Dynamics of diffusivity and pressure drop in flow-through and parallel-flow bioreactors during tissue regeneration

In this study, transport characteristics in flow-through and parallel-flow bioreactors used in tissue engineering were simulated using computational fluid dynamics. To study nutrient distribution and consumption by smooth muscle cells colonizing the 100 mm diameter and 2-mm thick scaffold, effective diffusivity of glucose was experimentally determined using a two-chambered setup. Three different concentrations of chitosan-gelatin scaffolds were prepared by freezing at -80°C followed by lyophilization. Experiments were performed in both bioreactors to measure pressure drop at different flow rates. At low flow rates, experimental results were in agreement with the simulation results for both bioreactors. However, increase in flow rate beyond 5 mL/min in flow-through bioreactor showed channeling at the circumference resulting in lower pressure drop relative to simulation results. The Peclet number inside the scaffold indicated nutrient distribution within the flow-through bioreactor to be convection-dependent, whereas the parallel-flow bioreactor was diffusion-dependent. Three alternative design modifications to the parallel-flow were made by (i) introducing an additional inlet and an outlet, (ii) changing channel position, and (iii) changing the hold-up volume. Simulation studies were performed to assess the effect of scaffold thickness, cell densities, and permeability. These new designs improved nutrient distribution for 2 mm scaffolds; however, parallel-flow configuration was found to be unsuitable for scaffolds more than 4-mm thick, especially at low porosities as tissues regenerate. Furthermore, operable flow rate in flow-through bioreactors is constrained by the mechanical strength of the scaffold. In summary, this study showed limitations and differences between flow-through and parallel-flow bioreactors used in tissue engineering.     

Flow rates in perfusion bioreactors to maximise mineralisation in bone tissue engineering in vitro

In bone tissue engineering experiments, fluid-induced shear stress is able to stimulate cells to produce mineralised extracellular matrix (ECM). The application of shear stress on seeded cells can for example be achieved through bioreactors that perfuse medium through porous scaffolds. The generated mechanical environment (i.e. wall shear stress: WSS) within the scaffolds is complex due to the complexity of scaffold geometry. This complexity has so far prevented setting an optimal loading (i.e. flow rate) of the bioreactor to achieve an optimal distribution of WSS for stimulating cells to produce mineralised ECM. In this study, we demonstrate an approach combining computational fluid dynamics (CFD) and mechano-regulation theory to optimise flow rates of a perfusion bioreactor and various scaffold geometries (i.e. pore shape, porosity and pore diameter) in order to maximise shear stress induced mineralisation. The optimal flow rates, under which the highest fraction of scaffold surface area is subjected to a wall shear stress that induces mineralisation, are mainly dependent on the scaffold geometries. Nevertheless, the variation range of such optimal flow rates are within 0.5–5 mL/min (or in terms of fluid velocity: 0.166–1.66 mm/s), among different scaffolds. This approach can facilitate the determination of scaffold-dependent flow rates for bone tissue engineering experiments in vitro, avoiding performing a series of trial and error experiments.     

Mathematical modelling of fibre-enhanced perfusion inside a tissue-engineering bioreactor

We develop a simple mathematical model for forced flow of culture medium through a porous scaffold in a tissue-engineering bioreactor. Porous-walled hollow fibres penetrate the scaffold and act as additional sources of culture medium. The model, based on Darcy's law, is used to examine the nutrient and shear-stress distributions throughout the scaffold. We consider several configurations of fibres and inlet and outlet pipes. Compared with a numerical solution of the full Navier-Stokes equations within the complex scaffold geometry, the modelling approach is cheap, and does not require knowledge of the detailed microstructure of the particular scaffold being used. The potential of this approach is demonstrated through quantification of the effect the additional flow from the fibres has on the nutrient and shear-stress distribution.     

