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Magnetic microposts for mechanical stimulation of biological cells: Fabrication, characterization, and analysis
Abstract
Cells use force as a mechanical signal to sense and respond to their microenvironment. Understanding how mechanical forces affect living cells requires the development of tool sets that can apply nanoscale forces and also measure cellular traction forces. However, there has been a lack of techniques that integrate actuation and sensing components to study force as a mechanical signal. Here, we describe a system that uses an array of elastomeric microposts to apply external forces to cells through cobalt nanowires embedded inside the microposts. We first biochemically treat the posts' surfaces to restrict cell adhesion to the posts' tips. Then by applying a uniform magnetic field (B<0.3 T), we induce magnetic torque on the nanowires that is transmitted to a cell's adhesion site as an external force. We have achieved external forces of up to 45 nN, which is in the upper range of current nanoscale force-probing techniques. Nonmagnetic microposts, similarly prepared but without nanowires, surround the magnetic microposts and are used to measure the traction forces and changes in cell mechanics. We record the magnitude and direction of the external force and the traction forces by optically measuring the deflection of the microposts, which linearly deflect as cantilever springs. With this approach, we can measure traction forces before and after force stimulation in order to monitor cellular response to forces. We present the fabrication methods, magnetic force characterization, and image analysis techniques used to achieve the measurements.
... For small deflections, the deformation of the microposts in response to cellular traction forces can be modeled by beam bending theory, which provides a method to directly transform the microposts deflections from their resting positions into the cellular traction force field (Fu et al., 2010;Tan et al., 2003). Further, embedding magnetic nanowires into the microposts provides an approach to applying mechanical perturbations to cells through these same focal adhesion linkages by magnetic actuation (Shi et al., 2019;Sniadecki et al., 2007;Sniadecki, Lamb, Liu, Chen, & Reich, 2008). Another widely used technique to probe cellular traction force is traction force microscopy (TFM), which employs flat substrates with embedded microbead tracers (Plotnikov et al., 2014). ...
... This protocol describes how to extend replica molding techniques for fabricating MPAD arrays (Fu et al., 2010;Yang et al., 2011) to embed magnetic Ni nanowires in individual microposts (Shi et al., 2019). The original work describing magnetic micropost array fabrication (Sniadecki et al., 2007;Sniadecki et al., 2008) used Co nanowires instead of Ni. While Co has a larger magnetic moment than Ni and hence can, in principle, provide larger magnetic actuation forces on individual microposts, some issues are associated with the use of Co that make Ni preferable. ...
... In addition, the magnetic properties of Co mean that Co nanowires do not form permanent magnets and so behave somewhat like superparamagnetic particles in a field perpendicular to their long axis (as one has here). As a result, microposts with Co nanowires can only be deflected in a single direction from their resting position, regardless of whether the applied magnetic field changes sign (Sniadecki et al., 2007;Sniadecki et al., 2008). Ni nanowires, in contrast, are good permanent magnets, so their actuation is bi-directional, following the direction of the applied field. ...
The dynamics of the cellular actomyosin cytoskeleton are crucial to many aspects of cellular function. Here, we describe techniques that employ active micropost array detectors (AMPADs) to measure cytoskeletal rheology and mechanical force fluctuations. The AMPADS are arrays of flexible poly(dimethylsiloxane) (PDMS) microposts with magnetic nanowires embedded in a subset of microposts to enable actuation of those posts via an externally applied magnetic field. Techniques are described to track the magnetic microposts’ motion with nanometer precision at up to 100 video frames per second to measure the local cellular rheology at well‐defined positions. Application of these high‐precision tracking techniques to the full array of microposts in contact with a cell also enables mapping of the cytoskeletal mechanical fluctuation dynamics with high spatial and temporal resolution. This article describes (1) the fabrication of magnetic micropost arrays, (2) measurement protocols for both local rheology and cytoskeletal force fluctuation mapping, and (3) special‐purpose software routines to reduce and analyze these data. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.
Basic Protocol 1 : Fabrication of magnetic micropost arrays
Basic Protocol 2 : Data acquisition for cellular force fluctuations on non‐magnetic micropost arrays
Basic Protocol 3 : Data acquisition for local cellular rheology measurements with magnetic microposts
Basic Protocol 4 : Data reduction: determining microposts’ motion
Basic Protocol 5 : Data analysis: determining local rheology from magnetic microposts
Basic Protocol 6 : Data analysis for force fluctuation measurements
Support Protocol 1 : Fabrication of magnetic Ni nanowires by electrodeposition
Support Protocol 2 : Configuring Streampix for magnetic rheology measurements
... As an extreme approximation, it was considered that the NP-loaded material of each pillar is much stiffer than the non-loaded counterpart, E PDMS+NP >> E PDMS . The spring constant of these structures in the absence of magnetic field was estimated using Castigliano's method (19), ...
... Finally, the induced moment of NP-loaded PDMS pillars in the presence of an applied magnetic field was estimated using the framework presented by Sniadecki et al. (19). In this case, a magnetic field (B, corresponding to B calib in Figure 1B) is applied tangential to the array (perpendicular to an individual pillar) as specified for the configuration of Sniadecki et al. ...
... The small diameter of these pillars made high-occupancy loading of pillars using the nanowire approach (19) or others for fabricating larger, magnetically actuated cilia (24,25) impractical. Instead, micro-scale magnetic structures were created by loading what will be the upper half of each elastomer with 10-nm superparamagnetic iron oxide nanoparticles (as detailed in 2.3 Magnetic Pillar Fabrication); coupling between NPs in the presence of an applied field recapitulates the behavior of the nanowires, leading to torque generation (26). ...
The ability of cells to recognize and respond to the mechanical properties of their environment is of increasing importance in T cell physiology. However, initial studies in this direction focused on planar hydrogel and elastomer surfaces, presenting several challenges in interpretation including difficulties in separating mechanical stiffness from changes in chemistry needed to modulate this property. We introduce here the use of magnetic fields to change the structural rigidity of microscale elastomer pillars loaded with superparamagnetic nanoparticles, independent of substrate chemistry. This magnetic modulation of rigidity, embodied as the pillar spring constant, changed the interaction of mouse naïve CD4⁺ T cells from a contractile morphology to one involving deep embedding into the array. Furthermore, increasing spring constant was associated with higher IL-2 secretion, showing a functional impact on mechanosensing. The system introduced here thus separates local substrate stiffness and long-range structural rigidity, revealing new facets of T cell interaction with their environment.
... To clarify the cellular mechanosensitive functions of physiological activities, cellular traction forces have been measured using various techniques, such as micro post arrays [7][8][9][10][11][12][13][14], fluorescent beads [15,16], atomic force microscopy (AFM) [17][18][19], and microelectromechanical systems (MEMS) force sensors [20,21]. Previous studies revealed that cells generate forces from nN to µN, according to the cell types and exper imental techniques used. ...
... Adhesive cells tend to form FAs in peripheral areas and attach to the substrate via these FAs, generating traction forces [13][14][15], as simply depicted in figure 1(a). The cytoskeleton of the cell pulls the substrate toward the cell nucleus (F 1 and F 2 ) and pushes the substrate under the nucleus (F 3 ). ...
... The cellular force measurements using piezoresistive cantilevers revealed that cellular adhesion on the sensor generated traction forces sufficient to bend the cantilever within 30 min and that the force increased up to 300 nN in 3 h, consistent with the traction forces estimated in previous studies [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. The force change per FA was estimated to be in the range 0.6-30.9 ...
Adhesive cells perceive the mechanical properties of the substrates to which they adhere, adjusting their cellular mechanical forces according to their biological characteristics. This mechanical interaction subsequently affects the growth, locomotion, and differentiation of the cell. However, little is known about the detailed mechanism that underlies this interaction between adherent cells and substrates because dynamically measuring mechanical phenomena is difficult. Here, we utilize microelectromechamical systems force sensors that can measure cellular traction forces with high temporal resolution (∼2.5 μs) over long periods (∼3 h). We found that the cellular dynamics reflected physical phenomena with time scales from milliseconds to hours, which contradicts the idea that cellular motion is slow. A single focal adhesion (FA) generates an average force of 7 nN, which disappears in ms via the action of trypsin-ethylenediaminetetraacetic acid. The force-changing rate obtained from our measurements suggests that the time required for an FA to decompose was nearly proportional to the force acting on the FA.
... Furthermore, polymer micropillar arrays do not have any known adverse side effects on cells and they can integrate individual actuators as small as a few microns (Moraes et al. 2006;Desmaële et al. 2011). It is for these reasons that over the past decade application of polymer micropillar arrays for cell-mechanics studies has attracted increasingly more attention from the scientific community (Zhang et al. 2014;Dickinson et al. 2012;Ghassemi et al. 2012;Pan et al. 2012;le Digabel et al. 2011;Schoen et al. 2010;Ghassemi et al. 2009a, b;Sniadecki et al. , 2008. ...
... some or all of the pillars to be actuated. Most of the active polymer micropillar arrays developed to date use magnetic actuation (le Digabel et al. 2011;Sniadecki et al. , 2008Zhu et al. 2014). To achieve this, functional magnetic particles are embedded in the structure of the micropillars which allow the micropillars to be actuated using external magnetic fields. ...
... The use of active micropillar structures in the field of cellmechanics has mostly been confined to the application of loads on individual cells as a means to study the intracellular response ). This has largely been due to limitations of the micropillar structures which generated relatively low actuation forces and only allowed uniform actuation over small regions comprised of tens of micropillars (Sniadecki et al. , 2008. Recent advances in microfabrication techniques however have allowed the fabrication of polymer sheets with surfaces containing tens of thousands of magnetic micropillar structures that can be remotely actuated in a uniform and controllable manner to apply mechanical stimuli on large groups of cells (Khademolhosseini and Chiao 2013). ...
We present a study on the application of magnetically actuated polymer micropillar surfaces in modifying the migration behaviour of cells. We show that micropillar surfaces actuated at a frequency of 1 Hz can cause more than a 5-fold decrease in cell migration rates compared to controls, whereas non-actuated micropillar surfaces cause no statistically significant alterations in cell migration rates. The effectiveness of the micropillar arrays in impeding cell migration depends on micropillar density and placement patterns, as well as the direction of micropillar actuation with respect to the direction of cell migration. Since the magnetic micropillar surfaces presented can be actuated remotely with small external magnetic fields, their integration with implants could provide new possibilities for in-vivo tissue engineering applications.
... By controlling the amplitude and the direction of the magnetic flux gradient generated at the center of the three micropoles, de Vries et al. validated actuation forces >100 pN on a magnetic microbead (∼500 nm radius) injected inside the nucleus of a HeLa cell [56]. Magnetic fields have also been used to actuate a dense array of vertical microposts [57,58]. Each post measured 1.5 µm in radius, 10 µm in height and had a low stiffness of 32 nN/µm. ...
... Magnetic cobalt nanowires (350 nm in diameter, 5-7 µm long) were incorporated within some posts during the fabrication process of the array (1 nanowire per 200 posts). External NdFeB [57,58]. magnets were used to generate a horizontal uniform magnetic field. ...
... By way of illustration, the MEMS cell puller of Scuor et al illustrated in Fig In addition, it is important to realize that for most MEMS cited, delicate and timeconsuming protocols are often required to properly prepare and place the cells prior to experiments. For instance, in [57,58,97,98,103], the authors functionalized the probe extremities to guarantee a firm attachment of the cells under tests. In [34,35], the authors used small drops of epoxy to attach a fibril between the two pads of their uniaxial cell tenser. ...
