Effects of aging on the biomechanical properties of the lung extracellular matrix: dependence on tissular stretch.

Introduction: Aging induces functional and structural changes in the lung, characterized by a decline in elasticity and diminished pulmonary remodeling and regenerative capacity. Emerging evidence suggests that most biomechanical alterations in the lung result from changes in the composition of the lung extracellular matrix (ECM), potentially modulating the behavior of pulmonary cells and increasing the susceptibility to chronic lung diseases. Therefore, it is crucial to investigate the mechanical properties of the aged lung. This study aims to assess the mechanical alterations in the lung ECM due to aging at both residual (RV) and functional (FV) lung volumes and to evaluate their effects on the survival and proliferation of mesenchymal stromal cells (MSCs). Methods: The lungs from young (4-6-month-old) and aged (20-24-month-old) mice were inflated with optimal cutting temperature compound to reach FV or non-inflated (RV). ECM proteins laminin, collagen I and fibronectin were quantified by immunofluorescence and the mechanical properties of the decellularized lung sections were assessed using atomic force microscopy. To investigate whether changes in ECM composition by aging and/or mechanical properties at RV and FV volumes affects MSCs, their viability and proliferation were evaluated after 72 h. Results: Laminin presence was significantly reduced in aged mice compared to young mice, while fibronectin and collagen I were significantly increased in aged mice. In RV conditions, the acellular lungs from aged mice were significantly softer than from young mice. By contrast, in FV conditions, the aged lung ECM becomes stiffer than that of in young mice, revealing that strain hardening significantly depends on aging. Results after MSCs recellularization showed similar viability and proliferation rate in all conditions. Discussion: This data strongly suggests that biomechanical measurements, especially in aging models, should be carried out in physiomimetic conditions rather than following the conventional non-inflated lung (RV) approach. The use of decellularized lung scaffolds from aged and/or other lung disease murine/human models at physiomimetic conditions will help to better understand the potential role of mechanotransduction on the susceptibility and progression of chronic lung diseases, lung regeneration and cancer.

Low-cost, open-source device for simultaneously subjecting rodents to different circadian cycles of light, food, and temperature.

Exposure of experimental rodents to controlled cycles of light, food, and temperature is important when investigating alterations in circadian cycles that profoundly influence health and disease. However, applying such stimuli simultaneously is difficult in practice. We aimed to design, build, test, and open-source describe a simple device that subjects a conventional mouse cage to independent cycles of physiologically relevant environmental variables. The device is based on a box enclosing the rodent cage to modify the light, feeding, and temperature environments. The device provides temperature-controlled air conditioning (heating or cooling) by a Peltier module and includes programmable feeding and illumination. All functions are set by a user-friendly front panel for independent cycle programming. Bench testing with a model simulating the CO2 production of mice in the cage showed: a) suitable air renewal (by measuring actual ambient CO2), b) controlled realistic illumination at the mouse enclosure (measured by a photometer), c) stable temperature control, and d) correct cycling of light, feeding, and temperature. The cost of all the supplies (retail purchased by e-commerce) was <300 US$. Detailed technical information is open-source provided, allowing for any user to reliably reproduce or modify the device. This approach can considerably facilitate circadian research since using one of the described low-cost devices for any mouse group with a given light-food-temperature paradigm allows for all the experiments to be performed simultaneously, thereby requiring no changes in the light/temperature of a general-use laboratory.

Assessment of the nano-mechanical properties of healthy and atherosclerotic coronary arteries by atomic force microscopy.

