A model for rouleaux pattern formation of red blood cells

Human red blood cells (RBCs) in a solution form rouleaux patterns under various conditions. The degree of rouleaux formation depends on, for example, the concentration and molecular weight of added large molecules. We present a two-dimensional discrete cellular space model in which an RBC is represented by a rectangle and differential adhesion is assumed among the longer (a-site), the shorter (b-site) sides of the rectangle and the solvent. The total sum of the adhesion energy is assumed to guide the step-by-step change of the model cell configuration and also define absolutely stable patterns. We compare the set of absolutely stable patterns and cell aggregate patterns for both actual and computer-simulated cases to obtain the basic validity of our framework. Then we proceed to assess the effects of added high polymers to the adhesion parameters. We first note that under suitable conditions, decrease in a-site-solvent affinity is necessary to have complex patterns rather than increase of a-a affinity. The hypothesis that addition of high polymers reduce the a-site-solvent affinity is concomitant with a newly proposed osmotic stress theory. The parameter fitting results for the experimental phase change curves can also be interpreted as supporting more the new theory than existing traditional explanations.     

Red blood cell rouleaux formation in dextran solution: dependence on polymer conformation

The velocity of rouleaux formation (RF), as previously shown, increases with increasing dextran concentration up to a critical concentration (C_a), beyond which the addition of dextran reduces the RF velocity (RFV). de Gennes' model for polymer solutions suggests that dextrans exist in two conformations: a coil structure at low concentrations, which changes to a network beyond a critical concentration (C^*). In the present study we examined the relation between C_a and C^* for dextrans of different molecular weight, and found that they coincide. This suggests that the change in dextran behavior, from increasing to decreasing RFV, occurs when their conformation changes from coil to network. In addition, it has been reported that in dilute dextran solutions the intercellular distance (D) between RBC in rouleaux increases with the molecular weight of the dextran. We found that D correlates with R_f, the end-to-end distance of the polymer molecule, and for all dextrans D ≤ 1.5 R_f. In accord with de Gennes' Model for polymers between surfaces, this corresponds to intercellular interaction with two overlapping surface-associated polymer layers, which may extend “tails” to interact with the opposing cells. Received: 8 August 1997 / Accepted: 28 November 1997     

Modes of rouleaux formation of human red blood cells in polyvinylpyrrolidone and dextran solutions

The mechanics by which normal human erythrocytes join on a plastic cover slip into two cell doublets and larger aggregates of rouleaux were studied microscopically. Polyvinylpyrrolidone (PVP-360) or dextran (DX-70 or DX-110) were used as the rouleau agents. The minimum concentration of the rouleau-inducing agents required to form doublets was 1 g/L for PVP-360 and 5 g/L for the DXs. Three modes of interaction were observed in Ringer's solution with PVP or DX, cresting and flipping (involving no intercellular sliding) and a sliding mode of doublet formation (involving less gravitational work and less cellular deformation). The sliding mechanism occurred in suspensions with the lower concentrations of the rouleau agent but was also observed when geometric constraints prevented the nonsliding interaction of larger groups of cells in the higher concentrations of the rouleau agent. The technique provides a sensitive index for studying the combined effect of cellular flexibility and intercellular adhesion. Significant changes were observed for reduced membrane surface charge or reduced ionic calcium.     

The equilibrium size distribution of rouleaux.

Rouleaux are formed by the aggregation of red blood cells in the presence of macromolecules that bridge the membranes of adherent erythrocytes. We compute the size and degree of branching of rouleaux for macroscopic systems in thermal equilibrium in the absence of fluid flow. Using techniques from statistical mechanics, analytical expressions are derived for (a) the average number of rouleaux consisting of n cells and having m branch points; (b) the average number of cells per rouleau; (c) the average number of branch points per rouleau; and (d) the number of rouleaux with n cells, n = 1, 2, ..., in a system containing a total of N cells. We also present the results of numerical evaluations to establish the validity of asymptotic expressions that simplify our formal analytic results.     

Pullulan-sodium alginate based edible films: Rheological properties of film forming solutions

Rheological properties of pullulan, sodium alginate and blend solutions were studied at 20 °C, using steady shear and dynamic oscillatory measurements. The intrinsic viscosity of pure sodium alginate solution was 7.340 dl/g, which was much higher than that of pure pullulan (0.436 dl/g). Pure pullulan solution showed Newtonian behavior between 0.1 and 100 s⁻¹ shear rate range. However, increasing sodium alginate concentration in pullulan-alginate blend solution led to a shear-thinning behavior. The effect of temperature on viscosities of all solutions was well-described by Arrhenius equation. Results from dynamical frequency sweep showed that pure sodium alginate and blend solutions at 4% (w/w) polymer concentration were viscoelastic liquid, whereas the pure pullulan exhibited Newtonian behavior. The mechanical properties of pure sodium alginate and pullulan-alginate mixture were analyzed using the generalized Maxwell model and their relaxation spectra were determined. Correlation between dynamic and steady-shear viscosity was analyzed with the empirical Cox-Merz rule.     

