Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.     

Growth rate control of adherent bacterial populations

We report a novel in vitro method which, through application of appropriate nutrient limitations, enables growth rate control of adherent bacterial populations. Exponentially growing cells are collected by pressure filtration onto cellulose acetate membranes. Following inversion into the bases of modified fermentors, membranes and bacteria are perfused with fresh medium. Newly formed and loosely attached cells are eluted with spent medium. Steady-state conditions (dependent upon the medium flow rate) at which the adherent bacterial biomass is constant and proportional to the limiting nutrient concentrations are rapidly achieved, and within limits, the growth rate is proportional to the medium flow rate. Scanning electron microscopic studies showed that such populations consist of individual cells embedded within an extracellular polymer matrix.     

Growth rate control of adherent bacterial populations.

We report a novel in vitro method which, through application of appropriate nutrient limitations, enables growth rate control of adherent bacterial populations. Exponentially growing cells are collected by pressure filtration onto cellulose acetate membranes. Following inversion into the bases of modified fermentors, membranes and bacteria are perfused with fresh medium. Newly formed and loosely attached cells are eluted with spent medium. Steady-state conditions (dependent upon the medium flow rate) at which the adherent bacterial biomass is constant and proportional to the limiting nutrient concentrations are rapidly achieved, and within limits, the growth rate is proportional to the medium flow rate. Scanning electron microscopic studies showed that such populations consist of individual cells embedded within an extracellular polymer matrix.     

Probing surface structures of Shewanella spp. by microelectrophoresis

Long-range electrostatic forces substantially influence bacterial interactions and bacterial adhesion during the preliminary steps of biofilm formation. The strength of these forces depends strongly on the structure of the bacterium surfaces investigated. The latter may be addressed from appropriate analysis of electrophoretic mobility measurements. Due to the permeable character of the bacterium wall and/or surrounding polymer layer, bacteria may be regarded as paradigms of soft bioparticles. The electrophoretic motion of such particles in a direct-current electric field differs considerably from that of their rigid counterparts in the sense that electroosmotic flow takes place around and within the soft surface layer. Recent developments of electrokinetic theories for soft particles now render possible the evaluation of the softness degree (or equivalently the hydrodynamic permeability) from the raw electrokinetic data. In this article, the electrophoretic mobilities of three Shewanella strains (MR-4, CN32, and BrY) presenting various and well-characterized phenotypes of polymer fringe are reported over a wide range of pH and ionic strength conditions. The data are quantitatively analyzed on the basis of a rigorous numerical evaluation of the governing electrostatic and hydrodynamic equations for soft particles. It is clearly shown how the peculiar surface structures of the bacteria investigated are reflected in their electrohydrodynamic properties.     

Assembly of collagen into microribbons: effects of pH and electrolytes

Collagen represents the major structural protein of the extracellular matrix. Elucidating the mechanism of its assembly is important for understanding many cell biological and medical processes as well as for tissue engineering and biotechnological approaches. In this work, conditions for the self-assembly of collagen type I molecules on a supporting surface were characterized. By applying hydrodynamic flow, collagen assembled into ultrathin ( approximately 3 nm) highly anisotropic ribbon-like structures coating the entire support. We call these novel collagen structures microribbons. High-resolution atomic force microscopy topographs show that subunits of these microribbons are built by fibrillar structures. The smallest units of these fibrillar structures have cross-sections of approximately 3 x 5nm, consistent with current models of collagen microfibril formation. By varying the pH and electrolyte of the buffer solution during the self-assembly process, the microfibril density and contacts formed within this network could be controlled. Under certain electrolyte compositions the microribbons and microfibers display the characteristic D-periodicity of approximately 65 nm observed for much thicker collagen fibrils. In addition to providing insight into the mechanism of collagen assembly, the ultraflat collagen matrices may also offer novel ways to bio-functionalize surfaces.     

