Advanced Materials

The yield stress at which biomaterials undergo plastic deformation limits the stresses that can be developed in encapsulated growing tissues. While mechanical properties of the matrix such as stiffness and viscoelasticity have a profound effect on cells, the role of yield stress has remained challenging to define. Here we design a self-healing granular hydrogel platform with supramolecular host-guest dynamic crosslinkers to precisely and quantitatively tune the stress at which the matrix repeatedly yields and reconfigures around tissues as they grow. Designed to provide similar mechanical constraints as a mesh stress ball, matrix yield stresses can be tuned between 12 and 370 Pa, while maintaining a storage modulus below [~]0.1kPa. We show that this range of yield stress is sufficient to promote or limit peripheral shedding in a model of non-adhesive cancer migration; and that early development of midbrain organoids is exquisitely sensitive to yield stress. Optimal yield stresses of only 25 Pa promoted budlike protrusions and large, lumenized neural rosettes, while variations as small as 10 Pa limited these phenotypes. These studies demonstrate that morphogenesis and tissue organization are exquisitely sensitive to yield stress, suggesting a new material property to target in designing biomaterials for disease modeling and regenerative medicine.

The ability to track the levels of specific molecules, such as drugs, metabolites, and biomarkers, in the living body, in real time and for long durations would improve our understanding of health and our ability to diagnose, treat and monitor disease. To this end, we are developing electrochemical aptamer-based (E-AB) biosensors, a general platform supporting high-frequency, real-time molecular measurements in the living body. Here we report that the addition of an agarose hydrogel protective layer to E-AB sensors significantly improves their baseline stability when deployed in the complex, highly time-varying environments found in vivo. The improved stability is sufficient that these hydrogel-protected sensors achieved good baseline stability when deployed in situ in the veins, muscles, bladder, or tumors of living rats without the use of the drift correction approaches traditionally required in such placements. Finally, this improved stability is achieved without any significant, associated "costs" in terms of detection limits, response times, or biocompatibility.

Brilliant structural colors originating from diverse photonic crystals are found across many phyla, including the striking iridescent colors of beetles and butterflies, produced by three-dimensional photonic crystal structures in the specialized cuticular scales. However, the precise composition of these structures remains largely unknown, although it is key to unravelling colour production mechanisms and morphogenesis. The longhorn beetle Doliops similis displays vibrant green patterns on its otherwise dark elytra. These patterns are formed by arrays of minute scales that encompass a three-dimensional photonic crystal made of orderly packed nanospheres. We found that these nanospheres are composed of carbonated amorphous calcium phosphate biomineral. By accurately parameterizing the structure and calculating the refractive index deduced from the relative fractions of the organic and inorganic phases, we derived reflection wavelengths that match the observed green hue, demonstrating the biominerals role in colour production. Our study further reveals that biomineralization is widespread in the Lamiinae subfamily, with colour diversity achieved through variations in nanosphere size, packing, and composition. This study opens new avenues for developing bioinspired mineral-based optical devices with high refractive indices and defect-resistance, overcoming the shortcomings of current polymer-based designs.

Bioelectronics have transformed our capacity to monitor and treat diseases; however, a lack of micrometer-scale, energy efficient communication options limit these devices from forming integrated networks that enable full-body, sensor driven, physiological control. Inspired by our nervous systems ability to transmit information via ionic conduction, we engineered a Smart Wireless Artificial Nervous System (SWANS) that utilizes the bodys own tissue to transmit signals between wearables and implantables. When SWANS emits signals, it generates voltage gradients throughout the body that selectively turn on implanted transistor switches when exceeding their gate threshold voltages. SWANS implantable communication components maintain syringe-injectable footprints and >15x greater power efficiencies than Bluetooth and Near Field Communication. In vivo studies in rats demonstrate SWANS ability to wirelessly regulate dual hind leg motor control by connecting electronic-skin sensors to implantable neural interfaces via ionic signaling as well as coordinate bioelectronics throughout the epidermal, subcutaneous, intraperitoneal, and gastrointestinal spaces.