Nano-indentation techniques might be better equipped to assess the heterogeneous material properties of plaques than macroscopic methods but there are no bespoke protocols for this kind of material testing for coronary arteries. Therefore, we developed a measurement protocol to extract mechanical properties from healthy and atherosclerotic coronary artery tissue sections. Young's modulus was derived from force-indentation data. Metrics of collagen fibre density were extracted from the same tissue, and the local material properties were co-registered to the local collagen microstructure with a robust framework. The locations of the indentation were retrospectively classified by histological category (healthy, plaque, lipid-rich, fibrous cap) according to Picrosirius Red stain and adjacent Hematoxylin & Eosin and Oil-Red-O stains. Plaque tissue was softer (p < 0.001) than the healthy coronary wall. Areas rich in collagen within the plaque (fibrous cap) were significantly (p < 0.001) stiffer than areas poor in collagen/lipid-rich, but less than half as stiff as the healthy coronary media. Young's moduli correlated (Pearson's ρ = 0.53, p < 0.05) with collagen content. Atomic force microscopy (AFM) is capable of detecting tissue stiffness changes related to collagen density in healthy and diseased cardiovascular tissue. Mechanical characterization of atherosclerotic plaques with nano-indentation techniques could refine constitutive models for computational modelling.

Opposing roles for ADAMTS2 and ADAMTS14 in myofibroblast differentiation and function.

Crosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess cooperative mechanisms in a 3D context, we generated chimeric spheroids using human and mouse cancer and stellate cells. Species-specific deconvolution of bulk-RNA sequencing data revealed cell type-specific transcriptomes underpinning invasion. This dataset highlighted stellate-specific expression of transcripts encoding the collagen-processing enzymes ADAMTS2 and ADAMTS14. Strikingly, loss of ADAMTS2 reduced, while loss of ADAMTS14 promoted, myofibroblast differentiation and invasion independently of their primary role in collagen-processing. Functional and proteomic analysis demonstrated that these two enzymes regulate myofibroblast differentiation through opposing roles in the regulation of transforming growth factor ß availability, acting on the protease-specific substrates, Serpin E2 and fibulin 2, for ADAMTS2 and ADAMTS14, respectively. Showcasing a broader complexity for these enzymes, we uncovered a novel regulatory axis governing malignant behaviour of the pancreatic cancer stroma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cryosectioning of Hydrogels as a Reliable Approach to Increase Yield and Further Tune Mechanical Properties.

Decellularized extracellular matrix (dECM) hydrogels have emerged as promising materials in tissue engineering. The steps to produce dECM hydrogels containing the bioactive epitopes found in the native matrix are often laborious, including the initial harvesting and decellularization of the animal organ. Furthermore, resulting hydrogels often exhibit weak mechanical properties that require the use of additional crosslinkers such as genipin to truly simulate the mechanical properties of the desired study tissue. In this work, we have developed a protocol to readily obtain tens of thin dECM hydrogel cryosections attached to a glass slide as support, to serve as scaffolds for two-dimensional (2D) or three-dimensional (3D) cell culture. Following extensive atomic force microscopy (AFM)-based mechanical characterization of dECM hydrogels crosslinked with increasing genipin concentrations (5 mM, 10 mM, and 20 mM), we provide detailed protocol recommendations for achieving dECM hydrogels of any biologically relevant stiffness. Given that our protocol requires hydrogel freezing, we also confirm that the approach taken can be further used to increase the mechanical properties of the scaffold in a controlled manner exhibiting twice the stiffness in highly crosslinked arrays. Finally, we explored the effect of ethanol-based short- and long-term sterilization on dECM hydrogels, showing that in some situations it may give rise to significant changes in hydrogel mechanical properties that need to be taken into account in experimental design. The hydrogel cryosections produced were shown to be biocompatible and support cell attachment and spreading for at least 72 h in culture. In brief, our proposed method may provide several advantages for tissue engineering: (1) easy availability and reduction in preparation time, (2) increase in the total hydrogel volume eventually used for experiments being able to obtain 15-22 slides from a 250 µL hydrogel) with a (3) reduction in scaffold variability (only a 17.5 ± 9.5% intraslide variability provided by the method), and (4) compatibility with live-cell imaging techniques or further cell characterization of cells.

Characterization of Bioinks Prepared via Gelifying Extracellular Matrix from Decellularized Porcine Myocardia.

