bims-ecemfi 2024-07-14 papers

bims-ecemfi

Biomed News

on ECM and fibroblasts

Issue of 2024‒07‒14
thirty-two papers selected by
Badri Narayanan Narasimhan, University of California, San Diego

Quantitative atlas of collagen hydrogels reveals mesenchymal cancer cell traction adaptation to the matrix nanoarchitecture.
Breast Cancer Cells Exhibit Mesenchymal-Epithelial Plasticity Following Dynamic Modulation of Matrix Stiffness.
Composite branched and linear F-actin maximize myosin-induced membrane shape changes in a biomimetic cell model.
Detection of fluorescent protein mechanical switching in cellulo.
Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel.
Structure-Mechanics Principles and Mechanobiology of Fibrocartilage Pericellular Matrix: A Pivotal Role of Type V Collagen.
Mechanically induced topological transition of spectrin regulates its distribution in the mammalian cell cortex.
Split Luciferase Molecular Tension Sensors for Bioluminescent Readout of Mechanical Forces in Biological Systems.
A 3D micro-printed single cell micro-niche with asymmetric niche signals - An in vitro model for asymmetric cell division study.
Modeling the mechanosensitive collective migration of cells on the surface and the interior of morphing soft tissues.
Soft Polyethylene Glycol Hydrogels Support Human PSC Pluripotency and Morphogenesis.
Tailored Physicochemical Cues Direct Human Mesenchymal Stem Cell Differentiation through Epigenetic Regulation Using Colloidal Self-Assembled Patterns.
Breast Tumor Cell Survival and Morphology in a Brain-like Extracellular Matrix Depends on Matrix Composition and Mechanical Properties.
Engineering the Cellular Microenvironment: Integrating Three-Dimensional Nontopographical and Two-Dimensional Biochemical Cues for Precise Control of Cellular Behavior.
Confinement, jamming, and adhesion in cancer cells dissociating from a collectively invading strand.
Probing Viscoelastic Properties and Interfaces in High-Density Polyethylene Vitrimers at the Nanoscale Using Dynamic Mode Atomic Force Microscopy.
Increased prevalence of hybrid epithelial/mesenchymal state and enhanced phenotypic heterogeneity in basal breast cancer.
Heterogeneous stiffness of the bone marrow microenvironment regulates the fate decision of haematopoietic stem and progenitor cells.
Mmp14-dependent remodeling of the pericellular-dermal collagen interface governs fibroblast survival.
Functional effects of the spatial-varying lens mechanical properties in accommodation.
Hard- and Soft-Coded Strain Stiffening in Metamaterials via Out-of-Plane Buckling Using Highly Entangled Active Hydrogel Elements.
Aging-related changes in the mechanical properties of single cells.
The characterization of cell traction force on nonflat surfaces with different curvature by elastic hydrogel microspheres.
Variations in fluid chemical potential induce fibroblast mechano-response in 3D hydrogels.
Size Matters: Rethinking Hertz Model Interpretation for Cell Mechanics Using AFM.
Dendritic cell force-migration coupling on aligned fiber networks.
Pulmonary Matrix-derived Hydrogels from Patients with Idiopathic Pulmonary Fibrosis Induce a Proinflammatory State in Lung Fibroblasts In Vitro.
Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.
Mechanical and biochemical feedback combine to generate complex contractile oscillations in cytokinesis.
Myo-optogenetics: optogenetic stimulation and electrical recording in skeletal muscles.
Turn-on protein switches for controlling actin binding in cells.
Fabricating oxygen self-supplying 3D printed bioactive hydrogel scaffold for augmented vascularized bone regeneration.

Acta Biomater. 2024 Jul 09. pii: S1742-7061(24)00364-7. [Epub ahead of print]

Quantitative atlas of collagen hydrogels reveals mesenchymal cancer cell traction adaptation to the matrix nanoarchitecture.

Pablo Blázquez-Carmona, Raquel Ruiz-Mateos, Jorge Barrasa-Fano, Apeksha Shapeti, José Enrique Martín-Alfonso, Jaime Domínguez, Hans Van Oosterwyck, Esther Reina-Romo, José Antonio Sanz-Herrera.

  Collagen-based hydrogels are commonly used in mechanobiology to mimic the extracellular matrix. A quantitative analysis of the influence of collagen concentration and properties on the structure and mechanics of the hydrogels is essential for tailored design adjustments for specific in vitro conditions. We combined focused ion beam scanning electron microscopy and rheology to provide a detailed quantitative atlas of the mechanical and nanoscale three-dimensional structural alterations that occur when manipulating different hydrogel's physicochemistry. Moreover, we study the effects of such alterations on the phenotype of breast cancer cells and their mechanical interactions with the extracellular matrix. Regardless of the microenvironment's pore size, porosity or mechanical properties, cancer cells are able to reach a stable mesenchymal-like morphology. Additionally, employing 3D traction force microscopy, a positive correlation between cellular tractions and ECM mechanics is observed up to a critical threshold, beyond which tractions plateau. This suggests that cancer cells in a stable mesenchymal state calibrate their mechanical interactions with the ECM to keep their migration and invasiveness capacities unaltered. STATEMENT OF SIGNIFICANCE: The paper presents a thorough study on the mechanical microenvironment in breast cancer cells during their interaction with collagen based hydrogels of different compositions. The hydrogels' microstructure were obtained using state-of-the-art 3D microscopy, namely focused ion beam-scanning electron microscope (FIB-SEM). FIB-SEM was originally applied in this work to reconstruct complex fibered collagen microstructures within the nanometer range, to obtain key microarchitectural parameters. The mechanical microenvironment of cells was recovered using Traction Force Microscopy (TFM). The obtained results suggest that cells calibrate tractions such that they depend on mechanical, microstructural and physicochemical characteristics of the hydrogels, hence revealing a steric hindrance. We hypothesize that cancer cells studied in this paper tune their mechanical state to keep their migration and invasiveness capacities unaltered.

Keywords:  FIB-SEM; breast cancer cells; cell morphology; mechanobiology; traction force microscopy; tumor microenvironment

DOI:  https://doi.org/10.1016/j.actbio.2024.07.002
Adv Biol (Weinh). 2024 Jul 08. e2400087

Breast Cancer Cells Exhibit Mesenchymal-Epithelial Plasticity Following Dynamic Modulation of Matrix Stiffness.

Chinmay S Sankhe, Jessica L Sacco, Jacob Lawton, Ryan A Fair, David Vidotto Rezende Soares, Mohammed K R Aldahdooh, Enrique D Gomez, Esther W Gomez.

  Mesenchymal-epithelial transition (MET) is essential for tissue and organ development and is thought to contribute to cancer by enabling the establishment of metastatic lesions. Despite its importance in both health and disease, there is a lack of in vitro platforms to study MET and little is known about the regulation of MET by mechanical cues. Here, hyaluronic acid-based hydrogels with dynamic and tunable stiffnesses mimicking that of normal and tumorigenic mammary tissue are synthesized. The platform is then utilized to examine the response of mammary epithelial cells and breast cancer cells to dynamic modulation of matrix stiffness. Gradual softening of the hydrogels reduces proliferation and increases apoptosis of breast cancer cells. Moreover, breast cancer cells exhibit temporal changes in cell morphology, cytoskeletal organization, and gene expression that are consistent with mesenchymal-epithelial plasticity as the stiffness of the matrix is reduced. A reduction in matrix stiffness attenuates the expression of integrin-linked kinase, and inhibition of integrin-linked kinase impacts proliferation, apoptosis, and gene expression in cells cultured on stiff and dynamic hydrogels. Overall, these findings reveal intermediate epithelial/mesenchymal states as cells move along a matrix stiffness-mediated MET trajectory and suggest an important role for matrix mechanics in regulating mesenchymal-epithelial plasticity.