Microinjuries to the lung epithelium are hypothesized to initiate maladaptive processes that lead to fibrosis. Human in vitro models remain a great need for studying this injury-initiation process for mechanistic understanding and therapeutic development. We established a photoresponsive synthetic extracellular matrix (ECM) inspired by the basement membrane that enables triggered injuries of defined size and frequency for probing cellular responses. The synthetic matrix integrated a photolabile bis-coumarin linker for light-triggered injury and relevant integrin-binding peptides for cell function. Bio-orthogonal chemistry was used to create hydrogel-based ECMs with tunable elasticity in transwells, which are traditionally used for epithelial cell culture. Integrin-binding peptide combinations synergistically promoted model epithelial cell layer formation with increased E-cadherin expression and barrier function. An accessible photomasking approach was established for selectively photodegrading the synthetic matrix with cytocompatible visible light and achieving different injury depths and widths. Following a critical injury size, cell responses recapitulated key features of dysregulated re-epithelialization with decreased E-cadherin, proliferation, and barrier function and increased apoptosis. This work provides a new materials-based tool for probing injury and repair processes with tunable control of both the ECM and injury to it with opportunities for future mechanistic and therapeutic insights to address maladaptive wound healing processes.

Transparent microelectrode arrays that enable multimodal investigation of spatiotemporal electrophysiological activity are critical tools for advancing the understanding of excitable tissues such as the brain, heart, and peripheral nerves. Traditional implantable devices are engineered for chronic use but require surgical removal when they fail or are no longer needed. In contrast, bioresorbable systems that naturally dissolve after serving temporary functions offer a compelling alternative, eliminating the risks and costs of extraction procedures. Here, we present the design, fabrication, and validation of a soft, fully bioresorbable, and optically transparent MEA platform for transient, bidirectional interfacing with living tissues. The device provides high-resolution electrical mapping of dynamic activity. We report precise characterization of electrochemical performance, mechanical properties, bioresorption kinetics, and biocompatibility. While validated in models of cardiac function, this platform establishes a versatile foundation for bioresorbable electrophysiological technologies with applications ranging from postsurgical monitoring of transient conditions to the study and treatment of neurological and neurodegenerative disorders.

For long-term, continuous operation of implantable biosensors and electrophysiological devices, the foreign body response (FBR) is a major obstacle that needs to be overcome. As the FBR progresses, any implanted device will become damaged and isolated from its physiological environment, due to encapsulation by fibrotic tissue and inflammatory immune cells. To achieve more compatible and low-impedance biointerfaces, conducting polymers, such as PEDOT:PSS, have been extensively explored as ideal materials. However, FBR on such conducting polymers remains an unmet challenge. We report a zwitteronic-hydrogel-based double-network design for PEDOT:PSS that can significantly suppress the FBR by 64%, in addition to improving conductivity by more than one order of magnitude. Surprisingly, the FBR level of this design is even lower than that of the parent zwitteronic hydrogel by 53%. Our further immunological investigations at the histological, cellular, and transcriptomic levels give deeper insights into the unique effects that come from the chemical heterogeneity. Furthermore, chronic electrocardiographic recording in mice demonstrate the benefit of this material design to long-term, implanted electrophysiology, which provides indications for the future development of immunocompatible electronic polymers.

