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Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such interactions can lead to the modulation of cellular activity. Collagen is often used in the culture of immune cells, but the effects of substrate functionalization conditions are not typically considered. Here, we show that the solvent system used to attach collagen onto a hydrogel surface affects its surface distribution and organization, and this can modulate the responses of macrophages subsequently cultured on these surfaces in terms of their inflammatory activation and expression of adhesion and mechanosensitive molecules. Collagen was solubilized in either acetic acid (Col-AA) or N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) (Col-HEP) solutions and conjugated onto soft and stiff polyacrylamide (PA) hydrogel surfaces. Bone marrow-derived macrophages cultured under standard conditions (pH 7.4) on the Col-HEP-derived surfaces exhibited stiffness-dependent inflammatory activation; in contrast, the macrophages cultured on Col-AA-derived surfaces expressed high levels of inflammatory cytokines and genes, irrespective of the hydrogel stiffness. Among the collagen receptors that were examined, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) was the most highly expressed, and knockdown of the Lair-1 gene enhanced the secretion of inflammatory cytokines. We found that the collagen distribution was more homogeneous on Col-AA surfaces but formed aggregates on Col-HEP surfaces. The macrophages cultured on Col-AA PA hydrogels were more evenly spread, expressed higher levels of vinculin, and exerted higher traction forces compared to those of cells on Col-HEP. These macrophages on Col-AA also had higher nuclear-to-cytoplasmic ratios of yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), key molecules that control inflammation and sense substrate stiffness. Our results highlight that seemingly slight variations in substrate deposition for immunobiology studies can alter critical immune responses, and this is important to elucidate in the broader context of immunomodulatory biomaterial design.

Chondrocytes, the resident cells of articular cartilage, play a vital role in tissue maintenance and regeneration. Recent studies have highlighted chondrocyte senescence as a key contributor to cartilage dysfunction. In this study, we investigated the influence of substrate stiffness on chondrocyte senescence using polydimethylsiloxane (PDMS) substrates with varying stiffness ratios (1:5, 1:20, and 1:60). After culturing chondrocytes on these substrates, we found that softer substrates (1:60) significantly reduced chondrocyte senescence, as evidenced by a decrease in senescence‐associated β‐galactosidase (SA‐β‐gal) activity by 33% compared with stiffer substrates (1:5). Furthermore, quantitative real‐time polymerase chain reaction (PCR) analysis revealed that chondrocytes cultured on softer substrates exhibited lower expression levels of senescence‐related genes (e.g., p16INK4a) and matrix‐degrading enzymes (e.g., MMP13). These findings suggest that substrate stiffness plays a critical role in regulating chondrocyte senescence and could potentially inform the design of scaffolds for cartilage tissue engineering. [ABSTRACT FROM AUTHOR]

Background: Neutrophils, the most abundant leukocytes circulating in blood, contribute to host defense and play a significant role in chronic inflammatory disorders. They can release their DNA in the form of extracellular traps (NETs), which serve as scaffolds for capturing bacteria and various blood cells. However, uncontrolled formation of NETs (NETosis) can lead to excessive activation of coagulation pathways and thrombosis. Once neutrophils are migrated to infected or injured tissues, they become exposed to mechanical forces from their surrounding environment. However, the impact of transient changes in tissue mechanics due to the natural process of aging, infection, tissue injury, and cancer on neutrophils remains unknown. To address this gap, we explored the interactive effects of changes in substrate stiffness and cyclic stretch on NETosis. Primary neutrophils were cultured on a silicon-based substrate with stiffness levels of 30 and 300 kPa for at least 3 h under static conditions or cyclic stretch levels of 5% and 10%, mirroring the biomechanics of aged and young arteries. Results: Using this approach, we found that neutrophils are sensitive to cyclic stretch and that increases in stretch intensity and substrate stiffness enhance nuclei decondensation and histone H3 citrullination (CitH3). In addition, stretch intensity and substrate stiffness promote the response of neutrophils to the NET-inducing agents phorbol 12-myristate 13-acetate (PMA), adenosine triphosphate (ATP), and lipopolysaccharides (LPS). Stretch-induced activation of neutrophils was dependent on calpain activity, the phosphatidylinositol 3-kinase (PI3K)/focal adhesion kinase (FAK) signalling and actin polymerization. Conclusions: In summary, these results demonstrate that the mechanical forces originating from the surrounding tissue influence NETosis, an important neutrophil function, and thus identify a potential novel therapeutic target. [ABSTRACT FROM AUTHOR]

Abstract Background Neutrophils, the most abundant leukocytes circulating in blood, contribute to host defense and play a significant role in chronic inflammatory disorders. They can release their DNA in the form of extracellular traps (NETs), which serve as scaffolds for capturing bacteria and various blood cells. However, uncontrolled formation of NETs (NETosis) can lead to excessive activation of coagulation pathways and thrombosis. Once neutrophils are migrated to infected or injured tissues, they become exposed to mechanical forces from their surrounding environment. However, the impact of transient changes in tissue mechanics due to the natural process of aging, infection, tissue injury, and cancer on neutrophils remains unknown. To address this gap, we explored the interactive effects of changes in substrate stiffness and cyclic stretch on NETosis. Primary neutrophils were cultured on a silicon-based substrate with stiffness levels of 30 and 300 kPa for at least 3 h under static conditions or cyclic stretch levels of 5% and 10%, mirroring the biomechanics of aged and young arteries. Results Using this approach, we found that neutrophils are sensitive to cyclic stretch and that increases in stretch intensity and substrate stiffness enhance nuclei decondensation and histone H3 citrullination (CitH3). In addition, stretch intensity and substrate stiffness promote the response of neutrophils to the NET-inducing agents phorbol 12-myristate 13-acetate (PMA), adenosine triphosphate (ATP), and lipopolysaccharides (LPS). Stretch-induced activation of neutrophils was dependent on calpain activity, the phosphatidylinositol 3-kinase (PI3K)/focal adhesion kinase (FAK) signalling and actin polymerization. Conclusions In summary, these results demonstrate that the mechanical forces originating from the surrounding tissue influence NETosis, an important neutrophil function, and thus identify a potential novel therapeutic target.

It is a long‐standing challenge to exploit cellular uptake mechanisms to deliver desired cargo into cells, for example, specific drugs or gene editing techniques. This study introduces a bioinspired material approach where nanoparticles are presented at the ventral side of cells adhering to engineered extracellular matrices. The effect of matrix stiffness on cell adhesion and mechanics, as well as on particle internalization by clathrin‐mediated endocytosis (CME), is investigated for varying particle size and surface functionalization. The results presented here show that substrate stiffness affects both cell adhesion and particle internalization, with softer substrates promoting higher levels of particle uptake. However, the activation of the CME pathway, either mechanically by particle size or functionally by receptor binding, regulates the sensitivity of cellular particle uptake to matrix stiffness. Finally, adeno‐associated viruses as the leading platform for therapeutic gene delivery are used as model cargo to showcase the importance of considering multiple components when designing delivery systems. These findings indicate that particle uptake is a multifaceted process that can be improved by the appropriate combination of extracellular environment mechanics and cargo properties. [ABSTRACT FROM AUTHOR]

