Publications by authors named "Paul A Janmey"

209 Publications

Conductive chitosan/polyaniline hydrogel with cell-imprinted topography as a potential substrate for neural priming of adipose derived stem cells.

RSC Adv 2021 Apr 28;11(26):15795-15807. Epub 2021 Apr 28.

Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences Tehran Iran.

Biophysical characteristics of engineered scaffolds such as topography and electroconductivity have shown potentially beneficial effects on stem cell morphology, proliferation, and differentiation toward neural cells. In this study, we fabricated a conductive hydrogel made from chitosan (CS) and polyaniline (PANI) with induced PC12 cell surface topography using a cell imprinting technique to provide both topographical properties and conductivity in a platform. The engineered hydrogel's potential for neural priming of rat adipose-derived stem cells (rADSCs) was determined . The biomechanical analysis revealed that the electrical conductivity, stiffness, and hydrophobicity of flat (F) and cell-imprinted (CI) substrates increased with increased PANI content in the CS/PANI scaffold. The conductive substrates exhibited a lower degradation rate compared to non-conductive substrates. According to data obtained from F-actin staining and AFM micrographs, both CI(CS) and CI(CS-PANI) substrates induced the morphology of rADSCs from their irregular shape (on flat substrates) into the elongated and bipolar shape of the neuronal-like PC12 cells. Immunostaining analysis revealed that both CI(CS) and CI (CS-PANI) significantly upregulated the expression of GFAP and MAP2, two neural precursor-specific genes, in rADSCs compared with flat substrates. Although the results reveal that both cell-imprinted topography and electrical conductivity affect the neural lineage differentiation, some data demonstrate that the topography effects of the cell-imprinted surface have a more critical role than electrical conductivity on neural priming of ADSCs. The current study provides new insight into the engineering of scaffolds for nerve tissue engineering.
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http://dx.doi.org/10.1039/d1ra00413aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9029165PMC
April 2021

Microindentation of Fluid-Filled Cellular Domes Reveals the Contribution of RhoA-ROCK Signaling to Multicellular Mechanics.

Small 2022 Apr 21:e2200883. Epub 2022 Apr 21.

Department of Biomedical Engineering, Rowan University, Glassboro, NJ, 08028, USA.

Cellular mechanics encompass both mechanical properties that resist forces applied by the external environment and internally generated forces applied at the location of cell-cell and cell-matrix junctions. Here, the authors demonstrate that microindentation of cellular domes formed by cell monolayers that locally lift off the substrate provides insight into both aspects of cellular mechanics in multicellular structures. Using a modified Hertz contact equation, the force-displacement curves generated by a micro-tensiometer are used to measure an effective dome stiffness. The results indicate the domes are consistent with the Laplace-Young relationship for elastic membranes, regardless of biochemical modulation of the RhoA-ROCK signaling axis. In contrast, activating RhoA, and inhibiting ROCK both alter the relaxation dynamics of the domes deformed by the micro-tensiometer, revealing an approach to interrogate the role of RhoA-ROCK signaling in multicellular mechanics. A finite element model incorporating a Mooney-Rivlin hyperelastic constitutive equation to describe monolayer mechanics predicts effective stiffness values that are consistent with the micro-tensiometer measurements, verifying previous measurements of the response of cell monolayers to tension. Overall, these studies establish microindentation of fluid-filled domes as an avenue to investigate the contribution of cell-generated forces to the mechanics of multicellular structures.
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http://dx.doi.org/10.1002/smll.202200883DOI Listing
April 2022

Glycosaminoglycans modulate long-range mechanical communication between cells in collagen networks.

Proc Natl Acad Sci U S A 2022 04 8;119(15):e2116718119. Epub 2022 Apr 8.

Center for Engineering MechanoBiology, University of Pennsylvania, Philadelphia, PA 19104.

Cells can sense and respond to mechanical forces in fibrous extracellular matrices (ECMs) over distances much greater than their size. This phenomenon, termed long-range force transmission, is enabled by the realignment (buckling) of collagen fibers along directions where the forces are tensile (compressive). However, whether other key structural components of the ECM, in particular glycosaminoglycans (GAGs), can affect the efficiency of cellular force transmission remains unclear. Here we developed a theoretical model of force transmission in collagen networks with interpenetrating GAGs, capturing the competition between tension-driven collagen fiber alignment and the swelling pressure induced by GAGs. Using this model, we show that the swelling pressure provided by GAGs increases the stiffness of the collagen network by stretching the fibers in an isotropic manner. We found that the GAG-induced swelling pressure can help collagen fibers resist buckling as the cells exert contractile forces. This mechanism impedes the alignment of collagen fibers and decreases long-range cellular mechanical communication. We experimentally validated the theoretical predictions by comparing the intensity of collagen fiber alignment between cellular spheroids cultured on collagen gels versus collagen–GAG cogels. We found significantly lower intensities of aligned collagen in collagen–GAG cogels, consistent with the prediction that GAGs can prevent collagen fiber alignment. The role of GAGs in modulating force transmission uncovered in this work can be extended to understand pathological processes such as the formation of fibrotic scars and cancer metastasis, where cells communicate in the presence of abnormally high concentrations of GAGs.
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http://dx.doi.org/10.1073/pnas.2116718119DOI Listing
April 2022

Materials science and mechanosensitivity of living matter.

