Different Levels Of Shear Stress Research Articles

Predicting the rutting parameters of nanosilica/waste denim fiber composite asphalt binders using the response surface methodology and machine learning methods

It is challenging to predict the mechanical properties of modified asphalt binders because of their complex non-linear viscoelastic behavior. This study evaluates and compares the feasibility of using the response surface methodology (RSM) and machine learning (ML) methods to predict the shear strain, accumulated shear strain, non-recoverable creep compliance (Jnr), and percentage of recovery (%R) of the base binder, nanosilica (NS)-modified, waste denim fiber (WDF)-modified, and NS/WDF composite asphalt binders. The study conducts an extensive investigation using ML algorithms to accurately predict the multiple stress creep recovery (MSCR) rutting parameters for the base and modified asphalt binders. The RSM statistical analysis revealed that the %NS and %WDF significantly affect the shear strain, accumulated shear strain, Jnr, and %R at different levels of shear stress within the 95% confidence interval. Besides, the RSM-based predictive models have correlation coefficients (R2) greater than 0.8 for all responses, indicating an adequate consistency between the predicted MSCR parameters by the developed models and the parameters from the experimental work. Analysis of the ML models shows that the Extreme Gradient Boosting regression (XGB regression) is among the most accurate models for predicting the shear strain and accumulated strain. Of the evaluated ML models, Decision Tree Regression (DTR) shows the best performance in predicting Jnr and %R, with the highest R2 of 0.99 and smallest root mean square error (RMSE) of <1%, which indicates its ability to represent the experimental MSCR parameters accurately. Evaluation of the XGB regression and DTR performance shows that the developed ML models outperform the RSM in predicting the MSCR rutting parameters.

On Escherichia coli Resistance to Fluid Shear Stress and Its Significance for Water Disinfection

Alternative water treatment techniques are needed to overcome the limitations of chemical disinfectants. Stemming from recent findings which point to high levels of shear stress induced by flow as the cause of microbial removal in water, we conducted systematic experiments on bacterial solutions in well-controlled hydrodynamic conditions to evaluate the effect of different levels of shear stress on the viability of Escherichia coli. We investigated a wide range of shear stresses (57–4240 Pa) using viscous substrates prepared by mixing a bacterial solution with thickeners (2-hydroxyethyl cellulose and/or guar gum). Substrate samples were tested for up to 60 min in a laminar shear flow at a constant temperature using a rotational rheometer equipped with a cone-plate measuring system so that the whole sampling volume was exposed to the same shear stress. Results show that, contrary to previous studies, high shear stresses (i.e., of order 103 Pa) do not induce inactivation or lysis of E. coli, even for prolonged exposure times. Stemming from our results and a thorough discussion of the literature on E. coli mechanical lysis and modeling cell dynamics, we infer that E. coli can resist high shear forces because of stress relaxation in a wide range of hydrodynamic conditions.

QS2: DIY Bioreactors for Tissue Engineering - An Ultra-low-cost Platform for Perfusion Cell Culture and Live Imaging