Keywords:  apoptosis; breast cancer; extracellular matrix; hyaluronic acid; integrin‐linked kinase

DOI:  https://doi.org/10.1002/adbi.202400087
Commun Biol. 2024 Jul 10. 7(1): 840

Composite branched and linear F-actin maximize myosin-induced membrane shape changes in a biomimetic cell model.

Ryota Sakamoto, Michael P Murrell.

The architecture of the actin cortex determines the generation and transmission of stresses, during key events from cell division to migration. However, its impact on myosin-induced cell shape changes remains unclear. Here, we reconstitute a minimal model of the actomyosin cortex with branched or linear F-actin architecture within giant unilamellar vesicles (GUVs, liposomes). Upon light activation of myosin, neither the branched nor linear F-actin architecture alone induces significant liposome shape changes. The branched F-actin network forms an integrated, membrane-bound "no-slip boundary" -like cortex that attenuates actomyosin contractility. By contrast, the linear F-actin network forms an unintegrated "slip boundary" -like cortex, where actin asters form without inducing membrane deformations. Notably, liposomes undergo significant deformations at an optimized balance of branched and linear F-actin networks. Our findings highlight the pivotal roles of branched F-actin in force transmission and linear F-actin in force generation to yield membrane shape changes.

DOI: https://doi.org/10.1038/s42003-024-06528-4
Cell Rep Methods. 2024 Jul 04. pii: S2667-2375(24)00180-2. [Epub ahead of print] 100815

Detection of fluorescent protein mechanical switching in cellulo.

T Curtis Shoyer, Kasie L Collins, Trevor R Ham, Aaron T Blanchard, Juilee N Malavade, Benjamin A Johns, Jennifer L West, Brenton D Hoffman.

  The ability of cells to sense and respond to mechanical forces is critical in many physiological and pathological processes. However, determining the mechanisms by which forces affect protein function inside cells remains challenging. Motivated by in vitro demonstrations of fluorescent proteins (FPs) undergoing reversible mechanical switching of fluorescence, we investigated whether force-sensitive changes in FP function could be visualized in cells. Guided by a computational model of FP mechanical switching, we develop a formalism for its detection in Förster resonance energy transfer (FRET)-based biosensors and demonstrate its occurrence in cellulo within a synthetic actin crosslinker and the mechanical linker protein vinculin. We find that in cellulo mechanical switching is reversible and altered by manipulation of cell force generation, external stiffness, and force-sensitive bond dynamics of the biosensor. This work describes a framework for assessing FP mechanical stability and provides a means of probing force-sensitive protein function inside cells.

Keywords:  CP: Cell biology; CP: Imaging; FRET; catch bond; fluorescent protein; mechanical switching; mechanosensitive; mechanotransduction; stiffness sensing; tension sensor

DOI:  https://doi.org/10.1016/j.crmeth.2024.100815
Front Ophthalmol (Lausanne). 2022 ;2 948397

Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel.

Tyler Bagué, Ayushi Singh, Rajanya Ghosh, Hannah Yoo, Curtis Kelly, Mitchell A deLong, Casey C Kopczynski, Samuel Herberg.

  Interactions between trabecular meshwork (TM) cells and their extracellular matrix (ECM) are critical for normal outflow function in the healthy eye. Multifactorial dysregulation of the TM is the principal cause of elevated intraocular pressure that is strongly associated with glaucomatous vision loss. Key characteristics of the diseased TM are pathologic contraction and actin stress fiber assembly, contributing to overall tissue stiffening. Among first-line glaucoma medications, the Rho-associated kinase inhibitor (ROCKi) netarsudil is known to directly target the stiffened TM to improve outflow function via tissue relaxation involving focal adhesion and actin stress fiber disassembly. Yet, no in vitro studies have explored the effect of netarsudil on human TM (HTM) cell contractility and actin remodeling in a 3D ECM environment. Here, we use our bioengineered HTM cell-encapsulated ECM hydrogel to investigate the efficacy of different netarsudil-family ROCKi compounds on reversing pathologic contraction and actin stress fibers. Netarsudil and all related experimental ROCKi compounds exhibited significant ROCK1/2 inhibitory and focal adhesion disruption activities. Furthermore, all ROCKi compounds displayed potent contraction-reversing effects on HTM hydrogels upon glaucomatous induction in a dose-dependent manner, relatively consistent with their biochemical/cellular inhibitory activities. At their tailored EC50 levels, netarsudil-family ROCKi compounds exhibited distinct effect signatures of reversing pathologic HTM hydrogel contraction and actin stress fibers, independent of the cell strain used. Netarsudil outperformed the experimental ROCKi compounds in support of its clinical status. In contrast, at uniform EC50-levels using netarsudil as reference, all ROCKi compounds performed similarly. Collectively, our data suggest that netarsudil exhibits high potency to rescue HTM cell pathobiology in a tissue-mimetic 3D ECM microenvironment, solidifying the utility of our bioengineered hydrogel model as a viable screening platform to further our understanding of TM pathophysiology in glaucoma.

Keywords:  ROCK inhibition; Rhopressa; cytoskeleton; primary open-angle glaucoma; tissue relaxation

DOI:  https://doi.org/10.3389/fopht.2022.948397
bioRxiv. 2024 Jun 30. pii: 2024.06.26.600498. [Epub ahead of print]

Structure-Mechanics Principles and Mechanobiology of Fibrocartilage Pericellular Matrix: A Pivotal Role of Type V Collagen.

Chao Wang, Mingyue Fan, Su-Jin Heo, Sheila M Adams, Thomas Li, Yuchen Liu, Qing Li, Claudia Loebel, Farid Alisafaei, Jason A Burdick, X Lucas Lu, David E Birk, Robert L Mauck, Lin Han.

The pericellular matrix (PCM) is the immediate microniche surrounding resident cells in various tissue types, regulating matrix turnover, cell-matrix cross-talk and disease initiation. This study elucidated the structure-mechanical properties and mechanobiological functions of the PCM in fibrocartilage, a family of connective tissues that sustain complex tensile and compressive loads in vivo. Studying the murine meniscus as the model tissue, we showed that fibrocartilage PCM contains thinner, random collagen fibrillar networks that entrap proteoglycans, a structure distinct from the densely packed, highly aligned collagen fibers in the bulk extracellular matrix (ECM). In comparison to the ECM, the PCM has a lower modulus and greater isotropy, but similar relative viscoelastic properties. In Col5a1 +/ D menisci, the reduction of collagen V, a minor collagen localized in the PCM, resulted in aberrant fibril thickening with increased heterogeneity. Consequently, the PCM exhibited a reduced modulus, loss of isotropy and faster viscoelastic relaxation. This disrupted PCM contributes to perturbed mechanotransduction of resident meniscal cells, as illustrated by reduced intracellular calcium signaling, as well as upregulated biosynthesis of lysyl oxidase and tenascin C. When cultured in vitro, Col5a1 +/ D meniscal cells synthesized a weakened nascent PCM, which had inferior properties towards protecting resident cells against applied tensile stretch. These findings underscore the PCM as a distinctive microstructure that governs fibrocartilage mechanobiology, and highlight the pivotal role of collagen V in PCM function. Targeting the PCM or its molecular constituents holds promise for enhancing not only meniscus regeneration and osteoarthritis intervention, but also addressing diseases across various fibrocartilaginous tissues.