Appl Phys Rev 2022 Mar;9(1):011320

Institute for Medicine and Engineering and Departments of Physiology and Physics & Astronomy, University of Pennsylvania, Philadelphia PA, 19104, USA.

Living systems are composed of molecules that are synthesized by cells that use energy sources within their surroundings to create fascinating materials that have mechanical properties optimized for their biological function. Their functionality is a ubiquitous aspect of our lives. We use wood to construct furniture, bacterial colonies to modify the texture of dairy products and other foods, intestines as violin strings, bladders in bagpipes, and so on. The mechanical properties of these biological materials differ from those of other simpler synthetic elastomers, glasses, and crystals. Reproducing their mechanical properties synthetically or from first principles is still often unattainable. The challenge is that biomaterials often exist far from equilibrium, either in a kinetically arrested state or in an energy consuming active state that is not yet possible to reproduce de novo. Also, the design principles that form biological materials often result in nonlinear responses of stress to strain, or force to displacement, and theoretical models to explain these nonlinear effects are in relatively early stages of development compared to the predictive models for rubberlike elastomers or metals. In this Review, we summarize some of the most common and striking mechanical features of biological materials and make comparisons among animal, plant, fungal, and bacterial systems. We also summarize some of the mechanisms by which living systems develop forces that shape biological matter and examine newly discovered mechanisms by which cells sense and respond to the forces they generate themselves, which are resisted by their environment, or that are exerted upon them by their environment. Within this framework, we discuss examples of how physical methods are being applied to cell biology and bioengineering.
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http://dx.doi.org/10.1063/5.0071648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8969880PMC
March 2022

Watching a cell sheet transform from soft to stiff.

Biophys J 2022 Apr 11;121(8):1349-1351. Epub 2022 Mar 11.

Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania; Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

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http://dx.doi.org/10.1016/j.bpj.2022.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9072575PMC
April 2022

N-Acetyl-Cysteine Increases Activity of Peanut-Shaped Gold Nanoparticles Against Biofilms Formed by Clinical Strains of Isolated from Sputum of Cystic Fibrosis Patients.

Infect Drug Resist 2022 5;15:851-871. Epub 2022 Mar 5.

Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Białystok, Białystok, 15-222, Poland.

Background: Extracellular polymeric substances (EPS) produced by bacteria, as they form a biofilm, determine the stability and viscoelastic properties of biofilms and prevent antibiotics from penetrating this multicellular structure. To date, studies demonstrated that an appropriate optimization of the chemistry and morphology of nanotherapeutics might provide a favorable approach to control their interaction with EPS and/or diffusion within the biofilm matrix. Targeting the biofilms' EPS, which in certain conditions can adopt liquid crystal structure, was demonstrated to improve the anti-biofilm activity of antibiotics and nanoparticles. A similar effect is achievable by interfering EPS' production by mucoactive agents, such as N-acetyl-cysteine (NAC). In our previous study, we demonstrated the nanogram efficiency of non-spherical gold nanoparticles, which due to their physicochemical features, particularly morphology, were noted to be superior in antimicrobial activity compared to their spherical-shaped counterparts.

Methods: To explore the importance of EPS matrix modulation in achieving a suitable efficiency of peanut-shaped gold nanoparticles (AuP NPs) against biofilms produced by strains isolated from cystic fibrosis patients, fluorescence microscopy, as well as resazurin staining were employed. Rheological parameters of AuP NPs-treated biofilms were investigated by rotational and creep-recovery tests using a rheometer in a plate-plate arrangement.

Results: We demonstrated that tested nanoparticles significantly inhibit the growth of mono- and mixed-species biofilms, particularly when combined with NAC. Notably, gold nanopeanuts were shown to decrease the viscosity and increase the creep compliance of biofilm, similarly to EPS-targeting NAC. Synergistic activity of AuP NPs with tobramycin was also observed, and the AuP NPs were able to eradicate bacteria within biofilms formed by tobramycin-resistant isolates.

Conclusion: We propose that peanut-shaped gold nanoparticles should be considered as a potent therapeutic agent against biofilms.
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http://dx.doi.org/10.2147/IDR.S348357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8906902PMC
March 2022

Liquid-Liver Phantom: Mimicking the Viscoelastic Dispersion of Human Liver for Ultrasound- and MRI-Based Elastography.

Invest Radiol 2022 Feb 21. Epub 2022 Feb 21.

From the Department of Radiology Institute of Medical Informatics, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin Department of Engineering and Natural Sciences, University of Applied Sciences Merseburg, Merseburg, Germany Medical Ultrasound Imaging Center, Department of Medical Imaging, Radboud University Medical Center, Nijmegen, the Netherlands Institute for Medicine and Engineering, Center for Engineering Mechanobiology, and Department of Physiology, University of Pennsylvania, Philadelphia, PA.

Objectives: Tissue stiffness can guide medical diagnoses and is exploited as an imaging contrast in elastography. However, different elastography devices show different liver stiffness values in the same subject, hindering comparison of values and establishment of system-independent thresholds for disease detection. There is a need for standardized phantoms that specifically address the viscosity-related dispersion of stiffness over frequency. To improve standardization of clinical elastography across devices and platforms including ultrasound and magnetic resonance imaging (MRI), a comprehensively characterized phantom is introduced that mimics the dispersion of stiffness of the human liver and can be generated reproducibly.

