Substrate Rigidity Research Articles

Effect of Dynamic Stiffness of the Substrates on Neurite Outgrowth by Using a DNA-Crosslinked Hydrogel

Central nervous system tissues, like other tissue types, undergo constant remodeling, which potentially leads to changes in their mechanical stiffness. Moreover, mechanical compliance of central nervous system tissues can also be modified under external load such as that experienced in traumatic brain or spinal cord injury, and during pathological processes. Thus, the neuronal responses to the dynamic stiffness of the microenvironment are of significance. In this study, we induced decrease in stiffness by using a DNA-crosslinked hydrogel, and subjected rat spinal cord neurons to such dynamic stiffness. The neurons respond to the dynamic cues as evidenced by the primary neurite structure, and the response from each neurite property (e.g., axonal length and primary dendrite number) is consistent with the behavior on static gels of same substrate rigidity, with one exception of mean primary dendrite length. The results on cell population distribution confirm the neuronal responses to the dynamic stiffness. Quantification on the focal adhesion kinase expression in the neuronal cell body on dynamic gels suggests that neurons also modify adhesion in coping with the dynamic stiffnesses. The results reported here extend the neuronal mechanosensing capability to dynamic stiffness of extracellular matrix, and give rise to a novel way of engineering neurite outgrowth in time dimension.

A Model of Fibroblast Motility on Substrates with Different Rigidities

To function efficiently in the body, the biological cells must have the ability to sense the external environment. Mechanosensitivity toward the extracellular matrix was identified as one of the sensing mechanisms affecting cell behavior. It was shown experimentally that a fibroblast cell prefers locomoting over the stiffer substrate when given a choice between a softer and a stiffer substrate. In this article, we develop a discrete model of fibroblast motility with substrate-rigidity sensing. Our model allows us to understand the interplay between the cell-substrate sensing and the cell biomechanics. The model cell exhibits experimentally observed substrate rigidity sensing, which allows us to gain additional insights into the cell mechanosensitivity.

Effects of substrate stiffness and cell density on primary hippocampal cultures

Previous studies have shown that dendrites are influenced by substrate stiffness when neurons are plated in either pure or mixed cultures. However, because substrate rigidity can also affect other aspects of culture development known to impact dendrite branching, such as overall cell number, it is unclear whether substrate stiffness exerts a direct or indirect effect on dendrite morphology. In this study, we determine whether substrate stiffness plays a critical role in regulating dendrite branching independent of cell number. We plated primary mixed hippocampal cultures on soft and stiff gels, with Young's moduli of 1 kPa and 7 kPa, respectively. We found that neurons plated on stiffer substrates showed increased branching relative to neurons grown on softer substrates at the same cell number. On the stiff gels, we also observed a cell number-dependent effect, in which increasing initial plating density decreased dendrite branching. This change correlates with an increase in extracellular glutamate. We concluded that both cell number and substrate stiffness play roles in determining dendrite branching, and that the two effects are independent of one another.

Dimensionality Controls Cytoskeleton Assembly and Metabolism of Fibroblast Cells in Response to Rigidity and Shape

BackgroundVarious physical parameters, including substrate rigidity, size of adhesive islands and micro-and nano-topographies, have been shown to differentially regulate cell fate in two-dimensional (2-D) cell cultures. Cells anchored in a three-dimensional (3-D) microenvironment show significantly altered phenotypes, from altered cell adhesions, to cell migration and differentiation. Yet, no systematic analysis has been performed that studied how the integrated cellular responses to the physical characteristics of the environment are regulated by dimensionality (2-D versus 3-D).Methodology/Principal FindingsArrays of 5 or 10 µm deep microwells were fabricated in polydimethylsiloxane (PDMS). The actin cytoskeleton was compared for single primary fibroblasts adhering either to microfabricated adhesive islands (2-D) or trapped in microwells (3-D) of controlled size, shape, and wall rigidity. On rigid substrates (Young's Modulus = 1 MPa), cytoskeleton assembly within single fibroblast cells occurred in 3-D microwells of circular, rectangular, square, and triangular shapes with 2-D projected surface areas (microwell bottom surface area) and total surface areas of adhesion (microwell bottom plus wall surface area) that inhibited stress fiber assembly in 2-D. In contrast, cells did not assemble a detectable actin cytoskeleton in soft 3-D microwells (20 kPa), regardless of their shapes, but did so on flat, 2-D substrates. The dependency on environmental dimensionality was also reflected by cell viability and metabolism as probed by mitochondrial activities. Both were upregulated in 3-D cultured cells versus cells on 2-D patterns when surface area of adhesion and rigidity were held constant.Conclusion/SignificanceThese data indicate that cell shape and rigidity are not orthogonal parameters directing cell fate. The sensory toolbox of cells integrates mechanical (rigidity) and topographical (shape and dimensionality) information differently when cell adhesions are confined to 2-D or occur in a 3-D space.

