Advanced Functional Materials

By decoupling biological function from cellular viability, fixed abiotic cellular materials have emerged as a promising platform for next-generation immunotherapies, preserving essential immunomodulatory activities while enabling unprecedented control over manufacturing, storage, and standardization. Nevertheless, the coupled effects of fabrication history and post-fabrication environmental cues on their biological functionality remain elusive. Here, we show that both preparation route and storage time critically affect the bioactivity of mesenchymal stromal cell (MSC)-derived microtissues. By exploring different devitalization agents, we demonstrate that an ethanol-based strategy provides a green fixation option with comparable preservation of volumetric and fixed cell architecture, as well as immunomodulatory function. Processing protocols did not merely substitute conventional fixation but tailored specific immune-related bioactivity. An unexpected processing protocol and time-dependent enhancement of immunomodulatory performance was observed, with ethanol-fixed microtissues maintaining a stable baseline bioactive profile while increasing functional potency over 8 weeks of storage. We establish a framework for the design of immunomodulatory abiotic cell-derived materials with potential for the treatment of immune-mediated diseases and regenerative therapies, and we hope that the principles uncovered here may contribute to a broader understanding of how devitalized tissues can be engineered, preserved, and functionally tuned, whether lab-made or derived from natural biological systems.

Type II photoinitiators are essential for numerous applications, including advanced 3D printing techniques such as Xolography. Despite offering tunable wavelengths and reaction mechanisms, their limited photoreactivity hinders widespread adoption. Solutions to increase reactivity, particularly in aqueous solutions and in a biocompatible manner, are urgently needed. Here, we report the surprising finding that iodixanol enhances reactivity of type II photoinitiation systems. Supplementing Xolographic print resins with iodixanol increases photoreactivity, which reduces print times and light dosage tenfold, and enables printing of previously unprintable material formulations. Surprisingly, iodixanol supplementation also improves cell viability in a dose-dependent manner, while reducing cell-mediated light scattering. Together, this enables accurate Xolographic printing of highly cell-dense bioresins, which facilitated the biofabrication of engineered living matter with up to 100 M cells mL-1 (>40% cells by volume) with a resolution of ~65 microns. These advances enabled the first demonstration of tissue function in Xolographically-printed materials. Human induced pluripotent stem cell-derived cardiomyocytes were printed into contractile cardiac-like engineered living matter, which exhibited enhanced cell-cell contact and synchronous contractions, confirming tissue level function. As a highly water soluble, cytocompatible, and clinically approved compound, iodixanol therefore represents a valuable and translatable solution to improve the reactivity of type II photoinitiation systems, which enables volumetric biofabrication of highly cell dense living matter. This unlocks a new role for iodixanol in a wide variety of fields including dentistry, chemistry, organic photovoltaics, tissue engineering, and 3D printing.

Recent efforts to reconstruct critical-sized craniomaxillofacial defects using biomaterials have increasingly sought strategies to accelerate proangiogenic processes immediately after surgical implantation to improve host-graft integration. A class of mineralized collagen scaffolds has recently been identified to promote osteogenesis of bone marrow and adipose-derived mesenchymal stem cells both in vivo and in vitro, motivating efforts to identify strategies to promote osteogenic-angiogenic coupling in order to support regenerative healing. Here, we report the first-time use of a glycoengineering approach to generate a population of engineered ASCs (eASCs) from human adipose-derived stem cells (hASCs). This effort inserts an azido sugar onto the cell membrane that can be selectively targeted using DBCO-tagged biomolecules to selectively boost eASCs activity. We report the effects of azido sugar labeling conditions (concentration and incubation time) on eASCs labeling efficiency. We show proliferation and pro-regenerative gene expression activity of eASCs in mineralized collagen scaffolds are largely similar to conventional hASCs. And we demonstrate that molecular cargo functionalized with dibenzocyclooctyne (DBCO) can be conjugated to azido groups on eASCs via click chemistry. Together, this work shows metabolically labeled eASCs have the potential to be used for selective delivery of activity-inducing bioactive cues to enhance regenerative potential.