In the future, credit-card sized and self-contained platforms that will be capable of measuring the Young's modulus of various types of cells in a high throughput manner could mark a new milestone in medicine and biomedical research. Indeed, the Young's modulus of cells appears today as a new meaningful marker for detecting several cell-based degenerative diseases at earlier stages. Besides, Young's modulus measurements may have the potential to disclose the specific effects of pharmaceuticals at the cellular level. Hence, measuring the Young's modulus of cells might also prove advantageous in drug development. However, exploiting the Young's modulus of cells as a reliable indicator still poses challenges. This doctoral dissertation reports the design, modeling and experimental validation of a novel force sensor aimed at bringing new solutions to problems encountered so far. Unlike most force sensitive systems intended to extract the Young's modulus of living cells, the force sensor presented in this work is based on a planar structure that exploits a resonant mode for achieving higher force sensitivity. In particular, the structure has been devised to maintain high dynamic performances even if cells are cultured in growth medium. Another key feature of the structure is that it has the potential to address both suspension and adherent cells. In addition, results reported in this work confirm that it can be used to rapidly estimate the Young's modulus of living cells without the need of a descriptive model and a microscope.
... The microposts' tips were coated with fibronectin to enable cells to form integrin-based focal adhesion linkages to the posts (23,25,28), which, in turn, mechanically coupled the microposts directly to the cells' actomyosin network. Magnetic nickel nanowires embedded in ∼1% of the microposts enabled actuation of those posts with an external magnetic field B(t) (25,40) to probe the cells' rheology via forces F mag (t) applied by the "magnetic" posts to the cell. Fig. 1B shows a confocal image of NIH 3T3 cells attached to a micropost array composed of 1.8-μm-diameter posts with 4-μm spacing in a hexagonal pattern. ...
... AMPAD devices were fabricated in PDMS using replica molding (23,28). Nickel nanowires with low-field magnetic moment of μ = 0.15 pA·m 2 were embedded in ∼1% of the posts during fabrication (25,40). The AMPADs were functionalized to restrict cell adhesion to the tips of the posts. ...
Significance
The cytoskeleton is a remarkable example of an active biomaterial and is at the heart of critically important problems in biology and medicine related to animal cells’ mechanical sensing and function. A satisfying physical understanding of the actomyosin cortex’s complex biophysics and mechanical properties has, however, proven elusive. We measure cytoskeletal motion, forces, and rheology using substrates containing arrays of flexible microscopic posts with high precision and statistical power. Surprisingly, the cytoskeletal networks display highly intermittent fluctuations whose energy content is dominated by rare large events, similar to what is seen in earthquakes or physical systems with avalanches. Our findings suggest that future cytoskeletal models must contain elements that self-organize onto a mechanically marginal state prone to avalanches.
... Previous studies have investigated the use of magnet-driven micropillar array devices. Sniadecki et al. (2007Sniadecki et al. ( , 2008 first showed that super-paramagnetic nanowires could be incorporated into PDMS micropillars to induce micropillar movement and attempted to apply mechanical stimulation to the adhesion sites of cells. However, the position of the nanowire-containing magnetic micropillars was uncontrollable; the nanowires, suspended in ethanol, were randomly drawn into the hole of the negative mold of micropillars. ...
... The average and maximum force of the magnetic pillars were $70 and $120 nN, respectively, comparable to the traction force at FAs of SMCs (10-80 nN) . Previously, researchers developed magnetic micropillars with cobalt nanowire, and tried to apply mechanical stimulation to sites of cell adhesion (Sniadecki et al., 2007(Sniadecki et al., , 2008. However, they were unable to control the position of the magnetic pillars in the substrate; the yield of the magnetic pillars and the maximum force generated by their pillar was only 0.5% and up to $45 nN, respectively. ...
Traction forces generated at cellular focal adhesions (FAs) play an essential role in regulating various cellular functions. These forces (1-100 nN) can be measured by observing the local displacement of a flexible substrate upon which cells have been plated. Approaches employing this method include using microfabricated arrays of poly(dimethylsiloxane) (PDMS) micropillars that bend by cellular traction forces. A tool capable of applying a force to FAs independently, by actively moving the micropillars, should become a powerful tool to delineate the cellular mechanotransduction mechanisms. Here, we developed a patterned magnetic micropillar array PDMS substrate that can be used for the mechanical stimulation of cellular FAs and the measurement of associated traction forces. The diameter, length, and center-to-center spacing of the micropillars were 3, 9, and 9 µm, respectively. Iron particles were embedded into the micropillars, enabling the pillars to bend in response to an external magnetic field, which also controlled their location on the substrate. Applying a magnetic field of 0.3 T bent the pillars by ∼4 µm and allowed transfer of external forces to the actin cytoskeleton through FAs formed on the pillar top. Using this approach, we investigated the traction force changes in cultured aortic smooth muscle cells (SMCs) after local compressive stimuli to release cell pretension. The mechanical responses of SMCs were roughly classified into two types: almost a half of the cells showed a little decrease of traction force at each pillar following compressive stimulation, although cell area increased significantly; and the rest showed the opposite, with increased forces and a simultaneous decrease in area. The traction forces of SMCs fluctuated markedly during the local compression. The root mean square of traction forces significantly increased during the compression, and returned to the baseline level after its release. These results suggest that the fluctuation of forces may be caused by active reorganization of the actin cytoskeleton and/or its dynamic interaction with myosin molecules. Thus, our magnetic micropillar substrate would be useful in investigating the mechanotransduction mechanisms of cells.
... The posts are made from PDMS using soft lithography and is similar to the fabrication of microfluidic devices. These arrays have been used to study cell migration [112], cell spreading [111], and traction forces [111,[113][114][115][116][117][118]. Forces within monolayers [112,[119][120][121][122] as well as tissue constructs [123], and cell-cell forces [124,125] have also been examined using this tool. ...
... Forces within monolayers [112,[119][120][121][122] as well as tissue constructs [123], and cell-cell forces [124,125] have also been examined using this tool. At the same time a variety of cell studies have been done using different types of cells, such as fibroblasts [111,116,117,126,127], smooth muscle cells [111,128], cardiomyocytes [129,130], epithelial cells [115,126], endothelial cells [128], and stem cells [114,131]. Micropost arrays are considered a novel tool for cell mechanic studies because they can be used to map the traction forces of cells spread over multiple posts. ...
A multi-physics model has been developed that closely matches with the biochemical regulation of platelet forces. The model is based on measurements of platelet forces using arrays of microposts. Different concentrations of thrombin or myosin inhibitors were added to the platelets to reduce their forces on the posts. The platelet forces obtained from the model have good agreement with those measured in the inhibition studies.
... The local focal adhesions growth was observed at magnetically actuated posts only, not at nonmagnetic sensor posts. Additionally, the system could be used to transmit magnetic torque served as the mechanical signal to a cell's adhesion site up to 45 nN [250], which was quite competitive with the upper limit of current force-probing system, such as optical tweezer [251]. Furthermore, in order to explore the mechanotransduction response of cells to different mechanical forces such as traction and compression stresses, some structure that could enable dynamic mechanical stimulation to cells cultured on the substrate were developed [252][253][254]. ...
Magnetic ordered materials based on magnetic nanoparticles (MNPs) possessing distinct and tunable morphological characteristics, magnetic property and excellent magnetic anisotropy, have received tremendous attentions, especially in biomedical fields. This review clearly organized the construction routes used to prepare magnetic ordered materials, including magnetic field-induced arrangement and orderly manufacturing-based construction. By focusing on the most relevant and the latest advances related to the biomedical applications in cell fate research, bio-inspired fabrication, magnetic hyperthermia, and magnetic resonance imaging, the unique advantages of magnetic ordered materials are presented and highlighted compared with magnetic unordered assemblies. Although the biosafety of many MNPs has been approved by Food and Drug Administration, future developments of magnetic ordered materials should be still centered on the safety, functional and structural diversity for interdisciplinary research. The review was concluded with a compelling perspective outlook and non-trivial challenges in future investigation of magnetic ordered materials.
... Micropost arrays can also be actuated to subject cells to external mechanical stimulation through local magnetic micropost deflections 115,[123][124][125][126] or through global mechanical 127,128 and vacuumdriven 129,130 stretching of the whole array. External application of tension by stretching provides a unique opportunity to probe at cellular tensegrity structures and quantify mechanical effects in the form of traction forces. ...
Cell-generated forces play a foundational role in tissue dynamics and homeostasis and are critically important in several biological processes, including cell migration, wound healing, morphogenesis, and cancer metastasis. Quantifying such forces in vivo is technically challenging and requires novel strategies that capture mechanical information across molecular, cellular, and tissue length scales, while allowing these studies to be performed in physiologically realistic biological models. Advanced biomaterials can be designed to non-destructively measure these stresses in vitro, and here, we review mechanical characterizations and force-sensing biomaterial-based technologies to provide insight into the mechanical nature of tissue processes. We specifically and uniquely focus on the use of these techniques to identify characteristics of cell and tissue “tensegrity:” the hierarchical and modular interplay between tension and compression that provide biological tissues with remarkable mechanical properties and behaviors. Based on these observed patterns, we highlight and discuss the emerging role of tensegrity at multiple length scales in tissue dynamics from homeostasis, to morphogenesis, to pathological dysfunction.
... 2) The Nanopost Module: Cells rely on their ability to produce forces not only for locomotion, but to mechanically probe their environment and maintain contractility, which provides feedback in regulating their function. To measure cellular forces, we have engineered a substrate that has vertical cantilevers [37]- [44]. We fabricated arrays of closely spaced, vertical polydimethylsiloxane (PDMS) posts such that individual cells can attach and spread across multiple posts (Fig. 11A). ...
... The positions of the micropillars were extracted using a centroid-based particle tracking algorithm (Crocker and Grier, 1996) in Igor Pro (Wavemetrics) (Shi et al., 2019). Static forces were calculated based on the displacement of the tips from their undeflected positions, which were determined via interpolation of the hexagonal grid based on the positions of posts not attached to cells (Sniadecki et al., 2008(Sniadecki et al., , 2007. The magnitude sum of static force from all micropillars underneath the cells was reported as the total force of each cell. ...
In this study, we investigated the biophysical interaction between cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and CD80. CTLA-4 is a key molecule in immunosuppression, and CD80 is a costimulatory receptor promoting T cell activation. We observed that after cell-cell contact was established between breast cancer cells and antigen presenting cells (APCs), CTLA-4 expressed on the breast cancer cells bind to CD80 expressed on the APCs, and underwent trans-endocytosis to deplete CD80. Force measurement and live cell imaging revealed that upon binding to CD80, forces generated by breast cancer cells and transmitted via CTLA-4 were sufficiently strong to displace CD80 from the surface of APCs to be internalized by breast cancer cells. We further demonstrated that because of the force-dependent trans-endocytosis of CD80, the capacity of APCs to activate T cells was significantly attenuated. Furthermore, inhibiting force generation in cancer cells would increase the T cell activating capacity of APCs. Our results provide a possible mechanism behind the immunosuppression commonly seen in breast cancer patients, and may lead to a new strategy to restore anti-tumor immunity by inhibiting pathways of force-generation.
... To date, the ability to study the effect of mechanical load on living single cells has been limited by the need for high resolution spatial and temporal optical imaging in a realistic myocyte configuration. Attempts have been made using magnetically actuated micropost surfaces to provide forces along the underlying surface of a cell, but these do not mimic the type of three-dimensional strain experienced by cells in vivo (Bidan et al. 2018;Sniadecki et al. 2008). Microgroove-aligned CMs were cyclically strained using the Flexcell device, but these cells could not be imaged while mechanically deformed (Motlagh et al. 2003;Senyo et al. 2007). ...
