Membrane Seal Research Articles

The importance of the degree of cataclasis in shear bands for fluid flow in porous sandstone, Provence, France

Determination of the membrane seal capacity of deformation bands is critical for managing geologic reservoirs in porous sandstones. In this study, we have analyzed a cataclastic shear-band network developed in uncemented porous sandstone in Provence, France. Geometrical analyses of the bands show significant differences between three types of bands (single strand, multistrand, and band cluster), sorted by their number of strands, their amount of shear displacement, and their thicknesses. At the microscopic scale, the image-analysis porosities and the grain-size distributions allow definition of three different types of microstructural deformation: damage zone, protocataclastic, and cataclastic. Whereas damage zone and protocataclastic deformations are observed in each type of band, cataclastic strands are observed in clusters and, sometimes, in multistrands. Cataclastic strands are characterized by a porosity reduction of 10 to 25% and a permeability reduction of three to five orders of magnitude compared to the host rock. Field observations of iron hydroxide precipitations around the bands suggest that cataclastic strands were membrane seals to water flow under vadose condition. This study therefore highlights the importance of the degree of cataclasis in shear bands as membrane seals to subsurface fluid flows in sandstone reservoirs.

Human telomerase reverse transcriptase and glucose-regulated protein 78 increase the life span of articular chondrocytes and their repair potential

BackgroundLike all mammalian cells, normal adult chondrocytes have a limited replicative life span, which decreases with age. To facilitate the therapeutic use of chondrocytes from older donors, a method is needed to prolong their life span.MethodsWe transfected chondrocytes with hTERT or GRP78 and cultured them in a 3-dimensional atelocollagen honeycomb-shaped scaffold with a membrane seal. Then, we measured the amount of nuclear DNA and glycosaminoglycans (GAGs) and the expression level of type II collagen as markers of cell proliferation and extracellular matrix formation, respectively, in these cultures. In addition, we allografted this tissue-engineered cartilage into osteochondral defects in old rabbits to assess their repair activity in vivo.ResultsOur results showed different degrees of differentiation in terms of GAG content between chondrocytes from old and young rabbits. Chondrocytes that were cotransfected with hTERT and GRP78 showed higher cellular proliferation and expression of type II collagen than those of nontransfected chondrocytes, regardless of the age of the cartilage donor. In addition, the in vitro growth rates of hTERT- or GRP78-transfected chondrocytes were higher than those of nontransfected chondrocytes, regardless of donor age. In vivo, the tissue-engineered cartilage implants exhibited strong repairing activity, maintained a chondrocyte-specific phenotype, and produced extracellular matrix components.ConclusionsFocal gene delivery to aged articular chondrocytes exhibited strong repairing activity and may be therapeutically useful for articular cartilage regeneration.

The effectiveness of total surface bearing compared to specific surface bearing prosthetic socket design on health outcomes of adults with a trans-tibial amputation: A systematic review.

Review objective The objective of this systematic review is to synthesise the best available evidence informing the effects of prosthetic socket design on outcomes of function, mobility, comfort and pain, energy expenditure, prosthetic and health related quality of life of adults with a trans-tibial amputation. Specifically, the review will consider studies evaluating the effects comparing the contemporary total surface bearing (TSB) socket design with the more traditional specific surface bearing (SSB) socket design Inclusion criteria Types of participants This review will consider adult trans-tibial amputees 18 years or older, with acquired or congenital limb loss irrespective of the aetiology, gender or the presence of co-morbidities using prostheses with either the contemporary total surface bearing (TSB) socket design or the traditional specific surface bearing (SSB) socket design. Each of the studies considered for inclusion will have documented study populations utilising both TSB and SSB socket designs within the study. Types of interventions The intervention of interest is the use of TSB socket. This is the ‘hydro static’ design or ‘limited load bearing’ often using silicon or gel liner interface, with suspension achieved via distal attachment of pin and lock or lanyard, expulsion valve and sleeve, vacuum assisted, integrated membrane seal or a combination of these. The comparative intervention will be the use of a SSB socket. This is often referred to as PTB, PTS, PTB-SC, PTK or KBM, typically utilising soft foam liner interface paired with supracondylar suspension. Types of outcomes This review will consider studies that include but will not be limited to the following outcomes: • Function and Mobility measured for example using Amputee Activity Score (AAS) or Locomotor Capabilities Index (LCI-5) or Timed Walk Tests (TWTs) or Step Activity Monitor (SAM); • Comfort and Pain measured for example using Socket Comfort Score (SCS) or Brief Pain Inventory (BPI); • Quality of Life including: Prosthetic Related Quality of Life (PRQoL) measured for example using Prosthetic Evaluation Questionnaire (PEQ) and Health Related Quality of Life (HRQoL) measured for example using Trinity Amputation & Prosthesis Experience Scales (TAPES) or Short Form 36 (SF-36); • Energy Expenditure measured for example using Physiological Cost Index (PCI).