Damage to the skin by trauma, burns, or surgical procedures often results in uncontrolled bleeding, which remains a leading cause of preventable death following injury, yet most conventional hemostatic materials are engineered solely to arrest bleeding and often adhere strongly to the wound bed, causing pain, rebleeding, and disruption of newly formed tissue upon removal. Here, we report a DNA hydrogel that structurally mimics neutrophil extracellular traps (NETs) and is crosslinked using a bioactive small molecule with potent autophagy-inducing, cardioprotective, anti-inflammatory, antioxidant, and mitochondria-protective properties, integrating rapid hemostasis with active support for tissue regeneration in a single biomaterial. The DNA network provides an intrinsically biocompatible, biodegradable scaffold capable of recruiting platelets and erythrocytes to achieve rapid clot formation, while the bioactive crosslinker is released as the network degrades, delivering a sustained cytoprotective and anti-inflammatory stimulus directly at the wound site. The hydrogel was characterised physiochemically and evaluated for cytocompatibility, hemolytic potential, hemostatic efficacy, and wound-healing performance in a murine model. Results demonstrate that the bioactive-crosslinked DNA hydrogel achieves rapid, effective hemostasis, while accelerating wound closure and supporting regenerative tissue remodelling. This dual-function platform offers a promising strategy for next-generation wound-care biomaterials that unite immediate bleeding control with accelerated, natural tissue healing.

Mechanotransduction plays a fundamental role in regulating immune cell function, yet how engineered virus-like nanospikes engage mechanosensitive signaling pathways to modulate innate immunity remains poorly understood. Here, we report virus-like nanotopography as a previously unrecognized regulator of Piezo1-mediated mechanotransduction in macrophages using virus-like mesoporous silica nanoparticles (VLPSi) with tunable rigid nanospike lengths. We demonstrate a direct structure-activity relationship between nanospike geometry and Piezo1-dependent Ca{superscript 2}+ influx, with longer nanospikes inducing significantly greater intracellular Ca{superscript 2}+ signaling. Building on this mechanistic insight, we developed biomimetic cancer cell membrane (CM)-coated, MSA-2-loaded VLPSi nanoparticle (CM/MSA-2@VLPSi) and investigate the combination of nanospikes-activated Piezo1 with STING signaling and CM antigens presentation in macrophage immune reprogramming. The resulting biomimetic nanoparticles robustly activate the STING-TBK1-IRF3/NF-{kappa}B axis, increase IFN-{beta} and pro-inflammatory cytokine production, and promote macrophage polarization toward M1 phenotype in a spike-length-dependent manner. Collectively, the present study provides a biomimetic strategy for enhancing the M1 polarization of macrophage through the coordinated regulation of mechanical, inflammatory, and antigenic signals.

Carbon is a versatile electrode material that is used in applications ranging from electrical energy storage and electrosynthesis to CO2 conversion, H2 production, and biosensing. The development of pyrolytic carbon electrodes derived from lithographically patterned photoresist structures has expanded the possibilities for creating diverse 2D and 3D electrode designs. In this paper, we present disc-shaped SU-8-derived pyrolytic carbon scaffold electrodes with an open hexagonal honeycomb lattice. The scaffolds were fabricated, using embedded 3D printing within a shear-thinning support bath composed of fused silica nanoparticles (7% w/v) in mineral oil. The rheological properties of the support bath were optimized to enable the printing of low-viscosity materials like SU-8, which would otherwise lack structural integrity in standard extrusion-based 3D printing. The SU-8 scaffolds were then pyrolyzed and characterized using SEM, Raman spectroscopy, Energy Dispersive X-ray, X-ray Photoelectron Spectrometry, and Transmission Electron Microscopy, which revealed graphene-like 'microflowers' on its surface. Electrochemical analysis demonstrated excellent electrochemical properties. The functionality of the scaffold electrodes was validated by differentiation of human neural stem cells into dopaminergic neurons on the scaffold surface, followed by real-time detection of dopamine exocytosis upon cell depolarization.

