Temporary network formation of hyaluronate under a physiological condition. 1. Molecular-weight dependence

The dynamic shear moduli at various frequencies and stress growth after the sudden start of steady shear were measured for 1% HA (hyaluronic acid) solutions with different molecular weights. From the results of the zero shear viscosity, eta 0, the steady shear compliance, J 0e, and delta eta defined as delta eta = eta 0 - eta a (infinity), where eta a (infinity) is the apparent viscosity at the steady state, it was shown that the molecular mechanism of flow of 1% HA solutions was classified into four regions with respect to molecular weight: (I) the viscosity-average molecular weight Mv less than 35 X 10(4), HA chains are dispersed molecularly in solution; (II) 35 X 10(4) less than Mv less than 100 X 10(4), the polymer chains form a weak entanglement network observed only through eta 0; (III) 100 X 10(4) less than Mv less than 160 X 10(4), the network is strengthened with increasing molecular weight and becomes detectable through both eta 0 and J 0e and also through the overshoot phenomenon; and (IV) 160 X 10(4) less than Mv, the network is "saturated" or "completed" dynamically. This is the conjecture presented for the first time by the present work.     

Evaluation of radius of gyration and intrinsic viscosity molar mass dependence and stiffness of hyaluronan

Nine hyaluronan (HA) samples were fractionated by size-exclusion chromatography, and molar mass (M), radius of gyration (Rg), and intrinsic viscosity ([eta]) were measured in 0.15 M NaCl at 37 degrees C by on-line multiangle light scattering and viscometer detectors. Using such method, we investigated the Rg and [eta] molar mass dependence for HA over a very wide range of molar masses: M ranging from 4 x 10(4) to 5.5 x 10(6) g/mol. The Rg and the [eta] molar mass dependence found for HA showed a meaningful difference. The Rg = f(M) power law was substantially linear in the whole range of molar masses explored with a constant slope of 0.6. In contrast, the [eta] = f(M) power law (Mark-Houwink-Sakurada plot) showed a marked curve shape, and a linear regression over the whole range of molar masses does not make sense. Also the persistence length (stiffness) for HA was estimated. The persistence length derived by using both the Odijk's model (7.5 nm from Rg vs M data) and the Bohdanecky's plot (6.8 nm from [eta] vs M data) were quite similar. These persistence length values are congruent with a semistiff conformation of HA macromolecules.     

Degradation of high-molecular-weight hyaluronan by hydrogen peroxide in the presence of cupric ions

Dynamic viscosity (eta) of the high-molecular-weight hyaluronan (HA) solution was measured by a Brookfield rotational viscometer equipped with a Teflon cup and spindle of coaxial cylindrical geometry. The decrease of eta of the HA solution, indicating degradation of the biopolymer, was induced by a system containing H2O2 alone or H2O2 plus CuCl2. The reaction system H2O2 plus CuCl2 as investigated by EPR spin-trapping technique revealed the formation of a four-line EPR signal characteristic of a *DMPO-OH spin adduct. Thus, hydroxyl radicals are implicated in degradation of high-molecular-weight HA by the system containing H2O2 and CuCl2.     

Solution structure and dynamics of cartilage aggrecan

We studied the structure and dynamics of porcine laryngeal aggrecan in solution using a range of noninvasive techniques: dynamic light scattering (DLS), small-angle neutron scattering (SANS), video particle tracking (VPT) microrheology, and diffusing wave spectroscopy (DWS). The data are analyzed within the framework of a combined static and dynamic scaling model, and evidence is found for reptation of the comb backbones with unentangled side-chain dynamics. Small-angle neutron scattering indicated standard polyelectrolyte scaling of the mesh size (xi) with concentration (c) in semidilute solutions for the whole aggrecan aggregate, xi = Ac(-0.47+/-0.04), with the prefactor (A) implying there is on average 60 nm between the aggrecan subunits along the backbone. VPT demonstrated large exponents for the power law dependence of the intrinsic viscosity (eta) on the polymer concentration in the semidilute concentration regime, eta approximately c(alpha); with alpha equal to 2.04 +/- 0.06 and 1.95 +/- 0.08 for the assembled and disassembled aggrecan aggregates, respectively. DWS at high frequencies (10(4)-10(5) Hz) gave evidence for internal Rouse modes of the aggrecan monomers, independent of the degree of self-assembly of the molecules.     