Cells interact intimately with complex microdomains in their extracellular matrix (ECM) and maintain a delicate balance of mechanical forces through mechanosensitive cellular components. Tissue injury results in acute degradation of the ECM and disruption of cell-ECM contacts, manifesting in loss of cytoskeletal tension, leading to pathological cell transformation and the onset of disease. Recently, microscale hydrogel constructs have been developed to provide cells with microdomains to form focal adhesion binding sites, which enable restoration of cytoskeletal tension. These synthetic anchors can recapitulate the complex 3D architecture of the native ECM to provide microtopographical cues. The mechanical deformation of proteins at the cell surface can activate signaling cascades to modulate downstream gene-level transcription, making this a unique materials-based approach for reprogramming cell behavior. An overview of the mechanisms underlying these mechanosensitive interactions in fibroblasts, stem and other cell types is provided to review their effects on cellular reprogramming. Recent investigations on the fabrication, functionalization and implementation of these materials and microtopographical features for drug testing and therapeutic applications are discussed.
... The driving force generated at the pillar tip was 68 ± 17 nN (mean ± SD, n = 1020), and the average and maximum forces of the magnetic pillars were~70 and~120 nN, respectively, comparable to the cellular traction force at FAs of SMCs (10-80 nN) . Previous studies developed magnetic micropillars with cobalt nanowires, and tried to apply mechanical stimulation to sites of cell adhesion; however, the yield of their magnetic pillars and the maximum force generated by their pillar was only 0.5% and up to~45 nN, respectively (Sniadecki et al. 2007;Sniadecki et al. 2008). These problems have been resolved in our study. ...
Cells change the traction forces generated at their adhesion sites, and these forces play essential roles in regulating various cellular functions. Here, we developed a novel magnetic-driven micropillar array PDMS substrate that can be used for the mechanical stimulation to cellular adhesion sites and for the measurement of associated cellular traction forces. The diameter, length, and center-to-center spacing of the micropillars were 3, 9, and 9 μm, respectively. Sufficient quantities of iron particles were successfully embedded into the micropillars, enabling the pillars to bend in response to an external magnetic field. We established two methods to apply magnetic fields to the micropillars (Suresh 2007). Applying a uniform magnetic field of 0.3 T bent all of the pillars by ~4 μm (Satcher et al. 1997). Creating a magnetic field gradient in the vicinity of the substrate generated a well-defined local force on the pillars. Deflection of the micropillars allowed transfer of external forces to the actin cytoskeleton through adhesion sites formed on the pillar top. Using the magnetic field gradient method, we measured the traction force changes in cultured vascular smooth muscle cells (SMCs) after local cyclic stretch stimulation at one edge of the cells. We found that the responses of SMCs were quite different from cell to cell, and elongated cells with larger pre-tension exhibited significant retraction following stretch stimulation. Our magnetic-driven micropillar substrate should be useful in investigating cellular mechanotransduction mechanisms.
... [297] It should be noted that for the mechanic transduction studies, the most commonly used magnetic particles are iron oxide particles because they are more easy to synthesize by coprecipitation from iron salts. [298] This approach allows the remote control of the mechanic transduction pathway with spatial and/or temporal precision in order to modulate the cell behavior, [296,298] such as by cell stretching, micropost manipulation, [299,300] localized stimulus at the single-cell level, [301] or cell receptors' activation. [298] These techniques that can apply mechanical forces directly to the cell or to the mechanoresponsive receptors of individual cells are important for a wide variety of tissue engineering applications, because they do not require the scaffold deformation. ...
Magnetic nanoparticles (NPs) are emerging as an important class of biomedical functional nanomaterials in areas such as hyperthermia, drug release, tissue engineering, theranostic, and lab-on-a-chip, due to their exclusive chemical and physical properties. Although some works can be found reviewing the main application of magnetic NPs in the area of biomedical engineering, recent and intense progress on magnetic nanoparticle research, from synthesis to surface functionalization strategies, demands for a work that includes, summarizes, and debates current directions and ongoing advancements in this research field. Thus, the present work addresses the structure, synthesis, properties, and the incorporation of magnetic NPs in nanocomposites, highlighting the most relevant effects of the synthesis on the magnetic and structural properties of the magnetic NPs and how these effects limit their utilization in the biomedical area. Furthermore, this review next focuses on the application of magnetic NPs on the biomedical field. Finally, a discussion of the main challenges and an outlook of the future developments in the use of magnetic NPs for advanced biomedical applications are critically provided.
... Thus, various techniques have been developed to introduce mechanical stimuli to the cellular microenvironment. Considering the complex in vivo microenvironment of the cells, the majority of the cell stretching approaches has been developed as in vitro platforms[14][15][16][17]. Most cell stretching approaches include the use of tweezers or micropipettes to induce the mechanical stimuli[18][19][20][21]. However, commercial cell stretching platforms such as Flexcell (Flexcell International Corporation, Burlington, NC, USA), Strex Systems for cell Stretching (STREX Inc., Osaka, Japan), and ElectroForce have been recently available[22][23][24]. ...
Cellular response tomechanical stimuli is an integral part of cell homeostasis. The interaction of the extracellular matrix with the mechanical stress plays an important role in cytoskeleton organisation and cell alignment. Insights from the response can be utilised to develop cell culture methods that achieve predefined cell patterns, which are critical for tissue remodelling and cell therapy. We report the working principle, design, simulation, and characterisation of a novel electromagnetic cell stretching platform based on the double-sided axial stretching approach. The device is capable of introducing a cyclic and static strain pattern on a cell culture. The platform was tested with fibroblasts. The experimental results are consistent with the previously reported cytoskeleton reorganisation and cell reorientation induced by strain. Our observations suggest that the cell orientation is highly influenced by external mechanical cues. Cells reorganise their cytoskeletons to avoid external strain and to maintain intact extracellular matrix arrangements.
... [8,9] Alternatively, the bending of these cilia under a static external field can serve as a mean to apply controlled stresses to soft materials deposited on the substrate such as tissues, films or cells in order to study their mechanical responses. [10][11][12] In the same line, free standing magnetic microrods can be used as active microrheometers to probe the viscoelastic properties of complex fluids, including living cells, with microscale resolution. [13,14] Many of these applications would benefit from the addition of a fluorescent functionality to the magnetic one. ...
This report presents the fabrication of bifunctional magnetic and fluorescent microneedles (µNDs) made of a ternary mixture of magnetic nanoparticles (NPs), quantum dots (QDs), and polyelectrolyte. The assembly relies on the electrostatic complexation of negatively charged NPs with positively charged polymer strands and is controlled by the charge ratio between the nanoparticle building blocks and the polymer mortar. The resulting 1D objects can be actuated using an external magnetic field and can be imaged using fluorescence microscopy, thanks to the fluorescent and superparamagnetic properties inherited from their NP constituents. Using a combination of core and surface characterizations and a state-of-the-art image analysis algorithm, the dependence of the brightness and length on the ternary composition is thoroughly investigated. In particular, statistics on hundreds of µNDs with a range of compositions show that the µNDs have a log-lormal length distribution and that their mean length can be robustly tuned in the 5–50 µm range to match the relevant length scales of various applications in micromixing, bioassays or biomechanics.
... Micropipettes or tweezers are two common in-vitro stretching methods used for introducing mechanical force into cells [10][11][12][13][14]. A number of commercial cell-stretching platforms are currently available. ...
This paper reports the modelling approach and optimization of a magnetically actuated cell-stretching device. The paper first describes the numerical simulation of the actuation system consisting of a permanent magnet and an electromagnet. The magnetic flux density and magnetic force were verified experimentally over the range of superimposed magnetic flux density from 186 mT to 204 mT. The relative errors for magnetic flux density and magnetic force are 5% and 15%, respectively. This systematic modelling approach provides a reasonable numerical model for optimizing the electromagnetic actuator of the cell-stretching device. The induced actuation force was then coupled with the structural analysis of the cell-stretching device to determine the acceptable distance between the two magnets. The results suggested that this actuation system is capable of precisely predicting the behavior of our existing cell-stretching device.
... Nevertheless, the complexity of mechanobiology associated with the in-vivo environment has led to the development of in-vitro systems. Cell stretching is the most common approach through the use of clinical tools such as micropipettes or tweezers (Sun et al. 2004; Nishimura et al. 2008; Sniadecki et al. 2008; Tan et al. 2010; Teitell et al. 2010). However, these techniques are not suitable for long-term experiments. ...
Olfactory ensheathing cells (OECs) are primary candidates for cell transplantation therapy to repair spinal cord injury (SCI). However, the post transplantation survival of these cells remains a major hurdle for a success using this therapy. Mechanical stimuli may contribute to the maintenance of these cells and thus, mechanotransduction studies of OECs may serve as a key benefit to identify strategies for improvement in cell transplantation. We developed an electromagnetic cell stretching device based on a single sided uniaxial stretching approach to apply tensile strain to OECs in culture. This paper reports the design, simulation and characterisation of the stretching device with preliminary experimental observations of OECs in vitro. The strain field of the deformable membrane was investigated both experimentally and numerically. Heterogeneity of the device provided an ideal platform for establishing strain requirement for the OEC culture. The cell stretching system developed may serve as a tool in exploring the mechanobiology of OECs for future SCI transplantation research.
... Our goal in this work was to measure the forces of neutrophil spreading on microfabricated post-array detectors (mPADs). Although mPADs have long been used to measure forces in mesenchymal cells (6)(7)(8)(9)(10), they have only recently been employed to study immune cell function. Ricart et al. (11) used mPADs to measure the traction stresses of dendritic cells undergoing chemotaxis and established that these cells migrate by a frontward pulling mechanism. ...
Human neutrophils are mediators of innate immunity and undergo dramatic shape changes at all stages of their functional life cycle. In this work, we quantified the forces associated with a neutrophil's morphological transition from a nonadherent, quiescent sphere to its adherent and spread state. We did this by tracking, with high spatial and temporal resolution, the cell's mechanical behavior during spreading on microfabricated post-array detectors printed with the extracellular matrix protein fibronectin. Two dominant mechanical regimes were observed: transient protrusion and steady-state contraction. During spreading, a wave of protrusive force (75 ± 8 pN/post) propagates radially outward from the cell center at a speed of 206 ± 28 nm/s. Once completed, the cells enter a sustained contractile state. Although post engagement during contraction was continuously varying, posts within the core of the contact zone were less contractile (-20 ± 10 pN/post) than those residing at the geometric perimeter (-106 ± 10 pN/post). The magnitude of the protrusive force was found to be unchanged in response to cytoskeletal inhibitors of lamellipodium formation and myosin II-mediated contractility. However, cytochalasin B, known to reduce cortical tension in neutrophils, slowed spreading velocity (61 ± 37 nm/s) without significantly reducing protrusive force. Relaxation of the actin cortical shell was a prerequisite for spreading on post arrays as demonstrated by stiffening in response to jasplakinolide and the abrogation of spreading. ROCK and myosin II inhibition reduced long-term contractility. Function blocking antibody studies revealed haptokinetic spreading was induced by β2 integrin ligation. Neutrophils were found to moderately invaginate the post arrays to a depth of ∼1 μm as measured from spinning disk confocal microscopy. Our work suggests a competition of adhesion energy, cortical tension, and the relaxation of cortical tension is at play at the onset of neutrophil spreading.
Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.
... Microposts have been used to elucidate the influence of substrate stiffness and spread area in the traction forces [27], and the effect of substrate rigidity on stem cell morphology, traction force generation, focal adhesion organization, and differentiation [28]. The use of magnetic micropost systems allows the study of mechanotransduction and the relation between traction forces exerted by the cells and external forces [29,30]. The measurements of traction forces using micropost have been mostly applied to mesenchymal migrating cells which exert high forces, such as fibroblasts [25,26,31], endothelial cells [27] and smooth muscle cells [30]. ...
