Flow-induced mechano-modulation of intestinal permeability on chip.

Endothelial cell (EC) morphology and functions can be highly impacted by the mechanical stresses that the cells experience in vivo. In most areas in the vasculature, ECs are continuously exposed to unsteady blood flow-induced shear stress and vasodilation-contraction-induced tensile stress/strain simultaneously. Investigations on how ECs respond to combined shear stress and tensile strain will help us to better understand how an altered mechanical environment affects EC mechanotransduction, dysfunction, and associated cardiovascular disease development. In the present study, a programmable shearing and stretching device that can apply dynamic fluid shear stress and cyclic tensile strain simultaneously to cultured ECs was developed. Flow and stress/strain conditions in the device were simulated using a fluid structure interaction (FSI) model. To characterize the performance of this device and the effect of combined shear stress-tensile strain on EC morphology, human coronary artery ECs (HCAECs) were exposed to concurrent shear stress and cyclic tensile strain in the device. Changes in EC morphology were evaluated through cell elongation, cell alignment, and cell junctional actin accumulation. Results obtained from the numerical simulation indicated that in the "in-plane" area of the device, both fluid shear stress and biaxial tensile strain were uniform. Results obtained from the in vitro experiments demonstrated that shear stress, alone or combined with cyclic tensile strain, induced significant cell elongation. While biaxial tensile strain alone did not induce any appreciable change in EC elongation. Fluid shear stress and cyclic tensile strain had different effects on EC actin filament alignment and accumulation. By combining various fluid shear stress and cyclic tensile strain conditions, this device can provide a physiologically relevant mechanical environment to study EC responses to physiological and pathological mechanical stimulation.

Ultrasound contrast agents inside a microvessel, when driven by ultrasound, oscillate and induce mechanical stresses on the vessel wall. These mechanical stresses can produce beneficial therapeutic effects but also induce vessel rupture if the stresses are too high. Therefore, it is important to use sufficiently low pressure amplitudes to avoid rupturing the vessels while still inducing the desired therapeutic effects. In this work, we developed a comprehensive three-dimensional model of a confined microbubble inside a vessel while considering the bubble shell properties, blood viscosity, vessel wall curvature and the mechanical properties of the vessel wall. Two bubble models with the assumption of a spherical symmetric bubble and a simple asymmetrical bubble were simulated. This work was validated with previous experimental results and enabled us to evaluate the microbubbles’ behaviour and the resulting mechanical stresses induced on the vessel walls. In this study, the fluid shear and circumferential stresses were evaluated as indicators of the mechanical stresses. The effects of acoustical parameters, vessel viscoelasticity and rigidity, vessel/bubble size and off-centre bubbles on bubble behaviour and stresses on the vessel were investigated. The fluid shear and circumferential stresses acting on the vessel varied with time and location. As the frequency changed, the microbubble oscillated with the highest amplitude at its resonance frequency which was different from the resonance frequency of an unbound bubble. The bubble resonance frequency increased as the rigidity of a flexible vessel increased. The fluid shear and circumferential stresses peaked at frequencies above the bubble's resonance frequency. The more rigid the vessels were, the more damped the bubble oscillations. The synergistic effect of acoustic frequency and vessel elasticity had also been investigated since the circumferential stress showed either an increasing trend or a decreasing one versus the vessel rigidity at different acoustic frequencies. When the acoustic pressure was increased from 52 to 680 kPa, the maximum bubble radius increase by 2.5 fold, and the maximum shear and circumferential stress increased by 15.7 and 18.3 fold, respectively. The shear stress was largest when the acoustic frequency was higher (3.25 MHz) and the ratio of the vessel radius to the bubble radius was lower. The circumferential stress was largest when the bubble wall was closer to the vessel wall. An oscillating off-centre bubble forms a mushroom shape with the most damping on the points closest to the vessel wall.