The intelligence of the human biological system is enabled by the highly distributed sensing receptors on soft skin that can distinguish various stimulations or environmental cues, thus establishing the fundamental logic of sensing and physiological regulation or response. To replicate biological perception, two approaches have emerged: artificial nervous systems that utilize soft electronics as biomimetic receptors to convert external stimuli into frequency-encoded signals, and biohybrid solutions that integrate living cells, plants, or even live animals with electronic components to decode environmental cues for life-like sensations. However, most current biohybrid approaches for artificial sensation are based on eukaryotic cells, which suffer from slow growth, stringent culture conditions, environmental susceptibility, and short lifespans, thus limiting their integration into practical wearables or robotic sensory skins. Here, we introduce fungi-based printable "Mycoelectronics", which are created by additive bioprinting of living fungal mycelium networks onto stretchable electronics, as a practical living thermo-responsive sensory platform. This Mycoelectronics approach leverages fungis capacity for rapid biological responsiveness, cultivability with exponential growth, stability and self-healing in ambient conditions, bioprintability for scalable manufacturing, and mechanical flexibility for seamless integration with soft electronics. Critically, we discovered that the thermal responsiveness of the fungal network arises from intrinsic cellular processes--specifically, heat-induced vacuole remodeling and fusion, which modulate ionic transport and thus the electrical conductivity of the mycelial cells and networks, enabling a rapid temperature response. By bridging the gap between cell biology and soft electronics, the Mycoelectronics device with a living mycelium network functions as a thermal sensation system with rapid response and intrinsic self-healing properties, autonomously restoring sensing capabilities after damage or autonomously establishing sensor pathways in hard-to-reach locations. Furthermore, by integrating fungal thermal sensing with electronic circuits, we established a hybrid bioelectronic reflex arc that can actuate muscles and initiate diverse actions, suggesting promising applications in future neurorobotics and neuroprosthetics.

Engineering physiologically relevant breast in vitro models remains challenging due to the glands complex three-dimensional microanatomy, together with the need for epithelial polarity and hormone responsiveness. To overcome these challenges, fabrication methods are needed that rapidly create alveoli-scale structures with efficient diffusion and sustained hormonal stimulation. Here, Filamented Light (FLight) biofabrication is leveraged to print highly porous, ECM-based hydrogel scaffolds directly within standard Transwell inserts with separate apical and basal access. FLights speckle-patterned laser generates multiscale scaffold architectures that integrate filament-derived microchannels ([~]15 m) to promote diffusion with alveoli-inspired cylindrical microwell arrays (O100, O150, O200 m) that impose geometric constraints to guide epithelial organization. Each insert is printed in <10 s and incorporates slow-release prolactin microcrystals to provide lactogenic stimulation in situ. Primary human milk-derived mammary epithelial cells (milk MECs) were seeded onto the constructs. There, milk MECs line the printed microwells, establish zona occludens-1-positive tight junctions, and express lactation-associated markers (prolactin receptor and {beta}-casein), alongside milk fat globules and intracellular lipid droplets. Collectively, this rapidly reconfigurable FLight platform enables high-throughput generation of hormone-responsive human mammary microtissues for lactation-focused studies and is adaptable to other lumen-forming epithelia.

Over 90% of deaths from cancer occur due to solid tumors, occurring at a rate of [~]1,500 deaths per day in the US, highlighting a profound and unmet need for new therapies. Solid tumors evade clearance by T cells due to a variety of immunosuppressive properties of the tumor microenvironment. However, this immunosuppression cannot be easily blocked on a global level because systemic activation of the immune system elicits a host of complications. An ideal therapy for solid tumors would act locally to activate the immune response without evoking global adverse effects. Here we present a biodegradable, macroporous scaffold that is implanted adjacent to the tumor and suppresses the main obstacle to cancer immunosurveillance: intratumoral regulatory T cells. The scaffold also promotes the recruitment and activation of T cell effectors into the tumor, resulting in clearance of otherwise aggressive and fatal tumors in mice. Unexpectedly, the local depletion of Tregs results in an "immunological abscopal effect" acting on distant tumors. We demonstrate that this versatile platform can also deliver tumor-antigen-specific T cells directly to the peri-tumoral environment, bypassing difficulties in intravenous delivery including the environmental barriers imposed by the tumors vasculature. By orchestrating multiple local immunomodulatory treatments, this scaffold offers a general approach to engineer T-cell responses to solid tumors without systemic toxicities.

