Abstract T-cells use molecular reactions with nonequilibrium error correction, i.e., proofreading, to discriminate between nearly identical antigens with high specificity and sensitivity. These receptor binding events are known to be force sensitive, yet traditional schemes of proofreading focus on reaction kinetics alone and do not consider the role of force dependent catch/slip bond behavior or interactions with mechanically engaged coreceptors such as adhesion molecules. To address this, we propose a minimal framework for proofreading of ligand discrimination by T-cell receptors (TCRs) that uses endogenous TCR mechanosensation and substrate-mediated mechanical interactions with adhesive proteins (load sharing) to improve recognition fidelity. We leverage the catch bond behavior of cognate antigens to delay decision making and amplify TCR signaling while discarding noncognate slip bond ligands in the presence of a force. By integrating our model with existing structural and molecular data, we show that substrate mechanics regulates the transmission of active cytoskeletal forces through a molecular clutch and controls the energization of bound TCRs needed for optimal proofreading. Our work demonstrates how mechanical forces and substrate properties can augment kinetic proofreading in T-cells, suggesting biomaterial design strategies for immunotherapies that tune the mechanical microenvironment of T-cells to achieve high fidelity TCR-ligand discrimination, antigen recognition, and activation.

Collagen molecules self-assemble into supramolecular fibers within a molecularly crowded, polysaccharide-rich extracellular matrix (ECM) that has fluid-like, viscoelastic properties. Here, we determine that the viscoelasticity of alginate networks regulates the assembly of type I collagen fibers. The viscoelasticity and shear moduli of the alginate network were tuned by the polymer weight percentage and degree of cooperative ionic and covalent norbornene-tetrazine cross-linking. Stepwise shear strain applied to covalently cross-linked hydrogels generated higher stress than in ionic hydrogels. Hydrogels with reduced viscoelasticity also showed reduced water permeability. Second-harmonic generation confocal imaging revealed that decreasing viscoelasticity significantly suppressed collagen fiber self-assembly. Simulations demonstrated mechanical coupling between the hydrogel network and the aggregate size of collagen molecules, which was consistent with experimental results showing impaired rate and magnitude of self-assembly in covalently cross-linked networks. These results provide a framework for understanding how ECM mechanical properties can influence the assembly and organization of fibrillar macromolecules.

Microbes are increasingly used as living therapeutics, yet their uncontrolled dissemination in the body has remained a clinical roadblock. Physical containment remains largely unattainable owing to eventual bacteria escape. In this work, we present an implantable material that encapsulates and confines bacteria, wherein synthetically engineered microbes produce therapeutic payloads from within. We developed a hydrogel scaffold with dual mechanical features: high stiffness to regulate bacterial proliferation and high toughness to resist material fracture under physiological stress. This design achieved complete bacterial containment for 6 months and withstood multiple forms of mechanical loading that otherwise caused catastrophic material failure. By genetically engineering embedded bacteria, we endowed the material with environmental sensing and on-demand therapeutic release capabilities and demonstrated autonomous treatment in a murine prosthetic joint infection model.

Background The scarcity of primary conventional dendritic cells (cDCs) and the limited functionality of monocyte-derived dendritic cells (moDCs) have long hindered mechanistic and translational studies in human dendritic cell (DC) biology and immunotherapy. Methods We developed a feeder-free differentiation platform to generate CD1c(+)CD141(+) human pluripotent stem cell-derived conventional dendritic cells (hPSC-cDCs) to provide a scalable source of DCs with defined properties. A Design-of-Experiments (DoE) optimization strategy was applied to refine cytokine and serum conditions, with the goal of enhancing differentiation efficiency while reducing cytokine demand. The resulting hPSC-cDCs were phenotypically, transcriptionally, and functionally characterized in comparison with primary cDC subsets and moDCs. Functional assays assessed antigen uptake, cytokine production, and the ability to prime antigen-specific CD8(+) T cells. Results The optimized protocol increased hPSC-cDC yield by more than twofold while reducing cytokine usage. hPSC-cDCs expressed canonical cDC2 markers and aligned transcriptionally with primary cDC2s. These cells exhibited efficient phagocytic activity, robust cytokine secretion in response to poly(I:C) or combined Toll-like receptors agonists, and a partially activated basal state resembling primary CD1c(+)CD141(+) DCs in human tissues. Functionally, hPSC-cDCs induced stronger antigen-specific CD8(+) T-cell proliferation, activation, and effector differentiation than moDCs. Conclusions This feeder-free and DoE-optimized system enables reproducible, large-scale generation of functional hPSC-cDCs that phenotypically and transcriptionally resemble primary cDC2s while exhibiting stronger T-cell priming capacity than moDCs. The platform provides a defined and scalable resource for mechanistic studies, vaccine development, and ex vivo T-cell expansion for cancer immunotherapy.

