The Effect of Shear on the Desorption of Liposomes Adsorbed to Bacterial Biofilms

Abstract

With the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,β[N-(N¹,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immoblized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome–biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition.     

Darcian permeability constant as indicator for shear stresses in regular scaffold systems for tissue engineering

The shear stresses in printed scaffold systems for tissue engineering depend on the flow properties and void volume in the scaffold. In this work, computational fluid dynamics (CFD) is used to simulate flow fields within porous scaffolds used for cell growth. From these models the shear stresses acting on the scaffold fibres are calculated. The results led to the conclusion that the Darcian (k ₁) permeability constant is a good predictor for the shear stresses in scaffold systems for tissue engineering. This permeability constant is easy to calculate from the distance between and thickness of the fibres used in a 3D printed scaffold. As a consequence computational effort and specialists for CFD can be circumvented by using this permeability constant to predict the shear stresses. If the permeability constant is below a critical value, cell growth within the specific scaffold design may cause a significant increase in shear stress. Such a design should therefore be avoided when the shear stress experienced by the cells should remain in the same order of magnitude.     

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.     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

Flow rates in perfusion bioreactors to maximise mineralisation in bone tissue engineering in vitro

In bone tissue engineering experiments, fluid-induced shear stress is able to stimulate cells to produce mineralised extracellular matrix (ECM). The application of shear stress on seeded cells can for example be achieved through bioreactors that perfuse medium through porous scaffolds. The generated mechanical environment (i.e. wall shear stress: WSS) within the scaffolds is complex due to the complexity of scaffold geometry. This complexity has so far prevented setting an optimal loading (i.e. flow rate) of the bioreactor to achieve an optimal distribution of WSS for stimulating cells to produce mineralised ECM. In this study, we demonstrate an approach combining computational fluid dynamics (CFD) and mechano-regulation theory to optimise flow rates of a perfusion bioreactor and various scaffold geometries (i.e. pore shape, porosity and pore diameter) in order to maximise shear stress induced mineralisation. The optimal flow rates, under which the highest fraction of scaffold surface area is subjected to a wall shear stress that induces mineralisation, are mainly dependent on the scaffold geometries. Nevertheless, the variation range of such optimal flow rates are within 0.5–5 mL/min (or in terms of fluid velocity: 0.166–1.66 mm/s), among different scaffolds. This approach can facilitate the determination of scaffold-dependent flow rates for bone tissue engineering experiments in vitro, avoiding performing a series of trial and error experiments.     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

A dynamical study of the mechanical stimuli and tissue differentiation within a CaP scaffold based on micro-CT finite element models

The control of the mechanical stimuli transmitted to the cells is critical for the design of functional scaffolds for tissue engineering. The objective of this study was to investigate the dynamics of the mechanical stimuli transmitted to the cells during tissue differentiation in an irregular morphology scaffold under compressive load and perfusion flow. A calcium phosphate-based glass porous scaffold was used. The solid phase and the fluid flow within the pores were modeled as linear elastic solid material and Newtonian fluid, respectively. In the fluid model, different levels of viscosity were used to simulate tissue differentiation. Compressive strain of 0.5% and fluid flow with constant inlet velocity of 10 μm/s or constant inlet pressure of 3 Pa were applied. Octahedral shear strain and fluid shear stress were used as mechano-regulatory stimuli. For constant inlet velocity, stimuli equivalent to bone were predicted in 80% of pore volume for the case of low tissue viscosity. For the cases of high viscosity, fluctuations between stimuli equivalent to tissue formation and cell death were predicted due to the increase in the fluid shear stress when tissue started to fill pores. When constant pressure was applied, stimuli equivalent to bone were predicted in 62% of pore volume when low tissue viscosity was used and 42% when high tissue viscosity was used. This study predicted critical variations of fluid shear stress when cells differentiated. If these variations are not controlled in vitro, they can impede the formation of new matured tissue.     