DOI: https://doi.org/10.1101/2024.06.26.600498
Nat Commun. 2024 Jul 08. 15(1): 5711

Mechanically induced topological transition of spectrin regulates its distribution in the mammalian cell cortex.

Andrea Ghisleni, Mayte Bonilla-Quintana, Michele Crestani, Zeno Lavagnino, Camilla Galli, Padmini Rangamani, Nils C Gauthier.

The cell cortex is a dynamic assembly formed by the plasma membrane and underlying cytoskeleton. As the main determinant of cell shape, the cortex ensures its integrity during passive and active deformations by adapting cytoskeleton topologies through yet poorly understood mechanisms. The spectrin meshwork ensures such adaptation in erythrocytes and neurons by adopting different organizations. Erythrocytes rely on triangular-like lattices of spectrin tetramers, whereas in neurons they are organized in parallel, periodic arrays. Since spectrin is ubiquitously expressed, we exploited Expansion Microscopy to discover that, in fibroblasts, distinct meshwork densities co-exist. Through biophysical measurements and computational modeling, we show that the non-polarized spectrin meshwork, with the intervention of actomyosin, can dynamically transition into polarized clusters fenced by actin stress fibers that resemble periodic arrays as found in neurons. Clusters experience lower mechanical stress and turnover, despite displaying an extension close to the tetramer contour length. Our study sheds light on the adaptive properties of spectrin, which participates in the protection of the cell cortex by varying its densities in response to key mechanical features.

DOI: https://doi.org/10.1038/s41467-024-49906-6
ACS Sens. 2024 Jul 07.

Split Luciferase Molecular Tension Sensors for Bioluminescent Readout of Mechanical Forces in Biological Systems.

Brian L Zhong, Jeandele M Elliot, Pengli Wang, Hongquan Li, R Nelson Hall, Bo Wang, Manu Prakash, Alexander R Dunn.

  The ability of proteins to sense and transmit mechanical forces underlies many biological processes, but characterizing these forces in biological systems remains a challenge. Existing genetically encoded force sensors typically rely on fluorescence or bioluminescence resonance energy transfer (FRET or BRET) to visualize tension. However, these force sensing modules are relatively large, and interpreting measurements requires specialized image analysis and careful control experiments. Here, we report a compact molecular tension sensor that generates a bioluminescent signal in response to tension. This sensor (termed PILATeS) makes use of the split NanoLuc luciferase and consists of the H. sapiens titin I10 domain with the insertion of a 10-15 amino acid tag derived from the C-terminal β-strand of NanoLuc. Mechanical load across PILATeS mediates exposure of this tag to recruit the complementary split NanoLuc fragment, resulting in force-dependent bioluminescence. We demonstrate the ability of PILATeS to report biologically meaningful forces by visualizing forces at the interface between integrins and extracellular matrix substrates. We further use PILATeS as a genetically encoded sensor of tension experienced by the mechanosensing protein vinculin. We anticipate that PILATeS will provide an accessible means of visualizing molecular-scale forces in biological systems.

Keywords:  bioluminescence; focal adhesion; force sensor; luciferase; mechanobiology; protein engineering

DOI:  https://doi.org/10.1021/acssensors.3c02664
Biomaterials. 2024 Jun 26. pii: S0142-9612(24)00218-7. [Epub ahead of print]311 122684

A 3D micro-printed single cell micro-niche with asymmetric niche signals - An in vitro model for asymmetric cell division study.

Nan Huang, Barbara Pui Chan.

  Intricate microenvironment signals orchestrate to affect cell behavior and fate during tissue morphogenesis. However, the underlying mechanisms on how specific local niche signals influence cell behavior and fate are not fully understood, owing to the lack of in vitro platform able to precisely, quantitatively, spatially, and independently manipulate individual niche signals. Here, microarrays of protein-based 3D single cell micro-niche (3D-SCμN), with precisely engineered biophysical and biochemical niche signals, are micro-printed by a multiphoton microfabrication and micropatterning technology. Mouse embryonic stem cell (mESC) is used as the model cell to study how local niche signals affect stem cell behavior and fate. By precisely engineering the internal microstructures of the 3D SCμNs, we demonstrate that the cell division direction can be controlled by the biophysical niche signals, in a cell shape-independent manner. After confining the cell division direction to a dominating axis, single mESCs are exposed to asymmetric biochemical niche signals, specifically, cell-cell adhesion molecule on one side and extracellular matrix on the other side. We demonstrate that, symmetry-breaking (asymmetric) niche signals successfully trigger cell polarity formation and bias the orientation of asymmetric cell division, the mitosis process resulting in two daughter cells with differential fates, in mESCs.

Keywords:  3D single cell micro-niche; Asymmetric cell division; Cell fate; Cell-cell interaction; Cell-matrix interaction; Mouse embryonic stem cell; Multiphoton microfabrication

DOI:  https://doi.org/10.1016/j.biomaterials.2024.122684
Biomech Model Mechanobiol. 2024 Jul 07.

Modeling the mechanosensitive collective migration of cells on the surface and the interior of morphing soft tissues.

Jaemin Kim, Mahmut Selman Sakar, Nikolaos Bouklas.

  Cellular contractility, migration, and extracellular matrix (ECM) mechanics are critical for a wide range of biological processes including embryonic development, wound healing, tissue morphogenesis, and regeneration. Even though the distinct response of cells near the tissue periphery has been previously observed in cell-laden microtissues, including faster kinetics and more prominent cell-ECM interactions, there are currently no models that can fully combine coupled surface and bulk mechanics and kinetics to recapitulate the morphogenic response of these constructs. Mailand et al. (Biophys J 117(5):975-986, 2019) had shown the importance of active elastocapillarity in cell-laden microtissues, but modeling the distinct mechanosensitive migration of cells on the periphery and the interior of highly deforming tissues has not been possible thus far, especially in the presence of active elastocapillary effects. This paper presents a framework for understanding the interplay between cellular contractility, migration, and ECM mechanics in dynamically morphing soft tissues accounting for distinct cellular responses in the bulk and the surface of tissues. The major novelty of this approach is that it enables modeling the distinct migratory and contractile response of cells residing on the tissue surface and the bulk, where concurrently the morphing soft tissues undergo large deformations driven by cell contractility. Additionally, the simulation results capture the changes in shape and cell concentration for wounded and intact microtissues, enabling the interpretation of experimental data. The numerical procedure that accounts for mechanosensitive stress generation, large deformations, diffusive migration in the bulk and a distinct mechanism for diffusive migration on deforming surfaces is inspired from recent work on bulk and surface poroelasticity of hydrogels involving elastocapillary effects, but in this work, a two-field weak form is proposed and is able to alleviate numerical instabilities that were observed in the original method that utilized a three-field mixed finite element formulation.