Materials And Methods: The phantom was made of linear polymerized polyacrylamide (PAAm) calibrated to the viscoelastic properties of healthy human liver in vivo as reported in the literature. Stiffness dispersion was analyzed using the 2-parameter springpot model fitted to the dispersion of shear wave speed of PAAm, which was measured by shear rheometry, ultrasound-based time-harmonic elastography, clinical magnetic resonance elastography (MRE), and tabletop MRE in the frequency range of 5 to 3000 Hz. Imaging parameters for ultrasound and MRI, reproducibility, aging behavior, and temperature dependency were assessed. In addition, the frequency bandwidth of shear wave speed of clinical elastography methods (Aplio i900, Canon; Acuson Sequoia, Siemens; FibroScan, EchoSense) was characterized.

Results: Within the entire frequency range analyzed in this study, the PAAm phantom reproduced well the stiffness dispersion of human liver in vivo despite its fluid properties under static loading (springpot stiffness parameter, 2.14 [95% confidence interval, 2.08-2.19] kPa; springpot powerlaw exponent, 0.367 [95% confidence interval, 0.362-0.373]). Imaging parameters were close to those of liver in vivo with only slight variability in stiffness values of 0.5% (0.4%, 0.6%), 4.1% (3.9%, 4.5%), and -0.63% (-0.67%, -0.58%), respectively, between batches, over a 6-month period, and per °C increase in temperature.

Conclusions: The liquid-liver phantom has useful properties for standardization and development of liver elastography. First, it can be used across clinical and experimental elastography devices in ultrasound and MRI. Second, being a liquid, it can easily be adapted in size and shape to specific technical requirements, and by adding inclusions and scatterers. Finally, because the phantom is based on noncrosslinked linear PAAm constituents, it is easy to produce, indicating potential widespread use among researchers and vendors to standardize liver stiffness measurements.
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http://dx.doi.org/10.1097/RLI.0000000000000862DOI Listing
February 2022

Extracellular Vimentin as a Target Against SARS-CoV-2 Host Cell Invasion.

Small 2022 02 5;18(6):e2105640. Epub 2021 Dec 5.

Physics Department and BioInspired Institute, Syracuse University, 140 Sims Dr, Syracuse, NY, 13244, USA.

Infection of human cells by pathogens, including SARS-CoV-2, typically proceeds by cell surface binding to a crucial receptor. The primary receptor for SARS-CoV-2 is the angiotensin-converting enzyme 2 (ACE2), yet new studies reveal the importance of additional extracellular co-receptors that mediate binding and host cell invasion by SARS-CoV-2. Vimentin is an intermediate filament protein that is increasingly recognized as being present on the extracellular surface of a subset of cell types, where it can bind to and facilitate pathogens' cellular uptake. Biophysical and cell infection studies are done to determine whether vimentin might bind SARS-CoV-2 and facilitate its uptake. Dynamic light scattering shows that vimentin binds to pseudovirus coated with the SARS-CoV-2 spike protein, and antibodies against vimentin block in vitro SARS-CoV-2 pseudovirus infection of ACE2-expressing cells. The results are consistent with a model in which extracellular vimentin acts as a co-receptor for SARS-CoV-2 spike protein with a binding affinity less than that of the spike protein with ACE2. Extracellular vimentin may thus serve as a critical component of the SARS-CoV-2 spike protein-ACE2 complex in mediating SARS-CoV-2 cell entry, and vimentin-targeting agents may yield new therapeutic strategies for preventing and slowing SARS-CoV-2 infection.
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http://dx.doi.org/10.1002/smll.202105640DOI Listing
February 2022

Programmable and contractile materials through cell encapsulation in fibrous hydrogel assemblies.

Sci Adv 2021 Nov 10;7(46):eabi8157. Epub 2021 Nov 10.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

The natural extracellular matrix (ECM) within tissues is physically contracted and remodeled by cells, allowing the collective shaping of functional tissue architectures. Synthetic materials that facilitate self-assembly similar to natural ECM are needed for cell culture, tissue engineering, and in vitro models of development and disease. To address this need, we develop fibrous hydrogel assemblies that are stabilized with photocrosslinking and display fiber density–dependent strain-responsive properties (strain stiffening and alignment). Encapsulated mesenchymal stromal cells locally contract low fiber density assemblies, resulting in macroscopic volumetric changes with increased cell densities and moduli. Because of properties such as shear-thinning and self-healing, assemblies can be processed into microtissues with aligned ECM deposition or through extrusion bioprinting and photopatterning to fabricate constructs with programmed shape changes due to cell contraction. These materials provide a synthetic approach to mimic features of natural ECM, which can now be processed for applications in biofabrication and tissue engineering.
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http://dx.doi.org/10.1126/sciadv.abi8157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8580309PMC
November 2021

Physics and Physiology of Cell Spreading in Two and Three Dimensions.

Physiology (Bethesda) 2021 11;36(6):382-391

Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.

