Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Integration of software tools for integrative modeling of biomolecular systems.

Integrative modeling computes a model based on varied types of input information, be it from experiments or prior models. Often, a type of input information will be best handled by a specific modeling software package. In such a case, we desire to integrate our integrative modeling software package, Integrative Modeling Platform (IMP), with software specialized to the computational demands of the modeling problem at hand. After several attempts, however, we have concluded that even in collaboration with the software's developers, integration is either impractical or impossible. The reasons for the intractability of integration include software incompatibilities, differing modeling logic, the costs of collaboration, and academic incentives. In the integrative modeling software ecosystem, several large modeling packages exist with often redundant tools. We reason, therefore, that the other development groups have similarly concluded that the benefit of integration does not justify the cost. As a result, modelers are often restricted to the set of tools within a single software package. The inability to integrate tools from distinct software negatively impacts the quality of the models and the efficiency of the modeling. As the complexity of modeling problems grows, we seek to galvanize developers and modelers to consider the long-term benefit that software interoperability yields. In this article, we formulate a demonstrative set of software standards for implementing a model search using tools from independent software packages and discuss our efforts to integrate IMP and the crystallography suite Phenix within the Bayesian modeling framework.

Effectiveness of the Activate injury prevention exercise programme to prevent injury in schoolboy rugby union.

OBJECTIVE: The efficacious Activate injury prevention exercise programme has been shown to prevent injuries in English schoolboy rugby union. There is now a need to assess the implementation and effectiveness of Activate in the applie setting. METHODS: This quasi-experimental study used a 24-hour time-loss injury definition to calculate incidence (/1000 hours) and burden (days lost/1000 hours) for individuals whose teams adopted Activate (used Activate during season) versus non-adopters. The dose-response relationship of varying levels of Activate adherence (median Activate sessions per week) was also assessed. Player-level rugby exposure, sessional Activate adoption and injury reports were recorded by school gatekeepers. Rate ratios (RR), adjusted by cluster (team), were calculated using backwards stepwise Poisson regression to compare rates between adoption and adherence groups. RESULTS: Individuals in teams adopting Activate had a 23% lower match injury incidence (RR 0.77, 95% CI 0.55 to 1.07), 59% lower training injury incidence (RR 0.41, 95% CI 0.17 to 0.97) and 26% lower match injury burden (95% CI 0.46 to 1.20) than individuals on non-adopting teams. Individuals with high Activate adherence (≥3 sessions per week) had a 67% lower training injury incidence (RR 0.33, 95% CI 0.12 to 0.91) and a 32% lower match injury incidence (RR 0.68, 95% CI 0.50 to 0.92) than individuals with low adherence (<1 session per week). While 65% of teams adopted Activate during the season, only one team used Activate three times per week, using whole phases and programme progressions. CONCLUSION: Activate is effective at preventing injury in English schoolboy rugby. Attention should focus on factors influencing programme uptake and implementation, ensuring Activate can have maximal benefit.

Exposure to Armed Conflict and Fertility in Sub-Saharan Africa.

Changes in fertility patterns are hypothesized to be among the many second-order consequences of armed conflict, but expectations about the direction of such effects are theoretically ambiguous. Prior research, from a range of contexts, has also yielded inconsistent results. We contribute to this debate by using harmonized data and methods to examine the effects of exposure to conflict on preferred and observed fertility outcomes across a spatially and temporally extensive population. We use high-resolution georeferenced data from 25 sub-Saharan African countries, combining records of violent events from the Armed Conflict Location and Event Data Project (ACLED) with data on fertility goals and outcomes from the Demographic and Health Surveys (n = 368,765 women aged 15-49 years). We estimate a series of linear and logistic regression models to assess the effects of exposure to conflict events on ideal family size and the probability of childbearing within the 12 months prior to the interview. We find that, on average, exposure to armed conflict leads to modest reductions in both respondents' preferred family size and their probability of recent childbearing. Many of these effects are heterogeneous between demographic groups and across contexts, which suggests systematic differences in women's vulnerability or preferred responses to armed conflict. Additional analyses suggest that conflict-related fertility declines may be driven by delays or reductions in marriage. These results contribute new evidence about the demographic effects of conflict and their underlying mechanisms, and broadly underline the importance of studying the second-order effects of organized violence on vulnerable populations.

