PIEZO1-mediated mechanosensing governs NK-cell killing efficiency and infiltration in three-dimensional matrices.

Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments are critical for their functions under physiological conditions. However, the role of mechanosensing in regulating NK-cell killing efficiency in physiologically relevant scenarios is poorly understood. Here, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK-cell killing efficiency in 3D is impaired against softened tumor cells, whereas it is enhanced against stiffened tumor cells. Notably, the durations required for NK-cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel among the examined candidates in NK cells. Perturbation of PIEZO1 abolishes stiffness-dependent NK-cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK-cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK-cell killing efficiency under 3D physiological conditions and the influence of environmental physical cues on NK-cell functions.

Rheological behavior of Pluronic/Pluronic diacrylate hydrogels used for bacteria encapsulation in engineered living materials.

Pluronic (Plu) hydrogels mixed with variable fractions of Pluronic diacrylate (PluDA) have become popular matrices to encapsulate bacteria and control their growth in engineered living materials. Here we study the rheological response of 30 wt% Plu/PluDA hydrogels with PluDA fraction between 0 and 1. We quantify the range of viscoelastic properties that can be covered in this system by varying in the PluDA fraction. We present stress relaxation and creep-recovery experiments and describe the variation of the critical yield strain/stress, relaxation and recovery parameters of Plu/PluDA hydrogels as function of the covalent crosslinking degree using the Burgers and Weilbull models. The analyzed hydrogels present two stress relaxations with different timescales which can be tuned with the covalent crosslinking degree. We expect this study to help users of Plu/PluDA hydrogels to estimate the mechanical properties of their systems, and to correlate them with the behaviour of bacteria in future Plu/PluDA devices of similar composition.

Active superelasticity in three-dimensional epithelia of controlled shape.

Fundamental biological processes are carried out by curved epithelial sheets that enclose a pressurized lumen. How these sheets develop and withstand three-dimensional deformations has remained unclear. Here we combine measurements of epithelial tension and shape with theoretical modelling to show that epithelial sheets are active superelastic materials. We produce arrays of epithelial domes with controlled geometry. Quantification of luminal pressure and epithelial tension reveals a tensional plateau over several-fold areal strains. These extreme strains in the tissue are accommodated by highly heterogeneous strains at a cellular level, in seeming contradiction to the measured tensional uniformity. This phenomenon is reminiscent of superelasticity, a behaviour that is generally attributed to microscopic material instabilities in metal alloys. We show that in epithelial cells this instability is triggered by a stretch-induced dilution of the actin cortex, and is rescued by the intermediate filament network. Our study reveals a type of mechanical behaviour-which we term active superelasticity-that enables epithelial sheets to sustain extreme stretching under constant tension.

Thiol-Methylsulfone-Based Hydrogels for Cell Encapsulation: Reactivity Optimization of Aryl-Methylsulfone Substrate for Fine-Tunable Gelation Rate and Improved Stability.

Hydrogels are widely used as hydrated matrices for cell encapsulation in a number of applications, spanning from advanced 3D cultures and tissue models to cell-based therapeutics and tissue engineering. Hydrogel formation in the presence of living cells requires cross-linking reactions that proceed efficiently under close to physiological conditions. Recently, the nucleophilic aromatic substitution of phenyl-oxadiazole (Ox) methylsulfones (MS) by thiols was introduced as a new cross-linking reaction for cell encapsulation. Reported poly(ethylene glycol) (PEG)-based hydrogels featured tunable gelation times within seconds to a few minutes within pH 8.0 to 6.6 and allowed reasonably good mixing with cells. However, their rapid degradation prevented cell cultures to be maintained beyond 1 week. In this Article, we present the reactivity optimization of the heteroaromatic ring of the MS partner to slow down the cross-linking kinetics and the degradability of the derived hydrogels. New MS substrates based on phenyl-tetrazole (Tz) and benzothiazole (Bt) rings, with lower electrophilicity than Ox, were synthesized by simple pathways. When mixed with PEG-thiol, the novel PEG-MS extended the working time of precursor mixtures and allowed longer term cell culture. The Tz-based MS substrate was identified as the best candidate, as it is accessible by simple chemical reactions from cost-effective reactants, hydrogel precursors show good stability in aqueous solution and keep high chemoselectivity for thiols, and the derived Tz gels support cell cultures for >2 weeks. The Tz system also shows tunable gelation kinetics within seconds to hours and allows comfortable manipulation and cell encapsulation. Our findings expand the toolkit of thiol-mediated chemistry for the synthesis of hydrogels with improved properties for laboratory handling and future automatization.

