Nanocellulose aerogels as 3D amyloid templates.

Proteins in solution tend to coat solid surfaces upon exposure. Depending on the nature of the surface, the environmental conditions, and the nature of the protein these adsorbed proteins may self-assemble into ordered, fibre-like structures called amyloids. Nanoparticulate surfaces, with their high surface to volume ratio, are particularly favourable to amyloid formation. Most prior research has focussed on either inorganic or organic nanoparticles in solution. In this research, we instead focus on aerogels created from TEMPO-oxidized cellulose nanofibers (TO-CNF) to serve as bio-based, three-dimensional amyloid templates with a tuneable surface chemistry. Previous research on the use of cellulose as a protein adsorption template has shown no evidence of a change in the secondary protein structure. Herein, however, with the aid of the reducing agent TCEP, we were able to induce the formation of amyloid-like 'worms' on the surface of TO-CNF aerogels. Furthermore, we demonstrate that the addition of the TO-CNF aerogel can also induce bulk aggregation under conditions where it previously did not exist. Finally, we show that the addition of the aerogel increases the rate of 'worm' formation in conditions where previous research has found a long lag-phase. Therefore, TO-CNF aerogels are shown to be excellent templates for inducing ordered protein aggregation.

2D foam film coating of antimicrobial lysozyme amyloid fibrils onto cellulose nanopapers.

Amyloid fibrils made from inexpensive hen egg white lysozyme (HEWL) are bio-based, bio-degradable and bio-compatible colloids with broad-spectrum antimicrobial activity, making them an attractive alternative to existing small-molecule antibiotics. Their surface activity leads to the formation of 2D foam films within a loop, similar to soap films when blowing bubbles. The stability of the foam was optimized by screening concentration and pH, which also revealed that the HEWL amyloid foams were actually stabilized by unconverted peptides unable to undergo amyloid self-assembly rather than the fibrils themselves. The 2D foam film was successfully deposited on different substrates to produce a homogenous coating layer with a thickness of roughly 30 nm. This was thick enough to shield the negative charge of dry cellulose nanopaper substrates, leading to a positively charged HEWL amyloid coating. The coating exhibited a broad-spectrum antimicrobial effect based on the interactions with the negatively charged cell walls and membranes of clinically relevant pathogens (Staphylococcus aureus, Escherichia coli and Candida albicans). The coating method presented here offers an alternative to existing techniques, such as dip and spray coating, in particular when optimized for continuous production. Based on the facile preparation and broad spectrum antimicrobial performance, we anticipate that these biohybrid materials could potentially be used in the biomedical sector as wound dressings.

Biotechnological development of Trichoderma-based formulations for biological control.

Trichoderma spp. are a genus of well-known fungi that promote healthy growth and modulate different functions in plants, as well as protect against various plant pathogens. The application of Trichoderma and its propagules as a biological control method can therefore help to reduce the use of chemical pesticides and fertilizers in agriculture. This review critically discusses and analyzes groundbreaking innovations over the past few decades of biotechnological approaches to prepare active formulations containing Trichoderma. The use of various carrier substances is covered, emphasizing their effects on enhancing the shelf life, viability, and efficacy of the final product formulation. Furthermore, the use of processing techniques such as freeze drying, fluidized bed drying, and spray drying are highlighted, enabling the development of stable, light-weight formulations. Finally, promising microencapsulation techniques for maximizing the performance of Trichoderma spp. during application processes are discussed, leading to the next-generation of multi-functional biological control formulations. KEY POINTS: • The development of carrier substances to encapsulate Trichoderma propagules is highlighted. • Advances in biotechnological processes to prepare Trichoderma-containing formulations are critically discussed. • Current challenges and future outlook of Trichoderma-based formulations in the context of biological control are presented.

Nanocomposites of cellulose nanofibers incorporated with carvacrol via stabilizing octenyl succinic anhydride-modified ɛ-polylysine.

