Tough and responsive hydrogels have recently attracted great research interestsfor potential applications in artifical muscles, soft robotics, and actuators,etc. This paper overviews our recent progresses in the design and synthesis ofhydrogels with very high strength and toughness, and actuators based on thesehydrogels. Inorganic nanospheres, nanorods, and nanosheets are exploited asmulti-functional crosslinkers to adsorb or bond with hydrophilic chains, leadingto hydrogels with very high strength, toughness, fatigue resistance, and/orself-healing. Introduction of functional groups including ionic monomers andamino groups results in hydrogels reponsive to pH, ionic strength and electricfield. Besides, ionoprinting has been used to change local crosslink densitybased on reversible chelating/decomposition of metal ions with functionalgroups. This process is rapid and thus enables reversible and rapid actuation ofhydrogel devices. Our studies will further aim to develop sophiscated devices byassembling hydrogel actuators.

Cartilage is a load bearing tissue that has multiple biological functions. Themajor proteoglycan in cartilage is the bottlebrush shaped aggrecan whosecomplexes with hyaluronic acid provide the compressive resistance of cartilage.The negatively charged aggrecan-hyaluronic acid complexes generate an osmoticswelling pressure within the tissue, which is balanced by the collagen network.To better understand the function of cartilage at the tissue level, we studyaggrecan assemblies using an array of microscopic and macroscopic techniques.The organization of aggrecan assemblies at the supramolecular level is probed bylight scattering, small-angle neutron scattering and small-angle X-rayscattering. Osmotic and rheological measurements are used to investigate themacroscopic physical properties.

The past few years have seen the introduction of a number of 3D and 4D printingtechniques used to process tough hydrogel materials. The use of‘color’ 3D printing technology where multiple inks areused in the one print allows for the production of composite materials andstructures that can further enhance the mechanical performance of the printedhydrogel. This article reviews a number of 3D and 4D printing techniques forfabricating functional hydrogel based devices.

We report on a hand-held reactive printing device used to pattern highlyconductive, edible hydrogel wires formed from gellan gum, gelatin, cross-linkersand a common salt (NaCl). The conductivity of the gels when printed (190± 20 mS/cm) closely matched the conductivity recorded for cast systems(200 ± 19 mS/cm). Printing was observed to reduce the elastic modulusand failure strains of hydrogels under compression, but printed gels retainedsufficient integrity for application as flexible conductive lines. Wedemonstrate that hand-held printing can utilize to pattern soft conductorelements within a simple electronic circuit.

Covalent functionalisation of collagen has been shown to be a promising strategyto adjust the mechanical properties of highly swollen collagen hydrogels. At thesame time, secondary interactions between for example, amino acidic terminationsor introduced functional groups also play an important role and are oftenchallenging to predict and control. To explore this challenge, 4-vinylbenzylchloride (4VBC) and methacrylic anhydride (MA) were reacted with type Icollagen, and the swelling and rheological properties of resultingphoto-activated hydrogel systems investigated. 4VBC-based hydrogels showedsignificantly increased swelling ratio, in light of the lower degree of collagenfunctionalisation, with respect to methacrylated collagen networks, whilstrheological storage moduli were found to be comparable between the two systems.To explore the role of benzyl groups in the mechanical properties of the4VBC-based collagen system, model chemical force microscopy (CFM) was carriedout in aqueous environment with an aromatised probe against an aromatisedgold-coated glass slide. A marked increase in adhesion force(F: 0.11±0.01 nN) was measured between aromatisedsamples, compared to the adhesion force observed between the non-modified probeand a glass substrate (F: 2.64±1.82 nN). These resultssuggest the formation of additional and reversible π-πstacking interactions in aromatic 4VBC-based networks and explain the remarkablerheological properties of this system in comparison to MA-based hydrogels.

