A New Flow-Regulating Cell Type in the Demosponge Tethya wilhelma – Functional Cellular Anatomy of a Leuconoid Canal System

Demosponges possess a leucon-type canal system which is characterized by a highly complex network of canal segments and choanocyte chambers. As sponges are sessile filter feeders, their aquiferous system plays an essential role in various fundamental physiological processes. Due to the morphological and architectural complexity of the canal system and the strong interdependence between flow conditions and anatomy, our understanding of fluid dynamics throughout leuconoid systems is patchy. This paper provides comprehensive morphometric data on the general architecture of the canal system, flow measurements and detailed cellular anatomical information to help fill in the gaps. We focus on the functional cellular anatomy of the aquiferous system and discuss all relevant cell types in the context of hydrodynamic and evolutionary constraints. Our analysis is based on the canal system of the tropical demosponge Tethya wilhelma, which we studied using scanning electron microscopy. We found a hitherto undescribed cell type, the reticuloapopylocyte, which is involved in flow regulation in the choanocyte chambers. It has a highly fenestrated, grid-like morphology and covers the apopylar opening. The minute opening of the reticuloapopylocyte occurs in an opened, intermediate and closed state. These states permit a gradual regulation of the total apopylar opening area. In this paper the three states are included in a theoretical study into flow conditions which aims to draw a link between functional cellular anatomy, the hydrodynamic situation and the regular body contractions seen in T. wilhelma. This provides a basis for new hypotheses regarding the function of bypass elements and the role of hydrostatic pressure in body contractions. Our study provides insights into the local and global flow conditions in the sponge canal system and thus enhances current understanding of related physiological processes.     

Microfluidic co-culture platform for investigating osteocyte-osteoclast signalling during fluid shear stress mechanostimulation

Bone cells exist in a complex environment where they are constantly exposed to numerous dynamic biochemical and mechanical stimuli. These stimuli regulate bone cells that are involved in various bone disorders, such as osteoporosis. Knowledge of how these stimuli affect bone cells have been utilised to develop various treatments, such as pharmaceuticals, hormone therapy, and exercise. To investigate the role that bone loading has on these disorders in vitro, bone cell mechanotransduction studies are typically performed using parallel plate flow chambers (PPFC). However, these chambers do not allow for dynamic cellular interactions among different cell populations to be investigated. We present a microfluidic approach that exposes different cell populations, which are located at physiologically relevant distances within adjacent channels, to different levels of fluid shear stress, and promotes cell-cell communication between the different channels. We employed this microfluidic system to assess mechanically regulated osteocyte-osteoclast communication. Osteoclast precursors (RAW264.7 cells) responded to cytokine gradients (e.g., RANKL, OPG, PGE-2) developed by both mechanically stimulated (fOCY) and unstimulated (nOCY) osteocyte-like MLO-Y4 cells simultaneously. Specifically, we observed increased osteoclast precursor cell densities and osteoclast differentiation towards nOCY. We also used this system to show an increased mechanoresponse of osteocytes when in co-culture with osteoclasts. We envision broad applicability of the presented approach for microfluidic perfusion co-culture of multiple cell types in the presence of fluid flow stimulation, and as a tool to investigate osteocyte mechanotransduction, as well as bone metastasis extravasation. This system could also be applied to any multi-cell population cross-talk studies that are typically performed using PPFCs (e.g. endothelial cells, smooth muscle cells, and fibroblasts).     

物质经皮转运的唯象理论

物质经皮渗透是一个非平衡态的热力学过程,驱使物质经皮转运的动力产生了物质经皮转运的通量,文章试图用唯象理论来阐述这种关系。假设扩散池系统恒温、无化学反应：溶液为非粘性流体,双组份、局域平衡。根据Gibbs方程,建立了扩散池系统的耗散函数,导出了其质量流与质量力、体积流与体积力：实验分对照组(被动扩散)和实验组(电脉冲扩散),分别做3次并以替硝唑为模式药物。实验结果表明：(1)扩散池系统中不但存在物质的经皮渗透,而且存在溶液的体积缺失现象;(2)根据实验数据,确定唯象系数具有时变性,并且推断当时间延迟时,质量力对质量流和体积力对体积流的影响减弱;(3)溶液对流在皮肤表面产生的速度梯度可能产生体积流。结论是扩散池药物经皮渗透系统是一个非线性时变系统。     

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 µm biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.     