Since the emergence of 3D bioprinting technology, both synthetic and natural materials have been used to develop bioinks for producing cell-laden cardiac grafts. To this end, extracellular-matrix (ECM)-derived hydrogels can be used to develop scaffolds that closely mimic the complex 3D environments for cell culture. This study presents a novel cardiac bioink based on hydrogels exclusively derived from decellularized porcine myocardium loaded with human-bone-marrow-derived mesenchymal stromal cells. Hence, the hydrogel can be used to develop cell-laden cardiac patches without the need to add other biomaterials or use additional crosslinkers. The scaffold ultrastructure and mechanical properties of the bioink were characterized to optimize its production, specifically focusing on the matrix enzymatic digestion time. The cells were cultured in 3D within the developed hydrogels to assess their response. The results indicate that the hydrogels fostered inter-cell and cell-matrix crosstalk after 1 week of culture. In conclusion, the bioink developed and presented in this study holds great potential for developing cell-laden customized patches for cardiac repair.

Lung Micrometastases Display ECM Depletion and Softening While Macrometastases Are 30-Fold Stiffer and Enriched in Fibronectin.

Mechanical changes in tumors have long been linked to increased malignancy and therapy resistance and attributed to mechanical changes in the tumor extracellular matrix (ECM). However, to the best of our knowledge, there have been no mechanical studies on decellularized tumors. Here, we studied the biochemical and mechanical progression of the tumor ECM in two models of lung metastases: lung carcinoma (CAR) and melanoma (MEL). We decellularized the metastatic lung sections, measured the micromechanics of the tumor ECM, and stained the sections for ECM proteins, proliferation, and cell death markers. The same methodology was applied to MEL mice treated with the clinically approved anti-fibrotic drug nintedanib. When compared to healthy ECM (~0.40 kPa), CAR and MEL lung macrometastases produced a highly dense and stiff ECM (1.79 ± 1.32 kPa, CAR and 6.39 ± 3.37 kPa, MEL). Fibronectin was overexpressed from the early stages (~118%) to developed macrometastases (~260%) in both models. Surprisingly, nintedanib caused a 4-fold increase in ECM-occupied tumor area (5.1 ± 1.6% to 18.6 ± 8.9%) and a 2-fold in-crease in ECM stiffness (6.39 ± 3.37 kPa to 12.35 ± 5.74 kPa). This increase in stiffness strongly correlated with an increase in necrosis, which reveals a potential link between tumor hypoxia and ECM deposition and stiffness. Our findings highlight fibronectin and tumor ECM mechanics as attractive targets in cancer therapy and support the need to identify new anti-fibrotic drugs to abrogate aberrant ECM mechanics in metastases.

Multi-Step Extracellular Matrix Remodelling and Stiffening in the Development of Idiopathic Pulmonary Fibrosis.

The extracellular matrix (ECM) of the lung is a filamentous network composed mainly of collagens, elastin, and proteoglycans that provides structural and physical support to its populating cells. Proliferation, migration and overall behaviour of those cells is greatly determined by micromechanical queues provided by the ECM. Lung fibrosis displays an aberrant increased deposition of ECM which likely changes filament organization and stiffens the ECM, thus upregulating the profibrotic profile of pulmonary cells. We have previously used AFM to assess changes in the Young's Modulus (E) of the ECM in the lung. Here, we perform further ECM topographical, mechanical and viscoelastic analysis at the micro- and nano-scale throughout fibrosis development. Furthermore, we provide nanoscale correlations between topographical and elastic properties of the ECM fibres. Firstly, we identify a softening of the ECM after rats are instilled with media associated with recovery of mechanical homeostasis, which is hindered in bleomycin-instilled lungs. Moreover, we find opposite correlations between fibre stiffness and roughness in PBS- vs bleomycin-treated lung. Our findings suggest that changes in ECM nanoscale organization take place at different stages of fibrosis, with the potential to help identify pharmacological targets to hinder its progression.

A Fast and Efficient Decellularization Method for Tissue Slices.

The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue's vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 µm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2-3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies. This protocol was validated in: Front Bioeng Biotechnol (2022), DOI: 10.3389/fbioe.2022.832178.

Lung Extracellular Matrix Hydrogels-Derived Vesicles Contribute to Epithelial Lung Repair.

