell below 40% of that in unperturbed membranes. Both anti-Wrb antibodies suppressed invasion completely at concentrations insufficient to affect significantly either membrane rigidity or intramembrane protein diffusion. A univalent anti-glycophorin A (Fab) fragment, the parent antibody of which was previously shown to inhibit invasion strongly, had only a modest effect on invasion and induced a correspondingly small change in the mobile fraction of band 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane rigidity of red blood cells parasitized by different strains of Plasmodium falciparum.

Changes in the structure of parasitized red blood cells may influence their ability to circulate. We have used a micropipette technique to examine the effects of invasion and maturation of Plasmodium falciparum on the membrane rigidity of red blood cells. In the presence of immature, ring form parasites from different laboratory strains, membrane rigidity remained unchanged as compared with uninfected red cells. However, development of more mature pigmented trophozoites caused a marked increase in membrane rigidity. Parasites from knobless strains caused a less-pronounced increase than parasites from knob-positive strains. Using closely synchronized cultures, the dependence of membrane rigidity on parasite maturation was studied in more detail for selected knob-positive and knobless strains. Over a period of 12 hours, while trophozoites developed into schizonts, no further rigidification of the red cell membrane occurred. The increase in membrane rigidity, occurring with the initial development of pigmented trophozoites, may be related to insertion of neoantigens into the red cell surface or modification of native membrane proteins that also occur at this time. In contrast to others, we found no effect of parasite-culture supernatant, harvested at different stages, on the rigidity of uninfected cells exposed to it. Interstrain variation of membrane rigidity could influence pathophysiology in several ways: by promoting margination and cytoadherence of knob-positive strains in the microcirculation, by modulating clearance of parasitized cells by the reticuloendothelial system, and by influencing ischemic complications of severe falciparum malaria.

[Assessment of whole cell deformability of individual erythrocytes with a capillary rigidometer--possibilities for standardization and modification]. / Bewertung der Ganzzelldeformierbarkeit einzelner Erythrozyten mittels Kapillar-Rigidometer--Möglichkeiten zur Standardisierung und Relativierung.

Human erythrocyte deformability is determined by cell geometry and volume, membrane elasticity and cytoplasmic viscosity. The deformability of red blood cells and their distribution among the total cell population, can be studied with the Capillary Rigidometer. This device is based on the kinetic measurement of red blood cell deformability, which has been developed, by modifying the micropipette aspiration technique. In order to investigate the validity of the method and the measuring parameters, a number of determining factors (heat treatment, osmolarity-changed cells) influencing the deformability were studied, and the sensitivity of the defined parameters for changes in deformability discussed. The results are examined in connection with different flow rates in the micropipette and show that the parameters are influenced by the flow conditions, so that they have to be related to these conditions. Initial studies using microspheres aimed at standardising the method are described.

[Results of the Berlin HOT/UVB comparative study in patients with peripheral arterial occlusive disorders]. / Ergebnisse der Berliner HOT/UVB-Vergleichstudie bei Patienten mit peripheren arteriellen Durchblutungsstörungen.

Both photobiological methods haematogenic oxydation therapy (HOT) and therapy by retransfused ultraviolet irradiated own blood (UVB) were compared with regard to its therapeutic efficacy in patients with peripheral arterial occulusive disease of lower extremities in stage II by Fontaine. In parallel to paraclinical and coagulation data, haemodynamic as well as haemorheological parameters were investigated to clarify possible mechanisms of action of these therapies. 15 male patients were enclosed in the corresponding patients groups with a mean walking distance of 178 +/- 108 m (HOT) and 213 +/- 147 m (UVI), respectively. The claudicatio-distances were significantly improved after 10 series of therapy by 94% in the HOT-group and by 83% in the UVI-group, respectively. A significant difference in the improvement of walking distances could not be detected between both therapeutical methods. Significant alterations in observed paraclinical parameters were not observed.

Elastic properties of passive leukemic white blood cells.

The key role of leukocytes (WBC) in pathologic alterations of microcirculation was repeatedly described in the literature. The aim of this study was to compare the mechanical characteristics of WBC of 4 healthy donors and 6 patients suffering from chronic myeloid leukemia (CML). The elastic properties of individual leukocytes in the passive state were studied by the micropipette technique (aspirating pressure delta P = 100 divided by 250 Pa; internal pipette radius Rp = 1.4 divided by 2.6 microns). The passive elastic rigidity of WBC (PER) from 30 cells/donor was characterized by the amplitude of their steady-state deformation (Dp) normalized for the pipette radius (Rp) against the stress parameter delta P * Rp. The effect of a CML-therapy with cytostatics was monitored by way of differential blood counts. A significantly higher rigidity of healthy lymphocytes (PER = 349.7 +/- 125.9 microN/m; n = 45) than that of polymorphonuclear granulocytes (PER = 226.5 +/- 86.7 microN/m; n = 62) was seen which appears to result from their large, solid nucleus. Control leukocytes have a significantly higher rigidity than leukemic WBC. Leukemic granulocytes (PER = 155.1 +/- 67.7 microN/m; n = 61) and lymphocytes (PER = 134.3 +/- 51.9 microN/m; n = 62) do not differ significantly in their values of rigidity.

