Microencapsulation and chondrogenic differentiation of human mesenchymal progenitor cells from subchondral bone marrow in Ca-alginate for cell injection.

The application of stem cells is a promising therapeutic approach for cartilage regeneration. For cell therapies, a biocompatible injectable carrier, which improves retention and cell distribution and enables cell differentiation, is a prerequisite. In this study, Ca-alginate microcapsules containing human subchondral cortico-spongious progenitor cells were prepared and the chondrogenic differentiation potential was verified by real-time reverse transcription-polymerase chain reaction analysis of typical chondrogenic marker genes. The results confirmed that these cells were able to differentiate along the chondrogenic lineage when encapsulated in Ca-alginate microcapsules with a mean diameter of 600-700microm and stimulated with TGF-beta3. Chondrogenic marker genes type II collagen, aggrecan and cartilage oligomeric matrix protein were induced together with type I collagen, whereas adipogenic and osteogenic marker genes showed no induction over 14 days. After 28 days, proteoglycans and type II collagen were evident histochemically and immunohistochemically. Mechanical stability as well as permeability of Ca-alginate capsules were analysed over the course of cultivation and found to be qualified for stable cell immobilization and sufficient exchange of solutes. Therefore, from the cell biology point of view, Ca-alginate, an established hydrogel scaffold material is suited for regenerative therapies of cartilage defects based on the injection of progenitor cells.

Resources and uncertainties in evaluation of chemicals: basic methodological developments.

Conscious handling of uncertainty is critical in the assessment of chemicals, where the demand of knowledge is high compared to the available amount of resources. Consequently weighing between the use of resources and the acceptance of uncertainty has to be a transparent process and this sets up a challenge in relation to the application of mathematical methods. The maximum entropy principle is a useful paradigm for setting up prior information that is as non-informative as possible. It is shown how the use of partial ordering for deriving ranking results can be considered as a maximum entropy strategy in relation to the model structure uncertainty. In this way it seems possible to solve central parts of the problem of resource optimising risk assessment by combining the precautionary principle and screening before more detailed risk assessment is undertaken. The non-parametric approach of partial order technique further appears to be more transparent than most other ranking methodologies for complex systems. Because in the partial order techniques, no more or less known uncertainty will be hidden in the application of specific weighting factors and functional relationships among the criteria.

Pre-selection of flocculants by the LUMiFuge separation analyser 114.

This paper reports on lab-scale investigations in relation to pre-selection of flocculants for sludge dewatering with decanter centrifuges. Results obtained were compared with CST-measurements and discussed in relation to findings under field conditions. Experiments were carried out with sewage sludges of different origin and characteristics and a number of commercial flocculants. Kinetics of sedimentation and clarification were measured as well as the compression behaviour and shear sensitivity of sludge sediments. To measure flocculant performance stability against intensive shearing, total solids in the sludge cake obtained and dewaterability of the sludge cake during the first 20-50 s of centrifugation were compared. A screening test procedure was developed. Efficient flocculants should produce high residual total solids and good initial compressibility. Lab-scale investigations deliver more reliable results if the dynamic behaviour of the sludge under centrifugal acceleration is also investigated. The separation analyser LUMiFuge 114 can provide results about the compression behaviour of sludges in the range between 10 and 100 s. So far no other method or device is known which can deliver such results.

Selecting chemical substances for the UN-ECE POP protocol.

In 1998 the UN-ECE POP Protocol was signed. Sixteen substances are included in the protocol. They can be characterised as persistent, bioaccumulating and toxic organic substances prone to long-range atmospheric transport. The Dutch Ministry of Housing, Physical Planning and the Environment started a project to select possible candidates for the protocol. In the first phase of the project possible candidates were selected using the so-called 'PTB-database' applying the criteria from the protocol. From the 12 substances that met the criteria four substances were selected for which preliminary risk profiles were drafted: polychlorinated naphthalenes, dicofol, hexachlorobutadiene and pentachlorobenzene. These profiles are presented. Revised profiles have to be prepared for the UN-ECE LRTAP Ad hoc Expert Group on persistent organic pollutants (POPs). Ultimately, the process should lead to a proposal to include additional POPs to the UN-ECE POP Protocol.