Purpose:While tissue engineering offers the promise of revolutionary innovation, scalable three-dimensional tissue culture is limited by the diffusion of nutrients and oxygen making media perfusion obligatory. Unfortunately, the cost of bioreactors for large construct tissue culture can be prohibitive, with a typical perfusion chamber costing several thousand dollars, and even small petri-dish-sized devices costing hundreds of dollars each. We have developed a low-cost perfusion setup that seals collagen-based perfusable cellular constructs within a sterile PDMS well between coverslips, allowing for repeated live-imaging of perfused 3D engineered tissues. Herein we describe fabrication of this novel system and validate its utility.Methods:Molds and frames were designed on 3D-modeling software (Fusion 360) and printed on a Prusa i3 MK3S 3D printer in poly(lactic acid) (PLA). Molds were filled with poly(dimethyl siloxane) (PDMS), which was cured to form chambers, bubble traps, mason jar lid chambers, and media reservoir lid adapters. In total, the tissue culture chamber device, mason jar lid inset, media reservoir lid, and bubble trap require 4, 1, 2, and 4 unique printed components, respectively.Results:Each perfusion chamber can be assembled for under 8 USD per device and reused repeatedly. The current model has a tissue chamber custom-built with 18x10x4 (LxWxH)mm3 dimensions, but this chamber can be readily customized to experiment-specific dimensions. These devices allow cellular hydrogel constructs to be maintained in a sterile environment after assembly, perfused at varying rates to expose cells to different levels of shear stress, and the cells can be intermittently imaged with light, fluorescent or confocal microscopy - an unparalleled benefit for monitoring of experiments and collection of timepoint imaging data. The perfusion circuit consists of autoclavable glass and PDMS components, including a bubble trap, a crucial component of the circuit for preventing air bubbles that can damage cells and block microchannels, and a lid adapter, which allows 50 mL conical tubes to serve as self-oxygenating media reservoirs. Media changes can be performed via peristaltic pump perfusion or with syringe-based cell culture techniques for static culture. Constructs have been perfused within standard incubators for up to 14 days demonstrating normal cell viability without contamination or evidence of infection, with longer perfusion culture intervals (>1 month) currently being tested.Conclusion:The increasing accessibility of 3D-modeling and 3D-printing has enabled rapid prototyping of devices to address the problems that we face as surgeon-scientists. We have developed a low-cost tissue-engineering perfusion circuit that facilitates 3D-tissue-culture while allowing for repeated live-imaging as the cultured tissue develops. The instructions for our setup can be utilized to replicate our devices in other labs, and these designs can be readily customized to meet the needs of specific experimental aims.

Effect of low wall shear stress on the morphology of endothelial cells and its evaluation indicators

ObjectiveThis study is designed to explore the morphological changes of endothelial cells (ECs) under different levels of shear stress and find the effective evaluation indicators with in vivo and in vitro experiments. MethodsHuman umbilical vein endothelial cells (HUVECs) and Sprague–Dawley rats were used to study the effect of different shear stress which applied by means of parallel plate-flow chamber and abdominal aorta banding model on the morphology of endothelial cells. Then, fluorescence images were acquired by means of a confocal laser-scanning microscope. Finally, Cell morphological indicators were extracted by SRAD-MCW computer image processing algorithm for quantitative analysis. Results1) The morphological changes of HUVECs were observed after exposure to shear stress for 6 h, the HUVECs were elongated and spindle-shaped. And the degree of cell deformability was different while the exposure time was different, then it became stable after 18 h. The HUVECs exposure to high shear stress (HSS) exhibited an ordered cell arrangement, while the HUVECs exposure to low shear stress (LSS) showed a disordered cell arrangement. 2) Traditional cell morphological indicators such as area, perimeter, long axis diameter, short axis diameter and orientation angle were not significantly different between the normal shear stress (NSS) group and the LSS group (P > 0.05), but the intercellular space characteristics such as the junction length per unit area and the triple points per unit area were significantly different (P < 0.05). ConclusionThese results demonstrate that the size and duration of shear stress can affect the morphology and arrangement of endothelial cells. The commonly used evaluation indicators for studying the effect of shear stress on the morphology of endothelial cells, including area, perimeter, long axis diameter, short axis diameter and orientation angle, etc., had no significant statistical significance, while the intercellular space characteristics including the junction length per unit area and the triple points per unit area can be used as effective indicator to study the effect of shear stress on the morphology of endothelial cells.

Characterization of the competing role of surface-contact and shear stress on platelet activation in the setting of blood contacting devices.

