Influence Of Substrate Stiffness Research Articles

Influence of Substrate Stiffness on iPSC-Derived Retinal Pigmented Epithelial Cells.

Retinal degenerative diseases are a major cause of blindness involving the dysfunction of photoreceptors, retinal pigmented epithelium (RPE), or both. A promising treatment approach involves replacing these cells via surgical transplantation, and previous work has shown that cell delivery scaffolds are vital to ensure sufficient cell survival. Thus, identifying scaffold properties that are conducive to cell viability and maturation (such as suitable material and mechanical properties) is critical to ensuring a successful treatment approach. In this study, we investigated the effect of scaffold stiffness on human RPE attachment, survival, and differentiation, comparing immortalized (ARPE-19) and stem cell-derived RPE (iRPE) cells. Polydimethylsiloxane was used as a model polymer substrate, and varying stiffness (~12 to 800 kPa) was achieved by modulating the cross-link-to-base ratio. Post-attachment changes in gene and protein expression were assessed using qPCR and immunocytochemistry. We found that while ARPE-19 and iRPE exhibited significant differences in morphology and expression of RPE markers, substrate stiffness did not have a substantial impact on cell growth or maturation for either cell type. These results highlight the differences in expression between immortalized and iPSC-derived RPE cells, and also suggest that stiffnesses in this range (~12-800 kPa) may not result in significant differences in RPE growth and maturation, an important consideration in scaffold design.

The influence of substrate stiffness on interfacial stresses for adhesive microfibrils

Bioinspired microfibrillar surfaces adhere by exploiting the presence of intermolecular forces at the contact interface. Experimental work has shown that compliant mushroom shaped fibrils can facilitate optimal adhesion across a range of substrates. This work evaluates numerically how fibril contact tip shape and the degree of elastic mismatch at the fibril-substrate interface influence adhesion. In a finite element simulation of fibrils subject to a remote stress, the interfacial stress distributions that define the detachment behaviour are computed for a range of fibril cap diameters, thicknesses, and substrate stiffnesses. Depending on the substrate stiffness, there is a singular stress field present at the edge of contact for smaller mushroom cap and straight punch fibrils while the stress at the corner for larger mushroom fibrils tends to zero. This variation in fibril-substrate interfacial stress has implications for the most probable defect type to initiate detachment and the stability of this crack growth. For fibrils within this corner singularity regime, the adhesion strength of a patch of fibrils is determined using interfacial fracture mechanics. The simulations are compared with experimental data of pull-off strengths and the biomedical applications of adhesive microfibrillar patches are considered.

Influence of Substrate Stiffness on Barrier Function in an iPSC-Derived In Vitro Blood-Brain Barrier Model.

Vascular endothelial cells respond to a variety of biophysical cues such as shear stress and substrate stiffness. In peripheral vasculature, extracellular matrix (ECM) stiffening alters barrier function, leading to increased vascular permeability in atherosclerosis and pulmonary edema. The effect of ECM stiffness on blood-brain barrier (BBB) endothelial cells, however, has not been explored. To investigate this topic, we incorporated hydrogel substrates into an in vitro model of the human BBB. Induced pluripotent stem cells were differentiated to brain microvascular endothelial-like (BMEC-like) cells and cultured on hydrogel substrates of varying stiffness. Cellular changes were measured by imaging, functional assays such as transendothelial electrical resistance (TEER) and p-glycoprotein efflux activity, and bulk transcriptome readouts. The magnitude and longevity of TEER in iPSC-derived BMEC-like cells is enhanced on compliant substrates. Quantitative imaging shows that BMEC-like cells form fewer intracellular actin stress fibers on substrates of intermediate stiffness (20 kPa relative to 1 and 150 kPa). Chemical induction of actin polymerization leads to a rapid decline in TEER, agreeing with imaging readouts. P-glycoprotein activity is unaffected by substrate stiffness. Modest differences in RNA expression corresponding to specific signaling pathways were observed as a function of substrate stiffness. iPSC-derived BMEC-like cells exhibit differences in passive but not active barrier function in response to substrate stiffness. These findings may provide insight into BBB dysfunction during neurodegeneration, as well as aid in the optimization of more complex three-dimensional neurovascular models utilizing compliant hydrogels. The online version contains supplementary material available at 10.1007/s12195-021-00706-8.