Gas-tight coke-oven doors

The gas-tightness of coke-oven doors may be improved by means of a flexible membrane seal in which the edge of the membrane is pressed against the perimeter by a spring; gas-exhaust channels; and separation of the door’s welded housing from the lining module using a sealing frame. This follows from many years of operational experience at two coke batteries equipped exclusively with such doors. A direct-action gate is proposed. The use of such gas-tight doors at coke plants within the Commonwealth of Independent States is recommended.

MEMBRANE SEAL LEAKAGE IN NON‐FRACTURED CAPROCKS BY THE FORMATION OF OIL‐WET FLOW PATHS

Leakage through non‐fractured shaly caprock sequences is often envisioned as hydrocarbon percolation through water‐wet pore networks. Leakage by this process requires that the buoyancy of the hydrocarbon column overcomes the capillary entry pressure of the caprock pores. If it does not, then leakage through caprocks that are not hydrofractured depends on diffusion of hydrocarbons, which is an extremely slow process. As a result, the hydrocarbon residence time would be almost endless.We suggest a novel model for leakage through non‐fractured caprock shales. Two lines of laboratory‐based research motivate our suggestion. One of these addresses wettability alterations, while the other addresses pore‐scale fluid flow in the presence of capillary sealing. The latter approach has demonstrated that water can flow vertically through water‐wet oil reservoirs and further through caprocks, whereas the oil remains in the reservoir. This recognition is a consequence of the observation that residual water in the reservoir can be both continuous and mobile.We suggest that such flow of residual water through reservoirs and into caprocks can lead to the establishment of oil‐wet flow paths in membrane seals. This process takes place in a sequence of events that leads to wettability alterations in the seals. Such wettability alterations allow seals to leak by multiphase Darcy flow, which would hardly happen if the seals were water wet and with sufficiently small pore throats.One implication of this model is that capillary sealing will be effective only for a limited period of time. The model also predicts that residual oil saturation in the shales as a whole remains low, as the suggested flow paths only develop locally and develop narrow leakage pathways.

Gigaohm resistance membrane seals with stealth probe electrodes

Direct electrical access into the cell interior is required for low-noise recording of ion channel activity, yet conventional patch clamp techniques are destructive, leading to rapid cell death, while on-chip devices have poor seal resistances. Here we report chip-based nanoscale electrodes that nondestructively incorporate into biological membranes. These consist of a metallic post with a hydrophobic band that mimics transmembrane proteins, driving insertion into the lipid membrane and forming a tight seal at the electrode-membrane interface. We demonstrate spontaneous gigaohm seals with an average seal resistance of 3.8±1.9 GΩ using red blood cells, and show the nanoband is the key attribute for high resistances.

The Plug Domain of the SecY Protein Stabilizes the Closed State of the Translocation Channel and Maintains a Membrane Seal

Proteins are translocated across membranes through a channel that is formed by the prokaryotic SecY or eukaryotic Sec61 complex. The crystal structure of the SecY channel from M. jannaschii revealed a plug domain that appears to seal the channel in its closed state. However, the role of the plug remains unclear, particularly because plug deletion mutants in S. cerevisiae are functional. Here, we demonstrate that plug deletion mutants in E. coli SecY are also functional and even efficiently translocate proteins with defective or missing signal sequences. The crystal structures of equivalent plug deletions in SecY of M. jannaschii show that, although the overall structures are maintained, new plugs are formed. These lack many interactions that normally stabilize the closed channel, explaining why the channels can open for proteins with signal-sequence mutations. Our data show that the plug domain is required to maintain a closed state of the channel and suggest a mechanism for channel gating.

