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Background Hemorrhage and ischemia after flow diverter stent (FDS) procedures for intracranial aneurysms are the most common complications and have been extensively described. Temporary brain edema (TBE) is an unknown complication that could be associated with particular FDS procedures. Objective To estimate the frequency, clinical presentation, imaging findings, and possible mechanisms associating TBE with FDS. Methods Unruptured aneurysms treated with FDS implantation performed in our service from June 2015 to March 2018 were reviewed. Medical antecedents, endovascular procedure, clinical assessments before and after treatment, aneurysm characteristics, and image records were collected. Artery diameters of patients in whom TEB developed were also calculated to investigate any correlation between TBE and anatomic descriptors. Results A total of 179 FDS procedures in 176 patients were reviewed. Six patients (3.4%) presented with symptomatic TBE, and all TBE patients had undergone FDS implantation from the middle cerebral artery (MCA) to the internal carotid artery (ICA). A Pearson product-moment correlation coefficient (PPCC) found smaller MCA diameters and MCA/ICA ratios in these 6 patients (respectively PPCC = −0.619, P < 0.04; PPCC = −0.647, P < 0.03). Hemorrhagic and ischemic complications were less frequent than TBE (2.3% and 1.1% vs. 3.4%). Conclusions TBE was more frequent than ischemic or hemorrhagic complications after FDS in this study. TBE seemed to be associated with a particular FDS positioning in small arteries, inducing flow changes and disruption of the blood–brain barrier.

Among the cues affecting cells behaviour, mechanical stimuli are known to have a key role in tissue formation and mineralization of bone cells. While soft scaffolds are better at mimicking the extracellular environment, they cannot withstand the high loads required to be efficient substitutes for bone in vivo. We propose a 3D hybrid scaffold combining the load-bearing capabilities of polycaprolactone (PCL) and the ECM-like chemistry of collagen gel to support the dynamic mechanical differentiation of human embryonic mesodermal progenitor cells (hES-MPs). In this study, hES-MPs were cultured in vitro and a BOSE Bioreactor was employed to induce cells differentiation by mechanical stimulation. From day 6, samples were compressed by applying a 5% strain ramp followed by peak-to-peak 1% strain sinewaves at 1 Hz for 15 min. Three different conditions were tested: unloaded (U), loaded from day 6 to day 10 (L1) and loaded as L1 and from day 16 to day 20 (L2). Cell viability, DNA content and osteocalcin expression were tested. Samples were further stained with 1% osmium tetroxide in order to investigate tissue growth and mineral deposition by micro-computed tomography (µCT). Tissue growth involved volumes either inside or outside samples at day 21 for L1, suggesting cyclic stimulation is a trigger for delayed proliferative response of cells. Cyclic load also had a role in the mineralization process preventing mineral deposition when applied at the early stage of culture. Conversely, cyclic load during the late stage of culture on pre-compressed samples induced mineral formation. This study shows that short bursts of compression applied at different stages of culture have contrasting effects on the ability of hES-MPs to induce tissue formation and mineral deposition. The results pave the way for a new approach using mechanical stimulation in the development of engineered in vitro tissue as replacement for large bone fractures.

International audience; Mechanical forces and 3D topological environment can be used to control differentiation of mesenchymal stem cells (MSCs). However, the effects of physical and mechanical cues of the microenvironment on MSC fate determination have not yet been fully understood. This study investigates and compares the effect of mechanical stimulations on soft cellular microspheres when subjected to dynamic fluid compression. Microspheres were produced by gelation of bovine collagen type I with concentrations of 2 mg/ml and 1000–2000 hES-MP cells per 5 μl droplet. A loading condition of 10% dynamic loading was applied by a BOSE BioDynamic bioreactor for 15 and 40 min/day for 5 and 10 days on the cell-seeded collagen microspheres. Cell viability and proliferation, alkaline phosphatase activity and mineralization were compared with controls. Monitoring alkaline phosphatase level reported a significant increase in the enzyme activity by day 14 in loaded samples of 40 min/day loading protocol compared with other experimental conditions. Mineralization was assessed by measuring calcium, phosphorous concentrations and intensity of H&E and alizarin red S staining and showed the highest mineral accumulation in the loaded samples on day 28 post encapsulation. This study indicated that loading of very low cell number seeded on soft natural scaffold can encourage osteogenesis of cells by enhancing both early stage bone marker and mineralization. Self-assembled cell/collagen microspheres present exceptional cell delivery model in bone healing/repair process and field of regenerative medicine.