Viscoelastic properties of an exopolysaccharide: Aeromonas gum, produced by Aeromonas nichidenii 5797

The viscoelastic properties of Aeromonas (A) gum in water were investigated by using the Rheometric Scientific ARES controlled strain rheometer. An intrinsic viscosity of 8336 ml/g was obtained according to the Fuoss-Straus equation. The effect of salt concentration on intrinsic viscosity revealed that the A gum exists as semiflexible chain. Typical shear-thinning (pseudoplastic) behavior was observed at concentrations higher than 0.52%. The zero shear viscosity (eta(0)) increased with increasing polysaccharide concentration (c) showing a gradient of approximately 1.0, 2.9 and 4.8 in different concentration domains. The critical concentrations c* and c**, at which the transitions from a dilute solution of independently moving chains to semidilute and then concentrated domains occurred, were determined roughly to be 1.2% and 3.5%. The results from dynamic experiments revealed that the A gum solution shows characteristics of polymer solutions without any evidence of gel-like character. All the results from steady and dynamic tests suggest that the A gum is a non-gelling polysaccharide. The temperature dependence of apparent viscosity was described by Arrhenius equation and the flow activation energy was estimated to be 45.2 kJ/mol, which is independent on polymer concentration.     

Molecular structure and rheological properties of short-side-chain heavily glycosylated porcine stomach mucin

The current accepted model for high-molecular-weight gastric mucins of the MUC family is that they adopt a polydisperse coil conformation in bulk solutions. We develop this model using well-characterized highly purified porcine gastric mucin Orthana that is genetically close to the human MUC6 type. It has short side chains and low levels of sialic acid residues and includes minute amounts of cysteine residues that, if abundant, can be responsible for the self-polymerization of mucin. We have established that the mucin structure in bulk solutions corresponds to a daisy-chain random coil. Dynamic light scattering experiments probe the internal dynamics of globular subunits (individual daisies) at the approximately 9 nm length scale, whereas viscosity and light scattering measurements indicate that the size of the whole mucin chains is much larger, approximately 50 nm. The bulk viscosity (eta) scales with mucin concentration (c) in a manner similar to that found for short-side-chain synthetic comb polyelectrolytes and is characterized by a transition between semidilute (eta approximately c1/2) and entangled (eta approximately c3/2) regimes.     

Thermally induced coil-to-helix transition of sodium gellan gum with different molar masses in aqueous salt solutions

Using 5 samples of well-purified Na-gellans (Na-gellans G1-G5, weight-average molar mass M(w) = 120 x 10(3)-32 x 10(3) at 40 degrees C), the effects of molar mass on the coil-to-double-helix transition in aqueous solutions with 25 mM NaCl were studied by light scattering and circular dichroism (CD) measurements, viscometry, and differential scanning calorimetry (DSC). From the temperature dependence of M(w), molar ellipticity at 201 nm [theta]201, intrinsic viscosity [eta], and DSC exothermic curves, it was found that the coil-to-double-helix transitions for G1-G5 samples took place at almost the same temperature. The [eta] and M(w) obtained in the temperature range from 40 to 25 degrees C can be explained by a simple coil/double-helix equilibrium model using the double-helix contents determined from CD data. The van't Hoff's transition enthalpy deltaH(vH) of Na-gellans depended on M(w). It is concluded that the coil-to-double-helix transitions of Na-gellans are all-or-none type transitions, and are accelerated with increasing M(w).     

