Bioinspired Lubricity from Surface Gel Layers.

Surface gel layers on commercially available contact lenses have been shown to reduce frictional shear stresses and mitigate damage during sliding contact with fragile epithelial cell layers in vitro. Spencer and co-workers recently demonstrated that surface gel layers could arise from oxygen-inhibited free-radical polymerization. In this study, polyacrylamide hydrogel shell probes (7.5 wt % acrylamide, 0.3 wt % N,N'-methylenebisacrylamide) were polymerized in three hemispherical molds listed in order of decreasing surface energy and increasing oxygen permeability: borosilicate glass, polyether ether ketone (PEEK), and polytetrafluoroethylene (PTFE). Hydrogel probes polymerized in PEEK and PTFE molds exhibited 100× lower elastic moduli at the surface (EPEEK* = 80 ± 31 and EPTFE* = 106 ± 26 Pa, respectively) than those polymerized in glass molds (Eglass* = 31,560 ± 1,570 Pa), in agreement with previous investigations by Spencer and co-workers. Biotribological experiments revealed that hydrogel probes with surface gel layers reduced frictional shear stresses against cells (τPEEK = 35 ± 15 and τPTFE = 22 ± 16 Pa) more than those without (τglass = 68 ± 15 Pa) and offered greater protection against cell damage when sliding against human telomerase-immortalized corneal epithelial (hTCEpi) cell monolayers. Our work demonstrates that the "mold effect" resulting in oxygen-inhibition polymerization creates hydrogels with surface gel layers that reduce shear stresses in sliding contact with cell monolayers, similar to the protection offered by gradient mucin gel networks across epithelial cell layers.

Block Polyelectrolyte Additives That Modulate the Viscoelasticity and Enhance the Printability of Gelatin Inks at Physiological Temperatures.

We demonstrate the utility of block polyelectrolyte (bPE) additives to enhance viscosity and resolve challenges with the three-dimensional (3D) printability of extrusion-based biopolymer inks. The addition of oppositely charged bPEs to solutions of photocurable gelatin methacryloyl (GelMA) results in complexation-driven self-assembly of the bPEs, leading to GelMA/bPE inks that are printable at physiological temperatures, representing stark improvements over GelMA inks that suffer from low viscosity at 37 °C, leading to low printability and poor structural stability. The hierarchical microstructure of the self-assemblies (either jammed micelles or 3D networks) formed by the oppositely charged bPEs, confirmed by small-angle X-ray scattering, is attributed to the enhancements in the shear strength and printability of the GelMA/bPE inks. Varying bPE concentration in the inks is shown to enable tunability of the rheological properties to meet the criteria of pre- and postextrusion flow characteristics for 3D printing, including prominent yielding behavior, strong shear thinning, and rapid recovery upon flow cessation. Moreover, the bPE self-assemblies also contribute to the robustness of the photo-cross-linked hydrogels; photo-cross-linked GelMA/bPE hydrogels are shown to exhibit higher shear strength than photo-cross-linked GelMA hydrogels. Last, the assessment of the printability of GelMA/bPE inks indicates excellent printing performance, including minimal swelling postextrusion, satisfactory retention of the filament shape upon deposition, and satisfactory shape fidelity of the various printed constructs. We envision this study to serve as a practical guide for the printing of bespoke extrusion inks where bPEs are used as scaffolds and viscosity enhancers that can be emulated in a range of photocurable precursors.

Molecular-scale substrate anisotropy, crowding and division drive collective behaviours in cell monolayers.