Temperature-controlled open-top chambers for global change research

To enable experiments on the interactive effects of elevated atmospheric CO₂ and increased air temperature on physiological processes in trees to be carried out, we altered the standard design of open-top chambers by replacing blowers with evaporative coolers and in-line heaters, with a feedback control system to maintain ambient or elevated air temperatures within the chambers. Ambient and elevated (+ 4 °C) temperature regimes were attained consistently and reliably throughout the growing season, with high reproducibility between chambers. From May through December the average of nearly 300,000 temperature measurements was 18.5 °C in ambient air, 18.9 ± 0.6 °C in six ambient chambers, and 22.4 ± 0.9 °C in six elevated temperature chambers. The difference in soil temperature between ambient and elevated chambers was 1.2 °C. Absolute humidity (vapour pressure) in the chambers was higher than that of ambient air, but it was generally similar between temperature treatments. Vapour pressure deficit therefore was higher in elevated temperature chambers than in ambient chambers, and this difference is considered an inseparable part of the temperature treatment. The addition of a temperature control system to open-top chambers removes what has been an important flaw in this important tool for global change research.     

Pressure gradient, not exposure duration, determines the extent of epithelial cell damage in a model of pulmonary airway reopening.

The reduction of tidal volume during mechanical ventilation has been shown to reduce mortality of patients with acute respiratory distress syndrome, but epithelial cell injury can still result from mechanical stresses imposed by the opening of occluded airways. To study these stresses, a fluid-filled parallel-plate flow chamber lined with epithelial cells was used as an idealized model of an occluded airway. Airway reopening was modeled by the progression of a semi-infinite bubble of air through the length of the channel, which cleared the fluid. In our laboratory's prior study, the magnitude of the pressure gradient near the bubble tip was directly correlated to the epithelial cell layer damage (Bilek AM, Dee KC, and Gaver DP III. J Appl Physiol 94: 770-783, 2003). However, in that study, it was not possible to discriminate the stress magnitude from the stimulus duration because the bubble propagation velocity varied between experiments. In the present study, the stress magnitude is modified by varying the viscosity of the occlusion fluid while fixing the reopening velocity across experiments. This approach causes the stimulus duration to be inversely related to the magnitude of the pressure gradient. Nevertheless, cell damage remains directly correlated with the pressure gradient, not the duration of stress exposure. The present study thus provides additional evidence that the magnitude of the pressure gradient induces cellular damage in this model of airway reopening. We explore the mechanism for acute damage and also demonstrate that repeated reopening and closure is shown to damage the epithelial cell layer, even under conditions that would not lead to extensive damage from a single reopening event.     

Cellular clot formation in a sipunculan worm: entrapment of foreign particles, cell death and identification of a PGRP-related protein

Clotting in animals having open or closed circulatory system comprises humoral and cellular mechanisms. Sipunculans are a small phylum of non-segmented marine worms that do not have a true circulatory system. These worms can form a cellular clot without transforming cell-free coelomic fluid into an insoluble mass. The clot may also contribute to immune response by entrapping foreign particles. We evaluated the formation of a cellular clot ex vivo in the sipunculan Themiste petricola after activation through glass contact and sea water, the ability to entrap magnetic beads and non-pathogen cyanobacteria particles within the clot, and the presence of a peptidoglycan recognition protein S (PGRP-S) antigen in cells forming the clot. Our results showed that the clot was formed by homotypic aggregation of large granular leukocytes (LGLs), a subtype of cells found in the coelomic fluid. Aggregated LGLs served to entrap magnetic beads and non-pathogen cyanobacteria particles, and PGRP-S antigen was detected both in non-activated LGLs and in activated homotypic aggregates through immunofluorescence, Western blot and flow cytometry. Cellular clots were found to be positive to Annexin V-FITC labelling. Complete disintegration of cytoplasm with shedding of microparticles, nuclear isolation and degradation were also observed by light microscopy and flow cytometry. In conclusion, cellular clot formation in Themiste petricola may serve both haemostatic and immune functions entailing rapid activation changes in LGLs, entrapment of foreign particles within a homotypic aggregate, and further cell death.     