Food packaging plays an extremely important role in the global food chain, allowing for products to be shipped across long distances without spoiling. However, there is an increased need to both reduce plastic waste caused by traditional single-use plastic packaging and improve the overall functionality of packaging materials to extend shelf-life even further. Herein, we investigate composite mixtures based on cellulose nanofibers and carvacrol via stabilizing octenyl-succinic anhydride-modified epsilon polylysine (MɛPL-CNF) for active food packaging applications. The effects of epsilon polylysine (εPL) concentration and modification with octenyl-succinic anhydride (OSA) and carvacrol are evaluated with respect to composites morphology, mechanical, optical, antioxidant, and antimicrobial properties. We find that both increased εPL concentration and modification with OSA and carvacrol lead to films with increased antioxidant and antimicrobial properties, albeit at the expense of reduced mechanical performance. Importantly, when sprayed onto the surface of sliced apples, MεPL-CNF-mixtures are able to successfully delay/hinder enzymatic browning, suggesting the potential of such materials for a range of active food packaging applications.

Biopolymer-based emulsions for the stabilization of Trichoderma atrobrunneum conidia for biological control.

Trichoderma spp. are ubiquitous soil-borne fungi that are widely used in biological control to promote and regulate healthy plant growth, as well as protect against plant pathogens. However, as with many biological materials, the relative instability of Trichoderma propagules limits its practical use in industrial applications. Therefore, there has been significant research interest in developing novel formulations with various carrier substances that are compatible with these fungal propagules and can enhance the shelf-life and overall efficacy of the Trichoderma. To this end, herein, we investigate the use of a variety of biopolymers and nanoparticles for the stabilization of Trichoderma atrobrunneum T720 conidia for biological control. The best-performing agents-agar and cellulose nanocrystals (CNC)-were then used in the preparation of oil-in-water emulsions to encapsulate conidia of T720. Emulsion properties including oil type, oil:water ratio, and biopolymer/particle concentration were investigated with respect to emulsion stability, droplet size, and viability of T720 conidia over time. Overall, agar-based formulations yielded highly stable emulsions with small droplet sizes, showing no evidence of drastic creaming, or phase separation after 1 month of storage. Moreover, agar-based formulations were able to maintain ~ 100% conidial viability of T720 after 3 months of storage, and over 70% viability after 6 months. We anticipate that the results demonstrated herein will lead to a new generation of significantly improved formulations for practical biological control applications. KEY POINTS: • Various biopolymers were evaluated for improving the stability of Trichoderma conidia • Oil in water emulsions was prepared using cellulose nanocrystals and agar as interface stabilizers • Agar-based emulsions showed ~ 100% viability for encapsulated conidia after 3 months of storage.

Phase Behavior, Self-Assembly, and Adhesive Potential of Cellulose Nanocrystal-Bovine Serum Albumin Amyloid Composites.

Structural organization is ubiquitous throughout nature and contributes to the outstanding mechanical/adhesive performance of organisms including geckoes, barnacles, and crustaceans. Typically, these types of structures are composed of polysaccharide and protein-based building blocks, and therefore, there is significant research interest in using similar building blocks in the fabrication of high-performance synthetic materials. Via evaporation-induced self-assembly, the organization of cellulose nanocrystals (CNCs) into a chiral nematic regime results in the formation of structured CNC films with prominent mechanical, optical, and photonic properties. However, there remains an important knowledge gap in relating equilibrium suspension behavior to dry film structuring and other functional properties of CNC-based composite materials. Herein, we systematically investigate the phase behavior of composite suspensions of rigid CNCs and flexible bovine serum albumin (BSA) amyloids in relation to their self-assembly into ordered films and structural adhesives. Increasing the concentration of BSA amyloids in the CNC suspensions results in a clear decrease in the anisotropic fraction volume percent via the preferential accumulation of BSA amyloids in the isotropic regime (as a result of depletion interactions). This translates to a blue shift or compression of the chiral nematic pitch in dried films. Finally, we also demonstrate the synergistic adhesive potential of CNC-BSA amyloid composites, with ultimate lap shear strengths in excess of 500 N/mg. We anticipate that understanding the systematic relationships between material interactions and self-assembly in suspension such as those investigated here will pave the way for a new generation of structured composite materials with a variety of enhanced functionalities.

Biohybrid Nanocellulose-Lysozyme Amyloid Aerogels via Electrostatic Complexation.