There are variety methods and materials for fabrication of the master mold for microfluidic system. In this report, two simple low-cost methods for fabrication of master molds for microfluidic chips are described and compared in quality of molds, thickness and resolution of micro-channels, design complexity, error tolerance and executive time. The first approach can create a master mold just in 5 min by using cutting plotter to cut the design out from vinyl adhesive paper based on the previous design; after that, the assembly attached into the petri dish to fabricate the complete mold. The second one is using nail polish meth(acrylates) (MA) gel as photoresist material to alter expensive SU-8 photoresist in soft photolithography technique and LED-UV light. These processes are simple, short time prototyping, inexpensive materials and no requirement for sophisticated equipment. Both methods can achieve the channels with the depth up to 80 μm. However, the channels of UV gel method are less affected by changes in temperature which enables more complex design as narrow as 200 μm in width compared to 500 μm of craft cutter method. The comparison of two proposed methods shows that UV gel satisfies the important demand for microfluidic master mold fabrication: low cost, possibility of more complex pattern in short time (15 min).

Early detection right at epidemic areas can prevent infectious diseases from propagation. Currently, the most common nucleic acid test—polymerase chain reaction (PCR) is time-consuming, complex, expensive and thermocycler required, thus limiting its utility in poor laboratory conditions or even non-laboratory condition of epidemic areas. Loop-mediated isothermal amplification (LAMP) is quick, cheap, sensitive and isothermal assay could be combined with a simple DNA extraction method to integrate into Lab-on-a-chip (LOC) device. Here, we attempted to improve LAMP method for malaria diagnosis on portable microfluidics chip platform by optimizing DNA extraction using boil and spin method and altering Tris-containing amplification buffer for ascertaining changing in pH of reaction solution. Basically, blood sample was mixed with extraction buffer containing Sodium Dedocyl Sulfate (SDS) concentration and treated under high temperature condition. Four concentrations of SDS (0, 0.4, 0.8 and 1%) were tested along with different temperature (65 and 95 °C) to adapt into LOC platform and avoid denaturation of LAMP reagent. All samples treated at 65 °C showed the presence of DNA after extraction. Furthermore, DNA amplification buffer was minimized Tris concentration to facilitate result read-out step. The releasing of hydrogen ion from amplification reaction causes increasing in pH which could be recognized by color of pH indicator paper or dye, for example, phenolphthalein. Throughout a series of experiments, LAMP is demonstrated that it can also occur in low-Tris buffer with pH indicator dye, efficiently. The positive sample will have a change from pink to transparent in solution color, otherwise, the negative sample will maintain pink. These improvements allowed us to adapt LAMP technique into Point-of-care (POC) devices in which the whole process run under isothermal condition (65 °C) and non-instrument required visual detection. The LAMP microfluidics chip will be potential tool for early detection infectious diseases and several other diseases in non-laboratory condition.

Tribology is an important factor (among other factors) during biological interactions of devices and tissues. The paper discusses how new computational and experimental methods can be used to understand and improve the design and development of medical devices at macro and micro scales to sustain life beyond 50 years. We have used pre-clinical experiments and computational methods to understand interactions between orthopaedic implants at the macro scale. The computational model has been validated with experiments. Now this computational model can predict damage in implants for different patients. One such application was successfully tried and tested in collaboration with University National Autonomous Mexico. This methodology can be used in future to design patient specific, affordable (using 3D printing) and robust implants which will be useful for developing countries like Vietnam, India and Mexico. Improvement of catheter designs is important to reduce damage to the internal tissues while being used for cardiovascular problems. We are developing new experimental techniques (in micro scale) that can be used to understand the interaction of cells with the catheter material. These will help reduce the hospital costs incurred during longer stay of the patients admitted for cardiovascular related problems.

Vascular medical devices, such as stents, catheters and more advanced devices inevitably interact with surfaces within the human body. These interactions and the underlying biological and tribological (friction) mechanisms and resulting implications are not well understood, currently. For the further optimisation of these devices and the development of new and safer devices, a deeper understanding of vascular biotribology is required. Studies about this topic are scarce and no review is available. This review paper introduces vascular physiology relevant to interaction with medical devices and highlights where tribological effects may come into play. Furthermore, implications with existing medical devices are investigated in the context of biotribology and relevant studies are discussed. The different approaches to study the interactions are compared, and the current state of the field is reviewed. The aim of this paper is to provide an introduction to this interdisciplinary field, for both researchers with an engineering background and those with a biological background, and to present the current state of the field of research.