Toward Magnetic Control of Cell Polarity

Cell polarity is involved in many aspects of cell and developmental biology. It is of fundamental importance for processes as diverse as cell motility, division, or differentiation. Cell polarization manifests itself through complex signaling and transport mechanisms by which molecules are asymmetrically localized within the cell. Whereas usual genetic and biochemical approaches are adapted to identify the elements in a transduction pathway which are necessary for the emergence of a cell polarity, they are not sufficient to know if the sole localized activity of a given effector is sufficient for the cell to acquire a polarity or to determine the kinetics of polarity formation. To address these important issues, we present a novel approach based on functionalized magnetic nanoparticles which are used to induce a localized signaling event of polarization. By doing so, we are able to monitor the cellular dynamic response to a local perturbation while preserving the complexity of the interaction feedbacks needed for the emergence of a global polarity. In our experiments, fluorescent magnetic nanoparticles (100-500 nm in size) are coated with purified constitutively active Cdc42 proteins, a key regulator of cell polarity. Once injected in the cytoplasm of live cells, these nanoparticles are manipulated using a customized magnetic setup able to exert forces on the order of 1-100 pN. We monitor at the single cell level the dynamics of nanoparticles and analyze the role of diverse factors (cytoskeleton, ER, substrate rigidity) on their intracellular mobility. Finally, we measure in different cell lines (Hela, 3T3) the effect of the local signalization on downstream effectors such as actin dynamics.

Modeling and Simulation of Soft Contact and Adhesion of Stem Cells

AbstractIn this paper, we briefly report our recent work on multiscale modeling and simulations of soft elasticity and focal adhesion of stem cells. In particular, our work is focused on modeling and simulation of contact and adhesion of stem cells on substrates with different rigidities. In order to understand the precise mechanical influences on cell contact/adhesion and to explain the possible mechanotransduction mechanism, we have developed a three-dimensional soft-matter cell model that uses liquid-crystal gel or liquid-crystal elastomer gel to model the overall constitutive relations of the cell, and we have simulated the responses of the cell to extra-cellular stimulus. The discussion here is specifically focused on the following issues: (1) how to model the overall myosin responses at the early stage of differentiation process of the stem cell, (2) the effects of both the adhesive force due to ligand-receptor interaction or focal adhesion and the surface tension, and (3) possible cell conformation and configuration changes triggered by substrate's rigidity.

Mimicking the Cellular Environment: Effects of Elastic Nanopatterned Substrates on Integrin-Mediated Cellular Interactions

An artificial substrate system, according to the biophysical and biochemical properties of the extracellular matrix in connective tissues, has been developed. The Young's moduli EY of poly(ethylene glycol)-diacrylate (PEG-DA) based hydrogel substrates span more than four orders of magnitude between 0,6kPa and 6MPa. Since PEG-DA substrates are protein repellent, they were decorated by extended gold nanoparticle arrays, manufactured by block copolymer micellar nanolithography. To provide bioactivity in terms of cell adhesion c(RGDfK) peptide, which is specific for αVβ3 integrins, was immobilized on the nanoparticles. The interparticle spacing and, hence, spacing of integrin binding sites ΔL could be precisely tuned, independently of the substrate rigidity between 20nm and 160nm. This system was used to investigate the behavior of fibroblasts as a function of changes within two-dimensional parameters space ΔL;EY). To this end, cell spreading area and cell-substrate interaction forces were determined by phase contrast microscopy and single cell force spectroscopy (SCFS), respectively. First, the effect of variation of ligand spacing on cellular behavior was investigated on hard substrates (EY>100kPa). We could demonstrate a strong increase in detachment force and spreading area on substrates featuring low ligand spacing. Than, substrate compliance was tuned whereas the ligand spacing was kept at approximately 50nm. This reveals a significant decrease in spreading area and detachment force on soft substrates (EY<8kPa). Additionally, both environmental parameters were varied simultaneously. Results from these experiments were determined as a function of hydrogel stiffness and integrin ligand distance. They revealed two tactile set points, thresholds in cellular sensing behavior, at EY=8kPa and ΔL=70nm, after 6, 12, and 24 hours of adhesion. Moreover, according to the hierarchical phase model in cellular behavior, elasticity was identified to be the dominant parameter in cellular sensing processes.