Physical changes in the tumor microenvironment, such as increased stiffness, regulate cancer hallmarks and play an essential role in gene expression, cell morphology, migration, and malignancy. However, the response of cancer cells to stiffness is not homogeneous and varies depending on the cell type and its mechanosensitivity. In this study, we investigated the differential responses of cervical (HeLa) and prostate (PC-3) cancer cell lines, as well as non-tumoral cell lines (HEK293 and HPrEC), to stiffness using polyacrylamide hydrogels mimicking normal and tumoral tissues. We analyzed cell morphology, migration, and the expression of neuropilin 1 (NRP1), a receptor involved in angiogenesis, cell migration, and extracellular matrix remodeling, known to be associated with cancer progression and poor prognosis. Our findings reveal that NRP1 expression increases on substrates mimicking the high stiffness characteristic of tumoral tissue in the non-tumoral cell lines HPrEC and HEK293. Conversely, in tumoral PC-3 cells, stiffness resembling normal prostate tissue induces an earlier and more sustained expression of NRP1. Furthermore, we observed that stiffness influences cell spreading, pseudopodia formation, and the mode of cell protrusion during migration. Soft substrates predominantly trigger bleb cell protrusion, while pseudopodia protrusions increase on substrates mimicking normal and tumor-like stiffnesses in HPrEC cells compared to PC-3 cells. Stiffer substrates also enhance the percentage of migratory cells, as well as their velocity and total displacement, in both non-tumoral and tumoral prostate cells. However, they only improve the persistence of migration in tumoral PC-3 cells. Moreover, we found that NRP1 co-localizes with actin, and its suppression impairs tumoral PC-3 spreading while decreasing pseudopodia protrusion mode. Our results suggest that the modulation of NRP1 expression by the stiffness can be a feedback loop to promote malignancy in non-tumoral and cancer cells, contingent upon the mechanosensitivity of the cells. [ABSTRACT FROM AUTHOR]

Purposes Osteoarthritis (OA) is a degenerative joint disease that commonly occurs in middle-aged and elderly people. Mechanical microenvironment is one of the most significant factors in the development of OA. However, when the mechanical microenvironment changes, the inflammatory response of chondrocyte is elusive. Methods By adopting polydimethylsiloxane (PDMS) substrates with varying stiffness which can mimic the physiological stiffness of chondrocyte pericellular matrix (PCM), influences of co-regulated substrate stiffness and inflammatory factors interleukin-1β (IL-1β) on chondrocyte morphology, inflammatory mediators, and PCM remodeling protein expression are quantitatively analyzed. First, prostaglandin E2 (PGE2) and nitric oxide (NO) released in different stiffness substrates and IL-1β stimulated substrates with chondrocytes are detected. Second, the changes in different stiffness substrates and IL-1β stimulated substrates through immunofluorescence technique are observed and recorded. Third, the protein expressions of type Ⅱ collagen (COLII) and matrix metalloproteinase-13 (MMP13) are measured by Western blot assay. Findings The experimental results identify that substrate stiffness regulates the response of chondrocyte to inflammatory signals. Soft substrate dramatically enhances the release of PGE2 and NO (P

Abstract Alterations in the structure and composition of Bruch’s membrane (BrM) and loss of retinal pigment epithelial (RPE) cells are associated with various ocular diseases, notably age-related macular degeneration (AMD) as well as several inherited retinal diseases (IRDs). We explored the influence of stiffness as a major BrM characteristic on the RPE transcriptome and morphology. ARPE-19 cells were plated on soft ( $$E={30}\,\hbox {kPa}$$ E = 30 kPa ) or stiff ( $$E={80}\,\hbox {kPa}$$ E = 80 kPa ) polyacrylamide gels (PA gels) or standard tissue culture plastic (TCP). Next-generation sequencing (NGS) data on differentially expressed small RNAs (sRNAs) and messenger RNAs (mRNAs) were validated by qPCR, immunofluorescence or western blotting. The microRNA (miRNA) fraction of sRNAs grew with substrate stiffness and distinct miRNAs such as miR-204 or miR-222 were differentially expressed. mRNA targets of differentially expressed miRNAs were stably expressed, suggesting a homeostatic effect of miRNAs. mRNA transcription patterns were substrate stiffness-dependent, including components of Wnt/beta-catenin signaling, Microphthalmia-Associated Transcription Factor (MITF) and Dicer. These findings highlight the relevance of mechanical properties of the extracellular matrix (ECM) in cell culture experiments, especially those focusing on ECM-related diseases, such as AMD.

Recognizing that cells "feel" and respond to their mechanical environment, recent studies demonstrate that many cells exhibit a phenomenon of "mechanical memory" in which features induced by prior mechanical cues persist after the mechanical stimulus has ceased. While there is a general recognition that different cell types exhibit different responses to changes in extracellular matrix stiffening, the phenomenon of mechanical memory within myocardial cell types has received little attention to date. To probe the dynamics of mechanical memory in cardiac fibroblasts (CFs) and cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs), we employed a magnetorheological elastomer (MRE) cell culture substrate with tunable and reversible stiffness spanning the range from normal to diseased myocardium. In CFs, using increased cell area and increases in α-smooth muscle actin as markers of cellular responses to matrix stiffening, we found that induction of mechanical memory required seven days of stiff priming. Both induction and maintenance of persistent CF activation were blocked with the F-actin inhibitor cytochalasin D, while inhibitors of microtubule detyrosination had no impact on CFs. In iPSC-CMs, mechanical memory was invoked after only 24 h of stiff priming. Moreover, mechanical memory induction and maintenance were microtubule-dependent in CMs with no dependence on F-actin. Overall, these results identify the distinct temporal dynamics of mechanical memory in CFs and iPSC-CMs with different cytoskeletal mediators responsible for inducing and maintaining the stiffness-activated phenotype. Due to its flexibility, this model is broadly applicable to future studies interrogating mechanotransduction and mechanical memory in the heart and might inform strategies for attenuating the impact of load-induced pathology and excess myocardial stiffness. [ABSTRACT FROM AUTHOR]

Mechanosensitive focal adhesion (FA) complexes mediate dynamic interactions between cells and substrates and regulate cellular function. Integrins in FA complexes link substrate ligands to stress fibers (SFs) and aid load transfer and traction generation. We developed a one-dimensional, multi-scale, stochastic finite element model of a fibroblast on a substrate that includes calcium signaling, SF remodeling, and FA dynamics. We linked stochastic dynamics, describing the formation and clustering of integrins to substrate ligands via motor-clutches, to a continuum level SF contractility model at various locations along the cell length. We quantified changes in cellular responses with substrate stiffness, ligand density, and cyclic stretch. Results show that tractions and integrin recruitments varied along the cell length; tractions were maximum at lamellar regions and reduced to zero at the cell center. Optimal substrate stiffness, based on maximum tractions exerted by the cell, shifted toward stiffer substrates at high ligand densities. Mean tractions varied biphasically with substrate stiffness and peaked at the optimal substrate stiffness. Cytosolic calcium increased monotonically with substrate stiffness and accumulated near lamellipodial regions. Cyclic stretch increased the cytosolic calcium, integrin concentrations, and tractions at lamellipodial and intermediate regions on compliant substrates. The optimal substrate stiffness under stretch shifted toward compliant substrates for a given ligand density. Stretch also caused cell deadhesions beyond a critical substrate stiffness. FA's destabilized on stiff substrates under cyclic stretch. An increase in substrate stiffness and cyclic stretch resulted in higher fibroblast contractility. These results show that chemomechanical coupling is essential in mechanosensing responses underlying cell–substrate interactions. [ABSTRACT FROM AUTHOR]

