Investigation of endotoxin adsorption with polyether polymer alloy dialysis membranes

Endotoxin (ET) in the dialysate is known to be adsorbed by dialysis membranes made of polyether polymer alloy (PEPA) and polymethylmethacrylate (PMMA). In the present study, the effect of polyvinylpyrrolidone (PVP) localization of the PEPA dialysis membrane on the adsorption of ET was investigated. The compounding of PVP in the PEPA membrane was changed, and hydrophobic membrane in both blood side and dialysate side, and hydrophilic membrane in only the blood side were used. Adsorption was evaluated by filling the contaminated dialysate inside and outside the membrane after priming with physiological saline, and determining the ET concentration in the blood side and dialysate side of dialysis membrane during the 240 min period from the start of filling the contaminated dialysate. With the PEPA membranes investigated, ET was significantly adsorbed to the hydrophobic side and was not adsorbed to the blood side of hydrophilic type membrane. These results suggest that in addition to electrostatic action attributable to the compounding of hydrophilic agent PVP to the dialysis membrane, the distribution of PVP that was compounded and the potential of the membrane itself may cause differences in adsorption of ET.     

Poly(N-vinyl-2-pyrrolidone) elution from polysulfone dialysis membranes by varying solvent and wall shear stress

Some dialysis patients are treated with post-hemodiafiltration (HDF); the blood viscosity of the patients who undergo post-HDF is higher than that of the patients who undergo conventional hemodialysis. This study aims to evaluate poly(N-vinyl-2-pyrrolidone) (PVP) elution from PSf dialysis membranes by varying solvents and high wall shear stress caused by blood viscosity. We tested three commercial membranes: APS-15SA (Asahi Kasei Kuraray), CX-1.6U (Toray) and FX140 (Fresenius). Dialysate and blood sides of the dialyzers were primed with reverse osmosis (RO) water and saline. RO water, saline and dextran solution (2.9 and 5.8 mPa s) were circulated in the blood side. The amount of eluted PVP was determined by 0.02 N iodometry. The hardness and adsorption force of human serum albumin (HSA) on the membrane surfaces were measured by the atomic force microscope. When wall shear stress was increased using dextran, the amount of PVP eluted by the 2.9 mPa s solution equaled that eluted by the 5.8 mPa s solution with APS-15SA and CX-1.6U sterilized by gamma rays. The amount of PVP eluted by the 5.8 mPa s solution was higher than that eluted by the 2.9 mPa s solution with FX140 sterilized by autoclaving. The wall shear stress increased the PVP elution from the surface, hardness and adsorption force of HSA. Sufficient gamma-ray irradiation is effective in decreasing PVP elution.     

New generation ceramic membranes have the potential of removing endotoxins from dialysis water and dialysate

Poor water properties, use of concentrated bicarbonate, and biofilm growth in pipes and storage tanks often cause dialysis water and dialysate contamination with bacteria and endotoxins. High-flux dialysis with bicarbonate may favor endotoxin transfer from the dialysate into the blood exposing patients to serious short-and long-term side effects. Ultrafiltration across hydrophobic synthetic membranes effectively removes endotoxins from dialysis water by combined filtration and adsorption. However, repeated sterilization worsens the membrane separation properties,and limits their use. Ceramic membranes are generally more resistant to harsh operating conditions than polymeric membranes, and may represent an alternative for endotoxin removal. Previously, we proved that the ceramic membranes commercially available at that time were not retentive enough to ensure production of endotoxin-free dialysis water. In this paper, we investigated the endotoxin removal capacity of new generation commercial ceramic membranes with nominal molecular weight cut-off down to 1,000. In dead-end filtration, all investigated membranes produced water meeting, the European standards, or close to,when challenged with low endotoxin concentrations, but only one membrane type succeeded at high endotoxin concentrations. In cross-flow filtration, none produced water meeting the European standard. Moreover, sterilization and rinsing procedures altered the separation properties of two out of three membrane types.     