High Resolution Force Distribution of Migrating and Spreading Cells

Cell motility is crucial for the immune system and its misregulation is associated with cancer and other diseases. Migration is a complex cellular activity with many proteins coordinating within a subcellular motility machine. The machine's parts conspire to propel the cell forward by exerting forces on the environment with spatial variations down to the nanoscale. In this work we measured and statistically characterized the force distributions with the aim of illuminating underlying mechanisms. We cultured mouse embryonic fibroblast cells expressing GFP-actin on dense arrays of flexible nanoengineered pillars. During cell spreading pillar deflections quantitatively revealed cell traction patterns on the substrate. Simultaneous actin imaging enabled the cellular force map to be visualized within the evolving cell contour. The greatest forces were exerted in a ∼10-20 micron strip behind the leading edge and are directed inward. We analyzed time profiles of forces exerted on single pillars as the cell passed over the pillar location. Forces were relatively short lived and showed more fluctuation for pillars with smaller diameter than for larger ones. When the protruding cell edge reaches a pillar a period of large traction force begins, typically lasting several minutes, with maximum stress on the order of several nN/μm2. Stiffer substrates provoked larger tractions than more compliant substrates. Once the front of the cell has passed by, a drastic reduction in traction occurs. Spatial force distribution and its evolution were characterized by correlation functions relative to the leading edge. By varying the geometric parameters of the pillars, we probed a range of effective substrate rigidities. In summary, we combined high resolution measurements, statistical analysis and quantitative modeling to characterize and interpret signature features of migrating and spreading cell force maps.

Dynamic Force Generation by Neural Stem Cells

Mechanical cues may have important roles in tissue morphogenesis; progression through complex functions like differentiation may be associated with changes in cellular force generation and mechanosensing. To explore this concept, we use elastomer pillar arrays to map forces generated by neural stem cells in vitro, and identify two distinct dynamics of force generation. First, cell generated forces decrease as cells transition from a proliferative mode to differentiation, a process covering several days. This change in force generation correlated with a loss of sensitivity to substrate rigidity over a series of polydimethylsiloxane substrates. Second, neural stem cells exhibit a faster pattern of localized contractions at the cell body and outlying processes; each lasts on the order of minutes, and is not synchronized across the cell. This faster process is reminiscent of migratory behavior observed in vivo, and may be involved in controlling the motion of internal structures such as the cell nucleus. These results together provide new clues into the role of forces during development, and may lead to design principles for materials targeted for use in the central nervous system.

PEGDA hydrogels with patterned elasticity: Novel tools for the study of cell response to substrate rigidity

The ability of cells to migrate in response to mechanical gradients (durotaxis) and differential cell behavior in adhesion, spreading, and proliferation in response to substrate rigidity are key factors both in tissue engineering, in which materials must be selected to provide the appropriate mechanical signals, and in studies of mechanisms of diseases such as cancer and atherosclerosis, in which changes in tissue stiffness may inform cell behavior. Using poly(ethylene glycol) diacrylate hydrogels with varying polymer chain length and photolithographic patterning techniques, we are able to provide substrates with spatially patterned, tunable mechanical properties in both gradients and distinct patterns. The hydrogels can be patterned to produce anisotropic structures and exhibit patterned strain under mechanical loading. These hydrogels may be used to study cell response to substrate rigidity in both two and three dimensions and can also be used as a scaffold in tissue-engineering applications.