Stereocomplex formation by enantiomeric poly(lactic acid) graft-type phospholipid polymers for tissue engineering

A porous scaffold as a cell-compatible material was designed and prepared using a phospholipid copolymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate, and enantiomeric macromonomers, the poly(L-lactic acid) (PLLA) macromonomer, and poly(D-lactic acid) (PDLA) macromonomer. On the basis of the wide-angle X-ray diffraction and differential scanning calorimetry measurements, the formation of a stereocomplex between the PLLA and PDLA segments of the copolymer was observed on the porous scaffold. The porous structure was prepared by a sodium chloride leaching technique, and the pore was linked to the scaffold. The pore size was confirmed by scanning electron microscopy and found to be ca. 200 microm. These observations suggest that the porous scaffold makes it possible to produce cell-compatible materials, which may involve the following advantages for tissue engineering: (i) cell compatibility using phospholipid copolymer, (ii) adequate cell adhesion by poly(lactic acid), and (iii) complete disappearance of scaffold by dissociation of stereocomplex. The cell experiment using the porous scaffold will be the next subject and reported in a forthcoming paper.     

Open carbon frameworks - a search for optimal geometry for hydrogen storage

Properties of a new class of hypothetical high-surface-area porous carbons (open carbon frameworks) have been discussed. The limits of hydrogen adsorption in these carbon porous structures have been analyzed in terms of competition between increasing surface accessible for adsorption and the lowering energy of adsorption. From an analysis of an analytical model and simulations of adsorption the physical limits of hydrogen adsorption have been defined: (i) higher storage capacities in slit-shaped pores can be obtained by fragmentation/truncation of graphene sheets into nano-metric elements which creates surface areas in excess of 2600 m²/g, the surface area for infinite graphene sheets; (ii) the positive influence of increasing surface area is compensated by the decreasing energy of adsorption in the carbon scaffolds of nano-metric sizes; (iii) for open carbon frameworks (OCF) built from coronene and benzene molecules with surface areas 6500 m² g^-1, we find an impressive excess adsorption of 75–110 g?H₂/kg C at 77 K, and high storage capacity of 110–150 g?H₂/kg C at 77 K and 100 bar; (iv) the new OCF, if synthesized and optimized, could lead to required hydrogen storage capacity for mobile applications.     

Flow modeling in a novel non-perfusion conical bioreactor

We have developed a bioreactor vessel design which has the advantages of simplicity and ease of assembly and disassembly, and with the appropriately determined flow rate, even allows for a scaffold to be suspended freely regardless of its weight. This article reports our experimental and numerical investigations to evaluate the performance of a newly developed non-perfusion conical bioreactor by visualizing the flow through scaffolds with 45 degrees and 90 degrees fiber lay down patterns. The experiments were conducted at the Reynolds numbers (Re) 121, 170, and 218 based on the local velocity and width of scaffolds. The flow fields were captured using short-time exposures of 60 microm particles suspended in the bioreactor and illuminated using a thin laser sheet. The effects of scaffold fiber lay down pattern and Reynolds number were obtained and correspondingly compared to results obtained from a computational fluid dynamics (CFD) software package. The objectives of this article are twofold: to investigate the hypothesis that there may be an insufficient exchange of medium within the interior of the scaffold when using our non-perfusion bioreactor, and second, to compare the flows within and around scaffolds of 45 degrees and 90 degrees fiber lay down patterns. Scaffold porosity was also found to influence flow patterns. It was therefore shown that fluidic transport could be achieved within scaffolds with our bioreactor design, being a non-perfusion vessel. Fluid velocities were generally same of the same or one order lower in magnitude as compared to the inlet flow velocity. Additionally, the 90 degrees fiber lay down pattern scaffold was found to allow for slightly higher fluid velocities within, as compared to the 45 degrees fiber lay down pattern scaffold. This was due to the architecture and pore arrangement of the 90 degrees fiber lay down pattern scaffold, which allows for fluid to flow directly through (channel-like flow).     