Action of rHuEpo on mechanical membrane properties of red blood cells in children with end-stage renal disease.

By means of a micropipette aspiration technique mechanical membrane properties of RBC in ten uraemic children undergoing haemodialysis were compared to those of nine healthy children. In contrast to the healthy reference group (mu = 4.01 +/- 0.71 microN/m), the mean apparent elastic shear modulus of RBC membrane mu as a parameter of static deformability was significantly increased to 9.05 +/- 1.61 microN/m in children with chronic renal failure measured in the pre-study period. At the 7th week of the correction period of rHuEpo therapy, the haematocrit of treated patients is enhanced to a mean of 0.30 +/- 0.03, whereas mean mu is decreased to 5.41 +/- 0.63 microN/m. Within the maintenance period of rHuEpo treatment (30th week), the parameter mu did not show significant differences to the reference value 3.78 +/- 0.31 microN/m. Additionally, a significant increase in red cell mean corpuscular volume was obtained during rHuEpo therapy. Improved deformability of uraemic RBC induced by the rHuEpo therapy can only be explained by the assumption that in the course of this therapy an increasing number of cells with normalised viscoelastic properties have been formed by stimulated erythropoiesis.

Freshwater fish diet affects lipid composition, deformability and aggregation properties of erythrocytes.

The effect of a 12-week diet of freshwater fish on fatty acid composition of the erythrocyte membrane, RBC deformability and artificial aggregation behavior of erythrocytes was studied in 20 healthy subjects. A different quantity of meals containing fish per week (control group, 1.5 and 3.8 fish meals per week) resulted in an increased content of n - 3 polyunsaturated fatty acids. The whole cell deformability of single erythrocytes characterized by a modified micropipette technique (capillary-rigidometer) was significantly increased dependent on fish intake. The parameter of entry time for cell deformability had a negative correlation with the n - 3/n - 6 ratio of fatty acids. No change was observed in MCV and MCHC of erythrocytes after the diet. The artificial aggregation behavior of washed erythrocytes in a suspension medium of low ionic strength and reduced pH was decreased depending on the number of fish meals eaten. The present results suggest that a relatively small shift in the profile of the polyunsaturated fatty acids causes changes in the viscoelastic properties of the erythrocyte membrane and in the artificially-induced aggregation of erythrocytes.

The effect of rimantadine on the structure of model and biological membranes.

The action of the antiviral drug rimantadine on the structure of bilayer lipid membranes (BLM) and RBC membranes was investigated. Structural changes in BLM were recorded by ionophore conductivity changes and by changes in the third harmonic of capacity current signal due to lateral compression of BLM in an electric field. It was shown that the adsorption of rimantadine on BLM results in an increase in ionophore mobility in bilayer membranes of dioleolyllecithin (DOL) and common lipids of bovine brain (CL) and in a decrease in those of azolectin (A). Relative changes in the third harmonic signal also depend on the membrane composition and have different signs. The results may be explained by the rimantadine action on the lipid bilayer structure: "rigidification" of A-membranes and "fluidization" of BLM from DOL and CL. Structural reorganization of RBC membranes as investigated by the ability of the cells to enter a micropipette (inner diameter greater than or equal to 3 microns) thereby undergoing deformation. It was shown that rimantadine influences RBC deformability due to drug induced inhomogenous mechanical membrane properties. Also, rimantadine accelerated the process of artificially induced aggregation of erythrocytes. The relation of the effects on artificial and biological membranes, and the structural changes in the lipid phase of membrane are discussed.

[The flow properties of blood and their characterization by hemorheologic methods]. / Die Fliesseigenschaften von Blut und ihre Charakterisierung mittels hämorheologischer Methoden.

In a number of supply disturbances the flow behaviour of blood plays an increasing role in modern therapy concepts. The present paper deals with representing factors, such as haematocrit, aggregation, deformation, which exercise an influence on the complex flow properties of blood under variable conditions. By referring to the example of deformability it is shown how and to what extent different mechanical parameters of individual erythrocytes participate in static or dynamic whole cell deformation. Starting from fundamental quality demands (sensitivity, specificity, value of prediction) to diagnostic measuring technique, haemorheological methods for determining complex flow properties, aggregation and deformation as single phenomena and mechanical properties of individual erythrocytes are presented. Selected examples of application for normal blood, cells altered in vitro or pathological changes measured ex vivo are referred to.