Cells spread on surfaces and within three-dimensional (3-D) matrixes as they grow, divide, and move. Both chemical and physical signals orchestrate spreading during normal development, wound healing, and pathological states such as fibrosis and tumor growth. Diverse molecular mechanisms drive different forms of cell spreading. This article discusses mechanisms by which cells spread in 2-D and 3-D and illustrates new directions in studies of this aspect of cell function.
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http://dx.doi.org/10.1152/physiol.00020.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8560373PMC
November 2021

A Novel Method to Make Polyacrylamide Gels with Mechanical Properties Resembling those of Biological Tissues.

Bio Protoc 2021 Aug 20;11(16):e4131. Epub 2021 Aug 20.

Department of Physiology, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA.

Studies characterizing how cells respond to the mechanical properties of their environment have been enabled by the use of soft elastomers and hydrogels as substrates for cell culture. A limitation of most such substrates is that, although their elastic properties can be accurately controlled, their viscous properties cannot, and cells respond to both elasticity and viscosity in the extracellular material to which they bind. Some approaches to endow soft substrates with viscosity as well as elasticity are based on coupling static and dynamic crosslinks in series within polymer networks or forming gels with a combination of sparse chemical crosslinks and steric entanglements. These materials form viscoelastic fluids that have revealed significant effects of viscous dissipation on cell function; however, they do not completely capture the mechanical features of soft solid tissues. In this report, we describe a method to make viscoelastic solids that more closely mimic some soft tissues using a combination of crosslinked networks and entrapped linear polymers. Both the elastic and viscous moduli of these substrates can be altered separately, and methods to attach cells to either the elastic or the viscous part of the network are described. Graphic abstract: Polyacrylamide gels with independently controlled elasticity and viscosity.
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http://dx.doi.org/10.21769/BioProtoc.4131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8413537PMC
August 2021

The Atr-Chek1 pathway inhibits axon regeneration in response to Piezo-dependent mechanosensation.

Nat Commun 2021 06 22;12(1):3845. Epub 2021 Jun 22.

Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.

Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. We show that in Drosophila sensory neurons removing Atr or Chek1, or overexpressing Cdc25 promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury. Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhances regeneration. Our findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma.
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http://dx.doi.org/10.1038/s41467-021-24131-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8219705PMC
June 2021

Membrane signalosome: where biophysics meets systems biology.

Curr Opin Syst Biol 2021 Mar 25;25:34-41. Epub 2021 Feb 25.

Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA.

We opine on the recent advances in experiments and modeling of modular signaling complexes assembled on mammalian cell membranes (membrane signalosomes) in the context of several applications including intracellular trafficking, cell migration, and immune response. Characterizing the individual components of the membrane assemblies at the nanoscale, ranging from protein-lipid and protein-protein interactions, to membrane morphology, and the energetics of emergent assemblies at the subcellular to cellular scales pose significant challenges. Overcoming these challenges through the iterative coupling of multiscale modeling and experiment can be transformative in terms of addressing the gaps between structural biology and super-resolution microscopy, as it holds the key to the discovery of fundamental mechanisms behind the emergence of function in the membrane signalosome.
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http://dx.doi.org/10.1016/j.coisb.2021.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8117111PMC
March 2021

Polyelectrolyte Gels Formed by Filamentous Biopolymers: Dependence of Crosslinking Efficiency on the Chemical Softness of Divalent Cations.

Gels 2021 Apr 8;7(2). Epub 2021 Apr 8.

Department of Physiology, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19063, USA.

Filamentous anionic polyelectrolytes are common in biological materials. Some examples are the cytoskeletal filaments that assemble into networks and bundled structures to give the cell mechanical resistance and that act as surfaces on which enzymes and other molecules can dock. Some viruses, especially bacteriophages are also long thin polyelectrolytes, and their bending stiffness is similar to those of the intermediate filament class of cytoskeletal polymers. These relatively stiff, thin, and long polyelectrolytes have charge densities similar to those of more flexible polyelectrolytes such as DNA, hyaluronic acid, and polyacrylates, and they can form interpenetrating networks and viscoelastic gels at volume fractions far below those at which more flexible polymers form hydrogels. In this report, we examine how different types of divalent and multivalent counterions interact with two biochemically different but physically similar filamentous polyelectrolytes: Pf1 virus and vimentin intermediate filaments (VIF). Different divalent cations aggregate both polyelectrolytes similarly, but transition metal ions are more efficient than alkaline earth ions and their efficiency increases with increasing atomic weight. Comparison of these two different types of polyelectrolyte filaments enables identification of general effects of counterions with polyelectrolytes and can identify cases where the interaction of the counterions and the filaments exhibits stronger and more specific interactions than those of counterion condensation.
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http://dx.doi.org/10.3390/gels7020041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167600PMC
April 2021

Dynamic Tuning of Viscoelastic Hydrogels with Carbonyl Iron Microparticles Reveals the Rapid Response of Cells to Three-Dimensional Substrate Mechanics.

ACS Appl Mater Interfaces 2021 May 28;13(18):20947-20959. Epub 2021 Apr 28.

Department of Biomedical Engineering, Rowan University, Glassboro, New Jersey 08028, United States.