Modeling evaluation of the fluid-dynamic microenvironment in tissue-engineered constructs: a micro-CT based model

Natural cartilage remodels both in vivo and in vitro in response to mechanical stresses, hence mechanical stimulation is believed to be a potential tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis in engineered cartilage constructs. The quantification of the hydrodynamic environment is a condition required to study the biochemical response to shear of 3D engineered cell systems. We developed a computational model of culture medium flow through the microstructure of a porous scaffold, during direct- perfused culture. The 3D solid model of the scaffold micro-geometry was reconstructed from 250 micro-computed tomography (micro-CT) images. The results of the fluid dynamic simulations were analyzed at the central portions of the fluid domain, to avoid boundary effects. The average, median and mode shear stress values calculated at the scaffold walls were 3.48, 2.90, and 2.45 mPa respectively, at a flow rate of 0.5 cm(3)/min, perfused through a 15 mm diameter scaffold, at an inlet fluid velocity of 53 microm/s. These results were compared to results estimated using a simplified micro-scale model and to results estimated using an analytical macro-scale porous model. The predictions given by the CT-based model are being used in conjunction with an experimental bioreactor model, in order to quantify the effects of fluid-dynamic shear on the growth modulation of tissue-engineered cartilage constructs, to potentially enhance tissue growth in vitro.     

A multiphase model for chemically- and mechanically- induced cell differentiation in a hollow fibre membrane bioreactor: minimising growth factor consumption

We present a simplified two-dimensional model of fluid flow, solute transport, and cell distribution in a hollow fibre membrane bioreactor. We consider two cell populations, one undifferentiated and one differentiated, with differentiation stimulated either by growth factor alone, or by both growth factor and fluid shear stress. Two experimental configurations are considered, a 3-layer model in which the cells are seeded in a scaffold throughout the extracapillary space (ECS), and a 4-layer model in which the cell–scaffold construct occupies a layer surrounding the outside of the hollow fibre, only partially filling the ECS. Above this is a region of free-flowing fluid, referred to as the upper fluid layer. Following previous models by the authors (Pearson et al. in Math Med Biol, 2013, Biomech Model Mechanbiol 1–16, 2014a, we employ porous mixture theory to model the dynamics of, and interactions between, the cells, scaffold, and fluid in the cell–scaffold construct. We use this model to determine operating conditions (experiment end time, growth factor inlet concentration, and inlet fluid fluxes) which result in a required percentage of differentiated cells, as well as maximising the differentiated cell yield and minimising the consumption of expensive growth factor.     

Production of PEX protein from QM7 cells cultured in polymer scaffolds in a Taylor–Couette bioreactor

In recent decades, many practical applications were developed with regard to the Taylor–Couette device, for example, reaction, filtration, extraction and bioreactor. In this study, the Taylor–Couette bioreactor was used to culture cells seeded in a biodegradable porous scaffold and produce PEX protein. Two different cell lines (NIH/3T3 and QM7) were seeded into PLGA sponges, which were fabricated using a solvent-free supercritical gas foaming method, and then cultured in the Taylor–Couette bioreactor. Cell proliferation was characterized using Quant-iT™ PicoGreen^® dsDNA assay and the results indicated that high mass transfer rate in the Taylor–Couette bioreactor enhanced cell proliferation. Qualitative distribution of live/dead cells was characterized using LIVE/DEAD^® Viability/Cytotoxicity assay and SEM and the results showed that cells cultured in static control mainly proliferated on the outer surface while the cells of Taylor-vortex bioreactor group could penetrate into the scaffold. The production yield of PEX protein, from QM7 cells transfected with pM9PEX, was quantified using PEX ELISA and the results showed a much higher PEX mass per scaffold for bioreactor than the control. As such, there is potential for the use of Taylor–Couette bioreactor in the mass production of PEX protein.     

Macro-scale topology optimization for controlling internal shear stress in a porous scaffold bioreactor

Shear stress is an important physical factor that regulates proliferation, migration, and morphogenesis. In particular, the homeostasis of blood vessels is dependent on shear stress. To mimic this process ex vivo, efforts have been made to seed scaffolds with vascular and other cell types in the presence of growth factors and under pulsatile flow conditions. However, the resulting bioreactors lack information on shear stress and flow distributions within the scaffold. Consequently, it is difficult to interpret the effects of shear stress on cell function. Such knowledge would enable researchers to improve upon cell culture protocols. Recent work has focused on optimizing the microstructural parameters of the scaffold to fine tune the shear stress. In this study, we have adopted a different approach whereby flows are redirected throughout the bioreactor along channels patterned in the porous scaffold to yield shear stress distributions that are optimized for uniformity centered on a target value. A topology optimization algorithm coupled to computational fluid dynamics simulations was devised to this end. The channel topology in the porous scaffold was varied using a combination of genetic algorithm and fuzzy logic. The method is validated by experiments using magnetic resonance imaging readouts of the flow field.     