Recent improvements in computational tools opened the possibility of patient-specific modeling to aid clinicians during diagnosis, treatment, and monitoring. One example is the modeling of blood flow for surgical planning, where modeling can help predict the prognosis. Computational analysis is used to extract hemodynamic information about the case; however, these methods are sensitive to assumptions on blood properties, boundary conditions, and appropriate geometry accuracy. When available, experimental measurements can be used to validate the results and, among the modalities, ultrasound-based methods are suitable due to their relative low cost and non-invasiveness. This work proposes a procedure to create accurate patient-specific silicone replicas of blood vessels and a power Doppler compatible experimental setup able to simulate and measure realistic flow conditions. The assessment of silicone model geometry shows small discrepancies between these and the target geometries (median of surface error lies within 57 μm and 82 μm). Power Doppler measurements were compared against computational fluid dynamics results, showing discrepancies within 10% near the wall. The experimental approach offers a setup to quantify flow in in vitro systems and provide more accurate results where other techniques (e.g., particle image velocimetry and particle tracking velocimetry) have shown limitations due to the interference of the interface. Fil: Narata, Ana Paula. Universite de Tours; Francia Fil: Silva de Moura, Fernando. Universidad Federal Do Abc; Brasil Fil: Patat, Fréderic. Universite de Tours; Francia Fil: Marzo, Alberto. The University Of Sheffield; Reino Unido Fil: Larrabide, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Exactas. Grupo de Plasmas Densos Magnetizados. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Grupo de Plasmas Densos Magnetizados; Argentina Fil: Gregoire, Jean Marc. Universite de Tours; Francia Fil: Perrault, Cecile. The University Of Sheffield; Reino Unido Fil: Sennoga, Charles A.. Universite de Tours; Francia Fil: Bouakaz, Ayache. Universite de Tours; Francia

Microfluidics are expected to revolutionize the healthcare industry especially in developing countries since it would bring portable, easy-to-use, self-contained diagnostic devices to places with limited access to healthcare. To date, however, microfluidics has not yet been able to live up to these expectations. One non-negligible factor can be attributed to inaccessible prototyping methods for researchers in low-resource settings who are unable to afford expensive equipment and/or obtain critical reagents and, therefore, unable to engage and contribute to microfluidics research. In this paper, we present methods to create microfluidic devices that reduce initial costs from hundreds of thousands of dollars to about $6000 by using readily accessible consumables and inexpensive equipment. By including the scientific community most embedded and aware of the requirements of healthcare in developing countries, microfluidics will be able to increase its reach in the research community and be better informed to provide relevant solutions to global healthcare challenges.

Mesenchymal stem cells (MSCs) are widely implicated for their potential use as a cell source for tissue engineering of skeletal tissue in regenerative medicine and tissue engineering. Mechanical forces from the microenvironment have a significant influence on differentiation of MSCs, and the resulting mechanotransduction would provide crucial adjuncts to standard biochemical signalling pathways. Combining microfluidic systems with mechanical stimulation for osteogenesis represents both a scientific and technological innovation that would greatly impact the field of regenerative medicine. We demonstrate a microfluidic chamber design for mechanical stimulation of flexible cellular microspheres and possibly a high-throughput microfluidic system for parallel processing of stem cell aggregation. We also showed that collagen microspheres serve an efficient cell delivery device supporting cell viability and migration post encapsulation.

The increased need to accelerate the healing process of critical size defects in the bone led to the study of optimal combination of cells, materials and external stimuli to obtain fully differentiated tissue to the injured site. Bioreactors play a crucial role in the control over the development of functional tissue allowing control over the surrounding chemical and mechanical environment. This chapter aims to review bioreactor systems currently available for monitoring mesenchymal stem cells (MSCs) behaviour under mechanical stimuli and to give an insight of their effect on cellular commitment. Shear stress, mechanical strain and pulsed electromagnetic field bioreactors are presented, and the effect of multiple conditions under varying parameters such as amplitude, frequency or duration of the stimuli on bone progenitor cells differentiation is considered and extensively discussed with particular focus on osteogenic and chondrogenic commitment.

Three-dimensional (3D) scaffolds are increasingly employed as support for studies on cellular activities. They are widely shown to enhance cell survival and are a promising approach to be employed to mimic the in vivo conditions due to their controlled architecture. Moreover, 3D stiff structures fabricated by additive manufacturing are able to bear mechanical stimuli finding a role in the investigation of the effect of mechanical forces on cell proliferation and commitment. With this purpose, we propose a combination of a 3D polycaprolactone (PCL) scaffold and collagen soft gel as support for studying the response of mesenchymal stem cells following mechanical compression. This chapter focuses on the characterization of 3D Insert® PCL scaffolds behaviour under mechanical compression. After defining mechanical properties and variability due to boundary effects, the focus moves on the development of a new composite scaffold made of a stiff PCL structure acting as support for cell activities and able to bear mechanical compression while embedding a soft collagen gel matrix responsible to provide an environment enhancing cellular activities as well as to transmit the stress resulting from the mechanical stimulation from the stiff matrix to the seeded cells. Finally, the last section focuses on the effect of low mechanical strain applied on seeded scaffolds and how the cellular response varies to bursts of compression applied at different time points.