Supraphysiological shear stress and surface-contact are recognized as driving mechanisms of platelet activation (PA) in blood contacting devices (BCDs). However, the competing role of these mechanisms in triggering thrombogenic events is poorly understood. Here, we characterized the dynamics of PA in response to the combined effect of shear stress and material exposure. Human platelets were stimulated with different levels of shear stress (500, 750, 1000 dynes/cm2) over a range of exposure times (10, 20, and 30 min) within capillary tubes made of various polymeric materials. Polyethylene (PE), polytetrafluoroethylene (PTFE), ethylene tetrafluoroethylene (ETFE), and polyether ether ketone (PEEK), used for BCDs fabrication, were investigated as compared to glass and thromboresistant Sigma™-coated glass. PA was quantified using the Platelet Activity State assay. Our results indicate that mechanical stimulation and polymer surface-contact both significantly contribute to PA. Notably, the contribution of the mechanical stimulus ranges between +36% and +43%, while that associated with polymer surface-contact ranges from +48% to +59%, depending on the exposure time. In more detail, our results indicate that: (i) PA increases with increasing shear stress magnitude; (ii) PA has a non-linear, time-dependent relationship to exposure time; (iii) PA is largely influenced by biomaterials, with PE and PEEK having respectively the lowest and highest prothrombotic potential; (iv) the effects of polymer surface-contact and shear stress are not correlated and can be studied separately. Our results suggest the importance of incorporating the evaluation of platelet activation driven by the combined effect of shear stress and polymer surface-contact for the comprehensive assessment, and eventually minimization, of BCDs thrombogenic potential.

Shear Stress-Dependent Targeting Efficiency Using Self-Assembled Gelatin-Oleic Nanoparticles in a Biomimetic Microfluidic System.

Cellular properties and microenvironments, as well as the characteristics of nanoparticles (NPs), affect the cellular uptake and cytotoxic effects of drug-loaded NPs. Since there is fluid flow in the human blood system, fluid flow also affects the drug delivery efficiency of NPs. This study aimed to evaluate the cellular behaviors of drug-loaded soft NPs on A549 cancer cells under different levels of shear stress (0.5, 5, and 50 dynes/cm2) in the biomimetic microfluidic system. The soft self-assembled NPs were formed by the gelatin–oleic conjugate (GOC). The poorly water-soluble coumarin-6 or paclitaxel (PTX) were used as model markers for encapsulation within self-assembled NPs (C-GONs or PTX-GONs, respectively). The cellular uptake of C-GONs was found to be improved with shear-stress dependence. The inhibitory concentration (IC50) of PTX-GONs at 0.5, 5, and 50 dynes/cm2 was 0.106 µg/mL, 0.108 µg/mL, and 0.091 µg/mL, respectively, as compared to 0.138 µg/mL in a static condition. The cell killing efficiency of PTX-GONs was increased in the highest shear stress of 50 dynes/cm2 in the static condition, and other levels of shear stress in dynamic conditions.

High-performance microwave dielectric composite ceramics sintered at low temperature without sintering-aids

(1-x)Li2(Mg0.96Ni0.04)SiO4+xLiZn0.93Co0.07PO4 composite ceramics were synthesised through solid-state reaction, and their sintering kinetics, microstructure and microwave dielectric properties were investigated. The perfect disconnection model was applied to explain the variation in grain growth with different x values and sintering temperatures. X-ray diffraction, scanning electron microscopy, thermo-mechanical analysis and network analysis were used to determine the macro and micro properties of the composite ceramics. Results showed that LiZn0.93Co0.07PO4 ceramic could promote grain growth and grain boundary modification, due to the different levels of shear stress and free energy density of the Si-rich and P-rich phases. The shrinkage onset point of the composite ceramics was adjusted to achieve full densification at 900 °C. The peak microwave dielectric properties are εr = 5.78, Q × f = 48,168 GHz at 16 GHz and τf = −44.7 ppm/°C when x = 0.5, sintering temperature is 900 °C and relative density is 96.1%. Therefore, mixing LiZn0.93Co0.07PO4 (pre-sintered) and Li2(Mg0.96Ni0.04)SiO4 (pre-sintered) ceramics is a feasible synthesis method for microwave dielectric materials with excellent sintering and dielectric properties.