The ability of cells to reorganize in response to external stimuli is important in areas ranging from morphogenesis to tissue engineering. While nematic order is common in biological tissues, it typically only extends to small regions of cells interacting via steric repulsion. On isotropic substrates, elongated cells can co-align due to steric effects, forming ordered but randomly oriented finite-size domains. However, we have discovered that flat substrates with nematic order can induce global nematic alignment of dense, spindle-like cells, thereby influencing cell organization and collective motion and driving alignment on the scale of the entire tissue. Remarkably, single cells are not sensitive to the substrate's anisotropy. Rather, the emergence of global nematic order is a collective phenomenon that requires both steric effects and molecular-scale anisotropy of the substrate. To quantify the rich set of behaviours afforded by this system, we analyse velocity, positional and orientational correlations for several thousand cells over days. The establishment of global order is facilitated by enhanced cell division along the substrate's nematic axis, and associated extensile stresses that restructure the cells' actomyosin networks. Our work provides a new understanding of the dynamics of cellular remodelling and organization among weakly interacting cells.

Tailoring Writability and Performance of Star Block Copolypeptides Hydrogels through Side-Chain Design.

Shear-recoverable hydrogels based on block copolypeptides with rapid self-recovery hold potential in extrudable and injectable 3D-printing applications. In this work, a series of 3-arm star-shaped block copolypeptides composed of an inner hydrophilic poly(l-glutamate) domain and an outer ß-sheet forming domain is synthesized with varying side chains and block lengths. By changing the ß-sheet forming domains, hydrogels with diverse microstructures and mechanical properties are prepared and structure-function relationships are determined using scattering and rheological techniques. Differences in the properties of these materials are amplified during direct-ink writing with a strong correlation observed between printability and material chemistry. Significantly, it is observed that non-canonical ß-sheet blocks based on phenyl glycine form more stable networks with superior mechanical properties and writability compared to widely used natural amino acid counterparts. The versatile design available through block copolypeptide materials provides a robust platform to access tunable material properties based solely on molecular design. These systems can be exploited in extrusion-based applications such as 3D-printing without the need for additives.

Silicone Implant Surface Roughness, Friction, and Wear.

Textured silicone breast implants with high average surface roughness ("macrotextured") have been associated with a rare cancer of the immune system, Breast Implant-Associated Anaplastic Large Cell Lymphoma (BIA-ALCL). Silicone elastomer wear debris may lead to chronic inflammation, a key step in the development of this cancer. Here, we model the generation and release of silicone wear debris in the case of a folded implant-implant ("shell-shell") sliding interface for three different types of implants, characterized by their surface roughness. The "smooth" implant shell with the lowest average surface roughness tested (Ra = 2.7 ± 0.6 µm) resulted in average friction coefficients of µavg = 0.46 ± 0.11 across 1,000 mm of sliding distance and generated 1,304 particles with an average particle diameter of Davg = 8.3 ± 13.1 µm. The "microtextured" implant shell (Ra = 32 ± 7.0 µm) exhibited µavg = 1.20 ± 0.10 and generated 2,730 particles with Davg = 4.7 ± 9.1 µm. The "macrotextured" implant shell (Ra = 80 ± 10 µm) exhibited the highest friction coefficients, µavg = 2.82 ± 0.15 and the greatest number of wear debris particles, 11,699, with an average particle size of Davg = 5.3 ± 3.3 µm. Our data may provide guidance for the design of silicone breast implants with lower surface roughness, lower friction, and smaller quantities of wear debris.

Design, Synthesis, and Application of a Water-soluble Photocage for Aqueous Cyclopentadiene-based Diels-Alder Photoclick Chemistry in Hydrogels.

Spatiotemporally functionalized hydrogels have exciting applications in tissue engineering, but their preparation often relies on radical-based strategies that can be deleterious in biological settings. Herein, the computationally guided design, synthesis, and application of a water-soluble cyclopentadienone-norbornadiene (CPD-NBD) adduct is disclosed as a diene photocage for radical-free Diels-Alder photopatterning. We show that this scalable CPD-NBD derivative is readily incorporated into hydrogel formulations, providing gels that can be patterned with dienophiles upon 365 nm uncaging of cyclopentadiene. Patterning is first visualized through conjugation of cyanine dyes, then biological utility is highlighted by patterning peptides to direct cellular adhesion. Finally, the ease of use and versatility of this CPD-NBD derivative is demonstrated by direct incorporation into a commercial 3D printing resin to enable the photopatterning of structurally complex, printed hydrogels.

