Mechanical Properties Of Substrate Research Articles

Integrins Direct Cell Adhesion in a Substrate-Dependent Manner

The relationship between substrate properties and cell behavior is complex, including roles for both mechanics and biochemistry. Here we investigate the role of viscous dissipation on cell adhesion behaviors, using polymer films of tunable lateral mobility. We find that fibroblasts selectively use \(\alpha _v \beta _3\) and \(\alpha _5 \beta _1\) integrin receptors to control their spreading area and polarization on low and high mobility films, respectively. In addition, the dynamics of cell spreading and polarization are well described by a semi-empirical sigmoidal relationship. Analysis of cell dynamic behavior reveals that spreading dynamics are controlled by the availability of integrins, whereas the polarization dynamics are controlled by intracellular signaling. The result that cells preferentially use specific integrin receptors in response to substrate mechanical properties has broad implications for processes in dynamic environments such as wound healing and cancer metastasis.

Understanding the Role of Substrate Elasticity on Intracellular Stresses during Cell Spreading

We present a two-dimensional mathematical model and finite element simulations of a cell interacting with a deformable substrate to better understand how substrate mechanical properties affect cellular mechanical response and spread areas. The cell is treated as an actively deforming hypoelastic material and the substrate is linearly elastic. The model predicts stress and displacement fields in both the cell and the substrate. This model of cell and substrate mechanics is coupled to models describing active cellular deformation due to stress fiber contraction and evolution of focal adhesions. In this work we compare cellular stresses and substrate displacements predicted by the model on substrates of increasing stiffness against two experimental works i) Hersh et. al. 20131 with cardiomyocytes and (ii) Marinkovic et. al. 20122 with hMSc's. The model predicts an increase in compressive stresses within the cell for the two cell types with increasing substrate stiffness between 1kPa-90kPa, which is in agreement with experimental results. Further model predictions of increase in substrate displacements with substrate stiffness are in good quantitative agreement with the experimental results. In addition on softer substrates we predict that increased stress fiber contraction due to a greater compliance of the substrate at the sites of attachment results in smaller spread areas.

The Mexican hat effect on the delamination buckling of a compressed thin film

Because of the interaction between film and substrate, the film buckling stress can vary significantly, depending on the delamination geometry, the film and substrate mechanical properties. The Mexican hat effect indicates such interaction. An analytical method is presented, and related dimensional analysis shows that a single dimensionless parameter can effectively evaluate the effect.

A universal model for the load–displacement relation in an elastic coated spherical contact

A finite element analysis was used in order to investigate the elastic contact of a coated sphere compressed by a rigid flat. Different coating and substrate geometrical and mechanical properties were analyzed to obtain a universal dimensionless model for the load–displacement relation. Both hard and soft coatings were considered. Dimensionless parameters, which control the behavior of the elastically loaded coated sphere, were identified. A proper normalization of the dimensional load and displacement was found resulting in a universal model. This model also provides a universal expression for the effective modulus of elasticity that is based only on mechanical properties of the coating and the substrate.

Strain and Stress in Magnetoelastic Co–Pt Multilayers

The influence of substrate strain on the magnetic properties of Co-Pt multilayers is investigated by density functional theory (DFT) and model calculations. The lattice parameters for strained Co-Pt multilayers, which are needed as an input for first-principle calculations, are determined by a hybrid approach where DFT is used to determine the lattice constants and atomic positions of the free multilayer, and the effect of the strained substrate is treated continuum mechanically. The lowest order anisotropy constants have been calculated for unstrained and strained hcp Co <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> Pt <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sub> (111) and fcc Co <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> Pt <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">5</sub> (111) multilayers on LiNbO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> , and in the strained case, the anisotropy is nonunixial with nonzero anisotropy constant K <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> and K <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> '. By involving both multilayer and substrate mechanical properties, our calculations go beyond the usually considered magnetoelastic anisotropy mechanisms.

The effect of substrate elasticity and actomyosin contractility on different forms of endocytosis.