Keywords:  Cell migration; Contractility; Mechanotransduction; Microtissue; Morphogenesis; Wound healing

DOI:  https://doi.org/10.1007/s10237-024-01870-2
ACS Biomater Sci Eng. 2024 Jul 08. 10(7): 4525-4540

Soft Polyethylene Glycol Hydrogels Support Human PSC Pluripotency and Morphogenesis.

Michael P Seitz, Yuanhui Song, Xiaojun Lance Lian, Zhen Ma, Era Jain.

  Lumenogenesis within the epiblast represents a critical step in early human development, priming the embryo for future specification and patterning events. However, little is known about the specific mechanisms that drive this process due to the inability to study the early embryo in vivo. While human pluripotent stem cell (hPSC)-based models recapitulate many aspects of the human epiblast, most approaches for generating these 3D structures rely on ill-defined, reconstituted basement membrane matrices. Here, we designed synthetic, nonadhesive polyethylene glycol (PEG) hydrogel matrices to better understand the role of matrix mechanical cues in iPSC morphogenesis, specifically elastic modulus. First, we identified a narrow range of hydrogel moduli that were conducive to the hPSC viability, pluripotency, and differentiation. We then used this platform to investigate the effects of the hydrogel modulus on lumenogenesis, finding that matrices of intermediate stiffness yielded the most epiblast-like aggregates. Conversely, stiffer matrices impeded lumen formation and apico-basal polarization, while the softest matrices yielded polarized but aberrant structures. Our approach offers a simple, modular platform for modeling the human epiblast and investigating the role of matrix cues in its morphogenesis.

Keywords:  PEG hydrogel; epiblast; hPSC; lumenogenesis; mechanobiology; morphogenesis

DOI:  https://doi.org/10.1021/acsbiomaterials.4c00923
ACS Appl Mater Interfaces. 2024 Jul 08.

Tailored Physicochemical Cues Direct Human Mesenchymal Stem Cell Differentiation through Epigenetic Regulation Using Colloidal Self-Assembled Patterns.

Javad Harati, Ping Du, Massimiliano Galluzzi, Xian Li, Jiao Lin, Haobo Pan, Peng-Yuan Wang.

  The extracellular matrix (ECM) shapes the stem cell fate during differentiation by exerting relevant biophysical cues. However, the mechanism of stem cell fate decisions in response to ECM-backed complex biophysical cues has not been fully understood due to the lack of versatile ECMs. Here, we designed two versatile ECMs using colloidal self-assembly technology to probe the mechanisms of their effects on mechanotransduction and stem cell fate regulation. Binary colloidal crystals (BCC) with a hexagonally close-packed structure, composed of silica (5 μm) and polystyrene (0.4 μm) particles as well as a polydimethylsiloxane-embedded BCC (BCCP), were fabricated. They have defined surface chemistry, roughness, stiffness, ion release, and protein adsorption properties, which can modulate the cell adhesion, proliferation, and differentiation of human adipose-derived stem cells (hASCs). On the BCC, hASCs preferred osteogenesis at an early stage but showed a higher tendency toward adipogenesis at later stages. In contrast, the results of BCCP diverged from those of BCC, suggesting a unique regulation of ECM-dependent mechanotransduction. The BCC-mediated cell adhesion reduced the size of the focal adhesion complex, accompanying an ordered spatial organization and cytoskeletal rearrangement. This morphological restriction led to the modulation of mechanosensitive transcription factors, such as c-FOS, the enrichment of transcripts in specific signaling pathways such as PI3K/AKT, and the activation of the Hippo signaling pathway. Epigenetic analyses showed changes in histone modifications across different substrates, suggesting that chromatin remodeling participated in BCC-mediated mechanotransduction. This study demonstrates that BCCs are versatile artificial ECMs that can regulate human stem cells' fate through unique biological signaling, which is beneficial in biomaterial design and stem cell engineering.

Keywords:  chromatin; differentiation; epigenetic state; mechanotransduction; stem cells

DOI:  https://doi.org/10.1021/acsami.4c02989
Adv Biol (Weinh). 2024 Jul 06. e2400184

Breast Tumor Cell Survival and Morphology in a Brain-like Extracellular Matrix Depends on Matrix Composition and Mechanical Properties.

Esra Türker, Mateo S Andrade Mier, Jessica Faber, Selma J Padilla Padilla, Nicoletta Murenu, Philipp Stahlhut, Gregor Lang, Zan Lamberger, Jeanette Weigelt, Natascha Schaefer, Jörg Tessmar, Pamela L Strissel, Torsten Blunk, Silvia Budday, Reiner Strick, Carmen Villmann.

  Triple-negative breast cancer (TNBC) is the most invasive type of breast cancer with high risk of brain metastasis. To better understand interactions between breast tumors with the brain extracellular matrix (ECM), a 3D cell culture model is implemented using a thiolated hyaluronic acid (HA-SH) based hydrogel. The latter is used as HA represents a major component of brain ECM. Melt-electrowritten (MEW) scaffolds of box- and triangular-shaped polycaprolactone (PCL) micro-fibers for hydrogel reinforcement are utilized. Two different molecular weight HA-SH materials (230 and 420 kDa) are used with elastic moduli of 148 ± 34 Pa (soft) and 1274 ± 440 Pa (stiff). Both hydrogels demonstrate similar porosities. The different molecular weight of HA-SH, however, significantly changes mechanical properties, e.g., stiffness, nonlinearity, and hysteresis. The breast tumor cell line MDA-MB-231 forms mainly multicellular aggregates in both HA-SH hydrogels but sustains high viability (75%). Supplementation of HA-SH hydrogels with ECM components does not affect gene expression but improves cell viability and impacts cellular distribution and morphology. The presence of other brain cell types further support numerous cell-cell interactions with tumor cells. In summary, the present 3D cell culture model represents a novel tool establishing a disease cell culture model in a systematic way.

Keywords:  Matrigel; PCL scaffolds; breast tumor cells; co‐cultures; hyaluronic acid

DOI:  https://doi.org/10.1002/adbi.202400184
ACS Nano. 2024 Jul 09.

Engineering the Cellular Microenvironment: Integrating Three-Dimensional Nontopographical and Two-Dimensional Biochemical Cues for Precise Control of Cellular Behavior.

Einollah Sarikhani, Dhivya Pushpa Meganathan, Anne-Kathrine Kure Larsen, Keivan Rahmani, Ching-Ting Tsai, Chih-Hao Lu, Abel Marquez-Serrano, Leah Sadr, Xiao Li, Mingdong Dong, Francesca Santoro, Bianxiao Cui, Lasse Hyldgaard Klausen, Zeinab Jahed.

