Magnitude Of Fluid Shear Stress Research Articles

A modular microfluidic platform to study how fluid shear stress alters estrogen receptor phenotype in ER+ breast cancer cells

Metastatic breast cancer leads to poor prognoses and worse outcomes in patients due to its invasive behavior and poor response to therapy. It is still unclear what biophysical and biochemical factors drive this more aggressive phenotype in metastatic cancer; however recent studies have suggested that exposure to fluid shear stress in the vasculature could cause this. In this study a modular microfluidic platform capable of mimicking the magnitude of fluid shear stress (FSS) found in human vasculature was designed and fabricated. This device provides a platform to evaluate the effects of FSS on MCF-7 cell line, an estrogen receptor positive (ER+) breast cancer cell line, during circulation in the vessels. Elucidation of the effects of FSS on MCF-7 cells was carried out utilizing two approaches: single cell analysis and bulk analysis. For single cell analysis, cells were trapped in a microarray after exiting the serpentine channel and followed by immunostaining on the device (on-chip). Bulk analysis was performed after cells were collected in a microtube at the outlet of the microfluidic serpentine channel for western blotting (off-chip). It was found that cells exposed to an FSS magnitude of 10 dyn/cm2 with a residence time of 60 s enhanced expression of the proliferation marker Ki67 in the MCF-7 cell line at a single cell level. To understand possible mechanisms for enhanced Ki67 expression, on-chip and off-chip analyses were performed for pro-growth and survival pathways ERK, AKT, and JAK/STAT. Results demonstrated that after shearing the cells phosphorylation of p-AKT, p-mTOR, and p-STAT3 were observed. However, there was no change in p-ERK1/2. AKT is a mediator of ER rapid signaling, analysis of phosphorylated ERα was carried out and no significant differences between sheared and non-sheared populations were observed. Taken together these results demonstrate that FSS can increase phosphorylation of proteins associated with a more aggressive phenotype in circulating cancer cells. These findings provide additional information that may help inform why cancer cells located at metastatic sites are usually more aggressive than primary breast cancer cells.

Human dermal microvascular endothelial cell morphological response to fluid shear stress

As the cells that line the vasculature, endothelial cells are continually exposed to fluid shear stress by blood flow. Recent studies suggest that the morphological response of endothelial cells to fluid shear stress depends on the endothelial cell type. Thus, the present study characterizes the morphological response of human dermal microvascular endothelial cells (HMEC-1) and nuclei to steady, laminar, and unidirectional fluid shear stress. Cultured HMEC-1 monolayers were exposed to shear stress of 0.3 dyn/cm2, 16 dyn/cm2, or 32 dyn/cm2 for 72 h with hourly live-cell imaging capturing both the nuclear and cellular morphology. Despite changes in elongation and alignment occurring with increasing fluid shear stress, there was a lack of elongation and alignment over time under each fluid shear stress condition. Conversely, changes in cellular and nuclear area exhibited dependence on both time and fluid shear stress magnitude. The trends in cellular morphology differed at shear stress levels above and below 16 dyn/cm2, whereas the nuclear orientation was independent of fluid shear stress magnitude. These findings show the complex morphological response of HMEC-1 to fluid shear stress.

Tubular flow activates magnesium transport in the distal convoluted tubule.

Magnesium (Mg2+) is an important cofactor of many enzymes crucial for life; therefore, maintaining a Mg2+ balance in the body is essential. In the kidney, the distal convoluted tubule (DCT) determines the final urinary Mg2+ excretion. The nephron is subjected to variable urinary flow, but little is known about the influence of flow on Mg2+ transport. Primary cilia, which are mechanosensory organelles that sense changes in flow, are expressed on tubular epithelial cells. This study aimed to elucidate whether urinary flow facilitates DCT Mg2+ transport. To this end, mouse DCT15 cells, with and without primary cilia, were exposed to physiologic fluid flow generating 0.3, 0.6, and 1.2 dyn/cm2 fluid shear stress (FSS). FSS stimulated Mg2+ uptake significantly. Net Mg2+ uptake ( i.e., the difference between static and FSS) followed a single component saturable first-order transport function and was independent of FSS magnitude and primary cilia. FSS did not affect the expression of magnesiotropic genes, including Cnnm2, Kcna1, Proegf, Trpm6, and Trpm7. Transient receptor potential cation channel subfamily melastatin (TRPM) member 7 (Trmp7) inhibition by 2-aminoethyl diphenyl borinate or knockout of TRPM6 did not alter net Mg2+ uptake, suggesting that TRPM6/TRPM7 homo/heterodimeric channels are not involved in FSS-activated Mg2+ transport. In summary, FSS generated by physiologic fluid flow is a new factor activating Mg2+ transport in DCT independent of primary cilia.-Verschuren, E. H. J., Hoenderop, J. G. J., Peters, D. J. M., Arjona, F. J., Bindels, R. J. M. Tubular flow activates magnesium transport in the distal convoluted tubule.

