Fluid Flow-induced Shear Stress Research Articles

A 96-wells fluidic system for high-throughput screenings under laminar high wall shear stress conditions

The ability of endothelial cells to respond to blood flow is fundamental for the correct formation and maintenance of a functional and hierarchically organized vascular network. Defective flow responses, in particular related to high flow conditions, have been associated with atherosclerosis, stroke, arteriovenous malformations, and neurodegenerative diseases. Yet, the molecular mechanisms involved in high flow response are still poorly understood. Here, we described the development and validation of a 96-wells fluidic system, with interchangeable cell culture and fluidics, to perform high-throughput screenings under laminar high-flow conditions. We demonstrated that endothelial cells in our newly developed 96-wells fluidic system respond to fluid flow-induced shear stress by aligning along the flow direction and increasing the levels of KLF2 and KLF4. We further demonstrate that our 96-wells fluidic system allows for efficient gene knock-down compatible with automated liquid handling for high-throughput screening platforms. Overall, we propose that this modular 96-well fluidic system is an excellent platform to perform genome-wide and/or drug screenings to identify the molecular mechanisms involved in the responses of endothelial cells to high wall shear stress.

Orthopedic Scaffolds: Evaluation of Structural Strength and Permeability of Fluid Flow via an Open Cell Neovius Structure for Bone Tissue Engineering.

The ability of bone to regenerate itself through mechanobiological responses is its dynamic property. Mechanical cues from a neighboring environment produce the structural strain to promote blood flow and bone marrow mobility that in turn aids the bone regeneration process. Occurrences of these phenomena are crucial for the success of metallic scaffolds implanted in the host bone tissue. Thus, permeability and fluid flow-induced wall shear stress (WSS) are two parameters that directly influence cell bioactivities inside a scaffold and are crucial for effective bone tissue regeneration. Given that the scaffolds shall be implanted in the body, permeability assessment was carried out using non-Newtonian fluid. In this work, the triply periodic minimal surface scaffolds with Neovius architectures were fabricated by using selective laser melting technology. The estimation of fluid flow was carried out using computational fluid dynamics (CFD) analysis with a non-Newtonian blood fluid model. Further, the structural strength of various open cell Neovius lattices was evaluated using a static compression test, and in vitro cell culture using Alamar blue assay was evaluated. Results revealed that the values of intrinsic blood flow permeability of the three-dimensional (3D)-printed open cell porous scaffold with Neovius architecture were of the same order of magnitude as those of human bone, ranging from 0.0025 × 10-9 to 0.0152 × 10-9 m2. The structural elastic modulus and compressive strength of NOCL40, NOCL50, and NOCL60 lattices range from 3.27 to 3.71 GPa and 194 to 205 MPa, respectively. All of the values are comparable to the human bone, thus making these lattices a suitable alternative for orthopedic applications.

Transient fluid flow improves photoimmunoconjugate delivery and photoimmunotherapy efficacy

Circulating drugs in the peritoneal cavity is an effective strategy for advanced ovarian cancer treatment. Photoimmunotherapy, an emerging modality with potential for the treatment of ovarian cancer, involves near-infrared light activation of antibody-photosensitizer conjugates (photoimmunoconjugates) to generate cytotoxic reactive oxygen species. Here, a microfluidic cell culture model is used to study how fluid flow-induced shear stress affects photoimmunoconjugate delivery to ovarian cancer cells. Photoimmunoconjugates are composed of the antibody, cetuximab, conjugated to the photosensitizer, and benzoporphyrin derivative. Longitudinal tracking of photoimmunoconjugate treatment under flow conditions reveals enhancements in subcellular photosensitizer accumulation. Compared to static conditions, fluid flow-induced shear stress at 0.5 and 1 dyn/cm2 doubled the cellular delivery of photoimmunoconjugates. Fluid flow-mediated treatment with three different photosensitizer formulations (benzoporphyrin derivative, photoimmunoconjugates, and photoimmunoconjugate-coated liposomes) led to enhanced phototoxicity compared to static conditions. This study confirms the fundamental role of fluid flow-induced shear stress in the anti-cancer effects of photoimmunotherapy.