Cultivation of cell-polymer tissue constructs in simulated microgravity

Tissue-engineered cartilage was cultivated under conditions of simulated microgravity using rotating bioreactors. Rotation randomized the effects of gravity on inoculated cells (chondrocytes) and permitted their attachment to three-dimensional (3D) synthetic, biodegradable polymer scaffolds that were freely suspended within the vessel. After 1 week of cultivation, the cells regenerated a cartilaginous extracellular matrix (ECM) consisting of glycosaminoglycan (GAG) and collagen types I and II. Tissue constructs grown in simulated microgravity had higher GAG contents and thinner outer capsules than control constructs grown in turbulent spinner flasks. Two fluid dynamic regimes of simulated microgravity were identified, depending on the vessel rotation speed: (i) a settling regime in which the constructs were maintained in a state of continuous free-fall close to a stationary point within the vessel and (ii) an orbiting regime in which the constructs orbited around the vessel spin axis. In the settling regime, the numerically calculated relative fluid-construct velocity was comparable to the experimentally measured construct settling velocity (2-3 cm/s). A simple mathematical model was used in conjunction with measured construct physical properties to determine the hydrodynamic drag force and to estimate the hydrodynamic stress at the construct surface (1.5 dyn/cm(2)). Rotating bioreactors thus provide a powerful research tool for cultivating tissue-engineered cartilage and studying 3D tissue morphogenesis under well-defined fluid dynamic conditions. (c) 1995 John Wiley & Sons, Inc.     

Electrospinning of collagen nanofibers

Electrospinning is a fabrication process that uses an electric field to control the deposition of polymer fibers onto a target substrate. This electrostatic processing strategy can be used to fabricate fibrous polymer mats composed of fiber diameters ranging from several microns down to 100 nm or less. In this study, we describe how electrospinning can be adapted to produce tissue-engineering scaffolds composed of collagen nanofibers. Optimizing conditions for calfskin type I collagen produced a matrix composed of 100 nm fibers that exhibited the 67 nm banding pattern that is characteristic of native collagen. The structural properties of electrospun collagen varied with the tissue of origin (type I from skin vs type I from placenta), the isotype (type I vs type III), and the concentration of the collagen solution used to spin the fibers. Electrospinning is a rapid and efficient process that can be used to selectively deposit polymers in a random fashion or along a predetermined and defined axis. Toward that end, our experiments demonstrate that it is possible to tailor subtle mechanical properties into a matrix by controlling fiber orientation. The inherent properties of the electrospinning process make it possible to fabricate complex, and seamless, three-dimensional shapes. Electrospun collagen promotes cell growth and the penetration of cells into the engineered matrix. The structural, material, and biological properties of electrospun collagen suggest that this material may represent a nearly ideal tissue engineering scaffold.     

Engineering superficial zone features in tissue engineered cartilage

A major challenge in cartilage tissue engineering is the need to recreate the native tissue's anisotropic extracellular matrix structure. This anisotropy has important mechanical and biological consequences and could be crucial for integrative repair. Here, we report that hydrodynamic conditions that mimic the motion‐induced flow fields in between the articular surfaces in the synovial joint induce the formation of a distinct superficial layer in tissue engineered cartilage hydrogels, with enhanced production of cartilage matrix proteoglycan and Type II collagen. Moreover, the flow stimulation at the surface induces the production of the surface zone protein Proteoglycan 4 (aka PRG4 or lubricin). Analysis of second harmonic generation signature of collagen in this superficial layer reveals a highly aligned fibrillar matrix that resembles the alignment pattern in native tissue's surface zone, suggesting that mimicking synovial fluid flow at the cartilage surface in hydrodynamic bioreactors could be key to creating engineered cartilage with superficial zone features. Biotechnol. Bioeng. 2013; 110: 1476–1486. © 2012 Wiley Periodicals, Inc.     

Hydrodynamic and Thermal Slip Effect on Double-Diffusive Free Convective Boundary Layer Flow of a Nanofluid Past a Flat Vertical Plate in the Moving Free Stream

The effects of hydrodynamic and thermal slip boundary conditions on the double-diffusive free convective flow of a nanofluid along a semi-infinite flat solid vertical plate are investigated numerically. It is assumed that free stream is moving. The governing boundary layer equations are non-dimensionalized and transformed into a system of nonlinear, coupled similarity equations. The effects of the controlling parameters on the dimensionless velocity, temperature, solute and nanofluid concentration as well as on the reduced Nusselt number, reduced Sherwood number and the reduced nanoparticle Sherwood number are investigated and presented graphically. To the best of our knowledge, the effects of hydrodynamic and thermal slip boundary conditions have not been investigated yet. It is found that the reduced local Nusselt, local solute and the local nanofluid Sherwood numbers increase with hydrodynamic slip and decrease with thermal slip parameters.     