Modern science is increasingly turning to nature for inspiration to design sustainable biomaterials in a smart and effective way. Herein, we describe biohybrid aerogels based on electrostatic complexation between cellulose and proteins-two of the most abundant natural polymers on Earth. The effects of both particle surface charge and particle size are investigated with respect to aerogel properties including the morphology, surface area, stability, and mechanical strength. Specifically, negatively charged nanocellulose (cellulose nanocrystals and cellulose nanofibers) and positively charged lysozyme amyloid fibers (full-length and shortened via sonication) are investigated in the preparation of fibrillar aerogels, whereby the nanocellulose component was found to have the largest effect on the resulting aerogel properties. Although electrostatic interactions between these two classes of charged nanoparticles allow us to avoid the use of any cross-linking agents, the resulting aerogels demonstrate a simple additive performance as compared to their respective single-component aerogels. This lack of synergy indicates that although electrostatic complexation certainly leads to the formation of local aggregates, these interactions alone may not be strong enough to synergistically improve bulk aerogel properties. Nevertheless, the results reported herein represent a critical step toward a broader understanding of biohybrid materials based on cellulose and proteins.

Melanized-Cationic Cellulose Nanofiber Foams for Bioinspired Removal of Cationic Dyes.

In recent years, water pollution has developed into a severe environmental and public health problem due to rapid urbanization and industrialization, especially in some developing countries. Finding solutions to tackle water pollution is urgently required and is of global importance. Currently, a range of water treatment methods are available; however, a water remediation process that is simple, inexpensive, eco-friendly, and effective for the removal of pollutants down to ppm/ppb concentrations has long been sought after. Herein, we describe a novel approach using fungal melanin for developing melanized-cationic cellulose nanofiber (melanized-C-CNF) foams that can successfully remove pollutants in water systems. The foam can be recycled several times while retaining its adsorption/desorption property, indicating high practicability for adsorbing the cationic dye crystal violet. This work highlights the opportunity to combine both the advanced features of sustainable polymers such as cellulose and the unique properties of fungal melanin to manufacture biohybrid composites for water purification.

Self-Assembly Pathways and Antimicrobial Properties of Lysozyme in Different Aggregation States.

Antimicrobial resistance in microorganisms will cause millions of deaths and pose a vast burden on health systems; therefore, alternatives to existing small-molecule antibiotics have to be developed. Lysozyme is an antimicrobial enzyme and has broad-spectrum antimicrobial activity in different aggregated forms. Here, we propose a reductive pathway to obtain colloidally stable amyloid-like worm-shaped lysozyme nanoparticles (worms) from hen egg white lysozyme (HEWL) and compare them to amyloid fibrils made in an acid hydrolysis pathway. The aggregation of HEWL into worms follows strongly pH-dependent kinetics and induces a structural transition from α-helices to ß-sheets. Both HEWL worms and amyloid fibrils show broad-spectrum antimicrobial activity against the bacteria Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-negative), and the fungus Candida albicans. The colloidal stability of the worms allows the determination of minimum inhibitory concentrations, which are lower than that for native HEWL in the case of S. aureus. Overall, amyloid fibrils have the strongest antimicrobial effect, likely due to the increased positive charge compared to native HEWL. The structural and functional characterizations of HEWL worms and amyloids investigated herein are critical for understanding the detailed mechanisms of antimicrobial activity and opens up new avenues for the design of broad-spectrum antimicrobial materials for use in various applications.

Chitin-amyloid synergism and their use as sustainable structural adhesives.

Structural adhesives are relevant to many engineering applications, especially those requiring load-bearing joints with high lap shear strength. Typical adhesives are synthesized from acrylics, epoxies, or urethanes, which may pose a burden to sustainability and the environment. In nature, the interfacial interactions between chitin and proteins are used for structural purposes and as a bio-cement, resulting in materials with properties unmatched by their man-made counterparts. Herein, we show that related supramolecular interactions can be harnessed to develop high strength green adhesives based on chitin nanocrystals (ChNCs), isolated from shrimp shells, and hen egg white lysozyme (HEWL) used in its monomeric or amyloid forms. Consolidation of the bicomponent suspensions, placed between glass substrates, results in long-range ordered superstructures. The formation of these structures is evaluated by surface energy considerations, followed by scanning electron, atomic force, and polarized microscopies of the consolidated materials. For 0.8 mg of bio-adhesive (lysozyme, ChNCs or their composites), lap shear loads of over 300 N are reached. Such remarkable adhesion reaches maximum values at protein-to-ChNC ratios below 1 : 4, reflecting the synergy established between the components (ca. 25% higher load compared to ChNCs, the strongest single component). We put the observed adhesive performance in perspective by comparing the lap-shear performance with current research on green supramolecular adhesives using natural biopolymers. The results are discussed in the context of current efforts to standardize the measurement of adhesive strength and bond preparation. The latter is key to formalizing the metrology and materials chemistry of bio-based adhesives. The proposed all-green system is expected to expand current developments in the design of bio-based adhesives.