There is an increasing need for novel biomaterials compatible with advanced biofabrication technologies, which also permit cells to remodel their microenvironment. This remodelling is crucial for maturing tissue constructs into fully functional tissue replacements. Recent progress in supramolecular chemistries has allowed for the production of dynamic biomaterials. Their properties enable bonds to be reversibly broken by cells, facilitating processes requiring morphological changes or migration, crucial for tissue development and homeostasis. Here, we present a one-of-its-kind gelatin-based hybrid covalent/supramolecular biomaterial. We demonstrate the advantage of adding supramolecular-reactive moieties on covalent materials, over covalent bonds alone, in facilitating processes such as cell growth, migration, spreading and organoid proliferation. This is exemplified by enhanced MSC and T cell migration and improved vascular network formation in hybrid hydrogels over covalent-only materials. The combination of supramolecular and covalent bonds further enabled control over photocrosslinking, allowing the use of the material in volumetric bioprinting of complex structures with high shape fidelity. As a proof-of-concept we bioprinted complex breast-like structures from encapsulated normal breast cell lines with a tumor organoid core. We demonstrated that engineered T cells can migrate large distances into the breast tissue, specifically targeting tumor cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/631505v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1e15084org.highwire.dtl.DTLVardef@16d1554org.highwire.dtl.DTLVardef@937d1aorg.highwire.dtl.DTLVardef@fa51f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Devices that can be remote-controlled under image guidance to precisely deliver biomedicines to sites of disease are a major goal of biomedical research. However, most existing externally triggered delivery systems are based on complex micromachines that are controlled with electromagnetic waves and require custom external instrumentation. Here we present a drug delivery platform comprising a simple protein-containing hydrogel that can be both imaged and triggered to release drugs at specific locations using widely available ultrasound imaging devices. This technology is based on the addition of air-filled protein nanostructures called gas vesicles (GVs) to hydrogel delivery vehicles. While intact, GVs sterically block the release of drug payloads and allow the vehicle to be imaged with ultrasound. An increase in ultrasound pressure causes the collapse of GVs within hydrogels present at the desired anatomical location, instantly creating percolation channels and triggering rapid drug release. Both the imaging and release are performed using a common diagnostic ultrasound probe. We implement this concept by establishing ultrasound-controlled drug diffusion and release from hydrogels in vitro and demonstrating targeted image-guided protein delivery in vivo following oral administration. We use this approach to deliver anti-inflammatory antibodies to treat gastrointestinal inflammation in a rat model of colitis. Targeted acoustic percolation switches (TAPS) open a conduit for local, image-guided drug delivery with a simple formulation and commonplace ultrasound equipment.

Anisotropic biological tissues contain hierarchical complexity from the nano to macro length scales. While novel fabrication strategies have advanced the creation of biomimetic architectures, most rely on biologically derived polymers that possess inherent batch-to-batch variability. Here, we fabricate omnidirectional anisotropic nanofibrous hydrogels using synthetic, self-assembling MultiDomain Peptides (MDPs). Using support bath-assisted extrusion 3D printing, MDP hydrogels are created with control over nanometer-scale fibrous alignment, ~150 {micro}m-scale print resolution, and centimeter-scale 3D architecture. Further, scaffold anisotropy is tuned by adjusting the ionic strength of the support bath, allowing fiber alignment to be decoupled from extrusion shear force and the ink used. Applying these hydrogels to in vitro tissue engineering, fabricated anisotropic hydrogels are shown to guide the alignment of multiple cell types within complex 3D prints. Furthermore, the gels are demonstrated to support the growth of human embryonic stem cell-derived cardiomyocytes into functional tissue. Collectively, this work introduces a platform for engineering anisotropic peptide hydrogels with hierarchical complexity, offering broad potential for bottom-up fabrication of functional human tissues in vitro.