Understanding how biophysical and biochemical microenvironmental cues together influence the regenerative activities of muscle stem cells and their progeny is crucial in strategizing remedies for pathological dysregulation of these cues in aging and disease. In this study, we investigated the cell-level influences of extracellular matrix (ECM) ligands and culture substrate stiffness on primary human myoblast contractility and proliferation within 16 h of plating and found that tethered fibronectin led to stronger stiffness-dependent responses compared to laminin and collagen. A proteome-wide analysis further uncovered cell metabolism, cytoskeletal and nuclear component regulation distinctions between cells cultured on soft and stiff substrates. Interestingly, we found that softer substrates increased the incidence of myoblasts with a wrinkled nucleus, and that the extent of wrinkling could predict Ki67 (also known as MKI67) expression. Nuclear wrinkling and Ki67 expression could be controlled by pharmacological manipulation of cellular contractility, offering a potential cellular mechanism. These results provide new insights into the regulation of human myoblast stiffness-dependent contractility response by ECM ligands and highlight a link between myoblast contractility and proliferation. [ABSTRACT FROM AUTHOR]

Since its invention in the late 1980s, the air-liquid-interface (ALI) culture system has been the standard in vitro model for studying human airway biology and pulmonary diseases. However, in a conventional ALI system, cells are cultured on a porous plastic membrane that is much stiffer than human airway tissues. Here, we develop a gel-ALI culture system by simply coating the plastic membrane with a thin layer of hydrogel with tunable stiffness matching that of healthy and fibrotic airway tissues. We determine the optimum gel thickness that does not impair the transport of nutrients and biomolecules essential to cell growth. We show that the gel-ALI system allows human bronchial epithelial cells (HBECs) to proliferate and differentiate into pseudostratified epithelium. Furthermore, we discover that HBECs migrate significantly faster on hydrogel substrates with stiffness matching that of fibrotic lung tissues, highlighting the importance of mechanical cues in human airway remodeling. The developed gel-ALI system provides a facile approach to studying the effects of mechanical cues in human airway biology and in modeling pulmonary diseases. NEW & NOTEWORTHY In a conventional ALI system, cells are cultured on a plastic membrane that is much stiffer than human airway tissues. We develop a gel-ALI system by coating the plastic membrane with a thin layer of hydrogel with tunable stiffness matching that of healthy and fibrotic airway tissues. We discover that human bronchial epithelial cells migrate significantly faster on hydrogel substrates with pathological stiffness, highlighting the importance of mechanical cues in human airway remodeling. [ABSTRACT FROM AUTHOR]

Physical changes in the tumor microenvironment, such as increased stiffness, regulate cancer hallmarks and play an essential role in gene expression, cell morphology, migration, and malignancy. However, the response of cancer cells to stiffness is not homogeneous and varies depending on the cell type and its mechanosensitivity. In this study, we investigated the differential responses of cervical (HeLa) and prostate (PC-3) cancer cell lines, as well as non-tumoral cell lines (HEK293 and HPrEC), to stiffness using polyacrylamide hydrogels mimicking normal and tumoral tissues. We analyzed cell morphology, migration, and the expression of neuropilin 1 (NRP1), a receptor involved in angiogenesis, cell migration, and extracellular matrix remodeling, known to be associated with cancer progression and poor prognosis. Our findings reveal that NRP1 expression increases on substrates mimicking the high stiffness characteristic of tumoral tissue in the non-tumoral cell lines HPrEC and HEK293. Conversely, in tumoral PC-3 cells, stiffness resembling normal prostate tissue induces an earlier and more sustained expression of NRP1. Furthermore, we observed that stiffness influences cell spreading, pseudopodia formation, and the mode of cell protrusion during migration. Soft substrates predominantly trigger bleb cell protrusion, while pseudopodia protrusions increase on substrates mimicking normal and tumor-like stiffnesses in HPrEC cells compared to PC-3 cells. Stiffer substrates also enhance the percentage of migratory cells, as well as their velocity and total displacement, in both non-tumoral and tumoral prostate cells. However, they only improve the persistence of migration in tumoral PC-3 cells. Moreover, we found that NRP1 co-localizes with actin, and its suppression impairs tumoral PC-3 spreading while decreasing pseudopodia protrusion mode. Our results suggest that the modulation of NRP1 expression by the stiffness can be a feedback loop to promote malignancy in non-tumoral and cancer cells, contingent upon the mechanosensitivity of the cells.

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Plastin-3 (PLS3) is a calcium-sensitive actin-bundling protein that has recently been linked to the development of childhood-onset osteoporosis. Clinical data suggest that PLS3 mutations lead to a defect in osteoblast function, however the underlying mechanism remains elusive. To investigate the role of PLS3 in bone mineralization, we generated MC3T3-E1 preosteoblast cells that are stably depleted of PLS3. Analysis of osteogenic differentiation of control and PLS3 knockdown (PLS3 KD) cells showed that depletion of PLS3 does not alter the first stage of osteoblast mineralization in which a collagen matrix is deposited, but severely affects the subsequent mineralization of that matrix. During this phase, osteoblasts heavily rely on mechanosensitive signaling pathways to sustain mineral deposition in response to increasing stiffness of the extracellular matrix (ECM). PLS3 prominently localizes to focal adhesions (FAs), which are intricately linked to mechanosensation. In line with this, we observed that depletion of PLS3 rendered osteoblasts unresponsive to changes in ECM stiffness and showed the same cell size, FA lengths and number of FAs when plated on soft (6 kPa) versus stiff (100 kPa) substrates in contrast to control cells, which showed an increased in each of these parameters when plated on 100 kPa substrates. Defective cell spreading of PLS3 KD cells on stiff substrates could be rescued by expression of wildtype PLS3, but not by expression of three PLS3 mutations that were identified in patients with early onset osteoporosis and that have aberrant actin-bundling activity. Altogether, our results show that actin-bundling by PLS3 is part of the mechanosensitive mechanism that promotes osteoblast mineralization and thus begins to elucidate how PLS3 contributes to the development of bone defects such as osteoporosis. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a chronic disease and is difficult to cure. Chondrocytes are highly mechanosensitive. Therefore, mechanical therapies have received attention as a therapeutic direction for OA. The stiffness, as a critical cue of the extracellular matrix (ECM), affects cell growth, development, and death. In this study, we use polydimethylsiloxane (PDMS) to create substrates with varying stiffness for chondrocyte growth, interleukin-1β (IL-1β) treatment to mimic the inflammatory environment, and Tubastatin A (Tub A) to inhibit histone deacetylase 6 (HDAC6). Our results show that stiff substrates can be anti-inflammatory and provide a better matrix environment than soft substrates. Inhibition of HDAC6 improves the inflammatory environment caused by IL-1β and coordinates with inflammation to spread the chondrocyte area and primary cilia elongation. Without IL-1β and Tub A treatments, the length of the primary cilia rather than frequency is stiffness-dependent, and their length on stiff substrates are greater than that on soft substrates. In conclusion, we demonstrate that stiff substrates, inflammation, and inhibition of HDAC6 enhance the mechanosensitivity of primary cilia and mediate substrate stiffness to suppress inflammation and protect the matrix.