  The development of biomaterials capable of regulating cellular processes and guiding cell fate decisions has broad implications in tissue engineering, regenerative medicine, and cell-based assays for drug development and disease modeling. Recent studies have shown that three-dimensional (3D) nanoscale physical cues such as nanotopography can modulate various cellular processes like adhesion and endocytosis by inducing nanoscale curvature on the plasma and nuclear membranes. Two-dimensional (2D) biochemical cues such as protein micropatterns can also regulate cell function and fate by controlling cellular geometries. Development of biomaterials with precise control over nanoscale physical and biochemical cues can significantly influence programming cell function and fate. In this study, we utilized a laser-assisted micropatterning technique to manipulate the 2D architectures of cells on 3D nanopillar platforms. We performed a comprehensive analysis of cellular and nuclear morphology and deformation on both nanopillar and flat substrates. Our findings demonstrate the precise engineering of single cell architectures through 2D micropatterning on nanopillar platforms. We show that the coupling between the nuclear and cell shape is disrupted on nanopillar surfaces compared to flat surfaces. Furthermore, our results suggest that cell elongation on nanopillars enhances nanopillar-induced endocytosis. We believe our platform serves as a versatile tool for further explorations into programming cell function and fate through combined physical cues that create nanoscale curvature on cell membranes and biochemical cues that control the geometry of the cell.

Keywords:  cell and nuclear deformation; cell and nuclear mechanics; cell and nuclear morphology; mechanobiology; micropatterning; nanopillar; nanostructures

DOI:  https://doi.org/10.1021/acsnano.4c03743
bioRxiv. 2024 Jun 29. pii: 2024.06.28.601053. [Epub ahead of print]

Confinement, jamming, and adhesion in cancer cells dissociating from a collectively invading strand.

Wei Wang, Robert A Law, Emiliano Perez Ipiña, Konstantinos Konstantopoulos, Brian A Camley.

When cells in a primary tumor work together to invade into nearby tissue, this can lead to cell dissociations-cancer cells breaking off from the invading front-leading to metastasis. What controls the dissociation of cells, and whether they break off singly or in small groups? Can this be determined by cell-cell adhesion or chemotactic cues given to cells? We develop a physical model for this question, based on experiments that mimic aspects of cancer cell invasion using microfluidic devices with microchannels of different widths. Experimentally, most dissociation events ("ruptures") involve single cells breaking off, but we observe some ruptures of large groups ( ∼ 20 cells) in wider channels. The rupture probability is nearly independent of channel width. We recapitulate the experimental results with a phase field cell motility model by introducing three different cell states (follower, guided, and high-motility metabolically active leader cells) based on their spatial position. These leader cells may explain why single-cell rupture is the universal most probable outcome. Our simulation results show that cell-channel adhesion is necessary for cells in narrow channels to invade, and strong cell-cell adhesion leads to fewer but larger ruptures. Chemotaxis also influences the rupture behavior: Strong chemotaxis strength leads to larger and faster ruptures. Finally, we study the relationship between biological jamming transitions and cell dissociations. Our results suggest unjamming is necessary but not sufficient to create ruptures.

DOI: https://doi.org/10.1101/2024.06.28.601053
ACS Appl Mater Interfaces. 2024 Jul 11.

Probing Viscoelastic Properties and Interfaces in High-Density Polyethylene Vitrimers at the Nanoscale Using Dynamic Mode Atomic Force Microscopy.

Lanti Yang, Pierre Nickmilder, Henk Verhoogt, Theo Hoeks, Philippe Leclère.

  Vitrimers are a new class of heterogeneous polymers that combine the best features of thermosets with those of thermoplastics. The introduction of cross-links strongly changes the viscoelastic behavior of vitrimer materials. However, the characterization and understanding of the nanostructures and interfaces in vitrimers resulting from dynamic cross-linking formation remain a major challenge. Here, using dynamic modes of atomic force microscopy (AFM), namely intermodulation AFM (ImAFM) and AFM-based dynamic mechanical analysis (AFM-nDMA), local viscoelastic properties and interfaces at the nanoscale length of high-density polyethylene (HDPE) vitrimer materials are reported. ImAFM imaging in combination with the k-means clustering algorithm clearly reveals two distinct phases in the vitrimer system with highly different viscoelastic properties. AFM-nDMA further provides quantitative nanoviscoelastic properties at the nanoscale to confirm that there is a cross-linking-rich aggregation area forming a nanosize network structure in the cross-linking-poor matrix phase. The cross-linking-rich region shows a similar elastic modulus but much higher adhesion force measured by AFM compared to the cross-linking-poor HDPE matrix. Furthermore, the frequency influence on the local viscoelastic properties of HDPE vitrimer at the nanoscale was initially screened. The observed HDPE vitrimer nanostructures and viscoelastic properties at the nanoscale also provide explanations on the observed bulk HDPE vitrimer crystallinity decrease and dimensional stability increase compared to HDPE. Therefore, probing the viscoelastic properties and interfaces of HDPE vitrimer provides important insights into understanding of the correlations between the vitrimer nanostructure and the bulk mechanical and rheological behaviors.

Keywords:  AFM-nDMA; HDPE vitrimer; ImAFM; cross-links; interface; viscoelasticity

DOI:  https://doi.org/10.1021/acsami.4c06809
iScience. 2024 Jul 19. 27(7): 110116

Increased prevalence of hybrid epithelial/mesenchymal state and enhanced phenotypic heterogeneity in basal breast cancer.

Sarthak Sahoo, Soundharya Ramu, Madhumathy G Nair, Maalavika Pillai, Beatriz P San Juan, Heloisa Zaccaron Milioli, Susmita Mandal, Chandrakala M Naidu, Apoorva D Mavatkar, Harini Subramaniam, Arpita G Neogi, Christine L Chaffer, Jyothi S Prabhu, Jason A Somarelli, Mohit Kumar Jolly.

  Intra-tumoral phenotypic heterogeneity promotes tumor relapse and therapeutic resistance and remains an unsolved clinical challenge. Decoding the interconnections among different biological axes of plasticity is crucial to understand the molecular origins of phenotypic heterogeneity. Here, we use multi-modal transcriptomic data-bulk, single-cell, and spatial transcriptomics-from breast cancer cell lines and primary tumor samples, to identify associations between epithelial-mesenchymal transition (EMT) and luminal-basal plasticity-two key processes that enable heterogeneity. We show that luminal breast cancer strongly associates with an epithelial cell state, but basal breast cancer is associated with hybrid epithelial/mesenchymal phenotype(s) and higher phenotypic heterogeneity. Mathematical modeling of core underlying gene regulatory networks representative of the crosstalk between the luminal-basal and epithelial-mesenchymal axes elucidate mechanistic underpinnings of the observed associations from transcriptomic data. Our systems-based approach integrating multi-modal data analysis with mechanism-based modeling offers a predictive framework to characterize intra-tumor heterogeneity and identify interventions to restrict it.

Keywords:  cancer; cancer systems biology; gene network; mathematical biosciences; molecular network

DOI:  https://doi.org/10.1016/j.isci.2024.110116
Cell Prolif. 2024 Jul 09. e13715

Heterogeneous stiffness of the bone marrow microenvironment regulates the fate decision of haematopoietic stem and progenitor cells.

Guolin Shi, Zhuo Chang, Pan Zhang, Xiaohang Zou, Xinmin Zheng, Xiru Liu, Jinxiao Yan, Huiyun Xu, Zhenhao Tian, Nu Zhang, Ning Cui, Leming Sun, Guangkui Xu, Hui Yang.