T-cell-based immunotherapies have revolutionized cancer treatment, yet their reliance on patient-derived T-cells limits scalability and accessibility. Engineering functional T-cells de novo from human hematopoietic stem cells (HSCs) represents a promising alternative toward a renewable and customizable source of therapeutic lymphocytes. Successful HSC-derived T-cell generation requires recapitulation of key signaling and adhesion cues of the thymic microenvironment, particularly Notch1-DLL-4 and α4β1-integrin-VCAM-1 interactions within ex vivo engineered thymic niche (ETN) systems. Notch1-DLL-4 and α4β1-integrin-VCAM-1 interactions are known to respond to mechanical forces that regulate their bond dissociation behaviors and downstream signal transduction, yet manipulating the mechanosensitive features of these key receptor-ligand interactions in thymopoiesis has been largely ignored in current ETN designs. Here, we demonstrate that human T-cell development from cord blood-derived CD34+ HSCs is regulated via molecular cooperativity in notch1 and integrin-mediated mechanotransduction. Mechanically confining interpenetrating network (IPN) hydrogel-based 3D cell culture, comprised of collagen type I and alginate polysaccharides functionalized with DLL-4 and VCAM-1 is used as a model viscoelastic 3D ETN to manipulate human progenitor (pro)T-cell differentiation. This ETN enables orthogonal control of the mechanical properties of the thymic niche, including storage modulus, and viscoelastic properties (e.g., stress relaxation kinetics). We identify that soft, viscous matrices that enhance activation of the notch1-pathway, and subsequently notch1 intracellular domain (NICD) nuclear import, sustain the T-cell development gene regulatory network during proT-cell differentiation. Conversely, stiff, elastic matrices inhibit HSC commitment to the T-lineage, and rather promotes Myeloid-cell differentiation. Our observations indicate mechanical reciprocity in signaling pathways indispensable to thymopoiesis and highlight extracellular matrix mechanics as a variable in controlling hematopoietic stem cell fate decisions.

The tumor microenvironment shapes immune surveillance through its mechanical properties, yet the role of matrix viscoelasticity remains unclear. Here, we used a tunable collagen system that models human tissue viscoelasticity to define how matrix relaxation directs dendritic cell (DC) behavior. Slow-relaxing, elastic networks restrict actomyosin-driven remodeling, limiting DC motility and reducing DC-T cell encounters and activation. Blocking DC migration in fast-relaxing matrices recapitulated key aspects of the impaired T cell priming seen in elastic networks, identifying migration as a mechanical checkpoint for immune activation. Prolonged confinement in elastic matrices induced a mechanomemory state, locking DCs into a state of reduced motility and altered chromatin accessibility. Studies using patient-derived ependymoma samples confirmed these findings, establishing viscoelastic relaxation as a key physical regulator of immune priming. Together, this tunable viscoelastic platform provides a defined, human-relevant model to dissect and model mechanical control of immunity for therapeutic design.

Hemorrhage from internal organs remains a critical challenge in both trauma care and surgical procedures, as existing hemostatic adjuncts frequently fail to provide consistent and effective bleeding control, particularly under conditions of active bleeding or impaired coagulation. Here, we develop and evaluate a Hemostatic Tough Adhesive (HTA) in controlled preclinical models of traumatic solid organ injury and compare its performance against leading commercially available hemostatic agents. The HTA consistently outperformed its counterparts, achieving 100% hemostasis in both liver and spleen injuries within an in vivo preclinical porcine model. In contrast, existing adjuncts exhibited variable and often incomplete efficacy. Beyond immediate hemostasis, the HTA demonstrated prolonged stability and biocompatibility during the postoperative wound healing phase. Notably, the HTA exhibited tissue surface adhesion energy several orders of magnitude greater than that of current hemostatic products, indicating its potential utility for surgical and trauma-related bleeding management.