Theoretical and experimental analysis of the sedimentation kinetics of concentrated red cell suspensions in a centrifugal field: determination of the aggregation and deformation of RBC by flux density and viscosity functions.

The flow properties of blood are mostly determined using various viscometric approaches, and described in terms of a shear rate or shear stress dependent apparent viscosity. The interpretation of results are rather difficult, especially at low shear rates when particle sedimentation and migration within the viscometer gap are significant. By contrast, analysing the separation process in concentrated RBC suspensions in a centrifugal field also yields information about the viscosity function, including particle-particle interaction and deformation parameters. In this paper, the sedimentation process is approached by means of the theory of kinematic waves and theoretically described by solving the corresponding one-dimensional quasi-linear partial differential equation based on viscosity/flow function as a function of volume concentration. The sedimentation kinetics of rigid spherical RBC suspended in saline and normal RBC suspended in Dx-saline solutions were investigated by means of a separation analyser (LUMiFuge 114). The instrument detects the light transmission over the total length of the cell containing the suspension. During centrifugation the analyser automatically determines the position of the particle free fluid/suspension interface or the sediment by means of a special algorithm. The data obtained with sedimentation of rigid spherical RBC at different volume concentrations demonstrate that, in the case of suspensions rotated in containers of constant cross section, there is good agreement between the theory of kinematic waves developed by Anestis and Schneider (1983) and the results of the experiments. Such good agreement was obtained even though a restrictive one-dimensional model was used to obtain the theoretically derived sedimentation time course. In addition, we describe an algorithm enabling the experimental determination of the viscosity and related flux density function to be made for any suspension. Through this approach, we investigated in detail the rheological behavior of suspended rigid spheres at low Reynolds numbers ranging from 10(-6) to 10(-3). The method here introduced also enabled us to investigate RBC suspensions with respect to the deformability and interactions of the cells by means of the separation analysis. Normal, rigid as well as aggregating RBC exhibited marked differences in the sedimentation kinetics, which were quantified by means of the flux and viscosity functions based on the theory of kinematic waves.

Oxygen radical generation of neutrophils: a reason for oxidative stress during marathon running?

Hematological parameters and blood markers that indicate oxidative stress, such as lipid peroxides (LPO), reduced and oxidized glutathione (GSH, GSSG), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), were measured in 18 marathon runners before, immediately after the race, and after 8 days of rest. In parallel, the oxygen radical generation of neutrophils (PMN) was measured by chemiluminescence in six randomly selected runners. After the race, a 4.4-fold enhanced PMN count and a 1.4-fold increased capacity to generate oxygen radicals of the PMN (2.20+/-0.38 vs. 3.12+/-0.69 arb. unit/10(6) cells) were found. Consequently, a 6.25-fold increased capacity to generate oxygen radicals of the post-run blood (7.26+/-1.3 vs. 45.40+/-10.3 arb. unit/ml blood) was calculated. This points to PMN as an important oxygen radical source established in the runners' blood, which could contribute to the oxidative stress indicated in the post-run blood by increased LPO (11.46+/-3.09 vs. 13.09+/-3.14 micromol/l plasma), GSSG (0.038+/-0.003 vs. 0.045+/-0. 005 mmol/l blood) and GSSG/GSH ratio (3.8+/-0.5 vs. 4.1+/-0.6%) and by decreased SOD (15.63+/-1.78 vs. 14.58+/-1.51 10(3)U/mmol Hb) and GSH-Px (485.1+/-107.1 vs. 434.9+/-101.7 U/mmol Hb). Despite the decline of the oxygen radical source during rest, the oxidative stress in the blood did not decrease in all runners.

The superposition of steady on oscillatory shear and its effect on the viscoelasticity of human blood and a blood-like model fluid.