Regulation of autocrine fibroblast growth factor‐2 signaling by perfusion flow in 3D human mesenchymal stem cell constructs

Perfusion bioreactor systems play a crucial role in mitigating nutrient limitation as well as providing biomechanical stimuli and redistributing regulatory macromolecules that influence human mesenchymal stem cells (hMSC) fate in three‐dimensional (3D) scaffolds. As fibroblast growth factor‐2 (FGF‐2) is known to regulate hMSC phenotype, understanding the role of autocrine FGF‐2 signaling in the 3D construct under the different perfusion flow provides important insight into an optimal bioreactor design. To investigate FGF‐2 signaling inhibition in hMSC cultured in the porous poly(ethylene terephthalate) (PET) scaffolds perfused under two flow configurations, PD173074, an FGFR1 inhibitor, was added in growth media after 7 day of pre‐culture and its impact on hMSC proliferation and clonogenicity during the subsequent 7 days of cultivation was analyzed. Compared with control constructs in growth media, the addition of PD173074 resulted in significant reduction in hMSC proliferation and colony formation in both constructs with a more dramatic reduction in the parallel flow constructs. The results demonstrate that autocrine FGF‐2 plays a significant role in 3D scaffold and suggest modulation of the perfusion flow in the bioreactor as a strategy to influence autocrine actions and cell fate in the 3D scaffold. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

In silico multi‐scale model of transport and dynamic seeding in a bone tissue engineering perfusion bioreactor

Computer simulations can potentially be used to design, predict, and inform properties for tissue engineering perfusion bioreactors. In this work, we investigate the flow properties that result from a particular poly‐L ‐lactide porous scaffold and a particular choice of perfusion bioreactor vessel design used in bone tissue engineering. We also propose a model to investigate the dynamic seeding properties such as the homogeneity (or lack of) of the cellular distribution within the scaffold of the perfusion bioreactor: a pre‐requisite for the subsequent successful uniform growth of a viable bone tissue engineered construct. Flows inside geometrically complex scaffolds have been investigated previously and results shown at these pore scales. Here, it is our aim to show accurately that through the use of modern high performance computers that the bioreactor device scale that encloses a scaffold can affect the flows and stresses within the pores throughout the scaffold which has implications for bioreactor design, control, and use. Central to this work is that the boundary conditions are derived from micro computed tomography scans of both a device chamber and scaffold in order to avoid generalizations and uncertainties. Dynamic seeding methods have also been shown to provide certain advantages over static seeding methods. We propose here a novel coupled model for dynamic seeding accounting for flow, species mass transport and cell advection‐diffusion‐attachment tuned for bone tissue engineering. The model highlights the timescale differences between different species suggesting that traditional homogeneous porous flow models of transport must be applied with caution to perfusion bioreactors. Our in silico data illustrate the extent to which these experiments have the potential to contribute to future design and development of large‐scale bioreactors. Biotechnol. Bioeng. 2013; 110: 1221–1230. © 2012 Wiley Periodicals, Inc.     

Modeling of flow‐induced shear stress applied on 3D cellular scaffolds: Implications for vascular tissue engineering

Novel tissue‐culture bioreactors employ flow‐induced shear stress as a means of mechanical stimulation of cells. We developed a computational fluid dynamics model of the complex three‐dimensional (3D) microstructure of a porous scaffold incubated in a direct perfusion bioreactor. Our model was designed to predict high shear‐stress values within the physiological range of those naturally sensed by vascular cells (1–10 dyne/cm²), and will thereby provide suitable conditions for vascular tissue‐engineering experiments. The model also accounts for cellular growth, which was designed as an added cell layer grown on all scaffold walls. Five model variants were designed, with geometric differences corresponding to cell‐layer thicknesses of 0, 50, 75, 100, and 125 µm. Four inlet velocities (0.5, 1, 1.5, and 2 cm/s) were applied to each model. Wall shear‐stress distribution and overall pressure drop calculations were then used to characterize the relation between flow rate, shear stress, cell‐layer thickness, and pressure drop. The simulations showed that cellular growth within 3D scaffolds exposes cells to elevated shear stress, with considerably increasing average values in correlation to cell growth and inflow velocity. Our results provide in‐depth analysis of the microdynamic environment of cells cultured within 3D environments, and thus provide advanced control over tissue development in vitro. Biotechnol. Bioeng. 2010; 105: 645–654. © 2009 Wiley Periodicals, Inc.     