Compromised bone marrow niches following irradiation limit hematopoietic stem cell transplantation (HSCT) success by delaying immune reconstitution. Strategies to rebuild functional marrow environments are essential to support hematopoietic stem cell (HSC) maintenance. However, the fundamental relationship between bone formation and bone marrow niche development, and how these processes are modulated by key biological variables, remains poorly understood. Here, we present an alginate-gelatin cryogel platform that provides sustained BMP-2 release for ectopic osteogenesis and use it to systematically investigate the effects of BMP-2 dose, host immune status, and biological sex. Within 2-4 weeks, the cryogels supported the formation of a cortical bone shell and an internal trabecular bone network populated by hematopoietic tissue. Increasing the BMP-2 dose accelerated mineralization and doubled the resident HSC population, whereas immunocompromised hosts displayed a two-week delay in niche development and a fourfold reduction in HSCs. Female mice exhibited enhanced niche formation relative to males under identical conditions. These findings establish BMP-2-releasing hybrid click cryogels as a tunable bone marrow niche platform and highlight how host- and dose-dependent parameters modulate engineered niche formation, providing insights that may inform future strategies to improve immune reconstitution following HSCT.

Adhesive hydrogels have the potential to address unmet clinical needs in the fields of wound closure, hemostasis, and soft-tissue reinforcement. However, commercial adhesives are limited by weak adhesion to internal organs and poor mechanical performance, especially in the fully swollen state. This study reports a bilayer hydrogel that combines an alginate-acrylamide double-network gel on one side and a highly entangled polyacrylamide gel on the other side to synergistically achieve robust mechanical and tissue-adhesive properties in the swollen state. Cohesion between both layers was achieved by controlling the diffusion of acrylamide between them during polymerization. The bilayer construct achieved stronger mechanical properties under tensile stress and higher performance in burst pressure tests than the double-network gel alone. The improved mechanical performance in the fully swollen state of bilayer hydrogels may expand the range of biomedical applications.

Supplementary Figure S3: Condensation of Oligonucleotide Process.

Adoptive T-cell therapies, and particularly CAR T cells and tumour-infiltrating lymphocytes, have transformed cancer treatment by selectively targeting malignant cells. Despite their clinical success, these therapies face substantial challenges, including costly manufacturing processes and tumour-imposed barriers that limit efficacy. Advances in understanding the nanoscale mechanisms governing T-cell activation and the role of the tumour microenvironment in restricting T-cell responses have driven the development of nanotechnology-based strategies that integrate key chemical and physical cues. Here we provide a brief overview of the current state of CAR T and tumour-infiltrating lymphocyte therapies and discuss nanotechnology strategies to enhance their ex vivo production, in vivo performance and the direct in vivo generation of CAR T cells. We highlight nanotechnology's transformative potential to overcome existing challenges, broaden therapeutic applications and identify factors that will shape the future of nanotechnology for CAR T and tumour-infiltrating lymphocyte therapies.