Oxygen gradients can determine epigenetic asymmetry and cellular differentiation via differential regulation of Tet activity in embryonic stem cells.

Graded levels of molecular oxygen (O2) exist within developing mammalian embryos and can differentially regulate cellular specification pathways. During differentiation, cells acquire distinct epigenetic landscapes, which determine their function, however the mechanisms which regulate this are poorly understood. The demethylation of 5-methylcytosine (5mC) is achieved via successive oxidation reactions catalysed by the Ten-Eleven-Translocation (Tet) enzymes, yielding the 5-hydroxymethylcytosine (5hmC) intermediate. These require O2 as a co-factor, and hence may link epigenetic processes directly to O2 gradients during development. We demonstrate that the activities of Tet enzymes display distinct patterns of [O2]-dependency, and that Tet1 activity, specifically, is subject to differential regulation within a range of O2 which is physiologically relevant in embryogenesis. Further, differentiating embryonic stem cells displayed a transient burst of 5hmC, which was both dependent upon Tet1 and inhibited by low (1%) [O2]. A GC-rich promoter region within the Tet3 locus was identified as a significant target of this 5mC-hydroxylation. Further, this region was shown to associate with Tet1, and display the histone epigenetic marks, H3K4me3 and H3K27me3, which are characteristic of a bivalent, developmentally 'poised' promoter. We conclude that Tet1 activity, determined by [O2] may play a critical role in regulating cellular differentiation and fate in embryogenesis.

Match injuries in English schoolboy rugby union.

Objective: This study describes the incidence, severity and burden of match injuries in schoolboy rugby union in England, across three age groups: under-13 (U13), under-15 (U15) and under-18 (U18). Methods: Data regarding 574 24-hour time-loss match injuries and 18 485 player-hours of match exposure were collected from a total of 35 schools (66 teams) in the 2017/18, 2018/19 and 2019/20 seasons. Injury incidence (injuries/1000 hours), severity (mean and median days lost) and burden (days lost/1000 hours) were calculated for each age group, injury region, event, playing position and match period and were compared using Z scores. Results: The U18 age group had a significantly higher injury incidence (34.6 injuries/1000 hours, 95% CI 31.5 to 38.1) and burden (941 days/1000 hours, 95% CI 856 to 1035) than both the U13 (incidence=20.7 injuries/1000 hours, 95% CI 14.1 to 30.3, p=0.03; burden=477 days lost/1000 hours, 95% CI 325 to 701, p<0.01) and U15 (incidence=24.6 injuries/1000 hours, 95% CI 20.6 to 29.5, p<0.01; burden=602 days lost/1000 hours, 95% CI 503 to 721, p<0.01) age groups, but no significant differences were found between the U13 and U15 age groups. Contact events accounted for 87% of known injury events, with the tackle responsible for 52% (U13), 48% (U15) and 62% (U18) of all injuries. Concussion was the most common injury type in all age groups (U13=4.8 injuries/1000 hours; U15=6.4 injuries/1000 hours; U18=9.2 injuries/1000 hours), but the incidence was not significantly different between age groups. Conclusion: Injury incidence and burden was higher in U18 than U13 and U15 age groups. Concussions and the tackle are priority areas at all age groups and should be the focus of injury prevention strategies.

Carboxylic acids and light interact to affect nanoceria stability and dissolution in acidic aqueous environments.

