Physiological Flow Rates Research Articles

In vitro controlled release of an anti-inflammatory from daily disposable therapeutic contact lenses under physiological ocular tear flow

Novel molecularly imprinted, therapeutic contact lenses capable of controlled release of the non-steroidal anti-inflammatory (NSAID) diclofenac sodium were synthesized, exploiting ionic non-covalent interactions. Poly(HEMA-co-DEAEM-co-PEG200DMA) soft contact lenses were prepared (105±5μm thickness, diameter 15.0±0.2mm, base curve of 8.6±0.2mm) with different monomer to template ratios and dynamic release studies were conducted in artificial lacrimal solution using two different in vitro methods. Under infinite sink conditions, imprinted contact lenses demonstrated concentration dependent release kinetics. Under physiological flow rates, by increasing the M/T ratio from 1 to 10.5, the release rate decreased from 11.72μg/h to 6.75μg/h during the first 48h. The release rate was more constant, moving toward zero-order release. To use these lenses as daily disposable lenses, the first 24h of release was studied and found to be linear with a rate of 17.27, 11.99, and 8.74μg/h for M/T ratios of 1, 3.5, and 10.5, respectively. Furthermore, the lenses prepared with a M/T ratio of 10.5 released diclofenac at a rate close to the maximum dose delivered by commercial eye drops, making them ideal for use as daily disposable lenses, and potentially leading to better patient benefit with substantially increased efficacy.

Direct numerical simulation of a 2D-stented aortic heart valve at physiological flow rates

We study the nonlinear interaction of an aortic heart valve, composed of hyperelastic corrugated leaflets of finite density attached to a stented vessel under physiological flow conditions. In our numerical simulations, we use a 2D idealised representation of this arrangement. Blood flow is caused by a time-varying pressure gradient that mimics that of the aortic valve and corresponds to a peak Reynolds number equal to 4050. Here, we fully account for the shear-thinning behaviour of the blood and large deformations and contact between the leaflets by solving the momentum and mass balances for blood and leaflets. The mixed finite element/Galerkin method along with linear discontinuous Lagrange multipliers for coupling the fluid and elastic domains is adopted. Moreover, a series of challenging numerical issues such as the finite length of the computational domain and the conditions that should be imposed on its inflow/outflow boundaries, the accurate time integration of the parabolic and hyperbolic momentum equations, the contact between the leaflets and the non-conforming mesh refinement in part of the domain are successfully resolved. Calculations for the velocity and the shear stress fields of the blood reveal that boundary layers appear on both sides of a leaflet. The one along the ventricular side transfers blood with high momentum from the core region of the vessel to the annulus or the sinusoidal expansion, causing the continuous development of flow instabilities. At peak systole, vortices are convected in the flow direction along the annulus of the vessel, whereas during the closure stage of the valve, an extremely large vortex develops in each half of the flow domain.

Microdrop Printing of Hydrogel Bioinks into 3D Tissue‐Like Geometries

An optimized 3D inkjet printing process is demonstrated for structuring alginate into a tissue-like microvasculature capable of supporting physiological flow rates. Optimizing the reaction at the single-droplet level enables wet hydrogel droplets to be stacked, thus overcoming their natural tendancy to spread and coalesce. Live cells can be patterned using this process and it can be extended to a range of other hydrogels.

Phenotypic and functional characterization of human bone marrow stromal cells in hollow-fibre bioreactors

The transplantation of human bone marrow stromal cells (BMSCs) is a novel immunotherapeutic approach that is currently being explored in many clinical settings. Evidence suggests that the efficacy of cell transplantation is directly associated with soluble factors released by human BMSCs. In order to harness these secreted factors, we integrated BMSCs into large-scale hollow-fibre bioreactor devices in which the cells, separated by a semipermeable polyethersulphone (PES) membrane, can directly and continuously release therapeutic factors into the blood stream. BMSCs were found to be rapidly adherent and exhibited long-term viability on PES fibres. The cells also preserved their immunophenotype under physiological fluid flow rates in the bioreactor, and exhibited no signs of differentiation during device operation, but still retained the capacity to differentiate into osteoblastic lineages. BMSC devices released growth factors and cytokines at comparable levels on a per-cell basis to conventional cell culture platforms. Finally, we utilized a potency assay to demonstrate the therapeutic potential of the collected secreted factors from the BMSC devices. In summary, we have shown that culturing BMSCs in a large-scale hollow-fibre bioreactor is feasible without deleterious effects on phenotype, thus providing a platform for collecting and delivering the paracrine secretions of these cells.