Measuring opening pressure in lumbar puncture--a new method]

Determination of the opening pressure (OP) during diagnostic lumbar puncture (LP) yields additional information that may impact on treatment and prognosis in disorders affecting the central nervous system (e.g. meningitis). Established methods contain systematic errors as well as risks to the patient. We therefore present a new procedure that allows measurement of the OP by timing the flow of cerebrospinal fluid through a capillary attached to an LP needle. A resistance located between needle and capillary slows down the flow of cerebrospinal fluid so that it becomes independent of the capillary forces acting on it. The time required for the fluid to travel between two marks on the capillary (defining a given volume) can be used to calculate the flow. Since the combined resistance of needle and resistance can be calibrated, the pressure driving the flow--in this case the opening pressure--can be calculated. A simple model was used to evaluate the impact of different resistances and different needles on OP determination. The effects of cellular elements and proteins in the CSF are discussed.     

Fluid pressure relaxation depends upon osteonal microstructure: modeling an oscillatory bending experiment.

When bone is mechanically loaded, bone fluid flow induces shear stresses on bone cells that have been proposed to be involved in bone's mechanosensory system. To investigate bone fluid flow and strain-generated potentials, several theoretical models have been proposed to mimic oscillatory four-point bending experiments performed on thin bone specimens. While these previous models assume that the bone fluid relaxes across the specimen thickness, we hypothesize that the bone fluid relaxes primarily through the vascular porosity (osteonal canals) instead and develop a new poroelastic model that integrates the microstructural details of the lacunar-canalicular porosity, osteonal canals, and the osteonal cement lines. Local fluid pressure profiles are obtained from the model, and we find two different fluid relaxation behaviors in the bone specimen, depending on its microstructure: one associated with the connected osteonal canal system, through which bone fluid relaxes to the nearby osteonal canals; and one associated with the thickness of a homogeneous porous bone specimen (approximately 1 mm in our model), through which bone fluid relaxes between the external surfaces of the bone specimen at relatively lower loading frequencies. Our results suggest that in osteonal bone specimens the fluid pressure response to cyclic loading is not sensitive to the permeability of the osteonal cement lines, while it is sensitive to the applied loading frequency. Our results also reveal that the fluid pressure gradients near the osteonal canals (and thus the fluid shear stresses acting on the nearby osteocytes) are significantly amplified at higher loading frequencies.     

Feedback-mediated dynamics in a model of coupled nephrons with compliant thick ascending limbs

The tubuloglomerular feedback (TGF) system in the kidney, a key regulator of glomerular filtration rate, has been shown in physiologic experiments in rats to mediate oscillations in thick ascending limb (TAL) tubular fluid pressure, flow, and NaCl concentration. In spontaneously hypertensive rats, TGF-mediated flow oscillations may be highly irregular. We conducted a bifurcation analysis of a mathematical model of nephrons that are coupled through their TGF systems; the TALs of these nephrons are assumed to have compliant tubular walls. A characteristic equation was derived for a model of two coupled nephrons. Analysis of that characteristic equation has revealed a number of parameter regions having the potential for differing stable dynamic states. Numerical solutions of the full equations for two model nephrons exhibit a variety of behaviors in these regions. Also, model results suggest that the stability of the TGF system is reduced by the compliance of TAL walls and by internephron coupling; as a result, the likelihood of the emergence of sustained oscillations in tubular fluid pressure and flow is increased. Based on information provided by the characteristic equation, we identified parameters with which the model predicts irregular tubular flow oscillations that exhibit a degree of complexity that may help explain the emergence of irregular oscillations in spontaneously hypertensive rats.     