The use of physiomimetic decellularized extracellular matrix-derived hydrogels is attracting interest since they can modulate the therapeutic capacity of numerous cell types, including mesenchymal stromal cells (MSCs). Remarkably, extracellular vesicles (EVs) derived from MSCs display similar functions as their parental cells, mitigating tissue damage in lung diseases. However, recent data have shown that ECM-derived hydrogels could release other resident vesicles similar to EVs. Here, we aim to better understand the contribution of EVs and ECM-vesicles released from MSCs and/or lung-derived hydrogel (L-HG) in lung repair by using an in vitro lung injury model. L-HG derived-vesicles and MSCs EVs cultured either in L-HG or conventional plates were isolated and characterized. The therapeutic capacity of vesicles obtained from each experimental condition was tested by using an alveolar epithelial wound-healing assay. The number of ECM-vesicles released from acellular L-HG was 10-fold greater than EVs from conventional MSCs cell culture revealing that L-HG is an important source of bioactive vesicles. MSCs-derived EVs and L-HG vesicles have similar therapeutic capacity in lung repair. However, when wound closure rate was normalized by total proteins, the MSCs-derived EVs shows higher therapeutic potential to those released by L-HG. The EVs released from L-HG must be considered when HG is used as substrate for cell culture and EVs isolation.

Development of a physiomimetic model of acute respiratory distress syndrome by using ECM hydrogels and organ-on-a-chip devices.

Acute Respiratory Distress Syndrome is one of the more common fatal complications in COVID-19, characterized by a highly aberrant inflammatory response. Pre-clinical models to study the effect of cell therapy and anti-inflammatory treatments have not comprehensively reproduced the disease due to its high complexity. This work presents a novel physiomimetic in vitro model for Acute Respiratory Distress Syndrome using lung extracellular matrix-derived hydrogels and organ-on-a-chip devices. Monolayres of primary alveolar epithelial cells were cultured on top of decellullarized lung hydrogels containing primary lung mesenchymal stromal cells. Then, cyclic stretch was applied to mimic breathing, and an inflammatory response was induced by using a bacteriotoxin hit. Having simulated the inflamed breathing lung environment, we assessed the effect of an anti-inflammatory drug (i.e., dexamethasone) by studying the secretion of the most relevant inflammatory cytokines. To better identify key players in our model, the impact of the individual factors (cyclic stretch, decellularized lung hydrogel scaffold, and the presence of mesenchymal stromal cells) was studied separately. Results showed that developed model presented a more reduced inflammatory response than traditional models, which is in line with what is expected from the response commonly observed in patients. Further, from the individual analysis of the different stimuli, it was observed that the use of extracellular matrix hydrogels obtained from decellularized lungs had the most significant impact on the change of the inflammatory response. The developed model then opens the door for further in vitro studies with a better-adjusted response to the inflammatory hit and more robust results in the test of different drugs or cell therapy.

Lung Extracellular Matrix Hydrogels Enhance Preservation of Type II Phenotype in Primary Alveolar Epithelial Cells.

One of the main limitations of in vitro studies on lung diseases is the difficulty of maintaining the type II phenotype of alveolar epithelial cells in culture. This fact has previously been related to the translocation of the mechanosensing Yes-associated protein (YAP) to the nuclei and Rho signaling pathway. In this work, we aimed to culture and subculture primary alveolar type II cells on extracellular matrix lung-derived hydrogels to assess their suitability for phenotype maintenance. Cells cultured on lung hydrogels formed monolayers and maintained type II phenotype for a longer time as compared with those conventionally cultured. Interestingly, cells successfully grew when they were subsequently cultured on a dish. Moreover, cells cultured on a plate showed the active form of the YAP protein and the formation of stress fibers and focal adhesions. The results of chemically inhibiting the Rho pathway strongly suggest that this is one of the mechanisms by which the hydrogel promotes type II phenotype maintenance. These results regarding protein expression strongly suggest that the chemical and biophysical properties of the hydrogel have a considerable impact on the transition from ATII to ATI phenotypes. In conclusion, culturing primary alveolar epithelial cells on lung ECM-derived hydrogels may facilitate the prolonged culturing of these cells, and thus help in the research on lung diseases.