Effects of growth factors on heparin‐carrying polystyrene‐coated atelocollagen scaffold for articular cartilage tissue engineering

The specific aim of our investigation is to study the potential use of a collagen/heparin-carrying polystyrene (HCPS) composite extracellular matrix for articular cartilage tissue engineering. Here, we created a high-performance extracellular matrix (HpECM) scaffold to build an optimal extracellular environment using an HCPS we originally developed, and an atelocollagen honeycomb-shaped-scaffold (ACHMS-scaffold) with a membrane seal. This scaffold was coated with HCPS to enable aggregation of heparin-binding growth factors such as FGF-2 and TGF-beta1 within the scaffold. Three-dimensional culture of rabbit articular chondrocytes within the HpECM-scaffold and subsequent preparation of a tissue-engineered cartilage were investigated. The results showed remarkably higher cell proliferative activity within the HpECM-pretreated-FGF-2 scaffold and the sustenance of phenotype within the HpECM-pretreated-TGF-beta1 scaffold. It was thought that both FGF-2 and TGF-beta1 were stably immobilized in the HpEMC-scaffold since HCPS generated an extracellular environment similar to that of heparan sulfate proteoglycan within the scaffold. These results suggest that an ACHMS-scaffold immobilized with HCPS can be a HpECM for cartilage regeneration to retain the heparin-binding growth factors within the scaffolds.

Hydrodynamics and membrane seal capacity

AbstractThe impact of hydrodynamic groundwater movement on the capacity of seals is currently in debate. There is an extensive record of publication on seals analysis and a similar history on petroleum hydrodynamics yet little work addresses the links between the two. Understanding and quantifying the effects of hydrodynamic flow has important implications for calibrating commonly used seal capacity estimation techniques. These are often based on measurements such as shale gouge, clay smear or mercury porosimitry where membrane sealing is thought to occur. For standard membrane seal analysis, seal capacity is estimated by quantifying capillary pressure‐related measurements and calibrating them with a large observational database of hydrocarbon column heights and measured buoyancy pressures. The seal capacity estimation process has historically been adjusted to account for a number of different generic trapping geometries. We define the characteristics of these geometries from a hydrodynamics viewpoint in order to fine‐tune the seal capacity calibration process. From theoretical analyses of several simplified trapping geometries, it can be concluded that generally, the high pressure side of the seal should be used as the water pressure gradient with which to calculate buoyancy pressure. Secondly, trap geometries where hydrocarbon is reservoired on both sides of a fault are not useful for estimating across fault seal capacity.

Deregulation of the SecYEG Translocation Channel upon Removal of the Plug Domain

Previous studies have shown that the SecY plug is displaced from the center of the SecYEG channel during polypeptide translocation. The structural and functional consequences of the deletion of the plug are now examined. Both in vivo and in vitro observations indicate that the plug domain is not essential to the function of the translocon. In fact, deletion of the plug confers to the cell and to the membranes a Prl-like phenotype: reduced proton-motive force dependence of translocation, increased membrane insertion of SecA, diminished requirement for functional leader peptide, and weakened SecYEG subunit association. Although the plug domain does not seem essential, locking the plug in the center of the channel inactivates the translocon. Thus, the SecY plug is important to regulate the activity of the channel and to confer specificity to the translocation reaction. We propose that the plug contributes to the gating mechanism of the channel by maintaining the structure of the SecYEG complex in a compact closed state.