Membrane fouling and dialysate flow pattern in an internal filtration-enhancing dialyzer

For efficient removal of large molecular weight solutes by dialysis, several types of internal filtration-enhancing dialyzers (IFEDs) are commercially available. However, in a pressure-driven membrane separation process (i.e., filtration), membrane fouling caused by adhesion of plasma proteins is a severe problem. The objective of the present study is to investigate the effects of internal filtration on membrane fouling based on the membrane's pure-water permeability, diffusive permeability, and sieving coefficient. Hemodialysis experiments were performed with two different dialyzers, IFEDs and non-IFEDs. Local membrane fouling in each dialyzer was evaluated by measuring the pure-water permeability, the diffusive permeability, and the sieving coefficient of native membranes and membranes treated with bovine blood. The effects of packing ratio on dialysate flow pattern were also evaluated by measuring the time required for an ion tracer to reach electrodes placed in the dialyzers. In the IFED, membrane fouling caused by protein adhesion is increased because of enhanced internal filtration only at the early stage of dialysis, and this fouling tends to occur only near the dialysate outlet port. However, enhanced internal filtration has little effect on measured membrane transfer parameters.     

Membrane plasma fractionation: effect of the surface area

The influence of the surface area on the performance of plasma filters for dead-end mode of filtration is presented in this paper. Theoretical analysis of the dead-end filtration was performed and verified experimentally (using ENKA cellulose-diacetate PF-100 membranes) in respect to beta-lipoprotein. The theoretical model allows to optimize the course of the transmembrane pressure during plasma fractionation procedure in dependence of the surface area, initial concentration of macromolecules, total volume of the feed and membrane structure. The results indicate that the surface area effect is an important factor in the operation of membrane plasma fractionation and should be considered in the design of the plasma fractionation filter.     

Longer storage of dialyzers increases elution of poly(N-vinyl-2-pyrrolidone) from polysulfone-group dialysis membranes

The objective of this study was to evaluate the effect of protracted storage of dialyzers on the amount of poly(N-vinyl-2-pyrrolidone) (PVP) eluted from polysulfone-group dialysis membranes. We tested five dialysis membranes: APS-15SA (Asahi Kasei Kuraray, wet), CX-1.6U (Toray, moist), FX140 (Fresenius, dry), PES-15Sα (Nipro, dry), and FDX-150GW (Nikkiso, wet). Each dialyzer was stored for 1, 3, 14, and 18 months after sterilization. The dialysis-fluid side compartment was primed with reverse osmosis (RO) water at 500 mL/min for 5 min at 310 K. The blood side compartment was primed with RO water at 200 mL/min for 5 min at 310 K. Finally, 1 L RO water was circulated through the blood side compartment at 200 mL/min for 4 h at 310 K. Eluted PVP was determined by use of the iodine method, using 0.02 N iodine solution. PVP was mainly eluted from wet-type dialyzers during priming. Thus, the standard 5 min priming of the wet-type dialyzer according to the maker manual inhibits PVP elution during circulation. PVP was eluted in the dialysis-fluid side of the moist-type dialyzer during priming but no PVP was eluted in the blood side. PVP was mainly eluted from dry-type dialyzers during circulation. We recommend more than the standard 5 min priming, particularly for dry-type dialyzers stored for protracted periods, because 5 min insufficient to inhibit PVP elution during circulation.     

Technical evaluation of newly-developed inorganic membranes for plasma fractionation

Constant transmembrane pressure experiments were made by crossflow filtration to clarify sieving characteristics of microporous glass membranes for plasma fractionation. The distribution of pore diameters is more limited in the microporous glass membranes than in currently utilized synthetic polymer membranes. The filtration resistance of the concentration polarization layer is the dominant factor in plasma fractionation. Proteins are separated more sharply with a higher wall shear rate because of destruction of the concentration polarization layer formed on membrane surfaces. Plasma fractionation using a microporous glass membrane with a pore diameter of 15 nm may allow separation of albumin and IgG at higher wall shear rates. Cascade filtration techniques using microporous glass membranes with various pore diameters may be suitable for plasma fractionation.     