To understand the pulsatility of human blood flow in vivo, it is necessary to separately investigate (1) steady shear and oscillatory flow, and (2) the superposition of steady shear flow on oscillatory flow performed under in vitro conditions. In this study a variable steady shear rate was superimposed in parallel on oscillatory shear at a constant frequency (0.5 Hz) for human blood (45% hematocrit), and an aqueous polyacrylamide polymer solution (AP 30E, concentration 300 ppm). The effect of superposition of the above two shear flows on the viscoelasticity of blood was more pronounced for the elastic (eta") than for the viscous (eta') component of viscoelasticity. With increasing superimposed shear rate, both eta' and eta" decreased, especially at the low shear region. This behavior can be explained by the viscoelastic properties of blood and the phenomenon of blood aggregation and disaggregation. Quantitatively, the dependence of the viscous component of complex viscosity on superimposed shear for both blood and polymer solution is described by a modified Carreau equation. The elastic component of complex viscosity decreased exponentially with increasing superimposed shear, and it is described by an exponential model.

[In vitro evaluation of hemolytic effects with use of infusion pumps with finger peristaltic action of blood pumping]. / In-vitro-Bewertung hämolytischer Effekte beim Einsatz von Infusions-pumpen mit Fingerperistaltik zur Blutförderung.

INTRODUCTION: Blood cells pumped by technical systems are exposed to non-physiological mechanical stress. Haemolysis or subhaemolytic damage may occur. Interaction between cells and the artificial material as well as the shear stress are the main reasons for this traumatisation. Up to now in vitro evaluation is the method of choice as no profound theory of mechanical cell damage has existed yet and, furthermore, haemolytic effects exhibit al large biological variability. METHODS: This study deals with the mechanically induced haemolysis due to blood pumping by infusion pumps (type INCA; Fresenius AG) in dependence on device related as well as blood preserve related (donor, storage time) factors. RBC count, MCV, MCHC, hematocrit, total and extracellular haemoglobin were obtained before and after pumping the blood sample. RESULTS: At a pump rate of 1000 ml/h, 0.06% of RBC (storage duration of 1-3 days) and 0.3% (storage duration of 34-36 days) were destroyed, respectively. Furthermore, it was shown that only gravity induced perfusion of the Intra-Air-system and the tubing system without any pumping lead to a minor, but significant elevation of extracellular haemoglobin concentration. In contrast, a volume flow rate of 2000 ml/h or the 10 times repeated pump process (1000 ml/h) increases haemolysis by only 27% or 520% respectively. CONCLUSION: It was concluded that any extrapolation from given experimental conditions to other ones must be performed with great caution due to the complex nature of the haemolysis process. Infusion pumps of the INCA-type stress RBC only slightly and may therefore be used for controlled blood transfusion.

In vitro testing of artificial heart valves: comparison between Newtonian and non-Newtonian fluids.

The in vitro testing of artificial heart valves is often performed with simple fluids like glycerol solutions. Blood, however, is a non-Newtonian fluid with a complex viscoelastic behavior, and different flow fields in comparable geometries may result. Therefore, we used different polymer solutions (Polyacrylamid, Xanthan gum) with blood-like rheological properties as well as various Newtonian fluids (water, glycerol solutions) in our heart valve test device. Hydrodynamic parameters of Björk-Shiley heart valves with a tissue annulus diameter (TAD) of 21-29 mm were investigated under aortic flow conditions. Major results can be summarized as follows. The mean systolic pressure differences depend on the model fluids tested. Closing time and closing volume are not influenced by the rheological behavior of fluids. These parameters depend on TAD and the pressure differences across the valve. In contrast, rheological behavior has a pronounced influence upon leakage flow and leakage volume, respectively. Results show furthermore that the apparent viscosity data as a function of shear rate are not sufficient to characterize the rheological fluid behavior relevant to hydrodynamic parameters of the heart valves investigated. Therefore, similarity in the yield curves of non-Newtonian test fluids mimicing blood is only a pre-requisite for a suitable test fluid. More information about the viscous and elastic component of the fluid viscosity is required, especially in geometries where a complex flow field exists as in the case of leakage flow.