Migrating cells exert traction forces when moving. Amoeboid cell migration is a common type of cell migration that appears in many physiological and pathological processes and is performed by a wide variety of cell types. Understanding the coupling of the biochemistry and mechanics underlying the process of migration has the potential to guide the development of pharmacological treatment or genetic manipulations to treat a wide range of diseases. The measurement of the spatiotemporal evolution of the traction forces that produce the movement is an important aspect for the characterization of the locomotion mechanics. There are several methods to calculate the traction forces exerted by the cells. Currently the most commonly used ones are traction force microscopy methods based on the measurement of the deformation induced by the cells on elastic substrate on which they are moving. Amoeboid cells migrate by implementing a motility cycle based on the sequential repetition of four phases. In this paper we review the role that specific cytoskeletal components play in the regulation of the cell migration mechanics. We investigate the role of specific cytoskeletal components regarding the ability of the cells to perform the motility cycle effectively and the generation of traction forces. The actin nucleation in the leading edge of the cell, carried by the ARP2/3 complex activated through the SCAR/WAVE complex, has shown to be fundamental to the execution of the cyclic movement and to the generation of the traction forces. The protein PIR121, a member of the SCAR/WAVE complex, is essential to the proper regulation of the periodic movement and the protein SCAR, also included in the SCAR/WAVE complex, is necessary for the generation of the traction forces during migration. The protein Myosin II, an important F-actin cross-linker and motor protein, is essential to cytoskeletal contractility and to the generation and proper organization of the traction forces during migration.
... With a similar focus, a study by Engler et al. provided information on how differing degrees of matrix elasticity/rigidity can effectively determine the phenotypes of na € ıve mesenchymal stem cells-that is whether they ultimately develop into bone, muscle or nervous tissues [24]. Meanwhile, Sniadecki et al. developed a system of elastomeric microposts embedded with cobalt nanowires to apply nanoscale traction forces to cells and then measure the resulting cellular responses, while a study by Munevar et al. used traction force microscopy and peptide-induced stress to investigate the mechanical interactions of fibroblasts migrating on a substrate, determining that leading edge and trailing edge adhesions exhibit distinct mechanical interactions [25,26]. Finally, Bellin et al. used elastomeric substrates to identify syndecan-4 as a non-integrin transmembrane protein that can also initiate mechanotransduction (an important finding insofar as the bulk of previous research has looked at the integrins as the main transmembrane protein means of starting mechanotransduction [27]. ...
Mechanotransduction plays a critical role in intracellular functioning—it allows cells to translate external physical forces into internal biochemical activities, thereby affecting processes ranging from proliferation and apoptosis to gene expression and protein synthesis in a complex web of interactions and reactions. Accordingly, aberrant mechanotransduction can either lead to, or be a result of, a variety of diseases or degenerative states. In this review, we provide an overview of mechanotransduction in the context of intervertebral discs, with a focus on the latest methods of investigating mechanotransduction and the most recent findings regarding the means and effects of mechanotransduction in healthy and degenerative discs. We also provide some discussion of potential directions for future research and treatments.
... 2) The Nanopost Module: Cells rely on their ability to produce forces not only for locomotion, but to mechanically probe their environment and maintain contractility, which provides feedback in regulating their function. To measure cellular forces, we have engineered a substrate that has vertical cantilevers [37]- [44]. We fabricated arrays of closely spaced, vertical polydimethylsiloxane (PDMS) posts such that individual cells can attach and spread across multiple posts (Fig. 11A). ...
Santosh Devasia is the Principal Investigator of a recently funded grant from the NSF Nanotechnology Undergraduate Education (NUE) Program, Grant # EEC 1042061; the proposed educational efforts under this NUE grant are described in this paper. Santosh Devasia received the B.Tech. (Hons) from the Indian Institute of Technology, Kharagpur, India, in 1988, and the M.S. and Ph.D. degrees in Mechanical Engineering from the University of California at Santa Barbara in 1990 and 1993 respectively. He is a Professor in the Mechanical Engineering Depart-ment at the University of Washington, Seattle where he joined in 2000. From 1994 to 2000, he taught in the Mechanical Engineering Department at the University of Utah, Salt Lake City. He has served as the Associate Editor for the ASME Journal of Dynamic Systems, Measurement and Control and the IEEE Transactions on Control Systems Technology. His current research interests include inversion-based con-trol theory and applications such as high-precision positioning systems for Atomic Force Microscopes and Scanning Tunneling Microscopes used in nanotechnology, biomedical applications such as the imag-ing of human cells to investigate cell locomotion, and control of distributed systems such as Air Traffic Management. Jim L Borgford-Parnell, University of Washington Dr. Jim Borgford-Parnell is Assistant Director and instructional consultant for the Center for Engineering Learning & Teaching at the University of Washington. He taught design drawing, and theory, research methods, educational theory, and adult and higher education pedagogy courses for over 25 years. Jim has been involved in instructional development more than ten years, and currently does both research and instructional development in engineering education.
... This is an important trend, since Engler et al. (2006) have recently shown that substrate elasticity, being one of the hallmarks of biomechanics, governs MSC differentiation and, thus, lineage determination to an optionally neurogenic, myogenic and/or osteogenic phenotype. In addition, previous studies elaborated on pillar arrays of distinct micropatterns have revealed the patterning of cell adhesion points, providing a further biomechanical hallmark (Sniadecki et al. 2008), having a regulative impact on hMSC behaviour (Proksch et al. 2012). The cell response to environmental biomechanics, i.e. mechanobiology, addresses the complex molecular constituents orchestrating the interplay of extracellular-environment-cell interactions. ...
Mechanobiology is a scientific interface discipline emerging from engineering and biology. With regard to tissue-regenerative cell-based strategies, mechanobiological concepts, including biomechanics as a target for cell and human mesenchymal stem cell behaviour, are on the march. Based on the periodontium as a paradigm, this mini-review discusses the key role of focal-adhesion kinase (FAK) in mechanobiology, since it is involved in mediating the transformation of environmental biomechanical signals into cell behavioural responses via mechanotransducing signalling cascades. These processes enable cells to adjust quickly to environmental cues, whereas adjustment itself relies on the specific intramolecular phosphorylation of FAK tyrosine residues and the multiple interactions of FAK with distinct partners. Furthermore, interaction-triggered mechanotransducing pathways govern the dynamics of focal adhesion sites and cell behaviour. Facets of behaviour not only include cell spreading and motility, but also proliferation, differentiation and apoptosis. In translational terms, identified and characterized biomechanical parameters can be incorporated into innovative concepts of cell- and tissue-tailored clinically applied biomaterials controlling cell behaviour as desired.
... These tools have helped to elucidate the mechanical behavior of cells, the nature of cellular forces, and mechanotransduction (Bhadriraju et al., 2007;Cai et al., 2006;du Roure et al., 2005;Fu et al., 2010;Ganz et al., 2006;Ghibaudo et al., 2008;Grashoff et al., 2010;Han, Bielawski, Ting, Rodriguez, & Sniadecki, 2012;Lemmon, Chen, & Romer, 2009;FIGURE 5.1 Stamping microposts for patterning cells. Lemmon et al., 2005;Liang, Han, Reems, Gao, & Sniadecki, 2010;Liu, Sniadecki, & Chen, 2010a;Liu, Tan, et al., 2010b;Nelson et al., 2005;Pirone et al., 2006;Rodriguez, Han, Regnier, & Sniadecki, 2011;Ruiz & Chen, 2008;Saez, Buguin, Silberzan, & Ladoux, 2005;Saez, Ghibaudo, Buguin, Silberzan, & Ladoux, 2007;Sniadecki, Lamb, Liu, Chen, & Reich, 2008;Tan et al., 2003;Tee, Fu, Chen, & Janmey, 2011;Ting et al., 2012;. ...
Micropatterning of cells can be used in combination with microposts to control cell shape or cell-to-cell interaction while measuring cellular forces. The protocols in this chapter describe how to make SU8 masters for stamps and microposts, how to use soft lithography to replicate these structures in polydimethylsiloxane, and how to functionalize the surface of the microposts for cell attachment.
Stimuli‐responsive micro‐pillared structures can perform complex and precise tasks at the microscale through dynamic and reversible deformation of the pillars in response to external triggers. Magnetic field is one of the most common actuation strategies due to its incomparable advantages such as instantaneous response, remote and nondestructive control, and superior biocompatibility. Over the past decade, many researches are attempted to design and optimize magnetically‐responsive micropillars for a wide range of applications and great progresses are accomplished. In this review, the most important aspects of recent progress of magnetically‐responsive micropillars are covered to give a comprehensive and systematical introduction to this new field, from the actuation mechanisms, fabrication methods, and deformation patterns to the practical applications. The increasingly maturing fabrication techniques can provide low‐cost and large‐scale magnetic micropillars with homogeneous responses. Some advanced techniques are developed to fabricate micropillars with programmable and reprogrammable responses for site‐specific and reconfigurable actuations. On the other hand, practical applications for particle/droplet/light manipulation, flow generation, miniature swimming/climbing/carrying microrobots, tunable adhesion, cellular probe, fog collector, and anti‐ice surfaces are also summarized. Finally, current challenges that limit the industrial implementation are discussed and the authors’ perspectives on the future directions of magnetically‐responsive micropillars are stated.
Organs-on-a-chip have emerged as next-generation tissue engineered models to accurately capture realistic human tissue behaviour, thereby addressing many of the challenges associated with using animal models in research. Mechanical features of the culture environment have emerged as being critically important in designing organs-on-a-chip, as they play important roles in both stimulating realistic tissue formation and function, as well as capturing integrative elements of homeostasis, tissue function, and tissue degeneration in response to external insult and injury. Despite the demonstrated impact of incorporating mechanical cues in these models, strategies to measure these mechanical tissue features in microfluidically-compatible formats directly on-chip are relatively limited. In this review, we first describe general microfluidically-compatible Organs-on-a-chip sensing strategies, and categorize these advances based on the specific advantages of incorporating them on-chip. We then consider foundational and recent advances in mechanical analysis techniques spanning cellular to tissue length scales; and discuss their integration into Organs-on-a-chips for more effective drug screening, disease modeling, and characterization of biological dynamics.
The skin is the largest organ that protects the body from the outside and is subjected to constant physical stimulation, such as stretching. Although many studies currently focus on UV radiation and skin aging, few studies have been reported on the effects of excessive physical stimulation on the skin. We have developed a magnetic stretching skin-on-chip (MSSC) with a built-in electromagnet to apply magnetic field-based tensile stimulation. According to the 12-h cycle circadian locomotor output cycles kaput (CLOCK) gene expression, 5% tensile stimulation was added at 0.01 Hz for 12 h per day. Physical stress was applied during the 28 days of the skin regeneration cycle, and the tissue morphological changes, protein expression, and gene expression of skin equivalents were compared to previous study results of compressive stimulation (opposite mode of tensile) to confirm the effects. Comprehensively report the skin reaction depending on the type of stimulation. The expression of genes related to the epidermal barrier showed a similar tendency for both stimulation in the case of filaggrin, but the opposite tendency appeared for involucrin and keratin 10. The proteins that make up the dermis and epidermis also showed opposite trends in expression.