Modeling and design of optimal flow perfusion bioreactors for tissue engineering applications

Perfusion bioreactors have been used in different tissue engineering applications because of their consistent distribution of nutrients and flow-induced shear stress within the tissue-engineering scaffold. A widely used configuration uses a scaffold with a circular cross-section enclosed within a cylindrical chamber and inlet and outlet pipes which are connected to the chamber on either side through which media is continuously circulated. However, fluid-flow experiments and simulations have shown that the majority of the flow perfuses through the center. This pattern creates stagnant zones in the peripheral regions as well as in those of high flow rate near the inlet and outlet. This non-uniformity of flow and shear stress, owing to a circular design, results in limited cell proliferation and differentiation in these areas. The focus of this communication is to design an optimized perfusion system using computational fluid dynamics as a mathematical tool to overcome the time-consuming trial and error experimental method. We compared the flow within a circular and a rectangular bioreactor system. Flow simulations within the rectangular bioreactor are shown to overcome the limitations in the circular design. This communication challenges the circular cross-section bioreactor configuration paradigm and provides proof of the advantages of the new design over the existing one.     

Laboratory-scale biofiltration of acrylonitrile by <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> DAP 96622 in a trickling bed bioreactor

Acrylonitrile (ACN), a volatile component of the waste generated during the production of acrylamide, also is often associated with aromatic contaminants such as toluene and styrene. Biofiltration, considered an effective technique for the treatment of volatile hydrocarbons, has not been used to treat volatile nitriles. An experimental laboratory-scale trickling bed bioreactor using cells of Rhodococcus rhodochrous DAP 96622 supported on granular activated carbon (GAC) was developed and evaluated to assess the ability of biofiltration to treat ACN. In addition to following the course of treatability of ACN, kinetics of ACN biodegradation during both recycle batch and open modes of operation by immobilized and free cells were evaluated. For fed-batch mode bioreactor with immobilized cells, almost complete ACN removal (>95%) was achieved at a flow rate of 0.1 μl/min ACN and 0.8 μl/min toluene (TOL) (for comparative purposes this is equivalent to 6.9 mg l^?1 h^?1 ACN and 83.52 mg l^?1 h^?1 TOL). In a single-pass mode bioreactor with immobilized cells, at ACN inlet loads of 100–200 mg l^?1 h^?1 and TOL inlet load of ~400 mg l^?1 h^?1, with empty bed retention time (EBRT) of 8 min, ACN removal efficiency was ~90%. The three-dimensional structure and characteristics of the biofilm were investigated using confocal scanning laser microscopy (CSLM). CLSM images revealed a robust and heterogeneous biofilm, with microcolonies interspersed with voids and channels. Analysis of the precise measurement of biofilm characteristics using COMSTAT^® agreed with the assumption that both biomass and biofilm thickness increased along the carbon column depth.     

A Novel Modular Bioreactor to In Vitro Study the Hepatic Sinusoid

We describe a unique, versatile bioreactor consisting of two plates and a modified commercial porous membrane suitable for in vitro analysis of the liver sinusoid. The modular bioreactor allows i) excellent control of the cell seeding process; ii) cell culture under controlled shear stress stimulus, and; iii) individual analysis of each cell type upon completion of the experiment. The advantages of the bioreactor detailed here are derived from the modification of a commercial porous membrane with an elastomeric wall specifically moulded in order to define the cell culture area, to act as a gasket that will fit into the bioreactor, and to provide improved mechanical robustness. The device presented herein has been designed to simulate the in vivo organization of a liver sinusoid and tested by co-culturing endothelial cells (EC) and hepatic stellate cells (HSC). The results show both an optimal morphology of the endothelial cells as well as an improvement in the phenotype of stellate cells, most probably due to paracrine factors released from endothelial cells. This device is proposed as a versatile, easy-to-use co-culture system that can be applied to biomedical research of vascular systems, including the liver.     