Bioinspired 3D microfluidic systems that combine vascularization with extracellular matrix architectures of organotypic geometry, composition and biophysical traits can help advance our understanding of microorgan physiology. Here, two-photon stereolithography is adopted to fabricate freestanding perfusable 3D cell scaffolds with micrometer resolution from gelatin methacryloyl hydrogel derived from extracellular matrix protein. As a proof of concept, we introduce an ultracompact bio-hybrid chip layout to demonstrate perfusion and cell seeding of double-digit m proteinaceous channels. This perfusion chip consists of a standardized microfluidic interface fabricated from standard resin and a GM10 bioink channel printed atop of this interface. In addition, we demonstrate that algorithmic design synthesis can recapitulate intact alveoli and capillary networks with tunable design parameters to implement vascularized alveolar tissue models. This approach will allow for a systematic investigation of cell-cell and tissue dynamics in response to defined structural, mechanical and bio-molecular cues and is ultimately scalable to fabricate organ-on-a-chip systems.

Biomechanical contributions of the ECM underpin cell growth and proliferation, differentiation, signal transduction, and other fate decisions. As such, biomaterials whose mechanics can be spatiotemporally altered - particularly in a reversible manner - are extremely valuable for studying these mechanobiological phenomena. Herein, we introduce a poly(ethylene glycol) (PEG)-based hydrogel model consisting of two interpenetrating step-growth networks that are independently formed via largely orthogonal bioorthogonal chemistries and sequentially degraded with distinct bacterial transpeptidases, affording reversibly tunable stiffness ranges that span healthy and diseased soft tissues (e.g., 500 Pa - 6 kPa) alongside terminal cell recovery for pooled and/or single-cell analysis in a near "biologically invisible" manner. Spatiotemporal control of gelation within the primary supporting network was achieved via mask-based and two-photon lithography; these stiffened patterned regions could be subsequently returned to the original soft state following sortase-based secondary network degradation. Using this approach, we investigated the effects of 4D-triggered network mechanical changes on human mesenchymal stem cell (hMSC) morphology and Hippo signaling, as well as Caco-2 colorectal cancer cell mechanomemory at the global transcriptome level via RNAseq. We expect this platform to be of broad utility for studying and directing mechanobiological phenomena, patterned cell fate, as well as disease resolution in softer matrices. TOC DescriptionBiomaterials that can dynamically change stiffnesses are essential in further understanding the role of extracellular matrix mechanics. Using independently formulated and subsequently degradable interpenetrating hydrogel networks, we reversibly and spatiotemporally trigger stiffening/softening of cell-laden matrices. Terminal cell recovery for pooled and/or single-cell analysis is permitted in a near "biologically invisible" manner. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/588191v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@d89309org.highwire.dtl.DTLVardef@9d6dc0org.highwire.dtl.DTLVardef@19065e6org.highwire.dtl.DTLVardef@1120aec_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biohybrid robots are emergent soft robots that combine engineered artificial structures and living biosystems to exploit unique characteristics of biological cells and tissues. Skeletal muscle tissue-based bio-actuators can respond to externally applied stimuli, such as electrical fields. However, current bio-actuation systems rely on open-loop control strategies that lack knowledge of the actuators state. The regulation of output force and position of bio-hybrid robots requires self-sensing control systems that combine bio-actuators with sensors and control paradigms. Here, we propose a soft, fiber-shaped mechanical sensor based on a composite with piezoresistive properties that efficiently integrates with engineered skeletal muscle tissue and senses its contracting states in a cell culture environment in the presence of applied electrical fields. After testing the sensors insulation and biocompatibility, we characterized its sensitivity for typical strains (<1%) and proved its ability to detect motions from contractile skeletal muscle tissue constructs. Finally, we showed that the sensor response can feed an autonomous control system, thus demonstrating the first proprioceptive bio-hybrid robot that can sense and respond to its contraction state. In addition to inspiring intelligent implantable systems, informative biomedical models, and other bioelectronic systems, the proposed technology will encourage strategies to exceed the durability, design, and portability limitations of biohybrid robots and confer them decisional autonomy, thus driving the paradigm shift between bio-actuators and intelligent bio-hybrid robots. One Sentence SummaryIntegrating soft mechanical sensors into engineered skeletal muscle tissue enables bio-hybrid robots with proprioception.

