Influence of glutaraldehyde fixation of cells adherent to solid substrata on their detachment during exposure to shear stress.

In order to determine the response of fixed and nonfixed cells adherent to a solid substratum to shear stress, human fibroblasts were allowed to adhere and spread on either hydrophilic glass or hydrophobic Fluoroethylene-propylene (FEP-Teflon) and fixed with glutaraldehyde. Then, the cells were exposed to an incrementally loaded shear stress in a parallel plate flow chamber up to shear stresses of about 500 dynes/cm2, followed by exposure to a liquid-air interface passage. The cellular detachment was compared with the one of nonfixed cells. In case of fixed cells, 50% of the adhering cells detached from FEP-Teflon at a shear stress of 350 dynes/cm2, whereas 50% of the adhering, nonfixed cells detached already at a shear stress of 20 dynes/cm2. No fixed cells detached from glass for shear stresses up to at least 500 dynes/cm2. More than 50% of the nonfixed cells were detached from glass at a shear stress of 350 dynes/cm2. Furthermore, the shape and morphology of fixed cells did not change during the incrementally loaded flow, in contrast to the ones of nonfixed cells, which clearly rounded up prior to detachment.     

利用转录组测序筛选低切应力作用下人脐静脉内皮细胞的差异表达基因

目的:通过转录组测序分析获得不同切应力作用下人脐静脉内皮细胞的基因的表达谱,为进一步探索切应力影响内皮细胞形态和功能的机制提供依据。方法:以人脐静脉内皮细胞为材料,通过Streamer系统建立6通道可调控切应力的流体动力学细胞模型,以层流切应力(15 dynes/cm~2)为对照,以低切应力(0.1 dynes/cm~2)为实验组,分别加载细胞18 h。提取总RNA逆转录合成cDNA,建立文库,以二代测序平台Illumina HiSeq中进行扩增和测序。结果:序列比对结果显示,有19986个基因比对上,新转录本分析显示各组新转录本数约占总转录本数的50%。基因表达差异分析显示,较对照组,低切应力组表达上调基因983个,表达下调基因701个。GO分析显示,有18499个基因得到了归类注释,绝大多数基因富集到生物学过程。KEGG分析显示,富集Top20的信号通路与细胞周期、DNA复制和细胞分裂、细胞应激和凋亡等生物学过程相关。结论:低切应力作用不仅仅激活内皮细胞中细胞的增殖相关基因,同时也涉及到DNA损伤修复和凋亡相关基因。     

Influence of Fluid Shear and Microbubbles on Bacterial Detachment from a Surface

Prevention of microbial adhesion and detachment of adhering microorganisms from surfaces is important in many environmental, industrial, and medical applications. Fluid shear is an obvious parameter for stimulating microbial detachment from surfaces, but recently it has been pointed out that a passing air-liquid interface also has potential in stimulating microbial detachment. In the present study, the ability of microbubbles to stimulate detachment of bacterial strains from a glass surface is compared with the effects of fluid flow. Adhesion and detachment of Actinomyces naeslundii T14V-J1, Streptococcus oralis J22, and their coadhering aggregates were studied on glass, mounted in a parallel plate flow chamber. High fluid wall shear rates (11,000 to 16,000 s⁻¹) were established in a laminar flow regime in the absence and presence of microbubbles. Wall shear rates stimulated detachment ranging from 70% to 30% for S. oralis and A. naeslundii, respectively. Coadhering aggregates were detached up to 54%. The presence of microbubbles in the flow increased the detachment of A. naeslundii within 2 min of flow from 40% in the absence of microbubbles to 98%, while detachment of neither S. oralis nor coadhering aggregates was affected by the presence of microbubbles. In summary, extremely high fluid flows can be effective in stimulating microbial detachment, depending on the strain involved. The addition of microbubbles to the flow allows the detachment of tenaciously adhering bacteria not detached by flow alone, but not of adhering coaggregates.     

Transglutaminase stabilizes melanoma adhesion under laminar flow

To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-crosslinkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (≥ 32.5 dynes/cm²). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm²). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked¹²⁵I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.     

