Membrane Tension Regulation is Required for Wound Repair.

Disruptions of the eukaryotic plasma membrane due to chemical and mechanical challenges are frequent and detrimental and thus need to be repaired to maintain proper cell function and avoid cell death. However, the cellular mechanisms involved in wound resealing and restoration of homeostasis are diverse and contended. Here, it is shown that clathrin-mediated endocytosis is induced at later stages of plasma membrane wound repair following the actual resealing of the wound.

A nanoporous hydrogel-based model to study chemokine gradient-driven angiogenesis under luminal flow.

The growth of new blood vessels through angiogenesis is a highly coordinated process, which is initiated by chemokine gradients that activate endothelial cells within a perfused parent vessel to sprout into the surrounding 3D tissue matrix. While both biochemical signals from pro-angiogenic factors, as well as mechanical cues originating from luminal fluid flow that exerts shear stress on the vessel wall, have individually been identified as major regulators of endothelial cell sprouting, it remains unclear whether and how both types of cues synergize. To fill this knowledge gap, here, we created a 3D biomimetic model of chemokine gradient-driven angiogenic sprouting, in which a micromolded tube inside a hydrogel matrix is seeded with endothelial cells and connected to a perfusion system to control fluid flow rates and resulting shear forces on the vessel wall.

A Protein-Adsorbent Hydrogel with Tunable Stiffness for Tissue Culture Demonstrates Matrix-Dependent Stiffness Responses.

Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, we present a novel hydrophobic hydrogel system chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates.

Stiffness Modulation and Pulsatile Release in Dual Responsive Hydrogels.

Inspired by nature, self-regulation can be introduced in synthetic hydrogels by incorporating chemo-mechanical signals or coupled chemical reactions to maintain or adapt the material's physico-chemical properties when exposed to external triggers. In this work, we present redox and light dual stimuli responsive hydrogels capable of rapidly adapting the polymer crosslinking network while maintaining hydrogel stability. Upon irradiation with UV light, polymer hydrogels containing redox responsive disulfide crosslinks and light responsive ortho-nitrobenzyl moieties show a release of payload accompanied by adaptation of the hydrogel network towards higher stiffness due to in situ crosslinking by S-nitrosylation.

Matrix Resistance Toward Proteolytic Cleavage Controls Contractility-Dependent Migration Modes During Angiogenic Sprouting.

Tissue homeostasis and disease states rely on the formation of new blood vessels through angiogenic sprouting, which is tightly regulated by the properties of the surrounding extracellular matrix. While physical cues, such as matrix stiffness or degradability, have evolved as major regulators of cell function in tissue microenvironments, it remains unknown whether and how physical cues regulate endothelial cell migration during angiogenesis. To investigate this, a biomimetic model of angiogenic sprouting inside a tunable synthetic hydrogel is created.

Tunable Synthetic Hydrogels to Study Angiogenic Sprouting.

Angiogenic sprouting, the formation of new blood vessels from pre-existing vasculature, is tightly regulated by the properties of the surrounding tissue microenvironment. Although the extracellular matrix has been shown to be a major regulator of this process, it is not clear how individual biochemical and mechanical properties influence endothelial cell sprouting. This information gap is largely due to the lack of suitable in vitro models that recapitulate angiogenic sprouting in a 3D environment with independent control over matrix properties.

3D biomimetic environment enabling ex utero trophoblast invasion and co-culture of embryos and somatic cells.

The first direct contact between the embryo and the mother is established during implantation. This process is inaccessible for direct studies as the implanting embryo is concealed by the maternal tissues. Here, we present a protocol for establishing a 3D biomimetic environment based on synthetic hydrogels which harbor key biomechanical properties of the uterine stroma.

Polarity inversion reorganizes the stem cell compartment of the trophoblast lineage.

The extra-embryonic tissues that form the placenta originate from a small population of trophectoderm cells with stem cell properties, positioned at the embryonic pole of the mouse blastocyst. During the implantation stages, the polar trophectoderm rapidly proliferates and transforms into extra-embryonic ectoderm. The current model of trophoblast morphogenesis suggests that tissue folding reshapes the trophoblast during the blastocyst to egg cylinder transition.

Primordial germ cells adjust their protrusion type while migrating in different tissue contexts in vivo.

In both physiological processes and disease contexts, migrating cells have the ability to adapt to conditions in their environment. As an in vivo model for this process, we use zebrafish primordial germ cells that migrate throughout the developing embryo. When migrating within an ectodermal environment, the germ cells form fewer and smaller blebs when compared with their behavior within mesodermal environment.

Nonswelling and Hydrolytically Stable Hydrogels Uncover Cellular Mechanosensing in 3D.

While matrix stiffness regulates cell behavior on 2D substrates, recent studies using synthetic hydrogels have suggested that in 3D environments, cell behavior is primarily impacted by matrix degradability, independent of stiffness. However, these studies did not consider the potential impact of other confounding matrix parameters that typically covary with changes in stiffness, particularly, hydrogel swelling and hydrolytic stability, which may explain the previously observed distinctions in cell response in 2D versus 3D settings. To investigate how cells sense matrix stiffness in 3D environments, a nonswelling, hydrolytically stable, linearly elastic synthetic hydrogel model is developed in which matrix stiffness and degradability can be tuned independently.

Force-induced changes of α-catenin conformation stabilize vascular junctions independently of vinculin.

Cadherin-mediated cell adhesion requires anchoring via the β-catenin-α-catenin complex to the actin cytoskeleton, yet, α-catenin only binds F-actin weakly. A covalent fusion of VE-cadherin to α-catenin enhances actin anchorage in endothelial cells and strongly stabilizes endothelial junctions in vivo, blocking inflammatory responses. Here, we have analyzed the underlying mechanism.