Experimental quantification of the fluid dynamics in blood-processing devices through 4D-flow imaging: A pilot study on a real oxygenator/heat-exchanger module

The performance of blood-processing devices largely depends on the associated fluid dynamics, which hence represents a key aspect in their design and optimization. To this aim, two approaches are currently adopted: computational fluid-dynamics, which yields highly resolved three-dimensional data but relies on simplifying assumptions, and in vitro experiments, which typically involve the direct video-acquisition of the flow field and provide 2D data only. We propose a novel method that exploits space- and time-resolved magnetic resonance imaging (4D-flow) to quantify the complex 3D flow field in blood-processing devices and to overcome these limitations.We tested our method on a real device that integrates an oxygenator and a heat exchanger. A dedicated mock loop was implemented, and novel 4D-flow sequences with sub-millimetric spatial resolution and region-dependent velocity encodings were defined. Automated in house software was developed to quantify the complex 3D flow field within the different regions of the device: region-dependent flow rates, pressure drops, paths of the working fluid and wall shear stresses were computed.Our analysis highlighted the effects of fine geometrical features of the device on the local fluid-dynamics, which would be unlikely observed by current in vitro approaches. Also, the effects of non-idealities on the flow field distribution were captured, thanks to the absence of the simplifying assumptions that typically characterize numerical models.To the best of our knowledge, our approach is the first of its kind and could be extended to the analysis of a broad range of clinically relevant devices.     

Flow through a rough,shallow reef

This paper presents a simple model of wave-driven flow through a coral reef that is characterized by a shallow, wide reef crest and a deeper, but frictional lagoon. Assuming that the dominant momentum balances are between quadratic drag and barotropic pressure gradients, along-lagoon and cross-reef flows are coupled through continuity and through a setup of the water surface in the lagoon that varies in the along-reef direction. Scaling of the governing equations shows that this flow is governed by a single parameter P that expresses the competing effects of cross-reef and along-lagoon drag. When P < 1, the cross-reef flow is nearly constant, whereas when P > 1, only that portion of the reef closest to the pass through the reef crest through which fluid exists the lagoon supports cross-reef flows.     

Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling

We recently modeled fluid flow through gap junction channels coupling the pigmented and nonpigmented layers of the ciliary body. The model suggested the channels could transport the secretion of aqueous humor, but flow would be driven by hydrostatic pressure rather than osmosis. The pressure required to drive fluid through a single layer of gap junctions might be just a few mmHg and difficult to measure. In the lens, however, there is a circulation of Na(+) that may be coupled to intracellular fluid flow. Based on this hypothesis, the fluid would cross hundreds of layers of gap junctions, and this might require a large hydrostatic gradient. Therefore, we measured hydrostatic pressure as a function of distance from the center of the lens using an intracellular microelectrode-based pressure-sensing system. In wild-type mouse lenses, intracellular pressure varied from ～330 mmHg at the center to zero at the surface. We have several knockout/knock-in mouse models with differing levels of expression of gap junction channels coupling lens fiber cells. Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels. When the lens' circulation of Na(+) was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally. These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium.     

Dynamic shear stress in parallel-plate flow chambers

An in vitro model using a parallel-plate fluid flow chamber is supposed to simulate in vivo fluid shear stresses on various cell types exposed to dynamic fluid flow in their physiological environment. The metabolic response of cells in vitro is associated with the wall shear stress. However, parallel-plate flow chambers have not been characterized for dynamic fluid flow experiments. We use a dimensionless ratio h / lambda(v), in determining the exact magnitude of the dynamic wall shear stress, with its oscillating components scaled by a shear factor T. It is shown that, in order to expose cells to predictable levels of dynamic fluid shear stress, two conditions have to be met: (1) h / lambda(v) < 2, where h is the distance between the plates and lambda(v) is the viscous penetration depth; and (2) f(0) < f(c) / m, where the critical frequency f(c) is the upper threshold for this flow regime, m is the highest harmonic mode of the flow, and f(0) is the fundamental frequency of fluid flow.     