3D In Vitro Platform for Cell and Explant Culture in Liquid-like Solids.

Existing 3D cell models and technologies have offered tools to elevate cell culture to a more physiologically relevant dimension. One mechanism to maintain cells cultured in 3D is by means of perfusion. However, existing perfusion technologies for cell culture require complex electronic components, intricate tubing networks, or specific laboratory protocols for each application. We have developed a cell culture platform that simply employs a pump-free suction device to enable controlled perfusion of cell culture media through a bed of granular microgels and removal of cell-secreted metabolic waste. We demonstrated the versatile application of the platform by culturing single cells and keeping tissue microexplants viable for an extended period. The human cardiomyocyte AC16 cell line cultured in our platform revealed rapid cellular spheroid formation after 48 h and ~90% viability by day 7. Notably, we were able to culture gut microexplants for more than 2 weeks as demonstrated by immunofluorescent viability assay and prolonged contractility.

Ex vivo SARS-CoV-2 infection of human lung reveals heterogeneous host defense and therapeutic responses.

Cell lines are the mainstay in understanding the biology of COVID-19 infection but do not recapitulate many of the complexities of human infection. The use of human lung tissue is one solution for the study of such novel respiratory pathogens. We hypothesized that a cryopreserved bank of human lung tissue would allow for the ex vivo study of the interindividual heterogeneity of host response to SARS-CoV-2, thus providing a bridge between studies with cell lines and studies in animal models. We generated a cryobank of tissues from 21 donors, many of whom had clinical risk factors for severe COVID-19. Cryopreserved tissues preserved 90% cell viability and contained heterogenous populations of metabolically active epithelial, endothelial, and immune cell subsets of the human lung. Samples were readily infected with HCoV-OC43 and SARS-CoV-2 and demonstrated comparable susceptibility to infection. In contrast, we observed a marked donor-dependent heterogeneity in the expression of IL6, CXCL8, and IFNB1 in response to SARS-CoV-2. Treatment of tissues with dexamethasone and the experimental drug N-hydroxycytidine suppressed viral growth in all samples, whereas chloroquine and remdesivir had no detectable effect. Metformin and sirolimus, molecules with predicted but unproven antiviral activity, each suppressed viral replication in tissues from a subset of donors. In summary, we developed a system for the ex vivo study of human SARS-CoV-2 infection using primary human lung tissue from a library of donor tissues. This model may be useful for drug screening and for understanding basic mechanisms of COVID-19 pathogenesis.

Hydration Control of Gel-Adhesion and Muco-Adhesion.

Protective mucin gel layers established by epithelial cell surfaces in biology have water contents above 90% and provide a low-shear stress nonadhesive interfacial boundary on epithelial surfaces throughout the body. Adhesion between gels and mucin layers, muco-adhesion, is an important aspect of drug delivery, biocompatibility, and the prevention of damage during insertion, use, and removal of medical devices in contact with moist epithelial surfaces. This manuscript develops a simple mathematical model to suggest that gel-adhesion and muco-adhesion are controlled by dehydration. For a fully swollen gel, the osmotic pressure is balanced by the elastic stress in the polymer gel, and differences in the elastic modulus are used to calculate dehydration stresses. A model based on Winkler contact mechanics gives a closed form expression for the force of adhesion that is dependent on the contact radius and gel thickness, inversely proportional to the mucin layer stiffness, and proportional to the square of the differences in elastic modulus. Submerged contact experiments conducted on Gemini gel interfaces of polyacrylamide aqueous gels showed increasing adhesion with increasing dehydration of the probe. Additionally, experiments conducted against mucinated epithelial cell monolayers found mucin transfer onto the most dehydrated gels and no transfer on swollen gels. The model and experiments reveal that high water content fully swollen gels are not intrinsically muco-adhesive, which is consistent with previous tribological experience showing increased lubricity with increasing water content and mesh size.

Polyelectrolyte scaling laws for microgel yielding near jamming.