Polymeric Nanoneedle Arrays Mediate Stiffness‐Independent Intracellular Delivery

AbstractTunable vertically aligned nanostructures, usually fabricated using inorganic materials, are powerful nanoscale tools for advanced cellular manipulation. However, nanoscale precision typically requires advanced nanofabrication machinery and involves high manufacturing costs. By contrast, polymeric nanoneedles (NNs) of precise geometry can be produced by replica molding or nanoimprint lithography—rapid, simple, and cost‐effective. Here, cytocompatible polymeric arrays of NNs are engineered with identical topographies but differing stiffness, using polystyrene (PS), SU8, and polydimethylsiloxane (PDMS). By interfacing the polymeric NN arrays with adherent and suspension mammalian cells, and comparing the cellular responses of each of the three polymeric substrates, the influence of substrate stiffness from topography on cell behavior is decoupled. Notably, the ability of PS, SU8, and PDMS NNs is demonstrated to facilitate mRNA delivery to GPE86 cells with 26.8% ± 3.5%, 33.2% ± 7.4%, and 30.1% ± 4.1% average transfection efficiencies, respectively. Electron microscopy reveals the intricacy of the cell–NN interactions; and immunofluorescence imaging demonstrates that enhanced endocytosis is one of the mechanisms of PS NN‐mediated intracellular delivery, involving the endocytic proteins caveolin‐1 and clathrin heavy chain. The results provide insights into the interfacial interactions between cells and polymeric NNs, and their related intracellular delivery mechanisms.

Substrate stiffness tunes the dynamics of polyvalent rolling motors.

Nature has evolved many mechanisms for achieving directed motion on the subcellular level. The burnt-bridges ratchet (BBR) is one mechanism used to achieve superdiffusive molecular motion over long distances through the successive cleavage of surface-bound energy-rich substrate sites. This mechanism has been associated with both nanoscale and microscale movement, with the latter accomplished through polyvalent interactions between a large hub (e.g. influenza virus) and substrate (e.g. cell surface receptors). Experimental successes in achieving superdiffusive motion by synthetic polyvalent BBRs have raised questions about the dynamics of their motility, including whether rolling or translation is better able to direct motion of microscale spherical hubs. Here we simulate the three-dimensional dynamics of a polyvalent sphere moving on and cleaving an elastic substrate. We find that substrate stiffness plays an important role in controlling both the motor's mode of motility and its directional persistence. As we tune lateral substrate stiffness from soft to stiff we find there exists an intermediate value that optimizes rolling behaviour. We also find that there is an optimal substrate stiffness for maximizing persistence length, while stiffness does not influence as strongly the superdiffusive dynamics of the particle. Lastly, we examine the effect of substrate density, and show that softer landscapes are better able to buffer against decreases in substrate occupancy, with the spherical motor maintaining superdiffusive motion more on softer landscapes than on stiff landscapes as occupancy drops. Our results highlight the importance of surface in controlling the motion of polyvalent BBRs.

The influence of substrate stiffness on osteogenesis of vascular smooth muscle cells

Vascular stiffening occurs with advanced age and under pathological conditions such as vascular calcification, during which the osteogenesis of smooth muscle cells (SMCs) plays a key role. However, whether the stiffness of cellular microenvironment influences osteogenic responses in vascular SMCs is not well understood. Here, we cultured SMCs on the poly(dimethylsiloxane) (PDMS) substrates with varying stiffness from 0.363 to 2.327 MPa. The cell osteogenic transdifferentiation was induced by β-glycerophosphate. Our findings demonstrated that the extent of osteogenesis in SMCs varied with the substrate stiffness. On three substrate stiffness, cells on the intermediate one (0.909 MPa) showed the highest extent of the osteogenesis based on the expression of osteogenic markers and calcium deposition. Transforming growth factor-β1 and autophagy were involved in this stiffness-dependent process. This work highlights the importance of substrate stiffness to the osteogenesis of vascular SMCs, giving new scientific information for understanding of SMCs-mediated vascular calcification and designing of vascular implants.