Osteogenesis and osteoclastogenesis on a chip: Engineering a self-assembling 3D coculture

Healthy bone is maintained by the process of bone remodeling. An unbalance in this process can lead to pathologies such as osteoporosis which are often studied with animal models. However, data from animals have limited power in predicting the results that will be obtained in human clinical trials. In search for alternatives to animal models, human in vitro models are emerging as they address the principle of reduction, refinement, and replacement of animal experiments (3Rs). At the moment, no complete in vitro model for bone-remodeling exists. Microfluidic chips offer great possibilities, particularly because of the dynamic culture options, which are crucial for in vitro bone formation. In this study, a scaffold free, fully human, 3D microfluidic coculture model of bone remodeling is presented. A bone-on-a-chip coculture system was developed in which human mesenchymal stromal cells differentiated into the osteoblastic lineage and self-assembled into scaffold free bone-like tissues with the shape and dimensions of human trabeculae. Human monocytes were able to attach to these tissues and to fuse into multinucleated osteoclast-like cells, establishing the coculture. Computational modeling was used to determine the fluid flow induced shear stress and strain in the formed tissue. Furthermore, a set-up was developed allowing for long-term (35 days) on-chip cell culture with benefits including continuous fluid-flow, low bubble formation risk, easy culture medium exchange inside the incubator and live cell imaging options. This on-chip coculture is a crucial advance towards developing in vitro bone remodeling models to facilitate drug testing.

PI3KC2\u03b1-dependent and VPS34-independent generation of PI3P controls primary cilium-mediated autophagy in response to shear stress

Cells subjected to stress situations mobilize specific membranes and proteins to initiate autophagy. Phosphatidylinositol-3-phosphate (PI3P), a crucial lipid in membrane dynamics, is known to be essential in this context. In addition to nutriments deprivation, autophagy is also triggered by fluid-flow induced shear stress in epithelial cells, and this specific autophagic response depends on primary cilium (PC) signaling and leads to cell size regulation. Here we report that PI3KC2α, required for ciliogenesis and PC functions, promotes the synthesis of a local pool of PI3P upon shear stress. We show that PI3KC2α depletion in cells subjected to shear stress abolishes ciliogenesis as well as the autophagy and related cell size regulation. We finally show that PI3KC2α and VPS34, the two main enzymes responsible for PI3P synthesis, have different roles during autophagy, depending on the type of cellular stress: while VPS34 is clearly required for starvation-induced autophagy, PI3KC2α participates only in shear stress-dependent autophagy.

Permeability and fluid flow-induced wall shear stress in bone scaffolds with TPMS and lattice architectures: A CFD analysis

Abstract Fluid flow dynamics within porous scaffolds for tissue engineering play a critical role in the transport of fundamental materials to the cells and in controlling the biocompatibility of the scaffold. Properties such as permeability and fluid flow-induced wall shear stress characterize the biological behavior of the scaffolds. Bioactivity depends on the diffusion of oxygen and other nutritious elements through the porous medium and fluid flow-induced shear stress is known as the dominant mechanical stimulant of cell differentiation and proliferation within the scaffolds. In this study, eight different bone scaffold models with a constant porosity of 80% were designed computationally using the TPMS and lattice-based structures. We investigated the fluid flow within the scaffolds using CFD analysis. The results of the work showed that scaffold architecture has a significant impact on the permeability and that scaffold permeability can vary up to three times depending on the architecture. The scaffolds with the minimal variation in their channel size exhibited the highest permeability. We investigated the distribution statistics of wall shear stress on the walls of the scaffolds and showed that a correlation between the architecture of the scaffolds and the distribution statistics of wall shear stress did not exist. The outcomings of this work can be promising in designing better scaffolds in tissue engineering from a biological point of view.