BACKGROUND AND PURPOSE: Treatment of intracranial bifurcation aneurysms with flow-diverter stents can lead to caliber changes of the distal vessels in a subacute phase. This study aims to evaluate whether local anatomy and flow disruption induced by flow-diverter stents are associated with vessel caliber changes in intracranial bifurcations. MATERIALS AND METHODS: Radiologic images and demographic data were acquired for 25 patients with bifurcation aneurysms treated with flow-diverter stents. Whisker plots and Mann-Whitney rank sum tests were used to evaluate if anatomic data and caliber changes could be linked. Symmetry/asymmetry were defined as diameter ratio 1 symmetric and diameter ratio 1 asymmetric. Computational fluid dynamics was performed on idealized and patient-specific anatomies to evaluate flow changes induced by flow-diverter stents in the jailed vessel. RESULTS: Statistical analysis identified a marked correspondence between asymmetric bifurcation and caliber change. Symmetry ratios were lower for cases showing narrowing or subacute occlusion (medium daughter vessel diameter ratio 0.59) compared with cases with posttreatment caliber conservation (medium daughter vessel diameter ratio 0.95). Computational fluid dynamics analysis in idealized and patient-specific anatomies showed that wall shear stress in the jailed vessel was more affected when flow-diverter stents were deployed in asymmetric bifurcations (diameter ratio 0.65) and less affected when deployed in symmetric anatomies (diameter ratio 1.00). CONCLUSIONS: Anatomic data analysis showed statistically significant correspondence between caliber changes and bifurcation asymmetry characterized by diameter ratio 0.7 (P .001). Similarly, computational fluid dynamics results showed the highest impact on hemodynamics when flow-diverter stents are deployed in asymmetric bifurcations (diameter ratio 0.65) with noticeable changes on wall sheer stress fields. Further research and clinical validation are necessary to identify all elements involved in vessel caliber changes after flow-diverter stent procedures. Fil: Narata, A. P.. Centre Hospitalier Regional Et Universitaire de Tours; Francia Fil: De Moura, F. S.. Universidade Federal Do Abc; Brasil Fil: Larrabide, Ignacio. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Exactas. Grupo de Plasmas Densos Magnetizados. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Grupo de Plasmas Densos Magnetizados; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil; Argentina Fil: Perrault, C. M.. University Of Sheffield; Reino Unido Fil: Patat, F.. Universite de Tours; Francia Fil: Bibi, R.. Centre Hospitalier Regional Et Universitaire de Tours; Francia Fil: Velasco, S.. Université de Poitiers; Francia Fil: Januel, A. C.. Centre Hospitalier Universitaire de Toulouse; Francia Fil: Cognard, C.. Centre Hospitalier Universitaire de Toulouse; Francia Fil: Chapot, R.. Alfried Krupp Krankenhaus; Alemania Fil: Bouakaz, A.. Universite de Tours; Francia Fil: Sennoga, C. A.. Universite de Tours; Francia Fil: Marzo, A.. University Of Sheffield; Reino Unido

Mesenchymal stem cells are widely implicated as a cell source for tissue engineering of skeletal tissue in cell-based therapy. Physical and mechanical cues are potent controlling factors in cell differentiation and can be implemented as a guide to study cellular response, matrix production and tissue regeneration. Microspheres were produced by gelation of bovine collagen type I with concentration of 2 mg/mL and 1,000-2,000 cells per droplet. Short and long term cell viability of human embryonic stem cell-derived mesenchymal progenitors (hES-MPs) and MG-63 osteoblastic cells as well as collagen microstructure and contraction were monitored during 28 days post encapsulation (pc). Results indicated that collagen concentration, hence mechanical properties of cell’s extracellular micro-environment are important in cell proliferation and differentiation. Contraction of cell-embedded microspheres was found to be vital in cell adaptation and the remodelling of their new environment. It was also found that collagen concentration of 2 mg/mL supports proliferation of hES-MPs while higher collagen concentration promoted the viability of MG-63s. Results of hES-MPs characterization in 3D soft environment and mechanically stimulated hES-MPs collagen microspheres can be used in cells/therapeutic carriers, implants in bone and cartilage healing applications. The microspheres developed in this study can also be used as a tool to build more optimised construct to transfer mechanically stimulated stem cells to the specific area of a defective bone which would add significant benefit to the field of bone regeneration.

Whereas the use of flow diverter stents (FDs) represents a powerful treatment alternative to surgical clipping of intracranial aneurysms (IAs) presenting at arterial bifurcations, endovascular deployment of FDs often involves the jailing of daughter vessels (DV), and can affect the patency of the jailed DV.