Action of hydroxyethyl starch on the flow properties of human erythrocyte suspensions

Hydroxyethyl starch (HES) has often been used as a plasma expander, but questions still remain concerning the mechanisms by which it produces changes in the rheological properties of blood and erythrocyte (RBC) suspensions under various flow conditions. The present investigation has shown that the dynamic viscosity of HES (232,000 and 565,000 daltons) solutions rises in a nonlinear fashion with increasing HES concentration, and for a given concentration of HES exhibits Newtonian behavior at shear rates between 0.15 to 124 sec-1. At low (less than 0.9 sec-1) shear rates the apparent viscosity of a 40% RBC suspension increases with lower concentrations of HES because of RBC aggregation. At higher concentrations of HES, increases in suspension viscosity are due to an increase in the viscosity of the continuous phase since the RBC are largely disaggregated. At high (greater than 36 sec-1) shear rates the relative viscosity (eta/eta O) of RBC suspensions slowly decreases with increasing HES concentration. At low shear rates eta/eta O increases and then decreases with increasing HES concentration. Evidence of the concentration-dependent effects of HES on RBC aggregation is provided not only by the viscometric analysis but also from measurements of erythrocyte sedimentation rate (ESR) and the zeta sedimentation ratio (ZSR). HES is a more potent aggregating agent in phosphate buffered saline (PBS) than it is in plasma. Polymer size has only a slight effect on the extent of RBC aggregation produced, but does have a significant effect on the concentration of polymer at which maximum aggregation occurs. The viscosity-corrected electrophoretic mobility of RBC in HES rises monotonically with the concentration of HES in the suspending medium. Decreases in the extent of RBC aggregation with increasing polymer concentrations probably result from an increase in the electrostatic repulsive forces between the cells.     

Friction coefficients for mechanically damaged bovine articular cartilage

We used a pin-on-disc tribometer to measure the friction coefficient of both pristine and mechanically damaged cartilage samples in the presence of different lubricant solutions. The experimental set up maximizes the lubrication mechanism due to interstitial fluid pressurization. In phosphate buffer solution (PBS), the measured friction coefficient increases with the level of damage. The main result is that when poly(ethylene oxide) (PEO) or hyaluronic acid (HA) are dissolved in PBS, or when synovial fluid (SF) is used as lubricant, the friction coefficients measured for damaged cartilage samples are only slightly larger than those obtained for pristine cartilage samples, indicating that the surface damage is in part alleviated by the presence of the various lubricants. Among the lubricants considered, 100 mg/mL of 100,000 Da MW PEO in PBS appears to be as effective as SF. We attempted to discriminate the lubrication mechanism enhanced by the various compounds. The lubricants viscosity was measured at shear rates comparable to those employed in the friction experiments, and a quartz crystal microbalance with dissipation monitoring was used to study the adsorption of PEO, HA, and SF components on collagen type II adlayers pre-formed on hydroxyapatite. Under the shear rates considered the viscosity of SF is slightly larger than that of PBS, but lower than that of lubricant formulations containing HA or PEO. Neither PEO nor HA showed strong adsorption on collagen adlayers, while evidence of adsorption was found for SF. Combined, these results suggest that synovial fluid is likely to enhance boundary lubrication. It is possible that all three formulations enhance lubrication via the interstitial fluid pressurization mechanism, maximized by the experimental set up adopted in our friction tests.     

Temperature transition of human hemoglobin at body temperature: effects of calcium

We studied the effects of calcium ion concentration on the temperature dependence of rheological behavior of human red blood cells (RBCs) and concentrated hemoglobin solutions. Our previous study (G. M. Artmann, C. Kelemen, D. Porst, G. Büldt, and S. Chien, 1998, Biophys. J., 75:3179-3183) showed a critical temperature (Tc) of 36.4 +/- 0.3 degrees C at which the RBCs underwent a transition from non-passage to passage through 1.3 microm micropipettes in response to an aspiration pressure of -2.3 kPa. An increase in intracellular Ca2+ concentration by using the ionophore A23187 reduced the passability of intact RBCs through small micropipettes above T(c); the micropipette diameter needed for >90% passage increased to 1.7 microm. Viscometry of concentrated hemoglobin solutions (45 and 50 g/dl) showed a sudden viscosity transition at 36 +/- 1 degrees C (Tc(eta)) at all calcium concentrations investigated. Below Tc(eta), the viscosity value of the concentrated hemoglobin solution at 1.8 mM Ca(2+) was higher than that at other concentrations (0.2 microM, 9 mM, and 18 mM). Above Tc(eta), the viscosity was almost Ca2+ independent. At 1.8 mM Ca2+ and 36 +/- 1 degrees C, the activation energy calculated from the viscometry data showed a strong dependence on the hemoglobin concentration. We propose that the transition of rheological behavior is attributable to a high-to-low viscosity transition mediated by a partial release of the hemoglobin-bound water.     