Cerium atoms on the surfaces of nanoceria (i.e., cerium oxide in the form of nanoparticles) can store or release oxygen, cycling between Ce3+ and Ce4+; therefore, they can cause or relieve oxidative stress within living systems. Nanoceria dissolution occurs in acidic environments. Nanoceria stabilization is a known problem even during its synthesis; in fact, a carboxylic acid, namely citric acid, is used in many synthesis protocols. Citric acid adsorbs onto nanoceria surfaces, limiting particle formation and creating stable dispersions with extended shelf life. To better understand factors influencing the fate of nanoceria, its dissolution and stabilization have been previously studied in vitro using acidic aqueous environments. Nanoceria agglomerated in the presence of some carboxylic acids over 30 weeks, and degraded in others, at pH 4.5 (i.e., the pH value in phagolysosomes). Plants release carboxylic acids, and cerium carboxylates are found in underground and aerial plant parts. To further test nanoceria stability, suspensions were exposed to light and dark conditions, simulating plant environments and biological systems. Light induced nanoceria agglomeration in the presence of some carboxylic acids. Nanoceria agglomeration did not occur in the dark in the presence of most carboxylic acids. Light initiates free radicals generated by ceria nanoparticles. Nanoceria completely dissolved in the presence of citric, malic, and isocitric acid when exposed to light, attributed to nanoceria dissolution, release of Ce3+ ions, and formation of cerium coordination complexes on the ceria nanoparticle surface that inhibit agglomeration. Key functional groups of carboxylic acids that prevented nanoceria agglomeration were identified. A long carbon chain backbone containing a carboxylic acid group geminal to a hydroxy group in addition to a second carboxylic acid group may optimally complex with nanoceria. The results provide mechanistic insight into the role of carboxylic acids in nanoceria dissolution and its fate in soils, plants, and biological systems.

Polymer/Nanoceria Hybrid Polyplexes for Gene and Antioxidant Delivery.

Various diseases, including cancers and inflammatory diseases, are characterized by a disruption of redox homeostasis, suggesting the need for synergistic treatments involving co-delivery of gene therapies and free radical scavengers. In this report, polyethylenimine (PEI), nanoceria (NC), and DNA were complexed to form nanoparticles providing simultaneous delivery of a gene and an antioxidant. NC was coated in citric acid to provide stable, 4 nm particles that electrostatically bound PEI/DNA polyplexes. The resulting ternary particles transfected HeLa cells with similar efficiency to that of ternary polyplexes comprising 15 kDa poly-l-α-glutamic acid/PEI/DNA while providing smaller particle sizes by more than 100 nm. NC/PEI/DNA polyplexes exhibited enhanced radical-scavenging activity compared to free NC, and oxidative stress from the superoxide-generating agent, menadione, could be completely reversed by the delivery of NC/PEI/DNA polyplexes. Transfection by NC/PEI/DNA polyplexes was demonstrated to occur efficiently through caveolin-mediated endocytosis and macropinocytosis. Co-delivery of genes encoding reactive oxygen species-scavenging proteins, transcription factors, growth factors, tumor suppressors, or anti-inflammatory genes with NC, therefore, may be a promising strategy in synergistic therapeutics.

Rodent Model for Orthotopic Implantation of Engineered Liver Devices.

This study presents a novel surgical model developed to provide hematological support for implanted cellularized devices augmenting or replacing liver tissue function. Advances in bioengineering provide tools and materials to create living tissue replacements designed to restore that lost to disease, trauma, or congenital deformity. Such substitutes are often assembled and matured in vitro and need an immediate blood supply upon implantation, necessitating the development of supporting protocols. Animal translational models are required for continued development of engineered structures before clinical implementation, with rodent models often playing an essential early role. Our long-term goal has been generation of living tissue to provide liver function, utilizing advances in additive manufacturing technology to create 3D structures with intrinsic micron to millimeter scale channels modeled on natural vasculature. The surgical protocol developed enables testing various design iterations in vivo by anastomosis to the host rat vasculature. Lobation of rodent liver facilitates partial hepatectomy and repurposing the remaining vasculature to support implanted engineered tissue. Removal of the left lateral lobe exposes the underlying hepatic vasculature and can create space for a device. A shunt is created from the left portal vein to the left hepatic vein by cannulating each with separate silicone tubing. The device is then integrated into the shunt by connecting its inflow and outflow ports to the tubing and reestablishing blood flow. Sustained anticoagulation is maintained with an implanted osmotic pump. In our studies, animals were freely mobile after implantation; devices remained patent while maintaining blood flow through their millifluidic channels. This vascular anastomosis model has been greatly refined during the process of performing over 200 implantation procedures. We anticipate that the model described herein will find utility in developing preclinical translational protocols for evaluation of engineered liver tissue. Impact statement Tissue and organ transplantation are often the best clinically effective treatments for a variety of human ailments. However, the availability of suitable donor organs remains a critical problem. Advances in biotechnology hold potential in alleviating shortages, yet further work is required to surgically integrate large engineered tissues to host vasculature. Improved animal models such as the one described are valuable tools to support continued development and evaluation of novel therapies.