A methodology for the design of scale-down bioreactors by the use of mixing and circulation stochastic models

Scale-up is traduced in practice by an increase of the dimensions of the bioreactors, leading to a modification of the time scale and thus of the process dynamics. In the present work, a methodology to study the effect of scale-up on bioreactors hydrodynamics and to put in place scale-down reactors representative of the flow properties encountered in real scales bioreactors is detailed.In order to simplify the analysis, we have proposed the use of a stochastic model which is directly affected by the time scale. Indeed, to run simulations with such models, we have to specify the time taken to achieve a transition Δt. Stochastic models are thus reliable to study scale-up effect on stirred reactors hydrodynamics. In addition, these models permit to have an insight on the internal dynamic of the process.In the case of the circulation process, qualitative aspects have to be taken into account and induce a modification of the flow regions arrangement of the model. The stochastic analysis of large-scale bioreactors permits to propose a translating methodology into a scale-down context. Optimised scale-down reactors can be used further to carry out fermentation tests with the hydrodynamic conditions of the industrial scale. In a general rule, the performances of stochastic model allow to facilitate greatly the analysis of the scale-up effect and the hydrodynamic characteristics of both large-scale and scale-down reactors.     

Staining kinetics in single cells. Part I. Influence of convective diffusion on the staining rate

The staining kinetics of single cells have been investigated using a perfusion cuvette in combination with a computer controlled microscope spectrometer. The physicochemical hydrodynamics of staining are characterized. Using a steady-state laminar flow parallel to the cell surface a hydrodynamic and a diffusional boundary layer are observed which are determined by the flow rate. The thickness of the diffusional boundary layer revealed by experimental data is in agreement with theoretically calculated values. At certain well-defined hydrodynamic conditions convective diffusion has no further effect on the staining rate.     

Microfluidic delivery of small molecules into mammalian cells based on hydrodynamic focusing

Microfluidics-based cell assays offer high levels of automation and integration, and allow multiple assays to be run in parallel, based on reduced sample volumes. These characteristics make them attractive for studies associated with drug discovery. Controlled delivery of drug molecules or other exogenous materials into cells is a critical issue that needs to be addressed before microfluidics can serve as a viable platform for drug screening and studies. In this study, we report the application of hydrodynamic focusing for controlled delivery of small molecules into cells immobilized on the substrate of a microfluidic device. We delivered calcein AM which was permeant to the cell membrane into cells, and monitored its enzymatic conversion into fluorescent calcein during and after the delivery. Different ratios of the sample flow to the side flow were tested to determine how the conditions of hydrodynamic focusing affected the delivery. A 3D numerical model was developed to help understand the fluid flow, molecular diffusion due to hydrodynamic focusing in the microfluidic channel. The results from the simulation indicated that the calcein AM concentration on the outer surface of a cell was determined by the conditions of hydrodynamic focusing. By comparing the results from the simulation with those from the experiment, we found that the calcein AM concentration on the cell outer surface correlated very well with the amount of the molecules delivered into the cell. This suggests that hydrodynamic focusing provides an effective way for potentially quantitative delivery of exogenous molecules into cells at the single cell or subcellular level. We expect that our technique will pave the way to high-throughput drug screening and delivery on a microfluidic platform.     

Experimental collision efficiencies of polymer-flocculated animal cells.

The determination of particle collision kinetics is useful to decouple the effects of process parameters on individual events in flocculation. This paper discusses the effects of flocculation conditions on the collision efficiency of ATCC strain CRL 1606 hybridomas flocculated with poly-L-histidine. Experimental determinations of the collision efficiency of cells in Couette flow are presented over a range of experimental conditions. The collision efficiency correlates with the cell zeta potential to the -2.4 power at high surface coverage, consistent with literature results in latex systems. At low coverage, accounting for the distribution of polymer on the cells corrects for deviation from the high-coverage behavior. Collision is dependent on the hydrodynamic environment as well. At high surface coverage, collision efficiency is weakly dependent on hydrodynamic conditions and follows a dependency on the shear rate and viscosity to the -0.32 power. This is consistent with ionic coagulation theory. At low surface coverage, the collision efficiency is strongly dependent on the viscous fluid forces. The results versus both dose and shear rate over the entire range of surface coverages are consistent with weak intercell bonding. Collision kinetics in the presence of high molecular weight dextrans show steric hindrance to cell collision.     

vpsA- and luxO-independent biofilms of Vibrio cholerae

The natural life cycle of Vibrio cholerae involves the transitioning of cells between different environmental surfaces such as the chitinous shell of Crustaceae and the epithelial layer of the human intestine. Previous studies using static biofilm systems showed a strict dependence of biofilm formation on the vps and lux genes, which are essential for exopolysaccharide formation and cell-cell signaling, respectively. The authors' report here that in biofilms grown under hydrodynamic conditions, DeltavpsA and DeltaluxO mutants of V. cholerae do form pronounced, three-dimensional biofilms that resemble all aspects of wild-type biofilms. By genetic experiments, it was shown that in hydrodynamically grown biofilms this independence of vpsA is due to the expression of rpoS, which is a negative regulator of vpsA expression. Biofilms also underwent substantial dissolution after 96 h that could be induced by a simple stop of medium flow. The studies indicate that metabolic conditions control the reversible attachment of cells to the biofilm matrix and are key in regulating biofilm cell physiology via RpoS. Furthermore, the results redefine the roles of vps and quorum-sensing in V. cholerae biofilms.     