Uterine leiomyomas, or fibroids, are common, benign tumors for which hysterectomy is the only definitive treatment. The extracellular matrix of fibroids is disorganized and stiffer than the surrounding myometrial tissue. To understand how stiffness affects fibroid cells, patient-matched fibroid and myometrial cells were cultured on substrates with stiffnesses varying from 0.2 to 150 kPa. Fibroid cells grew more slowly than myometrial cells overall, and only the myometrial cells altered their growth rate in response to stiffness. In both cell types, cell proliferation decreased with inhibition of PI3K and increased with inhibition of IGF-1. The cellular area was greater for the fibroid cells. The only significant effect of stiffness on the cell area was between the 0.2 and 64 kPa substrates, and this was true for both cell types. To investigate intracellular stiffness, intracellular particle tracking microrheology was used. Fibroid cells exhibited a more than 100-fold increase in elastic modulus at a frequency of 1 Hz in response to the addition of external stress, while myometrial cells showed little change in elastic modulus. Overall, the responses of both cells followed similar trends in response to stiffness and inhibitors, although the response was attenuated in the fibroid cells. The changes that were demonstrated by the change in intracellular stiffness with response to compression suggest that other mechanical forces may provide insight into differences in the two cell types. [ABSTRACT FROM AUTHOR]

Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and activation of mechanosensitive signaling pathways. To address the functional coupling of ECM composition and matrix rigidity on compliant substrates, we developed a matrix spot array system to screen cell phenotypes against different ECM mixtures on defined substrate stiffnesses at high resolution. We applied this system to both cancer and normal cells and surprisingly identified ECM mixtures that support stiffness-insensitive cell spreading on soft substrates. Employing the motor-clutch model to simulate cell adhesion on biochemically distinct soft substrates, with varying numbers of available ECM–integrin–cytoskeleton (clutch) connections, we identified conditions in which spreading would be supported on soft matrices. Combining simulations and experiments, we show that cell spreading on soft is supported by increased clutch engagement on specific ECM mixtures and even augmented by the partial inhibition of actomyosin contractility. Thus, “stiff-like” spreading on soft is determined by a balance of a cell’s contractile and adhesive machinery. This provides a fundamental perspective for in vitro mechanobiology studies, identifying a mechanism through which cells spread, function, and signal effectively on soft substrates [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension has characteristic changes to the mechanical environment, extracellular matrix, and cellular proliferation. In order to develop a culture system to investigate extracellular matrix (ECM) compositional-dependent changes in pulmonary arterial hypertension, we decellularized and characterized protein and lipid profiles from healthy and Sugen-Chronic Hypoxia rat lungs. Significant changes in lipid profiles were observed in intact Sugen-Hypoxia lungs compared with healthy controls. Decellularized lung matrix retained lipids in measurable quantities in both healthy and Sugen-Chronic Hypoxia samples. Proteomics revealed significantly changed proteins associated with pulmonary arterial hypertension in the decellularized Sugen-Chronic Hypoxia lung ECM. We then investigated the potential role of healthy vs. Sugen-Chronic Hypoxia ECM with controlled substrate stiffness to determine if the ECM composition regulated endothelial cell morphology and phenotype. CD117+ rat lung endothelial cell clones were plated on the variable stiffness gels and cellular proliferation, morphology, and gene expression were quantified. Sugen-Chronic Hypoxia ECM on healthy stiffness gels produced significant changes in cellular gene expression levels of Bmp2, Col1α1, Col3α1 and Fn1. The signaling and cell morphology observed at low substrate stiffness suggests early changes to the ECM composition can initiate processes associated with disease progression. These data suggest that Sugen-Chronic Hypoxia ECM can be used to investigate cell-ECM interactions relevant to pulmonary arterial hypertension. [ABSTRACT FROM AUTHOR]

Matrix mechanics regulate essential cell behaviors through mechanotransduction, and as one of its most important elements, substrate stiffness was reported to regulate cell functions such as viability, communication, migration, and differentiation. Neutrophils (Neus) predominate the early inflammatory response and initiate regeneration. The activation of Neus can be regulated by physical cues; however, the functional alterations of Neus by substrate stiffness remain unknown, which is critical in determining the outcomes of engineered tissue mimics. Herein, a three-dimensional (3D) culture system made of hydrogels was developed to explore the effects of varying stiffnesses (1.5, 2.6, and 5.7 kPa) on the states of Neus. Neus showed better cell integrity and viability in the 3D system. Moreover, it was shown that the stiffer matrix tended to induce Neus toward an anti-inflammatory phenotype (N2) with less adhesion molecule expression, less reactive oxygen species (ROS) production, and more anti-inflammatory cytokine secretion. Additionally, the aortic ring assay indicated that Neus cultured in a stiffer matrix significantly increased vascular sprouting. RNA sequencing showed that a stiffer matrix could significantly activate JAK1/STAT3 signaling in Neus and the inhibition of JAK1 ablated the stiffness-dependent increase in the expression of CD182 (an N2 marker). Taken together, these results demonstrate that a stiffer matrix promotes Neus to shift to the N2 phenotype, which was regulated by JAK1/STAT3 pathway. This study lays the groundwork for further research on fabricating engineered tissue mimics, which may provide more treatment options for ischemic diseases and bone defects. • The 3D culture system was more suitable for neutrophils (Neus), providing an opportunity to mimic the in-vivo experiments. • Substrate stiffness could alter neutrophil phenotypes and stiffer substrate promoted Neus to shift to the N2 phenotype, which was regulated by modulating the JAK1/STAT3 pathway. • This work lays the foundation for the application of hydrogel-based biomaterials, which provides more treatment options for ischemic diseases and bone defects. [Display omitted] [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) have significant therapeutic potential due to their ability to differentiate into musculoskeletal lineages suitable for tissue-engineering, as well as the immunomodulatory and pro-regenerative effects of the paracrine factors that these cells secrete. Cues from the extracellular environment, including physical stimuli such as substrate stiffness, are strong drivers of MSC differentiation, but their effects upon MSC paracrine activity are not well understood. This study, therefore sought to determine the impact of substrate stiffness on the paracrine activity of MSCs, analysing both effects on MSC fate and their effect on T-cell and macrophage activity and angiogenesis. The data show that conditioned medium (CM) from MSCs cultured on 0.2 kPa (soft) and 100 kPa (stiff) polyacrylamide hydrogels have differing effects on MSC proliferation and differentiation, with stiff CM promoting proliferation whilst soft CM promoted differentiation. There were also differences in the effects upon macrophage phagocytosis and angiogenesis, with the most beneficial effects from soft CM. Analysis of the media composition identified differences in the levels of proteins including IL-6, OPG, and TIMP-2. Using recombinant proteins and blocking antibodies, we confirmed a role for OPG in modulating MSC proliferation with a complex combination of factors involved in the regulation of MSC differentiation. Together the data confirm that the physical microenvironment has an important influence on the MSC secretome and that this can alter the differentiation and regenerative potential of the cells. These findings can be used to tailor the culture environment for manufacturing potent MSCs for specific clinical applications or to inform the design of biomaterials that enable the retention of MSC activity after delivery into the body. • MSCs cultured on 100 kPa matrices produce a secretome that boosts MSC proliferation • MSCs cultured on 0.2 kPa matrices produce a secretome that promotes MSC osteogenesis and adipogenesis, as well as angiogenesis and macrophage phagocytosis • IL-6 secretion is elevated in MSCs on 0.2 kPa substrates • OPG, TIMP-2, MCP-1, and sTNFR1 secretion are elevated in MSCs on 100 kPa substrates [Display omitted] [ABSTRACT FROM AUTHOR]

The infiltration and deposition of cholesterol in the arterial wall play an important role in the initiation and development of atherosclerosis. Smooth muscle cells (SMCs) are the major cell type in the intima. Upon exposure to cholesterol, SMCs may undergo a phenotype switching into foam cells. Meanwhile, the pathological processes of the blood vessel such as cholesterol deposition and calcification induce the changes in the substrate stiffness around SMCs. However, whether substrate stiffness affects the cholesterol accumulation in SMCs and the formation of foam cells is not well-understood. In this study, SMCs were cultured on the substrates with different stiffnesses ranging from 1 to 100 kPa and treated with cholesterol. We found that cholesterol accumulation in SMCs was higher on 1 and 100 kPa substrates than that on intermediate stiffness at 40 kPa; consistently, total cholesterol (TC) content on 1 and 100 kPa substrates was also higher. As a result, the accumulation of cholesterol increased the expression of macrophage marker CD68 and downregulated SMC contractile marker smooth muscle α-actin (ACTA2). Furthermore, the mRNA and protein expression level of cholesterol efflux gene ATP-binding cassette transporter A1 (ABCA1) was much higher on 40 kPa substrate. With the treatment of a liver X receptor (LXR) agonist GW3965, the expression of ABCA1 increased and cholesterol loading decreased, showing an additive effect with substrate stiffness. In contrast, inhibition of LXR decreased ABCA1 gene expression and increased cholesterol accumulation in SMCs. Consistently, when ABCA1 gene was knockdown, the cholesterol accumulation was increased in SMCs on all substrates with different stiffness. These results revealed that substrate stiffness played an important role on SMCs cholesterol accumulation by regulating the ABCA1 expression. Our findings on the effects of substrate stiffness on cholesterol efflux unravel a new mechanism of biophysical regulation of cholesterol metabolism and SMC phenotype, and provide a rational basis for the development of novel therapies.