Amount of adsorbed albumin loss by dialysis membranes with protein adsorption

Polymethylmethacrylate (PMMA) membrane is the first synthetic polymeric hollow fiber used in dialyzers that is known to adsorb β₂-microglobulin. Polyester polymer alloy (PEPA), a blend of two polymers, i.e., polyarylate and polyethersulfone, is another dialysis membrane material with adsorption characteristics. In this study, the adsorption and permeation characteristics of BG-1.6PQ (PMMA) and FLX-15GW (PEPA) dialyzers were investigated by performing ultrafiltration experiments using chymotrypsinogen (molecular weight 25 000) and albumin (molecular weight 66 000) as test solutes. Although PMMA and PEPA had the same sieving coefficient for chymotrypsinogen at steady state, PMMA showed approximately 20% higher fractional adsorption than PEPA under the same initial concentrations. The fractional adsorption for albumin was approximately 20% in PEPA regardless of the ultrafiltration flow rate. The fractional adsorption for albumin in PMMA, however, increased as the ultrafiltration flow rate increased and reached 50%–60% after 10 h. Since PEPA has two skins, one inside and one outside the hollow fiber, proteins may have been adsorbed mainly by these two layers. However, since PMMA is a uniform membrane and since the higher the ultrafiltration flow rate, the higher the fractional adsorption found in PMMA, adsorption may be the result of the occlusion of the dense structure of the membrane. The amount of albumin loss is often clinically evaluated by measuring the amount of permeated albumin in the dialysate. However, when dialyzers with adsorption characteristics are examined, the loss by adsorption should also be taken into account.     

Microscopy of internal structures of Sendai virus associated with the cytoplasmic surface of host membranes

The cytoplasmic surface (PS) of the plasma membrane of cells infected with Sendai virus was studied by immunofluorescence microscopy and freeze-drying electron microscopy. After cells had been attached to glass coverslips, they were subjected to a jet stream of physiological buffer which sheared off the upper portion of each cell, leaving the attached membrane with the PS exposed. This uncapping maneuver permitted direct examination of internal virus-specific elements associated with the inner surface of the host cell. At a stage of infection at which viral budding occurs, strands of nucleoprotein (RNP) were observed to be attached to the PS of plasma membranes. The sites at which RNP was adherent to the membrane were modified by virus-specific particles arranged in orthogonal patterns. The presence of the same crystalline structures in the hydrophobic domain of freeze-fractured membranes indicated that they were inserted into the inner lipid leaflet. The spatial association of the surface glycoprotein spikes and the internal RNP with this crystalline structure suggests its special relationship if not identity with the internal viral matrix (M) protein. The possible significance of the localization and crystalline nature of this structural element with respect to viral morphogenesis, hemolytic and cell-fusing activities is discussed. In contrast to the foregoing changes observed in the infected cell, no detectable viral antigens were found on the PS of normal cells to which exogenous virions had been fused. Absence of internalized antigen from the PS under these circumstances could indicate that infectious viral components are processed by the potential host cell in a manner which differs from what is observed with human erythrocytes. In the latter instance internalized antigens after fusion of virus to the cell remain associated with the PS.     

Differences in the adsorption of nafamostat mesilate between polyester-polymer alloy and polysulfone membranes