Extended release of high molecular weight hydroxypropyl methylcellulose from molecularly imprinted, extended wear silicone hydrogel contact lenses

Symptoms of contact lenses induced dry eye (CLIDE) are typically treated through application of macromolecular re-wetting agents via eye drops. Therapeutic soft contact lenses can be formulated to alleviate CLIDE symptoms by slowly releasing comfort agent from the lens. In this paper, we present an extended wear silicone hydrogel contact lens with extended, controllable release of 120 kDa hydroxypropyl methylcellulose (HPMC) using a molecular imprinting strategy. A commercial silicone hydrogel lens was tailored to release approximately 1000 μg of HPMC over a period of up to 60 days in a constant manner at a rate of 16 μg/day under physiological flowrates, releasing over the entire range of continuous wear. Release rates could be significantly varied by the imprinting effect and functional monomer to template ratio (M/T) with M/T values 0, 0.2, 2.8, 3.4 corresponding to HPMC release durations of 10, 13, 23, and 53 days, respectively. Lenses had high optical quality and adequate mechanical properties for contact lens use. This work highlights the potential of imprinting in the design and engineering of silicone hydrogel lenses to release macromolecules for the duration of wear, which may lead to decreased CLIDE symptoms and more comfortable contact lenses.

X-ray Velocimetry and Haemodynamic Forces Within a Stenosed Femoral Model at Physiological Flow Rates

High resolution in vivo velocity measurements within the cardiovascular system are essential for accurate calculation of vessel wall shear stress, a highly influential factor for the progression of arterial disease. Unfortunately, currently available techniques for in vivo imaging are unable to provide the temporal resolution required for velocity measurement at physiological flow rates. Advances in technology and improvements in imaging systems are allowing a relatively new technique, X-ray velocimetry, to become a viable tool for such measurements. This study investigates the haemodynamics of pulsatile blood flow in an optically opaque in vitro model at physiological flow rates using X-ray velocimetry. The in vitro model, an asymmetric stenosis, is designed as a 3:1 femoral artery with the diameter and flow rate replicating vasculature of a mouse. Velocity measurements are obtained over multiple cycles of the periodic flow at high temporal and spatial resolution (1ms and 29μm, respectively) allowing accurate measurement of the velocity gradients and calculation of the wall shear stress. This study clearly illustrates the capability of in vitro X-ray velocimetry, suggesting it as a possible measurement technique for future in vivo vascular wall shear stress measurement.

In Vitro Fluid Dynamics of the Ahmed Glaucoma Valve Modified with Expanded Polytetrafluoroethylene

Purpose: Long-term intraocular pressure reduction by glaucoma drainage devices (GDDs) is often limited by the fibrotic capsule that forms around them. Prior work demonstrates that modifying a GDD with a porous membrane promotes a vascularized and more permeable capsule. This work examines the in vitro fluid dynamics of the Ahmed valve after enclosing the outflow tract with a porous membrane of expanded polytetrafluoroethylene (ePTFE).Materials and Methods: The control and modified Ahmed implants (termed porous retrofitted implant with modified enclosure or PRIME-Ahmed) were submerged in saline and gelatin and perfused in a system that monitored flow (Q) and pressure (P). Flow rates of 1–50 μl/min were applied and steady state pressure recorded. Resistance was calculated by dividing pressure by flow.Results: Modifying the Ahmed valve implant outflow with expanded ePTFE increased pressure and resistance. Pressure at a flow of 2 μl/min was increased in the PRIME-Ahmed (11.6 ± 1.5 mm Hg) relative to the control implant (6.5 ± 1.2 mm Hg). Resistance at a flow of 2 μl/min was increased in the PRIME-Ahmed (5.8 ± 0.8 mm Hg/μl/min) when compared to the control implant (3.2 ± 0.6 mm Hg/μl/min).Conclusions: Modifying the outflow tract of the Ahmed valve with a porous membrane adds resistance that decreases with increasing flow. The Ahmed valve implant behaves as a variable resistor. It is partially open at low pressures and provides reduced resistance at physiologic flow rates.