Multi-scale structuring of cell-instructive cellulose nanocrystal composite hydrogel sheets via sequential electrospinning and thermal wrinkling.

Structured hydrogel sheets offer the potential to mimic the mechanics and morphology of native cell environments in vitro; however, controlling the morphology of such sheets across multiple length scales to give cells consistent multi-dimensional cues remains challenging. Here, we demonstrate a simple two-step process based on sequential electrospinning and thermal wrinkling to create nanocomposite poly(oligoethylene glycol methacrylate)/cellulose nanocrystal hydrogel sheets with a highly tunable multi-scale wrinkled (micro) and fibrous (nano) morphology. By varying the time of electrospinning, rotation speed of the collector, and geometry of the thermal wrinkling process, the hydrogel nanofiber density, fiber alignment, and wrinkle geometry (biaxial or uniaxial) can be independently controlled. Adhered C2C12 mouse myoblast muscle cells display a random orientation on biaxially wrinkled sheets but an extended morphology (directed preferentially along the wrinkles) on uniaxially wrinkled sheets. While the nanofiber orientation had a smaller effect on cell alignment, parallel nanofibers promoted improved cell alignment along the wrinkle direction while perpendicular nanofibers disrupted alignment. The highly tunable structures demonstrated are some of the most complex morphologies engineered into hydrogels to-date without requiring intensive micro/nanofabrication approaches and offer the potential to precisely regulate cell-substrate interactions in a "2.5D" environment (i.e. a surface with both micro- and nano-structured topographies) for in vitro cell screening or in vivo tissue regeneration. STATEMENT OF SIGNIFICANCE: While structured hydrogels can mimic the morphology of natural tissues, controlling this morphology over multiple length scales remains challenging. Furthermore, the incorporation of secondary morphologies within individual hydrogels via simple manufacturing techniques would represent a significant advancement in the field of structured biomaterials and an opportunity to study complex cell-biomaterial interactions. Herein, we leverage a two-step process based on electrospinning and thermal wrinkling to prepare structured hydrogels with microscale wrinkles and nanoscale fibers. Fiber orientation/density and wrinkle geometry can be independently controlled during the electrospinning and thermal wrinkling processes respectively, demonstrating the flexibility of this technique for creating well-defined multiscale hydrogel structures. Finally, we show that while wrinkle geometry is the major determinant of cell alignment, nanofiber orientation also plays a role in this process.

Functional Materials from Nanocellulose: Utilizing Structure-Property Relationships in Bottom-Up Fabrication.

It is inherently challenging to recapitulate the precise hierarchical architectures found throughout nature (such as in wood, antler, bone, and silk) using synthetic bottom-up fabrication strategies. However, as a renewable and naturally sourced nanoscale building block, nanocellulose-both cellulose nanocrystals and cellulose nanofibrils-has gained significant research interest within this area. Altogether, the intrinsic shape anisotropy, surface charge/chemistry, and mechanical/rheological properties are some of the critical material properties leading to advanced structure-based functionality within nanocellulose-based bottom-up fabricated materials. Herein, the organization of nanocellulose into biomimetic-aligned, porous, and fibrous materials through a variety of fabrication techniques is presented. Moreover, sophisticated material structuring arising from both the alignment of nanocellulose and via specific process-induced methods is covered. In particular, design rules based on the underlying fundamental properties of nanocellulose are established and discussed as related to their influence on material assembly and resulting structure/function. Finally, key advancements and critical challenges within the field are highlighted, paving the way for the fabrication of truly advanced materials from nanocellulose.

Nanocellulose-lysozyme colloidal gels via electrostatic complexation.