A prediction of cell differentiation and proliferation within a collagen–glycosaminoglycan scaffold subjected to mechanical strain and perfusive fluid flow

Mesenchymal stem cell (MSC) differentiation can be influenced by biophysical stimuli imparted by the host scaffold. Yet, causal relationships linking scaffold strain magnitudes and inlet fluid velocities to specific cell responses are thus far underdeveloped. This investigation attempted to simulate cell responses in a collagen–glycosaminoglycan (CG) scaffold within a bioreactor. CG scaffold deformation was simulated using μ-computed tomography (CT) and an in-house finite element solver (FEEBE/linear). Similarly, the internal fluid velocities were simulated using the afore-mentioned μCT dataset with a computational fluid dynamics solver (ANSYS/CFX). From the ensuing cell-level mechanics, albeit octahedral shear strain or fluid velocity, the proliferation and differentiation of the representative cells were predicted from deterministic functions. Cell proliferation patterns concurred with previous experiments. MSC differentiation was dependent on the level of CG scaffold strain and the inlet fluid velocity. Furthermore, MSC differentiation patterns indicated that specific combinations of scaffold strains and inlet fluid flows cause phenotype assemblies dominated by single cell types. Further to typical laboratory procedures, this predictive methodology demonstrated loading-specific differentiation lineages and proliferation patterns. It is hoped these results will enhance in-vitro tissue engineering procedures by providing a platform from which the scaffold loading applications can be tailored to suit the desired tissue.     

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity¹. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes². However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia^3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries^5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)⁷ for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.     

Spectrally decomposed dark-to-light transitions in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Photosynthetic activity and respiration share the thylakoid membrane in cyanobacteria. We present a series of spectrally resolved fluorescence experiments where whole cells of the cyanobacterium Synechocystis sp. PCC 6803 and mutants thereof underwent a dark-to-light transition after different dark-adaptation (DA) periods. Two mutants were used: (i) a PSI-lacking mutant (ΔPSI) and (ii) M55, a mutant without NAD(P)H dehydrogenase type-1 (NDH-1). For comparison, measurements of the wild-type were also carried out. We recorded spectrally resolved fluorescence traces over several minutes with 100 ms time resolution. The excitation light was at 590 nm so as to specifically excite the phycobilisomes. In ΔPSI, DA time has no influence, and in dichlorophenyl-dimethylurea (DCMU)-treated samples we identify three main fluorescent components: PB–PSII complexes with closed (saturated) RCs, a quenched or open PB–PSII complex, and a PB–PSII ‘not fully closed.’ For the PSI-containing organisms without DCMU, we conclude that mainly three species contribute to the signal: a PB–PSII–PSI megacomplex with closed PSII RCs and (i) slow PB → PSI energy transfer, or (ii) fast PB → PSI energy transfer and (iii) complexes with open (photochemically quenched) PSII RCs. Furthermore, their time profiles reveal an adaptive response that we identify as a state transition. Our results suggest that deceleration of the PB → PSI energy transfer rate is the molecular mechanism underlying a state 2 to state 1 transition.     

Effects of Na+ on the Predominant K+ Channel in the Tonoplast of Chara: Decrease of Conductance by Blocks in 100 Nanosecond Range and Induction of Oligo- or Poly-subconductance Gating Modes

We present three mechanisms by which Na⁺ inhibits the open channel currents of the predominant K⁺ channel in the tonoplast of Chara corallina: (i) Fast block, i.e., short (100 ns range) interruptions of the open channel current which are determined by open channel noise analysis, (ii): Oligo-subconductance mode, i.e., a gating mode which occurs preferentially in the presence of Na⁺; this mode comprises a discrete number (here 3) of open states with smaller conductances than normal, and (iii): Polysubconductance mode, i.e., a gating mode with a nondiscrete, large number (>30) of states with smaller conductances than the main open channel conductance. This novel mode has also been observed only in the presence of Na⁺. Received: 16 November 1999/Revised: 8 February 2000