Phenotyping polarization dynamics of immune cells using a lipid droplet-cell pairing microfluidic platform.

The immune synapse is the tight contact zone between a lymphocyte and a cell presenting its cognate antigen. This structure serves as a signaling platform and entails a polarization of intracellular components necessary to the immunological function of the cell. While the surface properties of the presenting cell are known to control the formation of the synapse, their impact on polarization has not yet been studied. Using functional lipid droplets as tunable artificial presenting cells combined with a microfluidic pairing device, we simultaneously observe synchronized synapses and dynamically quantify polarization patterns of individual B cells. By assessing how ligand concentration, surface fluidity, and substrate rigidity impact lysosome polarization, we show that its onset and kinetics depend on the local antigen concentration at the synapse and on substrate rigidity. Our experimental system enables a fine phenotyping of monoclonal cell populations based on their synaptic readout.

A 3D in Silico Multi-Tissue Evolution Model Highlights the Relevance of Local Strain Accumulation in Bone Fracture Remodeling.

Since 5-10% of all bone fractures result in non-healing situations, a thorough understanding of the various bone fracture healing phases is necessary to propose adequate therapeutic strategies. In silico models have greatly contributed to the understanding of the influence of mechanics on tissue formation and resorption during the soft and hard callus phases. However, the late-stage remodeling phase has not been investigated from a mechanobiological viewpoint so far. Here, we propose an in silico multi-tissue evolution model based on mechanical strain accumulation to investigate the mechanobiological regulation of bone remodeling during the late phase of healing. Computer model predictions are compared to histological data of two different pre-clinical studies of bone healing. The model predicted the bone marrow cavity re-opening and the resorption of the external callus. Our results suggest that the local strain accumulation can explain the fracture remodeling process and that this mechanobiological response is conserved among different mammal species. Our study paves the way for further understanding of non-healing situations that could help adapting therapeutic strategies to foster bone healing.

Prediction of Cortical Bone Thickness Variations in the Tibial Diaphysis of Running Rats.

A cell-mechanobiological model is used for the prediction of bone density variation in rat tibiae under medium and high mechanical loads. The proposed theoretical-numerical model has only four parameters that need to be identified experimentally. It was used on three groups of male Wistar rats under sedentary, moderate intermittent and continuous running scenarios over an eight week period. The theoretical numerical model was able to predict an increase in bone density under intermittent running (medium intensity mechanical load) and a decrease of bone density under continuous running (higher intensity mechanical load). The numerical predictions were well correlated with the experimental observations of cortical bone thickness variations, and the experimental results of cell activity enabled us to validate the numerical results predictions. The proposed model shows a good capacity to predict bone density variation through medium and high mechanical loads. The mechanobiological balance between osteoblast and osteoclast activity seems to be validated and a foreseen prediction of bone density is made available.

A Cellular Potts energy-based approach to analyse the influence of the surface topography on single cell motility.

The surface shape is an important aspect to take into account to ensure the success of an implant. At the cellular scale level, the cell behaviour, especially its migration, is affected by the specificities of the surface of the substrate, such as the stiffness of the surface and its roughness topography. The latter has been shown to have a great impact on various cell mechanisms, such as the cell adhesion, migration, or proliferation. In fact, the mere presence of micro roughness leads to an improvement of those mechanisms, with a better integration of the implants. However, the phenomena behind those improvements are still not clear. In this paper, we propose a three-dimensional (3D) model of a single cell migration using a Cellular Potts (CP) model to study the influence of the surface topography on cell motility. To do so, various configurations were tested, such as: (i) a substrate with a random roughness, (ii) a substrate with a rectangular groove pattern (parallel and perpendicular to the direction of motion), (ii) a substrate with a sinusoidal groove pattern. To evaluate the influence of the surface topography on cell motility, for each configuration, the cell speed and shape as well as the contact surface between the cell and the substrate have been quantified. Our numerical results demonstrate that, in agreement with the experimental observations of the literature, the substrate topography has an influence on the cell efficiency (i.e. cell speed), orientation and shape. Besides, we also show that the increase of the contact surface alone in presence of roughness is not enough to explain the improvement of cell migration on the various rough surfaces. Finally, we highlight the importance of the roughness dimension on cell motility. This could be a critical aspect to consider for further analyses and applications, such as surface treatments for medical applications.