Computational insights into the influence of substrate stiffness on collective cell migration

Critically important biological phenomena in health and disease, such as wound healing, cancer metastasis, and embryonic development, are governed by collective cell migration. This highly complex process depends not only on cellular features, but also on different stimuli from the local cell environment. Cell migration is promoted by the combination of physico-chemical cues, including the mechanical properties of the extracellular matrix (ECM). Stiffness gradients within ECM have recently been demonstrated to result into preferred directions of cell migration. However, the specific mechanisms driving this directed collective cell migration and their relative roles remain unclear. Here, we develop a continuum formulation and its finite element (FE) implementation to test different hypotheses on the cause of spatial heterogeneities during cell migration on heterogeneous-stiffness substrates. We evaluate two key hypotheses: (i) cell polarisation is promoted by stiffness gradients within the ECM and; (ii) propulsion forces are weighted by ECM stiffness. Ultimately, we provide a robust in silico framework to explain experimental observations and guide future research.

Inkjet‐Printed Soft Resistive Pressure Sensor Patch for Wearable Electronics Applications

AbstractSoft pressure sensors may find a wide range of applications in soft robotics, biomedical devices, and smart wearables. Here, an inkjet‐printed resistive pressure sensor that offers high sensitivity and can be fabricated using a very simple process is reported. The device is composed of a conductive silver nanoparticle (AgNP) layer directly printed onto a polydimethylsiloxane substrate and encapsulated by a VHB tape. The pressure is measured through change in electrical resistance caused by pressure‐induced strain in the printed AgNP thin film. The influence of substrate stiffness and thickness on the sensitivity and achieved sensors with an optimized configuration that exhibit highly repeatable response with a sensitivity of up to 0.48 kPa−1 is systematically studied. It is further demonstrated that such a printed soft sensor patch is capable of measuring arterial pulse waveforms or detecting acoustic vibrations under various sound pressure levels. With its simple and low‐cost fabrication process and high sensitivity, the inkjet‐printed resistive pressure sensor is promising for future biomedical and smart wearable device applications.

Influence of substrate stiffness on human induced pluripotent stem cells: preliminary results.

Skeletal muscle differentiation was proven to be influenced by changes in the substrate stiffness. However, a lack of knowledge features this field, concerning skeletal muscle tissues obtained from human induced pluripotent stem cells. Here we report the fabrication of polydimethylsiloxane-based substrates in a range of stiffness values from 3.5 to 141 kPa and the response of human induced pluripotent stem cells cultured on them for 5 days. The substrates were able to sustain cell adhesion and proliferation throughout the whole period. An inversely proportional relationship (although not significant) was found between the proliferation rate and the substrate stiffness. Initial analyses of iPSCs skeletal muscle differentiation shown no influences on markers of the early stages. These results lay the foundations for further studies on the influence of extrinsic mechanical stimuli on induced pluripotent stem cells-derived skeletal muscle tissues.

The matrix: building more bioactive extracellular matrices via tuning of substrate stiffness