Effect of scaffold architecture on cell seeding efficiency: A discrete phase model CFD analysis

Within perfusion cell culture systems, scaffold architecture is able to control important biological parameters such as permeability and fluid flow-induced shear stress. As well, one of the main factors affecting the final fate of this process as well as optimal cell differentiation and proliferation in these systems is initial adhesion of cells to scaffolds. In this study, the effect of scaffold architecture on the adhesion of the cells was computationally investigated. For this purpose, four scaffold models including double-diamond, gyroid, FR-D, and Schwarz-primitive were designed using triply periodic minimal surface (TPMS) geometry with a constant porosity of 80%. As well, the inlet velocity of zero to simulate static cell culture and three different inlet velocities for modeling the dynamic cell culture conditions were also selected. The results showed that cell culture efficiency of scaffolds could be changed up to seven times from architecture to architecture under the same conditions. The efficiency of cell culture in scaffolds with tortuous architecture was also reported higher than those with relatively straight microchannels. In terms of culture methods, unlike dynamic cell culture model in which almost a homogeneous cell distribution was observed in static cell culture simulation, more cells adhered, but they had agglomerated in the scaffold entrance regions and had failed to reach all regions. The results of this study shed more light on the selection and design of scaffold architecture for optimal cell culture in tissue engineering.

The primary cilium protein folliculin is part of the autophagy signaling pathway to regulate epithelial cell size in response to fluid flow.

Autophagy is a conserved molecular pathway directly involved in the degradation and recycling of intracellular components. Autophagy is associated with a response to stress situations, such as nutrients deficit, chemical toxicity, mechanical stress or microbial host defense. We have recently shown that primary cilium-dependent autophagy is important to control kidney epithelial cell size in response to fluid flow induced shear stress. Here we show that the ciliary protein folliculin (FLCN) actively participates to the signaling cascade leading to the stimulation of fluid flow-dependent autophagy upstream of the cell size regulation in HK2 kidney epithelial cells. The knockdown of FLCN induces a shortening of the primary cilium, inhibits the activation of AMPK and the recruitment of the autophagy protein ATG16L1 at the primary cilium. Altogether, our results suggest that FLCN is essential in the dialog between autophagy and the primary cilium in epithelial cells to integrate shear stress-dependent signaling.

A test method to assess the contribution of fluid shear stress to the cleaning of reusable device surfaces.

Adequate cleaning of reusable medical devices is critical for preventing cross-infection among patients. For reusable medical devices, cleaning using mechanical brushes and detergent may not be sufficient to completely remove the infectious contaminants from the surfaces. This study evaluates the role of fluid flow-induced shear stress in the detachment and removal of contaminants from device surfaces. A stainless-steel test coupon, acting as a surrogate for a device surface, was coated with artificial clot of varying mass. The test coupon was exposed to fluid shear stress both with and without an enzymatic detergent. The relationship between clot removal quantity and the applied shear stress was obtained for multiple clot masses. Our results showed that fluid shear increased the effectiveness of the cleaning process. In the absence of flow, soaking the clot surface in the enzymatic detergent removed 67%, 77%, and 95% of the clot for 16 mg, 6.8 mg, and 1 mg initial masses, respectively. In the presence of fluid shear (0.3 Pa for 5 min), approximately 85%, 97%, and 99% of the clot was removed from the surface. The clot mass removed followed a linear relationship (R2 = 0.98) versus the applied fluid shear stress. This study showed that different cleaning processes such as fluid shear and detergent action contribute to the soil removal process. This method could be used to evaluate cleaning protocols for minimizing contaminant residue after the reprocessing of medical devices. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1132-1140, 2019.

Computational Fluid Dynamics Study of the Effects of Surface Roughness on Permeability and Fluid Flow-Induced Wall Shear Stress in Scaffolds.

