Basic principles of hydrogel-based tissue transformation technologies and their applications.

Emerging tissue transformation technologies provide an unprecedented opportunity to investigate system-level molecular and anatomical features in situ. Hydrogel-based methods engineer physicochemical tissue properties to render intact organs optically transparent and size and shape adjustable while preserving biomolecules at their physiological locations. When combined with advanced molecular tools, labeling, and imaging techniques, tissue transformation enables three-dimensional (3D) mapping of molecules, cells, and their interrelationships at increasing speeds and resolutions. In this review, we discuss the basic engineering principles of tissue transformation and labeling techniques as well as their broad applications, current challenges, and future potential.

Expansion Microscopy: Scalable and Convenient Super-Resolution Microscopy.

Expansion microscopy (ExM) is a physical form of magnification that increases the effective resolving power of any microscope. Here, we describe the fundamental principles of ExM, as well as how recently developed ExM variants build upon and apply those principles. We examine applications of ExM in cell and developmental biology for the study of nanoscale structures as well as ExM's potential for scalable mapping of nanoscale structures across large sample volumes. Finally, we explore how the unique anchoring and hydrogel embedding properties enable postexpansion molecular interrogation in a purified chemical environment. ExM promises to play an important role complementary to emerging live-cell imaging techniques, because of its relative ease of adoption and modification and its compatibility with tissue specimens up to at least 200 µm thick.

Injectable ultrasonic sensor for wireless monitoring of intracranial signals.

Direct and precise monitoring of intracranial physiology holds immense importance in delineating injuries, prognostication and averting disease1. Wired clinical instruments that use percutaneous leads are accurate but are susceptible to infection, patient mobility constraints and potential surgical complications during removal2. Wireless implantable devices provide greater operational freedom but include issues such as limited detection range, poor degradation and difficulty in size reduction in the human body3. Here we present an injectable, bioresorbable and wireless metastructured hydrogel (metagel) sensor for ultrasonic monitoring of intracranial signals. The metagel sensors are cubes 2 × 2 × 2 mm3 in size that encompass both biodegradable and stimulus-responsive hydrogels and periodically aligned air columns with a specific acoustic reflection spectrum. Implanted into intracranial space with a puncture needle, the metagel deforms in response to physiological environmental changes, causing peak frequency shifts of reflected ultrasound waves that can be wirelessly measured by an external ultrasound probe. The metagel sensor can independently detect intracranial pressure, temperature, pH and flow rate, realize a detection depth of 10 cm and almost fully degrade within 18 weeks. Animal experiments on rats and pigs indicate promising multiparametric sensing performances on a par with conventional non-resorbable wired clinical benchmarks.

Mechanical release of homogenous proteins from supramolecular gels.

A long-standing challenge is how to formulate proteins and vaccines to retain function during storage and transport and to remove the burdens of cold-chain management. Any solution must be practical to use, with the protein being released or applied using clinically relevant triggers. Advanced biologic therapies are distributed cold, using substantial energy, limiting equitable distribution in low-resource countries and placing responsibility on the user for correct storage and handling. Cold-chain management is the best solution at present for protein transport but requires substantial infrastructure and energy. For example, in research laboratories, a single freezer at -80 °C consumes as much energy per day as a small household1. Of biological (protein or cell) therapies and all vaccines, 75% require cold-chain management; the cost of cold-chain management in clinical trials has increased by about 20% since 2015, reflecting this complexity. Bespoke formulations and excipients are now required, with trehalose2, sucrose or polymers3 widely used, which stabilize proteins by replacing surface water molecules and thereby make denaturation thermodynamically less likely; this has enabled both freeze-dried proteins and frozen proteins. For example, the human papilloma virus vaccine requires aluminium salt adjuvants to function, but these render it unstable against freeze-thaw4, leading to a very complex and expensive supply chain. Other ideas involve ensilication5 and chemical modification of proteins6. In short, protein stabilization is a challenge with no universal solution7,8. Here we designed a stiff hydrogel that stabilizes proteins against thermal denaturation even at 50 °C, and that can, unlike present technologies, deliver pure, excipient-free protein by mechanically releasing it from a syringe. Macromolecules can be loaded at up to 10 wt% without affecting the mechanism of release. This unique stabilization and excipient-free release synergy offers a practical, scalable and versatile solution to enable the low-cost, cold-chain-free and equitable delivery of therapies worldwide.