Biohybrid colloids were fabricated based on electrostatic complexation between anionic TEMPO-oxidized cellulose nanofibrils (TO-CNF) and cationic hen egg white lysozyme (HEWL). By altering the loading of HEWL, physical colloidal complexes can be obtained at a relatively low concentration of TO-CNF (0.1 wt%). At neutral pH, increasing the HEWL loading induces an increase in charge screening, as probed by zeta-potential, resulting in enhanced TO-CNF aggregation and colloidal gel formation. Systematic rheological testing shows that mechanical reinforcement of the prepared biohybrid gels is easily achieved by increasing the loading of HEWL. However, due to the relatively weak nature of electrostatic complexation, the formed colloidal gels exhibit partial destruction when subjected to cyclic shear stresses. Still, they resist thermo-cycling up to 90 °C. Finally, the pH responsiveness of the colloidal complex gels was demonstrated by adjusting pH to above and below the isoelectric point of HEWL, representing a facile mechanism to tune the gelation of TO-CNF/HEWL complexes. This work highlights the potential of using electrostatic complexation between HEWL and TO-CNF to form hybrid colloids, and demonstrates the tunability of the colloidal morphology and rheology by adjusting the ratio between the two components and the pH.

Assembly of Cellulose Nanocrystal-Lysozyme Composite Films with Varied Lysozyme Morphology.

In modern society, there is a constant need for developing reliable, sustainable, and cost-effective antibacterial materials. Here, we investigate the preparation of cellulose nanocrystal (CNC)-lysozyme composite films via the well-established method of evaporation-induced self-assembly. We consider the effects of lysozyme concentration and aggregation state (native lysozyme, lysozyme amyloid fibers, and sonicated lysozyme amyloid fibers) on suspension aggregation and film-forming ability. Although at higher lysozyme loading levels (ca. 10 wt %), composite films lost their characteristic chiral nematic structuring, these films demonstrated improved mechanical properties and antibacterial activity with respect to CNC-only films, regardless of lysozyme aggregation state. We anticipate that the results presented herein could also contribute to the preparation of other CNC-protein-based materials, including films, hydrogels, and aerogels, with improved mechanical performance and antibacterial activity.

Dual-porous cellulose nanofibril aerogels via modular drying and cross-linking.

Nanofibrillar foams and aerogels are traditionally either macroporous with low surface area and high mechanical strength, or mesoporous with high surface area and low mechanical strength. In this work, an anionic cellulose nanofibril (CNF)-based dual-porous aerogel with BET specific surface area up to 430 m2 g-1 was prepared via a modular process combining directional freeze-thawing (creating macro-pores, ca. 50-200 µm) and supercritical drying (creating meso-pores, ca. 2-50 nm). Furthermore, by optionally utilizing both physical and chemical cross-linking strategies, aerogels with a Young's modulus of up to 711 kPa and good stability in aqueous conditions were demonstrated. By altering cross-linking strategies, the properties of resulting aerogels, such as hydrophilicity, mechanical strength and stability in water, can be precisely controlled for different applications. As a result, cationic methylene blue (MB) and metal ions (Ag+) were chosen as model species to investigate the absorption properties of the physically cross-linked aerogels in water. The aerogels showed a maximum adsorption of MB up to 234 mg g-1 and of Ag+ up to 116 mg g-1 as a result of the high specific surface area of the aerogels and their strong electrostatic interaction with the model species. Importantly, the hierarchical dual porosity of the aerogels enabled fast adsorption kinetics combined with a considerable adsorption capacity overall. Finally, it was shown that the adsorbed Ag+ could be converted to metallic Ag, demonstrating the additional functionality of these dual porous hybrid aerogels for antibacterial or catalytic applications.

Dual physically and chemically crosslinked regenerated cellulose - Gelatin composite hydrogels towards art restoration.

Art restoration poses many challenges for scientists and conservators, as any restorative action can lead to lasting modification or damage to the original artefact. Recent interest in gel encapsulation has grown due to the ability to control the cleaning action; yet the restoration of modern paints such as acrylic-based systems still presents issues due to their extremely high sensitivity to most solvents. Herein, the preparation of dual physically and chemically crosslinked hydrogels based on regenerated cellulose and cinnamoyl-modified gelatin is demonstrated. These dual crosslinked hydrogels show increased mechanical strength and enhanced water retention compared to pure physically crosslinked hydrogels. When applied to acrylic-based paint surfaces, the dual crosslinked hydrogels extract a smaller amount of hydrophilic additives (albeit still leading to swelling within the paint film) versus physically crosslinked gels. It is anticipated that this dual crosslinking approach can be broadly applied to prepare gels for conservation of cultural heritage artefacts.

2.5D Hierarchical Structuring of Nanocomposite Hydrogel Films Containing Cellulose Nanocrystals.

