Tuneable Stiffness Research Articles

Substrate stiffness promotes vascular smooth muscle cell calcification by reducing the levels of nuclear actin monomers

BackgroundVascular calcification (VC) is a prevalent independent risk factor for adverse cardiovascular events and is associated with diabetes, hypertension, chronic kidney disease, and atherosclerosis. However, the mechanisms regulating the osteogenic differentiation of vascular smooth muscle cells (VSMC) are not fully understood. MethodsUsing hydrogels of tuneable stiffness and lysyl oxidase-mediated stiffening of human saphenous vein ex vivo, we investigated the role of substrate stiffness in the regulation of VSMC calcification. ResultsWe demonstrate that increased substrate stiffness enhances VSMC osteogenic differentiation and VSMC calcification. We show that the effects of substrate stiffness are mediated via a reduction in the level of actin monomer within the nucleus. We show that in cells interacting with soft substrate, elevated levels of nuclear actin monomer repress osteogenic differentiation and calcification by repressing YAP-mediated activation of both TEA Domain transcription factor (TEAD) and RUNX Family Transcription factor 2 (RUNX2). ConclusionThis work highlights for the first time the role of nuclear actin in mediating substrate stiffness-dependent VSMC calcification and the dual role of YAP-TEAD and YAP-RUNX2 transcriptional complexes.

TMIC-80. PERK CONTRIBUTES TO GLIOBLASTOMA PROGRESSION BY REGULATING GLIOBLASTOMA STEM CELL ADAPTATION TO EXTRA CELLULAR MATRIX STIFFENING THROUGH F-ACTIN REMODELING AND FOCAL ADHESION COMPLEX REGULATION

Abstract The association between a stiffening extracellular matrix (ECM) and increased aggressiveness of gliomas is well established. Originally recognized as a protective mechanism against endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) is primarily responsible for maintaining cellular protein homeostasis and triggering cell death under severe stress conditions. However, emerging evidence indicates that the ER transmembrane UPR sensors, IRE1 and PERK, possess additional functions beyond proteostasis, such as modulating cancer stem cell differentiation, cell adhesion, and migration, which also involve more recently identified protein scaffold functions. In this study, we uncover a novel role of PERK in cellular adaptation to matrix stiffening. Glioblastoma stem cells (GSCs), when cultured on human blood plasma/alginate hydrogels with tuneable stiffness, exhibited stiffness-dependent cellular responses, including cell flattening, differentiation, and enhanced migration. Through genetic depletion and pharmacological inhibition strategies combined with immunofluorescence microscopy, we identified the involvement of a previously found PERK/FLNA signaling mechanism in the adaptive response to matrix stiffness, where F-actin polymerization emerged as a key mechanism. To explore the possible involvement of PERK in sensing ECM stiffness, the expression of various proteins that constitute the focal adhesion complex were studied. Remarkably, PERK also influenced the maturation of focal adhesion complexes components know to be involved in sensing ECM stiffness. Finally, comparative transcriptional profiling of PERK proficient and deficient GSCs uncovered significant alterations in the expression of genes associated with cell adhesion and extracellular matrix (ECM)-related processes. These findings reveal a novel function of PERK in stiffness-dependent cellular adaptations, with ongoing investigations exploring the underlying molecular mechanisms and therapeutic implications for glioblastoma progression

Static and dynamic instability of functionally graded graphene origami-enabled auxetic metamaterial beams with variable thickness in fluid

Functionally graded (FG) graphene origami (GOri)-enabled auxetic metamaterial (GOEAM) structures have shown great potential for various engineering applications due to their exceptional mechanical and physical properties such as high strength-to-weight ratio, tuneable stiffness and strength, and negative Poisson's ratio (NPR). This paper aims to investigate the buckling and dynamic instability behaviours of FG-GOEAM beams with variable thickness immersed in a fluid, with a particular focus on the influence of NPR. The material properties such as Young's modulus and Poisson's ratio of the GOEAM are determined by using a genetic programming (GP)-based micromechanics model. Within the framework of the first-order shear deformation theory and by employing Hamilton's principle and modelling the fluid effect as added mass, the governing equations of motion are established and are then discretised by means of differential quadrature (DQ) method to obtain a linear system of Mathieu-Hill equations from which the principal instability regions of the FG-GOEAM beam are determined by Bolotin's method. A comprehensive parametric study is conducted to reveal the effects of GOri's folding degree, distribution, weight fraction, as well as fluid density and beam dimensions on the static and dynamic instability behaviours of the FG-GOEAM beam immersed in fluid. Numerical results show that the FG-GOEAM beam with NPR considerably outperforms its pristine metallic counterpart in terms of resistance against static buckling and dynamic stability.