Transient rheological behavior of blood in low-shear tube flow: velocity profiles and effective viscosity.

Velocity profiles of human blood flowing through vertical and horizontal glass tubes (25-100 microns ID) were measured as a function of time following a sudden reduction of wall shear stress (tau w) from a high value to values ranging from 2 to 100 mPa. Cell velocities at various radial positions were determined off-line from video recordings by digital image analysis. In vertical tubes, symmetric velocity profiles were obtained that developed increasing bluntness with time, particularly at lower tau w and in smaller tubes. In horizontal tubes, velocity profiles developed strong asymmetry as a function of time. Red blood cell (RBC) sedimentation was associated with uniform low flow velocities in the concentrating cell sediment, whereas faster flow and almost parabolic profiles were observed in the supernatant plasma region. Calculations of effective blood viscosity showed a decrease with time at low tau w in vertical tubes but an increase in horizontal tubes. The differences between profile shape and effective viscosity in vertical and horizontal tubes disappeared at tau w > 50 mPa. These findings are related to the cross-sectional distribution of RBC, which depends on RBC aggregation and sedimentation.

The mechanical properties of actin gels. Elastic modulus and filament motions.

To address large discrepancies reported in the literature, the viscoelastic properties of gels formed by purified actin filaments have been measured by five different techniques and five different instruments using actin preparations purified separately in four different laboratories. These measurements consistently showed that the elastic shear modulus of 2 mg/ml F-actin is on the order of several hundred pascals, and depends very strongly on the length of the filaments and on the history of the sample prior to measurement. Shortening of actin filaments with gelsolin and mechanical perturbations reduce the shear modulus to low values identical to some reported in the literature, indicating that such perturbations account for low shear moduli and poor responsiveness to filament modifying treatments reported previously. The structures of individual actin filaments within gels very similar or identical to those studied rheometrically were also examined by dynamic light scattering and fluorescence microscopy. Dynamic light scattering data were analyzed by a new method to confirm that actin filaments have no stable associations with each other and fluctuate in solution at a rate governed by the filament bending modulus or persistence length, determined to be approximately 10 microns. Fluorescence microscopy confirmed that applying even small shear stresses to F-actin can orient and rupture the filaments, and that in a minimally perturbed viscoelastic gel, long actin filaments are free to diffuse within a limit of constraints formed by their neighbors. These findings confirm that relatively isotropic F-actin networks are sufficiently strong to stabilize cells.

[Mechanical hemolysis caused by artificial organs--comparison of in vitro hemolysis studies and their application to in vivo conditions]. / Mechanische Hämolyse durch künstliche Organe--Vergleichbarkeit von In-vitro-Hämolyseuntersuchungen und ihre Ubertragbarkeit auf die Verhältnisse in vivo.

Changes of plasma concentration are often used for in vitro characterisation of the hemolytic potency of artificial organs and apparatus. Different indices of hemolysis are derived from Hb concentration, which, in general, depend on experimental conditions and cannot be compared quantitatively or used to describe the in vivo damage. In this paper we propose a similarity number called "lysis number" that is independent of experimental conditions. It describes the probability for a single blood cell to be completely destroyed in a single pass through the corresponding artificial assist system. The concept is based on the steps: 1. Definition of "lysis number" as an index of hemolytic performance of artificial organs or implants. 2. Description of more complex hemolytic damaging processes (different hemolytic steps) that may be in series or parallel and definition of an effective lysis number. 3. Experimental in vitro estimation of each of the processes in consecutive steps. 4. Calculation of total hemolysis of the complex system using the linkage rules. 5. Application to in vivo by an appropriate differential equation in RBC mas taking into account mechanically-induced hemolysis rate, survival time of normal RBC and erythropoetic generation rate.