Although two-dimensional hydrogel thin films have been applied across many biomedical applications, creating higher dimensionality structured hydrogel interfaces would enable potentially improved and more biomimetic hydrogel performance in biosensing, bioseparations, tissue engineering, drug delivery, and wound healing applications. Herein, we present a new and simple approach to control the structure of hydrogel thin films in 2.5D. Hybrid suspensions containing cellulose nanocrystals (CNCs) and aldehyde- or hydrazide-functionalized poly(oligoethylene glycol methacrylate) (POEGMA) were spin-coated onto prestressed polystyrene substrates to form cross-linked hydrogel thin films. The films were then structured via thermal shrinking, with control over the direction of shrinking leading to the formation of biaxial, uniaxial, or hierarchical wrinkles. Notably, POEGMA-only hydrogel thin films (without CNCs) did not form uniform wrinkles due to partial dewetting from the substrate during shrinking. Topographical feature sizes of CNC-POEGMA films could be tuned across 2 orders of magnitude (from ∼300 nm to 20 µm) by varying the POEGMA concentration, the length of poly(ethylene glycol) side chains in the polymer, and/or the overall film thickness. Furthermore, by employing adhesive masks during the spin-coating process, structured films with gradient wrinkle sizes can be fabricated. This precise control over both wrinkle size and wrinkle topography adds a level of functionality that to date has been lacking in conventional hydrogel networks.

Tissue Response and Biodistribution of Injectable Cellulose Nanocrystal Composite Hydrogels.

Interest in cellulose nanocrystal (CNC)-based hydrogels for drug delivery, tissue engineering, and other biomedical applications has rapidly expanded despite the minimal in vivo research reported to date. Herein, we assess both in vitro protein adsorption and cell adhesion as well as in vivo subcutaneous tissue responses and CNC biodistribution of injectable CNC-poly(oligoethylene glycol methacrylate) (POEGMA) hydrogels. Hydrogels with different PEG side chain lengths, CNC loadings, and with or without in situ magnetic alignment of the CNCs are compared. CNC loading has a minimal impact on protein adsorption but significantly increases cell adhesion. In vivo, both CNC-only and CNC-POEGMA injections largely stay at their subcutaneous injection site over one month, with minimal bioaccumulation of CNCs in any typical clearance organ. CNC-POEGMA hydrogels exhibit mild acute and chronic inflammatory responses, although significant fibroblast penetration was observed with the magnetically aligned hydrogels. Collectively, these results suggest that CNC-POEGMA hydrogels offer promise in practical biomedical applications.

Patterning of Structurally Anisotropic Composite Hydrogel Sheets.

Compositional and structural patterns play a crucial role in the function of many biological tissues. In the present work, for nanofibrillar hydrogels formed by chemically cross-linked cellulose nanocrystals (CNC) and gelatin, we report a microextrusion-based 3D printing method to generate structurally anisotropic hydrogel sheets with CNCs aligned in the direction of extrusion. We prepared hydrogels with a uniform composition, as well as hydrogels with two different types of compositional gradients. In the first type of gradient hydrogel, the composition of the sheet varied parallel to the direction of CNC alignment. In the second hydrogel type, the composition of the sheet changed orthogonally to the direction of CNC alignment. The hydrogels exhibited gradients in structure, mechanical properties, and permeability, all governed by the compositional patterns, as well as cytocompatibility. These hydrogels have promising applications for both fundamental research and for tissue engineering and regenerative medicine.

Structured Macroporous Hydrogels: Progress, Challenges, and Opportunities.

Structured macroporous hydrogels that have controllable porosities on both the nanoscale and the microscale offer both the swelling and interfacial properties of bulk hydrogels as well as the transport properties of "hard" macroporous materials. While a variety of techniques such as solvent casting, freeze drying, gas foaming, and phase separation have been developed to fabricate structured macroporous hydrogels, the typically weak mechanics and isotropic pore structures achieved as well as the required use of solvent/additives in the preparation process all limit the potential applications of these materials, particularly in biomedical contexts. This review highlights recent developments in the field of structured macroporous hydrogels aiming to increase network strength, create anisotropy and directionality within the networks, and utilize solvent-free or additive-free fabrication methods. Such functional materials are well suited for not only biomedical applications like tissue engineering and drug delivery but also selective filtration, environmental sorption, and the physical templating of secondary networks.