Novel Decellularization Method for Tissue Slices.

Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue's vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices-with an emphasis on lung-while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies.

Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion.

In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) can both promote and suppress tumor progression. Here, we show that the Rho effector protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 is associated with reduced PSC proliferation, contractility, and alpha-smooth muscle actin (α-SMA) stress fibers. In spheroid co-cultures with PDAC cells, loss of PKN2 prevents PSC invasion but, counter-intuitively, promotes invasive cancer cell outgrowth. PKN2 deletion induces a myofibroblast to inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo. Further, deletion of PKN2 in the pancreatic stroma induces more locally invasive, orthotopic pancreatic tumors. Finally, we demonstrate that a PKN2KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast function can limit important stromal tumor-suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.

Fast cycling of intermittent hypoxia in a physiomimetic 3D environment: A novel tool for the study of the parenchymal effects of sleep apnea.

Background: Patients with obstructive sleep apnea (OSA) experience recurrent hypoxemic events with a frequency sometimes exceeding 60 events/h. These episodic events induce downstream transient hypoxia in the parenchymal tissue of all organs, thereby eliciting the pathological consequences of OSA. Whereas experimental models currently apply intermittent hypoxia to cells conventionally cultured in 2D plates, there is no well-characterized setting that will subject cells to well-controlled intermittent hypoxia in a 3D environment and enable the study of the effects of OSA on the cells of interest while preserving the underlying tissue environment. Aim: To design and characterize an experimental approach that exposes cells to high-frequency intermittent hypoxia mimicking OSA in 3D (hydrogels or tissue slices). Methods: Hydrogels made from lung extracellular matrix (L-ECM) or brain tissue slices (300-800-µm thickness) were placed on a well whose bottom consisted of a permeable silicone membrane. The chamber beneath the membrane was subjected to a square wave of hypoxic/normoxic air. The oxygen concentration at different depths within the hydrogel/tissue slice was measured with an oxygen microsensor. Results: 3D-seeded cells could be subjected to well-controlled and realistic intermittent hypoxia patterns mimicking 60 apneas/h when cultured in L-ECM hydrogels ≈500 µm-thick or ex-vivo in brain slices 300-500 µm-thick. Conclusion: This novel approach will facilitate the investigation of the effects of intermittent hypoxia simulating OSA in 3D-residing cells within the parenchyma of different tissues/organs.

Baseline Stiffness Modulates the Non-Linear Response to Stretch of the Extracellular Matrix in Pulmonary Fibrosis.

Pulmonary fibrosis (PF) is a progressive disease that disrupts the mechanical homeostasis of the lung extracellular matrix (ECM). These effects are particularly relevant in the lung context, given the dynamic nature of cyclic stretch that the ECM is continuously subjected to during breathing. This work uses an in vivo model of pulmonary fibrosis to characterize the macro- and micromechanical properties of lung ECM subjected to stretch. To that aim, we have compared the micromechanical properties of fibrotic ECM in baseline and under stretch conditions, using a novel combination of Atomic Force Microscopy (AFM) and a stretchable membrane-based chip. At the macroscale, fibrotic ECM displayed strain-hardening, with a stiffness one order of magnitude higher than its healthy counterpart. Conversely, at the microscale, we found a switch in the stretch-induced mechanical behaviour of the lung ECM from strain-hardening at physiological ECM stiffnesses to strain-softening at fibrotic ECM stiffnesses. Similarly, we observed solidification of healthy ECM versus fluidization of fibrotic ECM in response to stretch. Our results suggest that the mechanical behaviour of fibrotic ECM under stretch involves a potential built-in mechanotransduction mechanism that may slow down the progression of PF by steering resident fibroblasts away from a pro-fibrotic profile.

Image-Based Method to Quantify Decellularization of Tissue Sections.