Background and Purpose: Treatment of intracranial aneurysms at bifurcations with flow-diverter stents (FDS) can lead to occlusion/narrowing of the distal vessels. This study investigated the role played by haemodynamics within different bifurcation types treated with FDS. Materials and Methods: Radiological images, demographic and outcome data were acquired for 25 bifurcating aneurysm treated with FDS. Statistical analysis was used to correlate the event of occlusion/narrowing with anatomical data. Computational Fluid Dynamics (CFD) study was performed on idealized and patient-specific anatomies to identify possible cause-effect mechanisms mediated by haemodynamics. Results: Statistical analysis identified marked correlation between asymmetric bifurcation and occlusion/narrowing (diameters ratio=DR=1 symmetric, Conclusion: Analysis of the anatomical data showed statistically significant correlations between occlusion/narrowing and bifurcation asymmetry characterized by DR

3D polymeric scaffolds are increasingly used for in vitro experiments aiming to mimic the environment found in vivo, to support for cellular growth and to induce differentiation through the application of external mechanical cues. In research, experimental results must be shown to be reproducible to be claimed as valid and the first clause to ensure consistency is to provide identical initial experimental conditions between trials. As a matter of fact, 3D structures fabricated in batch are supposed to present a highly reproducible geometry and consequently, to give the same bulk response to mechanical forces. This study aims to measure the overall mechanical response to compression of commercially available 3D Insert PCL scaffolds (3D PCL) fabricated in series by fuse deposition and evaluate how small changes in the architecture of scaffolds affect the mechanical response. The apparent elastic modulus (Ea) was evaluated by performing quasi-static mechanical tests at various temperatures showing a decrease in material stiffness from 5 MPa at 25 °C to 2.2 MPa at 37 °C. Then, a variability analysis revealed variations in Ea related to the repositioning of the sample into the testing machine, but also consistent differences comparing different scaffolds. To clarify the source of the differences measured in the mechanical response, the same scaffolds previously undergoing compression, were scanned by micro computed tomography (μCT) to identify any architectural difference. Eventually, to clarify the contribution given by differences in the architecture to the standard deviation of Ea, their mechanical response was qualitatively compared to a compact reference material such as polydimethylsiloxane (PDMS). This study links the geometry, architecture and mechanical response to compression of 3D PCL scaffolds and shows the importance of controlling such parameters in the manufacturing process to obtain scaffolds that can be used in vitro or in vivo under reproducible conditions.

International audience; One of the most important areas of research on microfluidic technologies focuses on the identification and characterisation of novel materials with enhanced properties and versatility. Here we present a fast, easy and inexpensive microstructuration method for the fabrication of novel, flexible, transparent and biocompatible microfluidic devices. Using a simple hot press, we demonstrate the rapid (30 s) production of various microfluidic prototypes embossed in a commercially available soft thermoplastic elastomer (sTPE). This styrenic block copolymer (BCP) material is as flexible as PDMS and as thermoformable as classical thermoplastics. It exhibits high fidelity of replication using SU-8 and epoxy master molds in a highly convenient low-isobar (0.4 bar) and iso-thermal process. Microfluidic devices can then be easily sealed using either a simple hot plate or even a room-temperature assembly, allowing them to sustain liquid pressures of 2 and 0.6 bar, respectively. The excellent sorption and biocompatibility properties of the microchips were validated via a standard rhodamine dye assay as well as a sensitive yeast cell-based assay. The morphology and composition of the surface area after plasma treatment for hydrophilization purposes are stable and show constant and homogenous distribution of block nanodomains (similar to 22 degrees after 4 days). These domains, which are evenly distributed on the nanoscale, therefore account for the uniform and convenient surface of a "microfluidic scale device". To our knowledge, this is the first thermoplastic elastomer material that can be used for fast and reliable fabrication and assembly of microdevices while maintaining a high and stable hydrophilicity.

The formation of new blood vessels from existing vasculature, angiogenesis, is driven by coordinated endothelial cell migration and matrix remodelling in response to local signals. Recently, a growing body of evidence has shown that mechanotransduction, along with chemotransduction, is a major regulator of angiogenesis. Mechanical signals, such as fluid shear stress and substrate mechanics, influence sprouting and network formation, but the mechanisms behind this relationship are still unclear. Here, we present cellular traction forces as possible effectors activated by mechanosensing to mediate matrix remodelling, and encourage the use of traction force microscopy to study mechanotransduction in angiogenesis. We also suggest that deciphering the response of endothelial cells to mechanical signals could reveal an optimal angiogenic mechanical environment, and provide insight into development, wound healing, the initiation and growth of tumours, and new strategies for tissue engineering. This article is protected by copyright. All rights reserved.