The endothelial monolayer is a signal transduction interface for blood-borne mechanical as well as chemical stimuli. The structural deformation arising from a mechanical stimulus, such as a change of hemodynamic shear stress, is conceptually different from the binding of a hormone or other agonist to its specific receptor, yet both elicit important endothelial signaling responses. Mechanical and chemical signaling appear to use common as well as unique pathways downstream of the initial stimulus. For example, signaling pathways arising from membrane deformation and hormone-receptor coupling may appear to converge through activation of phospholipases1 or nuclear factor κB transcription factor complex2 but later diverge at the level of DNA binding3 (Figure 1). However, simple interpretations of mechanotransduction are confounded by intracellular and pericellular force transfer, principally by the cytoskeleton,4,5⇓ that results in the generation of transduction pathways at multiple sites throughout the cell, ie, decentralized mechanotransduction.6,7⇓ Thus, although flow-induced shear stress acts at the luminal surface of the endothelial monolayer, the forces are transmitted to cell junctions, nuclear structures, basal adhesion sites, and organelles that are structurally interconnected. This spatial organization of intracellular signaling may result in the stimulation of multiple parallel, convergent, and/or divergent mechanotransduction pathways. The mechanism of transduction of a purely mechanical signal to second messenger pathways such as IP3-driven intracellular calcium mobilization8 or mitogen-activated protein kinases (MAPK) activation9 is unknown. Major efforts are therefore underway to understand endothelial mechanotransduction in terms of the initial stimulus, the generation of …

The mechanobiology of circulating tumor cells, detached from an extracellular matrix, is poorly understood. A longstanding idea in cancer biology is that during metastasis, cancer cells shed into the circulation from epithelial organs are inherently fragile and are mechanically destroyed by hemodynamic forces. To the contrary, we have recently shown that malignant cells, from diverse tissue types, are remarkably resistant to brief (millisecond) pulses of high-level fluid shear stress (FSS) that may be encountered in the microenvironment of the circulation as compared to benign cells [1]. FSS resistance is a phenotype conferred by a variety of oncogenic signaling pathways and involves both an enhanced ability of malignant cells to repair plasma membrane damage as well as to rapidly adapt to prior exposure to FSS to resist subsequent pulses of FSS. However, the mechanisms underlying the FSS resistance phenotype and whether these are relevant to cancer cells in tumor tissue or to circulating tumor cells was unknown. Here we show that cancer cells exposed to FSS exhibit an adaptive response, demonstrating an increased stiffness (Young's modulus) as measured by micropipette aspiration [2]. Increased stiffness of cancer cells is associated with reduced damage to the plasma membrane upon subsequent challenges with FSS. To examine the mechanisms underlying this response, we evaluated the Rho-dependent signaling pathway that controls cell contractility. Knockdown of RhoA, but not RhoC sensitize prostate cancer cells to FSS. Pharmacologic inhibition myosin II-based contractility also reduced prostate cancer cell resistance to FSS. To determine if cancer cells that exist in tumor tissue exhibit resistance to FSS, we isolated cell suspensions from mice bearing prostate-specific PTEN and/or p53 mutations and exposed them to repeated pulses of FSS. We found that, compared to wild-type control mice, prostate cells from tumor-bearing mice exhibited resistance to FSS characteristic of established human prostate cancer cell lines, indicating that our findings extend beyond established cell lines. Moreover, extending our findings to human tumors, we acutely isolated cells from PDX tumors from melanoma patients. 3 independently-derived tumors exhibited a characteristic biphasic response curve when exposed to multiple pulses of FSS. Interestingly, brief exposure to Vemurafenib altered the response profile of two of these isolates. To examine whether FSS impacts circulating tumor cells, we treated prostate cancer cell line acutely with blebbistatin, a myosin II inhibitor, and vehicle control and injected these differentially fluorescently-labeled cells along with 15 micron fluorescent beads. The beads lodge in the microvasculature and act as a reference for cell counts. Within one minute after injection, we euthanized the mice and isolated lungs. Labeled cells and beads were counted in frozen sections and the ratio of cells:beads was counted and compared to pre-injection ratios. ~60% of vehicle treated cancer cells survive intact in the lungs whereas only ~40% of blebbistatin-treated cells survive. This suggests that a single pass through the right mouse heart damages circulating tumor cells, but that a mechanism that depends on myosin II promotes survival of these cells. This is the first evidence that FSS resistance is a contributing factor for the survival of circulating tumor cells. This work is supported by NCI grants CA179981&CA196202.