Segregation into separate rouleaux of erythrocytes from different species. Evidence against the agglomerin hypothesis of rouleaux formation.

Erythrocytes from one species were labelled with fluorescein isothiocyanate and mixed with unlabelled erythrocytes from another species. Albumin polymers were added to generate rouleaux. The species of origin of erythrocytes in rouleaux was determined by fluorescence microscopy. Erythrocytes from different species segregated into independent rouleaux. However, fluorescent and non-fluorescent erythrocytes from one individual were mixed randomly in rouleaux. These results confirm, using a novel experimental approach, previous observations of Sewchand & Canham [(1976) Can. J. Physiol. Pharmacol. 54, 437-442]. Since rouleaugenic agents are not species-specific, under the 'agglomerin' hypothesis of rouleau formation they would be expected to form bridges between cells from different species. It follows that either the agglomerin hypothesis is incorrect, or additional species-specific surface components are involved in the aggregation of agglomerin-cross-bridged cells.     

Photopolymerization of bovine hemoglobin entrapped nanoscale hydrogel particles within liposomal reactors for use as an artificial blood substitute

Lipogel particles encapsulating bovine hemoglobin (BHb) were synthesized via photopolymerization of poly(N-isopropylacrylamide) (pNIPA) and poly(acrylamide) (pAAm) monomers within liposomal reactors. Nanoscale hydrogel particles (NHPs) encapsulating bovine hemoglobin, which represent a hybrid between acellular and cellular hemoglobin based oxygen carriers, were formed upon solubilization of the lipid bilayer of lipogel particles encapsulating BHb. Lipogels and NHPs encapsulating BHb constitute a new class of blood substitute that prevents both dissociation of hemoglobin (Hb) and in vivo exposure of acellular Hb, while allowing oxygen transport through the polymer matrix. pNIPA and pAAm particles encapsulating BHb displayed oxygen affinities ranging from 9.9 +/- 1.9 to 14.4 +/- 0.1 mmHg for lipogels, methemoglobin levels ranging from 9.3 +/- 3.7% to 26.0 +/- 5.0% for lipogels and NHPs, and encapsulation efficiencies ranging from 34.2 +/- 3.4% to 97.4 +/- 15.8% for lipogels and NHPs. Interestingly, the methemoglobin level of pNIPA particles was reduced 61% by coencapsulating the reducing agent, N-acetylcysteine. Fractionation and light scattering results showed that lipogels and NHPs were spherical and exhibited narrow size distributions. The colloidal osmotic pressure of pNIPA and pAAm lipogels ranged from 3.71 +/- 0.02 to 206.87 +/- 0.42 mmHg, depending on UV-irradiation time, type of buffer, and polymer composition. These results demonstrate that hemoglobin can be encapsulated within hydrogel based particles for use as an artificial blood substitute.     

Work behaviors of artificial muscle based on cation driven polypyrrole

A soft actuator mimicking natural muscles (artificial muscle) has been developed using a flexible conducting polymer of polypyrrole films, which were driven by electrical stimulus in a saline solution. The work characteristics were studied under various load stresses and found to behave like natural muscles. The artificial muscles shrunk and stiffened by the positive electrical stimulus by 2-3% at the maximum force of 5 MPa, and relaxed by application of negative voltages. At larger load stresses, the artificial muscle shrunk slowly as natural muscles do. The driving current also lasted longer at larger loads, indicating that the muscle sensed the magnitude of the load stress. During contraction of the muscle, the conversion efficiency from the electrical input and mechanical output energies was estimated to be around 0.06%. The maximum volumetric work was approximately estimated to be 100 kJ m(-3). These figures are unexpectedly small compared with those of natural muscles.     

Future prospects for artificial blood

Concern about potential infective agents in donated blood has stimulated the recent development of blood substitutes. Chemically cross-linked hemoglobins are already undergoing clinical trials and might soon be ready for routine use. New generations of modified hemoglobin are being prepared to modulate the effects of nitric oxide and oxygen radicals, and artificial red blood cells are also under development.