Microfluidic systems are increasingly being used for the culture and study of dissociated cells because they require only minute amounts of materials while enabling drug screening and chemotaxis studies down to the single cell level. However, the culture of organized tissue, such as brain slices, has been more difficult to adapt to microfluidic devices. Here, we present a microfluidic system, comprising (i) a perfusion chamber for the culture of organotypic slices that is compatible with high resolution imaging on inverted microscopes, and (ii) a novel transparent microfluidic probe (MFP) for the localized microperfusion of the brain tissue. The MFP is made in poly(dimethylsiloxane), features six micrometre-scale apertures and can be assembled within a few hours in a standard laboratory. Each aperture can indiscriminately be used either for the injection or aspiration of solutions, giving rise to many possible combinations. The MFP was successfully used for the perfusion of a small number of cells in a brain slice with concurrent confocal fluorescence imaging of the perfused dye and sub-cellular structures within the tissue.

Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeostasis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow regime in which endothelial cell mechanics is acutely responsive to shear stress.

Stromal-derived factor-1 (SDF-1) is a critical chemokine for endothelial progenitor cell (EPC) recruitment to areas of ischemia, allowing these cells to participate in compensatory angiogenesis. The SDF-1 receptor, CXCR4, is expressed in developing blood vessels as well as on CD34+ EPCs. We describe that picomolar and nanomolar concentrations of SDF-1 differentially influence neovascularization, inducing CD34+ cell migration and EPC tube formation. CD34+ cells isolated from diabetic patients demonstrate a marked defect in migration to SDF-1. This defect is associated, in some but not all patients, with a cell surface activity of CD26/dipeptidyl peptidase IV, an enzyme that inactivates SDF-1. Diabetic CD34+ cells also do not migrate in response to vascular endothelial growth factor and are structurally rigid. However, incubating CD34+ cells with a nitric oxide (NO) donor corrects this migration defect and corrects the cell deformability. In addition, exogenous NO alters vasodilator-stimulated phosphoprotein and mammalian-enabled distribution in EPCs. These data support a common downstream cytoskeletal alteration in diabetic CD34+ cells that is independent of growth factor receptor activation and is correctable with exogenous NO. This inability of diabetic EPCs to respond to SDF-1 may contribute to aberrant tissue vascularization and endothelial repair in diabetic patients.

Gastrin-releasing peptide (GRP) is typically viewed as a growth factor in cancer. However, we have suggested that in colon cancer, GRP acts primarily as a morphogen when it and its receptor (GRP-R) are aberrantly upregulated. As such, GRP/GRP-R act(s) primarily to modulate processes contributing to the assumption or maintenance of tumor differentiation. One of the most important such processes is the ability of tumor cells to achieve directed motility in the context of tissue remodeling. Yet the cellular conditions affecting GRP/GRP-R expression, and the biochemical pathways involved in mediating its morphogenic properties, remain to be established. To study this, we evaluated the human colon cancer cell lines Caco-2 and HT-29 cells. We found that confluent cells do not express GRP/GRP-R. In contrast, disaggreation and plating at subconfluent densities results in rapid GRP/GRP-R upregulation followed by their progressive decrease as confluence is achieved. GRP/GRP-R coexpression correlated with that of focal adhesion kinase (FAK) phosphorylation of Tyr397, Tyr407, Tyr861, and Tyr925 but not Tyr576 or Tyr577. To more specifically evaluate the kinetics of GRP/GRP-R upregulation, we wounded confluent cell monolayers. At t = 0 h GRP/GRP-R were not expressed, yet cells immediately began migrating into the gap created by the wound. GRP/GRP-R were first detected at ∼2 h, and maximal levels were observed at ∼6 h postwounding. The GRP-specific antagonist [d-Phe6]-labeled bombesin methyl ester had no effect on cell motility before GRP-R expression. In contrast, this agent increasingly attenuated cell motility with increasing GRP-R expression such that from t = 6 h onward no further cell migration into the gap was observed. Overall, these findings indicate the existence of GRP-independent and -dependent phases of tumor cell remodeling with the latter mediating colon cancer cell motility during remodeling via FAK.

The ability to model the mechanical responses of different cell types presents many opportunities to tissue engineering research to further identify changes from physiological conditions to disease. Using a previously validated finite element cell model we aim to show how variation of the material properties of the intracellular components affects cell response after compression and shearing. A parametric study was performed to understand the key mechanical features from different cell types, focussing on specific cytoskeleton components and prestress. Results show that actin cortex does not have a mechanical role in resisting shearing loading conditions. The sensitivity analysis predicted that cell force to compression and shearing is highly affected by changes in cortex thickness, cortex Young's modulus and rigidity of the remaining cytoplasm. Variation of prestress affects mainly the response of cells under shear loads and the model defines a relationship between cell force and prestress depending on the specific loading conditions, which is in good agreement with in vitro experiments. The results are used to make predictions that can relate mechanical properties with cell phenotype to be used as guidelines for individual cytoskeletal structures for future modelling efforts of the structure–function relationships of living cells.