Dynamic adhesion of umbilical cord blood endothelial progenitor cells under laminar shear stress

Late outgrowth endothelial progenitor cells (EPCs) represent a promising cell source for rapid reendothelialization of damaged vasculature after expansion ex vivo and injection into the bloodstream. We characterized the dynamic adhesion of umbilical-cord-blood-derived EPCs (CB-EPCs) to surfaces coated with fibronectin. CB-EPC solution density affected the number of adherent cells and larger cells preferentially adhered at lower cell densities. The number of adherent cells varied with shear stress, with the maximum number of adherent cells and the shear stress at maximum adhesion depending upon fluid viscosity. CB-EPCs underwent limited rolling, transiently tethering for short distances before firm arrest. Immediately before arrest, the instantaneous velocity decreased independent of shear stress. A dimensional analysis indicated that adhesion was a function of the net force on the cells, the ratio of cell diffusion to sliding speed, and molecular diffusivity. Adhesion was not limited by the settling rate and was highly specific to α₅β₁ integrin. Total internal reflection fluorescence microscopy showed that CB-EPCs produced multiple contacts of α₅β₁ with the surface and the contact area grew during the first 20 min of attachment. These results demonstrate that CB-EPC adhesion from blood can occur under physiological levels of shear stress.     

Vibration induced osteogenic commitment of mesenchymal stem cells is enhanced by cytoskeletal remodeling but not fluid shear

Consistent across studies in humans, animals and cells, the application of vibrations can be anabolic and/or anti-catabolic to bone. The physical mechanisms modulating the vibration-induced response have not been identified. Recently, we developed an in vitro model in which candidate parameters including acceleration magnitude and fluid shear can be controlled independently during vibrations. Here, we hypothesized that vibration induced fluid shear does not modulate mesenchymal stem cell (MSC) proliferation and mineralization and that cell's sensitivity to vibrations can be promoted via actin stress fiber formation. Adipose derived human MSCs were subjected to vibration frequencies and acceleration magnitudes that induced fluid shear stress ranging from 0.04 Pa to 5 Pa. Vibrations were applied at magnitudes of 0.15g, 1g, and 2g using frequencies of both 100 Hz and 30 Hz. After 14 d and under low fluid shear conditions associated with 100 Hz oscillations, mineralization was greater in all vibrated groups than in controls. Greater levels of fluid shear produced by 30 Hz vibrations enhanced mineralization only in the 2g group. Over 3 d, vibrations led to the greatest increase in total cell number with the frequency/acceleration combination that induced the smallest level of fluid shear. Acute experiments showed that actin remodeling was necessary for early mechanical up-regulation of RUNX-2 mRNA levels. During osteogenic differentiation, mechanically induced up-regulation of actin remodeling genes including Wiskott–Aldrich syndrome (WAS) protein, a critical regulator of Arp2/3 complex, was related to the magnitude of the applied acceleration but not to fluid shear. These data demonstrate that fluid shear does not regulate vibration induced proliferation and mineralization and that cytoskeletal remodeling activity may play a role in MSC mechanosensitivity.     

Fluid-induced low shear stress improves cartilage like tissue fabrication by encapsulating chondrocytes

In the recent years, there has been considerable development in the regenerative medicine, which aims to repair, regenerate, and improve injured articular cartilage. The aim of the present study was to investigate the effect of flow-induced shear stress in perfusion bioreactor on alginate encapsulating chondrocytes. The shear stress imposed on the cells in the culture chamber of bioreactor was predicted with computational fluid dynamic. Bovine nasal chondrocytes were isolated and expanded to obtain a pellet. The cell pellet was resuspends in alginate solution, transferred to the culture chamber, and dynamically cultured under direct perfusion. At the end of culture, tissue constructs were examined histologically and by immunohistochemistry. The results of computational fluid dynamic modeling revealed that maximum wall shear stress was 4.820 × 10^?3 Pascal. Macroscopic views of the alginate/chondrocyte beads suggested that it possessed constant shape but were flexible. Under inverted microscope, round shape of chondrocyte observed. Cell distribution was homogeneous throughout the scaffold. Tissue construct subjected to shear showed morphological features, which are characteristic for natural cartilage. Immunohistochemistry results revealed immunopositivity for type II collagens in tissue constructs samples. Flow induced shear stress in the perfusion bioreactor and chnondrocyte encapsulation provide environment to support cell growth, and tissue regeneration and improve cartilage like tissue fabrication.     