Background & Aim Mesenchymal stromal cells (MSCs) have long been at the forefront of cellular therapy and regenerative medicine, and they are being explored in more than 700 NIH-funded trials for their therapeutic potential. The low frequency of MSCs in the human body, poor yield of MSCs from tissue biopsies, and high number of MSCs required for clinical applications demand that tissue-isolated MSCs undergo a lengthy expansion ex vivo, during which they exhibit senescence and become unsuitable for therapeutic applications. Decellularized extracellular matrices (dECM) present a novel solution to this issue, as MSCs grown on dECM maintain greater viability as compared to those grown on conventional tissue culture substrates. The effect of environmental parameters on extracellular matrix bioactivity requires further elucidation. Particularly, we will investigate the influence of substrate stiffness on the composition of MSC-derived ECM, to produce customized dECM which enhances cellular growth and functionality. This is achieved by culturing MSCs on hydrogels of varying stiffnesses, and assessing the bioactivity of the ECM produced during culture. Methods, Results & Conclusion Mechanical properties of tunable stiffness polyacrylamide gels were verified using an Instron Microtester. Cells used were isolated from the decidua basalis, and ECM was sourced from decidua basalis hTERT-transfected MSCs. ECM deposition was induced by addition of ascorbic acid. Instron measurements confirm the elastic moduli of fully-hydrated polyacrylamide gels to be 4 kPa, 10 kPa, and 40 kPa, tailored to mimic the stiffness of adipose tissue, skeletal muscle tissue, and collagenous bone tissue, respectively. MSCs exhibit a spindle-shaped morphology when cultured on the hydrogels. Confocal microscopy reveals microarchitectural differences between ECM deposited on softer vs. stiffer hydrogels. We have shown that MSCs can be grown on hydrogels of varying stiffnesses, and can be induced to laydown extracellular matrix. The functionalization of the gels with collagen I to aid cell adhesion has been confirmed with immunofluorescence. The deposition of ECM has also been confirmed with confocal microscopy, and the alignment of ECM fibrils has been shown to depend on substrate stiffness. Further work will involve proteomic characterization of the dECMs, and culturing MSCs on the dECMs to assess bioactivity. We expect that the substrate stiffness will modulate the ECM bioactivity, allowing customization of tailored dECM for MSC expansion.

Inter‐ and intraspecific differences in leaf beetle attachment on rigid and compliant substrates

AbstractThe influence of substrate stiffness on the attachment ability of insects has been largely neglected so far. In the present study, traction experiments with adult beetles Gastrophysa viridula and Leptinotarsa decemlineata were carried out to study the influence of smooth, non‐structured surfaces, having different stiffness, on beetle attachment. Force measurements were performed with tethered walking adult insects, both males and females, intact and after removal of claws, on hydrophilic (normal) and hydrophobic (silanized) glass and four polydimethylsiloxane (PDMS) substrates ranging from 0.3 to 20 MPa in elastic modulus. Adult G. viridula generated higher safety factors (force/body weight) than L. decemlineata, 4.6–54.0 and 0.5–15.8 (min.–max.) respectively. The results show that the amputation of claws had no significant influence on the force generation by beetles on these smooth substrates. Males and females of both species performed best on stiffer surfaces having elastic moduli larger than 5 MPa. On the softer substrates, forces and safety factors significantly decreased. This effect was more prominent in L. decemlineata compared to the ten times lighter G. viridula. Since some natural substrates are rather soft, it is assumed that the effect of decrease in attachment ability of leaf beetles on soft substrata is of potential importance for the biology of leaf beetles.

Attachment and spatial organisation of human mesenchymal stem cells on poly(ethylene glycol) hydrogels.

Strategies that enable hydrogel substrates to support cell attachment typically incorporate either entire extracellular matrix proteins or synthetic peptide fragments such as the RGD (arginine-glycine-aspartic acid) motif. Previous studies have carefully analysed how material characteristics can affect single cell morphologies. However, the influence of substrate stiffness and ligand presentation on the spatial organisation of human mesenchymal stem cells (hMSCs) have not yet been examined. In this study, we assessed how hMSCs organise themselves on soft (E = 7.4-11.2 kPa) and stiff (E = 27.3-36.8 kPa) poly(ethylene glycol) (PEG) hydrogels with varying concentrations of RGD (0.05-2.5 mM). Our results indicate that hMSCs seeded on soft hydrogels clustered with reduced cell attachment and spreading area, irrespective of RGD concentration and isoform. On stiff hydrogels, in contrast, cells spread with high spatial coverage for RGD concentrations of 0.5 mM or higher. In conclusion, we identified that an interplay of hydrogel stiffness and the availability of cell attachment motifs are important factors in regulating hMSC organisation on PEG hydrogels. Understanding how cells initially interact and colonise the surface of this material is a fundamental prerequisite for the design of controlled platforms for tissue engineering and mechanobiology studies.