Several computational models based on experimental techniques and theories have been proposed to describe cytoskeleton (CSK) mechanics. Tensegrity is a prominent model for force generation, but it cannot predict mechanics of individual CSK components, nor explain the discrepancies from the different single cell stimulating techniques studies combined with cytoskeleton-disruptors. A new numerical concept that defines a multi-structural 3D finite element (FE) model of a single-adherent cell is proposed to investigate the biophysical and biochemical differences of the mechanical role of each cytoskeleton component under loading. The model includes prestressed actin bundles and microtubule within cytoplasm and nucleus surrounded by the actin cortex. We performed numerical simulations of atomic force microscopy (AFM) experiments by subjecting the cell model to compressive loads. The numerical role of the CSK components was corroborated with AFM force measurements on U2OS-osteosarcoma cells and NIH-3T3 fibroblasts exposed to different cytoskeleton-disrupting drugs. Computational simulation showed that actin cortex and microtubules are the major components targeted in resisting compression. This is a new numerical tool that explains the specific role of the cortex and overcomes the difficulty of isolating this component from other networks in vitro. This illustrates that a combination of cytoskeletal structures with their own properties is necessary for a complete description of cellular mechanics.

The microfluidic probe (MFP) consists of a flat, blunt tip with two apertures for the injection and reaspiration of a microjet into a solution--thus hydrodynamically confining the microjet--and is operated atop an inverted microscope that enables live imaging. By scanning across a surface, the microjet can be used for surface processing with the capability of both depositing and removing material; as it operates under immersed conditions, sensitive biological materials and living cells can be processed. During scanning, the MFP is kept immobile and centered over the objective of the inverted microscope, a few micrometers above a substrate that is displaced by moving the microscope stage and that is flushed continuously with the microjet. For consistent and reproducible surface processing, the gap between the MFP and the substrate, the MFP's alignment, the scanning speed, the injection and aspiration flow rates, and the image capture need all to be controlled and synchronized. Here, we present an automated MFP station that integrates all of these functionalities and automates the key operational parameters. A custom software program is used to control an independent motorized Z stage for adjusting the gap, a motorized microscope stage for scanning the substrate, up to 16 syringe pumps for injecting and aspirating fluids, and an inverted fluorescence microscope equipped with a charge-coupled device camera. The parallelism between the MFP and the substrate is adjusted using manual goniometer at the beginning of the experiment. The alignment of the injection and aspiration apertures along the scanning axis is performed using a newly designed MFP screw holder. We illustrate the integrated MFP station by the programmed, automated patterning of fluorescently labeled biotin on a streptavidin-coated surface.

We have shown that the microtopography (mT) underlying colon cancer changes as a tumor de-differentiates. We distinguish the well-differentiated mT based on the increasing number of "pits" and poorly differentiated mT on the basis of increasing number of "posts." We investigated Rho A as a mechanosensing protein using mT features derived from those observed in the ECM of colon cancer. We evaluated Rho A activity in less-tumorogenic (Caco-2 E) and more tumorigenic (SW620) colon cancer cell-lines on microfabricated pits and posts at 2.5 μm diameter and 200 nm depth/height. In Caco-2 E cells, we observed a decrease in Rho A activity as well as in the ratio of G/F actin on surfaces with either pits or posts but despite this low activity, knockdown of Rho A led to a significant decrease in confined motility suggesting that while Rho A activity is reduced on these surfaces it still plays an important role in controlling cellular response to barriers. In SW620 cells, we observed that Rho A activity was greatest in cells plated on a post microtopography which led to increased cell motility, and an increase in actin cytoskeletal turnover.

Stromal-derived factor-1 (SDF-1) is a critical chemokine for endothelial progenitor cell (EPC) recruitment to areas of ischemia, allowing these cells to participate in compensatory angiogenesis. The SDF-1 receptor, CXCR4, is expressed in developing blood vessels as well as on CD34+ EPCs. We describe that picomolar and nanomolar concentrations of SDF-1 differentially influence neovascularization, inducing CD34+ cell migration and EPC tube formation. CD34+ cells isolated from diabetic patients demonstrate a marked defect in migration to SDF-1. This defect is associated, in some but not all patients, with a cell surface activity of CD26/dipeptidyl peptidase IV, an enzyme that inactivates SDF-1. Diabetic CD34+ cells also do not migrate in response to vascular endothelial growth factor and are structurally rigid. However, incubating CD34+ cells with a nitric oxide (NO) donor corrects this migration defect and corrects the cell deformability. In addition, exogenous NO alters vasodilator-stimulated phosphoprotein and mammalian-enabled distribution in EPCs. These data support a common downstream cytoskeletal alteration in diabetic CD34+ cells that is independent of growth factor receptor activation and is correctable with exogenous NO. This inability of diabetic EPCs to respond to SDF-1 may contribute to aberrant tissue vascularization and endothelial repair in diabetic patients.