Substrate mechanical properties have emerged as potent determinants of cell functions and fate. We here tested the hypothesis that different forms of endocytosis are regulated by the elasticity of the synthetic hydrogels cells are cultured on. Towards this objective, we quantified cell-associated fluorescence of the established endocytosis markers transferrin (Tf) and cholera toxin subunit B (CTb) using a flow-cytometry based protocol, and imaged marker internalization using microscopy techniques. Our results demonstrated that clathrin-mediated endocytosis of Tf following a 10-minute incubation with a fibroblast cell line was lower on the softer substrates studied (5 kPa) compared to those with elasticities of 40 and 85 kPa. This effect was cancelled after 1-hour incubation revealing that intracellular accumulation of Tf at this time point did not depend on substrate elasticity. Lipid-raft mediated endocytosis of CTb, on the other hand, was not affected by substrate elasticity in the studied range of time and substrate elasticity. The use of pharmacologic contractility inhibitors revealed inhibition of endocytosis for both Tf and CTb after a 10-minute incubation and a dissimilar effect after 1 hour depending on the inhibitor type. Further, the internalization of fluorescent NPs, used as model drug delivery systems, showed a dependence on substrate elasticity, while transfection efficiency was unaffected by it. Finally, an independence on substrate elasticity of Tf and CTb association with HeLa cells indicated that there are cell-type differences in this respect. Overall, our results suggest that clathrin-mediated but not lipid-raft mediated endocytosis is potentially influenced by substrate mechanics at the cellular level, while intracellular trafficking and accumulation show a more complex dependence. Our findings are discussed in the context of previous work on how substrate mechanics affect the fundamental process of endocytosis and highlight important considerations for future studies.

Effects of substrate stiffness on adipogenic and osteogenic differentiation of human mesenchymal stem cells

Substrate mechanical properties, in addition to biochemical signals, have been shown to modulate cell phenotype. In this study, we inspected the effects of substrate stiffness on human mesenchymal stem cells (hMSCs) derived from adult human bone marrow differentiation into adipogenic and osteogenic cells. A chemically modified extracellular matrix derived and highly biocompatible hydrogel, based on thiol functionalized hyaluronic acid (HA-SH) and thiol functionalized recombinant human gelatin (Gtn-SH), which can be crosslinked by poly (ethylene glycol) tetra-acrylate (PEGTA), was used as a model system. The stiffness of the hydrogel was controlled by adjusting the crosslinking density. Human bone marrow MSCs were cultured on the hydrogels with different stiffness under adipogenic and osteogenic conditions. Oil Red O staining and F-actin staining were applied to assess the change of cell morphologies under adipogenic and osteogenic differentiation, respectively. Gene expression of cells was determined with reverse transcription polymerase chain reaction (RT-PCR) as a function of hydrogel stiffness. Results support the hypothesis that adipogenic and osteogenic differentiation of hMSCs are inclined to occur on substrate with stiffness similar to their in vivo microenvironments.

Percussion signals of Lygus rugulipennis Poppius (Heteroptera: Miridae)

Abstract The European tarnished plant bug (Lygus rugulipennis Poppius) is among the most serious pests in the family Miridae, and therefore there is increasing interest in understanding the behaviour of this species. In the present study, laboratory recordings were taken using a laser vibrometer on adult males and females to ascertain whether acoustic signals are involved in intraspecific communication. Recordings were both carried out on plant and loudspeaker membrane substrates. Males and females emitted vibratory signals and the present results indicate that these signals are important during courtship. The basic signal characteristics measured were the dominant frequency, pulse duration, repetition time and number of pulses per group within the signal. Male and female signals did not differ in respect to any of these characteristics. Plant recorded signals were longer because of different mechanical properties of substrates. Additionally, the high frequency components were attenuated due to the low-pass filtering properties of plants. As this is the first study on vibratory communication of the European tarnished plant bug, we believe these findings may contribute considerably to the better understanding of the mating behavior of this important pest species.