Seal capacity estimation from subsurface pore pressures

ABSTRACTA cap rock's capacity to seal hydrocarbons depends on its wettability and the sizes of the pore throats within the interconnected pore system that the leaking hydrocarbons must penetrate. These critical pore throat sizes are often poorly constrained in hydrocarbon exploration, partly because measurements of pore throat sizes have not been performed, and partly because pore throat measurements on a few individual samples in the cap rock may not be representative for the seal capacity of the top seal as a whole.To the contrary, the presence of formation overpressure can normally be estimated in drilled exploration targets. The presence of overpressure in reservoirs testifies to small pore throats in the cap rocks, as large pore throats will result in sufficiently high cap rock permeability to bleed off the overpressure.We suggest a stepwise procedure that enables quantification of top seal capacities of overpressured traps, based on subsurface pressure information. This procedure starts with the estimation of cap rock permeabilities, which are consistent with observed overpressure gradients across the top seals. Knowledge of burial histories is essential for these estimations. Relationships between pore throat size and permeability from laboratory experiments are then applied to estimate critical pore throat diameters in cap rocks. These critical pore throat diameters, combined with information of the physical properties of the pore fluids, are then used to calculate membrane seal capacity of cap rocks.Estimates of top seal capacity based on this procedure are rather sensitive to the vertical fluid velocity, but they are also to some extent sensitive to inaccuracies of the pore throat/permeability relationship, overpressure gradient, interfacial tensions between pore fluids, hydrocarbon density and water viscosity values. Despite these uncertainties, applications of the above‐mentioned procedure demonstrated that the mere presence of reservoir overpressures testifies to sufficient membrane seal capacity of cap rocks for most geological histories. Exempt from this statement are basins with rapid and substantial sediment compaction in the recent past.

Multi-disciplinary approach to fault and top seal appraisal; Pyrenees–Macedon oil and gas fields, Exmouth Sub-basin, Australian Northwest Shelf

The Pyrenees–Macedon (P–M) fields in the Exmouth Sub-basin of the Northern Carnarvon Basin, Australian Northwest Shelf are currently under-filled relative to available closure despite being a regional focal point for Cretaceous to recent charge. Late structural development of the P–M trap with respect to charge was thought to be the reason for under-filling. However, seismic amplitude anomalies and gas shows above the reservoir suggest vertical leakage may have controlled column heights. Hydrodynamic analysis of pressure data also suggests that faults separating the fields act as barriers to the migration of hydrocarbons and water, whilst faults within the Macedon Field do not. The reasons for hydrocarbon leakage and the difference in fault seal capacities are investigated by integrating field observations, analysis of pressure and stress data, the appraisal of top (mercury porosimetry measurements) and fault (Shale Gouge Ratios; SGR) membrane seal capacities, constraining geomechanical properties (top and fault seals) and well-based fracture analysis. The top seals are at a low risk of capillary failure, but vertical leakage is possible via dynamic failure along pre-existing faults and conductive fractures, and lateral leakage across reservoir against thief zone fault juxtapositions. The difference in observed fault seal capacities between different faults is explained by a combination of the spatial distributions of SGR and buoyancy pressure. The procedure presented delivers a robust description of the key risks concerning reservoir connectivity and the integrity and capacity of seals where static (geological timescale migration) and dynamic (tectonically related flow) conditions must be considered.

Capillary resistance and trapping of hydrocarbons: a laboratory experiment

Low permeability cap rocks retain oil by capillary forces when the pore throats of the seals are sufficiently small to prevent a flux of oil into the cap rock. In order to investigate the influence of aquifer overpressures on oil retention, water pressure was applied to a water-wet, highly permeable (1988 mD) core sample, which was oil-saturated to irreducible water saturation S wi and mounted with a low-permeability and water-wet membrane at the outlet. A water pressure difference of 0.5 MPa was applied across the core. This pressure was high enough to ensure fluid flow through the sample. The experiment was designed to see whether the water pressure would force oil through the membrane or if capillary forces at the sandstone–membrane interface would retain the oil, in which case water flow might take place in the (residual) water in the core and through the membrane. The experiment showed that oil was kept in place by capillary forces while water flowed through the core and the membrane. Accordingly, residual water can move through sandstones that are saturated to S wi . The experiments also demonstrated that the permeability associated with this residual water is high enough to prevent overpressures in the aquifer below the oil–water contact from pushing oil through a membrane seal. Thus, even for this highly permeable sandstone, the overpressure in the aquifer will not cause capillary seal failure.