The Sertoli cell ectoplasmic specialization (ES) is a specialized domain of the calcium-dependent Sertoli cell-spermatid junctional complex. Not only is it associated with the mechanical adhesion of the cells, but it also plays a role in the morphogenesis and differentiation of the developing germ cells. Abnormal or absent Sertoli ESs have been associated with step-8 spermatid sloughing and subsequent oligospermia. With a micropipette pressure transducing system (MPTS) to measure the force needed to detach germ cells from Sertoli cells, this study examined, for the first time, the strength of the junction between Sertoli cells and spermatids and between Sertoli cells and spermatocytes. The mean force needed to detach spermatocytes from Sertoli cells was 5.25 x 10(-7) pN, prestep-8 spermatids from Sertoli cells was 4.73 x 10(-7) pN, step-8 spermatids from Sertoli cells was 8.82 x 10(-7) pN, and spermatids plus EDTA was 2.16 x 10(-7) pN. These data confirm the hypothesis that step-8 spermatids are more firmly attached to Sertoli cells than are spermatocytes and pre-step-8 spermatids and that calcium chelation reduces binding strength between Sertoli cells and spermatids. The MPTS is a useful tool in studying the various molecular models of the Sertoli-germ cell junctional strength and the role of reproductive hormones and enzymes in coupling and uncoupling of germ cells from Sertoli cells.

Purpose: Stent deployment to treat coronary artery disease causes damage and loss of endothelial cells (EC). Repair of injured arteries by EC migration is co-ordinated by Rho family GTPases. It is also regulated by wall shear stress (WSS), a mechanical force exerted by flowing blood on the vessel wall, via mechanisms that are poorly understood. It was hypothesised that stent struts may impede repair of injured endothelium by inducing localised disturbances in WSS. Methods: To simulate a stented artery in vitro, chamber slides were fabricated with ridges (100 μm high) positioned perpendicularly to the flow direction. Confluent EC monolayers seeded on one side of either ridged or non-ridged (control) chambers were exposed to flowing culture medium (Ibidi® system). Flow patterns were determined by computational fluid dynamic (CFD) modelling and particle velocimetry. Live cell imaging and analysis using ImageJ® software enabled quantitation of EC migration velocity and directional persistence (DP). Results: CFD modelling and live cell imaging indicated that EC on the non-ridged chamber slide were exposed to a uniform WSS of 13 dyn/cm2 and migrated relatively uniformly in parallel with the flow direction (average velocity 1.13±0.20 μm/min; DP 0.59±0.14). By contrast, significant spatial differences in WSS were observed over the ridged slide in CFD, with significant spikes above physiological levels (>70 dyn/cm2) at the corners of the ridges and distinctive flow recirculation zone immediately upstream/ downstream from the ridge (-4 dyn/cm2). These features were verified by particle velocimetry using fluorescently labelled polystyrene beads (2 μm diameter). Time-lapse imaging further revealed interrupted EC migration at the ridges. Specifically, although EC could migrate over the ridges, those that reached the recirculation zone downstream from the ridge migrated with non-uniform directionality (DP 0.25±0.06) and displayed a reduction in velocity (0.78±0.18 μm/min). Inhibition of the RhoA/ROCK signalling pathway with ROCK inhibitors (Y27632 or HA1077) promoted EC forward migration within the recirculation zone by significantly elevating DP (0.41±0.04, p=0.01) and velocity (1.16±0.09 μm/min, p=0.01). Conclusions: Disturbed WSS downstream from stent strut-like ridges prevented the forward migration of EC. Inhibition of the RhoA/ROCK signaling pathway promoted EC migration and re-population of these sites. Our data suggest that treatment using a ROCK inhibitor may promote re-endothelialisation of stented arteries; a concept that is currently been tested using a porcine model.

A functional tissue engineered scaffold requires an optimum cell seeding process. Proper cell density and spatial distribution in a 3D scaffold are essential to morphogenetic development of an engineered tissue [Li et al, 2001]. A high number of cells and an even cell distribution in a scaffold are associated with better culture results [Wendt et al, 2006]. Since human cells are often available in short supply, maximization of the cell seeding process is necessary. The aim of the present study was to propose a complete method capable of predicting the cell seeding process under dynamic conditions for regular scaffolds.