Patterns of Living β-Actin Movement in Wounded Human Coronary Artery Endothelial Cells Exposed to Shear Stress

We previously demonstrated that physiologic levels of shear stress enhance endothelial repair. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate (Albuquerque et al., 2000, Am. J. Physiol. Heart Circ. Physiol. 279, H293–H302). However, the patterns and movements of β-actin filaments responsible for cell motility and translocation in human coronary artery endothelial cells (HCAECs) have not been previously investigated under physiologic flow. HCAECs transfected with β-actin-GFP were cultured on type I collagen-coated coverslips. Confluent cell monolayers were subjected to laminar shear stress of 12 dynes/cm² for 18 h in a parallel-plate flow chamber to attain cellular alignment and then wounded by scraping with a metal spatula and subsequently exposed to a laminar shear stress of 20 dynes/cm² (S-W-sH) or static (S-W-sT) conditions. Time-lapse imaging and deconvolution microscopy was performed during the first 3 h after imposition of S-W-sH or S-W-sT conditions. The spatial and temporal dynamics of β-actin-GFP motility and translocation during wound closure in HCAEC monolayers were analyzed under both conditions. Compared with HCAEC under S-W-sT conditions, our data show that HCAEC under S-W-sH conditions demonstrated greater β-actin-GFP motility, filament and clumping patterns, and filament arcs used during cellular attachment and detachment. These findings demonstrate intriguing patterns of β-actin organization and movement during wound closure in HCAEC exposed to physiological flow.     

Mitosis and cytokinesis in subconfluent endothelial cells exposed to increasing levels of shear stress

An in vitro flow apparatus in combination with cultured endothelium was used to determine the effects of fluid-generated shear stress on cells undergoing mitosis and cytokinesis. Cell responses were recorded by time-lapse video microscopy under phase contrast or Hoffman modulation contrast optics. Completion of cell division in mitotic cells was dependent upon both the initial presence of intercellular attachments and the magnitude of fluid wall shear stress. In nonisolated populations, 95.3%, 69.5%, and 57.1% of the cells completed cell division as opposed to 66.6%, 20.4%, and 11.9% in the isolated cell groups at 2.8, 14.1, and 33 dynes/cm², respectively. Prestressing cells for 14 h prior to monitoring failed to increase retention of isolated mitotic cells. The presence of neighboring cells facilitated replication by providing an anchoring attachment or a luminal surface for completion of division. Cell detachment most commonly occurred at the onset of cytokinesis when substrate contact areas were minimal and focal contacts were absent. A comparison between no flow controls and shear stress specimens indicated no significant differences in transit times for mitosis and cytokinesis. Thus, subconfluent endothelial cells may be more susceptible to detachment during cell division due to increases in shear stress, the absence of intercellular attachments, and reduced cell-substrate contacts. © 1994 wiley-Liss, Inc.     

Analysis of Bacterial Detachment from Substratum Surfaces by the Passage of Air-Liquid Interfaces

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (Streptococcus sobrinus HG1025, Streptococcus oralis J22, Actinomyces naeslundii T14V-J1, Bacteroides fragilis 793E, and Pseudomonas aeruginosa 974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 × 10⁶ cells cm⁻² was reached. For S. sobrinus HG1025, S. oralis J22, and A. naeslundii T14V-J1, the conditioning film consisted of adsorbed salivary components, while for B. fragilis 793E and P. aeruginosa 974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with P. aeruginosa 974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s⁻¹), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high air bubble velocities, and spherical strains (i.e., streptococci) detached more efficiently than rod-shaped organisms. The present results demonstrate that methodologies to study bacterial adhesion which include contact with a moving air-liquid interface (i.e., rinsing and dipping) yield detachment of an unpredictable number of adhering microorganisms. Hence, results of studies based on such methodologies should be referred as “bacterial retention” rather than “bacterial adhesion”.     