Biomembrane-mimicking lipid bilayer system as a mechanically tunable cell substrate

Cell behavior such as cell adhesion, spreading, and contraction critically depends on the elastic properties of the extracellular matrix. It is not known, however, how cells respond to viscoelastic or plastic material properties that more closely resemble the mechanical environment cells encounter in the body. In this report, we employ viscoelastic and plastic biomembrane-mimicking cell substrates. The compliance of the substrates can be tuned by increasing the number of polymer-tethered bilayers. This leaves the density and conformation of adhesive ligands on the top bilayer unaltered. We then observe the response of fibroblasts to these property changes. For comparison, we also study the cells on soft polyacrylamide and hard glass surfaces. Cell morphology, motility, cell stiffness, contractile forces and adhesive contact size all decrease on more compliant matrices but are less sensitive to changes in matrix dissipative properties. These data suggest that cells are able to feel and respond predominantly to the effective matrix compliance, which arises as a combination of substrate and adhesive ligand mechanical properties.

Study on the effect of DIC deformation sensor on mechanical property of substrate

The thin epoxy film with micro-scale speckle pattern as a digital image correlation (DIC) deformation sensor has been fabricated or transferred on the surface of sample in the previous study. When the thickness of the film cannot be ignored, it may have an influence on the validity of measurement results. And thus, the influence of the thin epoxy film on mechanical properties of substrates should be investigated. In this study, the mechanical behavior of thin film itself and surface strain of composite structure of thin film and substrate were measured using micro digital image correlation (MDIC) method. And theoretical and simulative results were also analyzed. From the comparison analysis of theoretical, simulative and experimental results, it is concluded that when the ratios of Young’s modulus and thickness between the film and substrate are smaller than 13 and 0.5 respectively, the influence from the thin film can be ignored, and thin epoxy film as DIC sensor can be used to measure the deformation of substrate.

Cellular mechanoadaptation to substrate mechanical properties: contributions of substrate stiffness and thickness to cell stiffness measurements using AFM

Mechanosensing by adherent cells is usually studied by quantifying cell responses on hydrogels that are covalently linked to a rigid substrate. Atomic force microscopy (AFM) represents a convenient way of characterizing the mechanoadaptation response of adherent cells on hydrogels of varying stiffness and thickness. Since AFM measurements reflect the effective cell stiffness, therefore, in addition to measuring real cytoskeletal alterations across different conditions, these measurements might also be influenced by the geometry and physical properties of the substrate itself. To better understand how the physical attributes of the gel influence AFM stiffness measurements of cells, we have used finite element analysis to simulate the indentation of cells of various spreads resting on hydrogels of varying stiffness and thickness. Consistent with experimental results, our simulation results indicate that for well spread cells, stiffness values are significantly over-estimated when experiments are performed on cells cultured on soft and thin gels. Using parametric studies, we have developed scaling relationships between the effective stiffness probed by AFM and the bulk cell stiffness, taking cell and tip geometry, hydrogel properties, nuclear stiffness and cell contractility into account. Finally, using simulated mechanoadaptation responses, we have demonstrated that a cell stiffening response may arise purely due to the substrate properties. Collectively, our results demonstrate the need to take hydrogel properties into account while estimating cell stiffness using AFM indentation.

Deformation Gradients Imprint the Direction and Speed of En Masse Fibroblast Migration for Fast Healing

En masse cell migration is more relevant than single cell migration in physiological processes of tissue formation, such as embryogenesis, morphogenesis and wound healing. In these situations, cells are influenced by the proximity of other cells including interactions facilitated by substrate mechanics. Here we found that when fibroblasts migrated en masse over a hydrogel, they established a well-defined deformation field by traction forces and migrated along a trajectory defined by field gradients. The mechanics of the hydrogel determined the magnitude of the gradient. For materials stiff enough to withstand deformation related to cellular traction forces, such patterns did not form. Furthermore, migration patterns functioned poorly on very soft matrices where only a minimal traction gradient could be established. The largest degree of alignment and migration velocity occurred on the gels with the largest gradients. Granulation tissue formation in punch wounds of juvenile pigs was correlated strongly with the modulus of the implanted gel in agreement with in vitro en masse cell migration studies. These findings provide basic insight into the biomechanical influences on fibroblast movement in early wounds and relevant design criteria for development of tissue-engineered constructs that aim to stimulate en masse cell recruitment for rapid wound healing.

Characterizing the effects of heparin gel stiffness on function of primary hepatocytes.