In this work, we investigated surface roughness effects on bone scaffold permeability and fluid flow-induced wall shear stress (WSS) using computational fluid dynamics (CFD) analysis. Scaffolds are made of interconnected microchannels, whose fluid flow can be examined from the perspective of fluid flow dynamics. Given that the roughness of microchannel surfaces serves a non-negligible function in the fluid dynamics within the channels, it is believed that the wall roughness of scaffolds can play an important role in their permeability and WSS. Given the criticality of permeability and WSS in the effective biological functioning of scaffolds, we investigated manufacturing-induced surface roughness effects on the two aforementioned biocompatibility characteristics. To this end, three scaffolds with square pores of different sizes (300, 600, and 900µm) and identical porosity (63%) were designed. Six roughness levels (0, 4, 8, 12, 16, and 20µm) were established for the scaffold walls, thus enabling us to develop 18 scaffold models. The pressure drop and WSS in the scaffolds were then measured by CFD. Scaffold permeability was calculated using Darcy's law, with reference to geometrical parameters and the pressure drop derived from the CFD analysis. In all the scaffolds, high roughness decreased permeability and WSS. A significant difference in WSS reduction was found between the models with smooth scaffolds and the models with scaffolds that had a roughness of 20µm. Except for the scaffold with a pore size of 300µm, all the others showed no considerable change in permeability at different roughness levels.

Permeability and fluid flow-induced wall shear stress of bone tissue scaffolds: Computational fluid dynamic analysis using Newtonian and non-Newtonian blood flow models

Among the factors that are important in successful bone tissue regeneration through scaffolds are permeability and fluid flow-induced wall shear stress (WSS) because of the direct contribution of these factors to cell bioactivities. The permeability of scaffolds is usually measured using fluids such as water, which are characterized as Newtonian materials with constant viscosity. However, using the fluid properties of blood as bases in measuring permeability can lead to more realistic results given that scaffolds are implanted in the body, where the only flowing fluid (i.e., blood) is a non-Newtonian fluid. Moreover, the linear relationship of WSS with fluid viscosity challenges the use of Newtonian fluids in determining WSS magnitude. With consideration for these issues, we investigated permeability and WSS through computational fluid dynamics (CFD) analyses of lattice-based and gyroid scaffold architectures with Newtonian and non-Newtonian blood flow properties. With reference to geometrical parameters and the pressure drops derived from the CFD analyses, the permeability levels of the Newtonian and non-Newtonian models were calculated by exploiting the classic and modified Darcy's equations, respectively. Results showed that both scaffold architectures were several times more permeable in the Newtonian blood flow models than in their non-Newtonian counterparts. Within the scaffolds, the non-Newtonian flow of blood caused almost twice the magnitude of WSS originating from Newtonian blood flow. These striking discrepancies in permeability and WSS between the two blood models were due to differences in their viscosity behaviors.

Functional and structural characterization of osteocytic MLO-Y4 cell proteins encoded by genes differentially expressed in response to mechanical signals in vitro

The anabolic response of bone to mechanical load is partially the result of osteocyte response to fluid flow-induced shear stress. Understanding signaling pathways activated in osteocytes exposed to fluid flow could identify novel signaling pathways involved in the response of bone to mechanical load. Bioinformatics allows for a unique perspective and provides key first steps in understanding these signaling pathways. We examined proteins encoded by genes differentially expressed in response to fluid flow in murine osteocytic MLO-Y4 cells. We considered structural and functional characteristics including putative intrinsic disorder, evolutionary conservation, interconnectedness in protein-protein interaction networks, and cellular localization. Our analysis suggests that proteins encoded by fluid flow activated genes have lower than expected conservation, are depleted in intrinsic disorder, maintain typical levels of connectivity for the murine proteome, and are found in the cytoplasm and extracellular space. Pathway analyses reveal that these proteins are associated with cellular response to stress, chemokine and cytokine activity, enzyme binding, and osteoclast differentiation. The lower than expected disorder of proteins encoded by flow activated genes suggests they are relatively specialized.