Influence of hydrodynamic imposed shear stress on the activation of factor X in the presence of tissue factor-factor VIIa complex in a continuous flow reactor

Summary The effect of shear stress on the ability of tissue factor-factor VIIa complex to activate factor X in a continuous flow reactor was studied. Tissue factor immobilized in a phospholipid bilayer on the inner surface of a capillary tube was exposed to a perfusate containing factors VIIa and X flowing at flow rates of 12.7, and 204 l/min, corresponding to wall shear rates of 100, and 1760 sec^-1. The maximum flux (moles formed per unit surface area per unit time) of factor Xa (activated form of factor X) produced at the wall decreased as the shear stress at the wall was increased from 1 to 3 dynes/cm² (3-fold) at a constant shear rate of 100 sec^-1. In contrast, at higher shear rate (1760 sec^-1), increasing shear stress from 16 to 48 dynes/cm² had no significant influence on factor Xa production. The decreased production of factor Xa at higher shear stress (low shear rate 100 sec^-1) probably reflects the transport limitation of factor X to the wall. Apparently shear stress can directly influence the activation of factor X at low shear rates.     

Relationships between Binding Quality of Meat and Myofibrillar Proteins

The viscosity change of myosin A concentrated solution with or without other components was measured as the incubation time elapsed at 30°C.

The viscosity of myosin A solution increased, but that of F-actin solution did not. The shear stress at 0.04 sec^?1 was not increased to 1.0 dyne/cm² in the former, but in the latter was below 0.5 dyne/cm².

The viscosity of myosin B solution increased slightly, but that of native tropomyosin-free myosin B solution decreased remarkably. In both the shear stress at 0.04 sec^?1 was greater than or equal to 15 dynes/cm².

The speed of the viscosity increase in the presence of 3 mm pyrophosphate and 3 mm MgCl₂ was higher in concentrated solution of myosin B than in that of native tropomysin-free myosin B. The shear stress at 0.04 sec^?1 after 6 hr at 30°C was 11.5 and 8.2 dynes/cm², respectively.

The effect of native tropomyosin and actin on the viscosity change was discussed.     

Shear sensitivity of cultured hybridoma cells (CRL-8018) depends on mode of growth,culture age and metabolite concentration

Samples of CRL-8018 hybridoma cultures were subjected to well-defined laminar shear in a Couette viscometer. Exposure of the samples to increasing levels of shear stress (0–50 dynes cm⁻² for 10 min) or times of exposure to shear (50 dynes cm⁻² for 0–10 min) resulted in higher levels of cellular damage and death. Cell death in the viscometer was shown to exhibit trends similar to cell death caused by excessive agitation in spinner flasks, suggesting that viscometric shear can be used to model in a more reproducible way some of the fluid mechanical aspects of damage to cells caused by agitation. Cells cultured with low levels of fluid stresses (T-flask and slowly stirred spinner cultures) were more sensitive to shear than cells from rapidly agitated cultures. Also, cells from either the lag or stationary phases of batch cultures were more sensitive to mechanical damage than exponentially growing cells. Accumulation of ammonia and changes in pH of the batch culture can contribute to this increase in shear sensitivity.     

流体剪切力对内皮细胞miR-21和miR-199a表达的影响

为探讨流体剪切力对内皮细胞micorRNAs表达的影响。采用旋转锥形圆盘剪切力系统对内皮细胞分别加载低(4dyn/cm2)、中(10 dyn/cm2)和高(15 dyn/cm2)3种不同梯度的剪切力作用24h。对照组未加载剪切力。采用高通量筛选芯片检测microRNAs表达变化,qRT-PCR验证,并进行生物信息学分析。与对照组比较,低剪切力组表达差异的microRNAs有33个(FC1.5或0.5倍,P0.05),其中28个上调,5个下调;中剪切力组表达差异的microRNAs有8个(FC1.5或0.5倍,P0.05),其中6个上调,2个下调;高剪切力组表达差异的microRNAs有31个(FC1.5或0.5倍,P0.05),其中25个上调,6个下调。miR-21在高剪切力组中上调最显著(FC=0.026),在低剪切力组中显著下调(FC=3.531)。miR-199a在低剪切力组中上调最显著(FC=0.075),在高剪切力组中显著下调(FC=3.031)。表达差异的microRNA的靶基因主要与内皮细胞的力学信号转导、细胞跨膜迁移、钙离子信号通路、细胞内吞作用等相关。流体剪切力可诱导内皮细胞miR-21和miR-199a表达发生改变。     

Use of moving aeration membrane bioreactor for the efficient production of tissue type plasminogen activator in serum free medium

A moving aeration-membrane (MAM) bioreactor was employed for the production of 2 μg/mL of tissue type Plasminogen Activator (tPA) in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating conditions, shear stress was as low as 0.65 dynes/cm² at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2 μg tPA/mL while maintaining a high cell density of 1.0×10⁷ viable cells/mL.     