Blood Pressure Predicts Endothelial Function and Effect of Ethinyl Estradiol in Young Obese and Lean Women

Obesity, mild hypertension and endothelial function are all predictors of cardiovascular disease in women, and we propose that these factors are interrelated. We hypothesized that obesity and high blood pressure are associated with endothelial dysfunction in young women. Further, we hypothesized that short‐term low‐dose ethinyl estradiol treatment would mitigate this dysfunction. We examined hyperemic responses before and during 30 μg/day oral ethinyl estradiol (EE) treatment for 7 days in 16 healthy women (mean 26 ± 5, 19–35 yrs) with normal (NBP; n=7, MAP range: 78–91 mmHg) or mildly elevated blood pressure (HBP; n=9, MAP range: 95–113 mmHg). We evaluated brachial vasodilatory responses following two stimuli designed to elicit different levels of shear stress: 5‐min occlusion (RH‐FMD), and 3‐min occlusion with ischemic handgrip exercise (IE‐FMD). Body composition did not impact RH‐FMD or IE‐FMD responses. In contrast, Pre‐EE, HBP had lower IE‐FMD responses relative to NBP (8.0 ± 3.5% vs. 12.3 ± 3.2%, respectively; P=0.05). During EE, IE‐FMD increased in HBP (12.8 ± 6.1%; P=0.02) and decreased in NBP (5.6 ± 2.4%, P&lt;0.01). The increase in shear stress (8659 ± 4964 vs. 14753 ± 8189 AUC, s−1; P=0.04) contributed to the increase in IE‐FMD in HBP subjects during EE, while shear stress did not change with EE in NBP (9740 ± 3222 vs. 10564 ± 2984 AUC, s−1; P=0.76). MAP did not predict RH‐FMD responses or associated shear stress, pre‐or during‐EE. Thus, 1) in young healthy women, moderately elevated blood pressure was a predictor of endothelial function whereas obesity was not; 2) EE mitigated endothelial dysfunction associated with elevated blood pressure; 3) the enhanced shear stress stimulus was able to subtle differences in endothelial function where the standard FMD stimulus did not. Because HBP women had only mildly high blood pressure, these data are of particular significance given the new American Heart Association guidelines for hypertension.Support or Funding InformationSupported by the APS Undergraduate Research Excellence Fellowship; the Paul Titus Fellowship Fund, Dept. Obstetrics Gynecology and Reproductive Sciences, Yale School of Medicine; and the AHA Postdoctoral Fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Assessment of ‘on-treatment platelet reactivity’ and relationship with cerebral micro-embolic signals in asymptomatic and symptomatic carotid stenosis

IntroductionThe relationship between on-treatment platelet reactivity and cerebral micro-embolic signals (MES) is unknown, and has not been previously simultaneously assessed in asymptomatic and symptomatic carotid stenosis patients. MethodsConsecutive eligible patients with ≥50% asymptomatic or recently symptomatic carotid stenosis (≤4weeks following TIA/ischaemic stroke) were recruited to this pilot study. Symptomatic patients were followed up to the ‘late’ phase (≥3months) following symptom onset or carotid intervention; longitudinal data were analysed from symptomatic patients with data available at both time-points. Platelet function/reactivity was assessed with the PFA-100® to measure collagen-ADP (C-ADP) and collagen-epinephrine (C-EPI) closure times in citrate-anticoagulated whole blood. Bilateral simultaneous 1-hour transcranial Doppler ultrasound (TCD) monitoring of the middle cerebral arteries was performed to classify patients as MES +ve or MES −ve. Results31 patients with ≥50% asymptomatic and 46 with early symptomatic carotid stenosis or occlusion were included. 35 symptomatic patients were followed up to the late phase (23 following carotid intervention). Prevalence of ‘high on-treatment platelet reactivity’ (HTPR) on the C-EPI cartridge did not differ between asymptomatic and symptomatic patients overall, but was lower in ‘symptomatic post-intervention’ than asymptomatic patients on aspirin monotherapy (10% vs. 50%; p=0.03). The prevalence of HTPR on the C-EPI cartridge decreased between the early and late phases in symptomatic patients (63% vs. 34%; p=0.017), including those on aspirin monotherapy (p=0.016). There were no significant differences in HTPR status between asymptomatic vs. early or late symptomatic MES +ve or MES −ve patients. DiscussionCarotid interventional treatment, presumably in combination with resolution of the acute phase response, may decrease the prevalence of HTPR in patients with recently symptomatic carotid stenosis over time. Preliminary subgroup analysis suggests that successful intervention may reduce the prevalence of aspirin-HTPR in symptomatic patients to lower levels than asymptomatic medically-treated patients on aspirin monotherapy. Larger, longitudinal studies are warranted to reassess the impact of more intensive secondary preventive treatment on ex vivo platelet function at different levels of shear stress in carotid stenosis patients.