3D biomimetic platform reveals the first interactions of the embryo and the maternal blood vessels.

The process of implantation and the cellular interactions at the embryo-maternal interface are intrinsically difficult to analyze, as the implanting embryo is concealed by the uterine tissues. Therefore, the mechanisms mediating the interconnection of the embryo and the mother are poorly understood. Here, we established a 3D biomimetic culture environment that harbors the key features of the murine implantation niche.

Synthetic extracellular matrices with tailored adhesiveness and degradability support lumen formation during angiogenic sprouting.

A major deficit in tissue engineering strategies is the lack of materials that promote angiogenesis, wherein endothelial cells from the host vasculature invade the implanted matrix to form new blood vessels. To determine the material properties that regulate angiogenesis, we have developed a microfluidic in vitro model in which chemokine-guided endothelial cell sprouting into a tunable hydrogel is followed by the formation of perfusable lumens. We show that long, perfusable tubes only develop if hydrogel adhesiveness and degradability are fine-tuned to support the initial collective invasion of endothelial cells and, at the same time, allow for matrix remodeling to permit the opening of lumens.

PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin.

Leukocyte extravasation is an essential step during the immune response and requires the destabilization of endothelial junctions. We have shown previously that this process depends in vivo on the dephosphorylation of VE-cadherin-Y731. Here, we reveal the underlying mechanism.

Ligand Diffusion Enables Force-Independent Cell Adhesion via Activating α5β1 Integrin and Initiating Rac and RhoA Signaling.

Cells reside in a dynamic microenvironment in which adhesive ligand availability, density, and diffusivity are key factors regulating cellular behavior. Here, the cellular response to integrin-binding ligand dynamics by directly controlling ligand diffusivity via tunable ligand-surface interactions is investigated. Interestingly, cell spread on the surfaces with fast ligand diffusion is independent of myosin-based force generation.

Pulmonary pericytes regulate lung morphogenesis.

Blood vessels are essential for blood circulation but also control organ growth, homeostasis, and regeneration, which has been attributed to the release of paracrine signals by endothelial cells. Endothelial tubules are associated with specialised mesenchymal cells, termed pericytes, which help to maintain vessel wall integrity. Here we identify pericytes as regulators of epithelial and endothelial morphogenesis in postnatal lung.

Matrix degradability controls multicellularity of 3D cell migration.

A major challenge in tissue engineering is the development of materials that can support angiogenesis, wherein endothelial cells from existing vasculature invade the surrounding matrix to form new vascular structures. To identify material properties that impact angiogenesis, here we have developed an in vitro model whereby molded tubular channels inside a synthetic hydrogel are seeded with endothelial cells and subjected to chemokine gradients within a microfluidic device. To accomplish precision molding of hydrogels and successful integration with microfluidics, we developed a class of hydrogels that could be macromolded and micromolded with high shape and size fidelity by eliminating swelling after polymerization.

Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments.

To investigate how cells sense stiffness in settings structurally similar to native extracellular matrices, we designed a synthetic fibrous material with tunable mechanics and user-defined architecture. In contrast to flat hydrogel surfaces, these fibrous materials recapitulated cell-matrix interactions observed with collagen matrices including stellate cell morphologies, cell-mediated realignment of fibres, and bulk contraction of the material. Increasing the stiffness of flat hydrogel surfaces induced mesenchymal stem cell spreading and proliferation; however, increasing fibre stiffness instead suppressed spreading and proliferation for certain network architectures.

Remodeling of fibrous extracellular matrices by contractile cells: predictions from discrete fiber network simulations.

Contractile forces exerted on the surrounding extracellular matrix (ECM) lead to the alignment and stretching of constituent fibers within the vicinity of cells. As a consequence, the matrix reorganizes to form thick bundles of aligned fibers that enable force transmission over distances larger than the size of the cells. Contractile force-mediated remodeling of ECM fibers has bearing on a number of physiologic and pathophysiologic phenomena.

A DNA-based molecular probe for optically reporting cellular traction forces.

We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can in principle be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.

Micropatterned multicolor dynamically adhesive substrates to control cell adhesion and multicellular organization.

We present a novel technique to examine cell-cell interactions and directed cell migration using micropatterned substrates of three distinct regions: an adhesive region, a nonadhesive region, and a dynamically adhesive region switched by addition of a soluble factor to the medium. Combining microcontact printing with avidin-biotin capture chemistry, we pattern nonadhesive regions of avidin that become adhesive through the capture of biotinylated fibronectin. Our strategy overcomes several limitations of current two-color dynamically adhesive substrates by incorporating a third, permanently nonadhesive region.

How cells sense extracellular matrix stiffness: a material's perspective.

The mechanical properties of the extracellular matrix (ECM) in which cells reside have emerged as an important regulator of cell fate. While materials based on natural ECM have been used to implicate the role of substrate stiffness for cell fate decisions, it is difficult in these matrices to isolate mechanics from other structural parameters. In contrast, fully synthetic hydrogels offer independent control over physical and adhesive properties.

Extracellular-matrix tethering regulates stem-cell fate.

To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating.

Mimicking normal tissue architecture and perturbation in cancer with engineered micro-epidermis.

Correct tissue architecture is essential for normal physiology, yet there have been few attempts to recreate tissues using micro-patterning. We have used polymer brush micro-engineering to generate a stratified micro-epidermis with fewer than 10 human keratinocytes. Epidermal stem cells are captured on 100 μm diameter circular collagen-coated disks.