High-throughput microfluidic micropipette aspiration device to probe time-scale dependent nuclear mechanics in intact cells.

The mechanical properties of the cell nucleus are increasingly recognized as critical in many biological processes. The deformability of the nucleus determines the ability of immune and cancer cells to migrate through tissues and across endothelial cell layers, and changes to the mechanical properties of the nucleus can serve as novel biomarkers in processes such as cancer progression and stem cell differentiation. However, current techniques to measure the viscoelastic nuclear mechanical properties are often time consuming, limited to probing one cell at a time, or require expensive, highly specialized equipment. Furthermore, many current assays do not measure time-dependent properties, which are characteristic of viscoelastic materials. Here, we present an easy-to-use microfluidic device that applies the well-established approach of micropipette aspiration, adapted to measure many cells in parallel. The device design allows rapid loading and purging of cells for measurements, and minimizes clogging by large particles or clusters of cells. Combined with a semi-automated image analysis pipeline, the microfluidic device approach enables significantly increased experimental throughput. We validated the experimental platform by comparing computational models of the fluid mechanics in the device with experimental measurements of fluid flow. In addition, we conducted experiments on cells lacking the nuclear envelope protein lamin A/C and wild-type controls, which have well-characterized nuclear mechanical properties. Fitting time-dependent nuclear deformation data to power law and different viscoelastic models revealed that loss of lamin A/C significantly altered the elastic and viscous properties of the nucleus, resulting in substantially increased nuclear deformability. Lastly, to demonstrate the versatility of the devices, we characterized the viscoelastic nuclear mechanical properties in a variety of cell lines and experimental model systems, including human skin fibroblasts from an individual with a mutation in the lamin gene associated with dilated cardiomyopathy, healthy control fibroblasts, induced pluripotent stem cells (iPSCs), and human tumor cells. Taken together, these experiments demonstrate the ability of the microfluidic device and automated image analysis platform to provide robust, high throughput measurements of nuclear mechanical properties, including time-dependent elastic and viscous behavior, in a broad range of applications.

In silico approach to quantify nucleus self-deformation on micropillared substrates.

Considering the major role of confined cell migration in biological processes and diseases, such as embryogenesis or metastatic cancer, it has become increasingly important to design relevant experimental set-ups for in vitro studies. Microfluidic devices have recently presented great opportunities in their respect since they offer the possibility to study all the steps from a suspended to a spread, and eventually crawling cell or a cell with highly deformed nucleus. Here, we focus on the nucleus self-deformation over a micropillared substrate. Actin networks have been observed at two locations in this set-up: above the nucleus, forming the perinuclear actin cap (PAC), and below the nucleus, surrounding the pillars. We can then wonder which of these contractile networks is responsible for nuclear deformation. The cytoplasm and the nucleus are represented through the superposition of a viscous and a hyperelastic material and follow a series of processes. First, the suspended cell settles on the pillars due to gravity. Second, an adhesive spreading force comes into play, and then, active deformations contract one or both actin domains and consequently the nucleus. Our model is first tested on a flat substrate to validate its global behaviour before being confronted to a micropillared substrate. Overall, the nucleus appears to be mostly pulled towards the pillars, while the mechanical action of the PAC is weak. Eventually, we test the influence of gravity and prove that the gravitational force does not play a role in the final deformation of the nucleus.

A numerical model suggests the interplay between nuclear plasticity and stiffness during a perfusion assay.

Cell deformability is a necessary condition for a cell to be able to migrate, an ability that is vital both for healthy and diseased organisms. The nucleus being the largest and stiffest organelle, it often is a barrier to cell migration. It is thus essential to characterize its mechanical behaviour. First, we numerically investigate the visco-elasto-plastic properties of the isolated nucleus during a compression test. This simulation highlights the impact of the mechanical behaviour of the nuclear lamina and the nucleoplasm on the overall plasticity. Second, a whole cell model is developed to simulate a perfusion experiment to study the possible interactions between the cytoplasm and the nucleus. We analyze and discuss the role of the lamina for a wild-type cell model, and a lamin-deficient one, in which the Young's modulus of the lamina is set to 1% of its nominal value. This simulation suggests an interplay between the cytoplasm and the nucleoplasm, especially in the lamin-deficient cell, showing the need of a stiffer nucleoplasm to maintain nuclear plasticity.