Cilia are microscopic hair-like external cell organelles that are ubiquitously present in nature, also within the human body. They fulfill crucial biological functions: motile cilia provide transportation of fluids and cells, and immotile cilia sense shear stress and concentrations of chemical species. Inspired by nature, scientists have developed artificial cilia mimicking the functions of biological cilia, aiming at application in microfluidic devices like lab-on-chip or organ-on-chip. By actuating the artificial cilia, for example by a magnetic field, an electric field, or pneumatics, microfluidic flow can be generated and particles can be transported. Other functions that have been explored are anti-biofouling and flow sensing. We provide a critical review of the progress in artificial cilia research and development as well as an evaluation of its future potential. We cover all aspects from fabrication approaches, actuation principles, artificial cilia functions - flow generation, particle transport and flow sensing - to applications. In addition to in-depth analyses of the current state of knowledge, we provide classifications of the different approaches and quantitative comparisons of the results obtained. We conclude that artificial cilia research is very much alive, with some concepts close to industrial implementation, and other developments just starting to open novel scientific opportunities.
Cells are sensitive to their surrounding microenvironment. The microscopic length scale of single cells makes it challenging to study how cells sense, interpret, and transform complex microenvironmental cues into cellular responses. The micropost and micropillar array fabricated using biocompatible elastomers has been shown as a useful tool to investigate cell-generated dynamic forces in response to microenvironmental cues. Such studies provide important insights into the underlying mechanism of cellular mechanotransduction. In this chapter, we will provide an overview of the fabrication and functionalization of micropillar and micropost arrays and highlight the utility of these systems for cell mechanics and mechanobiological studies.
Cellular response to mechanical stimuli is an integral part of cell homeostasis. The interaction of the extracellular matrix with the mechanical stress plays an important role in cytoskeleton organisation and cell alignment. Insights from the response can be utilised to develop cell culture methods that achieve predefined cell patterns, which are critical for tissue remodelling and cell therapy. We report the working principle, design, simulation and characterisation of a novel electromagnetic cell stretching platform based on the double-sided axial stretching approach. The device is capable of introducing a cyclic and static strain pattern on a cell culture. The platform was tested with fibroblasts. The experimental results are consistent with the previously reported cytoskeleton reorganisation and cell reorientation induced by strain. The orientation of the cells is highly influenced by external mechanical cues. Cells reorganise their cytoskeleton to avoid external strain and to maintain intact extracellular matrix arrangements.
Biological cilia that generate fluid flow or propulsion are often found to exhibit a collective wavelike metachronal motion, i.e. neighboring cilia beat slightly out-of-phase rather than synchronously. Inspired by this observation, this article experimentally demonstrates that microscopic magnetic artificial cilia (µMAC) performing a metachronal motion can generate strong microfluidic flows, though, interestingly, the mechanism is different from that in biological cilia, as is found through a systematic experimental study. The µMAC are actuated by a facile magnetic setup, consisting of an array of rod-shaped magnets. This arrangement imposes a time-dependent non-uniform magnetic field on the µMAC array, resulting in a phase difference between the beatings of adjacent µMAC, while each cilium exhibits a two-dimensional whip-like motion. By performing the metachronal 2D motion, the µMAC are able to generate a strong flow in a microfluidic chip, with velocities of up to 3000 µm s-1 in water, which, different from biological cilia, is found to be a result of combined metachronal and inertial effects, in addition to the effect of asymmetric beating. The pumping performance of the metachronal µMAC outperforms all previously reported microscopic artificial cilia, and is competitive with that of most of the existing microfluidic pumping methods, while the proposed platform requires no physical connection to peripheral equipment, reduces the usage of reagents by minimizing "dead volumes", avoids undesirable electrical effects, and accommodates a wide range of different fluids. The 2D metachronal motion can also generate a flow with velocities up to 60 μm s-1 in pure glycerol, where Reynolds number is less than 0.05 and the flow is primarily caused by the metachronal motion of the µMAC. These findings offer a novel solution to not only create on-chip integrated micropumps, but also design swimming and walking microrobots, as well as self-cleaning and antifouling surfaces.
Stimuli‐responsive micro/nanostructures that can dynamically and reversibly adapt their configurations according to external stimuli have stimulated a wide scope of engineering applications, ranging from material surface engineering to micromanipulations. However, it remains a challenge to achieve a precise local control of the actuation to realize applications that require heterogeneous and on‐demand responses. Here, a new experimental technique is developed for large arrays of hybrid magnetic micropillars and achieve precise local control of actuation using a simple magnetic field. By manipulating the spatial distribution of magnetic nanoparticles within individual elastomer micropillars, a wide range of the magnetomechanical responses from less than 5% to ≈50% for the ratio of the bending deflection to the original length of the pillars is realized. It is demonstrated that the micropillars with different degrees of bending deformation can be configured in any spatial pattern using a photomask‐assisted template‐casting technique to achieve heterogeneous, site‐specific, and programmed bending actuations. This unprecedented local control of the micropillars offers exciting novel applications, as demonstrated here in encryptable surface printing and stamping, direction‐ and track‐programmable microparticle/droplet transport, and smart magnetic micro‐tweezers. The hybrid magnetic micropillars reported here provide a versatile prototype for heterogeneous and on‐demand actuation using programmable stimuli‐responsive micro/nanostructures.
Magnetic micropillars that can dynamically and reversibly bend actuated by external magnetic field have been widely studied for surface engineering and micromanipulation applications. However, current micropillars usually exhibit uniform distribution of the magnetic media inside and thus respond essentially the same way to external stimuli. Here we report a new concept and a corresponding experimental technique for heterogeneous magnetic micropillars with the placement of the inside magnetic nanoparticles being precisely controlled. By manipulating the spatial distribution of the magnetic nanoparticles from the base region to the tip region within the micropillars, we show that the actuated bending deformation can be tuned by as large as one order of magnitude under the same actuation condition. The different bending responses are enabled by the resulting different distributions of the local stiffness and the actuation force along the micropillars, in consistent with the fundamental bending principles for cantilever beams. The heterogeneous magnetic micropillars reported here provide a prototype for regulated and on-demand actuations using stimuli-responsive materials/structures.
The ability for biological cells to produce mechanical forces is important for the development, function, and homeostasis of tissue. The measurement of cellular forces is not a straightforward task because individual cells are microscopic in size and the forces they produce are at the nanonewton scale. Consequently, studies in cell mechanics rely on advanced biomaterials or flexible structures that permit one to infer these forces by the deformation they impart on the material or structure. Herein, the scientific progression on the use of deformable materials and deformable structures to measure cellular forces are reviewed. The findings and insights made possible with these approaches in the field of cell mechanics are summarized.
Traction Force Microscopy (TFM) derives maps of cell-generated forces, typically in the nN range, transmitted to the extracellular environment upon actuation of complex biological processes. In traditional approaches, force rendering requires a terminal, time-consuming step of cell deadhesion to obtain a reference image. A conceptually opposite approach is provided by reference-free methods, opening to the on-the-fly generation of force maps from an ongoing experiment. This requires an image processing algorithm keeping the pace of the biological phenomena under investigation. Here, we introduce an integrated software pipeline rendering force maps from single reference-free TFM images seconds to minutes after their acquisition. The algorithm tackles image processing, reference image estimation, and finite element analysis as a single problem, yielding a robust and fully automatic solution. The method’s capabilities are demonstrated in two applications. First, the mechanical annihilation of cancer cells is monitored as a function of rising environmental temperature, setting a population threshold at 45°C. Second, the fast temporal correlation of forces produced across individual cells is used to map physically connected adhesion points, yielding typical lengths that vary as function of the cell cycle phase.
Physical forces and other mechanical stimuli are fundamental regulators of cell behaviour and function. Cells are also biomechanically competent: they generate forces to migrate, contract, remodel and sense their environment. As the knowledge of the mechanisms of mechanobiology increases, the need to resolve and probe increasingly small scales calls for novel technologies to mechanically manipulate cells, to examine forces exerted by cells and to characterise cellular biomechanics. Here, we review novel methods to quantify cellular force generation, to measure cell mechanical properties and to exert localised pico- and nanonewton forces on cells, receptors and proteins. The combination of these technologies will provide further insight on the effect of mechanical stimuli on cells and the mechanisms that convert these stimuli into biochemical and biomechanical activity.
A cellular response to mechanical stimuli is well-known phenomena known as a mechanotransduction. It is widely accepted that mechanotransduction plays an important role in cell alignment which is critical for cell homeostasis. Although many approaches have been developed in recent years to study the effect of external mechanical stimuli on cell behaviour, most of them have not explored the ability of the mechanical stimuli to engineer cell alignment to obtain patterned cell culture. This paper introduces a simple, yet effective pneumatically actuated 4x2 cell stretching array for concurrently inducing a range of cyclic normal strain onto cell cultures to achieve predefined cell alignment. We utilised ring-shaped normal strain pattern to demonstrate the growth of in vitro patterned cell culture with predefined circumferential cellular alignment. Furthermore, to ensure the compatibility of the developed cell stretching platform with general tools and existing protocols, dimensions of the developed cell-stretching platform follow the standard F bottom 96 well plate. In this study we report principle design, simulation and characterisation of the cell-stretching platform with the preliminary observations using fibroblast cells. Our experimental results of cytoskeleton reorganisation such as perpendicular cellular alignment of the cells to the direction of normal strain are consistent with those reported in the literature. After two hours of stretching, the circumferential alignment of fibroblast cells confirms the capability of the developed system to achieve a patterned cell culture. The cell-stretching platform reported is potentially a useful tool for drug screening in 2D mechanobiology experiments, tissue engineering and regenerative medicine.
A coupling model for a compound quartz crystal resonator (QCR) system, consisting of a QCR under the thickness-shear mode (TSM) vibrations and surface micro-beam arrays immersed in liquid, is developed using the modified couple stress theory and the Hamilton's variational principle. Couple stresses are included in both governing equations of micro-beams and the quartz crystal plate. Incorporating the boundary conditions, a set of homogeneous equations is numerically solved to get the frequency shift caused by micro-beams for different material parameters, such as Young's modulus and material characteristic length. The normalized beam deflections are plotted and discussed. Influence of couple stresses of both micro-beams and QCR together with their interaction on frequency shift is discussed in detail. The obtained results are useful in measuring geometrical and physical properties of micro-beams using plate-like acoustic wave sensors.
Migration of a fibroblast along a collagen fiber can be regarded as cell locomotion in one-dimension (1D). In this process, a cell protrudes forward, forms a new adhesion, produces traction forces, and releases its rear adhesion in order to advance itself along a path. However, how a cell coordinates its adhesion formation, traction forces, and rear release in 1D migration is unclear. Here, we studied fibroblasts migrating along a line of microposts. We found that when the front of a cell protruded onto a new micropost, the traction force produced at its front increased steadily, but did so without a temporal correlation in the force at its rear. Instead, the force at the front coordinated with a decrease in force at the micropost behind the front. A similar correlation in traction forces also occurred at the rear of a cell, where a decrease in force due to adhesion detachment corresponded to an increase in force at the micropost ahead of the rear. Analysis with a bio-chemo-mechanical model for traction forces and adhesion dynamics indicated that the observed relationship between traction forces at the front and back of a cell is possible only when cellular elasticity is lower than the elasticity of the cellular environment.
Cells within the human body are subjected to continuous, cyclic mechanical strain caused by various organ functions, movement, and growth. Cells are well known to have the ability to sense and respond to mechanical stimuli. This process is referred to as mechanotransduction. A better understanding of mechanotransduction is of great interest to clinicians and scientists alike to improve clinical diagnosis and understanding of medical pathology. However, the complexity involved in in vivo biological systems creates a need for better in vitro technologies, which can closely mimic the cells' microenvironment using induced mechanical strain. This technology gap motivates the development of cell stretching devices for better understanding of the cell response to mechanical stimuli. This review focuses on the engineering and biological considerations for the development of such cell stretching devices. The paper discusses different types of stretching concepts, major design consideration and biological aspects of cell stretching and provides a perspective for future development in this research area.