Relative roles of stream flow and sedimentary conditions in controlling hyporheic exchange

Hyporheic exchange is often controlled by subsurface advection driven by the interaction of the stream with sedimentary pore water. The nature and magnitude of the induced exchange flow is dependent on the characteristics of both the stream flow and the sediment bed. Fundamental hydrodynamic theory can be applied to determine general relationships between stream characteristics, sediment characteristics, and hyporheic exchange rates. When the stream bed is fine enough to allow application of Darcy's Law, as with sand beds, the induced advective exchange can be calculated from fundamental hydrodynamic principles. Comparison with a wide range of experimental results demonstrates the predictive capability of this theory. Coarser sediments such as gravels are more complex because they admit turbulent interactions between the stream and subsurface flows, which can produce considerable exchange even when the bed surface is flat and no flows are induced by the bed topography. Even for this case, however, scaling arguments can still be used to determine how exchange rates vary with stream and sedimentary conditions. Evaluation of laboratory flume experiments for a wide range of stream conditions, bed sediment types including sand and gravel, and bed geometries demonstrates that exchange scales with the permeability of the bed sediments and the square of the stream velocity. These relationships occur due to fundamental hydrodynamic processes, and were observed to hold over almost five orders of magnitude of exchange flux. Such scaling relationships are very useful in practice because they can be used to extend observed hyporheic exchange rates to different flow conditions and to uniquely identify the role of sedimentary conditions in controlling exchange flux.     

Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells

Fluorescence activated cell sorting, FACS, is a widely used method to sort subpopulations of cells to high purities. To achieve relatively high sorting speeds, FACS instruments operate by forcing suspended cells to flow in a single file line through a laser(s) beam(s). Subsequently, this flow stream breaks up into individual drops which can be charged and deflected into multiple collection streams. Previous work by Ma et al. (2002) and Mollet et al. (2007; Biotechnol Bioeng 98:772-788) indicates that subjecting cells to hydrodynamic forces consisting of both high extensional and shear components in micro-channels results in significant cell damage. Using the fluid dynamics software FLUENT, computer simulations of typical fluid flow through the nozzle of a BD FACSVantage indicate that hydrodynamic forces, quantified using the scalar parameter energy dissipation rate, are similar in the FACS nozzle to levels reported to create significant cell damage in micro-channels. Experimental studies in the FACSVantage, operated under the same conditions as the simulations confirmed significant cell damage in two cell lines, Chinese Hamster Ovary cells (CHO) and THP1, a human acute monocytic leukemia cell line.     

Swirling flow implementation in a photobioreactor for batch and continuous cultures of Porphyridium cruentum

Light is the main limiting factor in photoautotrophic-intensive production of microorganisms, and improvement of its use is an important concern for photobioreactor design and operation. Swirling flows, which are known to improve mass and photon transfers, were applied to annular light chambers of a photobioreactor and studied by simulation and microalgal culture. Two hydrodynamic conditions were compared: axial flow generating poor radial mixing, and tangential flow generating three-dimensional swirling motion. Batch and continuous cultures of the Rhodophyte Porphyridium cruentum were performed in a 100-L, 1.5-m(2), fully controlled photobioreactor with eight light chambers. The inlet design of these chambers was modified to create the hydrodynamic conditions for comparison. Various intensities of swirling motion were used, characterized by the velocity factor (VF), defined as the ratio between annular chamber flow and inlet aperture sections. Experiments were performed within the range of photon flux densities (PFD) optimizing the yield of light energy transformation into living substance for the species and the temperature used. Culture kinetics with swirling flows generated by apertures of VF = 2, 4, and 9 were compared with pseudoaxial VF = 2 chosen as reference. Batch cultures with VF = 4 swirling flow showed no significant difference, whereas continuous cultures proved more discriminating. Although no significant difference was obtained for VF = 2, a 7% increase of steady-state productivity and a 26% decrease in time required to reach this steady state were obtained with VF = 4 swirling flow. This beneficial effect of swirling flow could have accounted for increased mixing. Conversely, VF = 9 swirling flow resulted in a 9% decrease of steady-state productivity and a 9% increase in the time required to reach this steady state, a negative effect that could have accounted for increased shear stress. CO(2) bioconversion yield at steady state showed a 34% increase for VF = 4. These results suggest that swirling motion makes microalgal cultures more efficient, provided that the resulting adverse effects remain acceptable. Experimental investigation was completed by a theoretical approach in which simulation of continuous cultures of P. cruentum was based on the hydrodynamic conditions achieved in the photobioreactor. Although the results obtained with pseudoaxial flow were correctly predicted, simulations with swirling flow showed a marked enhancement of productivity not observed experimentally. The influence of side effects induced by increased mixing (particularly hydrodynamic shear stress) was considered with respect to modeling assumptions. Comparison of experimental results with theoretical simulation provided a better understanding of the mixing effect, a key factor in improving the efficiency of such bioprocesses.     