Immobilization into 3D matrices stabilizes proteins in comparison to flat planarsurfaces and facilitates the study of the biomolecular interactions as well asintegration into optrodes for cell physiology. Photocrosslinkable hydrogels havereceived significant attention in recent years as they provide not only a highlyhydrophilic 3D environment to promote protein stabilization and its interactionswith analyte molecules, but enable optically addressable patterning for spatialcontrol of protein localization. At the same time, the explosion of newgenetically encoded sensor proteins has greatly expanded the range of opticalmolecular sensors for cell physiology. Here we integrate a genetically encodedglutamate sensor protein into a photocrosslinkable hydrogel via covalentinteraction to create a novel glutamate sensor material. Protein immobilizationcan be achieved through covalent bonds, physical interactions, or physicalentrapment. Although physical entrapment without chemical modifications offers auniversal approach for protein immobilization, leaching of protein through thepores of the hydrogel is a significant challenge. Thus, here an alternativemethod is developed to provide better control of protein localization andimmobilization using naturally existing reactive groups of proteins. To thisend, a genetically encoded FRET based glutamate indicator protein (FLIPE) ismodified with diacrylated poly (ethylene glycol) (PEGDA) by Michael-typeaddition between acrylate groups and the thiol side chains of the cysteineresidues. We optimize the molecular weight of PEGDA (300, 740, and 3400 Da) aswell as concentrations of the photoinitiator (0.1, 0.5 and 1 % (w/w)) andmonomer (10, 20, and 30 % (w/w)) in the precursor solution. Next the precursorsolution is grown at the distal end of an optical fiber to test thespectroscopic properties and characteristic bioactivities of proteins in thehydrogel network. Optimization of the irradiation parameters, light intensityand exposure time, improves the spatial resolution of 3D hydrogel tips. Thisstudy examines the capability of fabricating 3D hydrogel sensors covalentlymodified with a member of recently growing genetically encoded fluorescentbiosensors, which can later be extended to all conformation-dependent proteinbiosensors and be used intracellularly for physiological and biological sensingpurposes.

Self-healing is one of the most desired material properties. Herein, we presentthe design and development of a new self-healing dental composite (SHDC) thatcan heal micro-cracks autonomously. The SHDC has two functional components inaddition to contemporary dental composites: healing powder (HP) and healingliquid (HL) encapsulated in silica microcapsules. The autonomous healing istriggered by micro-cracks which fracture microcapsules in their propagation pathand release the HL. As a consequence, the released HL dissolves and reacts withthe HP, and then fill the micro-cracks with a cement-like new material. This3-step crack-release-heal process prevents micro-cracks from causing restorationfailure, thus improving the service life of dental restorative material. Themechanical performance of the SHDC prepared were evaluated in terms of elasticmodulus and fracture toughness, which were in the upper level compared tocommercial dental restorative materials, and the self-healing capability wasconfirmed through fracture toughness recovery test. In addition, the SHDCs weremade with clinically-tested, biocompatible materials, which makes them readilyapplicable as medical devices.

Biological systems routinely produce nanoscopic molecular structures withconsiderably less dispersion in size and shape than encountered in mostmanufactured materials. Indeed, Biological structures are frequently andessentially monodisperse. An example of this uniformity, combined with anintriguing geometry, is the nanometer-scale protein nanorings produced byinteraction of the protein tubulin with certain hydrophobic tri-, tetra- andpentapeptides originally extracted as natural products from marine biosystems.Different peptides produce different sized nanorings, but we focus on thoseproduced by binding to tubulin of the cyclic depsipeptide cryptophycin. Thenanorings that form upon binding of this ligand show a sharp mass distributionindicating that the nanorings are made of 8 tubulin dimers of 100 kDa.

In this submission, we demonstrate how a combination of fluorescence correlationspectroscopy, dynamic light scattering, electron microscopy, analyticalultracentrifugation, small-angle neutron scattering, and modeling is applied toreveal interactions of tubulin and cryptophycin in solution and to characterizetheir structures. We find that the cryptophycin-tubulin nanorings(∼25 nm diameter) are single-walled, appear rigid, are composed of 8tubulin dimers in a single closed ring, and are stable upon dilution tonanomolar concentrations.

Similar studies with a different peptide, the linear pentapeptide dolastatin 10,demonstrated that binding of this peptide to tubulin produces larger nanorings(14 tubulin dimers, ∼45 nm diameter rings), with slightly differentproperties. The ability to adjust the ring size with different peptides, andproduce uniform nanorings with properties that differ slightly between sizeclasses, makes the tubulin-peptide ring structures an appealing structuralsystem.

Nacre (mother of pearl) is an attractive model for the development of newmaterials. Its sheet structure of alternating layers of calcium carbonate and anorganic matrix confers it highly desirable properties such as high toughness andstrength. In this study, we produce a nacre-inspired composite material usingonly bacterially-produced components. Calcium carbonate is crystallized via theaction of ureolytic bacteria. After each crystallization event, we applybacterially produced γ-polyglutamate (PGA) to the sample, whichpromotes layering compared to the PGA-free control. We show that the combinationof these two compounds yields a layered material reminiscent of nacre, showing away towards the biotechnological production of new, nacre-inspiredmaterials.