Plastin-3 (PLS3) is a calcium-sensitive actin-bundling protein that has recently been linked to the development of childhood-onset osteoporosis. Clinical data suggest that PLS3 mutations lead to a defect in osteoblast function, however the underlying mechanism remains elusive. To investigate the role of PLS3 in bone mineralization, we generated MC3T3-E1 preosteoblast cells that are stably depleted of PLS3. Analysis of osteogenic differentiation of control and PLS3 knockdown (PLS3 KD) cells showed that depletion of PLS3 does not alter the first stage of osteoblast mineralization in which a collagen matrix is deposited, but severely affects the subsequent mineralization of that matrix. During this phase, osteoblasts heavily rely on mechanosensitive signaling pathways to sustain mineral deposition in response to increasing stiffness of the extracellular matrix (ECM). PLS3 prominently localizes to focal adhesions (FAs), which are intricately linked to mechanosensation. In line with this, we observed that depletion of PLS3 rendered osteoblasts unresponsive to changes in ECM stiffness and showed the same cell size, FA lengths and number of FAs when plated on soft (6 kPa) versus stiff (100 kPa) substrates in contrast to control cells, which showed an increased in each of these parameters when plated on 100 kPa substrates. Defective cell spreading of PLS3 KD cells on stiff substrates could be rescued by expression of wildtype PLS3, but not by expression of three PLS3 mutations that were identified in patients with early onset osteoporosis and that have aberrant actin-bundling activity. Altogether, our results show that actin-bundling by PLS3 is part of the mechanosensitive mechanism that promotes osteoblast mineralization and thus begins to elucidate how PLS3 contributes to the development of bone defects such as osteoporosis.

Glycocalyx (GCX) is a carbohydrate-rich structure that coats the surface of endothelial cells (ECs) and lines the blood vessel lumen. Mechanical perturbations in the vascular environment, such as blood vessel stiffness, can be transduced and sent to ECs through mechanosensors such as GCX. Adverse stiffness alters GCX-mediated mechanotransduction and leads to EC dysfunction and eventually atherosclerotic cardiovascular diseases. To understand GCX-regulated mechanotransduction events, an in vitro model emulating in vivo vessel conditions is needed. To this end, we investigated the impact of matrix chemical and mechanical properties on GCX expression via fabricating a tunable non-swelling matrix based on the collagen-derived polypeptide, gelatin. To study the effect of matrix composition, we conducted a comparative analysis of GCX expression using different concentrations (60–25,000 μg/mL) of gelatin and gelatin methacrylate (GelMA) in comparison to fibronectin (60 μg/mL), a standard coating material for GCX-related studies. Using immunocytochemistry analysis, we showed for the first time that different substrate compositions and concentrations altered the overall GCX expression on human umbilical vein ECs (HUVECs). Subsequently, GelMA hydrogels were fabricated with stiffnesses of 2.5 and 5 kPa, representing healthy vessel tissues, and 10 kPa, corresponding to diseased vessel tissues. Immunocytochemistry analysis showed that on hydrogels with different levels of stiffness, the GCX expression in HUVECs remained unchanged, while its major polysaccharide components exhibited dysregulation in distinct patterns. For example, there was a significant decrease in heparan sulfate expression on pathological substrates (10 kPa), while sialic acid expression increased with increased matrix stiffness. This study suggests the specific mechanisms through which GCX may influence ECs in modulating barrier function, immune cell adhesion, and mechanotransduction function under distinct chemical and mechanical conditions of both healthy and diseased substrates.

Peripheral arterial disease (PAD) is an age-related medical condition affecting mostly muscular arteries of the limb. It is the 3rd leading cause of atherosclerotic morbidity. The mechanical environment of endothelial cells (ECs) in PAD is characterized by disturbed blood flow (d-flow) and stiff extracellular matrices. In PAD, the stiffness of arteries is due to decreased elastin function and increased collagen content. These flow and stiffness parameters are largely missing from current models of PAD. It has been previously proven that ECs exposed to d-flow or stiff substrates lead to proatherogenic pathways, but the effect of both, d-flow and stiffness, on EC phenotype has not been fully investigated. In this study, we sought to explore the effect of sex on proatherogenic pathways that could result from exposing endothelial cells to a d-flow and stiff environment. We utilized the scRNA-seq tool to analyze the gene expression of ECs exposed to the different mechanical conditions both in vitro and in vivo. We found that male ECs exposed to different mechanical stimuli presented higher expression of genes related to fibrosis and d-flow in vitro. We validated our findings in vivo by exposing murine carotid arteries to d-flow via partial carotid artery ligation. Since women have delayed onset of arterial stiffening and subsequent PAD, this work may provide a framework for some of the pathways in which biological sex interacts with sex-based differences in PAD.

Pulmonary arterial hypertension has characteristic changes to the mechanical environment, extracellular matrix, and cellular proliferation. In order to develop a culture system to investigate extracellular matrix (ECM) compositional-dependent changes in pulmonary arterial hypertension, we decellularized and characterized protein and lipid profiles from healthy and Sugen-Chronic Hypoxia rat lungs. Significant changes in lipid profiles were observed in intact Sugen-Hypoxia lungs compared with healthy controls. Decellularized lung matrix retained lipids in measurable quantities in both healthy and Sugen-Chronic Hypoxia samples. Proteomics revealed significantly changed proteins associated with pulmonary arterial hypertension in the decellularized Sugen-Chronic Hypoxia lung ECM. We then investigated the potential role of healthy vs. Sugen-Chronic Hypoxia ECM with controlled substrate stiffness to determine if the ECM composition regulated endothelial cell morphology and phenotype. CD117+ rat lung endothelial cell clones were plated on the variable stiffness gels and cellular proliferation, morphology, and gene expression were quantified. Sugen-Chronic Hypoxia ECM on healthy stiffness gels produced significant changes in cellular gene expression levels of Bmp2, Col1α1, Col3α1 and Fn1. The signaling and cell morphology observed at low substrate stiffness suggests early changes to the ECM composition can initiate processes associated with disease progression. These data suggest that Sugen-Chronic Hypoxia ECM can be used to investigate cell-ECM interactions relevant to pulmonary arterial hypertension.