NOTCH1 is a mechanosensor in adult arteries

Endothelial cells transduce mechanical forces from blood flow into intracellular signals required for vascular homeostasis. Here we show that endothelial NOTCH1 is responsive to shear stress, and is necessary for the maintenance of junctional integrity, cell elongation, and suppression of proliferation, phenotypes induced by laminar shear stress. NOTCH1 receptor localizes downstream of flow and canonical NOTCH signaling scales with the magnitude of fluid shear stress. Reduction of NOTCH1 destabilizes cellular junctions and triggers endothelial proliferation. NOTCH1 suppression results in changes in expression of genes involved in the regulation of intracellular calcium and proliferation, and preventing the increase of calcium signaling rescues the cell–cell junctional defects. Furthermore, loss of Notch1 in adult endothelium increases hypercholesterolemia-induced atherosclerosis in the descending aorta. We propose that NOTCH1 is atheroprotective and acts as a mechanosensor in adult arteries, where it integrates responses to laminar shear stress and regulates junctional integrity through modulation of calcium signaling.

Structural optimization design of perfusion bioreactors with multilayer circular parallel plates scaffold

Perfusion bioreactors with multilayer circular parallel plates scaffold have the potential to provide a considerable scalability in cells’ cultivation for tissue engineering. By now, relatively little attention has been focused on the uniform distribution of fluid shear stress (FSS) among the parallel plates. However, the magnitude of FSS has a significant effect on both proliferation and differentiation of cells. This paper analyses the relationship between the structural parameters of the scaffold and the distribution of FSS among the plates and then presents an approach to optimize the flow field within the bioreactors. By regulating the structural parameters of the scaffold, the uniform distribution of FSS on the 23-layers plates can be achieved. The resolution of FSS can be up to 0.001 Pa and the range of error restrained within ±0.00015 Pa. By means of structural optimization, the discrepancy of the flow field among the plates can be effectively diminished.

Alterations in cancer cell mechanical properties after fluid shear stress exposure: a micropipette aspiration study.

Over 90% of cancer deaths result not from primary tumor development, but from metastatic tumors that arise after cancer cells circulate to distal sites via the circulatory system. While it is known that metastasis is an inefficient process, the effect of hemodynamic parameters such as fluid shear stress (FSS) on the viability and efficacy of metastasis is not well understood. Recent work has shown that select cancer cells may be able to survive and possibly even adapt to FSS in vitro. The current research seeks to characterize the effect of FSS on the mechanical properties of suspended cancer cells in vitro. Nontransformed prostate epithelial cells (PrEC LH) and transformed prostate cancer cells (PC-3) were used in this study. The Young’s modulus was determined using micropipette aspiration. We examined cells in suspension but not exposed to FSS (unsheared) and immediately after exposure to high (6,400 dyn/cm2) and low (510 dyn/cm2) FSS. The PrEC LH cells were ~140% stiffer than the PC-3 cells not exposed to FSS. Post-FSS exposure, there was an increase of ~77% in Young’s modulus after exposure to high FSS and a ~47% increase in Young’s modulus after exposure to low FSS for the PC-3 cells. There was no significant change in the Young’s modulus of PrEC LH cells post-FSS exposure. Our findings indicate that cancer cells adapt to FSS, with an increased Young’s modulus being one of the adaptive responses, and that this adaptation is specific only to PC-3 cells and is not seen in PrEC LH cells. Moreover, this adaptation appears to be graded in response to the magnitude of FSS experienced by the cancer cells. This is the first study investigating the effect of FSS on the mechanical properties of cancer cells in suspension, and may provide significant insights into the mechanism by which some select cancer cells may survive in the circulation, ultimately leading to metastasis at distal sites. Our findings suggest that biomechanical analysis of cancer cells could aid in identifying and diagnosing cancer in the future.