Bioinspired Directional Surfaces for Adhesion, Wetting and Transport.

In Nature, directional surfaces on insect cuticle, animal fur, bird feathers, and plant leaves are comprised of dual micro-nanoscale features that tune roughness and surface energy. This feature article summarizes experimental and theoretical approaches for the design, synthesis and characterization of new bioinspired surfaces demonstrating unidirectional surface properties. The experimental approaches focus on bottom-up and top-down synthesis methods of unidirectional micro- and nanoscale films to explore and characterize their anomalous features. The theoretical component of the review focuses on computational tools to predict the physicochemical properties of unidirectional surfaces.

Designer hydrophilic regions regulate droplet shape for controlled surface patterning and 3D microgel synthesis.

A simple technique is presented for controlling the shapes of micro- and nanodrops by patterning surfaces with special hydrophilic regions surrounded by hydrophobic boundaries. Finite element method simulations link the shape of the hydrophilic regions to that of the droplets. Shaped droplets are used to controllably pattern planar surfaces and microwell arrays with microparticles and cells at the micro- and macroscales. Droplets containing suspended sedimenting particles, initially at uniform concentration, deposit more particles under deeper regions than under shallow regions. The resulting surface concentration is thus proportional to the local fluid depth and agrees well with the measured and simulated droplet profiles. A second application is also highlighted in which shaped droplets of prepolymer solution are crosslinked to synthesize microgels with tailored 3D geometry.

Fi-score: a novel approach to characterise protein topology and aid in drug discovery studies.

Target evaluation is at the centre of rational drug design and biologics development. In order to successfully engineer antibodies, T-cell receptors or small molecules it is necessary to identify and characterise potential binding or contact sites on therapeutically relevant target proteins. Currently, there are numerous challenges in achieving a better docking precision as well as characterising relevant sites. We devised a first-of-its-kind in silico protein fingerprinting approach based on the dihedral angle and B-factor distribution to probe binding sites and sites of structural importance. Our derived Fi-score can be used to classify protein regions or individual structural subsets of interest and the described scoring system could be integrated into other discovery pipelines, such as protein classification databases, or applied to investigate new targets. We further demonstrated how our method can be integrated into machine learning Gaussian mixture models to predict different structural elements. Fi-score, in combination with other biophysical analytical methods depending on the research goals, could help to classify and systematically analyse not only targets but also drug candidates that bind to specific sites. The described methodology could greatly improve pre-screening stage, target selection and drug repurposing efforts in finding other matching targets. HIGHLIGHTSDescription and derivation of a first-of-its-kind in silico protein fingerprinting method using B-factors and dihedral angles.Derived Fi-score allows to characterise the whole protein or selected regions of interest.Demonstration how machine learning using Gaussian mixture models on Fi-scores captures and allows to predict functional protein topology elements.Fi-score is a novel method to help evaluate therapeutic targets and engineer effective biologics.Communicated by Ramaswamy H. Sarma.

Surface-tension-driven gradient generation in a fluid stripe for bench-top and microwell applications.

A simple and inexpensive method is presented employing passive mechanisms to generate centimeters-long gradients of molecules and particles in under a second with only a coated glass slide and a micropipette. A drop of solution is pipetted onto a fluid stripe held in place on a glass slide by a hydrophobic boundary. The resulting difference in curvature pressure drives the flow and creates a concentration gradient by convection. Experiments and theoretical models characterize the flows and gradient profiles and their dependence on the fluid volumes, properties, and stripe geometry. A bench-top rapid prototyping method is outlined to allow the user to design and fabricate the coated slides using only tape and hydrophobic spray. The rapid prototyping method is compatible with microwell arrays, allowing soluble gradients to be applied to cells in shear-protected microwells. The method's simplicity makes it accessible to virtually any researcher or student and its use of passive mechanisms makes it ideal for field use and compatible with point-of-care and global health initiatives.