Controlling Shear Stress in 3D Bioprinting is a Key Factor to Balance Printing Resolution and Stem Cell Integrity.

A microvalve-based bioprinting system for the manufacturing of high-resolution, multimaterial 3D-structures is reported. Applying a straightforward fluid-dynamics model, the shear stress at the nozzle site can precisely be controlled. Using this system, a broad study on how cell viability and proliferation potential are affected by different levels of shear stress is conducted. Complex, multimaterial 3D structures are printed with high resolution. This work pioneers the investigation of shear stress-induced cell damage in 3D bioprinting and might help to comprehend and improve the outcome of cell-printing studies in the future.

Hydrodynamic Effects on Drug Dissolution and Deaggregation in the Small Intestine—A Study with Felodipine as a Model Drug

The aim of this study was to understand and predict the influence of hydrodynamic effects in the small intestine on dissolution of primary and aggregated drug particles. Dissolution tests of suspensions with a low-solubility drug, felodipine, were performed in a Couette cell under hydrodynamic test conditions corresponding to the fed small intestine. Dissolution was also performed in the USP II apparatus at two paddle speeds of 25 and 200 rpm and at different surfactant concentrations below critical micelle concentration. The experimental dissolution rates were compared with theoretical calculations. The different levels of shear stress in the in vitro tests did not influence the dissolution of primary or aggregated particles and experimental dissolution rates corresponded very well to calculations. The dissolution rate for the aggregated drug particles increased after addition of surfactant because of deaggregation, but there were still no effect of hydrodynamics. In conclusion, hydrodynamics do not influence dissolution and deaggregation of micronized drug particles in the small intestine of this model drug. Surface tension has a strong effect on the deaggregation and subsequent dissolution. Addition of surfactants at in vivo relevant surface tension levels is thus critical for in vivo predictive in vitro dissolution testing.

Arterial specification of endothelial cells derived from human induced pluripotent stem cells in a biomimetic flow bioreactor

Endothelial cells (ECs) exist in different microenvironments in vivo, including under different levels of shear stress in arteries versus veins. Standard stem cell differentiation protocols to derive ECs and EC-subtypes from human induced pluripotent stem cells (hiPSCs) generally use growth factors or other soluble factors in an effort to specify cell fate. In this study, a biomimetic flow bioreactor was used to subject hiPSC-derived ECs (hiPSC-ECs) to shear stress to determine the impacts on phenotype and upregulation of markers associated with an anti-thrombotic, anti-inflammatory, arterial-like phenotype. The in vitro bioreactor system was able to efficiently mature hiPSC-ECs into arterial-like cells in 24 h, as demonstrated by qRT-PCR for arterial markers EphrinB2, CXCR4, Conexin40 and Notch1, as well protein-level expression of Notch1 intracellular domain (NICD). Furthermore, the exogenous addition of soluble factors was not able to fully recapitulate this phenotype that was imparted by shear stress exposure. The induction of these phenotypic changes was biomechanically mediated in the shear stress bioreactor. This biomimetic flow bioreactor is an effective means for the differentiation of hiPSC-ECs toward an arterial-like phenotype, and is amenable to scale-up for culturing large quantities of cells for tissue engineering applications.