A microscale soft ionic power source modulates neuronal network activity.

Bio-integrated devices need power sources to operate1,2. Despite widely used technologies that can provide power to large-scale targets, such as wired energy supplies from batteries or wireless energy transduction3, a need to efficiently stimulate cells and tissues on the microscale is still pressing. The ideal miniaturized power source should be biocompatible, mechanically flexible and able to generate an ionic current for biological stimulation, instead of using electron flow as in conventional electronic devices4-6. One approach is to use soft power sources inspired by the electrical eel7,8; however, power sources that combine the required capabilities have not yet been produced, because it is challenging to obtain miniaturized units that both conserve contained energy before usage and are easily triggered to produce an energy output. Here we develop a miniaturized soft power source by depositing lipid-supported networks of nanolitre hydrogel droplets that use internal ion gradients to generate energy. Compared to the original eel-inspired design7, our approach can shrink the volume of a power unit by more than 105-fold and it can store energy for longer than 24 h, enabling operation on-demand with a 680-fold greater power density of about 1,300 W m-3. Our droplet device can serve as a biocompatible and biological ionic current source to modulate neuronal network activity in three-dimensional neural microtissues and in ex vivo mouse brain slices. Ultimately, our soft microscale ionotronic device might be integrated into living organisms.

Cartilage-like protein hydrogels engineered via entanglement.

Load-bearing tissues, such as muscle and cartilage, exhibit high elasticity, high toughness and fast recovery, but have different stiffness (with cartilage being significantly stiffer than muscle)1-8. Muscle achieves its toughness through finely controlled forced domain unfolding-refolding in the muscle protein titin, whereas articular cartilage achieves its high stiffness and toughness through an entangled network comprising collagen and proteoglycans. Advancements in protein mechanics and engineering have made it possible to engineer titin-mimetic elastomeric proteins and soft protein biomaterials thereof to mimic the passive elasticity of muscle9-11. However, it is more challenging to engineer highly stiff and tough protein biomaterials to mimic stiff tissues such as cartilage, or develop stiff synthetic matrices for cartilage stem and progenitor cell differentiation12. Here we report the use of chain entanglements to significantly stiffen protein-based hydrogels without compromising their toughness. By introducing chain entanglements13 into the hydrogel network made of folded elastomeric proteins, we are able to engineer highly stiff and tough protein hydrogels, which seamlessly combine mutually incompatible mechanical properties, including high stiffness, high toughness, fast recovery and ultrahigh compressive strength, effectively converting soft protein biomaterials into stiff and tough materials exhibiting mechanical properties close to those of cartilage. Our study provides a general route towards engineering protein-based, stiff and tough biomaterials, which will find applications in biomedical engineering, such as osteochondral defect repair, and material sciences and engineering.

Water-responsive supercontractile polymer films for bioelectronic interfaces.

Connecting different electronic devices is usually straightforward because they have paired, standardized interfaces, in which the shapes and sizes match each other perfectly. Tissue-electronics interfaces, however, cannot be standardized, because tissues are soft1-3 and have arbitrary shapes and sizes4-6. Shape-adaptive wrapping and covering around irregularly sized and shaped objects have been achieved using heat-shrink films because they can contract largely and rapidly when heated7. However, these materials are unsuitable for biological applications because they are usually much harder than tissues and contract at temperatures higher than 90 °C (refs. 8,9). Therefore, it is challenging to prepare stimuli-responsive films with large and rapid contractions for which the stimuli and mechanical properties are compatible with vulnerable tissues and electronic integration processes. Here, inspired by spider silk10-12, we designed water-responsive supercontractile polymer films composed of poly(ethylene oxide) and poly(ethylene glycol)-α-cyclodextrin inclusion complex, which are initially dry, flexible and stable under ambient conditions, contract by more than 50% of their original length within seconds (about 30% per second) after wetting and become soft (about 100 kPa) and stretchable (around 600%) hydrogel thin films thereafter. This supercontraction is attributed to the aligned microporous hierarchical structures of the films, which also facilitate electronic integration. We used this film to fabricate shape-adaptive electrode arrays that simplify the implantation procedure through supercontraction and conformally wrap around nerves, muscles and hearts of different sizes when wetted for in vivo nerve stimulation and electrophysiological signal recording. This study demonstrates that this water-responsive material can play an important part in shaping the next-generation tissue-electronics interfaces as well as broadening the biomedical application of shape-adaptive materials.