Therapeutic T-cell engineering ex vivo from human hematopoietic stem cells (HSCs) focuses on recapitulating notch1-signaling and α4β1-integrin-mediated adhesion within the thymic niche with supportive stromal cell feeder-layers or surface-immobilized recombinant protein-based engineered thymic niches (ETNs). The relevant Notch1-DLL-4 and α4β1-integrin-VCAM-1 interactions are known to respond to mechanical forces that regulate their bond dissociation behaviors and downstream signal transduction, yet manipulating the mechanosensitive features of these key receptor-ligand interactions in thymopoiesis has been largely ignored in current ETN designs. Here, we demonstrate that human T-cell development from cord blood-derived CD34 + HSCs is regulated via molecular cooperativity in notch1 and integrin-mediated mechanotransduction. Mechanically confining interpenetrating network (IPN) hydrogel-based 3D cell culture comprised of collagen type I and alginate polysaccharides functionalized with DLL-4 and VCAM-1 is used as a model viscoelastic 3D ETN to manipulate human progenitor (pro)T-cell differentiation. This ETN enables orthogonal control of the mechanical and biomolecular features of the thymic niche, including thymopoietic ligand density, modulus, and viscoelastic properties (e.g., stress relaxation kinetics). We identify that soft, viscous matrices that enhance activation of the notch1-pathway, and subsequently notch1 intracellular domain (NICD) nuclear import sustain the T-cell development gene regulatory network during proT-cell differentiation. Conversely, stiff, elastic matrices inhibit HSC commitment to the T-lineage, and rather promotes Myeloid-cell differentiation. Our observations indicate mechanical reciprocity in signaling pathways indispensable to thymopoiesis, and highlights extracellular matrix mechanics as a variable in controlling hematopoietic stem cell fate decisions.

The production of functional hierarchical architectures through the biomineralization of a continuously secreted protein matrix is prevalent in nature; however, it remains challenging to mimic this dynamic aspect of the biomineralization process, especially in biological systems. Here we report the use of dynamically generated supramolecular assemblies of peptides for in situ biomimetic mineralization in live cells. Specifically, by integrating enzymatic regulation of inorganic phosphate concentration and enzyme-instructed self-assembly, we demonstrate a phosphorylated tripeptide that self-assembles into dynamic supramolecular nanofibers via enzymatic dephosphorylation to template biomineralization with the inorganic phosphate. This biomimetic mineralization results in the formation of peptide-inorganic hybrid nanocrystals, with tunable crystal size and calcium-to-phosphorus (Ca:P) ratio, regulated by enzyme activity. Cellular enzymes can instruct in situ biomineralization around mammalian cells, inducing cell aggregation and osteogenic differentiation. This work presents a novel strategy for mimicking the dynamic biomineralization of a protein matrix and regulating biomimetic mineralization in live cells to control the cell fate.

Biochemical signals in native tissue microenvironments instruct cell behavior during many biological processes ranging from developmental morphogenesis and tissue regeneration to tumor metastasis and disease progression. The detection and characterization of these signals using spatial and highly resolved quantitative methods have revealed their existence as matricellular proteins in the matrisome, some of which are bound to the extracellular matrix while others are freely diffusing. Including these biochemical signals in engineered biomaterials can impart enhanced functionality and native-like complexity, ultimately benefiting efforts to understand, model, and treat various diseases. In this review, we discuss advances in characterizing, mimicking, and harnessing biochemical signals in developing advanced engineered biomaterials. An overview of the diverse forms in which these biochemical signals exist and their effects on intracellular signal transduction is also provided. Finally, we highlight the application of biochemically complex biomaterials in the three broadly defined areas of tissue regeneration, immunoengineering, and organoid morphogenesis.

The blood-brain barrier (BBB) plays a vital role in regulating the passage of biomolecules between the bloodstream and the central nervous system (CNS) while also protecting the CNS from pathogens. Pericytes reside at the interface between endothelial cells and the brain parenchyma. These cells are critical for maintaining BBB integrity and regulating vessel permeability, blood flow, and immune cell migration. In this study, we developed a serum-free protocol to generate neural crest cell-derived pericytes (NCC-PCs) from human pluripotent stem cells (hPSCs). These NCC-PCs can be co-cultured with hPSC-derived brain microvascular endothelial cells (BMECs) in a co-culture BBB model that recapitulates the in vivo cellular interactions at the BBB. We used this model to evaluate the pathological consequences of BBB exposure to highly neuroinvasive flaviviruses. Our results identify a previously undescribed role for NCC-PCs in maintaining BMEC barrier integrity during infection and reducing the spread of viral infection to the CNS.