Pulsatile ex vivo perfusion of human saphenous vein grafts under controlled pressure conditions increases MMP-2 expression

BackgroundThe use of human saphenous vein grafts (HSVGs) as a bypass conduit is a standard procedure in the treatment of coronary artery disease while their early occlusion remains a major problem.MethodsWe have developed an ex vivo perfusion system, which uses standardized and strictly controlled hemodynamic parameters for the pulsatile and non-static perfusion of HSVGs to guarantee a reliable analysis of molecular parameters under different pressure conditions. Cell viability of HSVGs (n = 12) was determined by the metabolic conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) into a purple formazan dye.ResultsUnder physiological flow rates (10 mmHg) HSVGs remained viable for two weeks. Their exposure to arterial conditions (100 mmHg) was possible for one week without important reduction in viability. Baseline expression of matrix metalloproteinase-2 (MMP-2) after venous perfusion (2.2 ± 0.5, n = 5) was strongly up-regulated after exposure to arterial conditions for three days (19.8 ± 4.3) or five days (23.9 ± 6.1, p < 0.05). Zymographic analyses confirmed this increase on the protein level. Our results suggest that expression and activity of MMP-2 are strongly increased after exposure of HSVGs to arterial hemodynamic conditions compared to physiological conditions.ConclusionTherefore, our system might be helpful to more precisely understand the molecular mechanisms leading to an early failure of HSVGs.

Establishment of steady-state metabolism of ethanol in perfused rat liver: the quantitative analysis using kinetic mechanism-based rate equations of alcohol dehydrogenase

Alcohol dehydrogenase (ADH) catalyzes oxidation of ingested ethanol to acetaldehyde, the first step in hepatic metabolism. The purpose of this study was to establish an ex vivo rat liver perfusion system under defined and verified steady states with respect to the metabolites and the metabolic rates, and to quantitatively correlate the observed rates with simulations based on the kinetic mechanism-based rate equations of rat liver ADH. Class I ADH1 was isolated from male Sprague–Dawley rats and characterized by steady-state kinetics in the Krebs–Ringer perfusion buffer with supplements. Nonrecirculating liver perfusion with constant input of ethanol at near physiological hepatic blood flow rate was performed in situ. Ethanol and the related metabolites acetaldehyde, acetate, lactate, and pyruvate in perfusates were determined. Results of the initial velocity, product, and dead-end inhibition studies showed that rat ADH1 conformed to the Theorell–Chance Ordered Bi Bi mechanism. Steady-state metabolism of ethanol in the perfused liver maintained up to 3 h as evidenced by the steady-state levels of ethanol and metabolites in the effluent, and the steady-state ethanol disappearance rates and acetate production rates. The changes of the metabolic rates were qualitatively and in general quantitatively correlated to the results from simulations with the kinetic rate equations of ADH1 under a wide range of ethanol, in the presence of competitive inhibitor 4-methylpyrazole and of uncompetitive inhibitor isobutyramide. Preliminary flux control analysis estimated that apparent C ADH J in the perfused liver may approximate 0.7 at constant infusion with 1–2 mM ethanol, suggesting that ADH plays a major but not the exclusive role in governing hepatic ethanol metabolism. The reported steady-state rat liver perfusion system may potentially be applicable to other drug or drug–ethanol interaction studies.