Current methods to dynamically tune three-dimensional hydrogel mechanics require specific chemistries and substrates that make modest, slow, and often irreversible changes in their mechanical properties, exclude the use of protein-based scaffolds, or alter the hydrogel microstructure and pore size. Here, we rapidly and reversibly alter the mechanical properties of hydrogels consisting of extracellular matrix proteins and proteoglycans by adding carbonyl iron microparticles (MPs) and applying external magnetic fields. This approach drastically alters hydrogel mechanics: rheology reveals that application of a 4000 Oe magnetic field to a 5 mg/mL collagen hydrogel containing 10 wt % MPs increases the storage modulus from approximately 1.5 to 30 kPa. Cell morphology experiments show that cells embedded within these hydrogels rapidly sense the magnetically induced changes in ECM stiffness. Ca transients are altered within seconds of stiffening or subsequent softening, and slower but still dynamic changes occur in YAP nuclear translocation in response to time-dependent application of a magnetic field. The near instantaneous change in hydrogel mechanics provides new insight into the effect of changing extracellular stiffness on both acute and chronic changes in diverse cell types embedded in protein-based scaffolds. Due to its flexibility, this method is broadly applicable to future studies interrogating cell mechanotransduction in three-dimensional substrates.
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http://dx.doi.org/10.1021/acsami.0c21868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8317442PMC
May 2021

Vimentin tunes cell migration on collagen by controlling β1 integrin activation and clustering.

J Cell Sci 2021 03 29;134(6). Epub 2021 Mar 29.

Faculty of Dentistry, University of Toronto, Toronto, ON M5G 1G6, Canada.

Vimentin is a structural protein that is required for mesenchymal cell migration and directly interacts with actin, β1 integrin and paxillin. We examined how these interactions enable vimentin to regulate cell migration on collagen. In fibroblasts, depletion of vimentin increased talin-dependent activation of β1 integrin by more than 2-fold. Loss of vimentin was associated with reduction of β1 integrin clustering by 50% and inhibition of paxillin recruitment to focal adhesions by more than 60%, which was restored by vimentin expression. This reduction of paxillin was associated with 65% lower Cdc42 activation, a 60% reduction of cell extension formation and a greater than 35% decrease in cell migration on collagen. The activation of PAK1, a downstream effector of Cdc42, was required for vimentin phosphorylation and filament maturation. We propose that vimentin tunes cell migration through collagen by acting as an adaptor protein for focal adhesion proteins, thereby regulating β1 integrin activation, resulting in well-organized, mature integrin clusters.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.254359DOI Listing
March 2021

In search of the correlation between nanomechanical and biomolecular properties of prostate cancer cells with different metastatic potential.

Arch Biochem Biophys 2021 01 6;697:108718. Epub 2020 Dec 6.

Institute of Nuclear Physics Polish Academy of Sciences, Krakow, PL-31342, Poland.

Nanomechanical properties of living cells, as measured with atomic force microscopy (AFM), are increasingly recognized as criteria that differentiate normal and pathologically altered cells. Locally measured cell elastic properties, described by the parameter known as Young's modulus, are currently proposed as a new diagnostic parameter that can be used at the early stage of cancer detection. In this study, local mechanical properties of normal human prostate (RWPE-1) cells and a range of malignant (22Rv1) and metastatic prostate cells (LNCaP, Du145 and PC3) were investigated. It was found that non-malignant prostate cells are stiffer than cancer cells while the metastatic cells are much softer than malignant cells from the primary tumor site. Next, the biochemical properties of the cells were measured using confocal Raman (RS) and Fourier-transform infrared (FT-IR) spectroscopies to reveal these cells' biochemical composition as malignant transformation proceeds. Nanomechanical and biochemical profiles of five different prostate cell lines were subsequently analyzed using partial least squares regression (PLSR) in order to identify which spectral features of the RS and FT-IR spectra correlate with the cell's elastic properties. The PLSR-based model could predict Young's modulus values based on both RS and FT-IR spectral information. These outcomes show not only that AFM, RS and FT-IR techniques can be used for discrimination between normal and cancer cells, but also that a linear correlation between mechanical response and biomolecular composition of the cells that undergo malignant transformation can be found. This knowledge broadens our understanding of how prostate cancer cells evolve thorough the multistep process of tumor pathogenesis.
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http://dx.doi.org/10.1016/j.abb.2020.108718DOI Listing
January 2021

Correction: Long-range mechanical signaling in biological systems.

Soft Matter 2021 Jan;17(2):410

Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA 19104, USA. and Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104, USA.

Correction for 'Long-range mechanical signaling in biological systems' by Farid Alisafaei et al., Soft Matter, 2020, DOI: 10.1039/d0sm01442g.
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http://dx.doi.org/10.1039/d0sm90235gDOI Listing
January 2021

Long-range mechanical signaling in biological systems.

Soft Matter 2021 Jan;17(2):241-253

Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA 19104, USA. and Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104, USA.