Design and Assessment of a Dynamic Perfusion Bioreactor for Large Bone Tissue Engineering Scaffolds

Bioreactors can be used to apply fluid flow in vitro to scaffolds to improve mass transport of media and apply mechanical forces to cells. In this study, we developed and tested an autoclavable, modular perfusion bioreactor suitable for large scaffolds. We investigated the effects of fluid flow induced shear stress (FFSS) on osteogenic differentiation of human embryonic stem cell-derived mesenchymal progenitors (hES-MP cells) cultured on large polyurethane (PU) scaffolds (30mm diameter × 5mm thickness) in osteogenesis induction media (OIM). After seeding, scaffolds wereeither maintained in static conditions or transferred to the bioreactor 3days post-seeding and a continuous flow rate of 3.47mL/min was applied. Alkaline phosphatase activity (ALP) was used to evaluate osteogenic differentiation and resazurin salt reduction (RR) to measure metabolic activity after 10days. Cultures subjected to flow contained significantly more metabolically active cells and higher total DNA content, as well as significantly higher ALP activity compared to scaffolds grown in static culture. These results confirm the responsiveness of hES-MP cells to fluid flow stimuli, and present a cost-effective, user-friendly bioreactor capable of supporting the growth and differentiation of mesenchymal progenitor cells within scaffolds capable of filling large bone defects.

Finite element analysis of mechanical behavior, permeability and fluid induced wall shear stress of high porosity scaffolds with gyroid and lattice-based architectures

Scaffold design necessitates the consideration of mechanical properties and fluid flow dynamics as the main factors in the development of such materials. The mechanical behavior of bone scaffolds is characterized by properties such as elastic modulus and compressive strength. In terms of fluid flow dynamics, within bone scaffolds, permeability is an important parameter that affects cells' biological activities, and flow-induced shear stress is used as a mechanical stimulant of cell growth. In this study, two scaffold architectures with gyroid and lattice-based rectangular unit cells were designed to analysis the effective elastic moduli, compressive strength, permeability and fluid flow-induced wall shear stress as functions of porosity. Six levels of porosity (65%, 70%, 75%, 80%, 85% and 90%) were assigned to the scaffold architectures, and 12 models were developed. Scaffold deformation under static loading, compressive strength based on von Mises criteria, pressure drop, and fluid flow-induced wall shear stress in the scaffolds were then determined by finite element analysis. In both the scaffold types, models with higher porosity exhibited lower mechanical properties. Under the same porosity, the lattice-based scaffolds exhibited a Young's modulus and a compressive strength higher than those achieved by the gyroid scaffolds. With reference to geometrical parameters and the derived pressure drop from the computational fluid dynamics (CFD) analysis, scaffolds permeability was calculated using Darcy's law. In both the scaffold architectures, high porosity increased permeability and decreased wall shear stress. In the same porosity, the lattice-based models exhibited higher permeability and lower wall shear stress than did the gyroid models. On the basis of the results on elastic modulus and permeability, the models that most effectively mimic the properties of cancellous bones were identified.

Fibroblast-like synoviocyte mechanosensitivity to fluid shear is modulated by interleukin-1α

Fibroblast-like synoviocytes (FLS) reside in the synovial membrane of diarthrodial joints and are exposed to a dynamic fluid environment that presents both physical and chemical stimuli. The ability of FLS to sense and respond to these stimuli plays a key role in their normal function, and is implicated in the alterations to function that occur in osteoarthritis (OA). The present work characterizes the response of FLS to fluid flow-induced shear stress via real-time calcium imaging, and tests the hypothesis that this response is modulated by interleukin-1α (IL-1α), a cytokine elevated in OA. FLS demonstrated a robust calcium signaling response to fluid shear that was dose dependent upon stress level and required both external and internal calcium sources. Preconditioning with 10ng/mL IL-1α for 24h heightened this shear stress response by significantly increasing the percent of responding cells and peak magnitude, while significantly decreasing the time for a peak to occur. Intercellular communication via gap junctions was found to account for a portion of the FLS population response in normal conditions, and was significantly increased by IL-1α preconditioning. IL-1α was also found to significantly increase average length and incidence of the primary cilium, an organelle commonly implicated in shear mechanosensing. These findings suggest that the elevated levels of IL-1α found in the OA environment heighten FLS sensitivity to fluid shear by altering both intercellular communication and individual cell sensitivity, which could affect downstream functions and contribute to progression of the disease state.