The formation of bone in a bone defect is accomplished by osteoblasts, while the over activation of fibroblasts promotes fibrosis. However, it is not clear how the extracellular matrix stiffness of the bone-regeneration microenvironment affects the function of osteoblasts and fibroblasts. This study aim to investigate the effect of bone-regeneration microenvironment stiffness on cell adhesion, cell proliferation, cell differentiation, synthesizing matrix ability and its potential mechanisms in mechanotransduction, in pre-osteoblasts and fibroblasts. Polyacrylamide substrates mimicking the matrix stiffness of different stages of the bone-healing process (15 kPa, mimic granulation tissue; 35 kPa, mimic osteoid; 150 kPa, mimic calcified bone matrix) were prepared. Mouse pre-osteoblasts MC3T3-E1 and mouse fibroblasts NIH3T3 were plated on three types of substrates, respectively. There were significant differences in the adhesion of pre-osteoblasts and fibroblasts on different polyacrylamide substrates. Runx2 expression increased with increasing substrate stiffness in pre-osteoblasts, while no statistical differences were found in the Acta2 expression in fibroblasts on three substrates. OPN expression in pre-osteoblasts, as well as Fn1 and Col1a1 expression in fibroblasts, decreased with increasing stiffness. The difference between the cell traction force generated by pre-osteoblasts and fibroblasts on substrates was also found. Our results indicated that substrate stiffness is a potent regulator of pre-osteoblasts and fibroblasts with the ability of promoting osteogenic differentiation of pre-osteoblasts, while having no effect on myofibroblast differentiation of fibroblasts. [ABSTRACT FROM AUTHOR]

Abstract The combined effect of surface topography and substrate rigidity in stem cell cultures is still under‐investigated, especially when biodegradable polymers are used. Herein, we assessed human bone marrow stem cell response on aliphatic polyester substrates as a function of anisotropic grooved topography and rigidity (7 and 12 kPa). Planar tissue culture plastic (TCP, 3 GPa) and aliphatic polyester substrates were used as controls. Cell morphology analysis revealed that grooved substrates caused nuclei orientation/alignment in the direction of the grooves. After 21 days in osteogenic and chondrogenic media, the 3 GPa TCP and the grooved 12 kPa substrate induced significantly higher calcium deposition and alkaline phosphatase (ALP) activity and glycosaminoglycan (GAG) deposition, respectively, than the other groups. After 14 days in tenogenic media, the 3 GPa TCP upregulated four and downregulated four genes; the planar 7 kPa substrate upregulated seven genes and downregulated one gene; and the grooved 12 kPa substrate upregulated seven genes and downregulated one gene. After 21 days in adipogenic media, the softest (7 kPa) substrates induced significantly higher oil droplet deposition than the other substrates and the grooved substrate induced significantly higher droplet deposition than the planar. Our data pave the way for more rational design of bioinspired constructs.

Cell-matrix interactions play a critical role in tissue repair and regeneration. With gradual uncovering of substrate mechanical characteristics that can affect cell-matrix interactions, much progress has been made to unravel substrate stiffness-mediated cellular response as well as its underlying mechanisms. Yet, as a part of cell-matrix interaction biology, this field remains in its infancy, and the detailed molecular mechanisms are still elusive regarding scaffold-modulated tissue regeneration. This review provides an overview of recent progress in the area of the substrate stiffness-mediated cellular responses, including 1) the physical determination of substrate stiffness on cell fate and tissue development; 2) the current exploited approaches to manipulate the stiffness of scaffolds; 3) the progress of recent researches to reveal the role of substrate stiffness in cellular responses in some representative tissue-engineered regeneration varying from stiff tissue to soft tissue. This article aims to provide an up-to-date overview of cell mechanobiology research in substrate stiffness mediated cellular response and tissue regeneration with insightful information to facilitate interdisciplinary knowledge transfer and enable the establishment of prognostic markers for the design of suitable biomaterials.

Gaining insight into the in situ receptor–ligand binding is pivotal for revealing the molecular mechanisms underlying the physiological and pathological processes and will contribute to drug discovery and biomedical application. An important issue involved is how the receptor–ligand binding responds to mechanical stimuli. This review aims to provide an overview of the current understanding of the effect of several representative mechanical factors, such as tension, shear stress, stretch, compression, and substrate stiffness on receptor–ligand binding, wherein the biomedical implications are focused. In addition, we highlight the importance of synergistic development of experimental and computational methods for fully understanding the in situ receptor–ligand binding, and further studies should focus on the coupling effects of these mechanical factors. [ABSTRACT FROM AUTHOR]

Cell contractility regulates epithelial tissue geometry development and homeostasis. The underlying mechanobiological regulation circuits are poorly understood and experimentally challenging. We developed an elastomeric pillar cage (EPC) array to quantify cell contractility as a mechanoresponse of epithelial microtissues to substrate stiffness and topography. The spatially confined EPC geometry consisted of 24 circularly arranged slender pillars (1.2 MPa, height: 50 µm; diameter: 10 µm, distance: 5 µm). These high-aspect-ratio pillars were confined at both ends by planar substrates with different stiffness (0.15–1.2 MPa). Analytical modeling and finite elements simulation retrieved cell forces from pillar displacements. For evaluation, highly contractile myofibroblasts and cardiomyocytes were assessed to demonstrate that the EPC device can resolve static and dynamic cellular force modes. Human breast (MCF10A) and skin (HaCaT) cells grew as adherence junction-stabilized 3D microtissues within the EPC geometry. Planar substrate areas triggered the spread of monolayered clusters with substrate stiffness-dependent actin stress fiber (SF)-formation and substantial single-cell actomyosin contractility (150–200 nN). Within the same continuous microtissues, the pillar-ring topography induced the growth of bilayered cell tubes. The low effective pillar stiffness overwrote cellular sensing of the high substrate stiffness and induced SF-lacking roundish cell shapes with extremely low cortical actin tension (11–15 nN). This work introduced a versatile biophysical tool to explore mechanobiological regulation circuits driving low- and high-tensional states during microtissue development and homeostasis. EPC arrays facilitate simultaneously analyzing the impact of planar substrate stiffness and topography on microtissue contractility, hence microtissue geometry and function. [ABSTRACT FROM AUTHOR]

Stiffness of the extracellular matrix regulates various biological responses, but the response mechanisms are poorly understood. Here, we found that the nuclear diphosphorylated myosin regulatory light chain (2P‐MRLC) is a critical mechanomediator that suppresses apoptosis in response to substrate stiffness. Stiff substrates promoted the nuclear localization of 2P‐MRLC. Zipper‐interacting protein kinase [ZIPK; also known as death‐associated protein kinase 3 (DAPK3)], a kinase for MRLC, was localized in the nucleus in response to stiff substrates and promoted the nuclear localization of 2P‐MRLC. Moreover, actin fiber formation induced by substrate stiffness promoted the nuclear localization of 2P‐MRLC via ZIPK. 2P‐MRLC in response to substrate stiffness suppressed the expression of MAF bZIP transcription factor B (MafB) and repressed apoptosis. These findings reveal a newly identified role of MRLC in mechanotransduction. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to explore the effect of biomaterials with different stiffness on Adipose Derived Mesenchymal Stem Cells (ADSC)–macrophage crosstalk in bone tissue engineering and its role in bone repair.Methods: Biomaterials with Young’s modulus of 64 and 0.2 kPa were selected, and the crosstalk between ADSCs and macrophages was investigated by means of conditioned medium treatment and cell co-culture, respectively. Polymerase chain reaction (PCR) and flow cytometry were used to evaluate the polarization of macrophages. Alkaline phosphatase (ALP) and alizarin red staining (ARS) solutions were used to evaluate the osteogenic differentiation of ADSCs. Transwell assay was used to evaluate the chemotaxis of ADSCs and macrophages. Moreover, mass spectrometry proteomics was used to analyze the secreted protein profile of ADSCs of different substrates and macrophages in different polarization states.Results: On exploring the influence of biomaterials on macrophages from ADSCs on different substrates, we found that CD163 and CD206 expression levels in macrophages were significantly higher in the 64-kPa group than in the 0.2-kPa group in conditioned medium treatment and cell co-culture. Flow cytometry showed that more cells became CD163+ or CD206+ cells in the 64-kPa group under conditioned medium treatment or cell co-culture. The Transwell assay showed that more macrophages migrated to the lower chamber in the 64-kPa group. The proteomic analysis found that ADSCs in the 64-kPa group secreted more immunomodulatory proteins, such as LBP and RBP4, to improve the repair microenvironment. On exploring the influence of biomaterials on ADSCs from macrophages in different polarization states, we found that ALP and ARS levels in ADSCs were significantly higher in the M2 group than in the other three groups (NC, M0, and M1 groups) in both conditioned medium treatment and cell co-culture. The Transwell assay showed that more ADSCs migrated to the lower chamber in the M2 group. The proteomic analysis found that M2 macrophages secreted more extracellular remodeling proteins, such as LRP1, to promote bone repair.Conclusion: In bone tissue engineering, the stiffness of repair biomaterials can affect the crosstalk between ADSCs and macrophages, thereby regulating local repair immunity and affecting bone repair.