In the liver, hepatocytes are exposed to a large array of stimuli that shape hepatic phenotype. This in vivo microenvironment is lost when hepatocytes are cultured in standard cell cultureware, making it challenging to maintain hepatocyte function in vitro. Our article focused on one of the least studied inducers of the hepatic phenotype-the mechanical properties of the underlying substrate. Gel layers comprised of thiolated heparin (Hep-SH) and diacrylated poly(ethylene glycol) (PEG-DA) were formed on glass substrates via a radical mediated thiol-ene coupling reaction. The substrate stiffness varied from 10 to 110 kPa by changing the concentration of the precursor solution. ELISA analysis revealed that after 5 days, hepatocytes cultured on a softer heparin gel were synthesizing five times higher levels of albumin compared to those on a stiffer heparin gel. Immunofluorescent staining for hepatic markers, albumin and E-cadherin, confirmed that softer gels promoted better maintenance of the hepatic phenotype. Our findings point to the importance of substrate mechanical properties on hepatocyte function.

Theoretical simulation and experimental verification of C/SiC joints with pins or bolts

C/SiC joints with pins or bolts were prepared by reaction joining process combined with precursor infiltration and pyrolysis (PIP) process. The influences of hole parameters including ratios of edge distance to diameter (E/D), width to diameter (W/D) and hole distance to diameter (H/D), on the mechanical properties of C/SiC substrates were studied by theoretical simulation and experimental methods. The results show that the experimental fracture loads and damage modes are consistent with the theoretical simulation values, and when E/D⩾3, W/D⩾3 and H/D⩾3 the effect of drilling on the mechanical properties of substrates could be neglected. More, the amendatory theoretical simulation can effectively predict the bearing loads and damage modes of the joints with different joining method (⩽13% of the error), which could provide theoretical guidance for the engineering application. The bearing loads of C/SiC joints increase almost linearly with the increase of number of C/SiC pins or bolts, and accordingly the damage mode changes from the pin/bolt rupture to the substrate rupture, thus the reliability of joints is also improved.

PEG-Phosphorylcholine Hydrogels As Tunable and Versatile Platforms for Mechanobiology

We report here the synthesis of a new class of hydrogels with an extremely wide range of mechanical properties suitable for cell studies. Mechanobiology has emerged as an important field in bioengineering, in part due to the development of synthetic polymer gels and fibrous protein biomaterials to control and quantify how cells sense and respond to mechanical forces in their microenvironment. To address the problem of limited availability of biomaterials, in terms of both mechanical range and optical clarity, we have prepared hydrogels that combine poly(ethylene glycol) (PEG) and phosphorylcholine (PC) zwitterions. Our goal was to create a hydrogel platform that exceeds the range of Young's moduli reported for similar hydrogels, while being simple to synthesize and manipulate. The Young's modulus of these "PEG-PC" hydrogels can be tuned over 4 orders of magnitude, much greater than commonly used hydrogels such as PEG-diacrylate, PEG-dimethacrylate, and polyacrylamide, with smaller average mesh sizes and optical clarity. We prepared PEG-PC hydrogels to study how substrate mechanical properties influence cell morphology, focal adhesion structure, and proliferation across multiple mammalian cell lines, as a proof of concept. These novel PEG-PC biomaterials represent a new and useful class of mechanically tunable hydrogels for mechanobiology.

The influence of substrate stiffness gradients on primary human dermal fibroblasts

Materials mechanical properties are known to be an important regulator of cellular processes such as proliferation, differentiation and migration, and have seen increasing attention in recent years. At present, there are only few approaches where the mechanical properties of thin films can be controllably varied across an entire surface. In this work, we present a technique for controlled generation of gradients of surface elastic moduli involving a weak polyelectrolyte multilayer (PEM) system of approximately 100 nm thickness and time dependent immersion in a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent. Uniform surface chemistry across the gradient and wettability was provided by the addition of a 10 nm thick plasma polymer layer deposited from vapour of either allylamine or acrylic acid. We used the resultant stiffness gradients (0.5–110 MPa in hydrated state) to investigate the adhesion, morphology and proliferation on human dermal fibroblasts (HDFs). We show that substrate mechanical properties strongly influence HDF cell fate. We also found that in the experimental range of surface properties used in this study, the surface stiffness was a stronger driving force to cells fate compared to chemistry and wettability.

Nanomechanics controls neuronal precursors adhesion and differentiation

The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat polydimethylsiloxane (PDMS) substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell-substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine.