Platelet aggregation inhibiting and anticoagulant effects of oligoamines, XXIII: Influence of the oligoamine RE 1492 on the deformability of red blood cells.

The influence of the oligoamine RE 1492 on the deformability of Red Blood Cells (RBCs) was measured with the micropipette aspiration technique and the capillary rigidometer. The maximal cell flow resistance (MCFR) and the apparent elastic membrane shear modulus mu were increased by 45% at RE 1492 concentrations > 10 mumol/L. There was a decrease of the surface/volume ratio of 6% at RE 1492 concentrations of 25 mumol/L. The qualitative analysis of the polypeptide pattern of membranes and extracted membranes of human RBCs suggests that the reduction of deformability is due to an increased affinity of skeletal proteins to the cytosolic part of the membrane. RE 1492 caused a decrease of osmotic hemolysis by 80% at concentrations of 8 mumol/L.

The viscosity of red blood cell membranes in patients with beta-thalassaemia.

The purpose of this work was to study the viscoelastic behaviour of the red blood cell membrane (RBCM) in cells from patients with beta-thalassaemia and to investigate whether the precipitated haemoglobin, which is one of the main features of thalassaemic syndromes, influences the membrane viscosity. RBCM viscosities were determined using the micropipet aspiration method. A negative pressure of about 50 Pa was applied in steps at the membrane surface so as to cause partial aspiration of the cell and the entry process was analyzed automatically by a TV-line analyzer. This analysis enabled estimation of the characteristic times (tau 1, tau 2) and corresponding values of the viscosity (eta 1, eta 2). Results were as follows: eta 1 = (1.87 +/- 0.55) microNs/m and eta 2 = (51.42 +/- 20) microNs/m for erythrocytes from normal donors; eta 1 = (3.97 +/- 0.98) microNs/m and eta 2 = (110.40 +/- 35) microNs/m for erythrocytes from patients. Inclusions (Heinz Bodies) were produced artificially in normal cells and the characteristic times (tau 1, tau 2) and corresponding viscosities (eta 1, eta 2) derived in the same manner. For three types of RBC containing increasing numbers of inclusions, the values were: eta 1 = (3.26 +/- 1.70) microNs/m and eta 2 = (77.33 +/- 46.96) microNs/m; eta 1 = (4.21 +/- 1.49) microNs/m and eta 2 = (129.60 +/- 47.90) microNs/m; eta 1 = (7.93 +/- 2.62) microNs/m and eta 2 = (206.60 +/- 93.19) microNs/m. It is concluded that the association of inclusion bodies with the membrane, either in disease or through artificial production, increases the membrane viscosity.

[Sedimentation analyser--calibration and testing of a newly developed device for recording separation behavior of blood and other dispersed systems]. / Sedimentationsanalysator--Eichung und Testung eines neuentwickelten Gerätes zur Aufzeichnung des Entmischungsverhaltens von Blut und anderen dispersen Systemen.

A computer-aided sedimentation analyser is described, which can be used for the continuous monitoring of the separation of blood and other dispersed systems by recording the separation-dependent infrared transmission in the range between 100 xg to 700 xg. Up to eight samples can be measured simultaneously within a short period of time, and only a small amount of suspension is required (15 minutes, 350 microliters). For centrifugal acceleration more than 100 xg and a haematocrit range of between 0.2 and 0.7, the evolution of the height of the plasma column (HPC) over time is expressed in non-linear regression form by HPC(t) = HPC infinity*(1-e-kt) + c. The separation constant k is influenced by plasma viscosity, haematocrit, aggregability and erythrocyte deformability, is directly proportional to centrifugal acceleration, and declines in hyperbolic fashion with increasing haematocrit between 300 xg and 650 xg. The separation constant is closely related to the maximum velocity which, in fact, represents the sensitive parameter of separation. Thus, the sedimentation analyser can be applied as an alternative to the traditional measurement of erythrocyte sedimentation rate according to Westergren.