Recoil and Stiffening by Adherent Leukocytes in Response to Fluid Shear

Although load-induced mechanical signals play a key role in bone formation and maintenance of bone mass and structure, the cellular mechanisms involved in the translation of these signals are still not well understood. Recent identification of a novel flow-induced mechanosignaling pathway involving VEGF in osteoblasts and the known VEGF regulation of actin reorganization in various cell types has led us to hypothesize that fluid shear stress-induced Vegf up-regulation underlies the actin cytoskeleton adaptation observed in osteoblasts during mechanotransduction. Our results show that MC3T3-E1 cells secrete significant VEGF in response to 5 h of pulsatile fluid shear stress (PFSS; 5 dynes/cm(2) at 1 Hz), whereas expression of VEGF receptors (VEGFR-1, VEGFR-2, or NRP1) is unaffected. These receptors, in particular VEGFR-2, participate in PFSS-induced VEGF release. Exposure to flow-conditioned medium or exogenous VEGF significantly induces stress fiber formation in osteoblasts that is comparable with PFSS-induced stress fiber formation, whereas VEGF knockdown abrogates this response to PFSS, thereby providing evidence that flow-induced VEGF release plays a role in actin polymerization. Using neutralizing antibodies against the receptors and VEGF isoforms, we found that soluble VEGFs, in particular VEGF(164), play a crucial role in transient stress fiber formation during osteoblast mechanotransduction, most likely through VEGFR-2 and NRP1. Based on these data we conclude that flow-induced VEGF release from osteoblasts regulates osteoblast actin adaptation during mechanotransduction and that VEGF paracrine signaling may provide potent cross-talk among bone cells and endothelial cells that is essential for fracture healing, bone remodeling, and osteogenesis.

Vascular endothelial cells are known to respond to fluid shear stress. To gain insights into the mechanism of flow response by these cells, various types of in vitro devices in which endothelial cells can be cultured under flowing culture medium have been designed. Using such a device, one can apply known levels of (usually laminar) fluid shear stress to cultured endothelial cells. We have made two types of devices: a viscometer-based cone-and-plate flow apparatus and a parallel plate chamber. The cone-and-plate apparatus is used to do biochemical analyses of flow effects on cells while the parallel plate chamber is used to observe dynamic behavior of endothelial cells under flow. We were able to maintain confluent endothelial cell cultures under flow for over a week in the parallel plate flow apparatus. Using this chamber and high resolution time-lapse video microscopy, we studied morphological changes of endothelial cells exposed to different levels of fluid shear stress. We found that endothelial cells in a confluent monolayer exhibited three types of fluid shear stress level-dependent morphological and motile responses within a narrow fluid shear stress range between 0.1–10 dyn/cm2. Endothelial cells cultured under no flow exhibited variable shapes and no preferred orientation of their long cell axes and showed a jiggling motion. When exposed to fluid shear stress levels of below 0.5 dyn/cm2, endothelial cell morphology and motility were not affected. However, when fluid shear stress levels were increased to 2–4 dyn/cm2, they became polygonal and showed increased random-walk activity. Fluid shear stress over 6 dyn/cm2 caused endothelial cells to initially become polygonal and increase their random-walk activity, but they soon became elongated and aligned in the direction of flow. As the cells elongated and aligned, they migrated in the direction of flow. The average velocity of this directed cell migration was less than that of cells moving randomly under the same flow condition at earlier times. These observations indicate that endothelial cells are able to detect and respond to a surprisingly small change in fluid shear stress. It is possible that endothelial cell physiology in vivo is also regulated by small changes in fluid shear stress and that a fluid shear stress change of a few dynes per cm2 within a certain region of an artery could trigger atherogenesis in that particular location.