Bone morphogenetic protein-4 and transforming growth factor-beta1 mechanisms in acute valvular response to supra-physiologic hemodynamic stresses.

To explore ex vivo the role of bone morphogenetic protein-4 (BMP-4) and transforming growth factor-beta1 (TGF-β1) in acute valvular response to fluid shear stress (FSS) abnormalities. Porcine valve leaflets were subjected ex vivo to physiologic FSS, supra-physiologic FSS magnitude at normal frequency and supra-physiologic FSS frequency at normal magnitude for 48 h in a double-sided cone-and-plate bioreactor filled with standard culture medium. The role of BMP-4 and TGF-β1 in the valvular response was investigated by promoting or inhibiting the downstream action of those cytokines via culture medium supplementation with BMP-4 or the BMP antagonist noggin, and TGF-β1 or the TGF-β1 inhibitor SB-431542, respectively. Fresh porcine leaflets were used as controls. Each experimental group consisted of six leaflet samples. Immunostaining and immunoblotting were performed to assess endothelial activation in terms of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expressions, paracrine signaling in terms of BMP-4 and TGF-β1 expressions and extracellular matrix (ECM) remodeling in terms of cathepsin L, cathepsin S, metalloproteinases (MMP)-2 and MMP-9 expressions. Immunostained images were quantified by normalizing the intensities of positively stained regions by the number of cells in each image while immunoblots were quantified by densitometry. Regardless of the culture medium, physiologic FSS maintained valvular homeostasis. Tissue exposure to supra-physiologic FSS magnitude in standard medium stimulated paracrine signaling (TGF-β1: 467% ± 22% vs 100% ± 6% in fresh controls, BMP-4: 258% ± 22% vs 100% ± 4% in fresh controls; P < 0.05) and ECM degradation (MMP-2: 941% ± 90% vs 100% ± 19% in fresh controls, MMP-9: 1219% ± 190% vs 100% ± 16% in fresh controls, cathepsin L: 1187% ± 175% vs 100% ± 12% in fresh controls, cathepsin S: 603% ± 88% vs 100% ± 13% in fresh controls; P < 0.05), while BMP-4 supplementation also promoted fibrosa activation and TGF-β1 inhibition reduced MMP-9 expression to the native tissue level (MMP-9: 308% ± 153% with TGF-β1 inhibition vs 100% ± 16% in fresh control; P > 0.05). Supra-physiologic FSS frequency had no effect on endothelial activation and paracrine signaling regardless of the culture medium but TGF-β1 silencing attenuated FSS-induced ECM degradation via MMP-9 downregulation (MMP-9: 302% ± 182% vs 100% ± 42% in fresh controls; P > 0.05). Valvular tissue is sensitive to FSS abnormalities. The TGF-β1 inhibitor SB-431542 is a potential candidate molecule for attenuating the effects of FSS abnormalities on valvular remodeling.

Defining the Role of Fluid Shear Stress in the Expression of Early Signaling Markers for Calcific Aortic Valve Disease

Calcific aortic valve disease (CAVD) is an active process presumably triggered by interplays between cardiovascular risk factors, molecular signaling networks and hemodynamic cues. While earlier studies demonstrated that alterations in fluid shear stress (FSS) on the fibrosa could trigger inflammation, the mechanisms of CAVD pathogenesis secondary to side-specific FSS abnormalities are poorly understood. This knowledge could be critical to the elucidation of key CAVD risk factors such as congenital valve defects, aging and hypertension, which are known to generate FSS disturbances. The objective of this study was to characterize ex vivo the contribution of isolated and combined abnormalities in FSS magnitude and frequency to early valvular pathogenesis. The ventricularis and fibrosa of porcine aortic valve leaflets were exposed simultaneously to different combinations of sub-physiologic/physiologic/supra-physiologic levels of FSS magnitude and frequency for 24, 48 and 72 hours in a double cone-and-plate device. Endothelial activation and paracrine signaling were investigated by measuring cell-adhesion molecule (ICAM-1, VCAM-1) and cytokine (BMP-4, TGF-β1) expressions, respectively. Extracellular matrix (ECM) degradation was characterized by measuring the expression and activity of the proteases MMP-2, MMP-9, cathepsin L and cathepsin S. The effect of the FSS treatment yielding the most significant pathological response was examined over a 72-hour period to characterize the time-dependence of FSS mechano-transduction. While cytokine expression was stimulated under elevated FSS magnitude at normal frequency, ECM degradation was stimulated under both elevated FSS magnitude at normal frequency and physiologic FSS magnitude at abnormal frequency. In contrast, combined FSS magnitude and frequency abnormalities essentially maintained valvular homeostasis. The pathological response under supra-physiologic FSS magnitude peaked at 48 hours but was then maintained until the 72-hour time point. This study confirms the sensitivity of valve leaflets to both FSS magnitude and frequency and suggests the ability of supra-physiologic FSS levels or abnormal FSS frequencies to initiate CAVD mechanisms.