Centriole splitting caused by loss of the centrosomal linker protein C-NAP1 reducescentriolar satellite density and impedescentrosome amplification.

Duplication of the centrosomes is a tightly regulated process. Abnormal centrosome numbers can impair cell division and cause changes in how cells migrate. Duplicated centrosomes are held together by a proteinaceous linker made up of rootletin filaments anchored to the centrioles by C-NAP1. This linker is removed in a NEK2A kinase-dependent manner as mitosis begins. To explore C-NAP1 activities in regulating centrosome activities, we used genome editing to ablate it. C-NAP1-null cells were viable and had an increased frequency of premature centriole separation, accompanied by reduced density of the centriolar satellites, with reexpression of C-NAP1 rescuing both phenotypes. We found that the primary cilium, a signaling structure that arises from the mother centriole docked to the cell membrane, was intact in the absence of C-NAP1, although components of the ciliary rootlet were aberrantly localized away from the base of the cilium. C-NAP1-deficient cells were capable of signaling through the cilium, as determined by gene expression analysis after fluid flow-induced shear stress and the relocalization of components of the Hedgehog pathway. Centrosome amplification induced by DNA damage or by PLK4 or CDK2 overexpression was markedly reduced in the absence of C-NAP1. We conclude that centriole splitting reduces the local density of key centriolar precursors to impede overduplication.

Delayed Microvascular Shear Adaptation in Pulmonary Arterial Hypertension. Role of Platelet Endothelial Cell Adhesion Molecule-1 Cleavage.

Altered pulmonary hemodynamics and fluid flow-induced high shear stress (HSS) are characteristic hallmarks in the pathogenesis of pulmonary arterial hypertension (PAH). However, the contribution of HSS to cellular and vascular alterations in PAH is unclear. We hypothesize that failing shear adaptation is an essential part of the endothelial dysfunction in all forms of PAH and tested whether microvascular endothelial cells (MVECs) or pulmonary arterial endothelial cells (PAECs) from lungs of patients with PAH adapt to HSS and if the shear defect partakes in vascular remodeling in vivo. PAH MVEC (n = 7) and PAH PAEC (n = 3) morphology, function, protein, and gene expressions were compared with control MVEC (n = 8) under static culture conditions and after 24, 72, and 120 hours of HSS. PAH MVEC showed a significantly delayed morphological shear adaptation (P = 0.03) and evidence of cell injury at sites of nonuniform shear profiles that are critical loci for vascular remodeling in PAH. In clear contrast, PAEC isolated from the same PAH lungs showed no impairments. PAH MVEC gene expression and transcriptional shear activation were not altered but showed significant decreased protein levels (P = 0.02) and disturbed interendothelial localization of the shear sensor platelet endothelial cell adhesion molecule-1 (PECAM-1). The decreased PECAM-1 levels were caused by caspase-mediated cytoplasmic cleavage but not increased cell apoptosis. Caspase blockade stabilized PECAM-1 levels, restored endothelial shear responsiveness in vitro, and attenuated occlusive vascular remodeling in chronically hypoxic Sugen5416-treated rats modeling severe PAH. Delayed shear adaptation, which promotes shear-induced endothelial injury, is a newly identified dysfunction specific to the microvascular endothelium in PAH. The shear response is normalized on stabilization of PECAM-1, which reverses intimal remodeling in vivo.

Transient receptor potential vanilloid 2-mediated shear-stress responses in C2C12 myoblasts are regulated by serum and extracellular matrix.