Numerical Simulations and Experiments on Changes in Erythrocyte Morphology under Continue Flow Ventricular Assisted Devices

Ventricular assist devices (VADs) implanted in human body produce different levels of shear stress due to mechanical structure, causing damage for erythrocytes. The study is focusing on the morphology changes of erythrocytes causing by continue flow VADs. This study used a Hemodynamic Shearing Device (Thermo Electron Corporation) to expose erythrocytes to shear stress produced by VADs. Then free hemoglobin of plasma is measured and blood smears are respectively made to count the number of abnormal erythrocytes. The results show that the correlation coefficient of the percent of abnormal erythrocytes (PAE) and shear stress is 0.725 (p=0.027, &lt;0.05). After blood shearing experiments the number of abnormal erythrocytes has increased with shear stress under constant exposure time, and both shear stress and exposure time contribute to morphology changes in erythrocytes. The conclusion indicates that low shear stress (lower than 450 Dynes/cm2) damage of erythrocyte is an important factor in the application of VADs.

Side-specific mechanical properties of valve endothelial cells.

Aortic valve endothelial cells (ECs) function in vastly different levels of shear stress. The biomechanical characteristics of cells on each side of valve have not been investigated. We assessed the morphology and mechanical properties of cultured or native valve ECs on intact porcine aortic valve cusps using a scanning ion conductance microscope (SICM). The autocrine influence of several endothelial-derived mediators on cell compliance and the expression of actin were also examined. Cells on the aortic side of the valve are characterized by a more elongated shape and were aligned along a single axis. Measurement of EC membrane compliance using the SICM showed that the cells on the aortic side of intact valves were significantly softer than those on the ventricular side. A similar pattern was seen in cultured cells. Addition of 10(-6) M of the nitric oxide donor sodium nitroprusside caused a significant reduction in the compliance of ventricular ECs but had no effect on cells on the aortic side of the valve. Conversely, endothelin-1 (10(-10)-10(-8) M) caused an increase in the compliance of aortic cells but had no effect on cells on the ventricular side of the valve. Aortic side EC compliance was also increased by 10(-4) M of the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester. Immunofluorescent staining of actin filaments revealed a great density of staining in ECs on the ventricular surface. The expression of actin and the relative membrane compliance of ECs on both side of the valve were not affected by ventricular and aortic patterns of flow. This study has shown side-specific differences in the biomechanics of aortic valve ECs. These differences can have important implications for valve function.

Examination of the role of transient receptor potential vanilloid type 4 in endothelial responses to shear forces.

Shear stress is the major mechanical force applied on vascular endothelial cells by blood flow, and is a crucial factor in normal vascular physiology and in the development of some vascular pathologies. The exact mechanisms of cellular mechano-transduction in mammalian cells and tissues have not yet been elucidated, but it is known that mechanically sensitive receptors and ion channels play a crucial role. This paper describes the use of a novel and efficient microfluidic device to study mechanically-sensitive receptors and ion channels in vitro, which has three independent channels from which recordings can be made and has a small surface area such that fewer cells are required than for conventional flow chambers. The contoured channels of the device enabled examination of a range of shear stresses in one field of view, which is not possible with parallel plate flow chambers and other previously used devices, where one level of flow-induced shear stress is produced per fixed flow-rate. We exposed bovine aortic endothelial cells to different levels of shear stress, and measured the resulting change in intracellular calcium levels ([Ca(2+)]i) using the fluorescent calcium sensitive dye Fluo-4AM. Shear stress caused an elevation of [Ca(2+)]i that was proportional to the level of shear experienced. The response was temperature dependant such that at lower temperatures more shear stress was required to elicit a given level of calcium signal and the magnitude of influx was reduced. We demonstrated that shear stress-induced elevations in [Ca(2+)]i are largely due to calcium influx through the transient receptor potential vanilloid type 4 ion channel.