Influence of substrate stiffness on plasma membrane fluidity in rabbit tenocytes

Influence of substrate stiffness on plasma membrane fluidity in rabbit tenocytes

Influence of substrate stiffness and of PVD parameters on the microstructure and tension fracture characteristics of TiN thin films

Titanium nitride is widely used as wear resistant coating thin films in industrial parts. Mechanically they correspond to nanometric 2D ceramic systems which are bound to a much thicker metallic substrate. Under uniaxial tension these films respond by forming a periodic array of cracks. The separation between the cracks is primarily defined by the strength and stiffness of the film. Secondary factors, as the elastic properties of the substrate, or the microstructure and/or induction of residual stresses during processing may also have and effect. In the present work PVD TiN films were deposited by triode magnetron sputtering in Brass and Aluminum substrates. The deposition characteristics of the films were varied by changing the bias potential and the nitrogen supply during deposition (constant or variable). Tension fractures were observed in situ using a traveling microscope and under nanoindentation/scratching tests. Energy Filtered TEM was used to access the nitrogen levels across film thickness. The results were correlated with the film residual stress levels obtained in the different deposition conditions.

Surface pattern formation on soft polymer substrate through photo‐initiated graft polymerization

Techniques for large‐area pattern formation on polymeric substrates are important for fabricating a large variety of functional devices, such as flexible electronics, tunable optical devices, adhesives, and so on. The present study demonstrates a method for pattern formation on poly(dimethylsiloxane) that involves grafting methacrylate polymers through photo‐initiated polymerization. The influence of substrate stiffness and monomers type on pattern formation was investigated. Firstly, the stiffness of the substrate was found to affect the topology of the patterns produced. The gap width of convex regions of the pattern was enlarged with decreasing stiffness. It was found that the gap width trended in a manner that was consistent with previous reports, but in this study, relatively large gap widths were observed compared with those from previous studies. Secondly, it was revealed that the solubility of the monomer in the poly(dimethylsiloxane) precursor was the dominant factor in determining whether or not pattern formation occurred. When using insoluble monomers (glycidyl methacrylate and benzyl methacrylate), characteristic patterns were observed. It is speculated that intermolecular attractive forces between the grafted polymers induce lateral aggregation on the substrate, resulting in buckling instability of the grafted polymer layer caused by a mismatch in the equilibrium between the grafted polymer layer and the substrate. Copyright © 2017 John Wiley & Sons, Ltd.

Stress reduction mechanisms during photopolymerization of functionally graded polymer nanocomposite coatings

From the experimental analysis of the photocuring process in terms of reaction kinetics as well as modulus and shrinkage build-up, the residual stresses arising during the photopolymerization of functionally graded composite coatings based on an acrylate matrix and Fe3O4@SiO2 core@shell nanoparticles are evaluated through a Finite Element Modeling approach. Owing to the monotonous variation of volume fraction of the constituent phases that influences the local conversion of the polymeric matrix, these coatings are able to decrease the residual stresseS at the coating/substrate interface by as much as approximate to 25% compared to those encountered in composites with homogeneous compositions, and by as much as approximate to 40% compared to those arising in the pure polymer. The influence of substrate stiffness, nanoparticle stiffness and conversion degree of the polymer matrix was also analyzed, providing further information for the optimization of the stress reduction mechanism in graded nanocomposite coatings. (C) 2015 Elsevier B.V. All rights reserved.

The Influence of Substrate Stiffness on the Performance of a Magnetoelastic Bilayer Gas Flow Sensor

A bilayer sensor consisting of a stress sensitive magnetoelastic layer mounted onto a support substrate has been developed utilizing its change in curvature when subjected to gas flow forces. Two different materials, aluminium and HAVAR, with various thicknesses were used as the support layer to investigate the effect of bilayer stiffness on sensor sensitivity. Selection of the support layer was also important in determining the location of the neutral bending axis of the composite bilayer ensuring maximum stress sensitivity within the magnetic layer. Change in magnetic permeability proportional to stress was detected by measuring the inductance of a pickup coil positioned around one end of the bilayer sensor. The sensor was subjected to various airflow speeds and delivered consistent signal outputs for flow speeds up to 25 ms $^{\mathrm {\mathbf {-1}}}$ while proving to be extremely robust in turbulent conditions.

