Focal adhesions (FAs) are mechanosensitive structures that mediate force transmission between cells and the extracellular matrix. While Traction Force Microscopy (TFM) quantifies cellular tractions exerted on deformable substrates, Förster Resonance Energy Transfer (FRET)-based tension probes, such as vinculin tension sensors, measure molecular-scale forces within FA proteins. Despite their potential synergy, these methods have rarely been combined to explore the interplay between molecular tension and cellular tractions. Here, we introduce a framework integrating TFM and FRET-based vinculin tension sensors to investigate FA mechanics across scales. At cell level, tractions and vinculin tension increased with substrate stiffness. At FA level, vinculin tension correlated solely with vinculin density, while tractions scaled with FA area, orientation, total vinculin content and vinculin density. Direct comparison of tractions to vinculin tension revealed a complex, heterogenous relationship between these forces, possibly linked to diverse cell and FA maturation states. Sub-FA analysis revealed conserved spatial patterns, with both tension and traction increasing towards the cell periphery. This multiscale approach provides an integrated workflow for studying focal adhesion forces, helping to bridge the gap between vinculin tension and cellular tractions.

Mechanical Memory and NF-κB Signaling in Dental Pulp Stem Cell Odontogenic Differentiation.

Adoptive T cell therapies (ACT) are an important class of oncology treatments that require ex vivo T cell expansion for clinical success. Technologies that can control both phenotype and yield in expanded cell products are highly desired. Here, we develop a new hydrogel scaffold for controlled T cell expansion with yields of up to 2000× fold in two weeks, compared to other hydrogel constructs (≈250×) and Dynabeads (≈1200×). Our 2D polyethylene glycol diacrylate (PEGDA) hydrogel scaffold is cross-linked with streptavidin moieties to present various biotinylated ligands to cells with controlled hydrogel stiffness (PEGDA-Strep). Using this platform, we demonstrate that combining substrate stiffness with adhesion receptor ligands (aLFA-1 or aCD2) dictates T cell activation and proliferation. On stiff substrates, these ligands drove expansions 49% (aLFA-1) and 68% (aCD2) greater than Dynabeads with comparable T cell products, preceded by elevated metabolic and transcriptional activity. Notably, while stiff substrates increased yield, soft substrates produced T cells with superior antigen-specific killing selectivity. These findings highlight the role of mechanical sensing in T cell-APC interactions and suggest improved manufacturing methods for adoptive T cell therapy (ACT).

Cancer cells adhere to the extracellular matrix, where they sense and respond to variations in substrate stiffness, influencing their proliferation and invasive potential. Numerous studies have examined the biological activities of cells in relation to mechanical forces; however, research addressing the combined effects of mechanical and chemical interactions on cancer cell behavior across different metastatic stages remains limited. Moreover, the influence of chemotherapeutic drugs in the context of specific cellular characteristics remains underexplored. Therefore, in this study, synthetic polyacrylamide gels with varying elastic moduli were utilized to effectively mimic the diversity of host tissue environments for prostate cancer cells. Additionally, cellular behavior of prostate cancer cells with differing metastatic potential—low (LNCaP), medium (DU145), and high (PC3)—was evaluated in response to anticancer drugs. Ultimately, effects of drug treatment were comprehensively examined using Docetaxel, Bicalutamide, and Abiraterone Acetate, which target distinct cellular components and activate diverse signaling pathways. The assessments were based on the analysis of actin filament content and organization, size of nucleus, and cellular elastic modulus. The results revealed that a soft substrate improves the medication efficacy, resulting in an enhanced cell death rate of 40–60% compared to 20–30% on a stiff substrate. Cells cultured on soft substrates exhibited lower phalloidin content (8–16%) compared to those on stiff substrates (18–32%). Additionally, drug treatments influenced cell mechanics, with Docetaxel reducing the elastic modulus, while Bicalutamide induced an increase. Based on these findings, a treatment strategy aimed at enhancing therapeutic efficacy can be proposed.