"Whither flows the fluid in bone?" An osteocyte's perspective

Bone represents a porous tissue containing a fluid phase, a solid matrix, and cells. Movement of the fluid phase within the pores or spaces of the solid matrix translates endogenous and exogenous mechanobiological, biochemical and electromechanical signals from the system that is exposed to the dynamic external environment to the cells that have the machinery to remodel the tissue from within. Hence, bone fluid serves as a coupling medium, providing an elegant feedback mechanism for functional adaptation. Until recently relatively little has been known about bone fluid per se or the influences governing the characteristics of its flow. This work is designed to review the current state of this emerging field. The structure of bone, as an environment for fluid flow, is discussed in terms of the properties of the spaces and channel walls through which the fluid flows and the influences on flow under physiological conditions. In particular, the development of the bone cell syncytium and lacunocanalicular system are presented, and pathways for fluid flow are described from the systemic to the organ, tissue, cellular and subcellular levels. Finally, exogenous and endogenous mechanisms for pressure-induced fluid movement through bone, including mechanical loading, vascular derived pressure gradients, and osmotic pressure gradients are discussed. The objective of this review is to survey the current understanding of the means by which fluid flow in bone is regulated, from the level of the skeletal system down to the level of osteocyte, and to provide impetus for future research in this area of signal transduction and coupling. An understanding of this important aspect of bone physiology has profound implications for restoration of function through innovative treatment modalities on Earth and in space, as well as for engineering of biomimetic replacement tissue.     

The imperative for controlled mechanical stresses in unraveling cellular mechanisms of mechanotransduction

Background  

In vitro mechanotransduction studies are designed to elucidate cell behavior in response to a well-defined mechanical signal that is imparted to cultured cells, e.g. through fluid flow. Typically, flow rates are calculated based on a parallel plate flow assumption, to achieve a targeted cellular shear stress. This study evaluates the performance of specific flow/perfusion chambers in imparting the targeted stress at the cellular level.     

Computational study of the role of fluid content and flow on the lumbar disc response in cyclic compression: Replication of in vitro and in vivo conditions

The intervertebral disc viscoelastic response is governed primarily by its fluid content and flow. In vivo measurements demonstrate that the disc volume, fluid content, height and nucleus pressure completely recover during resting even after diurnal loading with twice longer duration (16 vs. 8 h). In view of much longer periods required for the recovery of disc height and pressure in vitro, concerns have been raised on the fluid inflow through the endplates that might be hampered by clogged blood vessels post mortem. This in silico study aimed to identify fluid-flow dependent response of discs and conditions essential to replicate in vitro and in vivo observations.An osmo-poroelastic finite element model of the human lumbar L4-L5 disc-bone unit was used. Simulating earlier in vitro experiments on bovine discs, the loading protocol started with 8 h preload at 0.06 MPa followed by 30 high/low compression loading cycles each lasting 7.5 min at 0.5/0.06 MPa, respectively. Three different endplate configurations were investigated: free in- and outflow, no inflow and closed endplates with no flow. Additionally, the preload magnitude was increased from 0.06 MPa to 0.28 MPa and 0.50 MPa, or the initial nucleus hydration was reduced from 83% to 50%.For 0.06 MPa preload, the model with no inflow best matched in vitro trends. The model with free inflow increased segment height and nucleus pressure while the model with no fluid inflow resulted in a relatively small recovery in segment height and a rather constant nucleus pressure during unloading periods.Results highlight an excessive mobile fluid content as well as a restricted fluid inflow through endplates as likely causes of the discrepancies between in vivo and in vitro studies. To replicate in vivo conditions in vitro and in silico, disc hydration level should be controlled by adequate selection of preload magnitude/period and/or mobile fluid porosity.     