The engineering of therapeutic living cells through genetic programming is poised to transform medicine. Diverse living medicines, including mammalian cells, fungi, bacteria and viruses, are under development. However, for these medicines to progress in the clinic, new strategies are needed to successfully deliver them into the body. Unlike conventional small-molecule and protein-based biologics, living medicines present distinct challenges for delivery, including the need to maintain viability, control replication, manage metabolism and mitigate immunogenicity. This Review focuses on delivery strategies for living medicines, identifying key challenges and efforts to overcome them. We survey clinically adopted biomaterial strategies for delivering conventional drugs and explore how these approaches can be tailored for living medicines. Finally, we discuss remaining challenges and future directions towards next-generation living medicine delivery. As the field of engineered therapeutic living cells advances, new strategies to deliver them into the body are needed. This Review identifies current challenges in living medicine delivery and discusses how biomaterial strategies could be leveraged to overcome these clinical barriers.

Sustained release of bone morphogenetic protein 2 (BMP-2) is used to enhance bone regeneration, but immobilizing BMP-2 in three-dimensional scaffolds could enable spatial regulation of stem cell differentiation and bone formation. Here, we fabricate porous granular hydrogels presenting BMP-2 on the surface to regulate stem cell growth and differentiation. Immobilization of BMP-2 and cell-adhesive ligands is achieved by surface-specific functionalization of microgels, which are jammed to form microporous hydrogels. Varying surface ligand density regulated spreading, proliferation and differentiation of cells. In addition, modulating the distribution of cell-adhesive ligands and BMP-2 allowed spatial control over cell adhesion and osteogenic differentiation.

Chronic wounds affect millions worldwide and lead to pain, infection, and impaired quality of life. Current wound sealing technologies for chronic wound care remain largely palliative, with poor adhesion, weak mechanical properties, and limited ability to deliver therapeutics. Emerging technologies, including autologous blood products, growth factor-enhanced scaffolds, and artificial skin, although clinically successful, are limited from widespread adoption by the high cost of production and challenging clinical workflow. Here, we present a tough adhesive-elastomer wound care technology that is mechanically robust to approximate wound edges, can adhere strongly to wet and dynamic wound surfaces, is capable of delivering antimicrobials, and supports cell migration and proliferation. This technology has the potential to address an unmet clinical need in wound healing.

The optimal means to prime for effective antitumor immunity in a patient with cancer remain elusive in the current era of checkpoint blockade. Crafting a strategy to amplify the number and function of CD8+ T cells while blocking regulatory cells should increase immunotherapy efficacy. Biomaterial carriers have been demonstrated in preclinical studies to amplify the effects of immunomodulatory agents, synergistically integrate the effects of different agents, and concentrate and manipulate immune cells in vivo. Herein, we report data from a phase I trial in patients with metastatic melanoma who received the cytokine GM-CSF and the innate Toll-like receptor 9 agonist CpG oligonucleotide admixed with autologous tumor lysate onto a microporous poly-lactide-co-glycolide matrix polymer scaffold that achieves precise control over the spatial and temporal release of immunostimulatory agents in vivo. This materials system (WDVAX) served as a physical antigen-presenting structure to which dendritic cells and other immune-stimulating cells are recruited and activated. In this first clinical trial of a macroscale biomaterial-based vaccine, WDVAX treatment was found to be feasible and to induce immune activation in patients with melanoma.

Regulatory T cells (Tregs) maintain immune homeostasis and their adoptive transfer is being widely explored to mitigate inflammatory and autoimmune conditions. Here a biomaterial is developed to accumulate Tregs at a specific anatomic location to bypass the need for ex vivo Treg isolation and adoptive transfer. It is first shown that eliglustat, an FDA-approved inhibitor of UDP-glucose ceramide glucosyltransferase, promotes Tregs from both naïve and activated CD4+ T cells in vitro. Click-crosslinked cryogels fabricated from alginate and collagen allow for a sustained release of CXCL10 or CXCL11, and when injected in subcutaneous tissues led to the enrichment of effector and memory T cells to the scaffolds. Loading eliglustat into these cryogels significantly enhances the local accumulation of Tregs in vivo. These findings demonstrate that eliglustat-loaded cryogels offer a simple yet effective biomaterial strategy to boost Treg directly in vivo, potentially providing a targeted method to treat various inflammatory and autoimmune diseases.