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Gelatin methacryloyl (GelMA) is a versatile material for a wide range of bioapplications. There is an intense interest in developing effective chemical strategies to prepare GelMA with a high degree of batch-to-batch consistency and controllability in terms of methacryloyl functionalization and physiochemical properties. Herein, we systematically investigated the batch-to-batch reproducibility and controllability of producing GelMA (target highly and lowly substituted versions) via a one-pot strategy. To assess the GelMA product, several parameters were evaluated, including the degree of methacryloylation, secondary structure, and enzymatic degradation, along with the mechanical properties and cell viability of GelMA hydrogels. The results showed that two types of target GelMA with five batches exhibited a high degree of controllability and reproducibility in compositional, structural, and functional properties owing to the highly controllable one-pot strategy.

Traditional hydrogels are strong candidates for biomedical applications; however, they may suffer from drawbacks such as weak mechanics, static properties, and an inability to fully replicate aspects of the cellular microenvironment. These challenges can be addressed through the incorporation of second networks to form interpenetrating polymer network (IPN) hydrogels. The objective of this review is to establish clear trends on the enhanced functionality achieved by incorporating secondary networks into traditional, biopolymer-based hydrogels. These include mechanical reinforcement, ‘smart’ systems that respond to external stimuli, and the ability to tune cell–material interactions. Through attention to network structure and chemistry, IPN hydrogels may advance to meet challenging criteria for a wide range of biomedical fields. The extracellular matrix (ECM) is a complex assembly of biopolymers, the organization and composition of which combine to provide structural, mechanical, and biochemical signals to cells. Although single network hydrogels recapitulate features of the ECM, further advancements are needed to expand their functionality for many applications.Incorporation of secondary networks into biopolymer hydrogels imparts mechanical reinforcement, the ability to respond to stimuli, and increased mimicry of the ECM. These interpenetrating polymer network hydrogels are promising for tissue engineering, drug delivery, and in vitro disease models for drug discovery and screening.Addition of a second network makes conventional hydrogels amenable to many emerging biofabrication techniques geared towards achieving hierarchical architectures and personalized medicine.

Matrigel, a mouse tumor extracellular matrix protein mixture, is an indispensable component of most organoid tissue culture. However, it has limited the utility of organoids for drug development and regenerative medicine due to its tumor-derived origin, batch-to-batch variation, high cost, and safety issues. Here, we demonstrate that gastrointestinal tissue-derived extracellular matrix hydrogels are suitable substitutes for Matrigel in gastrointestinal organoid culture. We found that the development and function of gastric or intestinal organoids grown in tissue extracellular matrix hydrogels are comparable or often superior to those in Matrigel. In addition, gastrointestinal extracellular matrix hydrogels enabled long-term subculture and transplantation of organoids by providing gastrointestinal tissue-mimetic microenvironments. Tissue-specific and age-related extracellular matrix profiles that affect organoid development were also elucidated through proteomic analysis. Together, our results suggest that extracellular matrix hydrogels derived from decellularized gastrointestinal tissues are effective alternatives to the current gold standard, Matrigel, and produce organoids suitable for gastrointestinal disease modeling, drug development, and tissue regeneration. The culture of gastrointestinal organoids relies on Matrigel that has several drawbacks for clinical application. Here, the authors report the feasibility of gastrointestinal tissue-mimetic matrices as effective alternatives to Matrigel for organoid culture and transplantation.

Organoids have extensive therapeutic potential and are increasingly opening up new avenues within regenerative medicine. However, their clinical application is greatly limited by the lack of effective GMP-compliant systems for organoid expansion in culture. Here, we envisage that the use of extracellular matrix (ECM) hydrogels derived from decellularized tissues (DT) can provide an environment capable of directing cell growth. These gels possess the biochemical signature of tissue-specific ECM and have the potential for clinical translation. Gels from decellularized porcine small intestine (SI) mucosa/submucosa enable formation and growth of endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and SI. ECM gels can be used as a tool for direct human organoid derivation, for cell growth with a stable transcriptomic signature, and for in vivo organoid delivery. The development of these ECM-derived hydrogels opens up the potential for human organoids to be used clinically. Organoid cultures have been developed from multiple tissues, opening new possibilities for regenerative medicine. Here the authors demonstrate the derivation of GMP-compliant hydrogels from decellularized porcine small intestine which support formation and growth of human gastric, liver, pancreatic and small intestinal organoids.