Cells can respond to signals generated by other cells that are remarkably far away. Studies from at least the 1920's showed that cells move toward each other when the distance between them is on the order of a millimeter, which is many times the cell diameter. Chemical signals generated by molecules diffusing from the cell surface would move too slowly and dissipate too fast to account for these effects, suggesting that they might be physical rather than biochemical. The non-linear elastic responses of sparsely connected networks of stiff or semiflexible filament such as those that form the extracellular matrix (ECM) and the cytoskeleton have unusual properties that suggest multiple mechanisms for long-range signaling in biological tissues. These include not only direct force transmission, but also highly non-uniform local deformations, and force-generated changes in fiber alignment and density. Defining how fibrous networks respond to cell-generated forces can help design new methods to characterize abnormal tissues and can guide development of improved biomimetic materials.
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http://dx.doi.org/10.1039/d0sm01442gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8385661PMC
January 2021

Tissue Rheology as a Possible Complementary Procedure to Advance Histological Diagnosis of Colon Cancer.

ACS Biomater Sci Eng 2020 10 7;6(10):5620-5631. Epub 2020 Sep 7.

Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Bialystok, 15-222 Bialystok, Poland.

In recent years, rheological measurements of cells and tissues at physiological and pathological stages have become an essential method to determine how forces and changes in mechanical properties contribute to disease development and progression, but there is no standardization of this procedure so far. In this study, we evaluate the potential of nanoscale atomic force microscopy (AFM) and macroscopic shear rheometry to assess the mechanical properties of healthy and cancerous human colon tissues. The direct comparison of tissue mechanical behavior under uniaxial and shear deformation shows that cancerous tissues not only are stiffer compared to healthy tissue but also respond differently when shear and compressive stresses are applied. These results suggest that rheological parameters can be useful measures of colon cancer mechanopathology. Additionally, we extend the list of biological materials exhibiting compressional stiffening and shear weakening effects to human colon tumors. These mechanical responses might be promising mechanomarkers and become part of the new procedures in colon cancer diagnosis. Enrichment of histopathological grading with rheological assessment of tissue mechanical properties will potentially allow more accurate colon cancer diagnosis and improve prognosis.
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http://dx.doi.org/10.1021/acsbiomaterials.0c00975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549092PMC
October 2020

The vimentin cytoskeleton: when polymer physics meets cell biology.

Phys Biol 2020 12 1;18(1):011001. Epub 2020 Dec 1.

Institute for Medicine and Engineering, Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, USA.

The proper functions of tissues depend on the ability of cells to withstand stress and maintain shape. Central to this process is the cytoskeleton, comprised of three polymeric networks: F-actin, microtubules, and intermediate filaments (IFs). IF proteins are among the most abundant cytoskeletal proteins in cells; yet they remain some of the least understood. Their structure and function deviate from those of their cytoskeletal partners, F-actin and microtubules. IF networks show a unique combination of extensibility, flexibility and toughness that confers mechanical resilience to the cell. Vimentin is an IF protein expressed in mesenchymal cells. This review highlights exciting new results on the physical biology of vimentin intermediate filaments and their role in allowing whole cells and tissues to cope with stress.
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http://dx.doi.org/10.1088/1478-3975/abbcc2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240483PMC
December 2020

Mechanical and Non-Mechanical Functions of Filamentous and Non-Filamentous Vimentin.

Bioessays 2020 11 6;42(11):e2000078. Epub 2020 Sep 6.

Department of Physiology, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Intermediate filaments (IFs) formed by vimentin are less understood than their cytoskeletal partners, microtubules and F-actin, but the unique physical properties of IFs, especially their resistance to large deformations, initially suggest a mechanical function. Indeed, vimentin IFs help regulate cell mechanics and contractility, and in crowded 3D environments they protect the nucleus during cell migration. Recently, a multitude of studies, often using genetic or proteomic screenings show that vimentin has many non-mechanical functions within and outside of cells. These include signaling roles in wound healing, lipogenesis, sterol processing, and various functions related to extracellular and cell surface vimentin. Extracellular vimentin is implicated in marking circulating tumor cells, promoting neural repair, and mediating the invasion of host cells by viruses, including SARS-CoV, or bacteria such as Listeria and Streptococcus. These findings underscore the fundamental role of vimentin in not only cell mechanics but also a range of physiological functions. Also see the video abstract here https://youtu.be/YPfoddqvz-g.
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http://dx.doi.org/10.1002/bies.202000078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8349470PMC
November 2020

Effects of extracellular matrix viscoelasticity on cellular behaviour.

Nature 2020 08 26;584(7822):535-546. Epub 2020 Aug 26.

Center for Engineering Mechanobiology and Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, PA, USA.

Substantial research over the past two decades has established that extracellular matrix (ECM) elasticity, or stiffness, affects fundamental cellular processes, including spreading, growth, proliferation, migration, differentiation and organoid formation. Linearly elastic polyacrylamide hydrogels and polydimethylsiloxane (PDMS) elastomers coated with ECM proteins are widely used to assess the role of stiffness, and results from such experiments are often assumed to reproduce the effect of the mechanical environment experienced by cells in vivo. However, tissues and ECMs are not linearly elastic materials-they exhibit far more complex mechanical behaviours, including viscoelasticity (a time-dependent response to loading or deformation), as well as mechanical plasticity and nonlinear elasticity. Here we review the complex mechanical behaviours of tissues and ECMs, discuss the effect of ECM viscoelasticity on cells, and describe the potential use of viscoelastic biomaterials in regenerative medicine. Recent work has revealed that matrix viscoelasticity regulates these same fundamental cell processes, and can promote behaviours that are not observed with elastic hydrogels in both two- and three-dimensional culture microenvironments. These findings have provided insights into cell-matrix interactions and how these interactions differentially modulate mechano-sensitive molecular pathways in cells. Moreover, these results suggest design guidelines for the next generation of biomaterials, with the goal of matching tissue and ECM mechanics for in vitro tissue models and applications in regenerative medicine.
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http://dx.doi.org/10.1038/s41586-020-2612-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676152PMC
August 2020

Compression stiffening of fibrous networks with stiff inclusions.