The bone marrow (BM) niches are the complex microenvironments that surround cells, providing various external stimuli to regulate a range of haematopoietic stem cell (HSC) behaviours. Recently, it has been proposed that the fate decision of HSCs is often correlated with significantly altered biophysical signals of BM niches. To thoroughly elucidate the effect of mechanical microenvironments on cell fates, we constructed 2D and 3D cell culture hydrogels using polyacrylamide to replicate the mechanical properties of heterogeneous sub-niches, including the inherent rigidity of marrow adipose tissue (2 kPa), perivascular tissue (8 kPa) and endosteum region (35 kPa) in BM. Our observations suggest that HSCs can respond to the mechanical heterogeneity of the BM microenvironment, exhibiting diversity in cell mechanics, haematopoietic pool maintenance and differentiated lineages. Hydrogels with higher stiffness promote the preservation of long-term repopulating HSCs (LT-HSCs), while those with lower stiffness support multi-potent progenitors (MPPs) viability in vitro. Furthermore, we established a comprehensive transcriptional profile of haematopoietic subpopulations to reflect the multipotency of haematopoietic stem and progenitor cells (HSPCs) that are modulated by niche-like stiffness. Our findings demonstrate that HSPCs exhibit completely distinct downstream differentiated preferences within hydrogel systems of varying stiffness. This highlights the crucial role of tissue-specific mechanical properties in HSC lineage decisions, which may provide innovative solutions to clinical challenges.

DOI: https://doi.org/10.1111/cpr.13715
J Cell Biol. 2024 Sep 02. pii: e202312091. [Epub ahead of print]223(9):

Mmp14-dependent remodeling of the pericellular-dermal collagen interface governs fibroblast survival.

Farideh Sabeh, Xiao-Yan Li, Adam W Olson, Elliot Botvinick, Abhishek Kurup, Luis E Gimenez, Jung-Sun Cho, Stephen J Weiss.

Dermal fibroblasts deposit type I collagen, the dominant extracellular matrix molecule found in skin, during early postnatal development. Coincident with this biosynthetic program, fibroblasts proteolytically remodel pericellular collagen fibrils by mobilizing the membrane-anchored matrix metalloproteinase, Mmp14. Unexpectedly, dermal fibroblasts in Mmp14-/- mice commit to a large-scale apoptotic program that leaves skin tissues replete with dying cells. A requirement for Mmp14 in dermal fibroblast survival is recapitulated in vitro when cells are embedded within, but not cultured atop, three-dimensional hydrogels of crosslinked type I collagen. In the absence of Mmp14-dependent pericellular proteolysis, dermal fibroblasts fail to trigger β1 integrin activation and instead actuate a TGF-β1/phospho-JNK stress response that leads to apoptotic cell death in vitro as well as in vivo. Taken together, these studies identify Mmp14 as a requisite cell survival factor that maintains dermal fibroblast viability in postnatal dermal tissues.

DOI: https://doi.org/10.1083/jcb.202312091
JPhys Photonics. 2024 Jul 01. 6(3): 035021

Functional effects of the spatial-varying lens mechanical properties in accommodation.

Justin Schumacher, Raymundo Rodriguez Lopez, Kirill Larin, Fabrice Manns, Giuliano Scarcelli.

  Lens biomechanical properties are critical for our eyes to accommodate. While it is well understood that lens mechanical properties change with age, different experimental techniques have been used over the years, with varying results on how the lens modulus changes. In this study, we developed a spatial-varying elasticity model to characterize the overall elastic modulus of the lens and establish its effect on accommodation. First, to validate the model, ex vivo porcine lenses underwent compression testing using biopsy punches of different diameters to change the percentage of nucleus within samples. Importantly, we found that, indeed, changing nucleus/cortex spatial ratio produces dramatic (∼7-fold) increase in overall sample modulus. Comparing the model with human lens spatial ratios, we demonstrate how changing spatial mechanics are more influential than peak modulus changes on overall elastic modulus. Next, in vivo clinical measurements of the spatial-varying lens modulus were used to generate a simplified mechanical-optical model of accommodation. We defined an ellipsoid lens with patient-derived modulus and geometry measurements, and a statics simulation and ray tracing analysis were performed through the deformed and undeformed lens. The resulting accommodation estimates agree with general accommodation expectations.

Keywords:  lens biomechanics; ophthalmology; presbyopia

DOI:  https://doi.org/10.1088/2515-7647/ad3e55
ACS Appl Mater Interfaces. 2024 Jul 09.

Hard- and Soft-Coded Strain Stiffening in Metamaterials via Out-of-Plane Buckling Using Highly Entangled Active Hydrogel Elements.

Oliver Skarsetz, Robin Mathes, Ricarda Sophia Schmidt, Moritz Simon, Viacheslav Slesarenko, Andreas Walther.

  Metamaterials show elaborate mechanical behavior such as strain stiffening, which stems from their unit cell design. However, the stiffening response is typically programmed in the design step and cannot be adapted postmanufacturing. Here, we show hydrogel metamaterials with highly programmable strain-stiffening responses by exploiting the out-of-plane buckling of integrated pH-switchable hydrogel actuators. The stiffening upon reaching a certain extension stems from the initially buckled active hydrogel beams. At low strain, the beams do not contribute to the mechanical response under tension until they straighten with a resulting step-function increase in stiffness. In the hydrogel actuator design, the acrylic acid concentration hard-codes the configuration of the metamaterial and range of possible stiffening onsets, while the pH soft-codes the exact stiffening onset point after fabrication. The utilization of out-of-plane buckling to realize subsequent stiffening without the need to deform the passive structure extends the application of hydrogel actuators in mechanical metamaterials. Our concept of out-of-plane buckled active elements that stiffen only under tension enables strain-stiffening metamaterials with high programmability before and after fabrication.

Keywords:  actuators; highly entangled hydrogels; out-of-plane buckling; programmable metamaterials; strain stiffening

DOI:  https://doi.org/10.1021/acsami.4c06610
Heliyon. 2024 Jun 30. 10(12): e32974

Aging-related changes in the mechanical properties of single cells.

Amarnath Singam, Chandrabali Bhattacharya, Seungman Park.

  Mechanical properties, along with biochemical and molecular properties, play crucial roles in governing cellular function and homeostasis. Cellular mechanics are influenced by various factors, including physiological and pathological states, making them potential biomarkers for diseases and aging. While several methods such as AFM, particle-tracking microrheology, optical tweezers/stretching, magnetic tweezers/twisting cytometry, microfluidics, and micropipette aspiration have been widely utilized to measure the mechanical properties of single cells, our understanding of how aging affects these properties remains limited. To fill this knowledge gap, we provide a brief overview of the commonly used methods to measure single-cell mechanical properties. We then delve into the effects of aging on the mechanical properties of different cell types. Finally, we discuss the importance of studying cellular viscous and viscoelastic properties as well as aging induced by different stressors to gain a deeper understanding of the aging process and aging-related diseases.

Keywords:  Aging; Cell; Cytoskeleton; Elastic property; F-actin; Mechanical property; Stress fiber; Viscous property

DOI:  https://doi.org/10.1016/j.heliyon.2024.e32974
Biotechnol Bioeng. 2024 Jul 08.

The characterization of cell traction force on nonflat surfaces with different curvature by elastic hydrogel microspheres.

Yuqing Dong, Cong Wang, Xin Ding, Xingquan Ma, Rong Huang, Moxiao Li, Qingzhen Yang.