The blood–brain barrier (BBB) is a mechanically responsive interface that protects the central nervous system. Brain tissue exhibits region-specific stiffness that evolves throughout development and is altered in aging and various neurological diseases. These stiffness changes are increasingly recognized as key modulators of endothelial cell behavior and BBB integrity. However, the mechanisms by which brain endothelial cells sense and adapt to variations in their mechanical microenvironment remain poorly defined. Moreover, how mechanical cues interact with cellular signals from astrocytes and pericytes to modulate endothelial mechanics and junctional organization has been largely unexplored. Here, we demonstrate spatial regulation of subcellular mechanics in human iPSC-derived brain microvascular endothelial cells (iBMECs) in response to physiologically and pathologically relevant substrate stiffness (1–194 kPa). Using atomic force microscopy, we quantified Young’s modulus at three distinct cellular regions—tricellular junctions, bicellular junctions, and cell bodies. iBMECs cultured on compliant substrates (1, 2.5, and 15 kPa) exhibited pronounced mechanical polarization, characterized by significantly elevated stiffness at tricellular regions compared with bicellular regions and cell bodies. This spatial organization was lost on supraphysiological stiffness (194 kPa), which reduced overall cell stiffness and eliminated regional distinctions. Co-culture with astrocytes and pericytes decreased global stiffness but preserved the dominant reinforcement at tricellular regions. In contrast, exposure to metastatic breast cancer cells abolished junction polarization at tricellular regions and suppressed stiffness across all regions, particularly on soft substrates. These findings reveal that BBB endothelial mechanics are regulated by both matrix stiffness and BBB cell context in a region-specific manner. This work provides new insight into how physical and cellular cues shape BBB structure and function, with implications for understanding barrier disruption in neurological disease and metastasis.

Transient Receptor Potential Vanilloid 4 in Macrophages Mediates TGF-β Activation to Drive Myofibroblast Differentiation and Pulmonary Fibrosis.

Integrin β1 mediates mechanosensitive regulation of human trabecular meshwork cell functions in response to substrate stiffness.

Substrate stiffness attenuates cardiomyocyte depolarization slope via sodium channel kinetics modulation.

Biophysical cues of edible microcarrier scaffolds regulate muscle satellite cell behavior and nutrient deposition in Larimichthys crocea.

Matrix stiffening-driven hepatocellular carcinoma progression through OASL-mediated cGAS-STING repression and subsequent macrophage activation.

Mechanical memory of cells to fluid shear stress and substrate stiffness revealed by atomic force microscopy.

BackgroundDuring the development of idiopathic pulmonary fibrosis (IPF), elastin is broken down and replaced with a stiffer substrate which interferes with normal breathing. This remodeling releases elastin degradation products (EDPs), or elastokines, which can then be measured. We examined the association between elastokine concentrations and disease severity to determine if EDP concentrations are associated with clinically relevant markers in IPF.MethodsConcentrations of elastokines were measured via enzyme linked immunosorbent assay (ELISA). Samples were obtained from a single institution’s Interstitial Lung Disease (ILD) registry/biorepository (n = 81 with IPF and 24 healthy volunteers). We used linear and logistic regression modeling to assess the association between EDP concentrations at the time of diagnosis, lung function, and clinical outcomes.ResultsPatients with IPF were older, more likely to be male, had ever smoked, and had worse lung function compared to healthy volunteers (p value ≤ 0.02 for all parameters). Patients with IPF had higher concentrations of elasotkines (p < 0.001), and the highest elastokine concentrations were associated with reduced forced vital capacity (FVC, p = 0.026), and decreased three-year transplant-free survival (p = 0.0005). These findings suggest that elastokines are biomarkers of matrix turnover and are associated with relevant clinical outcomes in patients with IPF.

Stiffening of the extracellular matrix underlying the epithelial cells of the large intestine is associated with aging as well as many diseases. Yet the impact of the stiffened matrix on epithelial physiology remains poorly understood. A 2D and 3D microphysiological model of the large intestine was developed using a collagen scaffold with a physiologic or excessive stiffness (Young's moduli of 2.84 ± 0.85 kPa and 15.9 ± 0.73 kPa) by altering the collagen concentration within the substrate. Diffusion of a 10 and 40 kDa fluorescent dextran was significantly different between the physiologic and stiff scaffold (97.8 vs 79.8µm2s-1[10 kDa] and 68.2 vs 56.8µm2s-1[40 kDa], respectively). When primary human epithelial cells of the large intestine were grown as a 2D monolayer, cultures on the physiologic scaffold grew to a significantly higher density with more proliferative and fewer differentiated cells than cultures on the stiffened scaffold. Three-dimensional crypt arrays were also fabricated with the physiologic and stiff substrates, populated with cells, and a growth factor gradient applied. The cell density, proliferation, and height-to-width ratio was significantly greater for cells on the physiologic scaffold relative to that of cells on the stiffened scaffolds. Placement of a layer of intestinal fibroblasts below the epithelium on the crypt arrays did not mitigate the impact of the stiffened substrate. Bulk-RNA sequencing revealed 378 genes that were significantly upregulated and 385 genes significantly downregulated in the stiffened vs physiologic scaffolds. This work demonstrates that a molded collagen hydrogel can be used to mimic the biophysical characteristics of a stiffened intestinal stroma, recapitulating physiology observedin vivo. Thisin vitromodel of polarized crypts with a tunable underlying substrate will enable an improved understanding of intestinal epithelial cell morphology, stem cell maintenance and lineage allocation within a stiffened environment.