Structure, composition, and strength of nitrifying membrane-aerated biofilms

Membrane-aerated biofilm reactors (MABRs) are a novel technology based on the growth of biofilms on oxygen-permeable membranes. Hereby, MABRs combine all the advantages of biofilm growth with a more flexible and efficient control of the oxygen load. In the present work, MABR flow cells were operated to achieve full nitrification. MABR biofilms had a significantly different structure than co-diffusion biofilms reported in the literature. Different levels of shear stress and oxygen loading during MABR operation also affected the biofilm parameters. Furthermore, reactor operation at higher oxygen loads resulted in an increased biofilm cohesiveness, which depended on the EPS mass in the biofilms and the type of stress applied (more cohesive against normal than shear stresses). The EPS in the strongest biofilms had a higher content of proteins and a lower level of carbohydrates. Staining analyses revealed that the outermost EPS in the stronger biofilm regions was of hydrophilic nature and distributed around dense microbial aggregates, whereas it was homogeneously distributed in the weaker strata. Overall, the obtained results provide input parameters to future modelling efforts and operating conditions to support more robust autotrophic N conversions in MABRs.

Shear stress modulates macrophage-induced urokinase plasminogen activator expression in human chondrocytes

IntroductionSynovial macrophages, which can release proinflammatory factors, are responsible for the upregulation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). In addition, shear stress exerts multifunctional effects on chondrocytes by inducing the synthesis of catabolic or anabolic genes. However, the interplay of macrophages, chondrocytes, and shear stress during the regulation of cartilage function remains poorly understood. We investigated the mechanisms underlying the modulation of human chondrocyte urokinase plasminogen activator (uPA) expression by macrophages and shear stress.MethodsHuman chondrocytes were stimulated by peripheral blood-macrophage- conditioned medium (PB-MCM), or exposure of chondrocytes cultured in PB-MCM to different levels of shear stress (2 to 20 dyn/cm2). Real-time polymerase chain reaction was used to analyze uPA gene expression. Inhibitors and small interfering RNA were used to investigate the mechanism for the effects of PB-MCM and shear stress in chondrocytes.ResultsStimulation of human chondrocytes with PB-MCM was found to induce uPA expression. We demonstrated that activation of the JNK and Akt pathways and NF-κB are critical for PB-MCM-induced uPA expression. Blocking assays by using IL-1ra further demonstrated that IL-1β in PB-MCM is the major mediator of uPA expression in chondrocytes. PB-MCM-treated chondrocytes subjected to a lower level of shear stress showed inhibition of MCM-induced JNK and Akt phosphorylation, NF-κB activation, and uPA expression. The PB-MCM-induced uPA expression was suppressed by AMP-activated protein kinase (AMPK) agonist. The inhibitor or siRNA for AMPK abolished the shear-mediated inhibition of uPA expression.ConclusionsThese data support the hypothesis that uPA upregulation stimulated by macrophages may play an active role in the onset of OA and in the shear-stress protection against this induction.

Gene expression of single human mesenchymal stem cell in response to fluid shear

Stem cell therapy may rely on delivery and homing through the vascular system to reach the target tissue. An optical tweezer model has been employed to exert different levels of shear stress on a single non-adherent human bone marrow–derived mesenchymal stem cell to simulate physiological flow conditions. A single-cell quantitative polymerase chain reaction analysis showed that collagen type 1, alpha 2 (COL1A2), heat shock 70-kDa protein 1A (HSPA1A) and osteopontin (OPN) are expressed to a detectable level in most of the cells. After exposure to varying levels of shear stress, there were significant variations in gene transcription levels across human mesenchymal stem cells derived from four individual donors. Significant trend towards upregulation of COL1A2 and OPN gene expression following shear was observed in some donors with corresponding variations in HSPA1A gene expression. The results indicate that shear stress associated with vascular flow may have the potential to significantly direct non-adherent stem cell expression towards osteogenic phenotypic expression. However, our results demonstrate that these results are influenced by the selection process and donor variability.