  It is of great importance to study the detachment/attachment behaviors of cells (cancer cell, immune cell, and epithelial cell), as they are closely related with tumor metastasis, immunoreaction, and tissue development at variety scales. To characterize the detachment/attachment during the interaction between cells and substrate, some researchers proposed using cell traction force (CTF) as the indicator. To date, various strategies have been developed to measure the CTF. However, these methods only realize the measurements of cell passive forces on flat cases. To quantify the active CTF on nonflat surfaces, which can better mimic the in vivo case, we employed elastic hydrogel microspheres as a force sensor. The microspheres were fabricated by microfluidic chips with controllable size and mechanical properties to mimic substrate. Cells were cultured on microsphere and the CTF led to the deformation of microsphere. By detecting the morphology information, the CTF exerted by attached cells can be calculated by the in-house numerical code. Using these microspheres, the CTF of various cells (including tumor cell, immunological cell, and epithelium cell) were successfully obtained on nonflat surfaces with different curvature radii. The proposed method provides a versatile platform to measure the CTF with high precision and to understand the detachment/attachment behaviors during physiology processes.

Keywords:  cell traction force; force sensor; microsphere; tumor microenvironment

DOI:  https://doi.org/10.1002/bit.28802
Biomater Adv. 2024 Jun 28. pii: S2772-9508(24)00176-6. [Epub ahead of print]163 213933

Variations in fluid chemical potential induce fibroblast mechano-response in 3D hydrogels.

Lorenza Garau Paganella, Asia Badolato, Céline Labouesse, Gabriel Fischer, Catharina S Sänger, Andreas Kourouklis, Costanza Giampietro, Sabine Werner, Edoardo Mazza, Mark W Tibbitt.

  Mechanical deformation of skin creates variations in fluid chemical potential, leading to local changes in hydrostatic and osmotic pressure, whose effects on mechanobiology remain poorly understood. To study these effects, we investigate the specific influences of hydrostatic and osmotic pressure on primary human dermal fibroblasts in three-dimensional hydrogel culture models. Cyclic hydrostatic pressure and hyperosmotic stress enhanced the percentage of cells expressing the proliferation marker Ki67 in both collagen and PEG-based hydrogels. Osmotic pressure also activated the p38 MAPK stress response pathway and increased the expression of the osmoresponsive genes PRSS35 and NFAT5. When cells were cultured in two-dimension (2D), no change in proliferation was observed with either hydrostatic or osmotic pressure. Furthermore, basal, and osmotic pressure-induced expression of osmoresponsive genes differed in 2D culture versus 3D hydrogels, highlighting the role of dimensionality in skin cell mechanotransduction and stressing the importance of 3D tissue-like models that better replicate in vivo conditions. Overall, these results indicate that fluid chemical potential changes affect dermal fibroblast mechanobiology, which has implications for skin function and for tissue regeneration strategies.

Keywords:  3D culture; Fibroblasts; Hydrogels; Matrix; Mechanobiology

DOI:  https://doi.org/10.1016/j.bioadv.2024.213933
Int J Mol Sci. 2024 Jun 29. pii: 7186. [Epub ahead of print]25(13):

Size Matters: Rethinking Hertz Model Interpretation for Cell Mechanics Using AFM.

Katarína Mendová, Martin Otáhal, Mitja Drab, Matej Daniel.

  Cell mechanics are a biophysical indicator of cell state, such as cancer metastasis, leukocyte activation, and cell cycle progression. Atomic force microscopy (AFM) is a widely used technique to measure cell mechanics, where the Young modulus of a cell is usually derived from the Hertz contact model. However, the Hertz model assumes that the cell is an elastic, isotropic, and homogeneous material and that the indentation is small compared to the cell size. These assumptions neglect the effects of the cytoskeleton, cell size and shape, and cell environment on cell deformation. In this study, we investigated the influence of cell size on the estimated Young's modulus using liposomes as cell models. Liposomes were prepared with different sizes and filled with phosphate buffered saline (PBS) or hyaluronic acid (HA) to mimic the cytoplasm. AFM was used to obtain the force indentation curves and fit them to the Hertz model. We found that the larger the liposome, the lower the estimated Young's modulus for both PBS-filled and HA-filled liposomes. This suggests that the Young modulus obtained from the Hertz model is not only a property of the cell material but also depends on the cell dimensions. Therefore, when comparing or interpreting cell mechanics using the Hertz model, it is essential to account for cell size.

Keywords:  Hertz contact model; atomic force microscopy (AFM); cell mechanics; cell stiffness

DOI:  https://doi.org/10.3390/ijms25137186
Biophys J. 2024 Jul 10. pii: S0006-3495(24)00450-8. [Epub ahead of print]

Dendritic cell force-migration coupling on aligned fiber networks.

Christian Hernandez-Padilla, Ben Joosten, Aime Franco, Alessandra Cambi, Koen van den Dries, Amrinder S Nain.

Dendritic cells (DCs) are antigen-presenting cells that reside in peripheral tissues and are responsible for initiating adaptive immune responses. As gatekeepers of the immune system, DCs need to continuously explore their surroundings, for which they can rapidly move through various types of connective tissue and basement membranes. DC motility has been extensively studied on flat 2D surfaces, yet the influences of a contextual 3D fibrous environment still need to be described. Using ECM-mimicking suspended fiber networks, we show how immature DCs (iDCs) engage in migratory cycles that allow them to transition from persistent migration to slow migratory states. For a subset of iDCs with high migratory potential, we report the organization of protrusions at the front of the cell body, which reverses upon treatment with inflammation agent PGE2. We identify an unusual migratory response to aligned fiber networks, whereby iDCs use filamentous protrusions to attach laterally and exert forces on fibers to migrate independent of fiber alignment. Increasing the fiber diameter from 200 nm to 500 nm does not significantly affect the migratory response, however, iDCs respond by forming denser actin bundles around larger diameters. Overall, the correlation between force-coupling and random migration of iDCs in aligned fibrous topography offers new insights into how iDCs might move in fibrous environments in vivo.

DOI: https://doi.org/10.1016/j.bpj.2024.07.011
Mol Biol Cell. 2024 Jul 10. mbcE23110428

Pulmonary Matrix-derived Hydrogels from Patients with Idiopathic Pulmonary Fibrosis Induce a Proinflammatory State in Lung Fibroblasts In Vitro.

J G Fernandez Davila, A K Singh, D W Moore, J Kim, J A Khan, M Lemma, C S King, S D Nathan, L R Rodriguez, G M Grant, J L Moran.

Idiopathic pulmonary fibrosis (IPF), one of the most common forms of interstitial lung disease, is a poorly understood, chronic, and often fatal fibroproliferative condition with only two FDA-approved medications. Understanding the pathobiology of the fibroblast in IPF is critical to evaluating and discovering novel therapeutics. Using a decellularized lung matrix derived from IPF patients, we generate three-dimensional (3D) hydrogels as in vitro models of lung physiology and characterize the phenotype of fibroblasts seeded into the hydrogels. When cultured in IPF ECM hydrogels, IPF fibroblasts display differential contractility compared to their normal counterparts, lose the classical myofibroblast marker α-smooth muscle actin, and increase expression of proinflammatory cytokines compared to fibroblasts seeded two-dimensionally (2D) on tissue culture dishes. We validate this proinflammatory state in fibroblast-conditioned media studies with monocytes and monocyte-derived macrophages. These findings add to a growing understanding of the lung microenvironment effect on fibroblast phenotypes, shed light on the potential role of fibroblasts as immune signaling hubs during lung fibrosis, and suggest intervention in fibroblast-immune cell crosstalk as a possible novel therapeutic avenue.