Injectable tissue prosthesis for instantaneous closed-loop rehabilitation.

To construct tissue-like prosthetic materials, soft electroactive hydrogels are the best candidate owing to their physiological mechanical modulus, low electrical resistance and bidirectional stimulating and recording capability of electrophysiological signals from biological tissues1,2. Nevertheless, until now, bioelectronic devices for such prostheses have been patch type, which cannot be applied onto rough, narrow or deep tissue surfaces3-5. Here we present an injectable tissue prosthesis with instantaneous bidirectional electrical conduction in the neuromuscular system. The soft and injectable prosthesis is composed of a biocompatible hydrogel with unique phenylborate-mediated multiple crosslinking, such as irreversible yet freely rearrangeable biphenyl bonds and reversible coordinate bonds with conductive gold nanoparticles formed in situ by cross-coupling. Closed-loop robot-assisted rehabilitation by injecting this prosthetic material is successfully demonstrated in the early stage of severe muscle injury in rats, and accelerated tissue repair is achieved in the later stage.

Phosphorylation-regulated binding of RNA polymerase II to fibrous polymers of low-complexity domains.

The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD.

De novo design of modular protein hydrogels with programmable intra- and extracellular viscoelasticity.

Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in viscoelastic biomaterials exhibiting fluid-like properties under rest and low shear, but solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly in a manner similar to formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology.

Instant tough adhesion of polymer networks.

Generating strong rapid adhesion between hydrogels has the potential to advance the capabilities of modern medicine and surgery. Current hydrogel adhesion technologies rely primarily on liquid-based diffusion mechanisms and the formation of covalent bonds, requiring prolonged time to generate adhesion. Here, we present a simple and versatile strategy using dry chitosan polymer films to generate instant adhesion between hydrogel-hydrogel and hydrogel-elastomer surfaces. Using this approach we can achieve extremely high adhesive energies (>3,000 J/m2), which are governed by pH change and non-covalent interactions including H-bonding, Van der Waals forces, and bridging polymer entanglement. Potential examples of biomedical applications are presented, including local tissue cooling, vascular sealing, prevention of surgical adhesions, and prevention of hydrogel dehydration. We expect these findings and the simplicity of this approach to have broad implications for adhesion strategies and hydrogel design.

Light management by algal aggregates in living photosynthetic hydrogels.

Rapid progress in algal biotechnology has triggered a growing interest in hydrogel-encapsulated microalgal cultivation, especially for the engineering of functional photosynthetic materials and biomass production. An overlooked characteristic of gel-encapsulated cultures is the emergence of cell aggregates, which are the result of the mechanical confinement of the cells. Such aggregates have a dramatic effect on the light management of gel-encapsulated photobioreactors and hence strongly affect the photosynthetic outcome. To evaluate such an effect, we experimentally studied the optical response of hydrogels containing algal aggregates and developed optical simulations to study the resultant light intensity profiles. The simulations are validated experimentally via transmittance measurements using an integrating sphere and aggregate volume analysis with confocal microscopy. Specifically, the heterogeneous distribution of cell aggregates in a hydrogel matrix can increase light penetration while alleviating photoinhibition more effectively than in a flat biofilm. Finally, we demonstrate that light harvesting efficiency can be further enhanced with the introduction of scattering particles within the hydrogel matrix, leading to a fourfold increase in biomass growth. Our study, therefore, highlights a strategy for the design of spatially efficient photosynthetic living materials that have important implications for the engineering of future algal cultivation systems.