Good vs Poor Results After Total Hip Arthroplasty: An Analysis Method Using Implant and Anatomic Parameters With the EOS Imaging System.

BACKGROUND: Existing imaging techniques and single-parameter analyses, in nonfunctional positions, fail to detect the differences between patients with good vs poor results after total hip arthroplasty. METHODS: The present study developed an analysis method using the EOS full-body, low-dose, biplanar, weightbearing imaging system to compare good vs poor patients after total hip arthroplasty and to report on our preliminary experiences (17 good, 18 poor). RESULTS: All revision cases were found to have at least 4 high or low implant or anatomic parameters relative to the good group. These included acetabular cup orientation, sagittal pelvic tilt, sacral slope, femoral offset, and neck-shaft angle. Acetabular cup orientation differed significantly between groups. CONCLUSION: With the EOS system, a large cohort can be studied relatively quickly and at low dose, which could lead to patient-specific guidelines.

Identification of anisotropic tensile strength of cortical bone using Brazilian test.

For a proper analysis of cortical bone behaviour, it is essential to take into account both the elastic stiffness and the failure criteria. While ultrasound methods allow complete identification of the elastic orthotropic coefficients, tests used to characterise the various failure mechanisms and to identify the brittle tensile strength in all directions are currently inadequate. In the present work we propose the Brazilian test as a complement to conventional tensile tests. In fact, this experimental technique, rarely employed in the biomechanics field, has the potential to provide an accurate description of the anisotropic strength of cortical bone. Additionally, it allows us to assess the scale influence on failure behaviour which may be attributed to an intrinsic length in correlation with the cortical bone microstructure. In order to correctly set up the Brazilian test, several aspects such as the machining, the geometrical parameters of the specimen and the loading conditions were determined. The finite element method was used to evaluate the maximal tensile stress at the centre of a 2D anisotropic elastic specimen as a simple function of the loading. To validate the protocol, the Brazilian test was carried out on 29 cortical bovine cylindrical specimens with diameters ranging from 10mm to 4mm.

Cell migration with multiple pseudopodia: temporal and spatial sensing models.

Cell migration triggered by pseudopodia (or "false feet") is the most used method of locomotion. A 3D finite element model of a cell migrating over a 2D substrate is proposed, with a particular focus on the mechanical aspects of the biological phenomenon. The decomposition of the deformation gradient is used to reproduce the cyclic phases of protrusion and contraction of the cell, which are tightly synchronized with the adhesion forces at the back and at the front of the cell, respectively. First, a steady active deformation is considered to show the ability of the cell to simultaneously initiate multiple pseudopodia. Here, randomness is considered as a key aspect, which controls both the direction and the amplitude of the false feet. Second, the migration process is described through two different strategies: the temporal and the spatial sensing models. In the temporal model, the cell "sniffs" the surroundings by extending several pseudopodia and only the one that receives a positive input will become the new leading edge, while the others retract. In the spatial model instead, the cell senses the external sources at different spots of the membrane and only protrudes one pseudopod in the direction of the most attractive one.

'Run-and-tumble' or 'look-and-run'? A mechanical model to explore the behavior of a migrating amoeboid cell.

Single cell migration constitutes a fundamental phenomenon involved in many biological events. Amoeboid cells are single cell organisms that migrate in a cyclic manner like worms. In this paper, we propose a 3D finite element model of an amoeboid cell migrating over a 2D surface. In particular, we focus on the mechanical aspect of the problem. The cell is able to generate cyclic active deformations, such as protrusion and contraction, in any direction. The progression of the cell is governed by a tight synchronization between the adhesion forces, which are alternatively applied at the front and at the rear edges of the cell, and the protrusion-contraction phases of the cell body. Finally, two important aspects have been taken into account: (1) the external stimuli in response to which the cell migrates (e.g. need to feed, morphogenetic events, normal or abnormal environment cues), (2) the heterogeneity of the 2D substrate (e.g. obstacles, rugosity, slippy regions) for which two distinct approaches have been evaluated: the 'run-and-tumble' strategy and the 'look-and-run' strategy. Overall, the results show a good agreement with respect to the experimental observations and the data from the literature (e.g. velocity and strains). Therefore, the present model helps, on one hand, to better understand the intimate relationship between the deformation modes of a cell and the adhesion strength that is required by the cell to crawl over a substrate, and, on the other hand, to put in evidence the crucial role played by mechanics during the migration process.