Piconewton (pN) receptor forces govern many biological processes, but measuring these forces remains challenging. Molecular tension probes (MTPs) provide a sensitive means to measure pN cellular forces via fluorescence microscopy; however, MTPs are challenging to use and only forces transmitted through the probes are reported, complicating their use in heterogenous environments. Here, we present Tension Deep Learning (TensionDL), which leverages convolutional neural networks and image-to-image translation to predict pN receptor tension maps from images of cell morphology and the force-transducing protein vinculin. We validate the accuracy of TensionDL at the subcellular and cellular scales, demonstrate model accuracy across different substrate stiffnesses and cell types, and leverage TensionDL to make semi-quantitative predictions of cell mechanical output. Finally, TensionDL enables long-term mapping of pN receptor tension and infers tension distributions in heterogeneous environments in which some forces are not transduced through MTPs.

Bone fractures often involve both bone and muscle damage, representing a critical need for regenerative strategies that can repair both tissues. We developed a gelatin-based hydrogel system with tunable stiffness to guide spheroids formed from induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) for simultaneous muscle and bone repair. Hydrogels were formed using hydrogen peroxide with horseradish peroxidase as a catalyst and substrate stiffness ranged from 1.6 to 9.3 kPa. Hydrogels entrapping iMSC spheroids enabled localized release of the iMSC secretome that exhibited biological effects. iMSC spheroids in soft hydrogels enhanced myogenic differentiation of C2C12 myoblasts via paracrine signaling. Conversely, iMSC spheroids exposed to the myoblast secretome and cultured in stiffer hydrogels exhibited enhanced osteogenic differentiation. This study demonstrates the synergistic influence of stiffness and reciprocal secretome signaling on iMSC behavior, offering a clinically relevant "two-in-one" platform for musculoskeletal regeneration.

Manufacturing clinical grade mesenchymal stromal cells (MSCs) remains a major bottleneck for cell-based therapies, as extensive in-vitro expansion on standard tissue-culture plastic (TCP) drives loss of stemness, reduced immunomodulatory activity, and diminished therapeutic efficacy. Although substrate stiffness is known to influence MSC fate through mechanotransduction, the epigenetic mechanisms linking mechanical stress to progressive phenotypic drift remain poorly defined. A mechano-epigenetic pathway is identified in this work, centered on the histone methyltransferase EZH2 - that governs chromatin remodeling and loss of stemness during serial passaging. Multi-omics, high-resolution imaging, and functional assays show that MSCs expanded on mechanically stiff TCP accumulate H3K27me3 repressive chromatin mark, lose SWI/SNF -ARID1A chromatin-remodeling foci, and exhibit an altered chromatin accessibility profile.Pharmacological inhibition of EZH2 with GSK343 selectively reduced H3K27me3, restored ARID1A-containing SWI/SNF organization, and preserved MSC morphology and expression of canonical stemness markers (CD73, CD90, CD105) even at later passage. ATAC-seq analysis revealed that GSK343 rebalanced chromatin accessibility, reopening TEAD/YAP-responsive regulatory regions while repressing accessibility at lineage-priming and senescence-associated sites. RNA-seq demonstrated that GSK343 maintained transcriptional programs associated with immunomodulation, migration, and trophic signaling, while suppressing hyperproliferative and senescence-associated genes that are characteristic of late-passage MSCs. Proteomic profiling of MSC secretomes further showed that GSK343 attenuated pro-fibrotic ECM factors and senescence-linked proteins while enhancing angiogenic and reparative mediators. Functionally, conditioned media from GSK343-treated MSCs significantly increased primary chondrocyte proliferation, demonstrating preserved therapeutic potency.Together, these findings establish EZH2 as a central mediator of stiffness-induced epigenetic drift in human MSCs and demonstrate that EZH2 inhibition can maintain the stemness without compromising their expansion ability. This work provides a foundational strategy for mechano-epigenetic engineering of MSCs and highlights EZH2 inhibition as a scalable, manufacturing-compatible approach to preserve potency for regenerative medicine applications.