A novel approach to blood plasma viscosity measurement using fluorescent molecular rotors

Molecular rotors, a group of fluorescent molecules with viscosity-dependent quantum yield, were tested for their suitability to act as fluorescence-based plasma viscometers. The viscosity of samples of human plasma was modified by the addition of pentastarch (molecular mass 260 kDa, 10% solution in saline) and measured with a Brookfield viscometer. Plasma viscosity was 1.6 mPa x s, and the mixtures ranged up to 4.5 mPa x s (21 degrees C). The stimulated light emission of the molecular rotors mixed in the plasma samples yielded light intensity that was nonoverlapping and of significantly different intensity for viscosity steps down to 0.3 mPa x s (n = 5, P < 0.0001). The mathematical relationship between intensity (I) and viscosity (eta) was found to be eta = (kappaI)(nu). After calibration and scaling the fluorescence based measurement had an average deviation versus the conventional viscometric measurements that was <1.8%. These results show the suitability of molecular rotors for fast, low-volume biofluid viscosity measurements achieving accuracy and precision comparable to mechanical viscometers.     

Effect of insulin on lipid bilayer viscoelasticity

Changes in the Young elasticity modulus in perpendicular direction to the membrane surface E perpendicular, in the coefficient of dynamic viscosity eta, in the electric capacitance C, in the surface charge U1, in the conductivity g and in the coefficient of non-linearity beta of current-voltage characteristic caused by insulin were studied in bilayer lipid membranes (BLM) prepared from a mixture of egg lecithin and cholesterol (4:1, w/w) in n-heptane. Even relatively small concentrations of insulin in electrolyte (ci approximately 4.8 x 10(-11) mol/l) caused a diminution in parameters E perpendicular and eta. Negative surface charge emerged on the membrane due to the insulin absorption, and U1 gradually increased depending on the concentration of the hormone in the electrolyte. Addition of insulin was also followed by an increase in membrane conductivity and affected the value of the coefficient of non-linearity beta of current-voltage characteristic. The effect of insulin on the BLM structure was discussed on the basis of the results obtained.     

DWS microrheology of a linear polysaccharide

Diffusing wave spectroscopy has been used to measure the rheological behavior of pullulan (M(w) = 1 x 10(5)) aqueous solutions up to concentration of 40 g/dL. It was found that these solutions were mainly viscous, with the loss modulus G' higher than the elastic modulus G'. The plot of the specific viscosity eta(sp) as a function of pullulan concentration showed two critical concentrations c = 4 g/dL and c = 15 g/dL. For c < c, eta( sp) approximately c(1.25+/-0.05); for c < c < c, eta( sp) approximately c(2+/-0.05); and for c > c, eta( sp) approximately c(4.5+/-0.5). These results are in very good agreement with those reported in the literature.     

On-line monitoring of the viscosity in dextran fermentation using piezoelectric quartz crystal

A novel viscous sensor utilizing AT-cut quartz crystal to monitor the viscosity of fermentation broth was developed. The sensor system was constructed from the piezoelectric quartz crystal fixed to the cell, exposing only one side of the quartz crystal electrode, an oscillating circuit, a peak level meter, and a personal computer. In order to investigate the characteristics of the sensor system, a sensor signal relating to the resonant resistance of the quartz crystal was measured using dextran solutions with different molecular weights. The linear relationship was obtained between the sensor signal and the (rhoeta)(1/2) of the liquid, where rho and eta are the density and viscosity, respectively. The sensor signal was dependent not only on the viscosity of the liquid but also on the molecular weight of dextran, because dextran solution shows a non-Newtonian property. The sensor system was applied for the on-line monitoring of the viscosity in dextran fermentation. A good correlation was observed between the sensor signal and the viscosity value measured with a rotational viscometer for the fermentation broth. Little bubbling effect and agitation of the sensor signal were observed, showing that this system can be utilized for viscosity monitoring in a bioprocess.     