ATP is the energy currency of life and overabundant in the tumor microenvironment, where it has been suggested as a target for cancer therapy. We introduce ATP-dissipative delivery of DNA signals from synthetic cells to living cells by exploiting an ATP-driven reaction network that transiently ejects DNA Signal strands from the shielded synthetic cell interior to the extracellular medium of living cells. We customize the Signal for intracellular uptake or for extracellular instruction using a cytokine-ssDNA chimera that can trigger efficient intracellular downstream signaling programs. Our study discusses details of system design on a timer circuit and synthetic cell level, system integration challenges, and how ATP concentrations regulate the transient delivery. The strategy can be extended to deliver therapeutic oligonucleotides for applications in gene therapy and gene silencing. For cancer therapy, it can use naturally enhanced ATP levels to induce selective delivery of therapeutic oligonucleotides.

Microbes are increasingly utilized as living therapeutic vehicles, yet their uncontrolled dissemination in the body has long remained a roadblock to clinical development. Physical containment, while widely used for mammalian cells, remains largely unattainable due to eventual bacteria escape. Here, we present an implantable material platform that encapsulates and confines bacteria, wherein synthetically engineered microbes produce therapeutic payloads from within. To prevent microbial escape, 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 over six 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. This multimodal strategy provides a safe and generalizable framework for deploying microbial medicines in vivo and supports their use as autonomous drug depots across a range of disease settings.

An advanced protein nanopatterned cell culture platform is engineered to emulate the extracellular matrixs complexity, enabling precise nanoscale biomolecule copatterning to mimic environments analogous to native tissue for cellular assays. Nanopatterns fabricated through sparse colloidal lithography, with 100 nm to 800 nm features in separate wells, are seamlessly integrated into standard microplate formats (96-well/384-well). Robust patterns are built from fully PEGylated, passivated thin glass coverslips optimized for minimal nonspecific interactions. Biotin-avidin binding and click chemistry to ensure the accurate and robust localization of bioligands. The transparent, metal-free substrates are free of topographical interference, rendering them ideal for diverse fluorescence microscopy techniques encompassing single-molecule TIRFM and extensive high-throughput imaging. The structural stability of these nanopatterns persists beyond a year in storage and long-term in cell culture conditions, endorsing their application for prolonged experimental studies and potential for widespread academic and industrial use. The platform has been demonstrated for nanopatterning an array of biomolecules, from small molecules to proteins, DNA, and extracellular matrix components, instrumental for studying cell signaling. Experiments with C2C12 cells demonstrated the exceptional specificity of the nanopatterned microplates, with nonspecific adhesion remaining below 2% and the platforms ability to elicit size-dependent cellular reactions when interfaced with nanopatterned fibronectin.

We present a proof-of-concept platform in which amyloids are displayed on the surface of engineered Bacillus subtilis spores for bioengineered materials. Amyloids possess high tensile strength, elasticity, and tunable assembly, but their use is limited by inaccessible native sources and low-yield or toxic heterologous expression. Here, spores were engineered to display the native amyloid TasA and Humboldt squid suckerins 9 and 10 as fusions to the spore coat protein CotY. Amyloid production and fibril formation were confirmed by Western blot and X-34 staining, and quantitative analysis indicated mg/L-level yields. Atomic force microscopy revealed altered stiffness and surface ultrastructure, and incorporation of amyloid-displaying spores into resin-based 3D printing modified tensile strength. These findings highlight spore-based amyloid display as a scalable, modular platform for materials applications, leveraging established industrial spore production.