Outflow Conditions for Image-Based Hemodynamic Models of the Carotid Bifurcation: Implications for Indicators of Abnormal Flow

Computational fluid dynamics (CFD) models have become very effective tools for predicting the flow field within the carotid bifurcation, and for understanding the relationship between local hemodynamics, and the initiation and progression of vascular wall pathologies. As prescribing proper boundary conditions can affect the solutions of the equations governing blood flow, in this study, we investigated the influence to assumptions regarding the outflow boundary conditions in an image-based CFD model of human carotid bifurcation. Four simulations were conducted with identical geometry, inlet flow rate, and fluid parameters. In the first case, a physiological time-varying flow rate partition at branches along the cardiac cycle was obtained by coupling the 3D model of the carotid bifurcation at outlets with a lumped-parameter model of the downstream vascular network. Results from the coupled model were compared with those obtained by imposing three fixed flow rate divisions (50/50, 60/40, and 70/30) between the two branches of the isolated 3D model of the carotid bifurcation. Three hemodynamic wall parameters were considered as indicators of vascular wall dysfunction. Our findings underscore that the overall effect of the assumptions done in order to simulate blood flow within the carotid bifurcation is mainly in the hot-spot modulation of the hemodynamic descriptors of atherosusceptible areas, rather than in their distribution. In particular, the more physiological, time-varying flow rate division deriving from the coupled simulation has the effect of damping wall shear stress (WSS) oscillations (differences among the coupled and the three fixed flow partition models are up to 37.3% for the oscillating shear index). In conclusion, we recommend to adopt more realistic constraints, for example, by coupling models at different scales, as in this study, when the objective is the outcome prediction of alternate therapeutic interventions for individual patients, or to test hypotheses related to the role of local fluid dynamics and other biomechanical factors in vascular diseases.

Elongational Flow Induces the Unfolding of von Willebrand Factor at Physiological Flow Rates

The unfolding of von Willebrand Factor (vWF), one of the largest multimeric proteins in our body, has been shown to be a crucial step in the process of blood clotting. Here we show that elongational flows, which appear during vasoconstriction or stenosis, are the primary activation mechanisms of vWF, and unfold the multimeric protein at flow rates that are two orders-of -magnitude below those corresponding to pure shear. The findings presented here complement the current understanding of blood clotting from the molecular to the physiological level, and provide new physical insights into the connection between clotting anomalies, such as Heyde's syndrome and stenosis. These findings also represent a new paradigm in the function and activation of vWF.

Polypyrrole solid phase microextraction: A new approach to rapid sample preparation for the monitoring of antibiotic drugs

Simple or even rapid bioanalytical methods are rare, since they generally involve complicated, time-consuming sample preparation from the biological matrices like LLE or SPE. SPME provides a promising approach to overcome these limitations. The full potential of this innovative technique for medical diagnostics, pharmacotherapy or biochemistry has not been tapped yet. In-house manufactured SPME probes with polypyrrole (PPy) coating were evaluated using three antibiotics of high clinical relevance – linezolid, daptomycin, and moxifloxacin – from PBS, plasma, and whole blood. The PPy coating was characterised by scanning electron microscopy. Influences of pH, inorganic salt, and blood anticoagulants were studied for optimum performance. Extraction yields were determined from stagnant media as well as re-circulating human blood using the heart-and-lung machine model system. The PPy-SPME fibres showed high extraction yields, particularly regarding linezolid. The reproducibility of the method was optimised to achieve RSDs of 9% or 17% and 7% for SPME from stagnant or re-circulating blood using fresh and re-used fibres, respectively. The PPy-SPME approach was demonstrated to meet the requirements of therapeutic monitoring of the drugs tested, even from re-circulating blood at physiological flow rates. SPME represents a rapid and simple dual-step procedure with potency to significantly reduce the effort and expenditure of complicated sample preparations in biomedical analysis.