溶液黏度对相转化水凝胶微针制备的影响

目的:选择黏度适宜的高分子溶液,制备相转化水凝胶微针。方法:通过考察两种型号的聚乙烯醇本身的黏度性质、溶液温度、几种不同性质的高分子材料,包括透明质酸、预胶化淀粉等可能影响聚乙烯醇溶液黏度的因素,使用粘度计测定其黏度值并作图比较,观察溶液黏度变化的规律。将所制溶液分别制备微针以观察针形的好坏,选择合适制备相转化水凝胶微针的溶液黏度范围。结果:实验表明黏度范围在2.5万至13万m Pa·s之间的聚合物溶液较为粘稠且流动性适宜。结论:在该黏度范围内的聚合物溶液可用以制备相转化水凝胶微针,适用于工业化生产。     

Viscoelastic and rheological properties of concentrated solutions of proteoglycan subunit and proteoglycan aggregate

The oscillatory and steady shear rheological properties of concentrated solutions of proteoglycan subunit (PGS) and aggregate (PGA) from bovine articular cartilage have been studied using a Rheometrics fluids spectrometer. At comparable concentrations in the physiological range tan delta increases from 0.5 to 1.0 for PGA as the oscillation frequency (omega) increases from 10(-1) to 10(2) rads/s, compared to a decrease from 40 to 5 for PGS. Thus PGA solutions exhibit predominantly elastic response whereas those of PGS exhibit primarily viscous behavior. PGA solutions show pronounced shear-thinning behavior at all shear rates (gamma) in the range 10(-2) less than gamma (s-1) less than 10(2), whereas PGS solutions exhibit predominantly Newtonian flow. For PGA, the small-strain complex viscosity eta* (omega) is substantially smaller than the steady-flow viscosity eta(gamma) at comparable values of omega and gamma. These observations indicate that the presence of proteoglycan aggregates leads to formation of a transient or weak-gel network. Since aggregation leads to a large increase in molecular hydrodynamic volume and hence in the relaxation times for macromolecular rotation, it appears that role of aggregate formation is to shift the linear viscoelastic response from the terminal viscous flow into the plateau elastomeric regime of relaxational behavior. Normal or pathological changes that produce a decrease in aggregation will result in a loss of elastomeric behavior of the proteoglycan matrix.     

The rotational diffusion of chloroplast phosphate translocator and of lipid molecules in bilayer membranes

The rotational mobility of the phosphate translocator from the chloroplast envelope and of lipid molecules in the membrane of unilamellar azolectin liposomes has been investigated. The rotational dynamics of the liposome membrane were investigated by measuring the rotational diffusion of eosin-5-isothiocyanate(EITC)-labeled L-alpha-dipalmitoylglycerophosphoethanolamine (Pam2 GroPEtn) in the lipid phase of the vesicles, either in the presence or absence of the reconstituted phosphate translocator. The temperature dependence of the anisotropy decay showed that above 25 degrees C the main contribution to the anisotropy decay was caused by uniaxial anisotropic rotation of the labelled lipid molecules around the axis normal to the membrane plane. The rate of rotation of the labelled lipid molecules was strongly dependent on the viscosity of the medium (eta 1). Extrapolation to eta 1 = 0 Pa.s yielded a correlation time of phi = 20 +/- 5 ns, t = 30 degrees C, for lipid rotation with respect to the membrane normal. The rotational diffusion coefficient of the lipid molecules was calculated to be Dr = 2.0 x 10(9) rad2.s-1 and the apparent microviscosity in the vesicle membrane, as derived from the rotational correlation time, was eta 2 approximately 12 mPa.s. The rotational correlation time of the phosphate translocator in the membrane was only slightly dependent on the viscosity of the medium. The temperature dependence of the protein rotation also indicated that the rotation of the protein in the membrane was largely restricted and occurred mainly about the axis normal to the membrane plane. Measurements at a medium viscosity of eta 1 = 1 mPa.s yielded a value of phi r approximately 450 ns corresponding to Dr = 8.8 x 10(7) rad2.s-1 for protein rotation with respect to the membrane normal. From this value and the data of the lipid rotation, the cross-sectional area of the protein part embedded in the membrane was calculated to be approximately 9 nm2. This cross-sectional area is large enough to include at most 14 membrane-spanning helices. Our results also indicated that at lipid/protein molar ratios greater than or equal to 1.5 x 10(4): 1 aggregation occurred in the model membranes below 30 degrees C. However, above 30 degrees C and at a high dilution of the protein in the membrane it appeared that the membrane viscosity monitored by lipid and protein rotational diffusion were identical.     