Design and analysis of an auxetic metamaterial with tuneable stiffness

Auxetic materials have attracted a considerable attention due to their excellent properties, e.g., fracture resistance, shear resistance, energy dissipation, etc. However, the stiffness of auxetics tends to be much weaker than solid structure because of the existence of internal holes. Inspired by tuning the compacted point of auxetic structures to enhance their stiffness, a systematic methodology for defining a single parametric of variable stiffness scale factor (VSF) to generate auxetic unit cell with variable stiffness has been proposed and verified in this study. Two models with different VSF proportions were investigated experimentally. Different centres of rotation, heights of deformation area, and VSF percentages were analyzed to prove the effectiveness of the method numerically. The results indicate that the compacted strain can be tuned effectively using the designed VSF proportions, and the difference between the designed VSF and real VSF could be reduced by slightly changing the height of deformation area. These desirable characteristics provide a new idea for the optimal design of auxetics, with potential application in protective structures.

Frequency-Adaptable Tuned Mass Damper Using Metal Cushions

A frequency-adaptable tuned mass damper (FATMD) using metal cushions as tuneable stiffness components is presented. The dynamic properties of the cushions with respect to stiffness and damping are investigated experimentally in this context. The natural frequency of the experimental FATMD is found to be dependent on the precompression of the metal cushions, which behave like nonlinear springs, yielding an adjustable frequency range from 67 to 826 Hz. As the precompression is increased, the stiffness increases while the damping characteristics decrease, the effect of which was quantified using a viscous mass damper model as a first approximation. Measurements have been carried out under five different excitation amplitudes to investigate the amplitude dependency of the resonance frequency. The FATMD was largely unaffected by changes in input amplitude. It was concluded that metal cushions show great potential for use in FATMDs, surpassing the utility of elastomers, especially with respect to their temperature stability.

Stiffness memory nanohybrid scaffolds generated by indirect 3D printing for biologically responsive soft implants

Cell and tissue stiffness is an important biomechanical signalling parameter for dynamic biological processes; responsive polymeric materials conferring responsive functionality are therefore appealing for in vivo implants. We have developed thermoresponsive poly(urea-urethane) nanohybrid scaffolds with ‘stiffness memory’ through a versatile 3D printing-guided thermally induced phase separation (3D-TIPS) technique. 3D-TIPS, a combination of 3D printing with phase separation, allows uniform phase-separation and phase transition of the polymer solution at a large interface of network within the printed sacrificial preform, leading to the creation of full-scale scaffolds with bespoke anatomical complex geometry. A wide range of hyperelastic mechanical properties of the soft elastomer scaffolds with interconnected pores at multi-scale, controlled porosity and crystallinity have been manufactured, not previously achievable via direct printing techniques or phase-separation alone. Semi-crystalline polymeric reverse self-assembly to a ground-stated quasi-random nanophase structure, throughout a hierarchical structure of internal pores, contributes to gradual stiffness relaxation during in vitro cell culture with minimal changes to shape. This ‘stiffness memory’ provides initial mechanical support to surrounding tissues before gradually softening to a better mechanical match, raising hopes for personalized and biologically responsive soft tissue implants which promote human fibroblast cells growth as model and potential scaffold tissue integration. Statement of SignificanceBiological processes are dynamic in nature, however current medical implants are often stronger and stiffer than the surrounding tissue, with little adaptability in response to biological and physical stimuli. This work has contributed to the development of a range of thermoresponsive nanohybrid elastomer scaffolds, with tuneable stiffness and hierarchically interconnected porous structure, manufactured by a versatile indirect 3D printing technique. For the first time, stiffness memory of the scaffold was observed to be driven by phase transition and a reverse self-assembly from a semicrystalline phase to a quasi-random nanostructured rubber phase. Early insight into cell response during the stiffness relaxation of the scaffolds in vitro holds promise for personalized biologically responsive soft implants.