[Metabolic and rheologic changes in long-term hypothermia of erythrocyte concentrates over 15 weeks]. / Metabolische und rheologische Veränderungen bei der hypothermen Langzeitlagerung von Erythrozytenkonzentraten über 15 Wochen.

With a multiple washing procedure in a chloride ion-free citrate-phosphate-glucose-adenine additive solution or with storage of red blood cells in a large volume of this solution, erythrocytes can be stored for 15 weeks at 4 degrees C. After 12 weeks' storage in this chloride-free solution the red blood cell parameters (2,3-DPG, glucose, lactose) measured are as good as stored erythrocytes resuspended in SAG-Mannitol after 21 days. Rheological parameters (morphology, filterability) show a maintenance of deformability of such preserved cells up to 84 days of storage. The preservation of erythrocytes by this method has the advantage that beside the hypothermic long-term storage (potential second aHIV screening of blood donors) the blood is of better quality in the first weeks of storage.

[Rejuvenation of autologous blood preserved by washing in glucose- adenine- phosphate- and citrate-containing solution]. / Rejuvenation von Eigenblutkonserven durch Waschen mit glukose-adenin-phosphat-citrathaltiger Lösung.

Outdated red cell concentrates (RCC-SAGM) were washed with a chloride-free, glucose-adenine-phosphate-citrate preservation solution. After resuspension of the red cells in this solution a hypothermic storage for additional 63 days was performed. Quality parameters (2,3-DPG, glucose, lactose, free hemoglobin, pH, chloride, pO50, shape quality index, filtration index) of the preserved red blood cells became normal within 7-11 days after the rejuvenation procedure. The data prove that a rejuvenation of outdated blood by a simple washing procedure allows a post-storage time of 21 days.

Improved red cell quality after erythroplasmapheresis with MCS-3P.

The cell separator MCS-3P is an apheresis system offering the flexibility to collect standardized red blood cells, plasma, and/or platelets from one donor. Two different programs were used for the red cell apheresis--RBCP (collection of one unit of red cells and two units of plasma) and RBCPS (one unit of red cells and one unit of plasma). The quality of the red cell concentrates (RCC resuspended in SAG-Mannitol) during the storage time of 42 days was measured by biochemical (ATP, 2,3-DPG, pH, free Hb, free potassium, glucose, lactose, p50, hemoglobin derivatives) and rheological (morphological index, filtration/rigidity index) parameters. The donation time with 53 donors was 20 min for 355 ml RCC-SAGM and 440 ml plasma and 7 min for 335 ml RCC-SAGM and 239 ml plasma. The donor tolerance was analogous to plateletpheresis or plasmapheresis. Twenty units of the RCC-SAGM were in-line filtered within 6 or 24 hours after donation. The results obtained for red blood cell storage are at least as good as with standard collection (free hemoglobin, free potassium, glucose, lactose, hemolysis) or better (ATP, 2,3-DPG, p50, hemoglobin derivatives, filtration/rigidity index) owing to prevention of collection lesion. All blood preparations were sterile after storage (red cells 42 days, plasma after freezing). The erythroplasmapheresis with MCS-3P can be especially recommended for application in an autologous blood program because the application of autologous blood donation in hospitals is often limited by the preconditions of component separation. The erythroplasmapheresis data with MCS-3P are encouraging for the development of a new blood collection methodology.

[In vitro modification of plasma viscosity and erythrocyte aggregation by four plasma substitutes]. / Zur In-vitro-Beeinflussung von Plasmaviskosität und Erythrozytenaggregation durch vier Plasmaersatzmittel.

We tested the haemorheological effects of the addition of 3.5% oxypolygelatine, 10% dextran 40, 6% dextran 75 or 5% albumin, respectively, to fresh donor blood of 25 healthy young persons. We examined the aggregation and the viscosity of substituent plasma mixtures in such various, but constant volume relations as may be present during intravenous infusion. Albumin reduced viscosity and aggregation, but especially dextran 75 increased both parameters significantly.

[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.