Differential adhesion,activity, and carbohydrate: Protein ratios ofPseudomonas atlantica monocultures attaching to stainless steel in a linear shear gradient

Biofilm formation on metallic surfaces in marine and freshwater environments often precedes corrosion and other biofouling conditions. Attachment is mediated by such environmental factors as the presence of surface conditioning films, fluid dynamics, bulk-phase nutrient levels, and surface chemistry. In this study, we utilized a Fowler Cell Adhesion Measurement Module to demonstrate that the changes in cellular concentration and composition of a monoculture ofPseudomonas atlantica biofilms on stainless steel were a function of the applied shear force. At shear forces in the range of 3–10 dynes cm^–2 (1.0 liter min^–1), attachment as measured by acridine orange direct microscopic counts was greatest at the higher shear forces.¹⁴C-Acetate uptake activity on the stainless steel surfaces increased with shear stress. Acetate incorporation ranged from 1×10^–5 to 19×10^–5 mol cm^–2 between 0.15 and 30 dynes cm^–2 for 30 min uptake periods. On a per cell basis, however, activity decreased with shear, indicating a shift in metabolism. Fourier transform infrared spectroscopy revealed that protein and carbohydrate concentrations also increased with the applied shear. Increased biofilm CN ratios and total fatty acids were associated with the higher shear stresses. Neither radius of interaction nor biofilm age appeared to significantly influence the relationship between fluid shear and attachment and cellular composition ofP. atlantica biofilms in the range of 1–10 dynes cm^–2.     

Fibroblast growth factor-2 binding to the endothelial basement membrane peaks at a physiologically relevant shear stress

Fibroblast growth factor-2 (FGF2) is produced and released by endothelial cells and binds to heparan sulfate proteoglycans in the endothelial basement membrane (BM), an important FGF2 storage reservoir. Experimental and computational models of FGF2 binding kinetics to both cells and BM under static conditions are well established in the literature but remain largely unexplored under flow. We now examine BM-FGF2 binding kinetics in fluid flow conditions. We hypothesized that FGF2 binding to the endothelial BM would decrease as fluid shear stress increased. To investigate this, BM-FGF2 equilibrium, associative, and dissociative bindings were measured at various shear stresses. Surprisingly, FGF2 binding increased up to a physiological arterial shear stress of 25 dynes/cm², after which it decreased to a level similar to the 1 dyne/cm² condition. Both BM-FGF2 dissociation and BM binding site availability increased with flow, while association remained constant. This suggests that force-dependent FGF2 equilibrium binding varies with shear stress due to a combination of an increase in binding site availability and FGF2 dissociation with flow. This improved understanding of BM-FGF2 binding with flow enriches current knowledge of FGF2 binding kinetics under physiologic conditions, which may contribute to improved growth factor therapy development.     

Influence of fluid shear and microbubbles on bacterial detachment from a surface

Prevention of microbial adhesion and detachment of adhering microorganisms from surfaces is important in many environmental, industrial, and medical applications. Fluid shear is an obvious parameter for stimulating microbial detachment from surfaces, but recently it has been pointed out that a passing air-liquid interface also has potential in stimulating microbial detachment. In the present study, the ability of microbubbles to stimulate detachment of bacterial strains from a glass surface is compared with the effects of fluid flow. Adhesion and detachment of Actinomyces naeslundii T14V-J1, Streptococcus oralis J22, and their coadhering aggregates were studied on glass, mounted in a parallel plate flow chamber. High fluid wall shear rates (11,000 to 16,000 s(-1)) were established in a laminar flow regime in the absence and presence of microbubbles. Wall shear rates stimulated detachment ranging from 70% to 30% for S. oralis and A. naeslundii, respectively. Coadhering aggregates were detached up to 54%. The presence of microbubbles in the flow increased the detachment of A. naeslundii within 2 min of flow from 40% in the absence of microbubbles to 98%, while detachment of neither S. oralis nor coadhering aggregates was affected by the presence of microbubbles. In summary, extremely high fluid flows can be effective in stimulating microbial detachment, depending on the strain involved. The addition of microbubbles to the flow allows the detachment of tenaciously adhering bacteria not detached by flow alone, but not of adhering coaggregates.     