Micro-scale hydrogel particles, known as microgels, are used in industry to control the rheology of numerous different products, and are also used in experimental research to study the origins of jamming and glassy behavior in soft-sphere model systems. At the macro-scale, the rheological behaviour of densely packed microgels has been thoroughly characterized; at the particle-scale, careful investigations of jamming, yielding, and glassy-dynamics have been performed through experiment, theory, and simulation. However, at low packing fractions near jamming, the connection between microgel yielding phenomena and the physics of their constituent polymer chains has not been made. Here we investigate whether basic polymer physics scaling laws predict macroscopic yielding behaviours in packed microgels. We measure the yield stress and cross-over shear-rate in several different anionic microgel systems prepared at packing fractions just above the jamming transition, and show that our data can be predicted from classic polyelectrolyte physics scaling laws. We find that diffusive relaxations of microgel deformation during particle re-arrangements can predict the shear-rate at which microgels yield, and the elastic stress associated with these particle deformations predict the yield stress.

Publisher's Note: Multicellular density fluctuations in epithelial monolayers [Phys. Rev. E 92, 032729 (2015)].

This corrects the article DOI: 10.1103/PhysRevE.92.032729.

Liquid-like Solids Support Cells in 3D.

The demands of tissue engineering have driven a tremendous amount of research effort in 3D tissue culture technology and, more recently, in 3D printing. The need to use 3D tissue culture techniques more broadly in all of cell biology is well-recognized, but the transition to 3D has been impeded by the convenience, effectiveness, and ubiquity of 2D culture materials, assays, and protocols, as well as the lack of 3D counterparts of these tools. Interestingly, progress and discoveries in 3D bioprinting research may provide the technical support needed to grow the practice of 3D culture. Here we investigate an integrated approach for 3D printing multicellular structures while using the same platform for 3D cell culture, experimentation, and assay development. We employ a liquid-like solid (LLS) material made from packed granular-scale microgels, which locally and temporarily fluidizes under the focused application of stress and spontaneously solidifies after the applied stress is removed. These rheological properties enable 3D printing of multicellular structures as well as the growth and expansion of cellular structures or dispersed cells. The transport properties of LLS allow molecular diffusion for the delivery of nutrients or small molecules for fluorescence-based assays. Here, we measure viability of 11 different cell types in the LLS medium, we 3D print numerous structures using several of these cell types, and we explore the transport properties in molecular time-release assays.

Kinetics of aqueous lubrication in the hydrophilic hydrogel Gemini interface.

The exquisite sliding interfaces in the human body share the common feature of hydrated dilute polymer mesh networks. These networks, especially when they constitute a sliding interface such as the pre-corneal tear film on the ocular interface, are described by the molecular weight of the polymer chains and a characteristic size of a minimum structural unit, the mesh size, ξ. In a Gemini interface where hydrophilic hydrogels are slid against each other, the aqueous lubrication behavior has been shown to be a function of sliding velocity, introducing a sliding timescale competing against the time scales of polymer fluctuation and relaxation at the surface. In this work, we examine two recent studies and postulate that when the Gemini interface slips faster than the single-chain relaxation time, chains must relax, suppressing the amplitude of the polymer chain thermal fluctuations.

Multicellular density fluctuations in epithelial monolayers.

Changes in cell size often accompany multicellular motion in tissue, and cell number density is known to strongly influence collective migration in monolayers. Density fluctuations in other forms of active matter have been explored extensively, but not the potential role of density fluctuations in collective cell migration. Here we investigate collective motion in cell monolayers, focusing on the divergent component of the migration velocity field to probe density fluctuations. We find spatial patterns of diverging and converging cell groups throughout the monolayers, which oscillate in time with a period of approximately 3-4 h. Simultaneous fluorescence measurements of a cytosol dye within the cells show that fluid passes between groups of cells, facilitating these oscillations in cell density. Our findings reveal that cell-cell interactions in monolayers may be mediated by intercellular fluid flow.