Tissue decellularization is typically assessed through absorbance-based DNA quantification after tissue digestion. This method has several disadvantages, namely its destructive nature and inadequacy in experimental situations where tissue is scarce. Here, we present an image processing algorithm for quantitative analysis of DNA content in (de)cellularized tissues as a faster, simpler and more comprehensive alternative. Our method uses local entropy measurements of a phase contrast image to create a mask, which is then applied to corresponding nuclei labelled (UV) images to extract average fluorescence intensities as an estimate of DNA content. The method can be used on native or decellularized tissue to quantify DNA content, thus allowing quantitative assessment of decellularization procedures. We confirm that our new method yields results in line with those obtained using the standard DNA quantification method and that it is successful for both lung and heart tissues. We are also able to accurately obtain a timeline of decreasing DNA content with increased incubation time with a decellularizing agent. Finally, the identified masks can also be applied to additional fluorescence images of immunostained proteins such as collagen or elastin, thus allowing further image-based tissue characterization.

Research Techniques Made Simple: Analysis of Skin Cell and Tissue Mechanics Using Atomic Force Microscopy.

Atomic force microscopy (AFM) is a powerful technique for nanoscale imaging and mechanical analysis of biological specimens. It is based on the highly sensitive detection of forces and displacement of a sharp-tipped cantilever as it scans the surface of an object. Because it requires minimal sample processing and preparation, AFM is particularly advantageous for the analysis of cells and tissues in their near-native state. Moreover, recent advances in Bio-AFM systems and the combination with light microscopy imaging have greatly enhanced the application of AFM in biological research. In the field of dermatology, the method has led to important insights into our understanding of the biomechanics of normal healthy skin and the pathogenesis of a variety of skin diseases. In this Research Techniques Made Simple article, we review the fundamental principles of AFM, how AFM can be applied to the analysis of cell and tissue mechanics, and recent applications of AFM in skin science and dermatology.

The Mechanical Interplay Between Differentiating Mesenchymal Stem Cells and Gelatin-Based Substrates Measured by Atomic Force Microscopy.

Traditional methods to assess hMSCs differentiation typically require long-term culture until cells show marked expression of histological markers such as lipid accumulation inside the cytoplasm or mineral deposition onto the surrounding matrix. In parallel, stem cell differentiation has been shown to involve the reorganization of the cell's cytoskeleton shortly after differentiation induced by soluble factors. Given the cytoskeleton's role in determining the mechanical properties of adherent cells, the mechanical characterization of stem cells could thus be a potential tool to assess cellular commitment at much earlier time points. In this study, we measured the mechanical properties of hMSCs cultured on soft gelatin-based hydrogels at multiple time points after differentiation induction toward adipogenic or osteogenic lineages. Our results show that the mechanical properties of cells (stiffness and viscosity) and the organization of the actin cytoskeleton are highly correlated with lineage commitment. Most importantly, we also found that the mechanical properties and the topography of the gelatin substrate in the vicinity of the cells are also altered as differentiation progresses toward the osteogenic lineage, but not on the adipogenic case. Together, these results confirm the biophysical changes associated with stem cell differentiation and suggest a mechanical interplay between the differentiating stem cells and their surrounding extracellular matrix.

The keratin network of intermediate filaments regulates keratinocyte rigidity sensing and nuclear mechanotransduction.

The keratin network of intermediate filaments provides keratinocytes with essential mechanical strength and resilience, but the contribution to mechanosensing remains poorly understood. Here, we investigated the role of the keratin cytoskeleton in the response to altered matrix rigidity. We found that keratinocytes adapted to increasing matrix stiffness by forming a rigid, interconnected network of keratin bundles, in conjunction with F-actin stress fiber formation and increased cell stiffness. Disruption of keratin stability by overexpression of the dominant keratin 14 mutation R416P inhibited the normal mechanical response to substrate rigidity, reducing F-actin stress fibers and cell stiffness. The R416P mutation also impaired mechanotransduction to the nuclear lamina, which mediated stiffness-dependent chromatin remodeling. By contrast, depletion of the cytolinker plectin had the opposite effect and promoted increased mechanoresponsiveness and up-regulation of lamin A/C. Together, these results demonstrate that the keratin cytoskeleton plays a key role in matrix rigidity sensing and downstream signal transduction.