不同流速下水华生消的模拟

河道型水库建成后,支流库湾水动力学条件改变,水流减缓,遇到适宜环境条件和充足营养条件后,易产生水华,从而给水生态系统带来不利影响.为调查不同水动力学条件对水华生消的影响,以流速为表征指标,在封闭循环模型中设置不同水体流速,控制环境条件和营养条件,开展室内水华生消模拟试验.结果表明：在一定范围内(<0.4 m·s^-1),流速增大有利于藻类生长和水华暴发;不同流速下,藻类生长均符合微生物生长的一般规律,即经迟滞期、指数期到稳定期,最后达衰退期,水华相应出现暴发高峰和消退低谷.表明一定范围内(<0.4m·s^-1)水流流速的提高并不能抑制藻类生长和水华暴发,与水华暴发有关的水动力学条件的表征指标有待深入研究.     

The nature of the fluid boundary layer and the selection of parameters for benthic ecology

1. The nature of flow structure close to a river bed is reviewed and suggestions made as to appropriate equations to apply to given hydrodynamic regimes. 2. It is emphasized that in natural rivers the laminar sublayer observed immediately above the bed in some experimental studies is completely disrupted for flows characterized by high turbulence levels. 3. Instead of resorting to direct topographical measurement of bed undulations, the roughness of the river bed also can be quantified using hydraulic data obtained from velocity profiles. 4. Ambuhl's experimental findings of 1959 underpin modern ecological research into the nature of the benthic boundary layer. Common misconceptions concerning Ambuhl's contribution are corrected and it is shown that his results only apply to certain prescribed hydrodynamic conditions. 5. The adoption of a consistent approach to describing the benthic boundary layer is to be preferred, so that diverse studies can be usefully compared.     

Hydrodynamic modes of arbitrarily shaped flexible macromolecules: Influence on dynamic light scattering

The diffusional motions of flexible macromolecules are analyzed with an increasingly realistic Rouse–Zimm model, i.e., by modeling the molecule as an arbitrary set of spheres connected by nearly harmonic springs. New features include (1) nearly arbitrary arrangements of spheres, (2) arbitrary arrangements of translational and torsional springs, (3) significant anharmonic corrections to the elastic potential surface, and (4) inclusion of torsional damping and various hydrodynamic cross-coupling effects (including two types of translational-rotational coupling) with no additional fitted parameters. The hydrodynamic interactions [R. F. Goldstein (1985) Journal of Chemical Physics, Vol. 83, pp. 2390–2397] contain no adjustable parameters other than temperature, viscosity, and the radii and positions of the spheres. These hydrodynamic interactions allow accurate calculations of rigid body diffusion as well as flexible motions. Given the positions, radii, and spring constant matrix, one can calculate a full set of three-dimensional diffusional modes. Because one uses an off-diagonal hydrodynamic resistance matrix instead of a diagonal mass matrix, the diffusional modes are different in structure from vacuum normal modes, and give rise to different rms motions in the laboratory frame. These hydrodynamic modes include the effects of vibrational-translational cross-coupling (i.e., motion along a vibrational coordinate may give rise to a translational force, and vice versa). The diffusional modes are used to simulate dynamic light scattering (DLS). I examine various molecules with different shapes, flexibilities, and with different scattering vectors. Radial and angular motions influence DLS decays differently. These effects are dependent upon the molecular shape (straight, bent, or curved) and type of flexibility (stretching or bending). Furthermore, small cubic corrections to the potential surface can be significant for DLS of certain geometries such as straight rods and semicircles. © 1993 John Wiley & Sons, Inc.