P 7 Fluid shear stress promotes placental growth factor upregulation in human syncytiotrophoblast through the camp-pka signaling pathway

Background: Interleukin-8 (IL-8) released from mechanosensitive cancer cells plays a key role in the inflammation and metastasis of solid carcinomas. In this study, we have explored IL-8 and its receptors signal transduction process of human ovarian cancer cells under conditions of FSS. Methods: After the fluid shear stress was loaded, LightCycler™ system and quantitative sandwich ELISA were employed to assay the IL-8 mRNA expression and protein production, respectively. IL-8 reporter gene pEGFP1-IL8USCS was constructed for determining IL-8 gene transcriptional activation through gene transfer and flow cytometric analysis. NF-κB nuclear translocation was observed by immunocytofluorescent staining. Western blot was used to examine IκB phosphorylation and degradation. RT-PCR, Northern blot and immunofluorescence were used to determine the expression of a cell-surface chemokine receptor CXCR2 at mRNA and protein levels. Results: IL-8 mRNA expression and protein production had biphasic responses to low shear stress (1.5 dyne/cm 2 ), with the peaks at 1 and 2 h respectively. There was an increase in enhanced green fluorescent protein expression in pEGFP1-IL8USCS-transfected SKOV3 cells subjected to a fluid shear stress of 1.5 dyne/cm 2 for 2 h. Following the application of shear stress of 1.5 dyne/cm 2 , NF-κB p65 became detectable in the cell nuclei, and Phosphorylated IκB in cell lysates increased significantly. CXCR2, which was constitutively present on the surface of SKOV3 cells, increased following exposure to fluid shear stress for 60 min. Conclusions: Fluid shear stress triggered IL-8/CXCR2 signaling of SKOV3 cells is an early gene activation, and the activation can be mediated through NF-κB. This observation suggested that fluid shear stress-induced IL-8 activation and the downstream signal pathways may have an important contribution to the pathogenesis and development of both inflammation and metastasis of ovarian carcinomas.

Fluid shear stress has been shown to be an important regulator of vascular structure and function through its effect on the endothelial cell. We have explored the effect of shear stress on the expression of the heparin-binding growth factors platelet-derived growth factor B chain (PDGF-B) and basic fibroblast growth factor (bFGF) in bovine aortic endothelial cells using a purpose-built cone-plate viscometer. Using morphometric analysis, we have mimicked the endothelial cell shape changes encountered in vivo in response to shear stress and correlated these with changes in gene expression. Steady laminar shear stress of 15 and 36 dyn/cm2 both resulted in endothelial cell shape change, but the higher shear stress induced greater and more uniform alignment in the direction of flow and nuclear protrusion after 24 h. Steady laminar shear stress of both 15 and 36 dyn/cm2 induced a significant 3.9- and 4.2-fold decrease, respectively, in PDGF-B mRNA at 9 h. In contrast, steady laminar shear of 15 dyn/cm2 induced a mild and transient 1.5-fold increase in bFGF mRNA while shear of 36 dyn/cm2 induced a significant 4.8-fold increase at 6 h of shear which remained at 2.9-fold at 9 h. Pulsatile and turbulent shear stress showed the same effect as steady laminar shear stress (all at 15 dyn/cm2 time-average magnitude) on PDGF-B and bFGF mRNA content. Cyclic stretch (20% strain, 20/min) of cells grown on silicone substrate did not significantly affect either PDGF-B or bFGF mRNA levels. These results suggest that expression of each peptide growth factor gene is differentially regulated by fluid shear stress in the vascular endothelial cell. These results may have implications on vascular structure and function in response to hemodynamic forces and present a model for the study of transduction of mechanical stimuli into altered gene expression.