This chapter highlights the use of biological microelectromechanical systems (BioMEMS) to better understand the physical and chemical interactions that cells have with their surroundings. These tools have provided significant insights into the cues from a cell's microenvironment and how they affect cell function. This information is useful in the design and development of new biomaterials and tissue-engineered constructs. BioMEMS devices and techniques are tools that are well-suited for a researcher to examine cell cytoskeleton and traction forces. Additionally, many of the tools presented can also be used to transmit forces to the cell in examining biochemical or biomechanical response. Measurement tools include membrane wrinkling, traction force microscopy, elastomer micropost arrays, extracellular matrix patterning. Micromanipulation techniques include traction force microscopy, magnetic twisting cytometry, optical traps and stretchers, and traditional MEMs fabricated tools. This chapter also covers microfluidic systems to exert physiologically relevant forces such as hemodynamic or interstitial shear forces to better match cell environments in living tissue. Increasingly, researchers have discovered that mechanical forces influence cell behavior in profound ways, and a better understanding through BioMEMS devices is important for future technology and therapy development.
Cells are constantly subjected to a variety of chemical and physico-mechanical cues (e.g. substrate stiffness, geometrical confinements and external mechanical forces) within their microenvironment. In turn, cells and tissues respond to these cues by altering their morphology (e.g. size and shape) and behavior (migration, proliferation and differentiation). While the role of chemical cues (growth factors etc.) in regulating cell behavior has been well studied, the importance of mechanical cues in regulating cell behavior is being understood only recently. In particular, microfabrication technologies have provided us with a number of versatile tools to effectively discriminate between the contribution of mechanical and chemical cues in regulating cell behavior. Originally developed for semiconductor industry, microfabrication tools have garnered significant interest by researchers working in the area of cell mechanics, cancer biology and regenerative medicine. These tools not only help in addressing specific questions in cell mechanics but also provide us with miniaturized high-throughput platforms for various diagnostic as well as therapeutic interventions. In this chapter, we give a general overview of some of the popular and important applications of microfabrication technologies to address various questions in cell and tissue mechanics.
As physical entities, living cells can sense and respond to various stimulations within and outside the body through cellular mechanotransduction. Any deviation in cellular mechanotransduction will not only undermine the orchestrated regulation of mechanical responses, but also lead to the breakdown of their physiological function. Therefore, a quantitative study of cellular mechanotransduction needs to be conducted both in experiments and in computational simulations to investigate the underlying mechanisms of cellular mechanotransduction. In this review, we present an overview of the current knowledge and significant progress in cellular mechanotransduction via micropost substrates. In the aspect of experimental studies, we summarize significant experimental progress and place an emphasis on the coupled relationship among cellular spreading, focal adhesion and contractility as well as the influence of substrate properties on force-involved cellular behaviors. In the other aspect of computational investigations, we outline a coupled framework including the biochemically motivated stress fiber model and thermodynamically motivated adhesion model and present their predicted biomechanical responses and then compare predicted simulation results with experimental observations to further explore the mechanisms of cellular mechanotransduction. At last, we discuss the future perspectives both in experimental technologies and in computational models, as well as facing challenges in the area of cellular mechanotransduction.
Traction force generated at focal adhesions (FAs) of cells plays an essential role in regulating various cellular functions. The force can be measured by plating cells on a flexible substrate to observe local displacement of the substrate caused by the forces (1–100 nN) [1]. Approaches employing this method include using microfabricated arrays of poly(dimethylsiloxane) (PDMS) micropillars that bend by cellular traction forces [2]. If you could apply forces to individual FAs independently by actively moving micropillars, it should become a powerful tool to delineate the cellular mechanotransduction mechanisms.
Arrays of nanowires are fabricated with alternating segments of the magnetostrictive alloy Fe1–x Gax and Cu using electrochemical deposition in nanoporous anodic aluminium oxide (AAO) templates. The difficult nature of Ga‐alloy electrochemistry is overcome by controlling mass‐transfer and hydrodynamic conditions using novel rotating disk electrode templates to obtain highly uniform segment lengths throughout the arrays. Extensive structural characterization by XRD, EBSD and TEM reveals a strong textured Fe1–x Gax growth. Furthermore, using vibrating sample magnetometry (VSM), we demonstrate that control of magnetization reversal processes is possible once uniform aspect ratios are obtained for both the Fe–Ga and Cu segments.
Arrays of actuated magnetic micropillars that can be tilted, twisted and rotated in the presence of a magnetic field gradient were obtained. The type and extent of the movements are dependent on the distribution (isotropic, anisotropic) of the magnetisable particles inside the pillars, the strength and the direction of the magnetic field gradient. Independent motion of groups of pillars in the same or opposite directions, or homogeneous motion of the whole pattern are realized. The change in the pattern geometry causes changes in the roll off angle (ROA) of water droplets on the surface. We show magnetically induced changes in the ROA, and direction-dependent ROAs as a consequence of the anisotropy of tilted patterns. We also demonstrate transfer of microparticles between magnetically actuated neighbouring pillars.
In this review we summarize current data on the mechanics of synthetic and naturally occurring biopolymers that are routinely employed in examination of contractility and cellular mechanosensation. We discuss the effect of physical boundaries on the mechanical behaviors of cell substrates and cellular mechanosensation. The application of contractile forces to underlying substrates enables anchorage-dependent cells to probe the physical properties of their microenvironment. Compliant substrates deform as a result of contractile forces generated by adherent cells and, in turn, the mechanical response of substrates influences numerous cellular processes. Unlike synthetic polymers that exhibit linear elastic responses to forces applied by adherent cells, naturally-occurring biopolymers exhibit non-linear, viscoelastic behavior. In turn, the viscoelastic behavior of fibrillar biopolymers may contribute to irreversible network compaction after application of cell-derived forces. Comprehensive characterization of the unusual mechanical properties of extracellular matrix proteins like collagen has provided novel insights into cell contractility and mechanosensation. We suggest that in the future, fabrication and application of novel substrates with fibrillar structures and non-linear viscoelastic behavior will be needed for a better understanding of the role of mechanosensation in many physiological and pathological processes.
Traction forces produced by moving fibroblasts have been observed as distortions in flexible substrata including wrinkling of thin, silicone rubber films. Traction forces generated by fibroblast lamellae were thought to represent the forces required to move the cell forwards. However, traction forces could not be detected with faster moving cell types such as leukocytes and growth cones (Harris, A. K., D. Stopak, and P. Wild. 1981. Nature (Lond.). 290:249-251). We have developed a new assay in which traction forces produced by rapidly locomoting fish keratocytes can be detected by the two-dimensional displacements of small beads embedded in the plane of an elastic substratum. Traction forces were not detected at the rapidly extending front edge of the cell. Instead the largest traction forces were exerted perpendicular to the left and right cell margins. The maximum traction forces exerted by keratocytes were estimated to be approximately 2 x 10(-8) N. The pattern of traction forces can be related to the locomotion of a single keratocyte in terms of lamellar contractility and area of close cell-substratum contact.
Integrin-mediated cell adhesions provide dynamic, bidirectional links between the extracellular matrix and the cytoskeleton. Besides having central roles in cell migration and morphogenesis, focal adhesions and related structures convey information across the cell membrane, to regulate extracellular-matrix assembly, cell proliferation, differentiation, and death. This review describes integrin functions, mechanosensors, molecular switches and signal-transduction pathways activated and integrated by adhesion, with a unifying theme being the importance of local physical forces.
Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 +/- 2 nNmicrom(-2). The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.
Traction forces produced by moving fibroblasts have been observed as distortions in flexible substrata including wrinkling of thin, silicone rubber films. Traction forces generated by fibroblast lamellae were thought to represent the forces required to move the cell forwards. However, traction forces could not be detected with faster moving cell types such as leukocytes and growth cones (Harris, A. K., D. Stopak, and P. Wild. 1981. Nature (Lond.). 290:249-251). We have developed a new assay in which traction forces produced by rapidly locomoting fish keratocytes can be detected by the two-dimensional displacements of small beads embedded in the plane of an elastic substratum. Traction forces were not detected at the rapidly extending front edge of the cell. Instead the largest traction forces were exerted perpendicular to the left and right cell margins. The maximum traction forces exerted by keratocytes were estimated to be approximately 2 x 10(-8) N. The pattern of traction forces can be related to the locomotion of a single keratocyte in terms of lamellar contractility and area of close cell-substratum contact.
The cytoskeletal activity of motile or adherent cells is frequently seen to induce detectable displacements of sufficiently compliant substrata. The physics of this phenomenon is discussed in terms of the classical theory of small-strain, plane-stress elasticity. The main results of such analysis is a transform expressing the displacement field of the elastic substrate as an integral over the traction field. The existence of this transform is used to derive a Bayesian method for converting noisy measurements of substratum displacement into "images" of the actual traction forces exerted by adherent or locomoting cells. Finally, the Monte Carlo validation of the statistical method is discussed, some new rheological studies of films are presented, and a practical application is given.
To survive in a mechanically active environment, cells must adapt to variations of applied membrane tension. A collagen-coated magnetic bead model was used to apply forces directly to the actin cytoskeleton through integrin receptors. We demonstrate here that by a calcium-dependent mechanism, human fibroblasts reinforce locally their connection with extracellular adhesion sites by inducing actin assembly and by recruiting actin-binding protein 280 (ABP-280) into cortical adhesion complexes. ABP-280 was phosphorylated on serine residues as a result of force application. This phosphorylation and the force-induced actin reorganization were largely abrogated by inhibitors of protein kinase C. In a human melanoma cell line that does not express ABP-280, actin accumulation could not be induced by force, whereas in stable transfectants expressing ABP-280, force-induced actin accumulation was similar to human fibroblasts. Cortical actin assembly played a role in regulating the activity of stretch-activated, calcium-permeable channels (SAC) since sustained force application desensitized SAC to subsequent force applications, and the decrease in stretch sensitivity was reversed after treatment with cytochalasin D. ABP-280-deficient cells showed a > 90% increase in cell death compared with ABP-280 +ve cells after force application. We conclude that ABP-280 plays an important role in mechanoprotection by reinforcing the membrane cortex and desensitizing SACs.
The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136-143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.
Mechanical stresses modulate cell function by either activating or tuning signal transduction pathways. Mechanotransduction, the process by which cells convert mechanical stimuli into a chemical response, occurs both in cells specialized for sensing mechanical cues and in parenchymal cells whose primary function is not mechanosensory. However, common among the various responses to mechanical stress is the importance of direct or indirect connections between the internal cytoskeleton, the extracellular matrix (ECM), and traditional signal transducing molecules. In many instances, these elements converge at focal adhesions, sites of structural attachment between the cytoskeleton and ECM that are anchored by cell surface integrin receptors. Alenghat and Ingber discuss the accumulating evidence for the central role of cytoskeleton, ECM, and integrin-anchored focal adhesions in several mechanotransduction pathways.
To adhere and migrate, cells must be capable of applying cytoskeletal force to the extracellular matrix (ECM) through integrin receptors. However, it is unclear if connections between integrins and the ECM are immediately capable of transducing cytoskeletal contraction into migration force, or whether engagement of force transmission requires maturation of the adhesion. Here, we show that initial integrin-ECM adhesions become capable of exerting migration force with the recruitment of vinculin, a marker for focal complexes, which are precursors of focal adhesions. We are able to induce the development of focal complexes by the application of mechanical force to fibronectin receptors from inside or outside the cell, and we are able to extend focal complex formation to vitronectin receptors by the removal of c-Src. These results indicate that cells use mechanical force as a signal to strengthen initial integrin-ECM adhesions into focal complexes and regulate the amount of migration force applied to individual adhesions at localized regions of the advancing lamella.