Macroscopic Self-Evolution of Dynamic Hydrogels to Create Hollow Interiors.

A solid-to-hollow evolution in macroscopic structures is challenging in synthetic materials. A fundamentally new strategy is reported for guiding macroscopic, unidirectional shape evolution of materials without compromising the material's integrity. This strategy is based on the creation of a field with a "swelling pole" and a "shrinking pole" to drive polymers to disassemble, migrate, and resettle in the targeted region. This concept is demonstrated using dynamic hydrogels containing anchored acrylic ligands and hydrophobic long alkyl chains. Adding water molecules and ferric ions (Fe3+ ) to induce a swelling-shrinking field transforms the hydrogels from solid to hollow. The strategy is versatile in the generation of various closed hollow objects (for example, spheres, helix tubes, and cubes with different diameters) for different applications.

Optoregulated Drug Release from an Engineered Living Material: Self-Replenishing Drug Depots for Long-Term, Light-Regulated Delivery.

On-demand and long-term delivery of drugs are common requirements in many therapeutic applications, not easy to be solved with available smart polymers for drug encapsulation. This work presents a fundamentally different concept to address such scenarios using a self-replenishing and optogenetically controlled living material. It consists of a hydrogel containing an active endotoxin-free Escherichia coli strain. The bacteria are metabolically and optogenetically engineered to secrete the antimicrobial and antitumoral drug deoxyviolacein in a light-regulated manner. The permeable hydrogel matrix sustains a viable and functional bacterial population and permits diffusion and delivery of the synthesized drug to the surrounding medium at quantities regulated by light dose. Using a focused light beam, the site for synthesis and delivery of the drug can be freely defined. The living material is shown to maintain considerable levels of drug production and release for at least 42 days. These results prove the potential and flexibility that living materials containing engineered bacteria can offer for advanced therapeutic applications.

Goosebumps-Inspired Microgel Patterns with Switchable Adhesion and Friction.

A substrate mimicking the surface topography and temperature sensitivity of skin goosebumps is fabricated. Close-packed arrays of thermoresponsive microgel particles undergo topographical changes in response to temperature changes between 25 and 37 °C, resembling the goosebump structure that human skin develops in response to temperature changes or other circumstances. Specifically, positively charged poly[2-(methacryloyloxy)ethyltrimethylammonium chloride] (PMETAC) brushes serve as an anchoring substrate for negatively charged poly(NIPAm-co-AA) microgels. The packing density and particle morphology can be tuned by brush layer thickness and pH of the microgel suspension. For brush layer thickness below 50 nm, particle monolayers are observed, with slightly flattened particle morphology at pH 3 and highly collapsed particles at pH above 7. Polymer brush films with thickness above 50 nm lead to the formation of particle multilayers. The temperature responsiveness of the monolayer assemblies allows reversible changes in the film morphology, which in turn affects underwater adhesion and friction at 25 and 37 °C. These results are promising for the design of new functional materials and may also serve as a model for biological structures and processes.

Four and a Half LIM Domains 1b (Fhl1b) Is Essential for Regulating the Liver versus Pancreas Fate Decision and for ß-Cell Regeneration.