Engineered Caf1 Protein Polymers form Tuneable Bioactive Hydrogel Scaffolds

Here, we describe a new animal-free biomaterial with potential uses in 3D-tissue culture and regenerative medicine due to its low cost, high stability and definable bioactivity. Capsular antigen fraction1 (Caf1) is a protein from the plague bacterium Yersinia pestis that is secreted via the chaperone-usher pathway and protects the pathogen from phagocytosis by forming a non-stick protective layer around the cell. The 15.5kDa monomer has an Ig-like fold and resembles the extracellular matrix protein fibronectin. The subunits polymerise via donor-strand complementation, forming a highly stable non-covalent polymer. In this work, we demonstrate the production of recombinant Caf1 polymers via batch fermentation using Escherichia coli. These are secreted by the bacterium into a flocculent layer above the cell pellet, and can be easily extracted and purified in large quantities. We show the polymer retains its robust thermostability in a variety of chemical conditions by circular dichroism and SDS-PAGE, and observe the large size of polymers by electron microscopy and SEC-MALS. This analysis revealed polymers containing up to 250 subunits, with lengths >1.5 µm and weights in the MDa range. Additionally, we have selectively reversed the natural non-stick behaviour of the WT polymer by introducing an integrin binding sequence, RGDS, into loop5 that can promote fibroblast adhesion to the polymer surface. Additional bioactive peptides (up to 19 amino acids in length), including bone morphogenic protein2, collagen and laminin motifs, were then introduced at different positions within Caf1. Finally, PEG based chemical cross linkers were used to form stable 3D hydrogels with designed porosities and tuneable stiffness, ideal for use in cell culture and drug delivery applications. The combination of these motifs into tuneable Caf1 hydrogels will help expand the functionality of this exciting new biomaterial for use in a variety of biomedical applications.

Photoresponsive Hydrogels with Photoswitchable Stiffness: Emerging Platforms to Study Temporal Aspects of Mesenchymal Stem Cell Responses to Extracellular Stiffness Regulation.

An extensive number of cell-matrix interaction studies have identified matrix stiffness as a potent regulator of cellular properties and behaviours. Perhaps most notably, matrix stiffness has been demonstrated to regulate mesenchymal stem cell (MSC) phenotype and lineage commitment. Given the therapeutic potential for MSCs in regenerative medicine, significant efforts have been made to understand the molecular mechanisms involved in stiffness regulation. These efforts have predominantly focused on using stiffness-defined polyacrylamide (PA) hydrogels to culture cells in 2D and have enabled elucidation of a number of mechano-sensitive signalling pathways. However, despite proving to be a valuable tool, these stiffness-defined hydrogels do not reflect the dynamic nature of living tissues, which are subject to continuous remodelling during processes such as development, ageing, disease and regeneration. Therefore, in order to study temporal aspects of stiffness regulation, researchers have developed and exploited novel hydrogel substrates with in situ tuneable stiffness. In particular, photoresponsive hydrogels with photoswitchable stiffness are emerging as exciting platforms to study MSC stiffness regulation. This chapter provides an introduction to the use of PA hydrogel substrates, the molecular mechanisms of mechanotransduction currently under investigation and the development of these emerging photoresponsive hydrogel platforms.

Polyethylene glycol-gelatin hydrogels with tuneable stiffness prepared by horseradish peroxidase-activated tetrazine-norbornene ligation.

Tetrazine-norbornene ligation has previously been applied in bioorthognal polymer crosslinking to form hydrogels suitable for 3D cell culture. However, the tetrazine group is prone to reduction by the free thiol in a biological environment, reducing the crosslinking efficiency and shortening the storage of tetrazine containing linkers. Here, we introduce a method to form a tetrazine group in situ by catalytic oxidation of the dihydrogen tetrazine using horse radish peroxidase (HRP). Enzymatic oxidation is highly efficient at a low HRP concentration and does not require hydrogen peroxide, allowing for rapid gelation when HRP was added to an aqueous solution of 4-arm PEG dihydrogentetrazine and gelatin norbornene. The storage modulus of the resultant gels can be varied by changing the concentration of the crosslinker, which is in the range of 1.2-3.8 kPa. Human mesenchymal stem cells encapsulated within these gels, with varying stiffness, display varied interactions and morphologies and can be maintained with prolonged culture periods of at least 32 days of 3D culture. The enzymatic activation of tetrazine-norbornene is therefore an attractive addition to the tetrazine ligation that is highly suitable for cell related studies in tissue engineering.

A Geometry Deformation Model for Braided Continuum Manipulators

Continuum manipulators have gained significant attention in the robotic community due to their high dexterity, deformability and reachability. Modeling of such manipulators has been shown to be very complex and challenging. Despite many research attempts, a general and comprehensive modeling method is yet to be established. In this paper, for the first time, we introduce the bending effect in the model of a braided extensile pneumatic actuator with both stiff and bendable threads. Then, the effect of the manipulator cross section deformation on the constant curvature and variable curvature models is investigated using simple analytical results from a novel geometry deformation method and is compared to experimental results. We achieve 24% simulation accuracy using our constant curvature model for a braided continuum manipulator in presence of body load and 10% error using our variable curvature model in presence of extensive external loads. With proper model assumptions and taking to account the cross section deformation, an 8-20% increase in the simulation accuracy is achieved compared to a fixed cross section model. The presented models can be used for the exact modeling and design optimization of compound continuum manipulators by providing an analytical tool for the sensitivity analysis of the manipulator performance. Our main aim is the application in minimal invasive manipulation with limited workspaces and manipulators with regional tuneable stiffness in their cross section.