An engineered anisotropic nanofilm with unidirectional wetting properties.

Anisotropic textured surfaces allow water striders to walk on water, butterflies to shed water from their wings and plants to trap insects and pollen. Capturing these natural features in biomimetic surfaces is an active area of research. Here, we report an engineered nanofilm, composed of an array of poly(p-xylylene) nanorods, which demonstrates anisotropic wetting behaviour by means of a pin-release droplet ratchet mechanism. Droplet retention forces in the pin and release directions differ by up to 80 µN, which is over ten times greater than the values reported for other engineered anisotropic surfaces. The nanofilm provides a microscale smooth surface on which to transport microlitre droplets, and is also relatively easy to synthesize by a bottom-up vapour-phase technique. An accompanying comprehensive model successfully describes the film's anisotropic wetting behaviour as a function of measurable film morphology parameters.

Microfluidic synthesis of composite cross-gradient materials for investigating cell-biomaterial interactions.

Combinatorial material synthesis is a powerful approach for creating composite material libraries for the high-throughput screening of cell-material interactions. Although current combinatorial screening platforms have been tremendously successful in identifying target (termed "hit") materials from composite material libraries, new material synthesis approaches are needed to further optimize the concentrations and blending ratios of the component materials. Here we employed a microfluidic platform to rapidly synthesize composite materials containing cross-gradients of gelatin and chitosan for investigating cell-biomaterial interactions. The microfluidic synthesis of the cross-gradient was optimized experimentally and theoretically to produce quantitatively controllable variations in the concentrations and blending ratios of the two components. The anisotropic chemical compositions of the gelatin/chitosan cross-gradients were characterized by Fourier transform infrared spectrometry and X-ray photoelectron spectrometry. The three-dimensional (3D) porous gelatin/chitosan cross-gradient materials were shown to regulate the cellular morphology and proliferation of smooth muscle cells (SMCs) in a gradient-dependent manner. We envision that our microfluidic cross-gradient platform may accelerate the material development processes involved in a wide range of biomedical applications.

Bioinspired design and optimization for thin film wearable and building cooling systems.

In this work, we report a paradigmatic shift in bioinspired microchannel heat exchanger design toward its integration into thin film wearable devices, thermally active surfaces in buildings, photovoltaic devices, and other thermoregulating devices whose typical cooling fluxes are below 1 kW m-2. The transparent thermoregulation device is fabricated by bonding a thin corrugated elastomeric film to the surface of a substrate to form a microchannel water-circuit with bioinspired unit cell geometry. Inspired by the dynamic scaling of flow systems in nature, we introduce empirically derived sizing rules and a novel numerical optimization method to maximize the thermoregulation performance of the microchannel network by enhancing the uniformity of flow distribution. The optimized network design results in a 25% to 37% increase in the heat flux compared to non-optimized designs. The study demonstrates the versatility of the presented design and architecture by fabricating and testing a scaled-up numerically optimized heat exchanger device for building-scale and wearable applications.

The preparation temperature influences the physicochemical nature and activity of nanoceria.

Cerium oxide nanoparticles, so-called nanoceria, are engineered nanomaterials prepared by many methods that result in products with varying physicochemical properties and applications. Those used industrially are often calcined, an example is NM-212. Other nanoceria have beneficial pharmaceutical properties and are often prepared by solvothermal synthesis. Solvothermally synthesized nanoceria dissolve in acidic environments, accelerated by carboxylic acids. NM-212 dissolution has been reported to be minimal. To gain insight into the role of high-temperature exposure on nanoceria dissolution, product susceptibility to carboxylic acid-accelerated dissolution, and its effect on biological and catalytic properties of nanoceria, the dissolution of NM-212, a solvothermally synthesized nanoceria material, and a calcined form of the solvothermally synthesized nanoceria material (ca. 40, 4, and 40 nm diameter, respectively) was investigated. Two dissolution methods were employed. Dissolution of NM-212 and the calcined nanoceria was much slower than that of the non-calcined form. The decreased solubility was attributed to an increased amount of surface Ce4+ species induced by the high temperature. Carboxylic acids doubled the very low dissolution rate of NM-212. Nanoceria dissolution releases Ce3+ ions, which, with phosphate, form insoluble cerium phosphate in vivo. The addition of immobilized phosphates did not accelerate nanoceria dissolution, suggesting that the Ce3+ ion release during nanoceria dissolution was phosphate-independent. Smaller particles resulting from partial nanoceria dissolution led to less cellular protein carbonyl formation, attributed to an increased amount of surface Ce3+ species. Surface reactivity was greater for the solvothermally synthesized nanoceria, which had more Ce3+ species at the surface. The results show that temperature treatment of nanoceria can produce significant differences in solubility and surface cerium valence, which affect the biological and catalytic properties of nanoceria.