The stiffness of the extracellular matrix of tumour cells plays a key role in tumour cell metastasis. However, it is unclear how mechanical properties regulate the cellular response to the environmental matrix. In this study, atomic force microscopy (AFM) and laser confocal imaging were used to qualitatively evaluate the relationship between substrate stiffness and migration of prostate cancer (PCa) cells. Cells cultured on stiff substrates (35 kPa) undergone several interesting phenomena compared to those on soft substrates (3 kPa). Here, the stimulation generated by the stiff substrates triggered the F-actin skeleton to bundle its filaments, increasing the polarity index of the external contour of PCa cells. Analysis of AFM force–distance curves indicated that the elasticity of the cells cultured on 35 kPa substrates increased while the viscosity decreased. Wound-healing experiments showed that PCa cells cultured on 35 kPa substrates have higher migration potential. These phenomena suggested that the mechanical properties may be correlated with the migration of PCa cells. After actin depolymerisation, the elasticity of the PCa cells decreased while the viscosity increased, and the migration ability was correspondingly decreased. In conclusion, this study clearly demonstrated the relationship between substrate stiffness and the mechanical properties of cells in prostate tumour metastasis, providing a basis for understanding the changes in the biomechanical properties at a single-cell level.

Surface-associated lifestyles dominate in the bacterial world. Large multicellular assemblies, called biofilms, are essential to the survival of bacteria in harsh environments and are closely linked to antibiotic resistance in pathogenic strains. Biofilms stem from the surface colonization of a wide variety of substrates encountered by bacteria, from living tissues to inert materials. Here, we demonstrate experimentally that the promiscuous opportunistic pathogen Pseudomonas aeruginosa explores substrates differently based on their rigidity, leading to striking variations in biofilm structure, exopolysaccharides (EPS) distribution, strain mixing during co-colonization and phenotypic expression. Using simple kinetic models, we show that these phenotypes arise through a mechanical interaction between the elasticity of the substrate and the type IV pilus (T4P) machinery, that mediates the surface-based motility called twitching. Together, our findings reveal a new role for substrate softness in the spatial organization of bacteria in complex microenvironments, with far-reaching consequences on efficient biofilm formation.

The aim of the study was to confirm whether cell substrate stiffness may participate in the regulation of fibrosis. The involvement of integrin α2β1, focal adhesion kinase (FAK) and Src kinase in signal transmission was investigated. Human atrial fibroblasts and myofibroblasts were cultured in both soft (2.23 ± 0.8 kPa) and stiff (8.28 ± 1.06 kPa) polyacrylamide gels. The cells were derived from the right atrium of patients with aortal stenosis undergoing surgery. The isolated cells, identified as fibroblasts or myofibroblasts, were stained positively with α smooth muscle actin, vimentin and desmin. The cultures settled on stiff gel demonstrated lower intracellular collagen and collagen type I telopeptide (PICP) levels; however, no changes in α1 chain of procollagen type I and III expression were noted. Inhibition of α2β1 integrin by TC-I 15 (10−7 and 10−8 M) or α2 integrin subunit silencing augmented intracellular collagen level. Moreover, FAK or Src kinase inhibitors increased collagen content within the culture. Lower TIMP4 secretion was reported within the stiff gel cultures but neither MMP 2 nor TIMP-1, 2 or 3 release was altered. The stiff substrate cultures also demonstrated lower interleukin-6 release. Substrate stiffness modified collagen deposition within the atrial fibroblast and myofibroblast cultures. The elasticity of the cellular environment exerts a regulatory influence on both synthesis and breakdown of collagen. Integrin α2β1, FAK and Src kinase activity participates in signal transmission, which may influence fibrosis in the atria of the human heart.

• Parent and recurrent GBM cells show different morphologies on brain-like soft substrate. • Cell migration, invasion, and survival of recurrent GBM cells is augmented on softer substrate. • Parent and recurrent GBM cells show differential response to EGFR inhibitor on softer substrate. • Total RNA-seq identified PLEKHA7 as a potential mechanoresponsive target specific for recurrent GBM. Recurrent glioblastoma is highly aggressive with currently no specific treatment regime. Therefore, to identify novel therapeutic targets for recurrent GBM, we used a cellular model developed in our lab from commercially available cell line U87MG and patient-derived cultures that allows the comparison between radiation naïve (Parent) and recurrent GBM cells generated after parent cells are exposed to lethal dose of radiation. Total RNA-seq of parent and recurrent population revealed significant upregulation of cell-ECM interactions pathway in the recurrent population. These results led us to hypothesize that the physical microenvironment contributes to the aggressiveness of recurrent GBM. To verify this, we cultured parent and recurrent GBM cells on collagen-coated polyacrylamide gels mimicking the stiffness of normal brain (Young's modulus E = 0.5kPa) or tumorigenic brain (E = 10kPa) and tissue culture plastic dishes (E ∼ 1 GPa). We found that compared to parent cells, recurrent cells showed higher proliferation, invasion, migration, and resistance to EGFR inhibitor. Using orthotopic GBM mouse model and resection model, we demonstrate that recurrent cells cultured on 0.5kPa had higher in vivo tumorigenicity and recurrent disease progression than parent cells, whereas these differences were insignificant when parent and recurrent cells were cultured on plastic substrates. Furthermore, recurrent cells on 0.5kPa showed high expression of ECM proteins like Collagen, MMP2 and MMP9. These proteins were also significantly upregulated in recurrent patient biopsies. Additionally, the brain of mice injected with recurrent cells grown on 0.5kPa showed higher Young's moduli suggesting the ability of these cells to make the surrounding ECM stiffer. Total RNA-seq of parent and recurrent cells grown on plastic and 0.5kpa identified PLEKHA7 significantly upregulated specifically in recurrent cells grown on 0.5 kPa substrate. PLEKHA7 was also found to be high in recurrent GBM patient biopsies. Accordingly, PLEKHA7 knockdown reduced invasion and survival of recurrent GBM cells. Together, these data provide an in vitro model system that captures the observed in vivo and clinical behavior of recurrent GBM by mimicking mechanical microenvironment and identifies PLEKHA7 as a novel potential target for recurrent GBM. [ABSTRACT FROM AUTHOR]

Commercial static cell culture substrates can usually not change their physical properties over time, resulting in a limited representation of the variation in biomechanical cues in vivo. To overcome this limitation, approaches incorporating gold nanoparticles to act as transducers to external stimuli have been employed. In this work, gold nanorods were embedded in an elastomeric matrix and used as photothermal transducers to fabricate biocompatible light-responsive substrates. The nanocomposite films analysed by lock-in thermography and nanoindentation show a homogeneous heat distribution and a greater stiffness when irradiated with NIR light. After irradiation, the initial stiffness values were recovered. In vitro experiments performed during NIR irradiation with NIH-3T3 fibroblasts demonstrated that these films were biocompatible and cells remained viable. Cells cultured on the light stiffened nanocomposite exhibited a greater proliferation rate and stronger focal adhesion clustering, indicating increased cell-surface binding strength. [ABSTRACT FROM AUTHOR]