A prediction of cell differentiation and proliferation within a collagen–glycosaminoglycan scaffold subjected to mechanical strain and perfusive fluid flow

Mesenchymal stem cell (MSC) differentiation can be influenced by biophysical stimuli imparted by the host scaffold. Yet, causal relationships linking scaffold strain magnitudes and inlet fluid velocities to specific cell responses are thus far underdeveloped. This investigation attempted to simulate cell responses in a collagen–glycosaminoglycan (CG) scaffold within a bioreactor. CG scaffold deformation was simulated using μ-computed tomography (CT) and an in-house finite element solver (FEEBE/linear). Similarly, the internal fluid velocities were simulated using the afore-mentioned μCT dataset with a computational fluid dynamics solver (ANSYS/CFX). From the ensuing cell-level mechanics, albeit octahedral shear strain or fluid velocity, the proliferation and differentiation of the representative cells were predicted from deterministic functions. Cell proliferation patterns concurred with previous experiments. MSC differentiation was dependent on the level of CG scaffold strain and the inlet fluid velocity. Furthermore, MSC differentiation patterns indicated that specific combinations of scaffold strains and inlet fluid flows cause phenotype assemblies dominated by single cell types. Further to typical laboratory procedures, this predictive methodology demonstrated loading-specific differentiation lineages and proliferation patterns. It is hoped these results will enhance in-vitro tissue engineering procedures by providing a platform from which the scaffold loading applications can be tailored to suit the desired tissue.     

Modeling evaluation of the fluid-dynamic microenvironment in tissue-engineered constructs: a micro-CT based model

Natural cartilage remodels both in vivo and in vitro in response to mechanical stresses, hence mechanical stimulation is believed to be a potential tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis in engineered cartilage constructs. The quantification of the hydrodynamic environment is a condition required to study the biochemical response to shear of 3D engineered cell systems. We developed a computational model of culture medium flow through the microstructure of a porous scaffold, during direct- perfused culture. The 3D solid model of the scaffold micro-geometry was reconstructed from 250 micro-computed tomography (micro-CT) images. The results of the fluid dynamic simulations were analyzed at the central portions of the fluid domain, to avoid boundary effects. The average, median and mode shear stress values calculated at the scaffold walls were 3.48, 2.90, and 2.45 mPa respectively, at a flow rate of 0.5 cm(3)/min, perfused through a 15 mm diameter scaffold, at an inlet fluid velocity of 53 microm/s. These results were compared to results estimated using a simplified micro-scale model and to results estimated using an analytical macro-scale porous model. The predictions given by the CT-based model are being used in conjunction with an experimental bioreactor model, in order to quantify the effects of fluid-dynamic shear on the growth modulation of tissue-engineered cartilage constructs, to potentially enhance tissue growth in vitro.     

Focal Adhesion Kinase Plays a Role in Osteoblast Mechanotransduction In Vitro but Does Not Affect Load-Induced Bone Formation In Vivo

A healthy skeleton relies on bone''s ability to respond to external mechanical forces. The molecular mechanisms by which bone cells sense and convert mechanical stimuli into biochemical signals, a process known as mechanotransduction, are unclear. Focal adhesions play a critical role in cell survival, migration and sensing physical force. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that controls focal adhesion dynamics and can mediate reparative bone formation in vivo and osteoblast mechanotransduction in vitro. Based on these data, we hypothesized that FAK plays a role in load-induced bone formation. To test this hypothesis, we performed in vitro fluid flow experiments and in vivo bone loading studies in FAK−/− clonal lines and conditional FAK knockout mice, respectively. FAK−/− osteoblasts showed an ablated prostaglandin E₂ (PGE₂) response to fluid flow shear. This effect was reversed with the re-expression of wild-type FAK. Re-expression of FAK containing site-specific mutations at Tyr-397 and Tyr-925 phosphorylation sites did not rescue the phenotype, suggesting that these sites are important in osteoblast mechanotransduction. Interestingly, mice in which FAK was conditionally deleted in osteoblasts and osteocytes did not exhibit altered load-induced periosteal bone formation. Together these data suggest that although FAK is important in mechanically-induced signaling in osteoblasts in vitro, it is not required for an adaptive response in vivo, possibly due to a compensatory mechanism that does not exist in the cell culture system.     