We previously experienced severe clot formation in a polyester-polymer alloy (PEPA) dialyzer and hemodialysis (HD) circuit with nafamostat mesilate (NM) as an anticoagulant. The possibility of NM adsorption to the PEPA membrane was taken into consideration, but there was not enough information. In the present study, we evaluated differences in the adsorption of NM between a PEPA membrane (FDX-120 GW, Nikkiso, Tokyo, Japan) and two different polysulfone membranes (FX-140, Fresenius Medical Care, Tokyo, Japan; NV-15U, Toray Medical, Tokyo, Japan). We calculated the NM concentration by measuring absorbance at 241 nm using a spectrometer. NM adsorption was evaluated in three ways. First, we evaluated NM adsorption to hollow fibers. Then, we passed an NM solution through dialyzers and evaluated its adsorption in a single-pass examination. Finally, we circulated an NM solution in an HD circuit using a blood pump and evaluated NM adsorption. In all the experiments, NM adsorption to the PEPA membrane was greater than that to the polysulfone membranes examined. In the blood pump experiment, the estimated adsorption quantities of NM to the PEPA membrane and the FX-140 and NV-15U polysulfone membranes were 12.0 ± 0.1, 1.0 ± 0.1, and 4.1 ± 0.4 mg/m², respectively. NM adsorption was confirmed, especially in the early phase, and the PEPA membrane adsorbed greater amounts of NM than the polysulfone membranes. We should pay attention to the choice of dialyzer as well as the appropriate dose of NM administration during the preparation of HD circuits.     

Development of vitamin E-modified polysulfone membrane dialyzers

Oxygen radicals have recently been attracting close attention because of their involvement in tissue damage and their close relationship to various clinical conditions. It has been suggested that hemodialysis increases oxidative stress, triggering the development of complications such as atherosclerosis and dialysis-related amyloidosis. We recently developed a dialyzer containing a highly functional polysulfone membrane on which vitamin E had been bonded (PS-ViE). The present study was undertaken to evaluate the biocompatibility of this membrane and to conduct other experiments on the membrane in vitro. Human blood was dialyzed with minidialyzers (300–600 cm² membrane area) made of PS-ViE, cellulose, or untreated polusulfone (PS), and the effects of the dialyzers on complements (C3a, C4a, and C5a), cytokines (IL-1β and IL-8), and granulocyte elastase as well as their anti-oxidative activity were investigated (n = 6). The effect of PS-ViE on complement activation and its effects on cytokines were comparable to those of PS membrane, whereas granulocyte elastase following dialysis with the PS-ViE membrane tended to be lower than that seen with PS membrane. The effects of PS-ViE-induced methemoglobin, lipid peroxide, and oxygen radicals were significantly less than those of PS membrane, indicating the antioxidative activity of PS-ViE. Vitamin E-modified polysulfone membrane dialyzers were found to have favorable effects on the immune system and to express antithrombotic and antioxidative effects.     

Computational modeling of effects of mechanical shaking on hemodynamics in hollow fibers

Introduction: Blood-membrane interaction during hemodialysis develops a secondary protein layer on the dialysis membrane surface, resulting in reduction of hemodialyzer performance. Wall shear stress at the surface of the hollow-fiber membrane is one of the determinant factors able to influence dialysis efficiency. Shaking of hemodialyzer during treatment could increase the wall shear stress of the membrane, which could enhance hemodialyzer performance. Methods: In this study, hemodynamic changes in hollow fibers were analyzed using computational fluid dynamics software for various shaking conditions of hemodialyzer (longitudinal, transverse, rotational motions). Results: Longitudinal motion induced reverse flow, while transverse motion induced symmetric swirling inside the hollow fiber. During rotational motions, nonuniform vortices were developed according to the rotational radius of the hollow fiber. These changes in flow pathlines induced by different shaking profiles increased the relative motion of blood, transmembrane pressure, and wall shear stress on dialysis membrane surfaces. Both longitudinal and transverse shaking profiles showed a linear relationship between shaking velocity (the product of amplitude and frequency) and wall shear stress. Conclusion: Performance of hemodialyzer can be enhanced with simple mechanical shaking motions, and optimal shaking profiles for clinical application can be investigated and predicted with the computational fluid dynamics model proposed in this study.     