Integrated Isogenic Human Induced Pluripotent Stem Cell-Based Liver and Heart Microphysiological Systems Predict Unsafe Drug-Drug Interaction.

Three-dimensional (3D) microphysiological systems (MPSs) mimicking human organ function in vitro are an emerging alternative to conventional monolayer cell culture and animal models for drug development. Human induced pluripotent stem cells (hiPSCs) have the potential to capture the diversity of human genetics and provide an unlimited supply of cells. Combining hiPSCs with microfluidics technology in MPSs offers new perspectives for drug development. Here, the integration of a newly developed liver MPS with a cardiac MPS-both created with the same hiPSC line-to study drug-drug interaction (DDI) is reported. As a prominent example of clinically relevant DDI, the interaction of the arrhythmogenic gastroprokinetic cisapride with the fungicide ketoconazole was investigated. As seen in patients, metabolic conversion of cisapride to non-arrhythmogenic norcisapride in the liver MPS by the cytochrome P450 enzyme CYP3A4 was inhibited by ketoconazole, leading to arrhythmia in the cardiac MPS. These results establish integration of hiPSC-based liver and cardiac MPSs to facilitate screening for DDI, and thus drug efficacy and toxicity, isogenic in the same genetic background.

Interface-directed self-assembly of cell-laden microgels.

Cell-laden hydrogels show great promise for creating engineered tissues. However, a major shortcoming with these systems has been the inability to fabricate structures with controlled micrometer-scale features on a biologically relevant length scale. In this Full Paper, a rapid method is demonstrated for creating centimeter-scale, cell-laden hydrogels through the assembly of shape-controlled microgels or a liquid-air interface. Cell-laden microgels of specific shapes are randomly placed on the surface of a high-density, hydrophobic solution, induced to aggregate and then crosslinked into macroscale tissue-like structures. The resulting assemblies are cell-laden hydrogel sheets consisting of tightly packed, ordered microgel units. In addition, a hierarchical approach creates complex multigel building blocks, which are then assembled into tissues with precise spatial control over the cell distribution. The results demonstrate that forces at an air-liquid interface can be used to self-assemble spatially controllable, cocultured tissue-like structures.

BIOMIMETIC GRADIENT HYDROGELS FOR TISSUE ENGINEERING.

During tissue morphogenesis and homeostasis, cells experience various signals in their environments, including gradients of physical and chemical cues. Spatial and temporal gradients regulate various cell behaviours such as proliferation, migration, and differentiation during development, inflammation, wound healing, and cancer. One of the goals of functional tissue engineering is to create microenvironments that mimic the cellular and tissue complexity found in vivo by incorporating physical, chemical, temporal, and spatial gradients within engineered three-dimensional (3D) scaffolds. Hydrogels are ideal materials for 3D tissue scaffolds that mimic the extracellular matrix (ECM). Various techniques from material science, microscale engineering, and microfluidics are used to synthesise biomimetic hydrogels with encapsulated cells and tailored microenvironments. In particular, a host of methods exist to incorporate micrometer to centimetre scale chemical and physical gradients within hydrogels to mimic the cellular cues found in vivo. In this review, we draw on specific biological examples to motivate hydrogel gradients as tools for studying cell-material interactions. We provide a brief overview of techniques to generate gradient hydrogels and showcase their use to study particular cell behaviours in two-dimensional (2D) and 3D environments. We conclude by summarizing the current and future trends in gradient hydrogels and cell-material interactions in context with the long-term goals of tissue engineering.