Observation of Bacterial Type I Pili Extension and Contraction under Fluid Flow

Type I pili are proteinaceous tethers that mediate bacterial adhesion of uropathogenic Escherichia coli to surfaces and are thought to help bacteria resist drag forces imparted by fluid flow via uncoiling of their quaternary structure. Uncoiling and recoiling have been observed in force spectroscopy experiments, but it is not clear if and how this process occurs under fluid flow. Here we developed an assay to study the mechanical properties of pili in a parallel plate flow chamber. We show that pili extend when attached E. coli bacteria are exposed to increasing shear stresses, that pili can help bacteria move against moderate fluid flows, and characterize two dynamic regimes of this displacement. The first regime is consistent with entropic contraction as modeled by a freely jointed chain, and the second with coiling of the quaternary structure of pili. These results confirm that coiling and uncoiling happen under flow but the observed dynamics are different from those reported previously. Using these results and those from previous studies, we review the mechanical properties of pili in the context of other elastic proteins such as the byssal threads of mussels. It has been proposed that the high extensibility of pili may help recruit more pili into tension and lower the force acting on each one by damping changes in force due to fluid flow. Our analysis of the mechanical properties suggests additional functions of pili; in particular, their extensibility may reduce tension by aligning pili with the direction of flow, and the uncoiled state of pili may complement uncoiling in regulating the force of the terminal adhesin.     

Amyloid Oligomers and Mature Fibrils Prepared from an Innocuous Protein Cause Diverging Cellular Death Mechanisms

Despite significant advances, the molecular identity of the cytotoxic species populated during in vivo amyloid formation crucial for the understanding of neurodegenerative disorders is yet to be revealed. In this study lysozyme prefibrillar oligomers and fibrils in both mature and sonicated states have been isolated through an optimized ultrafiltration/ultracentrifugation method and characterized with various optical spectroscopic techniques, atomic force microscopy, and transmission electron microscopy. We examined their level and mode of toxicity on rat pheochromocytoma (PC12) cells in both differentiated and undifferentiated states. We find that oligomers and fibrils display cytotoxic capabilities toward cultured cells in vitro, with oligomers producing elevated levels of cellular injury toward undifferentiated PC12 cells (PC12^undiff). Furthermore, dual flow cytometry staining experiments demonstrate that the oligomers and mature fibrils induce divergent cellular death pathways (apoptosis and secondary necrosis, respectively) in these PC12 cells. We have also shown that oligomers but not sonicated mature fibrils inhibit hippocampal long term potentiation, a form of synaptic plasticity implicated in learning and memory, in vivo. We conclude that our in vitro and in vivo findings confer a level of resistance toward amyloid fibrils, and that the PC 12-based comparative cytotoxicity assay can provide insights into toxicity differences between differently aggregated protein species.     

Cells in 3D matrices under interstitial flow: Effects of extracellular matrix alignment on cell shear stress and drag forces

Interstitial flow is an important regulator of various cell behaviors both in vitro and in vivo, yet the forces that fluid flow imposes on cells embedded in a 3D extracellular matrix (ECM), and the effects of matrix architecture on those forces, are not well understood. Here, we demonstrate how fiber alignment can affect the shear and pressure forces on the cell and ECM. Using computational fluid dynamics simulations, we show that while the solutions of the Brinkman equation accurately estimate the average fluid shear stress and the drag forces on a cell within a 3D fibrous medium, the distribution of shear stress on the cellular surface as well as the peak shear stresses remain intimately related to the pericellular fiber architecture and cannot be estimated using bulk-averaged properties. We demonstrate that perpendicular fiber alignment of the ECM yields lower shear stress and pressure forces on the cells and higher stresses on the ECM, leading to decreased permeability, while parallel fiber alignment leads to higher stresses on cells and increased permeability, as compared to a cubic lattice arrangement. The Spielman–Goren permeability relationships for fibrous media agreed well with CFD simulations of flow with explicitly considered fibers. These results suggest that the experimentally observed active remodeling of ECM fibers by fibroblasts under interstitial flow to a perpendicular alignment could serve to decrease the shear and drag forces on the cell.