Multiscale embedded printing of engineered human tissue and organ equivalents.

Creating tissue and organ equivalents with intricate architectures and multiscale functional feature sizes is the first step toward the reconstruction of transplantable human tissues and organs. Existing embedded ink writing approaches are limited by achievable feature sizes ranging from hundreds of microns to tens of millimeters, which hinders their ability to accurately duplicate structures found in various human tissues and organs. In this study, a multiscale embedded printing (MSEP) strategy is developed, in which a stimuli-responsive yield-stress fluid is applied to facilitate the printing process. A dynamic layer height control method is developed to print the cornea with a smooth surface on the order of microns, which can effectively overcome the layered morphology in conventional extrusion-based three-dimensional bioprinting methods. Since the support bath is sensitive to temperature change, it can be easily removed after printing by tuning the ambient temperature, which facilitates the fabrication of human eyeballs with optic nerves and aortic heart valves with overhanging leaflets on the order of a few millimeters. The thermosensitivity of the support bath also enables the reconstruction of the full-scale human heart on the order of tens of centimeters by on-demand adding support bath materials during printing. The proposed MSEP demonstrates broader printable functional feature sizes ranging from microns to centimeters, providing a viable and reliable technical solution for tissue and organ printing in the future.

Matrix stiffness-dependent regulation of immunomodulatory genes in human MSCs is associated with the lncRNA CYTOR.

Cell-matrix interactions in 3D environments significantly differ from those in 2D cultures. As such, mechanisms of mechanotransduction in 2D cultures are not necessarily applicable to cell-encapsulating hydrogels that resemble features of tissue architecture. Accordingly, the characterization of molecular pathways in 3D matrices is expected to uncover insights into how cells respond to their mechanical environment in physiological contexts, and potentially also inform hydrogel-based strategies in cell therapies. In this study, a bone marrow-mimetic hydrogel was employed to systematically investigate the stiffness-responsive transcriptome of mesenchymal stromal cells. High matrix rigidity impeded integrin-collagen adhesion, resulting in changes in cell morphology characterized by a contractile network of actin proximal to the cell membrane. This resulted in a suppression of extracellular matrix-regulatory genes involved in the remodeling of collagen fibrils, as well as the upregulation of secreted immunomodulatory factors. Moreover, an investigation of long noncoding RNAs revealed that the cytoskeleton regulator RNA (CYTOR) contributes to these 3D stiffness-driven changes in gene expression. Knockdown of CYTOR using antisense oligonucleotides enhanced the expression of numerous mechanoresponsive cytokines and chemokines to levels exceeding those achievable by modulating matrix stiffness alone. Taken together, our findings further our understanding of mechanisms of mechanotransduction that are distinct from canonical mechanotransductive pathways observed in 2D cultures.

Hydrogel biomaterials that stiffen and soften on demand reveal that skeletal muscle stem cells harbor a mechanical memory.

Muscle stem cells (MuSCs) are specialized cells that reside in adult skeletal muscle poised to repair muscle tissue. The ability of MuSCs to regenerate damaged tissues declines markedly with aging and in diseases such as Duchenne muscular dystrophy, but the underlying causes of MuSC dysfunction remain poorly understood. Both aging and disease result in dramatic increases in the stiffness of the muscle tissue microenvironment from fibrosis. MuSCs are known to lose their regenerative potential if cultured on stiff plastic substrates. We sought to determine whether MuSCs harbor a memory of their past microenvironment and if it can be overcome. We tested MuSCs in situ using dynamic hydrogel biomaterials that soften or stiffen on demand in response to light and found that freshly isolated MuSCs develop a persistent memory of substrate stiffness characterized by loss of proliferative progenitors within the first three days of culture on stiff substrates. MuSCs cultured on soft hydrogels had altered cytoskeletal organization and activity of Rho and Rac guanosine triphosphate hydrolase (GTPase) and Yes-associated protein mechanotransduction pathways compared to those on stiff hydrogels. Pharmacologic inhibition identified RhoA activation as responsible for the mechanical memory phenotype, and single-cell RNA sequencing revealed a molecular signature of the mechanical memory. These studies highlight that microenvironmental stiffness regulates MuSC fate and leads to MuSC dysfunction that is not readily reversed by changing stiffness. Our results suggest that stiffness can be circumvented by targeting downstream signaling pathways to overcome stem cell dysfunction in aged and disease states with aberrant fibrotic tissue mechanics.