On the determination of the film hardness in hard film/substrate composites using depth-sensing indentation

Abstract The main difficulty in the mechanical characterisation of thin films using depth-sensing indentation is the determination of the relative substrate and film contributions to the measured properties of the film/substrate composite. In this study, a three-dimensional numerical simulation of the Vickers hardness test is used to study the influence of the substrate and film mechanical properties on the composite's behaviour under depth-sensing indentation. The particular case of hard films on soft substrates is analysed. In order to understand the behaviour of the composite, a study of the plastic strain distribution under indentation of several composites is performed. A methodology to determine the relative film hardness, i.e. H F / H S ratio, is proposed. The methodology is successfully verified using fictitious and real composite materials.

Variation in Foot Strike Patterns during Running among Habitually Barefoot Populations

Endurance running may have a long evolutionary history in the hominin clade but it was not until very recently that humans ran wearing shoes. Research on modern habitually unshod runners has suggested that they utilize a different biomechanical strategy than runners who wear shoes, namely that barefoot runners typically use a forefoot strike in order to avoid generating the high impact forces that would be experienced if they were to strike the ground with their heels first. This finding suggests that our habitually unshod ancestors may have run in a similar way. However, this research was conducted on a single population and we know little about variation in running form among habitually barefoot people, including the effects of running speed, which has been shown to affect strike patterns in shod runners. Here, we present the results of our investigation into the selection of running foot strike patterns among another modern habitually unshod group, the Daasanach of northern Kenya. Data were collected from 38 consenting adults as they ran along a trackway with a plantar pressure pad placed midway along its length. Subjects ran at self-selected endurance running and sprinting speeds. Our data support the hypothesis that a forefoot strike reduces the magnitude of impact loading, but the majority of subjects instead used a rearfoot strike at endurance running speeds. Their percentages of midfoot and forefoot strikes increased significantly with speed. These results indicate that not all habitually barefoot people prefer running with a forefoot strike, and suggest that other factors such as running speed, training level, substrate mechanical properties, running distance, and running frequency, influence the selection of foot strike patterns.

Effects of Copper Pattern Density and Orientation on the Modulus of BGA Substrates

Substrate material properties directly impact package mechanical performance. Estimation of substrate mechanical properties based on each component layer provides maximum flexibility and better accuracy. The component materials of a BGA substrate are the fiberglass-reinforced epoxy layers, copper and soldermask. Even when the material properties (modulus and CTE) of each component are known, the behavior of the final substrate is not easily determined because factors such as copper density and pattern have significant effects. For example, the copper pattern may be oriented such that conductor traces run in one direction for a considerable length. The modulus in the direction parallel to the traces will be different from the modulus in the direction perpendicular to the traces. Package mechanical simulations typically do not mesh the details of the copper patterns, but rather average within each layer, thus neglecting the orientation effects. Therefore FEM (Finite Element Model) simulations may yield inaccurate stress and warpage results. This study utilized a test vehicle (TV) to study the copper pattern density and orientation effect on substrate modulus. Each TV sample was a two-metal-layer copper-clad laminate designed with one of eight simple geometric patterns. One set of patterns had Cu traces running parallel in one direction, with varied metal densities of 0%, 25%, 50%, 75% and 100% respectively. Measurements were made both parallel and perpendicular to the trace orientation. Also studied were a pattern of holes (continuous metal plane with unconnected holes) and a dot pattern (unconnected circular metal pads). Substrate flexural modulus measurements were made using two types of instruments: DMA and Instron. Data was collected from −65 to 260°C. FEM simulations which meshed the details of copper patterns were used to calculate the overall substrate modulus. These correlated better with the empirical data than simulations utilizing traditional layer averaging. The copper pattern had significant effects; the substrate modulus was always much lower in the direction perpendicular to the traces than the modulus in the direction parallel to the traces. The amount varied depending on temperature, but could be as high as 35%. When measured parallel to the copper traces, the substrate modulus was observed to increase with copper pattern density as predicted by theoretical mixture rules. However, the substrate modulus in the direction perpendicular to the traces was not easily predicted theoretically. Therefore, more sophisticated averaging techniques and simulations are needed.