Asymmetric triacetate membrane keeps high water flux during ultrafiltration: in vitro study

Membrane fouling is a primary challenge encountered during the administration of hemodialysis (HD) and hemodiafiltration (HDF). A high-flux membrane is suitable for dialyzer reuse, since it is used repeatedly. Water flux is a benchmark used to assess the effectiveness of the dialysis membrane during treatment and it is usually evaluated to determine whether membrane fouling has occurred. Polysulfone (PS) membrane has good biocompatibility and solute permeability; however, polyethersulfone (PES) is often used as a hemodiafilter membrane because of better hydrophilicity compared to PS. We evaluated water flux across hemodiafilters using newly developed asymmetric triacetate (ATA) and PES as conventional membranes in vitro. Water flux of across ATA and PES membranes significantly decreased 30 min after the start of the experiments and thereafter showed stabilization. Water flux across the ATA membrane consistently showed significantly higher values of greater than 100 mL/m²/h/mmHg, compared to lower values observed across the PES membrane. These results suggest that the ATA membrane has a potential use not only for HDF, but also for long-time therapies of HD and HDF.     

Influence of membranes on generation of beta 2 M and release of leukocyte lysosomal enzymes

Normal leukocyte functional capacity was investigated by evaluation of phagocytosis of opsonised yeast cells in a radiometric test system. After incubation with dialysis membranes (different cellulosic membranes, polysulfon membrane (PS), polymethylmetacrylate membrane (PMMN), the phagocytosis index, expressed as percent decrease with respect to initial values without membrane, decreased by 10%-25%. The most pronounced effect was observed with PS, cuprophane, modified cellulose and PMMA. The results are not related to differences in the viability of PMN during the test procedure; dead PMN amounted to about 4-6.5%. A significant increase in beta-NAG and beta-Gluc activities was released in the supernatants of the phagocytosis suspensions. This increase activity can be explained by the phagocytosis of PMN but it was not influenced by membrane contact. There was no influence of membrane contact or phagocytosis activity of PMN on the beta 2 M concentration in the supernatant demonstrating that no in vitro generation during incubation with either membrane exists.     

Force spectroscopy study of the adhesion of plasma proteins to the surface of a dialysis membrane: role of the nanoscale surface hydrophobicity and topography

A mechanochemical study of the process of adhesion of plasma proteins to the surface of dialysis membranes was carried out with a scanning force microscope (SFM) in the force spectroscopy mode. Three representative blood plasma proteins (fibronectin, fibrinogen, and albumin) covalently were grafted to a SFM probe, and the adhesion forces of these proteins to cellulosic and synthetic dialysis membranes were measured. The experiment was tailored to apply a controlled load on the protein molecules adsorbed onto the surface in order to simulate the squeezing forces exerted on them during blood filtration. The de-adhesion forces, measured using this new approach for studying the interaction between a protein and dialysis membranes, suggest that the membrane's topography, at a nanometer scale, plays a critical role in the adhesion process. This result was strongly supported by parallel experiments performed on a flattened glass surface with the same dominant hydrophilic character as dialysis membranes. In contrast, a hydrophobic polystyrene surface led to de-adhesion forces at least one order of magnitude greater, overwhelming any possible shape recognition process between the protein molecules and the surface.     

成骨细胞的体外剪切应力加载实验的变形分析及信号转导区域探讨

目的 根据已有体外培养鼠成骨细胞的参数实验数据,估算剪切应力加载实验中细胞整体剪切形变,借以研究细胞的主要转导区域.方法 计算过程采用黏弹性力学理论,对细胞运用了标准黏弹性模型,并简化其膜所受剪切力为均匀.结果 细胞剪切力产生的细胞变形大约是引起成骨细胞相同生物学响应的拉伸加载变形的十分之一.结论 从细胞总的力学刺激生物学响应来看,剪切应力加载实验中细胞的整体变形所产生的力学转导是可以忽略的,主要转导区域在承受剪切应力的细胞膜.     