Viscosity and molecular weight of hyaluronic acids.

The intrinsic viscosity ([eta]) and the molecular weight (M) by sedimentation equilibrium were determined for hyaluronic acids of low (M=104--7.2X10(4)) and high (M=3.1X10(5)--1.5X10(6)) molecular weights. Double logarithmic plot of [eta] against M gave different lines for the two groups. The relationship between [eta] and M was [eta]=3.0X10(6)XM1,20 for the former and [eta]=5.7X10(-4)XM0.46 for the latter group. The molecular weight at the point of intersection of the two lines was about 1.5X10(5). The rheological behavior of the hyaluronic acids below M=2.1X10(4), for which the value of reduced viscosity was independent of concentration, was different from that of the hyaluronic acids above M=5.1X10(4), for which the value of reduced viscosity increased with concentration.     

Complex viscosity of bovine red blood cells in suspensions

The complex viscosity eta* has been measured of bovine red blood cells suspended in a medium of isotonic NaCl solutions including dextran and buffered with potassium phosphate at pH 7.0. A multiple lumped resonator apparatus was used at the frequencies of 144, 572, 1491, 3742, and 8026 Hz at 20.0 degrees C. Due to the high molecular weight of dextran the medium also exhibited some visco-elasticity eta s*. So we adopted the complex specific viscosity eta sp* = (eta*-eta s*)/[eta s*]. At 20.0 degrees C eta sp* decreased with the frequency where the hematocrit was 0.233 and eta s 0.34 poise. The measurements were made for the medium with different viscosity at 5.0 degrees C and 25.0 degrees C. The results are compared with the theory of elastic shells.     

Viscoelastic properties for kappa- and iota-carrageenan in aqueous NaI from the liquid-like to the solid-like behaviour

The dynamic mechanical behaviour of a series of concentrations of kappa-carrageenan (KC; 0.35-1.6% w/w) and iota-carrageenan (IC; 0. 2-1% w/w) in 0.2 M NaI has been investigated. The flow behaviour of KC within the concentration range 0.004-0.8% (w/v) was also described. The high intrinsic viscosity of KC in 0.2 M NaI (23.4 dl g(-1)) and the great increase in viscosity with increasing concentration, in comparison with linear flexible polysaccharides, is well consistent with the stiffness of KC helices in NaI. The variation of the 'zero-shear' specific viscosity of KC in 0.2 M NaI with the degree of space-occupancy (c[eta]) displays two critical concentrations at c* approximately 0.09% w/v (c*[eta] approximately 2) and c** approximately 0.4% w/v (c**[eta] approximately 10). Different viscoelastic behaviours were exhibited from the liquid-like to the solid-like depending upon the type of carrageenan. From the application of the time-temperature superposition, classical frequency-temperature master curves could be obtained for KC, but not for IC. Moreover, for KC, a concentration-frequency master curve could be constructed for the concentrations below 1.5%, indicating a 'solution-like' behaviour in this entire concentration range, although systems above 0.8% were visually gel-like. It is proposed that the rigidity of the KC helices is responsible for the slow relaxation rates of the gel-like samples. At higher concentrations (beyond 1.6%) a frequency-temperature superposition was no longer possible. In contrast to KC, IC behaved as a typical viscoelastic gel with a very weak frequency dependence of the storage modulus at all temperatures. This indicates the existence of associations beyond simple entanglements for IC. Creep experiments performed at higher carrageenan concentrations in 0.1 M NaI further corroborated the differences in the viscoelastic behaviour between KC and IC.