LFA-1/ICAM-3 mediates neutrophil homotypic aggregation under fluid shear stress

We found that human neutrophils undergo homotypic aggregation by loading the physiological range of fluid shear stress (12–30 dynes/cm²). Under the fluid shear stress, an increase of intracellular Ca²⁺ concentration of neutrophils was observed. This increase of intracellular Ca²⁺ concentration was caused by Ca²⁺ influx, and the blockage of the flux by NiCl₂ suppressed the neutrophil homotypic aggregation. Furthermore, this neutrophil aggregation under fluid shear stress was completely inhibited by pretreatment with antibody against LFA-1 or ICAM-3. These results suggested that NiCl₂-sensitive Ca²⁺ channel played an important role in LFA-1/ICAM-3-mediated neutrophil homotypic aggregation under fluid shear stress. © 1996 Wiley-Liss, Inc.     

The responses of osteoblasts to fluid shear stress depend on substrate chemistries

Natural bone tissue receives chemical and mechanical stimuli in physiological environment. The effects of material chemistry alone and mechanical stimuli alone on osteoblasts have been widely investigated. This study reports the synergistic influences of material chemistry and flow shear stress (FSS) on biological functions of osteoblasts. Self-assembled monolayers (SAMs) on glass slides with functional groups of OH, CH₃, and NH₂ were employed to provide various material chemistries, while FSS (12 dynes/cm²) was produced by a parallel-plate fluid flow system. Material chemistry alone had no obvious effects on the expressions of ATP, nitric oxide (NO), and prostaglandin E₂ (PGE₂), whereas FSS stimuli alone increased the production of those items. When both material chemistry and FSS were loaded, cell proliferation and the expressions of ATP, NO and PGE₂ were highly dependent on the material chemistry. Examination of the focal adhesion (FA) formation and F-actin organization of osteoblasts before FSS exposure indicates that the FA formation and F-actin organization followed similar chemistry-dependence. The inhibition of FAs and/or disruption of F-actins eliminated the material dependence of FSS-induced ATP, PGE₂ and NO release. A possible mechanism is proposed: material chemistry controls the F-actin organization and FA formation of osteoblasts, which further modulates FSS-induced cellular responses.     

Fluid shear stress stimulates breast cancer cells to display invasive and chemoresistant phenotypes while upregulating PLAU in a 3D bioreactor

Breast cancer cells experience a range of shear stresses in the tumor microenvironment (TME). However most current in vitro three-dimensional (3D) models fail to systematically probe the effects of this biophysical stimuli on cancer cell metastasis, proliferation, and chemoresistance. To investigate the roles of shear stress within the mammary and lung pleural effusion TME, a bioreactor capable of applying shear stress to cells within a 3D extracellular matrix was designed and characterized. Breast cancer cells were encapsulated within an interpenetrating network hydrogel and subjected to shear stress of 5.4 dynes cm⁻² for 72 hr. Finite element modeling assessed shear stress profiles within the bioreactor. Cells exposed to shear stress had significantly higher cellular area and significantly lower circularity, indicating a motile phenotype. Stimulated cells were more proliferative than static controls and showed higher rates of chemoresistance to the anti-neoplastic drug paclitaxel. Fluid shear stress-induced significant upregulation of the PLAU gene and elevated urokinase activity was confirmed through zymography and activity assay. Overall, these results indicate that pulsatile shear stress promotes breast cancer cell proliferation, invasive potential, chemoresistance, and PLAU signaling.     

Shear inactivation of cellulase of Trichoderma reesei

Inactivation of the cellulase of Trichoderma reesei (EC 3.2.1.4) by shear, is of sufficient magnitude to merit consideration in the design of equipment for the enzymatic hydrolysis of cellulose. The inac inactivation constant, k_d, is a function of the flow rate of the enzyme solution through a fine capillary tube. k_d increased slowly at low shear stress, and much more rapidly when the shear stress was greater than 15 dynes cm^?2.