SummaryBreast cancer progression is strongly influenced by the mechanical properties of the tumor microenvironment, yet how extracellular matrix stiffness coordinates signaling between YAP and β-catenin remains unclear. Using breast cancer cells cultured on soft and stiff 2D substrates and 3D Matrigel spheroids, we show that the rigid matrices drive joint nuclear localization of YAP and β-catenin, whereas compliant environments reveal a compensatory increase in β-catenin nuclear entry following YAP depletion. However, this increase is insufficient to activate canonical Wnt targets, and AXIN2 emerges as a stiffness-sensitive gene requiring cooperative input from both regulators. Cytoskeletal tension and cell density further tune this interplay, indicating that mechanical and architectural cues jointly govern nuclear signaling. In 3D culture, YAP loss reduces β-catenin nuclear localization and spheroid viability in a stiffness-dependent manner. These findings identify a mechanically gated YAP-β-catenin axis that integrates multiple microenvironmental cues to shape transcriptional programs in metastatic breast cancer.

Abstract Contact printing, calendaring, and coating of packaging paperboard are standard industry processes that utilize rolling nips. The pressure pulse, maximum pressure, and duration have been extensively studied regarding its effect on the substrate and how it can be changed and controlled to achieve the desired effect. The present study considers lateral variations of the stress in a rolling process. A parametric study of the surface roughness, substrate stiffness, cylinder cover stiffness, and changed nip engagement or impression is performed using Finite Element Modelling. The simulation shows that a smooth surface does not completely negate the effects of the structural thickness. The impression has the most significant impact, and the combination of roughness and non-linear material means that the pressure distribution can change drastically, not just the maximum pressure pulse. Additionally, different combinations of settings can achieve the same mean pressure pulse but have very different stress distributions. E.g. changing the surface roughness will have a significant effect on the pressure variations, but the effect on the pressure profile shape is negligible.

Purpose:Fuchs’ Endothelial Corneal Dystrophy (FECD), a degenerative corneal disorder, is marked by the thickening of Descemet’s membrane and a progressive loss of corneal endothelial cells, ultimately leading to vision loss. A feature associated with the disease is the reduced stiffness of Descemet’s membrane. However, the effects of this change in Descemet’s membrane, on corneal endothelial cell health are not well understood. To explore this, we used in vitro, in vivo, and ex vivo studies to investigate how changes in substrate stiffness and the signaling pathways associated with these changes influence corneal endothelial functions.Methods:For in-vitro studies, we cultured bovine corneal endothelial cells for 96 hours on stiff (32 kPa) and soft (8 kPa) substrate CytoSoft plates. By using Jess immunoassay and traditional western blotting, we evaluated changes in integrin signaling components, endothelial-to-mesenchymal transition, apoptosis, autophagy, and ubiquitin-proteasome pathway markers. Mitochondrial health and mitochondrial superoxide levels were assessed using commercial kits. We assessed the protein levels of the above-mentioned markers in the Col8a2Q455K/Q455K FECD mouse model. To evaluate whether FAK signaling contributes to the FECD pathogenesis, we injected the Col8a2Q455K/Q455K mice with an FAK inhibitor and assessed the corneal phenotypes.Results:We observed increased levels of phosphorylated FAK, integrins α4 and α5 in bovine corneal endothelial cells cultured on soft substrate. We also found upregulated endothelial-to-mesenchymal transition (EndMT) markers, mitochondrial dysfunction, and apoptosis in cells grown on soft substrate. In the Col8a2Q455K/Q455K mouse model of FECD, there was increased pFAK Y397 levels coincident with the onset of phenotypes. Intraperitoneal injections of a pFAK inhibitor improved antioxidant protein expression and decreased EndMT; however, it did not improve FECD-associated disease progression.Conclusion:In this study, we explored how changes in the physical characteristics of the Descemet’s membrane impact corneal endothelial cell health. While we discovered activation of Focal adhesion kinase as a result of stiffness changes, its inhibition alone was insufficient to improve cell health in an FECD mouse model.

Decoding vascular aging: Substrate stiffness and shear stress orchestrate endothelial inflammation and remodelling via mechanosensitive pathways.