切应力对内皮细胞膜流动性的影响

目的：探讨切应力对内皮细胞膜流动性的影响。方法：选择１５ｄｙｎｅ／ｃｍ＾２、５０ｄｙｎｅ／ｃｍ＾２、３００ｄｙｎｅ／ｃｍ＾２三种切应力,分别模拟大中动脉处,大中动脉分叉处及病理状态下的切应力,以体外培养的人脐静脉内皮细胞作为实验对象,分别测定三组切应力作用０ｈ、１ｈ、２ｈ、４ｈ、６ｈ、１０ｈ的内皮细胞膜流动性。结果：切应力可降低内皮细胞膜液动性,且切应力越大,作用越强。切应力作用４ｈ,内皮细胞膜流     

Characterization of membrane materials and membrane coatings for bioreactor units of bioartificial kidneys

The bioreactor unit of bioartificial kidneys contains porous membranes seeded with renal cells. For clinical applications, it is mandatory that human primary renal proximal tubule cells (HPTCs) form differentiated epithelia on the membranes. Here, we show that HPTCs do not grow and survive on a variety of polymeric membrane materials. This applies also to membranes consisting of polysulfone/polyvinylpyrrolidone (PSF/PVP), which have been used in the bioreactor unit of bioartificial kidneys after coating with an extracellular matrix (ECM). Our data reveal that coating with just an ECM does not sufficiently improve HPTC performance on non-HPTC-compatible membrane materials. On the other hand, we have characterized the effects of a variety of surface treatments and coatings, and found that double coating with 3,4-dihydroxy-l-phenylalanine and an ECM markedly improves HPTC performance and results in the formation of differentiated epithelia on PSF/PVP membranes. We have also synthesized alternative membrane materials, and characterized membranes consisting of polysulfone and Fullcure. We found that these membranes sustain proper HPTC performance without the need for surface treatments or coatings. Together, our data reveal that the materials that have been previously applied in bioartificial kidneys are not suitable for applications with HPTCs. This study elucidates the types of membrane materials and coatings that are favorable for the bioreactor unit of bioartificial kidneys.     

Surface Modification of Ceramic‐Supported Polyethersulfone Membranes by Interfacial Polymerization for Reduced Membrane Fouling

Summary: This study reports a simple and effective route for the hydrophilic surface modification of ceramic‐supported polyethersulfone membranes by synthesizing a poly(vinyl alcohol)/polyamide composite thin surface layer with an interfacial polymerization method. The fabricated membranes were characterized with XPS, SEM, and contact angle measurements, and the effects of hydrophilic surface modification on the membrane flux and oil rejection in the treatment of oil‐in‐water (O/W) microemulsions were experimentally studied. All the characterizations and filtration results showed that, the hydrophilic surface modification was achieved successfully and consequently reduced the membrane fouling effectively. The presented route is valuable for developing robust membranes with a low level of membrane fouling in the separation of O/W microemulsions.

Hydrophilic surface modification resulted in enhanced membrane performance.     

Fluid shear induced endothelial cell detachment from glass - influence of adhesion time and shear stress

In this study, human umbilical vein and human saphenous vein endothelial cells were seeded on glass and exposed to fluid shear in a parallel-plate flow chamber. Cell retention, morphology and migration were studied as a function of shear stress and of adhesion time prior to exposure to shear. Three-hour and 24-h adhesion times gave rise to comparable cell retention values after 2 h of flow for both cell types. Cell retention decreased from 85 to 20% as shear stress increased from 88 to 264 dynes cm⁻² (8.8 to 26 Pa). Mean spreading areas decreased after the onset of flow, but subsequently stabilized to plateau values, which were smaller at higher shear stresses. Shape factors increased faster to higher values as cells were exposed to higher shear stresses, without any obvious preference in orientation of the cells with respect to the direction of flow. Migration was unidirectional with flow and linear with time. Migration was faster for cells which had adhered for 24 h than for cells which had adhered for 3 h and was accompanied by the presence of fibrillar structures left behind on the surface upstream of migrating cells. It is concluded that after 3 h adhesion to glass, cells have adhered with an adhesion strength that does not substantially increase during the next 21 h. However, during this time changes in cell-substratum interactions seem to occur judging by the differences in, e.g., migration rates.