The relationship between fibroblast growth and the dynamic stiffnesses of a DNA crosslinked hydrogel

The microenvironment of cells is dynamic and undergoes remodeling with time. This is evident in development, aging, pathological processes, and at tissue-biomaterial interfaces. But in contrast, the majority of the biomimetic materials have static properties. Here, we show that a previously developed DNA crosslinked hydrogel circumvents the need of environmental factors and undergoes controlled stiffness change via DNA delivery, a feasible approach to initiate property changes in vivo, different from previous attempts. Two types of fibroblasts, L929 and GFP, were subject to the alterations in substrate rigidity presented in the hydrogels. Our results show that exogenous DNA does not cause appreciable cell shape change. Cells do respond to mechanical alterations as demonstrated in the cell projection area and polarity (e.g., Soft vs. Soft → Medium), and the responses vary depending on magnitude (e.g., Soft → Medium vs. Soft → Stiff) and range of stiffness changes (e.g., Soft → Medium vs. Medium → Stiff). The two types of fibroblasts share specific responses in common (e.g., Soft → Medium), while differ in others (e.g., Medium → Stiff). For each cell type, the projection area and polarity respond differently. This approach provides insight into pathology (e.g., cancer) and tissue functioning, and assists in designing biomaterials with controlled dynamic stiffness by choosing the range and magnitude of stiffness change.

Single-cell response to stiffness exhibits muscle-like behavior

Living cells sense the rigidity of their environment and adapt their activity to it. In particular, cells cultured on elastic substrates align their shape and their traction forces along the direction of highest stiffness and preferably migrate towards stiffer regions. Although numerous studies investigated the role of adhesion complexes in rigidity sensing, less is known about the specific contribution of acto-myosin based contractility. Here we used a custom-made single-cell technique to measure the traction force as well as the speed of shortening of isolated myoblasts deflecting microplates of variable stiffness. The rate of force generation increased with increasing stiffness and followed a Hill force-velocity relationship. Hence, cell response to stiffness was similar to muscle adaptation to load, reflecting the force-dependent kinetics of myosin binding to actin. These results reveal an unexpected mechanism of rigidity sensing, whereby the contractile acto-myosin units themselves can act as sensors. This mechanism may translate anisotropy in substrate rigidity into anisotropy in cytoskeletal tension, and could thus coordinate local activity of adhesion complexes and guide cell migration along rigidity gradients.

Synthetic Materials in the Study of Cell Response to Substrate Rigidity

While it has long been understood that cells can sense and respond to a variety of stimuli, including soluble and insoluble factors, light, and externally applied mechanical stresses, the extent to which cells can sense and respond to the mechanical properties of their environment has only recently begun to be studied. Cell response to substrate stiffness has been suggested to play an important role in processes ranging from developmental morphogenesis to the pathogenesis of disease states and may have profound implications for cell and tissue culture and tissue engineering. Given the importance of this phenomenon, there is a clear need for systems for cell study in which substrate mechanics can be carefully defined and varied independently of biochemical and other signals. This review will highlight past work in the field of cell response to substrate rigidity as well as areas for future study.

A photo-modulatable material for probing cellular responses to substrate rigidity.

Recent studies indicate that extracellular mechanical properties, including rigidity, profoundly affect cellular morphology, growth, migration, and differentiation [R. J. Pelham, Jr. and Y. Wang, Proc. Natl. Acad. Sci. U. S. A., 1997, 94(25), 13661-13665; H. B. Wang, M. Dembo and Y. L. Wang, Am. J. Physiol. Cell Physiol., 2000, 279(5), C1345-C1350; P. C. Georges, and P. A. Janmey, J. Appl. Physiol., 2005, 98(4), 1547-1553; C. M. Lo, H. B. Wang, M. Dembo and Y. L. Wang, Biophys. J., 2000. 79(1), 144-152; D. E. Discher, P. Janmey and Y. L. Wang, Science, 2005, 310(5751), 1139-1143; A. J. Engler, M. A. Griffin, S. Sen, C. G. Bonnemann, H. L. Sweeney and D. E. Discher, J. Cell Biol., 2004, 166(6), 877-887]. However, most studies involving rigidity sensing have been performed by comparing cells on separate substrata of fixed stiffness. To allow spatial and/or temporal manipulation of mechanical properties, we have developed a modulatable hydrogel by reacting linear polyacrylamide (PA) with a photosensitive crosslinker. This material allows UV-mediated control of rigidity, softening by 20-30% upon irradiation at a dose tolerated by live cells. Global UV irradiation induces an immediate recoiling of 3T3 fibroblasts and a reduced spread area at steady state. Furthermore, localized softening of the posterior substratum of polarized cells causes no apparent effect, while softening of the anterior substratum elicits pronounced retraction, indicating that rigidity sensing is localized to the frontal region. This type of material allows precise spatial and temporal control of mechanical signals for both basic research and regenerative medicine.