The developmental sensitivity of skeletal muscle to mechanical forces is unparalleled in other tissues. Calcium entry via reputedly mechanosensitive transient receptor potential (TRP) channel classes has been shown to play an essential role in both the early proliferative stage and subsequent differentiation of skeletal muscle myoblasts, particularly TRP canonical (TRPC) 1 and TRP vanilloid (TRPV) 2. Here we show that C2C12 murine myoblasts respond to fluid flow-induced shear stress with increments in cytosolic calcium that are largely initiated by the mechanosensitive opening of TRPV2 channels. Response to fluid flow was augmented by growth in low extracellular serum concentration (5 vs. 20% fetal bovine serum) by greater than 9-fold and at 18 h in culture, coincident with the greatest TRPV2 channel expression under identical conditions (P < 0.02). Fluid flow responses were also enhanced by substrate functionalization with laminin, rather than with fibronectin, agreeing with previous findings that the gating of TRPV2 is facilitated by laminin. Fluid flow-induced calcium increments were blocked by ruthenium red (27%) and SKF-96365 (38%), whereas they were unaltered by 2-aminoethoxydiphenyl borate, further corroborating that TRPV2 channels play a predominant role in fluid flow mechanosensitivity over that of TRPC1 and TRP melastatin (TRPM) 7.

Human periosteal-derived cell expansion in a perfusion bioreactor system: proliferation, differentiation and extracellular matrix formation.

Perfusion bioreactor systems have shown to be a valuable tool for the in vitro development of three-dimensional (3D) cell-carrier constructs. Their use for cell expansion, however, has been much less explored. Since maintenance of the initial cell phenotype is essential in this process, it is imperative to obtain insight into the bioreactor-related variables determining cell fate. Therefore, this study investigated the influence of fluid flow-induced shear stress on the proliferation, differentiation and matrix deposition of human periosteal-derived cells in the absence of additional differentiation-inducing stimuli; 120 000 cells were seeded on additive manufactured 3D Ti6Al4V scaffolds and cultured for up to 28 days at different flow rates in the range 0.04-6 ml/min. DNA measurements showed, on average, a three-fold increase in cell content for all perfused conditions in comparison to static controls, whereas the magnitude of the flow rate did not have an influence. Contrast-enhanced nanofocus X-ray computed tomography showed substantial formation of an engineered neotissue in all perfused conditions, resulting in a filling (up to 70%) of the total internal void volume, and no flow rate-dependent differences were observed. The expression of key osteogenic markers, such as RunX2, OCN, OPN and Col1, did not show any significant changes in comparison to static controls after 28 days of culture, with the exception of OSX at high flow rates. We therefore concluded that, in the absence of additional osteogenic stimuli, the investigated perfusion conditions increased cell proliferation but did not significantly enhance osteogenic differentiation, thus allowing for this process to be used for cell expansion. Copyright © 2014 John Wiley & Sons, Ltd.

Regulation of cell migration and osteogenic differentiation in mesenchymal stem cells under extremely low fluidic shear stress

Mesenchymal stem cells (MSCs) are multipotent stem cells predominantly obtained from bone marrow, which are sensitive to mechanical loadings in physiological microenvironment. However, how the MSCs sense and respond to extremely low fluidic shear stress analogous to interstitial flow in vivo is poorly understood. In this work, we present a functional microfluidic device to examine the migration and differentiation behaviors of MSCs in response to multiple orders of physiologically relevant interstitial flow levels. The different magnitudes of fluid flow-induced shear stress were produced by a hydraulic resistance-based microfluidic perfusion system consisting of a microchannel network and a parallel of uniform cell culture chambers. By changing the length and width of the flow-in channels, the multiple magnitudes of low shear stresses could be generated ranging from ∼10−5 to ∼10−2 dyne/cm2. We demonstrated enhanced significant F-actin expression and cell migration in MSCs under applied fluidic shear stress at ∼10−2 dyne/cm2. We also demonstrated a significant osteogenic differentiation under this interstitial level of slow flows from ∼10−2 to ∼10−4 dyne/cm2 in MSCs by analyzing alkaline phosphatase activity and osteopontin staining. Moreover, cytochalasin D and Rho-inhibitor Y-27632 significantly reduced the cytoskeleton F-actin expression and osteogenic differentiation in MSCs, indicating the mediated mechanical responses of MSCs under extremely low fluidic shear stress, possibly as a consequence of Rho-associated kinase pathway. The established microfluidic perfusion system with multiple shear-flow capabilities is simple and easy to operate, providing a flexible platform for studying the responses of diverse types of cells to the multiple interstitial flow levels in a single assay.