IQ Domain GTPase-Activating Protein 1 is Involved in Shear Stress-Induced Progenitor-Derived Endothelial Cell Alignment

Shear stress is one of mechanical constraints which are exerted by blood flow on endothelial cells (ECs). To adapt to shear stress, ECs align in the direction of flow through adherens junction (AJ) remodeling. However, mechanisms regulating ECs alignment under shear stress are poorly understood. The scaffold protein IQ domain GTPase activating protein 1 (IQGAP1) is a scaffold protein which couples cell signaling to the actin and microtubule cytoskeletons and is involved in cell migration and adhesion. IQGAP1 also plays a role in AJ organization in epithelial cells. In this study, we investigated the potential IQGAP1 involvement in the endothelial cells alignment under shear stress. Progenitor-derived endothelial cells (PDECs), transfected (or not) with IQGAP1 small interfering RNA, were exposed to a laminar shear stress (1.2 N/m²) and AJ proteins (VE-cadherin and β-catenin) and IQGAP1 were labeled by immunofluorescence. We show that IQGAP1 is essential for ECs alignment under shear stress. We studied the role of IQGAP1 in AJs remodeling of PDECs exposed to shear stress by studying cell localization and IQGAP1 interactions with VE-cadherin and β-catenin by immunofluorescence and Proximity Ligation Assays. In static conditions, IQGAP1 interacts with VE-cadherin but not with β-catenin at the cell membrane. Under shear stress, IQGAP1 lost its interaction from VE-cadherin to β-catenin. This “switch” was concomitant with the loss of β-catenin/VE-cadherin interaction at the cell membrane. This work shows that IQGAP1 is essential to ECs alignment under shear stress and that AJ remodeling represents one of the mechanisms involved. These results provide a new approach to understand ECs alignment under to shear stress.     

Fluid Shear Stress Upregulates E-Tmod41 via miR-23b-3p and Contributes to F-Actin Cytoskeleton Remodeling during Erythropoiesis

The membrane skeleton of mature erythrocyte is formed during erythroid differentiation. Fluid shear stress is one of the main factors that promote embryonic hematopoiesis, however, its effects on erythroid differentiation and cytoskeleton remodeling are unclear. Erythrocyte tropomodulin of 41 kDa (E-Tmod41) caps the pointed end of actin filament (F-actin) and is critical for the formation of hexagonal topology of erythrocyte membrane skeleton. Our study focused on the regulation of E-Tmod41 and its role in F-actin cytoskeleton remodeling during erythroid differentiation induced by fluid shear stress. Mouse erythroleukemia (MEL) cells and embryonic erythroblasts were subjected to fluid shear stress (5 dyn/cm²) and erythroid differentiation was induced in both cells. F-actin content and E-Tmod41 expression were significantly increased in MEL cells after shearing. E-Tmod41 overexpression resulted in a significant increase in F-actin content, while the knockdown of E-Tmod41 generated the opposite result. An E-Tmod 3’UTR targeting miRNA, miR-23b-3p, was found suppressed by shear stress. When miR-23b-3p level was overexpressed / inhibited, both E-Tmod41 protein level and F-actin content were reduced / augmented. Furthermore, among the two alternative promoters of E-Tmod, P_E0 (upstream of exon 0), which mainly drives the expression of E-Tmod41, was found activated by shear stress. In conclusion, our results suggest that fluid shear stress could induce erythroid differentiation and F-actin cytoskeleton remodeling. It upregulates E-Tmod41 expression through miR-23b-3p suppression and P_E0 promoter activation, which, in turn, contributes to F-actin cytoskeleton remodeling.     

Fluid shear stress regulates the invasive potential of glioma cells via modulation of migratory activity and matrix metalloproteinase expression

Background

Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate.

Methodology/Principal Findings

A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.