Substrate rigidity regulates Ca2+ oscillation via RhoA pathway in stem cells

Substrate rigidity plays crucial roles in regulating cellular functions, such as cell spreading, traction forces, and stem cell differentiation. However, it is not clear how substrate rigidity influences early cell signaling events such as calcium in living cells. Using highly sensitive Ca(2+) biosensors based on fluorescence resonance energy transfer (FRET), we investigated the molecular mechanism by which substrate rigidity affects calcium signaling in human mesenchymal stem cells (HMSCs). Spontaneous Ca(2+) oscillations were observed inside the cytoplasm and the endoplasmic reticulum (ER) using the FRET biosensors targeted at subcellular locations in cells plated on rigid dishes. Lowering the substrate stiffness to 1 kPa significantly inhibited both the magnitudes and frequencies of the cytoplasmic Ca(2+) oscillation in comparison to stiffer or rigid substrate. This Ca(2+) oscillation was shown to be dependent on ROCK, a downstream effector molecule of RhoA, but independent of actin filaments, microtubules, myosin light chain kinase, or myosin activity. Lysophosphatidic acid, which activates RhoA, also inhibited the frequency of the Ca(2+) oscillation. Consistently, either a constitutive active mutant of RhoA (RhoA-V14) or a dominant negative mutant of RhoA (RhoA-N19) inhibited the Ca(2+) oscillation. Further experiments revealed that HMSCs cultured on gels with low elastic moduli displayed low RhoA activities. Therefore, our results demonstrate that RhoA and its downstream molecule ROCK may mediate the substrate rigidity-regulated Ca(2+) oscillation, which determines the physiological functions of HMSCs.

Analysis of film strain and stress in a film-substrate cantilever system

The bending problem of a magnetic film-nonmagnetic substrate cantilever system is studied by using the principle of energy minimization. Emphasis is placed on the analysis of geometrical and physical parameter dependence of the neutral plane, internal film stress and strain of the cantilever system, and then the influence of such a parameter on the bending characteristic is presented. The results indicate, owing to the anisotropic expanding feature of the magnetostriction, that the neutral plane is generally anisotropic, and moves downwards rapidly with the increasing thickness ratio. Meanwhile, the bounding rigidity of substrate on the film will decrease with the increasing thickness ratio, and thus release the film stress, i.e., it decreases, but the film strain increases. The effect of Poisson’s ratio of the materials on the film strain, the stress and the neutral plane in the direction transverse to the magnetization is prominent. For the strain and the stress in the magnetization, however, the role of Poisson’s ratio is inconspicuous. This property is due to the initiative elongating (or contracting) feature of the magnetic film along its magnetization.

Mechanical Forces Induced by the Transendothelial Migration of Human Neutrophils

The mechanisms regulating neutrophil transmigration of vascular endothelium are not fully elucidated, but involve neutrophil firm attachment and passage through endothelial cell–cell junctions. The goal of this study was to characterize the tangential forces exerted by neutrophils during transendothelial migration at cell–cell junctions using an in vitro laminar shear flow model in which confluent activated endothelium is grown on a microfabricated pillar substrate. The tangential forces are deduced from the measurement of pillar deflection beneath the endothelial cell–cell junction as neutrophils transmigrate. The force diagram displays an initial force increase, which coincides with neutrophil penetration into the intercellular space and formation of a gap in VE-cadherin staining. This is followed by a rapid and large increase of traction forces exerted by endothelial cells on the substrate in response to the transmigration process and the disruption of cell–cell contacts. The average maximum force exerted by an actively transmigrating neutrophil is three times higher than the force generated by an adherent neutrophil that does not transmigrate. Furthermore, we show that substrate rigidity can modify the mechanical forces induced by the transmigration of a neutrophil through the endothelium. Our data suggest that the force induced by neutrophil transmigration plays a key role in the disruption of endothelial adherens junctions.