Shear stress influences the pluripotency of murine embryonic stem cells in stirred suspension bioreactors

Pluripotent embryonic stem cells (ESCs) have been used increasingly in research as primary material for various tissue-engineering applications. Pluripotency, or the ability to give rise to all cells of the body, is an important characteristic of ESCs. Traditional methods use leukaemia inhibitory factor (LIF) to maintain murine embryonic stem cell (mESC) pluripotency in static and bioreactor cultures. When LIF is removed from mESCs in static cultures, pluripotency genes are downregulated and the cultures will spontaneously differentiate. Recently we have shown the maintenance of pluripotency gene expression of mESCs in stirred suspension bioreactors during differentiation experiments in the absence of LIF. This is undesired in a differentiation experiment, where the goal is downregulation of pluripotency gene expression and upregulation of gene expression characteristic to the differentiation. Thus, the objective of this study was to examine how effectively different levels of shear stress [100 rpm (6 dyne/cm(2) ), 60 rpm (3 dyne/cm(2) )] maintained and influenced pluripotency in suspension bioreactors. The pluripotency markers Oct-4, Nanog, Sox-2 and Rex-1 were assessed using gene expression profiles and flow-cytometry analysis and showed that shear stress does maintain and influence the gene expression of certain pluripotency markers. Some significant differences between the two levels of shear stress were seen and the combination of shear stress and LIF was observed to synergistically increase the expression of certain pluripotency markers. Overall, this study provides a better understanding of the environmental conditions within suspension bioreactors and how these conditions affect the pluripotency of mESCs.

Abstract 4566: Dynamic biochemical tissue analysis: A novel in situ assay that demonstrates potential as a diagnostic/prognostic for cancer

Abstract Work in the field of cell adhesion has undeniably shown that the full spectrum of molecular bond properties is germane to biological recognition and communication in physiologic and pathologic conditions, including cancer. For example, the unique kinetic and tensile properties of E-selectin bonds allow it to mediate cancer cell adhesion to the endothelium and leukocyte recruitment to sites of inflammation. Typically in a biochemical tissue analysis (e.g., immunohistochemistry), a molecular probe (e.g., antibody) detects antigens by utilizing only one property of the probe-antigen bond, specifically affinity, and other aspects of the bond (e.g., dynamic biophysical properties) remain unexploited. Therefore, we developed a dynamic biochemical tissue analysis (DBTA) that allows exquisite control over the interaction between the probe and the antigen to enhance characterization of molecular recognition in situ. Specifically, we conjugated a recombinant E-selectin construct to polystyrene microspheres and used the resulting microspheres as the probe to investigate E-selectin ligand activity on colon cancer, breast cancer, and normal tissues using DBTA. When E-selectin microspheres were perfused over invasive colon adenocarcinoma tissue sections at a wall shear stress of 0.80 dynes/ cm2, the microspheres exhibited robust adhesion with the tissue. The adhesion of E-selectin microspheres was significantly higher than that of negative control (IgG conjugated) microspheres, demonstrating the specificity of interaction of E-selectin microspheres with the tissue sections. To demonstrate that modulating easily controlled assay parameters results in discernible changes in adhesion, E-selectin microspheres of 10 and 15 µm diameters were perfused over the colon adenocarcinoma tissue sections at different levels of shear stress, achieved by varying the volumetric flow rate. The results showed that with increasing shear stress or particle diameter, the adhesion of E-selectin microspheres to the tissue sections decreased as predicted. To demonstrate that DBTA can be used for high throughput analysis, colon and breast cancer tissue microarrays were used. These microarray slides consisted of tissues from various histopathological classifications. E-selectin microspheres exhibited higher adhesion than the IgG microspheres to the majority of the cancer tissues, showing the preferential expression of functional E-selectin ligands on cancer tissues. Combined the results (i) convincingly demonstrate the specificity of the DBTA assay, (ii) reveal the critical role physical factors play in tissue analysis and (iii) demonstrate that DBTA allows facile control over these key physical factors. Thus, we have established a new approach for characterizing tissue that will lead to novel diagnostic and prognostic assays for a large spectrum of pathologies including cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4566. doi:1538-7445.AM2012-4566