In vitro model to study the effects of matrix stiffening on Ca2+ handling and myofilament function in isolated adult rat cardiomyocytes

Key points This paper describes a novel model that allows exploration of matrix‐induced cardiomyocyte adaptations independent of the passive effect of matrix rigidity on cardiomyocyte function.Detachment of adult cardiomyocytes from the matrix enables the study of matrix effects on cell shortening, Ca2+ handling and myofilament function.Cell shortening and Ca2+ handling are altered in cardiomyocytes cultured for 24 h on a stiff matrix.Matrix stiffness‐impaired cardiomyocyte contractility is reversed upon normalization of extracellular stiffness.Matrix stiffness‐induced reduction in unloaded shortening is more pronounced in cardiomyocytes isolated from obese ZSF1 rats with heart failure with preserved ejection fraction compared to lean ZSF1 rats. Extracellular matrix (ECM) stiffening is a key element of cardiac disease. Increased rigidity of the ECM passively inhibits cardiac contraction, but if and how matrix stiffening also actively alters cardiomyocyte contractility is incompletely understood. In vitro models designed to study cardiomyocyte–matrix interaction lack the possibility to separate passive inhibition by a stiff matrix from active matrix‐induced alterations of cardiomyocyte properties. Here we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to matrix stiffening. Adult rat cardiomyocytes were cultured for 24 h on matrices of tuneable stiffness representing the healthy and the diseased heart and detached from their matrix before functional measurements. We demonstrate that matrix stiffening, independent of passive inhibition, reduces cell shortening and Ca2+ handling but does not alter myofilament‐generated force. Additionally, detachment of adult cultured cardiomyocytes allowed the transfer of cells from one matrix to another. This revealed that stiffness‐induced cardiomyocyte changes are reversed when matrix stiffness is normalized. These matrix stiffness‐induced changes in cardiomyocyte function could not be explained by adaptation in the microtubules. Additionally, cardiomyocytes isolated from stiff hearts of the obese ZSF1 rat model of heart failure with preserved ejection fraction show more pronounced reduction in unloaded shortening in response to matrix stiffening. Taken together, we introduce a method that allows evaluation of the influence of ECM properties on cardiomyocyte function separate from the passive inhibitory component of a stiff matrix. As such, it adds an important and physiologically relevant tool to investigate the functional consequences of cardiomyocyte–matrix interactions.

On the Free Vibration Modeling of Spindle Systems: A Calibrated Dynamic Stiffness Matrix

The effect of bearings on the vibrational behavior of machine tool spindles is investigated. This is done through the development of a calibrated dynamic stiffness matrix (CDSM) method, where the bearings flexibility is represented by massless linear spring elements with tuneable stiffness. A dedicated MATLAB code is written to develop and to assemble the element stiffness matrices for the system’s multiple components and to apply the boundary conditions. The developed method is applied to an illustrative example of spindle system. When the spindle bearings are modeled as simply supported boundary conditions, the DSM model results in a fundamental frequency much higher than the system’s nominal value. The simply supported boundary conditions are then replaced by linear spring elements, and the spring constants are adjusted such that the resulting calibrated CDSM model leads to the nominal fundamental frequency of the spindle system. The spindle frequency results are also validated against the experimental data. The proposed method can be effectively applied to predict the vibration characteristics of spindle systems supported by bearings.

Magnetorheological brush – a soft structure with highly tuneable stiffness

By combining the field-stiffening effect of magnetorheological (MR) elastomers and the Euler buckling mechanism, we developed a brush-like magneto-active structure with highly tuneable stiffness. When the applied mechanical load is within a certain range, the effective stiffness of the structure can be tuned by several orders of magnitude with the applied magnetic field. The performance of the structure and its dependence on various synthesis parameters, such as the curing field and filler concentration, were investigated experimentally. It is found that the increase in the critical load for buckling is more than the contribution from the stiffening of the MR elastomer. To unravel the relationship between the stiffness increase and the applied field, a theoretical model with coupled mechanical deformation and magnetic field is established. The prediction of the model agrees well with experimental results. The theory may also be used to model the behaviour of other similar materials, such as MR gels. The MR brush developed in this research holds promise for potential applications in smart structures or devices that require mechanical stiffness to be tuneable in a relatively large range. As the amplification mechanism is independent of the base material, it could be used in conjunction with emerging MR materials for further enhanced performance.