Development of porcine skeletal muscle extracellular matrix-derived hydrogels with improved properties and low immunogenicity.

Hydrogels derived from decellularized extracellular matrices (ECM) of animal origin show immense potential for regenerative applications due to their excellent cytocompatibility and biomimetic properties. Despite these benefits, the impact of decellularization protocols on the properties and immunogenicity of these hydrogels remains relatively unexplored. In this study, porcine skeletal muscle ECM (smECM) underwent decellularization using mechanical disruption (MD) and two commonly employed decellularization detergents, sodium deoxycholate (SDC) or Triton X-100. To mitigate immunogenicity associated with animal-derived ECM, all decellularized tissues were enzymatically treated with α-galactosidase to cleave the primary xenoantigen-the α-Gal antigen. Subsequently, the impact of the different decellularization protocols on the resultant hydrogels was thoroughly investigated. All methods significantly reduced total DNA content in hydrogels. Moreover, α-galactosidase treatment was crucial for cleaving α-Gal antigens, suggesting that conventional decellularization methods alone are insufficient. MD preserved total protein, collagen, sulfated glycosaminoglycan, laminin, fibronectin, and growth factors more efficiently than other protocols. The decellularization method impacted hydrogel gelation kinetics and ultrastructure, as confirmed by turbidimetric and scanning electron microscopy analyses. MD hydrogels demonstrated high cytocompatibility, supporting satellite stem cell recruitment, growth, and differentiation into multinucleated myofibers. In contrast, the SDC and Triton X-100 protocols exhibited cytotoxicity. Comprehensive in vivo immunogenicity assessments in a subcutaneous xenotransplantation model revealed MD hydrogels' biocompatibility and low immunogenicity. These findings highlight the significant influence of the decellularization protocol on hydrogel properties. Our results suggest that combining MD with α-galactosidase treatment is an efficient method for preparing low-immunogenic smECM-derived hydrogels with enhanced properties for skeletal muscle regenerative engineering and clinical applications.

Structurally and mechanically tuned macroporous hydrogels for scalable mesenchymal stem cell-extracellular matrix spheroid production.

Mesenchymal stem cells (MSCs) are essential in regenerative medicine. However, conventional expansion and harvesting methods often fail to maintain the essential extracellular matrix (ECM) components, which are crucial for their functionality and efficacy in therapeutic applications. Here, we introduce a bone marrow-inspired macroporous hydrogel designed for the large-scale production of MSC-ECM spheroids. Through a soft-templating approach leveraging liquid-liquid phase separation, we engineer macroporous hydrogels with customizable features, including pore size, stiffness, bioactive ligand distribution, and enzyme-responsive degradability. These tailored environments are conducive to optimal MSC proliferation and ease of harvesting. We find that soft hydrogels enhance mechanotransduction in MSCs, establishing a standard for hydrogel-based 3D cell culture. Within these hydrogels, MSCs exist as both cohesive spheroids, preserving their innate vitality, and as migrating entities that actively secrete functional ECM proteins. Additionally, we also introduce a gentle, enzymatic harvesting method that breaks down the hydrogels, allowing MSCs and secreted ECM to naturally form MSC-ECM spheroids. These spheroids display heightened stemness and differentiation capacity, mirroring the benefits of a native ECM milieu. Our research underscores the significance of sophisticated materials design in nurturing distinct MSC subpopulations, facilitating the generation of MSC-ECM spheroids with enhanced therapeutic potential.

Layered nanocomposites by shear-flow-induced alignment of nanosheets.