The liver and pancreas originate from overlapping embryonic regions, and single-cell lineage tracing in zebrafish has shown that Bone morphogenetic protein 2b (Bmp2b) signaling is essential for determining the fate of bipotential hepatopancreatic progenitors towards the liver or pancreas. Despite its pivotal role, the gene regulatory networks functioning downstream of Bmp2b signaling in this process are poorly understood. We have identified four and a half LIM domains 1b (fhl1b), which is primarily expressed in the prospective liver anlage, as a novel target of Bmp2b signaling. fhl1b depletion compromised liver specification and enhanced induction of pancreatic cells from endodermal progenitors. Conversely, overexpression of fhl1b favored liver specification and inhibited induction of pancreatic cells. By single-cell lineage tracing, we showed that fhl1b depletion led lateral endodermal cells, destined to become liver cells, to become pancreatic cells. Reversely, when fhl1b was overexpressed, medially located endodermal cells, fated to differentiate into pancreatic and intestinal cells, contributed to the liver by directly or indirectly modulating the discrete levels of pdx1 expression in endodermal progenitors. Moreover, loss of fhl1b increased the regenerative capacity of ß-cells by increasing pdx1 and neurod expression in the hepatopancreatic ductal system. Altogether, these data reveal novel and critical functions of Fhl1b in the hepatic versus pancreatic fate decision and in ß-cell regeneration.

A Photoactivatable α5 ß1 -Specific Integrin Ligand.

The integrin α5 ß1 is overexpressed in colon, breast, ovarian, lung and brain tumours, and has been identified as key component in mechanosensing. In order to study how dynamic changes in α5 ß1 engagement affect cellular behaviour, photoactivatable derivatives of α5 ß1 -specific ligands are presented in this article. A photoremovable protecting group (PRPG) was introduced into the ligand structure at a relevant position for integrin recognition. The presence of the chromophore temporarily inhibited ligand bioactivity. Light exposure at a cell-compatible dose efficiently cleaved the protecting group and restored functionality. The photoactive ligand had an azide end-functional group for covalent immobilisation onto biomaterials by click chemistry. Selective cell responses (attachment, spreading, migration) to the activated ligand on the surface are achieved by controlled exposure to light, at similar levels to the native ligand. Spatial and temporal control of the cellular response is demonstrated, including the possibility of in situ activation. Photoactivatable integrin-selective ligands in model microenvironments will allow the study of cellular behaviour in response to changes in the activation of individual integrins as consequence of dynamic variations in matrix composition.

Photoactivatable Adhesive Ligands for Light-Guided Neuronal Growth.

Neuro-regeneration after trauma requires growth and reconnection of neurons to reestablish information flow in particular directions across the damaged tissue. To support this process, biomaterials for nerve tissue regeneration need to provide spatial information to adhesion receptors on the cell membrane and to provide directionality to growing neurites. Here, photoactivatable adhesive peptides based on the CASIKVAVSADR laminin peptidomimetic are presented and applied to spatiotemporal control of neuronal growth to biomaterials in vitro. The introduction of a photoremovable group [6-nitroveratryl (NVOC), 3-(4,5-dimethoxy-2-nitrophenyl)butan-2-yl (DMNPB), or 2,2'-((3'-(1-hydroxypropan-2-yl)-4'-nitro-[1,1'-biphenyl]-4-yl)azanediyl)bis(ethan-1-ol) (HANBP)] at the amino terminal group of the K residue temporally inhibited the activity of the peptide. The bioactivity was regained through controlled light exposure. When used in neuronal culture substrates, the peptides allowed light-based control of the attachment and differentiation of neuronal cells. Site-selective irradiation activated adhesion and differentiation cues and guided seeded neurons to grow in predefined patterns. This is the first demonstration of ligand-based light-controlled interaction between neuronal cells and biomaterials.

Optoregulated Biointerfaces to Trigger Cellular Responses.

Optoregulated biointerfaces offer the possibility to manipulate the interactions between cell membrane receptors and the extracellular space. This Invited Feature Article summarizes recent efforts by our group and others during the past decade to develop light-responsive biointerfaces to stimulate cells and elicit cellular responses using photocleavable protecting groups (PPG) as our working tool. This article begins by providing a brief introduction to available PPGs, with a special focus on the widely used o-nitrobenzyl family, followed by an overview of molecular design principles for the control of bioactivity in the context of cell-material interactions and the characterization methods to use in following the photoreaction at surfaces. We present various light-guided cellular processes using PPGs, including cell adhesion, release, migration, proliferation, and differentiation, both in vitro and in vivo. Finally, this Invited Feature Article closes with our perspective on the current status and future challenges of this topic.