The Exploitation and Simulation of a Microfluidic Device Research on the Response of Cell Fluid Shear Stress

This paper provides an economical and high efficient kind of microfluidic chip device which can investigate cells' response under different magnitude of fluid shear stress (FSS) at the same time. The experimental results of a large number of test groups in the same environmental conditions are more persuasive for conclusion. Cells are simultaneously cultured in different width of micro channel, and we verified the cell's shear force with a COMSOL simulation. In the experiment, Human vascular endothelial cell which possess apparent FSS response is selected to verify the reliability of the device. The results of the experiment show that cell response to FSS has obvious morphological differences in different test group.

Examining the Lateral Displacement of HL60 Cells Rolling on Asymmetric P-Selectin Patterns

The lateral displacement of cells orthogonal to a flow stream by rolling on asymmetrical receptor patterns presents a new opportunity for the label-free separation and analysis of cells. Understanding the nature of cell rolling trajectories on such substrates is necessary to the engineering of substrates and the design of devices for cell separation and analysis. Here, we investigate the statistical nature of cell rolling and the effect of pattern geometry and flow shear stress on cell rolling trajectories using micrometer-scale patterns of biomolecular receptors with well-defined edges. Leukemic myeloid HL60 cells expressing the PSGL-1 ligand were allowed to flow across a field of patterned lines fabricated using microcontact printing and functionalized with the P-selectin receptor, leveraging both the specific adhesion of this ligand-receptor pair and the asymmetry of the receptor pattern inclination angle with respect to the fluid shear flow direction (α = 5, 10, 15, and 20°). The effects of the fluid shear stress magnitude (τ = 0.5, 1, 1.5, and 2.0 dyn/cm(2)), α, and P-selectin incubation concentration were quantified in terms of the rolling velocity and edge tracking length. Rolling cells tracked along the inclined edges of the patterned lines before detaching and reattaching on another line. The detachment of rolling cells after tracking along the edge was consistent with a Poisson process of history-independent interactions. Increasing the edge inclination angle decreased the edge tracking length in an exponential manner, contrary to the shear stress magnitude and P-selectin incubation concentration, which did not have a significant effect. On the basis of these experimental data, we constructed an empirical model that predicted the occurrence of the maximum lateral displacement at an edge angle of 7.5°. We also used these findings to construct a Monte Carlo simulation for the prediction of rolling trajectories of HL60 cells on P-selectin-patterned substrates with a specified edge inclination angle. The prediction of lateral displacement in the range of 200 μm within a 1 cm separation length supports the feasibility of label-free cell separation via asymmetric receptor patterns in microfluidic devices.

A quantitative study on morphological responses of osteoblastic cells to fluid shear stress

Fluid shear stress (FSS) is widely explored regarding its influence on osteoblasts. In vitro studies have shown that the cytoskeleton is very important in cellular responses to FSS. However, morphological changes, which would reflect the cytoskeleton changes as well as other cellular responses, were rarely quantitatively studied in the past years. Therefore, FSS-induced morphological changes in osteoblasts were quantified in this study. Real-time rapid morphological responses were observed by exposing osteoblasts to FSS with magnitude of 1.2, 1.6, and 1.9 Pa for 1 h. Afterward, osteoblast actin cytoskeleton was labeled with rhodamine phalloidin and observed using fluorescence microscopy. The results showed that 1.6 and 1.9 Pa FFS resulted in significant cellular elongation and reorientation along the direction of fluid flow. Besides, along with the enhancement of FSS magnitude, cytoskeleton aggregated more remarkably. Furthermore, extracellular Ca(2+)-depleted fluid flow was also used to stimulate osteoblasts for 1 h with magnitude of 1.6 and 1.9 Pa. No morphological change was observed after removing extracellular calcium. Our study suggested that the level of FSS from 1.2 to 1.9 Pa is capable of influencing cellular morphology, and extracellular calcium might play a role in osteoblasts' response to FSS stimulation.