Fault intersections as critical hydrocarbon leakage zones: integrated field study and numerical modelling of an example from the Timor Sea, Australia

Fault intersections are identified as important sites for hydrocarbon leakage from the Skua oil field in the Timor Sea, Australia. Integrated structural and fluid history data sets suggest that these fault intersections may be efficient and long-lived fluid conduits. Three-dimensional (3D) numerical modelling, based on fault patterns observed in the Skua Field, generated zones of high dilation in the vicinity of fault intersections during contraction, even at low bulk strain values. In nature, these dilational zones are likely to be sites of high structural permeability containing concentrated open fracture networks ideal for high fluid flux. The potential for fluid leakage from these zones may be further enhanced where low shear strain occurs due to mechanical locking at the fault intersection. Although not tested in the numerical experiments, fault gouge development is likely to be less extensive in these zones of low shear strain, reducing the probability of forming membrane seals. The modelling results support previously published charge and leakage history studies of the Skua Field and highlight the potential for large volumes of hydrocarbons to be lost where fault intersection zones breach the top seal. Fault intersections may therefore play a significant role in influencing trap integrity conditions in other areas.

Tissue engineering of the intervertebral disc with cultured annulus fibrosus cells using atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS scaffold).

The objective of the study was to investigate the regeneration of intervertebral discs after laser discectomy using tissue engineering procedures. Annulus fibrosus (AF) cells from the intervertebral discs of Japanese white rabbits were cultured in an atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS scaffold), to produce a high-density, three-dimensional culture for up to 3 weeks. Although the DNA content in the scaffold increased at a lower rate than that in the monolayer culture, expression of type II collagen and glycosaminoglycan accumulation in the scaffold were at higher levels than in the monolayer. The AF cells that had been cultured in the scaffold for 7 days were allografted into the lacunae of intervertebral discs of recipients (40 rabbits, 14-16 weeks old; average weight, 3.2 kg), whose nucleus pulposus (NP) had been vaporised with an ICG dye-enhanced laser. The allografted cultured AF cells survived and produced hyaline-like cartilage. Furthermore, the narrowing of the intervertebral disc space of the cell-containing scaffold insertion groups was significantly inhibited after 12 post-operative weeks.

An Experimental Study of the Regeneration of the Intervertebral Disc With an Allograft of Cultured Annulus Fibrosus Cells Using a Tissue-Engineering Method

Cultured annulus fibrosus cells within an atelocollagen honeycomb-shaped scaffold with a membrane seal were allografted into the lacunas of intervertebral discs of which the nucleus pulposus had been vaporized using an indocyanine green dye-enhanced laser. Regeneration of the intervertebral disc was assessed based on the viability and histologic status of the allografted annulus fibrosus cells, as well as the prevention of narrowing disc space. To study the regeneration of intervertebral disc after laser discectomy using tissue-engineering methods. Intervertebral disc is the most avascular tissue in the human body, and its ability to regenerate is as low as that of articular cartilage. When nucleotomy is carried out, little regeneration of the annulus fibrosus is observed; consequently, intervertebral disc degeneration is inevitable. Annulus fibrosus cells isolated from 20 Japanese white rabbits were labeled with a PKH-26 fluorescent dye and seeded within an atelocollagen honeycomb-shaped scaffold with a membrane seal. Annulus fibrosus cells cultured in atelocollagen honeycomb-shaped scaffold with a membrane seal for 1 week were allografted into the lacunas of intervertebral discs of recipient rabbit of which the nucleus pulposus had been vaporized using an ICG dye-enhanced laser. Soft radiograph photographs of the lumbar spine of these anesthetized rabbits were taken, the disc space measured, and the lumbar spine extracted 2, 4, 8, and 12 weeks after the operation. The proliferation of allografted annulus fibrosus cells with 5-bromo-2'-deoxyuridine/PKH-26 fluorescent labels was assessed using consecutive frozen sections, and safranin-O staining carried out for histologic evaluation. The allografted annulus fibrosus cells were viable and showed proliferation activity with a hyaline-like cartilage being produced. The narrowing of the intervertebral disc space of the cell translation group was significantly prevented, as shown, up to 12 postoperative weeks. The annulus fibrosus cells cultured in an ACHMS-scaffold were allografted into the lacunae of nucleus pulposus (obtained using laser vaporization), as well as the hole of annulus fibrosus (obtained by laser fiber insertion) of rabbit intervertebral discs. These cells were viable and showed cell proliferation in the disc tissues of recipients.