Multifunctional Thin Films and Coatings from Caffeic Acid and a Cross-Linking Diamine.

The exploitation of easily accessible and nontoxic natural catechol compounds for surface functionalization and coating is attracting growing interest for biomedical applications. We report herein the deposition on different substrates of chemically stable thin films by autoxidation of 1 mM caffeic acid (CA) solutions at pH 9 in the presence of equimolar amounts of hexamethylenediamine (HMDA). UV-visible, mass spectrometric, and solid state 13C and 15N NMR analysis indicated covalent incorporation of the amine during CA polymerization to produce insoluble trioxybenzacridinium scaffolds decorated with carboxyl and amine functionalities. Similar coatings are obtained by replacing CA with 4-methylcatechol (MC) in the presence of HMDA. No significant film deposition was detected in the absence of HMDA nor by replacing it with shorter chain ethylenediamine, or with monoamines. The CA/HMDA-based films resisted oxidative and reductive treatments, displayed efficient Fe(II) and Cu(II) binding capacity and organic dyes adsorption, and provided an excellent cytocompatible platform for growing embryonic stem cells. These results pointed to HMDA as an efficient cross-linking mediator of film deposition from natural catechols for surface functionalization and coatings.

Bifunctional Poly(acrylamide) Hydrogels through Orthogonal Coupling Chemistries.

Biomaterials for cell culture allowing simple and quantitative presentation of instructive cues enable rationalization of the interplay between cells and their surrounding microenvironment. Poly(acrylamide) (PAAm) hydrogels are popular 2D-model substrates for this purpose. However, quantitative and reproducible biofunctionalization of PAAm hydrogels with multiple ligands in a trustable, controlled, and independent fashion is not trivial. Here, we describe a method for bifunctional modification of PAAm hydrogels with thiol- and amine- containing biomolecules with controlled densities in an independent, orthogonal manner. We developed copolymer networks of AAm with 9% acrylic acid and 2% N-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)acrylamide. The covalent binding of thiol- and amine-containing chromophores at tunable concentrations was demonstrated and quantified by UV spectroscopy. The morphology, mechanical properties, and homogeneity of the copolymerized hydrogels were characterized by scanning electron microscopy, dynamic mechanical analysis, and confocal microscopy studies. Our copolymer hydrogels were bifunctionalized with polylysine and a laminin-mimetic peptide using the specific chemistries. We analyzed the effect of binding protocol of the two components in the maturation of cultured postmitotic cortical neurons. Our substrates supported neuronal attachment, proliferation, and neuronal differentiation. We found that neurons cultured on our hydrogels bifunctionalized with ligand-specific chemistries in a sequential fashion exhibited higher maturation at comparable culture times than using a simultaneous bifunctionalization strategy, displaying a higher number of neurites, branches, and dendritic filopodia. These results demonstrate the relevance of quantitative and optimized coupling chemistries for the performance of simple biomaterials and with sensitive cell types.

Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials.

Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.

Stick-Slip Friction of PDMS Surfaces for Bioinspired Adhesives.

Friction plays an important role in the adhesion of many climbing organisms, such as the gecko. During the shearing between two surfaces, periodic stick-slip behavior is often observed and may be critical to the adhesion of gecko setae and gecko-inspired adhesives. Here, we investigate the influence of short oligomers and pendent chains on the stick-slip friction of polydimethylsiloxane (PDMS), a commonly used material for bioinspired adhesives. Three different stick-slip patterns were observed on these surfaces (flat or microstructured) depending on the presence or absence of oligomers and their ability to diffuse out of the material. After washing samples to remove any untethered oligomeric chains, or after oxygen plasma treatment to convert the surface to a thin layer of silica, we decouple the contributions of stiffness, oligomers, and pendant chains to the stick-slip behavior. The stick phase is mainly controlled by the stiffness while the amount of untethered oligomers and pendant chains available at the contact interface defines the slip phase. A large amount of oligomers and pendant chains resulted in a large slip time, dominating the period of stick-slip motion.