We describe an approach to manipulate and measure mechanical interactions between cells and their underlying substrates by using microfabricated arrays of elastomeric, microneedle-like posts. By controlling the geometry of the posts, we varied the compliance of the substrate while holding other surface properties constant. Cells attached to, spread across, and deflected multiple posts. The deflections of the posts occurred independently of neighboring posts and, therefore, directly reported the subcellular distribution of traction forces. We report two classes of force-supporting adhesions that exhibit distinct force-size relationships. Force increased with size of adhesions for adhesions larger than 1 microm(2), whereas no such correlation existed for smaller adhesions. By controlling cell adhesion on these micromechanical sensors, we showed that cell morphology regulates the magnitude of traction force generated by cells. Cells that were prevented from spreading and flattening against the substrate did not contract in response to stimulation by serum or lysophosphatidic acid, whereas spread cells did. Contractility in the unspread cells was rescued by expression of constitutively active RhoA. Together, these findings demonstrate a coordination of biochemical and mechanical signals to regulate cell adhesion and mechanics, and they introduce the use of arrays of mechanically isolated sensors to manipulate and measure the mechanical interactions of cells.
We describe a novel synchronous detection approach to map the transmission of mechanical stresses within the cytoplasm of an adherent cell. Using fluorescent protein-labeled mitochondria or cytoskeletal components as fiducial markers, we measured displacements and computed stresses in the cytoskeleton of a living cell plated on extracellular matrix molecules that arise in response to a small, external localized oscillatory load applied to transmembrane receptors on the apical cell surface. Induced synchronous displacements, stresses, and phase lags were found to be concentrated at sites quite remote from the localized load and were modulated by the preexisting tensile stress (prestress) in the cytoskeleton. Stresses applied at the apical surface also resulted in displacements of focal adhesion sites at the cell base. Cytoskeletal anisotropy was revealed by differential phase lags in X vs. Y directions. Displacements and stresses in the cytoskeleton of a cell plated on poly-L-lysine decayed quickly and were not concentrated at remote sites. These data indicate that mechanical forces are transferred across discrete cytoskeletal elements over long distances through the cytoplasm in the living adherent cell.
Cells face not only a complex biochemical environment but also a diverse biomechanical environment. How cells respond to variations in mechanical forces is critical in homeostasis and many diseases. The mechanisms by which mechanical forces lead to eventual biochemical and molecular responses remain undefined, and unraveling this mystery will undoubtedly provide new insight into strengthening bone, growing cartilage, improving cardiac contractility, and constructing tissues for artificial organs. In this article we review the physical bases underlying the mechanotransduction process, techniques used to apply controlled mechanical stresses on living cells and tissues to probe mechanotransduction, and some of the important lessons that we are learning from mechanical stimulation of cells with precisely controlled forces.
We measure dynamic traction forces exerted by epithelial cells on a substrate. The force sensor is a high-density array of elastomeric microfabricated pillars that support the cells. Traction forces induced by cell migration are deduced from the measurement of the bending of these pillars and are correlated with actin localization by fluorescence microscopy. We use a multiple-particle tracking method to estimate the mechanical activity of cells in real time with a high-spatial resolution (down to 2 μm) imposed by the periodicity of the post array. For these experiments, we use differentiated Madin-Darby canine kidney (MDCK) epithelial cells. Our data provide definite information on mechanical forces exerted by a cellular assembly. The maximum intensity of the forces is localized on the edge of the epithelia. Hepatocyte growth factor promotes cell motility and induces strong scattering activity of MDCK cells. Thus, we compare forces generated by MDCK cells in subconfluent epithelia versus isolated cells after hepatocyte growth factor treatment. Maximal-traction stresses at the edge of a monolayer correspond to higher values than those measured for a single cell and may be due to a collective behavior.
• cell mechanics
• microfabrication
• traction forces
• multiple particle tracking
The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin-cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean +/- s.e.m.) of 18.1 +/- 1.7 nm s(-1). This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.
Normal tissue cells are generally not viable when suspended in a fluid and are therefore said to be anchorage dependent. Such
cells must adhere to a solid, but a solid can be as rigid as glass or softer than a baby's skin. The behavior of some cells
on soft materials is characteristic of important phenotypes; for example, cell growth on soft agar gels is used to identify
cancer cells. However, an understanding of how tissue cells—including fibroblasts, myocytes, neurons, and other cell types—sense
matrix stiffness is just emerging with quantitative studies of cells adhering to gels (or to other cells) with which elasticity
can be tuned to approximate that of tissues. Key roles in molecular pathways are played by adhesion complexes and the actinmyosin
cytoskeleton, whose contractile forces are transmitted through transcellular structures. The feedback of local matrix stiffness
on cell state likely has important implications for development, differentiation, disease, and regeneration.
In the pursuit to understand the interaction between cells and their underlying substrates, the life sciences are beginning to incorporate micro- and nanotechnology-based tools to probe and measure cells. The development of these tools portends endless possibilities for new insights into the fundamental relationships between cells and their surrounding microenvironment that underlie the physiology of human tissue. Here, we review techniques and tools that have been used to study how a cell responds to the physical factors in its environment. We also discuss unanswered questions that could be addressed by these approaches to better elucidate the molecular processes and mechanical forces that dominate the interactions between cells and their physical scaffolds.
The shapes of eukaryotic cells and ultimately the organisms that they form are defined by cycles of mechanosensing, mechanotransduction and mechanoresponse. Local sensing of force or geometry is transduced into biochemical signals that result in cell responses even for complex mechanical parameters such as substrate rigidity and cell-level form. These responses regulate cell growth, differentiation, shape changes and cell death. Recent tissue scaffolds that have been engineered at the micro- and nanoscale level now enable better dissection of the mechanosensing, transduction and response mechanisms.
The interplay of mechanical forces between the extracellular environment and the cytoskeleton drives development, repair, and senescence in many tissues. Quantitative definition of these forces is a vital step in understanding cellular mechanosensing. Microfabricated post array detectors (mPADs) provide direct measurements of cell-generated forces during cell adhesion to extracellular matrix. A new approach to mPAD post labeling, volumetric imaging, and an analysis of post bending mechanics determined that cells apply shear forces and not point moments at the matrix interface. In addition, these forces could be accurately resolved from post deflections by using images of post tops and bases. Image analysis tools were then developed to increase the precision and throughput of post centroid location. These studies resulted in an improved method of force measurement with broad applicability and concise execution using a fully automated force analysis system. The new method measures cell-generated forces with less than 5% error and less than 90 seconds of computational time. Using this approach, we demonstrated direct and distinct relationships between cellular traction force and spread cell surface area for fibroblasts, endothelial cells, epithelial cells and smooth muscle cells.
Elastomeric stamps and molds provide a great opportunity to eliminate some of the disadvantages of photolithograpy, which is currently the leading technology for fabricating small structures. In the case of "soft lithography" there is no need for complex laboratory facilities and high-energy radiation. Therefore, this process is simple, inexpensive, and accessible even to molecular chemists. The current state of development in this promising area of research is presented here.
The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.
The influence of the pore size, the ratio of length to diameter, the microstructure of the initial growth part of the Fe cylinder and the packing density of the Fe needles on the magnetic behaviour of alumite media are reviewed. It is found that the magnetization reversal is controlled by curling rotation, if the applied field lies along the film normal. The Hc is mainly determined by the diameter of the needles, but it slightly decreases with increasing packing density. If the applied field deviates from the film normal, the magnetic behaviour of alumite media is strongly affected by the packing density. For alumite with a low packing density, the magnetization reversal is controlled by a cos-type of incoherent rotation and a demagnetizing field. For high packing densities, the reversal can be considered as the superposition of a cos-type of incoherent rotation with perpendicular anisotropy and in-plane domain-wall motion. The alumite media can exhibit both pe rpendicular as well as longitudinal anisotropy by an appropriately controlled aspect ratio and the morphology of initial growth part of the iron needles.
Using a planar microstrip transmission line, the dipolar interactions in electrodeposited Ni nanowires arrays are characterized as a function of the membrane porosity (4% to 38%) and the wire diameter (56 to 250 nm). The dipolar interactions between the wires can be modeled in a mean-field approach as an effective uniaxial anisotropy field oriented perpendicular to the wire axis and proportional to the membrane porosity. The dipolar interaction field opposes the self-demagnetization field of an isolated single wire which keeps the magnetization parallel to the wire axis. An increase in the porosity therefore induces a switching of the effective anisotropy easy axis from parallel to perpendicular to the wire axis above a critical porosity of 35-38% independent of the wire diameter.
The use of magnetic nanowires is demonstrated as a method for the application of force to mammalian cells. Magnetic separations were carried out on populations of NIH-3T3 mouse fibroblast cells using ferromagnetic Ni wires 350 nm in diameter and 35 μm long. Separation purities in excess of 90% and yields of 49% are obtained. The nanowires are shown to outperform magnetic beads of comparable volume. © 2003 American Institute of Physics.
The IEEE Press is pleased to reissue this essential book for understanding the basis of modern magnetic materials. Diamagnetism, paramagnetism, ferromagnetism, ferrimagnetism, and antiferromagnetism are covered in an integrated manner -- unifying subject matter from physics, chemistry, metallurgy, and engineering. Magnetic phenomena are discussed both from an experimental and theoretical point of view. The underlying physical principles are presented first, followed by macroscopic or microscopic theories. Although quantum mechanical theories are given, a phenomenological approach is emphasized. More than half the book is devoted to a discussion of strongly coupled dipole systems, where the molecular field theory is emphasized. The Physical Principles of Magnetism is a classic "must read" for anyone working in the magnetics, electromagnetics, computing, and communications fields. © 2001 by the Institute of Electrical and Electronics Engineers, Inc. All rights reserved.
The magnetic anisotropy and domain structure of electrodeposited cylindrical Co nanowires with length of 10 or 20 μm and diameters
ranging from 30 to 450 nm are studied by means of magnetization and magnetic torque measurements, as well as magnetic force
microscopy. Experimental results reveal that crystal anisotropy either concurs with shape anisotropy in maintaining the Co
magnetization aligned along the wire or favours an orientation of the magnetization perpendicular to the wire, hence competing
with shape anisotropy, depending on whether the diameter of the wires is smaller or larger than a critical diameter of 50
nm. This change of crystal anisotropy, originating in changes in the crystallographic structure of Co, is naturally found
to strongly modify the zero (or small) field magnetic domain structure in the nanowires. Except for nanowires with parallel-to-wire
crystal anisotropy (very small diameters) where single-domain behaviour may occur, the formation of magnetic domains is required
to explain the experimental observations. The geometrical restriction imposed on the magnetization by the small lateral size
of the wires proves to play an important role in the domain structures formed.
When tissue cells are cultured on very thin sheets of cross-linked silicone fluid, the traction forces the cells exert are
made visible as elastic distortion and wrinkling of this substratum. Around explants this pattern of wrinkling closely resembles
the "center effects" long observed in plasma clots and traditionally attributed to dehydration shrinkage.
Mechanical stresses were applied directly to cell surface receptors with a magnetic twisting device. The extracellular matrix receptor, integrin beta 1, induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhesion receptors did not. The cytoskeletal stiffness (ratio of stress to strain) increased in direct proportion to the applied stress and required intact microtubules and intermediate filaments as well as microfilaments. Tensegrity models that incorporate mechanically interdependent struts and strings that reorient globally in response to a localized stress mimicked this response. These results suggest that integrins act as mechanoreceptors and transmit mechanical signals to the cytoskeleton. Mechanotransduction, in turn, may be mediated simultaneously at multiple locations inside the cell through force-induced rearrangements within a tensionally integrated cytoskeleton.