Conclusions/Significance

Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.     

Ranolazine inhibits shear sensitivity of endogenous Na+ current and spontaneous action potentials in HL-1 cells

Na_V1.5 is a mechanosensitive voltage-gated Na⁺ channel encoded by the gene SCN5A, expressed in cardiac myocytes and required for phase 0 of the cardiac action potential (AP). In the cardiomyocyte, ranolazine inhibits depolarizing Na⁺ current and delayed rectifier (I_Kr) currents. Recently, ranolazine was also shown to be an inhibitor of Na_V1.5 mechanosensitivity. Stretch also accelerates the firing frequency of the SA node, and fluid shear stress increases the beating rate of cultured cardiomyocytes in vitro. However, no cultured cell platform exists currently for examination of spontaneous electrical activity in response to mechanical stimulation. In the present study, flow of solution over atrial myocyte-derived HL-1 cultured cells was used to study shear stress mechanosensitivity of Na⁺ current and spontaneous, endogenous rhythmic action potentials. In voltage-clamped HL-1 cells, bath flow increased peak Na⁺ current by 14 ± 5%. In current-clamped cells, bath flow increased the frequency and decay rate of AP by 27 ± 12% and 18 ± 4%, respectively. Ranolazine blocked both responses to shear stress. This study suggests that cultured HL-1 cells are a viable in vitro model for detailed study of the effects of mechanical stimulation on spontaneous cardiac action potentials. Inhibition of the frequency and decay rate of action potentials in HL-1 cells are potential mechanisms behind the antiarrhythmic effect of ranolazine.     

Fluid-Flow Induced Wall Shear Stress and Epithelial Ovarian Cancer Peritoneal Spreading

Epithelial ovarian cancer (EOC) is usually discovered after extensive metastasis have developed in the peritoneal cavity. The ovarian surface is exposed to peritoneal fluid pressures and shear forces due to the continuous peristaltic motions of the gastro-intestinal system, creating a mechanical micro-environment for the cells. An in vitro experimental model was developed to expose EOC cells to steady fluid flow induced wall shear stresses (WSS). The EOC cells were cultured from OVCAR-3 cell line on denuded amniotic membranes in special wells. Wall shear stresses of 0.5, 1.0 and 1.5 dyne/cm² were applied on the surface of the cells under conditions that mimic the physiological environment, followed by fluorescent stains of actin and β-tubulin fibers. The cytoskeleton response to WSS included cell elongation, stress fibers formation and generation of microtubules. More cytoskeletal components were produced by the cells and arranged in a denser and more organized structure within the cytoplasm. This suggests that WSS may have a significant role in the mechanical regulation of EOC peritoneal spreading.     

Modeling of flow‐induced shear stress applied on 3D cellular scaffolds: Implications for vascular tissue engineering

Novel tissue‐culture bioreactors employ flow‐induced shear stress as a means of mechanical stimulation of cells. We developed a computational fluid dynamics model of the complex three‐dimensional (3D) microstructure of a porous scaffold incubated in a direct perfusion bioreactor. Our model was designed to predict high shear‐stress values within the physiological range of those naturally sensed by vascular cells (1–10 dyne/cm²), and will thereby provide suitable conditions for vascular tissue‐engineering experiments. The model also accounts for cellular growth, which was designed as an added cell layer grown on all scaffold walls. Five model variants were designed, with geometric differences corresponding to cell‐layer thicknesses of 0, 50, 75, 100, and 125 µm. Four inlet velocities (0.5, 1, 1.5, and 2 cm/s) were applied to each model. Wall shear‐stress distribution and overall pressure drop calculations were then used to characterize the relation between flow rate, shear stress, cell‐layer thickness, and pressure drop. The simulations showed that cellular growth within 3D scaffolds exposes cells to elevated shear stress, with considerably increasing average values in correlation to cell growth and inflow velocity. Our results provide in‐depth analysis of the microdynamic environment of cells cultured within 3D environments, and thus provide advanced control over tissue development in vitro. Biotechnol. Bioeng. 2010; 105: 645–654. © 2009 Wiley Periodicals, Inc.