Quantifying fluid shear stress in a rocking culture dish

Fluid shear stress (FSS) is an important stimulus for cell functions. Compared with the well established parallel-plate and cone-and-plate systems, a rocking “see-saw” system offers some advantages such as easy operation, low cost, and high throughput. However, the FSS spatiotemporal pattern in the system has not been quantified. In the present study, we developed a lubrication-based model to analyze the FSS distributions in a rocking rectangular culture dish. We identified an important parameter (the critical flip angle) that dictates the overall FSS behaviors and suggested the right conditions to achieving temporally oscillating and spatially relatively uniform FSS. If the maximal rocking angle is kept smaller than the critical flip angle, which is defined as the angle when the fluid free surface intersects the outer edge of the dish bottom, the dish bottom remains covered with a thin layer of culture medium. The spatial variations of the peak FSS within the central 84% and 50% dish bottom are limited to 41% and 17%, respectively. The magnitude of FSS was found to be proportional to the fluid viscosity and the maximal rocking angle, and inversely proportional to the square of the fluid depth-to-length ratio and the rocking period. For a commercial rectangular dish (length of 37.6mm) filled with ∼2mL culture medium, the FSS at the center of the dish bottom is expected to be on the order of 0.9dyn/cm2 when the dish is rocked +5° at 1cycle/s. Our analysis suggests that a rocking “see-saw” system, if controlled well, can be used as an alternative method to provide low-magnitude, dynamic FSS to cultured cells.

Fluid Shear Stress Magnitude, Duration, and Total Applied Load Regulate Gene Expression and Nitric Oxide Production in Primary Calvarial Osteoblast Cultures

Successful bone engineering requires an understanding of the effects of mechanical stress on osteoblast differentiation. Therefore, we examined the effects of varying magnitude and duration of fluid shear stress on factors associated with osteoblastic differentiation. Using a cone viscometer, primary neonatal rat calvarial osteoblasts were exposed to continuous fluid shear stress at varying doses: 0.21, 0.43, and 0.85 Pa for varying time periods. Gene expression was analyzed using Northern blots and nitric oxide production was quantified with the colorimetric Griess reaction. Fluid shear stress stimulated comparable transient increases in TGF-beta1 and TGF-beta3 expression by 3 hours. TGF-beta1 expression returned to baseline by 12 hours at all shear doses. In contrast, TGF-beta3 expression decreased by 22 percent and 47 percent at 12 hours in response to 0.43 Pa and 0.85 Pa, respectively. Osteopontin and Msx-2 expression patterns were consistent with a more differentiated phenotype at all shear levels. The maximum level of shear stress increased nitric oxide production 2.5-fold at 12 hours and 6.0-fold at 24 hours. These data demonstrate differential regulation of TGF-beta1 and TGF-beta3 isoforms with fluid shear stress. Furthermore, because osteopontin and Msx-2 changes were consistent with progressive differentiation at all levels of shear stress, dosage appears to be less important than the presence of an effective physical stimulus. Lastly, nitric oxide does not appear to be the primary regulator of early transcriptional changes found in this study.

Secondary current effects on cohesive river bank erosion

The emphasis of this research was placed on the development of a methodology that allows (1) an adequate representation of the distribution of the near‐bank shear stress τs when secondary currents are present and (2) estimation of the critical erosional strength τcr and other sediment erodibility parameters for fluvial erosion. These two factors combine to form the overarching objective of this investigation, which was carried out in Union Flat Creek, a stream with pronounced cross‐sectional irregularities located within the loess region of the Palouse basin of Washington State, United States. The study has shown that the presence of the secondary currents increases the magnitude of the depth‐averaged sidewall shear stress at least by a factor of 2.0. It was also found that the ratio of the maximum to the depth‐averaged sidewall shear stress is greater than 5. These findings suggest therefore that use of the may be a good approximation of the fluid shear stress magnitude for simple channel geometries but not for natural channels characterized by complex geometries. The measured critical erosional strength (τcr = 4.16 Pa) was comparable with the findings of other laboratory and field studies that also focused on Palouse sediments.