Synthetic hydrogels are here created by enzyme-induced mineralization of hydrogel networks, yielding materials that are tough yet impressively stiff, with calcium phosphate particles distributed homogeneously throughout the network. Synthetic hydrogels get tough and stiff Natural materials such as cartilage and skin have a combination of toughness (meaning they are hard to fracture) and stiffness (meaning they are resistant to bending) that is difficult to emulate in synthetic hydrogels. Previously reported tough hydrogels owed their toughness to their ability to deform by stretching, but they lacked stiffness. Here Joerg Tiller and colleagues create hydrogels that are both tough and stiff by generating in situ amorphous calcium phosphate nanoparticles that are homogenously distributed throughout the hydrogel matrix. The resulting structures are tougher than most water-swollen synthetic hydrogels, and are stiffer than their natural counterparts. The highly filled composite materials can even be designed to be optically transparent, and they remain stretchable even when notched with a razor blade. The researchers attribute the stiffness of these materials to the formation of a percolated network of the calcium phosphate nanoparticles throughout the hydrogel. The cartilage and skin of animals, which are made up of more than fifty per cent water, are rather stiff (having elastic moduli of up to 100 megapascals) 1 , 2 as well as tough and hard to break (with fracture energies of up to 9,000 joules per square metre) 3 , 4 . Such features make these biological materials mechanically superior to existing synthetic hydrogels. Lately, progress has been made in synthesizing tough hydrogels, with double-network hydrogels achieving the toughness of skin 5 and inorganic–organic composites showing even better performance 6 . However, these materials owe their toughness to high stretchability; in terms of stiffness, synthetic hydrogels cannot compete with their natural counterparts, with the best examples having elastic moduli of just 10 megapascals or less 7 , 8 , 9 , 10 , 11 . Previously, we described the enzyme-induced precipitation and crystallization of hydrogels containing calcium carbonate, but the resulting materials were brittle 12 . Here we report the enzyme-induced formation of amorphous calcium phosphate nanostructures that are homogenously distributed within polymer hydrogels. Our best materials have fracture energies of 1,300 joules per square metre even in their fully water-swollen state—a value superior to that of most known water-swollen synthetic materials. We are also able to modulate their stiffness up to 440 megapascals, well beyond that of cartilage and skin. Furthermore, the highly filled composite materials can be designed to be optically transparent and to retain most of their stretchability even when notched. We show that percolation drives the mechanical properties, particularly the high stiffness, of our uniformly mineralized hydrogels.

Studies of cancer cell migration have found two modes: one that is protease-independent, requiring micron-sized pores or channels for cells to squeeze through, and one that is protease-dependent, relevant for confining nanoporous matrices such as basement membranes (BMs). However, many extracellular matrices exhibit viscoelasticity and mechanical plasticity, irreversibly deforming in response to force, so that pore size may be malleable. Here we report the impact of matrix plasticity on migration. We develop nanoporous and BM ligand-presenting interpenetrating network (IPN) hydrogels in which plasticity could be modulated independent of stiffness. Strikingly, cells in high plasticity IPNs carry out protease-independent migration through the IPNs. Mechanistically, cells in high plasticity IPNs extend invadopodia protrusions to mechanically and plastically open up micron-sized channels and then migrate through them. These findings uncover a new mode of protease-independent migration, in which cells can migrate through confining matrix if it exhibits sufficient mechanical plasticity. In order to metastasize, cancer cells must migrate through basement membranes and dense stroma, and proteases are thought to be required due to the confining nature of these matrices. Here the authors use synthetic matrices to show that cells can migrate through confining matrices using force generation alone, rather than protease degradation, if the matrices exhibit mechanical plasticity.