The pericellular matrix stiffness is strongly associated with its biochemical and structural changes during the aging and osteoarthritis progress of articular cartilage. However, how substrate stiffness modulates the chondrocyte regulatory volume decrease (RVD) and calcium signaling in chondrocytes remains unknown. This study aims to investigate the effects of substrate stiffness on the chondrocyte RVD and calcium signaling by recapitulating the physiologically relevant substrate stiffness. Our results showed that substrate stiffness induces completely different dynamical deformations between the cell swelling and recovering progresses. Chondrocytes swell faster on the soft substrate but recovers slower than the stiff substrate during the RVD response induced by the hypo-osmotic challenge. We found that stiff substrate enhances the cytosolic Ca2+ oscillation of chondrocytes in the iso-osmotic medium. Furthermore, chondrocytes exhibit a distinctive cytosolic Ca2+ oscillation during the RVD response. Soft substrate significantly improves the Ca2+ oscillation in the cell swelling process whereas stiff substrate enhances the cytosolic Ca2+ oscillation in the cell recovering process. Our work also suggests that the TRPV4 channel is involved in the chondrocyte sensing substrate stiffness by mediating Ca2+ signaling in a stiffness-dependent manner. This helps to understand a previously unidentified relationship between substrate stiffness and RVD response under the hypo-osmotic challenge. A better understanding of substrate stiffness regulating chondrocyte volume and calcium signaling will aid the development of novel cell-instructive biomaterial to restore cellular functions.

Due to the limited capacity of corneal endothelial cells (CECs) division, corneal endothelial diseases have become a great challenge. The cornea is subjected to various mechanical stimuli in vivo, which may have a positive or negative influence. Thus, it is significant to gain an insight into the mechanism of mechanobiology of CECs for seeking more possible treatment. The purpose of this study was to determine the impacts of mechanical stretch and substrate stiffness on the morphology and fundamental cell behavior of CECs. Rabbit corneal endothelial cells (RCECs) were subjected to a 5% mechanical stretch or cultured on substrates of different stiffness. The impacts of mechanical stimulus on cell area, aspect ratio, circularity, cell density, nuclear shape, cytoskeleton, and cell viability were investigated. The expressions of the corneal endothelium-related markers ZO-1 and Na+/K+-ATPase were also evaluated by confocal immunofluorescence microscopy in the stiffness group. Our results suggested that mechanical stretch promoted the rearrangement of the cytoskeleton while decreasing the cell circularity, nuclear area, and cell density as well as cell viability. RCECs cultured on 10 ​kPa substrates, which was close to the physiological stiffness of rabbit Descemet's membrane (DM), showed better cell morphology, more stable actin cytoskeleton assembly, and more robust expression of the functional marker compared with other softer or stiffer substrates. In summary, mechanical stretch and substrate stiffness have profound influences on the morphology and function of CECs, which may have implications for the understanding and possible treatment of corneal endothelial diseases.

Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increasedmaturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calciumcontent and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis). [ABSTRACT FROM AUTHOR]

BackgroundTo maximize the translational utility of human induced pluripotent stem cells (iPSCs), the ability to precisely modulate the differentiation of iPSCs to target phenotypes is critical. Although the effects of the physical cell niche on stem cell differentiation are well documented, current approaches to direct step-wise differentiation of iPSCs have been typically limited to the optimization of soluble factors. In this regard, we investigated how temporally varied substrate stiffness affects the step-wise differentiation of iPSCs towards various lineages/phenotypes.MethodsElectrospun nanofibrous substrates with different reduced Young's modulus were utilized to subject cells to different mechanical environments during the differentiation process towards representative phenotypes from each of three germ layer derivatives including motor neuron, pancreatic endoderm, and chondrocyte. Phenotype-specific markers of each lineage/stage were utilized to determine differentiation efficiency by reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence imaging for gene and protein expression analysis, respectively.ResultsThe results presented in this proof-of-concept study are the first to systematically demonstrate the significant role of the temporally varied mechanical microenvironment on the differentiation of stem cells. Our results demonstrate that the process of differentiation from pluripotent cells to functional end-phenotypes is mechanoresponsive in a lineage- and differentiation stage-specific manner.ConclusionsLineage/developmental stage-dependent optimization of electrospun substrate stiffness provides a unique opportunity to enhance differentiation efficiency of iPSCs for their facilitated therapeutic applications.

The combined effect of surface topography and substrate rigidity in stem cell cultures is still under‐investigated, especially when biodegradable polymers are used. Herein, we assessed human bone marrow stem cell response on aliphatic polyester substrates as a function of anisotropic grooved topography and rigidity (7 and 12 kPa). Planar tissue culture plastic (TCP, 3 GPa) and aliphatic polyester substrates were used as controls. Cell morphology analysis revealed that grooved substrates caused nuclei orientation/alignment in the direction of the grooves. After 21 days in osteogenic and chondrogenic media, the 3 GPa TCP and the grooved 12 kPa substrate induced significantly higher calcium deposition and alkaline phosphatase (ALP) activity and glycosaminoglycan (GAG) deposition, respectively, than the other groups. After 14 days in tenogenic media, the 3 GPa TCP upregulated four and downregulated four genes; the planar 7 kPa substrate upregulated seven genes and downregulated one gene; and the grooved 12 kPa substrate upregulated seven genes and downregulated one gene. After 21 days in adipogenic media, the softest (7 kPa) substrates induced significantly higher oil droplet deposition than the other substrates and the grooved substrate induced significantly higher droplet deposition than the planar. Our data pave the way for more rational design of bioinspired constructs. [ABSTRACT FROM AUTHOR]

Platelets play vital roles in vascular repair, especially in primary hemostasis, and have been widely used in transfusion to prevent bleeding or manage active bleeding. Recently, platelets have been used in tissue repair (e.g. , bone, skin, and dental alveolar tissue) and cell engineering as drug delivery carriers. However, the biomedical applications of platelets have been associated with platelet storage lesions (PSLs), resulting in poor clinical outcomes with reduced recovery, survival, and hemostatic function after transfusion. Accumulating evidence has shown that biophysical cues play important roles in platelet lesions, such as granule secretion caused by shear stress, adhesion affected by substrate stiffness, and apoptosis caused by low temperature. This review summarizes four major biophysical cues (i.e. , shear stress, substrate stiffness, hydrostatic pressure, and thermal microenvironment) involved in the platelet preparation and storage processes, and discusses how they may synergistically induce PSLs such as platelet shape change, activation, apoptosis and clearance. We also review emerging methods for studying these biophysical cues in vitro and existing strategies targeting biophysical cues for mitigating PSLs. We conclude with a perspective on the future direction of biophysics-based strategies for inhibiting PSLs. Platelet storage lesions (PSLs) involve a series of structural and functional changes. It has long been accepted that PSLs are initiated by biochemical cues. Our manuscript is the first to propose four major biophysical cues (shear stress, substrate stiffness, hydrostatic pressure, and thermal microenvironment) that platelets experience in each operation step during platelet preparation and storage processes in vitro , which may synergistically contribute to PSLs. We first clarify these biophysical cues and how they induce PSLs. Strategies targeting each biophysical cue to improve PSLs are also summarized. Our review is designed to draw the attention from a broad range of audience, including clinical doctors, biologists, physical scientists, engineers and materials scientists, and immunologist, who study on platelets physiology and pathology. [Display omitted] [ABSTRACT FROM AUTHOR]