Bioconjugating Thiols to Poly(acrylamide) Gels for Cell Culture Using Methylsulfonyl Co-monomers.

Poly(acrylamide) P(AAm) gels have become relevant model substrates to study cell response to the mechanical and biochemical properties of the cellular microenvironment. However, current bioconjugation strategies to functionalize P(AAm) gels, mainly using photoinduced arylazide coupling, show poor specificity and hinder conclusive studies of material properties and cellular responses. We describe methylsulfonyl-containing P(AAm) hydrogels for cell culture. These gels allow easy, specific and functional covalent coupling of thiol containing bioligands in tunable concentrations under physiological conditions, while retaining the same swelling, porosity, cytocompatibility, and low protein adsorption of P(AAm) gels. These materials allow quantitative and standardized studies of cell-materials interactions with P(AAm) gels.

Gauging and Tuning Cross-Linking Kinetics of Catechol-PEG Adhesives via Catecholamine Functionalization.

The curing time of an adhesive material is determined by the polymerization and cross-linking kinetics of the adhesive formulation and needs to be optimized for the particular application. Here, we explore the possibility of tuning the polymerization kinetics and final mechanical properties of tissue-adhesive PEG gels formed by polymerization of end-functionalized star-PEGs with catecholamines with varying substituents. We show strong differences in cross-linking time and cohesiveness of the final gels among the catecholamine-PEG variants. Installation of an electron-withdrawing but π-electron donating chloro substituent on the catechol ring resulted in faster and more efficient cross-linking, while opposite effects were observed with the strongly electron-withdrawing nitro group. Chain substitution slowed down the kinetics and hindered cross-linking due either to chain breakdown (ß-OH group, in norepinephrine) or intramolecular cyclization (α-carboxyl group, in DOPA). Interesting perspectives derive from use of mixtures of catecholamine-PEG precursors offering further opportunities for fine-tuning of the curing parameters. These are interesting properties for the application of catecholamine-PEG gels as tissue glues or biomaterials for cell encapsulation.

Bioinspired orientation-dependent friction.

Spatular terminals on the toe pads of a gecko play an important role in directional adhesion and friction required for reversible attachment. Inspired by the toe pad design of a gecko, we study friction of polydimethylsiloxane (PDMS) micropillars terminated with asymmetric (spatular-shaped) overhangs. Friction forces in the direction of and against the spatular end were evaluated and compared to friction forces on symmetric T-shaped pillars and pillars without overhangs. The shape of friction curves and the values of friction forces on spatula-terminated pillars were orientation-dependent. Kinetic friction forces were enhanced when shearing against the spatular end, while static friction was stronger in the direction toward the spatular end. The overall friction force was higher in the direction against the spatula end. The maximum value was limited by the mechanical stability of the overhangs during shear. The aspect ratio of the pillar had a strong influence on the magnitude of the friction force, and its contribution surpassed and masked that of the spatular tip for aspect ratios of >2.

A Polyurethane-Based Positive Photoresist.

Polyurethane (PU) monomer mixtures containing commercially available o-nitrobenzyl-based photocleavable monomers have been formulated and tested as low-cost positive tone photoresists. The photolysis reaction is studied by UV spectroscopy. Well-defined micropatterns on 2 µm thick photodegradable PU films are obtained using 365 nm light exposure. This strategy is also extended to improved formulations based on synthesized o-nitrobiphenylpropyl derivatives with enhanced photochemical properties for single photon excitation and high two-photon absorption cross-sections. Improved pattern resolution in 2D and the capability of 3D resolution using a scanning laser at 780 nm is demonstrated. This work demonstrates the potential of PUs as readily available, versatile, and easy-to-use photoresist materials for low-cost lithography applications.