To move forward, migrating cells must generate traction forces through surface receptors bound to extracellular matrix molecules coupled to a rigid structure. We investigated whether cells sample and respond to the rigidity of the anchoring matrix. Movement of beads coated with fibronectin or an anti-integrin antibody was restrained with an optical trap on fibroblasts to mimic extracellular attachment sites of different resistance. Cells precisely sense the restraining force on fibronectin beads and respond by a localized, proportional strengthening of the cytoskeleton linkages, allowing stronger force to be exerted on the integrins. This strengthening was absent or transient with antibody beads, but restored with soluble fibronectin. Hence, ligand binding site occupancy was required. Finally, phenylarsine oxide inhibited strengthening of cytoskeletal linkages, indicating a role for dephosphorylation. Thus, the strength of integrin-cytoskeleton linkages is dependent on matrix rigidity and on its biochemical composition. Matrix rigidity may, therefore, serve as a guidance cue in a process of mechanotaxis.
Directional cell locomotion is critical in many physiological processes, including morphogenesis, the immune response, and wound healing. It is well known that in these processes cell movements can be guided by gradients of various chemical signals. In this study, we demonstrate that cell movement can also be guided by purely physical interactions at the cell-substrate interface. We cultured National Institutes of Health 3T3 fibroblasts on flexible polyacrylamide sheets coated with type I collagen. A transition in rigidity was introduced in the central region of the sheet by a discontinuity in the concentration of the bis-acrylamide cross-linker. Cells approaching the transition region from the soft side could easily migrate across the boundary, with a concurrent increase in spreading area and traction forces. In contrast, cells migrating from the stiff side turned around or retracted as they reached the boundary. We call this apparent preference for a stiff substrate "durotaxis." In addition to substrate rigidity, we discovered that cell movement could also be guided by manipulating the flexible substrate to produce mechanical strains in the front or rear of a polarized cell. We conclude that changes in tissue rigidity and strain could play an important controlling role in a number of normal and pathological processes involving cell locomotion.
By modulating adhesion signaling and cytoskeletal organization, mechanical forces play an important role in various cellular functions, from propelling cell migration to mediating communication between cells. Recent developments have resulted in several new approaches for the detection, analysis and visualization of mechanical forces generated by cultured cells. Combining these methods with other approaches, such as green-fluorescent protein (GFP) imaging and gene manipulation, proves to be particularly powerful for analyzing the interplay between extracellular physical forces and intracellular chemical events.
Cell motility is driven by the sum of asymmetric traction forces exerted on the substrate through adhesion foci that interface with the actin cytoskeleton. Establishment of this asymmetry involves microtubules, which exert a destabilising effect on adhesion foci via targeting events. Here, we demonstrate the existence of a mechano-sensing mechanism that signals microtubule polymerisation and guidance of the microtubules towards adhesion sites under increased stress. Stress was applied either by manipulating the body of cells moving on glass with a microneedle or by stretching a flexible substrate that cells were migrating on. We propose a model for this mechano-sensing phenomenon whereby microtubule polymerisation is stimulated and guided through the interaction of a microtubule tip complex with actin filaments under tension.
The skeleton adapts to its mechanical usage, although at the cellular level, the distribution and magnitude of strains generated and their detection are ill-understood. The magnitude and nature of the strains to which cells respond were investigated using an atomic force microscope (AFM) as a microindentor. A confocal microscope linked to the setup enabled analysis of cellular responses. Two different cell response pathways were identified: one, consequent upon contact, depended on activation of stretch-activated ion channels; the second, following stress relaxation, required an intact microtubular cytoskeleton. The cellular responses could be modulated by selectively disrupting cytoskeletal components thought to be involved in the transduction of mechanical stimuli. The F-actin cytoskeleton was not required for responses to mechanical strain, whereas the microtubular and vimentin networks were. Treatments that reduced membrane tension, or its transmission, selectively reduced contact reactions. Immunostaining of the cell cytoskeleton was used to interpret the results of the cytoskeletal disruption studies. We provide an estimate of the cellular strain magnitude needed to elicit intracellular calcium responses and propose a model that links single cell responses to whole bone adaptation. This technique may help to understand adaptation to mechanical usage in other organs.
The current focus of medicine on molecular genetics ignores the physical basis of disease even though many of the problems that lead to pain and morbidity, and bring patients to the doctor's office, result from changes in tissue structure or mechanics. The main goal of this article is therefore to help integrate mechanics into our understanding of the molecular basis of disease. This article first reviews the key roles that physical forces, extracellular matrix and cell structure play in the control of normal development, as well as in the maintenance of tissue form and function. Recent insights into cellular mechanotransduction--the molecular mechanism by which cells sense and respond to mechanical stress--also are described. Re-evaluation of human pathophysiology in this context reveals that a wide range of diseases included within virtually all fields of medicine and surgery share a common feature: their etiology or clinical presentation results from abnormal mechanotransduction. This process may be altered by changes in cell mechanics, variations in extracellular matrix structure, or by deregulation of the molecular mechanisms by which cells sense mechanical signals and convert them into a chemical or electrical response. Molecules that mediate mechanotransduction, including extracellular matrix molecules, transmembrane integrin receptors, cytoskeletal structures and associated signal transduction components, may therefore represent targets for therapeutic intervention in a variety of diseases. Insights into the mechanical basis of tissue regulation also may lead to development of improved medical devices, engineered tissues, and biologically-inspired materials for tissue repair and reconstruction.
The ability to spatially control cell adhesion and multicellular organization is critical to many biomedical and tissue-engineering applications. This work describes a straightforward method to micropattern cells onto glass, silicone rubber, and polystyrene using commercially available reagents. An elastomeric polydimethylsiloxane stamp is used to contact-transfer extracellular matrix protein onto a surface followed by blocking cell adhesion in the surrounding regions by the physisorption of Pluronic surfactants. Using self-assembled monolayers of alkanethiols on gold as model surfaces to control surface wettability, we found that protein printing was most effective at intermediate to highly wetting surfaces whereas Pluronic adsorption occurred at intermediate to low wetting surfaces. Within a regimen of intermediate wettability both techniques were applied in conjunction to restrict cell adhesion to specified patterns. Adjusting the wettability of common tissue culture substrates to the same intermediate range again allowed the micropatterning of cells, suggesting that this approach is likely to be generally applicable to many types of materials. This technique therefore may allow for wider adoption of cell patterning.
To understand how cells sense and adapt to mechanical stress, we applied tensional forces to magnetic microbeads bound to cell-surface integrin receptors and measured changes in bead displacement with sub-micrometer resolution using optical microscopy. Cells exhibited four types of mechanical responses: (1) an immediate viscoelastic response; (2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces; (3) later adaptive cell stiffening with sustained (>15 second) static stresses; and (4) a large-scale repositioning response with prolonged (>1 minute) stress. Importantly, these adaptation responses differed biochemically. The immediate and early responses were affected by chemically dissipating cytoskeletal prestress (isometric tension), whereas the later adaptive response was not. The repositioning response was prevented by inhibiting tension through interference with Rho signaling, similar to the case of the immediate and early responses, but it was also prevented by blocking mechanosensitive ion channels or by inhibiting Src tyrosine kinases. All adaptive responses were suppressed by cooling cells to 4 degrees C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.
Cell forces define cell morphology, alterations in which are caused by tyrosine kinase and phosphatase mutations, which implies a causal linkage. Recent studies have shown that phosphotyrosine signaling is involved in force sensing for cells on flat surfaces. Early force-dependent activation of Src family kinases by phosphatases or cytoskeleton stretch leads to the activation of downstream signaling. In addition, force generation by cells depends on a feedback mechanism between matrix rigidity or force generation and myosin contractility. Components of the force-sensing pathway are linked to the integrin-cytoskeleton complex at sites of force application and serve as scaffolds for signaling processes. Thus, early events in force detection are mechanically induced cytoskeletal changes that result in biochemical signals to mechanoresponsive pathways that then regulate cell form.
A new model is proposed for force transmission through the cytoskeleton (CSK). A general discussion is first presented on the physical principles that underlie the modeling of this phenomenon. Some fundamental problems of conventional models--continuous and discrete--are examined. It is argued that mediation of focused forces is essential for good control over intracellular mechanical signals. The difficulties of conventional continuous models in describing such mediation are traced to a fundamental assumption rather than to their being continuous. Relevant advantages and disadvantages of continuous and discrete modeling are discussed. It is concluded that favoring discrete models is based on two misconceptions, which are clarified. The model proposed here is based on the idea that focused propagation of mechanical stimuli in frameworks over large distances (compared to the mesh size) can only occur when considerable regions of the CSK are isostatic. The concept of isostaticity is explained and a recently developed continuous isostaticity theory is briefly reviewed. The model enjoys several advantages: it leads to good control over force mediation; it explains nonuniform stresses and action at a distance; it is continuous, making it possible to model force propagation over long distances; and it enables prediction of individual force paths. To be isostatic, or nearly so, CSK networks must possess specific structural characteristics, and these are quantified. Finally, several experimental observations are interpreted using the new model and implications are discussed. It is also suggested that this approach may give insight into the dynamics of reorganization of the CSK. Many of the results are amenable to experimental measurements, providing a testing ground for the proposed picture, and generic experiments are suggested.
Adhesion-mediated signaling provides cells with information about multiple parameters of their microenvironment, including mechanical characteristics. Often, such signaling is based on a unique feature of adhesion structures: their ability to grow and strengthen when force is applied to them, either from within the cell or from the outside. Such adhesion reinforcement is characteristic of integrin-mediated cell-matrix adhesions, but may also operate in other types of adhesion structures. Though the amount of knowledge about adhesion-mediated signaling is growing rapidly, the mechanisms underlying force-dependent regulation of junction assembly are largely unknown. Experiments have been carried out that have started to uncover the major signaling pathways involved in the response of adhesion sites to force. Theoretical models have also been used to address the physical mechanisms underlying adhesion-mediated mechanosensing.
Nonmuscle cells exert biomechanical forces known as traction forces on the extracellular matrix (ECM). Spatial coordination of these traction forces against the ECM is in part responsible for directing cell migration, for remodeling the surrounding tissue scaffold, and for the folds and rearrangements seen during morphogenesis. The traction forces are applied through a number of discrete adhesions between a cell and the ECM. We have developed a device consisting of an array of flexible, microfabricated posts capable of measuring these forces under an adherent cell. Functionalizing the top of each post with ECM protein allows cells to attach and spread across the tops of the posts. Deflection of the tips of the posts is proportional to cell-generated traction forces during cell migration or contraction. In this chapter, we describe the microfabrication, preparation, and experimental use of such microfabricated post array detector system (mPADs).
Cells respond to mechanical forces whether applied externally or generated internally via the cytoskeleton. To study the cellular response to forces separately, we applied external forces to cells via microfabricated magnetic posts containing cobalt nanowires interspersed among an array of elastomeric posts, which acted as independent sensors to cellular traction forces. A magnetic field induced torque in the nanowires, which deflected the magnetic posts and imparted force to individual adhesions of cells attached to the array. Using this system, we examined the cellular reaction to applied forces and found that applying a step force led to an increase in local focal adhesion size at the site of application but not at nearby nonmagnetic posts. Focal adhesion recruitment was enhanced further when cells were subjected to multiple force actuations within the same time interval. Recording the traction forces in response to such force stimulation revealed two responses: a sudden loss in contractility that occurred within the first minute of stimulation or a gradual decay in contractility over several minutes. For both types of responses, the subcellular distribution of loss in traction forces was not confined to locations near the actuated micropost, nor uniformly across the whole cell, but instead occurred at discrete locations along the cell periphery. Together, these data reveal an important dynamic biological relationship between external and internal forces and demonstrate the utility of this microfabricated system to explore this interaction.
• focal adhesions
• magnetic nanowires
• mechanotransduction
• microfabrication
• traction forces
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