Optimization of axial-pump pressure sensitivity for a continuous-flow total artificial heart

In this study, we describe the potential advantages of a continuous-flow total artificial heart (CFTAH) comprising two small, non-pulsatile pumps with optimized responsiveness to the pressure gradient. We modified a MicroMed DeBakey axial-flow pump by increasing its inducer-impeller inlet angle, thereby increasing its pressure responsivity. We obtained the in vitro pressure gradient response and compared it with those of the clinically used, unmodified MicroMed DeBakey pump, Jarvik 2000 FlowMaker and HeartMate II. The modified pump showed an increased response to changes in the pressure gradient at pump flow rates of between 2 and 4 liters/min. The maximum pressure responsivity of the modified pump was 2.5 liters/min/mm Hg; the corresponding maximum responsivities of the Jarvik 2000, HeartMate II and MicroMed DeBakey ventricular assist devices (VADs) were 0.12, 0.09 and 0.38 liters/min/mm Hg, respectively. Because of the inherent properties of non-pulsatile pumps, the CFTAH may potentially respond to changes in inflow and outflow pressures while maintaining physiologic flow rates sufficient for normal daily activity. In addition, the hemodynamic interplay between the two optimized pumps should allow a physiologic response to normal flow imbalances between the pulmonary and systemic circulations. Improved responsiveness to inflow pressure may further simplify and improve the CFTAH and affect its potential clinical use as a meaningful therapy for terminal heart failure.

Hemodynamic Comparison of Differing Anastomotic Geometries Using Magnetic Resonance Velocimetry

Hemodynamic factors at the distal anastomosis play an important role in prosthetic graft performance. A new magnetic resonance imaging (MRI) technique was used to determine the effect of anastomotic geometry on hemodynamic flow patterns. Four dimensional (4D) magnetic resonance velocimetry (4D-MRV) is a noninvasive method of analyzing pulsatile flow in three dimensions (3D). End-to-side anastomotic models were constructed by suturing 6 mm polytetrafluoroethylene (ePTFE) grafts to silicone tubing (4 mm i.d.). The models included straight ePTFE, precuffed ePTFE, and patched ePTFE configurations in a pulsatile system, which created flow consistent with physiologic flow rates and pressures. Blood was simulated by a solution of 40% glycerol in distilled water with trace gadolinium. The different models were imaged using MRV techniques in a three-dimensional (3D) coronal slab (0.5 mm thick coronal slices, in-plane field of view (FOV) 18 cm.) The data were reconstructed, resulting in an interpolated resolution of 0.35 mm in each coronal plane. The 3D flow fields were represented as isosurfaces, visualizing the internal geometry of the models with streamlines tangent to the velocity vectors identifying the path of the fluid. Volumetric flow rates for each time phase were calculated by integrating the flow through cross sections of each anastomotic model. Analysis of the flow patterns focused on the anastomotic regions prone to the development of intimal hyperplasia and graft failure as identified in the literature; the toe, floor, heel, and hood. Conventional end-to-side geometry resulted in uniform flow with a low angle of impingement on the recipient vessel floor. A small vortex at the anastomotic heel created minimal recirculation. The precuffed geometry resulted in a large recirculation vortex of chaotic, low flow that increased throughout the pulsatile cycle. Regions of low flow velocity were noted in a substantial portion of the precuffed anastomotic configuration. Flow separation distal to the toe occurred in both geometries, but was more apparent in the precuffed configuration. The patch model had flow characteristics similar to the straight end-to-side geometry. Magnetic resonance velocimetry produces 3D, time varying velocity measurements with sufficient accuracy and resolution to analyze hemodynamics in anastomotic geometries. Flow structures in different graft configurations were effectively captured with marked differences noted between standard and precuffed anastomotic geometries. The findings support a conventional end-to-side anastomosis with a low incidence angle using a straight graft as producing favorable hemodynamics as compared to a cuffed configuration. The vein patch configuration closely approximates the conventional, straight anastomotic pattern. We believe the MRV technique has been sufficiently developed to warrant additional in vitro and in vivo studies providing insight into hemodynamic implications for the development of optimal prosthetic graft performance.