Size Distribution and Molecular Associations of Plasma Fibronectin and Fibronectin Crosslinked by Transglutaminase 2

Fibronectin (FN) is a ubiquitously expressed cell adhesion protein capable of assembling into large, extended fibrillar networks as part of an extracellular matrix (ECM) that regulates cell behavior. FN is a substrate for certain members of the transglutaminase family of protein-crosslinking enzymes-enzymes which can modify the ability of FN to support cell adhesion. In this study, we have analyzed the thermo-chemical stability of plasma FN in its noncrosslinked form, and after crosslinking by transglutaminase 2 (TG2), using dynamic light scattering. We report that FN is found in a generally globular (8.7 nm hydrodynamic radius), dimerized form in aqueous solutions, but unfolds into a linear arrangement at high ionic (1 M NaCl) and chaotropic (5 M urea) environments. FN conformation remained stable after multiple heating and cooling cycles ranging from 4 to 60 degrees C. Crosslinking of FN with TG2 formed large, multimeric complexes having high chemical stability in aqueous, high ionic and chaotropic environments, demonstrating that this covalent modification stabilizes FN. Given recent data that substrate (e.g. ECM) rigidity profoundly affects cell differentiation and behavior, we further studied how TG2 crosslinking affects the molecular rigidity of FN by obtaining atomic force microscopy nanoindentation measurements from untreated and crosslinked FN samples embedded in acrylamide gels. We demonstrate that TG2-mediated crosslinking of FN significantly increases Young's modulus (of elasticity), an observation of increased rigidity having important implications with respect to the biological role of ECM protein-crosslinking in cell signaling and guiding cell differentiation.

Control of integrin‐mediated platelet adhesion by rigidity sensing at the surface of fibrin(ogen) matrix

Recent evidence suggests that substrate rigidity controls cell adhesion through integrin‐mediated signaling. We have found that binding of soluble fibrinogen to the surface of insoluble fibrin matrix inhibits integrin‐mediated cell adhesion by creating an anti‐adhesive fibrinogen layer. To gain understanding of the mechanism underlying the differential ability of cells to adhere to the naked fibrin gel (rigid substrate) and to the loose fibrinogen layer formed on the surface of fibrin (soft substrate), we examined integrin αIIbβ3‐mediated signaling in platelets. Platelet adhesion to fibrin clots induced strong phosphorylation of FAK and Syk kinases as compared to adhesion on soft substrates. Also, the rigid, but not soft substrate induced recruitment of signaling molecules talin and skelemin to integrin‐containing focal adhesions. Consistent with their limited ability to induce sufficient signaling, soft substrates do not support platelet spreading. Differential platelet signaling was also observed when fibrinogen was adsorbed on plastic at low and high densities, a condition which mimics rigid and soft substrates, respectively. These data suggest that soluble fibrinogen prevents stable platelet adhesion to the fibrin clot by modifying its mechanical properties which results in insufficient signaling and reduced force generation.NIH HL 63199

The motility of normal and cancer cells in response to the combined influence of the substrate rigidity and anisotropic microstructure

Cell adhesion and migration are strongly influenced by extracellular matrix (ECM) architecture and rigidity, but little is known about the concomitant influence of such environmental signals to cell responses, especially when considering cells of similar origin and morphology, but exhibiting a normal or cancerous phenotype. Using micropatterned polydimethylsiloxane substrates (PDMS) with tunable stiffness (500 kPa, 750 kPa, 2000 kPa) and topography (lines, pillars or unpatterned), we systematically analyse the differential response of normal (3T3) and cancer (SaI/N) fibroblastic cells. Our results demonstrate that both cells exhibit differential morphology and motility responses to changes in substrate rigidity and microtopography. 3T3 polarisation and spreading are influenced by substrate microtopography and rigidity. The cells exhibit a persistent type of migration, which depends on the substrate anisotropy. In contrast, the dynamic of SaI/N spreading is strongly modified by the substrate topography but not by substrate rigidity. SaI/N morphology and migration seem to escape from extracellular cues: the cells exhibit uncorrelated migration trajectories and a large dispersion of their migration speed, which increases with substrate rigidity.