Proc Natl Acad Sci U S A 2020 09 17;117(35):21037-21044. Epub 2020 Aug 17.

Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX 77005;

Tissues commonly consist of cells embedded within a fibrous biopolymer network. Whereas cell-free reconstituted biopolymer networks typically soften under applied uniaxial compression, various tissues, including liver, brain, and fat, have been observed to instead stiffen when compressed. The mechanism for this compression-stiffening effect is not yet clear. Here, we demonstrate that when a material composed of stiff inclusions embedded in a fibrous network is compressed, heterogeneous rearrangement of the inclusions can induce tension within the interstitial network, leading to a macroscopic crossover from an initial bending-dominated softening regime to a stretching-dominated stiffening regime, which occurs before and independently of jamming of the inclusions. Using a coarse-grained particle-network model, we first establish a phase diagram for compression-driven, stretching-dominated stress propagation and jamming in uniaxially compressed two- and three-dimensional systems. Then, we demonstrate that a more detailed computational model of stiff inclusions in a subisostatic semiflexible fiber network exhibits quantitative agreement with the predictions of our coarse-grained model as well as qualitative agreement with experiments.
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http://dx.doi.org/10.1073/pnas.2003037117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474641PMC
September 2020

Evaluation of active Rac1 levels in cancer cells: A case of misleading conclusions from immunofluorescence analysis.

J Biol Chem 2020 10 14;295(40):13698-13710. Epub 2020 Aug 14.

Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. Electronic address:

A large number of aggressive cancer cell lines display elevated levels of activated Rac1, a small GTPase widely implicated in cytoskeleton reorganization, cell motility, and metastatic dissemination. A commonly accepted methodological approach for detecting Rac1 activation in cancer cells involves the use of a conformation-sensitive antibody that detects the active (GTP-bound) Rac1 without interacting with the GDP-bound inactive form. This antibody has been extensively used in fixed cell immunofluorescence and immunohistochemistry. Taking advantage of prostate and pancreatic cancer cell models known to have high basal Rac1-GTP levels, here we have established that this antibody does not recognize Rac1 but rather detects the intermediate filament protein vimentin. Indeed, Rac1-null PC3 prostate cancer cells or cancer models with low levels of Rac1 activation still show a high signal with the anti-Rac1-GTP antibody, which is lost upon silencing of vimentin expression. Moreover, this antibody was unable to detect activated Rac1 in membrane ruffles induced by epidermal growth factor stimulation. These results have profound implications for the study of this key GTPase in cancer, particularly because a large number of cancer cell lines with characteristic mesenchymal features show simultaneous up-regulation of vimentin and high basal Rac1-GTP levels when measured biochemically. This misleading correlation can lead to assumptions about the validity of this antibody and inaccurate conclusions that may affect the development of appropriate therapeutic approaches for targeting the Rac1 pathway.
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http://dx.doi.org/10.1074/jbc.RA120.013919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7535912PMC
October 2020

Elasticity-dependent response of malignant cells to viscous dissipation.

Biomech Model Mechanobiol 2021 Feb 12;20(1):145-154. Epub 2020 Aug 12.

Institute for Medicine and Engineering and Center for Engineering MechanoBiology, University of Pennsylvania, Philadelphia, PA, USA.

The stiffness of the cellular environment controls malignant cell phenotype and proliferation. However, the effect of viscous dissipation on these parameters has not yet been investigated, in part due to the lack of in vitro cell substrates reproducing the mechanical properties of normal tissues and tumors. In this article, we use a newly reported viscoelastic polyacrylamide gel cell substrate, and we characterize the impact of viscous dissipation on three malignant cell lines: DU145 and PC3 derived from prostate and LN229 from brain. The spreading, motility and proliferation rates of these cells were analyzed on 1 kPa and 5 kPa elastic and viscoelastic gels. Surprisingly, the effect of substrate viscous dissipation on cell behavior depended on substrate stiffness for the three cell types tested. We conclude that viscoelasticity controls the spreading, proliferation and migration of malignant cells in vitro. These results highlight the critical role of viscous dissipation in the phenotype and proliferation of malignant cells, especially in stiff tumor environments.
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http://dx.doi.org/10.1007/s10237-020-01374-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892690PMC
February 2021

Multiscale modeling of protein membrane interactions for nanoparticle targeting in drug delivery.

Curr Opin Struct Biol 2020 10 27;64:104-110. Epub 2020 Jul 27.

Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, United States; Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

Nanoparticle (NP)-based imaging and drug delivery systems for systemic (e.g. intravenous) therapeutic and diagnostic applications are inherently a complex integration of biology and engineering. A broad range of length and time scales are essential to hydrodynamic and microscopic molecular interactions mediating NP (drug nanocarriers, imaging agents) motion in blood flow, cell binding/uptake, and tissue accumulation. A computational model of time-dependent tissue delivery, providing in silico prediction of organ-specific accumulation of NPs, can be leveraged in NP design and clinical applications. In this article, we provide the current state-of-the-art and future outlook for the development of predictive models for NP transport, targeting, and distribution through the integration of new computational schemes rooted in statistical mechanics and transport. The resulting multiscale model will comprehensively incorporate: (i) hydrodynamic interactions in the vascular scales relevant to NP margination; (ii) physical and mechanical forces defining cellular and tissue architecture and epitope accessibility mediating NP adhesion; and (iii) subcellular and paracellular interactions including molecular-level targeting impacting NP uptake.
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http://dx.doi.org/10.1016/j.sbi.2020.06.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666034PMC
October 2020

A novel method to make viscoelastic polyacrylamide gels for cell culture and traction force microscopy.

APL Bioeng 2020 Sep 2;4(3):036104. Epub 2020 Jul 2.

Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.

Polyacrylamide hydrogels are commonly used in cell biology, notably to cultivate cells on soft surfaces. Polyacrylamide gels are purely elastic and well adapted to cell culture as they are inert and can be conjugated with adhesion proteins. Here, we report a method to make viscoelastic polyacrylamide gels with mechanical properties more closely resembling biological tissues and suitable for cell culture . We demonstrate that these gels can be used for traction force microscopy experiments. We also show that multiple cell types respond to the viscoelasticity of their substrate and that viscous dissipation has an influence on cell spreading, contractility, and motility. This new material provides new opportunities for investigating how normal or malignant cells sense and respond to viscous dissipation within the extra-cellular matrix.
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http://dx.doi.org/10.1063/5.0002750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334032PMC
September 2020

Divalent cations bind to phosphoinositides to induce ion and isomer specific propensities for nano-cluster initiation in bilayer membranes.

R Soc Open Sci 2020 May 20;7(5):192208. Epub 2020 May 20.

Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

We report all-atom molecular dynamics simulations of asymmetric bilayers containing phosphoinositides in the presence of monovalent and divalent cations. We have characterized the molecular mechanism by which these divalent cations interact with phosphoinositides. Ca desolvates more readily, consistent with single-molecule calculations, and forms a network of ionic-like bonds that serve as a 'molecular glue' that allows a single ion to coordinate with up to three phosphatidylinositol-(4,5)-bisphosphate (PI(4, 5)P) lipids. The phosphatidylinositol-(3,5)-bisphosphate isomer shows no such effect and neither does PI(4, 5)P in the presence of Mg. The resulting network of Ca-mediated lipid-lipid bonds grows to span the entire simulation space and therefore has implications for the lateral distribution of phosophoinositides in the bilayer. We observe context-specific differences in lipid diffusion rates, lipid surface densities and bilayer structure. The molecular-scale delineation of ion-lipid arrangements reported here provides insight into similar nanocluster formation induced by peripheral proteins to regulate the formation of functional signalling complexes on the membrane.
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http://dx.doi.org/10.1098/rsos.192208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7277276PMC
May 2020

Lipid droplets disrupt mechanosensing in human hepatocytes.

Am J Physiol Gastrointest Liver Physiol 2020 07 28;319(1):G11-G22. Epub 2020 May 28.

Departments of Medicine and Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania.

Hepatocellular carcinoma (HCC) is the fourth-leading cause of cancer death in the world. Although most cases occur in stiff, cirrhotic livers, and stiffness is a significant risk factor, HCC can also arise in noncirrhotic livers in the setting of nonalcoholic fatty liver disease (NAFLD). We hypothesized that lipid droplets in NAFLD might apply mechanical forces to the nucleus, functioning as mechanical stressors akin to stiffness. We investigated the effect of lipid droplets on cellular mechanosensing and found that primary human hepatocytes loaded with the fatty acids oleate and linoleate exhibited decreased stiffness-induced cell spreading and disrupted focal adhesions and stress fibers. The presence of large lipid droplets in hepatocytes resulted in increased nuclear localization of the mechano-sensor Yes-associated protein (YAP). In cirrhotic livers from patients with NAFLD, hepatocytes filled with large lipid droplets showed significantly higher nuclear localization of YAP as compared with cells with small lipid droplets. This work suggests that lipid droplets induce a mechanical signal that disrupts the ability of the hepatocyte to sense its underlying matrix stiffness and that the presence of lipid droplets can induce intracellular mechanical stresses. This work examines the impact of lipid loading on mechanosensing by human hepatocytes. In cirrhotic livers, the presence of large (although not small) lipid droplets increased nuclear localization of the mechanotransducer YAP. In primary hepatocytes in culture, lipid droplets led to decreased stiffness-induced cell spreading and disrupted focal adhesions and stress fibers; the presence of large lipid droplets resulted in increased YAP nuclear localization. Collectively, the data suggest that lipid droplets induce intracellular mechanical stress.
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http://dx.doi.org/10.1152/ajpgi.00098.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468756PMC
July 2020
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