Morphologic adaptation of arterial endothelial cells to longitudinal stretch in organ culture

Arteries in vivo are subjected to large longitudinal stretch, which changes significantly due to vascular disease and surgery. However, little is known about the effect of longitudinal stretch on arterial endothelium. The aim of this study was to determine the morphologic adaptation of arterial endothelial cells (ECs) to elevated axial stretch. Porcine carotid arteries were stretched 20% more than their in vivo length while being maintained at physiological pressure and flow rate in an organ culture system. The ECs were elongated with the application of the axial stretch (aspect ratio 2.81±0.25 versus 3.65±0.38, n=8, p<0.001). The elongation was slightly decreased after three days and the ECs recovered their normal shape after seven days, as measured by the shape index and aspect ratio (0.55±0.03 versus 0.56±0.04, and 2.93±0.28 versus 2.88±0.20, respectively, n=5). Cell proliferation was increased in the intima of stretched arteries in three days as compared to control arteries but showed no difference after seven days in organ culture. These results demonstrate that the ECs adapt to axial stretch and maintain their normal shape.

Development of a Composite Degradable/Nondegradable Tissue‐engineered Vascular Graft

The present study aimed to determine the feasibility of constructing a reinforced autologous vascular graft by combining the advantages of fibrin gel as an autologous cell carrier material with the inherent mechanical strength of an integrated mesh structure. It was hypothesized that the mesh and dynamic culture conditions could be combined to generate mechanically stable and implantable vascular grafts within a shorter cultivation period than traditional methods. A two-step moulding technique was developed to integrate a polyvinylidene fluoride (PVDF) mesh (pore size: 1-2 mm) in the wall of a fibrin-based vascular graft (I.D. 5 mm) seeded with carotid myofibroblasts. The graft was cultured under increasing physiological flow conditions for 2 weeks. Histology, burst strength, and suture retention strength were evaluated. Cell growth and tissue development was excellent within the fibrin gel matrix surrounding the PVDF fibers, and tissue structure demonstrated remarkable similarity to native tissue. The grafts were successfully subjected to physiological flow rates and pressure gradients from the outset, and mechanical properties were enhanced by the mesh structure. Mean suture retention strength of the graft tissue was 6.3 N and the burst strength was 236 mm Hg. Using the vascular composite graft technique, the production of tissue engineered, small-caliber vascular grafts with good mechanical properties within a conditioning period of 14 days is feasible.

Use of an Ultrasound Blood-Mimicking Fluid for Doppler Investigations of Turbulence In Vitro

Turbulence is an important factor in the assessment of stenotic disease and a possible causative mechanism for thromboembolism. Previous Doppler studies of turbulence have typically used whole-blood preparations or suspensions of erythrocytes. Recently, a water-glycerol based blood-mimicking fluid (BMF) has been developed for use in Doppler ultrasound studies. This fluid has desirable ultrasound properties but it has not previously been described during in vitro investigations of turbulence intensity. We report on investigations of grid-generated and constrained-jet turbulence in an in vitro test system. The BMF was found to generate significant levels of turbulence during steady flow at physiological flow rates, producing turbulent patterns in the distal region that were consistent with previous studies. Turbulence intensity increased significantly with flow rate ( p < 0.005) for both the constrained jet and the constrained grid. Based on our observations, we conclude that a water-glycerol based BMF provides a suitable working fluid during in vitro investigations of turbulence using Doppler ultrasound. (E-mail: david.holdsworth@imaging.robarts.ca)

Medium with blood-analog mechanical properties for cardiovascular tissue culturing

Physiological wall shear rates and stresses in vessel culture or tissue engineering are relevant for maintaining endothelial cell (EC) integrity. To this end, the culture medium should have an appropriate viscosity. The viscosity of a standard culture medium was increased using xanthan gum (XG) and compared with literature data on whole blood, resulting in a medium with blood-analog shear-thinning behavior (XG-medium). The measured osmolality of the XG-medium was 285+/-2 mOsm kg(-1), which is within a physiologically acceptable range. The XG-medium was compared to standard medium to verify whether XG alters vascular cell function. First, the effect of XG on the growth of human EC monolayers was determined. In addition, to study whether XG changes drug-induced vasoconstriction or endothelium-dependent vasodilation, different drugs were administered to porcine coronary artery rings in a solution with or without XG, measuring the isometric force developed. XG did not influence EC growth, nor did it change drug-induced vascular tone. Moreover, the ECs aligned in the direction of flow after 24 h of physiological shearing with XG-medium. We conclude that, unlike standard culture media, XG-medium as a blood-analog culture medium has rheological properties suitable for use in vessel culture and tissue engineering to induce physiological wall shear stresses at physiological flow rates.