Biological materials, such as bones, teeth and mollusc shells, are well known for their excellent strength, modulus and toughness1-3. Such properties are attributed to the elaborate layered microstructure of inorganic reinforcing nanofillers, especially two-dimensional nanosheets or nanoplatelets, within a ductile organic matrix4-6. Inspired by these biological structures, several assembly strategies-including layer-by-layer4,7,8, casting9,10, vacuum filtration11-13 and use of magnetic fields14,15-have been used to develop layered nanocomposites. However, how to produce ultrastrong layered nanocomposites in a universal, viable and scalable manner remains an open issue. Here we present a strategy to produce nanocomposites with highly ordered layered structures using shear-flow-induced alignment of two-dimensional nanosheets at an immiscible hydrogel/oil interface. For example, nanocomposites based on nanosheets of graphene oxide and clay exhibit a tensile strength of up to 1,215 ± 80 megapascals and a Young's modulus of 198.8 ± 6.5 gigapascals, which are 9.0 and 2.8 times higher, respectively, than those of natural nacre (mother of pearl). When nanosheets of clay are used, the toughness of the resulting nanocomposite can reach 36.7 ± 3.0 megajoules per cubic metre, which is 20.4 times higher than that of natural nacre; meanwhile, the tensile strength is 1,195 ± 60 megapascals. Quantitative analysis indicates that the well aligned nanosheets form a critical interphase, and this results in the observed mechanical properties. We consider that our strategy, which could be readily extended to align a variety of two-dimensional nanofillers, could be applied to a wide range of structural composites and lead to the development of high-performance composites.

Soft, strong, tough, and durable protein-based fiber hydrogels.

Load-bearing soft tissues normally show J-shaped stress-strain behaviors with high compliance at low strains yet high strength at high strains. They have high water content but are still tough and durable. By contrast, naturally derived hydrogels are weak and brittle. Although hydrogels prepared from synthetic polymers can be strong and tough, they do not have the desired bioactivity for emerging biomedical applications. Here, we present a thermomechanical approach to replicate the combinational properties of soft tissues in protein-based photocrosslinkable hydrogels. As a demonstration, we create a gelatin methacryloyl fiber hydrogel with soft tissue-like mechanical properties, such as low Young's modulus (0.1 to 0.3 MPa), high strength (1.1 ± 0.2 MPa), high toughness (9,100 ± 2,200 J/m3), and high fatigue resistance (2,300 ± 500 J/m2). This hydrogel also resembles the biochemical and architectural properties of native extracellular matrix, which enables a fast formation of 3D interconnected cell meshwork inside hydrogels. The fiber architecture also regulates cellular mechanoresponse and supports cell remodeling inside hydrogels. The integration of tissue-like mechanical properties and bioactivity is highly desirable for the next-generation biomaterials and could advance emerging fields such as tissue engineering and regenerative medicine.

Nanocolloidal hydrogel mimics the structure and nonlinear mechanical properties of biological fibrous networks.

Fibrous networks formed by biological polymers such as collagen or fibrin exhibit nonlinear mechanical behavior. They undergo strong stiffening in response to weak shear and elongational strains, but soften under compressional strain, in striking difference with the response to the deformation of flexible-strand networks formed by molecules. The nonlinear properties of fibrous networks are attributed to the mechanical asymmetry of the constituent filaments, for which a stretching modulus is significantly larger than the bending modulus. Studies of the nonlinear mechanical behavior are generally performed on hydrogels formed by biological polymers, which offers limited control over network architecture. Here, we report an engineered covalently cross-linked nanofibrillar hydrogel derived from cellulose nanocrystals and gelatin. The variation in hydrogel composition provided a broad-range change in its shear modulus. The hydrogel exhibited both shear-stiffening and compression-induced softening, in agreement with the predictions of the affine model. The threshold nonlinear stress and strain were universal for the hydrogels with different compositions, which suggested that nonlinear mechanical properties are general for networks formed by rigid filaments. The experimental results were in agreement with an affine model describing deformation of the network formed by rigid filaments. Our results lend insight into the structural features that govern the nonlinear biomechanics of fibrous networks and provide a platform for future studies of the biological impact of nonlinear mechanical properties.