Bioreactor systems, for example, spinner flask and perfusion bioreactors, and cell-seeded three-dimensional (3D)-printed scaffolds are used in bone tissue engineering strategies to stimulate cells and produce bone tissue suitable for implantation into the patient. The construction of functional and clinically relevant bone graft using cell-seeded 3D-printed scaffolds within bioreactor systems is still a challenge. Bioreactor parameters, for example, fluid shear stress and nutrient transport, will crucially affect cell function on 3D-printed scaffolds. Therefore, fluid shear stress induced by spinner flask and perfusion bioreactors might differentially affect osteogenic responsiveness of pre-osteoblasts inside 3D-printed scaffolds. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine fluid shear stress and osteogenic responsiveness of MC3T3-E1 pre-osteoblasts seeded on the scaffolds in the bioreactors using finite element (FE)-modeling and experiments. FE-modeling was used to quantify wall shear stress (WSS) distribution and magnitude inside 3D-printed PCL scaffolds within spinner flask and perfusion bioreactors. MC3T3-E1 pre-osteoblasts were seeded on NaOH surface-modified 3D-printed PCL scaffolds, and cultured in customized static, spinner flask, and perfusion bioreactors up to 7 days. The scaffolds' physicochemical properties and pre-osteoblast function were assessed experimentally. FE-modeling showed that spinner flask and perfusion bioreactors locally affected WSS distribution and magnitude inside the scaffolds. The WSS distribution was more homogeneous inside scaffolds in perfusion than in spinner flask bioreactors. The average WSS on scaffold-strand surfaces ranged from 0 to 6.5 mPa for spinner flask bioreactors, and from 0 to 4.1 mPa for perfusion bioreactors. Surface modification of scaffolds by NaOH resulted in a surface with a honeycomb-like pattern and increased surface roughness (1.6-fold), but decreased water contact angle (0.3-fold). Both spinner flask and perfusion bioreactors increased cell spreading, proliferation, and distribution throughout the scaffolds. Perfusion, but not spinner flask bioreactors more strongly enhanced collagen (2.2-fold) and calcium deposition (2.1-fold) throughout the scaffolds after 7 days compared with static bioreactors, likely due to uniform WSS-induced mechanical stimulation of the cells revealed by FE-modeling. In conclusion, our findings indicate the importance of using accurate FE models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems. Impact Statement The success of cell-seeded three-dimensional (3D)-printed scaffolds depends on cell stimulation by biomechanical/biochemical factors to produce bone tissue suitable for implantation into the patient. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine wall shear stress (WSS) and osteogenic responsiveness of pre-osteoblasts seeded on the scaffolds using finite element (FE)-modeling and experiments. We found that cell-seeded 3D-printed PCL scaffolds within perfusion bioreactors more strongly enhanced osteogenic activity than within spinner flask bioreactors. Our results indicate the importance of using accurate FE-models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems.

Mechanical loads are essential towards maintaining bone mass and skeletal integrity. Such loads generate various stimuli at the cellular level, including cyclic hydraulic pressure (CHP) and fluid shear stress (FSS). To gain insight into the anabolic responses of osteoblasts to CHP and FSS, we subjected MC3T3-E1 preosteoblasts to either FSS (12 dynes/cm(2)) or CHP varying from 0 to 68 kPa at 0.5 Hz. As with FSS, CHP produced a significant increase in ATP release over static controls within 5 min of onset. Cell stiffness examined by atomic force microscopy increased after 15 min of either CHP or FSS stimulation, which was attenuated when extracellular ATP was hydrolyzed with apyrase. As previously shown FSS induced polymerization of actins into stress fibers. However, the microtubule network was completely disrupted under FSS. In contrast, CHP appeared to maintain strong microtubule and f-actin networks. The purinergic signaling was found to be involved in the remodeling of f-actin, but not microtubule. Both CHP and FSS applied for 1 hour increased expression of COX-2. These data indicate that, while CHP and FSS produce similar anabolic responses, these stimuli have very different effects on the cytoskeleton remodeling and could contribute to loss of mechanosensitivity with extended loading.

Quantifying fluid shear stress in a rocking culture dish

Objective Objective To study the expression of several bone markers in osteocytes (MLO-Y4) and osteoblast (MOB) also the effect from fluid shear stress in osteocytes. Methods Reverse transcription-polymerase chain reaction analysis indicate the expression differences between MLO-Y4 cells and MOB cells also before and after fluid shear stress in MLO-Y4 cells. Results MLO-Y4 cells appear to express relatively greater amounts of mRNA for osteocalsin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) but less amounts for osteoprotegerin (OPG) than MOB cells. Although the receptor activator of nuclear factor kappa B ligand (RANKL) mRNA expression is more in MOB, the ratio of RANKL to OPG mRNA is greater in the MLO-Y4 cells. Fluid shear stress up regulates the expression of RANKL and OPN mRNA but down regulates ALP and OCN mRNA expression significantly in MLO-Y4 cells and also make some changes in OPG. Conclusions The properties of MLO-Y4 cell's are more like osteocytes than osteobiast. The expression of ALP,OCN,OPN and the ratio of RANKL/OPG in MLO-Y4 ceils changed when exposed to fluid shear stress, which suggest osteocytes play a very important role in bone remodlling.

Effect of fluid shear stress on the internalization of kidney-targeted delivery systems in renal tubular epithelial cells