DOI: https://doi.org/10.1091/mbc.E23-11-0428
Curr Protoc. 2024 Jul;4(7): e1096

Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.

Ivan Krupa, Niall J Treacy, Shane Clerkin, Jessica L Davis, Aline F Miller, Alberto Saiani, Jacek K Wychowaniec, Emmanuel G Reynaud, Dermot F Brougham, John Crean.

  With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.

Keywords:  GelMA; gelatin methacryloyl; hiPSC‐derived kidney organoids; human induced pluripotent stem cells; hydrogels; self‐assembling peptide hydrogels; stiffness

DOI:  https://doi.org/10.1002/cpz1.1096
Curr Biol. 2024 Jul 04. pii: S0960-9822(24)00821-2. [Epub ahead of print]

Mechanical and biochemical feedback combine to generate complex contractile oscillations in cytokinesis.

Michael E Werner, Dylan D Ray, Coleman Breen, Michael F Staddon, Florian Jug, Shiladitya Banerjee, Amy Shaub Maddox.

  The actomyosin cortex is an active material that generates force to drive shape changes via cytoskeletal remodeling. Cytokinesis is the essential cell division event during which a cortical actomyosin ring closes to separate two daughter cells. Our active gel theory predicted that actomyosin systems controlled by a biochemical oscillator and experiencing mechanical strain would exhibit complex spatiotemporal behavior. To test whether active materials in vivo exhibit spatiotemporally complex kinetics, we imaged the C. elegans embryo with unprecedented temporal resolution and discovered that sections of the cytokinetic cortex undergo periodic phases of acceleration and deceleration. Contractile oscillations exhibited a range of periodicities, including those much longer periods than the timescale of RhoA pulses, which was shorter in cytokinesis than in any other biological context. Modifying mechanical feedback in vivo or in silico revealed that the period of contractile oscillation is prolonged as a function of the intensity of mechanical feedback. Fast local ring ingression occurs where speed oscillations have long periods, likely due to increased local stresses and, therefore, mechanical feedback. Fast ingression also occurs where material turnover is high, in vivo and in silico. We propose that downstream of initiation by pulsed RhoA activity, mechanical feedback, including but not limited to material advection, extends the timescale of contractility beyond that of biochemical input and, therefore, makes it robust to fluctuations in activation. Circumferential propagation of contractility likely allows for sustained contractility despite cytoskeletal remodeling necessary to recover from compaction. Thus, like biochemical feedback, mechanical feedback affords active materials responsiveness and robustness.

Keywords:  C. elegans; active gel theory; active matter; cell division; contractility; cytokinesis; mathematical modeling; microscopy; oscillations; wavelet analysis

DOI:  https://doi.org/10.1016/j.cub.2024.06.037
bioRxiv. 2024 Jun 25. pii: 2024.06.21.600113. [Epub ahead of print]

Myo-optogenetics: optogenetic stimulation and electrical recording in skeletal muscles.

Jeong Jun Kim, Isis S Wyche, William Olson, Jiaao Lu, Muhannad S Bakir, Samuel J Sober, Daniel H O'Connor.

Complex movements involve highly coordinated control of local muscle elements. Highly controlled perturbations of motor outputs can reveal insights into the neural control of movements. Here we introduce an optogenetic method, compatible with electromyography (EMG) recordings, to perturb muscles in transgenic mice. By expressing channelrhodopsin in muscle fibers, we achieved noninvasive, focal activation of orofacial muscles, enabling detailed examination of the mechanical properties of optogenetically evoked jaw muscle contractions. We demonstrated simultaneous EMG recording and optical stimulation, revealing the electrophysiological characteristics of optogenetically triggered muscle activity. Additionally, we applied optogenetic activation of muscles in physiologically and behaviorally relevant settings, mapping precise muscle actions and perturbing active behaviors. Our findings highlight the potential of muscle optogenetics to precisely manipulate muscle activity, offering a powerful tool for probing neuromuscular control systems and advancing our understanding of motor control.

DOI: https://doi.org/10.1101/2024.06.21.600113
Nat Commun. 2024 Jul 11. 15(1): 5840

Turn-on protein switches for controlling actin binding in cells.

Unyime M Effiong, Hannah Khairandish, Isabela Ramirez-Velez, Yanran Wang, Brian Belardi.

Within a shared cytoplasm, filamentous actin (F-actin) plays numerous and critical roles across the cell body. Cells rely on actin-binding proteins (ABPs) to organize F-actin and to integrate its polymeric characteristics into diverse cellular processes. Yet, the multitude of ABPs that engage with and shape F-actin make studying a single ABP's influence on cellular activities a significant challenge. Moreover, without a means of manipulating actin-binding subcellularly, harnessing the F-actin cytoskeleton for synthetic biology purposes remains elusive. Here, we describe a suite of designed proteins, Controllable Actin-binding Switch Tools (CASTs), whose actin-binding behavior can be controlled with external stimuli. CASTs were developed that respond to different external inputs, providing options for turn-on kinetics and enabling orthogonality and multiplexing. Being genetically encoded, we show that CASTs can be inserted into native protein sequences to control F-actin association locally and engineered into structures to control cell and tissue shape and behavior.

DOI: https://doi.org/10.1038/s41467-024-49934-2
Bioact Mater. 2024 Oct;40 227-243

Fabricating oxygen self-supplying 3D printed bioactive hydrogel scaffold for augmented vascularized bone regeneration.

Yang Yang, Wanmeng Wang, Qianrui Zeng, Ning Wang, Wenbo Li, Bo Chen, Qingxin Guan, Changyi Li, Wei Li.

  Limited cells and factors, inadequate mechanical properties, and necrosis of defects center have hindered the wide clinical application of bone-tissue engineering scaffolds. Herein, we construct a self-oxygenated 3D printed bioactive hydrogel scaffold by integrating oxygen-generating nanoparticles and hybrid double network hydrogel structure. The hydrogel scaffold possesses the characteristics of extracellular matrix; Meanwhile, the fabricated hybrid double network structure by polyacrylamide and CaCl2-crosslinked sodium carboxymethylcellulose endows the hydrogel favorable compressive strength and 3D printability. Furthermore, the O2 generated by CaO2 nanoparticles encapsulated in ZIF-8 releases steadily and sustainably because of the well-developed microporous structure of ZIF-8, which can significantly promote cell viability and proliferation in vitro, as well as angiogenesis and osteogenic differentiation with the assistance of Zn2+. More significantly, the synergy of O2 and 3D printed pore structure can prevent necrosis of defects center and facilitate cell infiltration by providing cells the nutrients and space they need, which can further induce vascular network ingrowth and accelerate bone regeneration in all areas of the defect in vivo. Overall, this work provides a new avenue for preparing cell/factor-free bone-tissue engineered scaffolds that possess great potential for tissue regeneration and clinical alternative.

Keywords:  3D printing; Bioactive hydrogel; Bone tissue engineering; Long-term oxygen-generating; Vascularized bone regeneration

DOI:  https://doi.org/10.1016/j.bioactmat.2024.06.016