An integrated, quantitative approach to assessing fault-seal risk

Fault sealing is one of the key factors controlling hydrocarbon accumulations and trap volumetrics and can be a significant influence on reservoir performance during production. Fault seal is, therefore, a major exploration and production uncertainty. We introduce a systematic framework in which the geologic risk of faults trapping hydrocarbons may be assessed.A fault may seal if deformation processes have created a membrane seal, or if it juxtaposes sealing rocks against reservoir rocks, and the fault has not been reactivated subsequent to hydrocarbons charging the trap. It follows from this statement that the integrated probability of fault seal can be expressed as {1 − [(1 − a)(1 − b)]} (1 − c), where a, b, and c are the probabilities of deformation process sealing, juxtaposition sealing, and of the fault being reactivated subsequent to charge, respectively. This relationship provides an assessment of fault-seal risk that integrates results from the critical parameters of fault-seal analysis that can be incorporated into standard exploration procedures for estimating the probability for geologic success. The integrated probability of fault seal for each prospect can be visualized using the fault-seal risk web, which allows rapid comparison of success and failure cases through construction of prospect risk web profiles.The impact of uncertainty (U) and the value of information (VOI) for each aspect of fault sealing on the overall fault-seal risk may be determined via the construction of data webs and the relation U = [1 − {(nw) / n}] 100, where nw is the value given to each data web parameter and n is the number of data web components. For example, the data web components required to assess fault reactivation risk are the orientation and magnitude of the in-situ principal stresses, pore pressure, fault architecture, and the geomechanical properties of the fault.Risking of the Apollo prospect, Dampier subbasin, North West shelf, Australia is presented as a worked example. Fault-seal risking for the Apollo prospect has been conducted on 10- and 100-ft oil columns to allow integration with volumetric probabilistic statements. The critical parameter for fault-seal risking at the Apollo prospect is the ability of disaggregation zone faults (low shale gouge ratio fault gouge) to support increasingly large hydrocarbon columns. Evaluation of the individual components for Apollo fault sealing indicates a = 0.3 (10-ft column) and a = 0.1 (100-ft column), b = 0.2, and c = 0.1. The overall probability of the Apollo trap-bounding fault sealing a 10-ft oil column is 0.4 or 40% (seal condition moderately unlikely). The likelihood that the fault seals oil columns greater than 100 ft is 0.3 (seal condition unlikely). Data web error margins for the Apollo prospect are 20% (juxtaposition uncertainty), 26% (fault-rock process uncertainty), and 27% (fault reactivation uncertainty). Recalculating each parameter by its uncertainty, for a 10-ft oil column, the upper value of integrated fault-seal risk is 0.5 (seal condition intermediate), and the lower value is 0.3 (seal condition unlikely). The upper value of integrated fault-seal risk for a 100-ft oil column is 0.3 (seal condition unlikely), and the lower value is 0.2 (seal condition very unlikely). The variation in the Apollo final risk calculation reflects the lack of prospect-specific data. The greatest VOI benefit for Apollo fault-seal prospectivity is sedimentary architecture data.