Influence of the PDMS substrate stiffness on the adhesion of Acanthamoeba castellanii.

Background: Mechanosensing of cells, particularly the cellular response to substrates with different elastic properties, has been discovered in recent years, but almost exclusively in mammalian cells. Much less attention has been paid to mechanosensing in other cell systems, such as in eukaryotic human pathogens.Results: We report here on the influence of substrate stiffness on the adhesion of the human pathogen Acanthamoebae castellanii (A. castellanii). By comparing the cell adhesion area of A. castellanii trophozoites on polydimethylsiloxane (PDMS) substrates with different Young’s moduli (4 kPa, 29 kPa, and 128 kPa), we find significant differences in cell adhesion area as a function of substrate stiffness. In particular, the cell adhesion area of A. castellanii increases with a decreasing Young’s modulus of the substrate.Conclusion: The dependence of A. castellanii adhesion on the elastic properties of the substrate is the first study suggesting a mechanosensory effect for a eukaryotic human pathogen. Interestingly, the main targets of A. castellanii infections in the human body are the eye and the brain, i.e., very soft environments. Thus, our study provides first hints towards the relevance of mechanical aspects for the pathogenicity of eukaryotic parasites.

Kinetic behaviour of the cells touching substrate: the interfacial stiffness guides cell spreading

To describe detailed behaviour of cell spreading under the influence of substrate stiffness, A549 cells cultured on the surfaces of polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) with bulk rigidities ranging from 0.1 kPa to 40 kPa were in situ observed. The spreading behaviour of cells on PAAm presented a positive correlation between spreading speed and substrate stiffness. After computing the deformations of PAAm gels and collagen, the bulk stiffness of PAAm, rather than matrix tethering, determined the cell behaviour. On the other hand, spreading behaviour of the cells was unaffected by varying the bulk stiffness of PDMS. Based on simulation analyses, the elasticity of silica-like layer induced by UV radiation on PDMS surface dominated cell-substrate interaction, rather than the bulk stiffness of the material, indicating that it is the interfacial stiffness that mainly guided the cell spreading. And then the kinetics of cell spreading was for the first time modeled based on absolute rate theory.

Influence of Substrate Stiffness and Thickness on Cell Traction Forces

It is known that various cell types can sense and respond to the mechanical properties of their microenvironment. Specifically, cells have been known to spread more when cultured on stiff substrates [1-3] and are able to match their internal stiffness to that of the substrate [2, 3]. Recent works have reported on dynamics of cellular properties such as cell shape, cell spread area, and focal adhesion area, as functions of environmental properties such as substrate stiffness, thickness, and chemistry. Building on earlier models [4, 5, 6], we present mathematical models that enable us to replicate some aspects of experimentally reported time-dependent cell behavior. Our models investigate the adaptation of internal cell stiffness through increase in number of focal adhesion complexes and temporal build-up of traction force. Our models crucially invoke the ability of some cell types to adapt their internal stiffness and show that substrate stiffness and thickness can strongly assist in rapid build-up of traction forces and formation of multiple cooperative focal adhesion complexes. Further using our models we generate some mechanistic insights into why certain cell types under the influence of specific substrate properties exhibit the kind of dynamics that has been experimentally reported. We attempt to establish correlation between the mechanistic models presented here and an earlier heuristic model that we have developed [6].Reference:1. R. J. Pelham, Jr., and Y. L. Wang, Proc. Natl. Acad. Sci. USA, 94, 13661 (1997).2. J. Solon et al, Biophys. J., 93, 4453 (2007).3. S. Tee et al, Biophys. J., 100, L25 (2011).4. U. S. Schwarz et al, Biosystems, 83, 225 (2006).5. J. M. Maloney et al, Phys. Rev. E, 78, 041923 (2008).6. S. Raghavan, A. R. Rammohan, and M. Hervy, Open J. Biophys, 3, (2013).