Bioreactor for Application of Subatmospheric Pressure to Three-Dimensional Cell Culture

Vacuum-assisted closure (VAC) negative pressure wound therapy (NPWT) is a highly successful and widely used treatment modality for wound healing, although no apparatus exists to monitor the effects of subatmospheric pressure application in vitro. Such an apparatus is desirable to better understand the biological effects of this therapy and potentially improve upon them. This article describes the development and validation of a novel bioreactor that permits such study. Tissue analogues consisting of 3-dimensional fibroblast-containing fibrin clots were cultured in off-the-shelf disposable cell culture inserts and multi-well plates that were integrated into the bioreactor module. Negative pressure dressings, commercialized for wound therapy, were placed on top of the culture, and subatmospheric pressure was applied to the dressing. Cultures were perfused with media at controlled physiologic wound exudate flow rates. The design of this bioreactor permits observation of the culture using an inverted microscope in brightfield and fluorescence modes and sustained incubation of the system in a 5% carbon dioxide atmosphere. This closed-system mimics the wound micro-environment under VAC NPWT. Matrix compression occurs as the subatmospheric pressure draws the dressing material down. At the contact zone, surface undulations were clearly evident on the fibroblast-containing tissue analogues at 24 h and appeared to correspond to the dressing microstructure. The bioreactor design, consisting of sterilizable machined plastics and disposable labware, can be easily scaled to multiple units. Validation experiments show that cell survival in this system is comparable with that seen in cells grown in static tissue culture. After application of VAC NPWT, cell morphology changed, with cells appearing thicker and with an organized actin cytoskeleton. The development and validation of this new culture system establishes a stable platform for in vitro investigations of subatmospheric pressure application to tissues.

A Heat Shock Protein 90 Binding Domain in Endothelial Nitric-oxide Synthase Influences Enzyme Function

Previous reports suggest heat shock protein 90 (hsp90) associates with endothelial nitric-oxide synthase (eNOS) to increase nitric oxide (*NO) generation. Ansamycin inhibition of chaperone-dependent activity increases eNOS generation of superoxide anion (O(2)(*)) upon enzyme activation. In the present study we identify where hsp90 binds to eNOS using overlapping decoy peptides based on the amino acid (aa) sequence of eNOS (291-420). B1, B2, and B3 peptides inhibited hsp90 association with eNOS in cell lysates from proliferating bovine aortic endothelial cells. B2 (aa 301-320), common to both B1 and B3, decreased stimulated *NO production and hsp90 association in bovine aortic endothelial cells. The B2/B3 peptide was redesigned to TSB2 that includes a TAT protein transduction domain and shortened to 14 aa. TSB2 impaired vasodilation of isolated facialis arteries in vitro and in vivo and increased eNOS-dependent O(2)(*) generation in native endothelial cells on mouse aortas, whereas a control peptide, TSB(Ctr), which has the four glutamic acids in TSB2 substituted with alanine, showed no such effects. Site-directed mutagenesis of eNOS at 310, 314, 318, and 323 Glu to Ala yields an eNOS mutant that exhibited reduced hsp90 association and generated O(2)(*) rather than *NO upon activation. Together, these data demonstrate that hsp90 associates with eNOS at aa 310-323. Moreover, a decoy peptide based on this sequence is sufficient to displace hsp90 from eNOS and uncouple eNOS activity from *NO generation. Thus, Glu-310, Glu-314, Glu-318, and Glu-323 in eNOS, although each does not do much by itself, synergistically they increase "cooperativity" in the association step that is critical for maintaining hsp90-eNOS interactions and promoting coupled eNOS activity. Such chaperone-dependent signaling may play an important role in modulating the balance of *NO and O(2)(*) generation from eNOS and, therefore, vascular function.