A device for automated control of pipette internal pressure for patch-clamp recording

Formation of a high-resistance seal between the tip of a glass recording pipette and the membrane of the recorded cell is the crucial step in patch clamping, or whole cell recording with patch pipettes. Formation of the seal, and subsequent rupture of the membrane for whole cell recording, requires a specific sequence of changes in pipette internal hydrostatic pressure. Generating this sequence of pressure changes adds to the complexity of setting up, gaining proficiency, and performing experiments. Automation of routine pipette pressure manipulations would simplify seal formation, and benefit productivity. Here we describe a device that automates control of patch pipette internal pressure. Solenoid valves sequentially operated by manual switching, or external electronic control, automatically provide the necessary sequence of connections to the pipette interior. This greatly simplifies the operations performed to obtain membrane seals and whole cell recordings and improves standardization and reproducibility in patch recording.

An atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS-scaffold) for the culture of annulus fibrosus cells from an intervertebral disc.

The aim of this study was to investigate the possibility of using the atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS-scaffold) for the culture of annulus fibrosus (AF) cells in tissue engineering procedures of intervertebral disc repair. AF cells from the intervertebral discs of Japanese white rabbits were cultured for up to 3 weeks in the ACHMS-scaffold to allow a high density, three-dimensional culture. Although the DNA content in the scaffold increased at a lower rate than in the monolayer culture, scanning electron microscopy data showed that the scaffold was filled with the grown AF cells and produced extracellular matrix on day 21. The amount of type II collagen and its mRNA expression by the scaffold cultured cells were determined using Western blotting and Northern blotting analyses, respectively, and remained at a higher level than in the monolayer cultured cells. Furthermore, glycosaminoglycan (GAG) accumulation in the scaffold culture was at a higher level than in the monolayer culture. Western blot analysis for extracted proteoglycans from the scaffold culture also exhibited a much higher proteoglycan accumulation than the monolayer culture. These results indicate that the AF cells are able to grow and remain phenotypically stable in the scaffold.

Chloride and non-selective cation channels in unstimulated trout red blood cells.

1. The cell-attached and excised inside-out configurations of the patch-clamp technique were used to demonstrate the presence of two different types of ion channels in the membrane of trout red blood cells under isotonic and normoxic conditions, in the absence of hormonal stimulation. The large majority (93%) of successful membrane seals allowed observation of at least one channel type. 2. In the cell-attached mode with Ringer solution in the bath and Ringer solution, 145 mM KCl or 145 NaCl in the pipette, a channel of intermediate conductance (15-25 pS at clamped voltage, Vp = 0 mV) was present in 85% of cells. The single channel activity reversed between 5 and 7 mV positive to the spontaneous membrane potential. A small conductance channel of 5-6 pS and +5 mV reversal potential was also present in 62% of cells. 3. After excision into the inside-out configuration (with 145 mM KCl or NaCl, pCa 8 in the bath, 145 mM KCl or NaCl, pCa 3 in the pipette) the intermediate conductance channel was present in 439 out of 452 successful seals. This channel was spontaneously active in 90% of patches and in the other 10% of patches the channel was activated by suction. The current-voltage relationship showed slight inward rectification. The channel conductance was in the range 15-20 pS between -60 and 0 mV and increased to 25-30 pS between 0 and 60 mV, with a reversal potential close to zero. Substitution of K+ for Na+ in the pipette or in the bath did not significantly change the single channel conductance. Dilution of the bathing solution KCl concentration shifted the reversal potential towards the Nernst equilibrium for cations. Substitution of N-methyl-D-glucamine (NMDG) for K+ or Na+ in the bath almost abolished the outward current whilst the divalent cation Ca2+ permeated the channel with a higher permeability than K+ and Na+. Inhibition of channel openings was obtained with flufenamic acid, quinine, gadolinium or barium. Taken together these data demonstrate that the intermediate conductance channel belongs to a class of non-selective cation (NSC) channels. 4. In excised patches, under the same control conditions, the conductance of the small conductance non-rectifying channel was 8.6 +/- 0.8 pS (n = 12) between -60 and +60 mV and the reversal potential was close to 0 mV. This channel could be blocked by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) but not by flufenamic acid, DIDS, barium or gadolinium. Selectivity and substitution experiments made it possible to identify this channel as a non-rectifying small conductance chloride (SCC) channel.