Although cell–matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular traction, independently of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). Moreover, switching the permissive hydrogel to a restrictive state through delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Furthermore, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment. Adhesive interactions between stem cells and the extracellular matrix are known to regulate stem cell differentiation, yet the underlying mechanisms are not well understood. It is now shown that fate decisions of stem cells encapsulated in covalently crosslinked hydrogels are regulated, independently of matrix mechanics and cell morphology, by the cellular tension generated from cell-induced degradation of the hydrogels.

Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules ⩾30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98‐derived hydrogel. It fully blocks entry of GFP‐sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β‐type NTRs at its surface. O‐GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid‐state NMR spectroscopy revealed that the O‐GlcNAc‐modified Nup98 gel lacks amyloid‐like β‐structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC‐like permeability. The phenylalanine‐glycine (FG) domains of vertebrate nucleoporins assemble into hydrogels with different sieving characteristics for macromolecules. Nup98 forms the tightest filter, which is relieved by O‐linked glycosylation.

Size, surface charge, and material compositions are known to influence cell uptake of nanoparticles. However, the effect of particle geometry, i.e., the interplay between nanoscale shape and size, is less understood. Here we show that when shape is decoupled from volume, charge, and material composition, under typical in vitro conditions, mammalian epithelial and immune cells preferentially internalize disc-shaped, negatively charged hydrophilic nanoparticles of high aspect ratios compared with nanorods and lower aspect-ratio nanodiscs. Endothelial cells also prefer nanodiscs, however those of intermediate aspect ratio. Interestingly, unlike nanospheres, larger-sized hydrogel nanodiscs and nanorods are internalized more efficiently than their smallest counterparts. Kinetics, efficiency, and mechanisms of uptake are all shape-dependent and cell type-specific. Although macropinocytosis is used by both epithelial and endothelial cells, epithelial cells uniquely internalize these nanoparticles using the caveolae-mediated pathway. Human umbilical vein endothelial cells, on the other hand, use clathrin-mediated uptake for all shapes and show significantly higher uptake efficiency compared with epithelial cells. Using results from both upright and inverted cultures, we propose that nanoparticle internalization is a complex manifestation of three shape- and size-dependent parameters: particle surface-to-cell membrane contact area, i.e., particle–cell adhesion, strain energy for membrane deformation, and sedimentation or local particle concentration at the cell membrane. These studies provide a fundamental understanding on how nanoparticle uptake in different mammalian cells is influenced by the nanoscale geometry and is critical for designing improved nanocarriers and predicting nanomaterial toxicity.

Pore-forming Gasdermin protein-induced pyroptosis in tumor cells promotes anti-tumor immune response through the release of pro-inflammatory cytokines and immunogenic substances after cell rupture. However, endosomal sorting complexes required for transport (ESCRT) III-mediated cell membrane repair significantly diminishes the tumor cell pyroptosis by repairing and subsequently removing gasdermin pores. Here, we show that blocking calcium influx-triggered ESCRT III-dependent membrane repair through a biodegradable nanoparticle-mediated sustained release of calcium chelator (EI-NP) strongly enhances the intracellularly delivered GSDMD-induced tumor pyroptosis via a bacteria-based delivery system (VNP-GD). An injectable hydrogel and a lyophilized hydrogel-based cell patch are developed for peritumoral administration for treating primary and metastatic tumors, and implantation for treating inoperable tumors respectively. The hydrogels, functioning as the local therapeutic reservoirs, can sustainedly release VNP-GD to effectively trigger tumor pyroptosis and EI-NP to prevent the ESCRT III-induced plasma membrane repair to boost the pyroptosis effects, working synergistically to augment the anti-tumor immune response. Activation of ESCRT-mediated cell membrane repair can reduce the extent of tumor cell pyroptosis. Here the authors develop two formulations (an injectable hydrogel and a cell patch) for the sustained release of a Gasdermin-D bacteria-based delivery system and of biodegradable nanoparticles loaded with an ESCRT inhibitor, triggering pyroptosis and antitumor immune responses in preclinical cancer models.