Chronic diabetic wounds resist healing due to impaired angiogenesis, stalled cellular migration, and persistent inflammation, a challenge further compounded by the rapid degradation of therapeutic growth factors in the proteolytic wound bed. To overcome this, Traditional Chinese Medicine (TCM) compounds are employed not as standalone drugs, but as biomolecular stimuli to precondition the secretome of Wharton's Jelly-derived mesenchymal stem cells (WJMSCs). To overcome these critical translational barriers, this study engineers a core-shell coaxial electrospun nanofibrous scaffold (polyvinyl alcohol core/gelatin shell) designed for the stabilizing and sustained dual-delivery of biologics. We introduce a novel synergistic payload with WJMSCs conditioned medium (WJMSCs-CM) uniquely primed by two specific chinese herbal compounds, Astragaloside IV (AS-IV) and Formononetin (FMN). This core-shell architecture provides native-like contact guidance for cells while converting conventional burst release into a sustained, weeks-long elution. In vitro, this functionalized scaffold restores Akt/eNOS signaling, rescues cellular viability, and promotes robust tube formation in high-glucose-stressed fibroblasts and endothelial cells. In vivo, within an STZ-induced diabetic rat model, the application of this WJMSCs-CM-loaded coaxial scaffold actively inhibits early inflammation and comprehensively accelerates healing, driving near-complete wound closure (98.2 by day 21), mature collagen deposition, and hair follicle neogenesis. Ultimately, this bio-instructive platform successfully integrates physical structural cues with sustained biochemical signaling, offering a potent, multifaceted strategy for chronic wound regeneration.

Engineering biomimetic tissues with dynamically evolving 3D architectures represents an important direction for next-generation tissue engineering, as it enables recapitulation of the continuous morphogenesis of native tissues during development and regeneration. Here, a self-actuating 4D cell-strand bioprinting platform is developed to engineer complex tissue architectures through autonomous cell contractile force (CCF)-driven morphing without requiring external stimuli. The platform integrates a mechanically compliant and self-softening base hydrogel with embedded high-density cell strands printed using a fast-degrading carrier bioink. During culture, the carrier bioink rapidly degrades while the encapsulated cells proliferate and establish connected cellular networks, generating localized contraction that drives programmable shape transformation. Through spatial patterning of embedded cell strands, constructs with diverse morphologies, including V-shaped, helical, folded, and tubular architectures, are generated via controllable self-actuated morphogenesis. The platform further enables engineering of cartilage-like and bone-like tissues with well-defined curvature configurations and mechanically robust tissue matrices. In addition, programmable multi-tissue engineering is demonstrated through fabrication of a muscle-tendon junction-mimicking construct containing spatially organized fibroblast and myoblast compartments. This self-actuating 4D bioprinting strategy enables highly programmable and directionally controlled morphogenesis using a simple construct design with low cell amount requirements, providing a versatile platform for engineering dynamic tissue architectures.

Despite their promise, lipid nanoparticle gene delivery systems have repeatedly failed clinical trials and struggle to achieve efficient, localized transfection in target tissues. The majority of endocytosed nanoparticles are degraded before nucleic acid release, and an inability to track particle distribution in vivo prevents validation of successful delivery. Alternatively, nanobubbles (NBs) are lipid-shelled, gas-core preclinical ultrasound contrast agents and stimuli-responsive drug delivery vehicles. Under varying acoustic pressures, NBs expand, contract, and burst, releasing cargo in an externally controlled, site-specific manner while scattering unique echoes for simultaneous ultrasound visualization. Here, we introduce a cationic nanobubble (CNB) formulation with a +42.3 mV zeta potential, 265 nm diameter, and 2.43x1011 NBs/mL concentration. CNBs produce stable ultrasound contrast, electrostatically load plasmid DNA onto their surface, and internalize into >99% of human prostate cancer cells within 15 minutes in vitro. CNBs remain brightly echogenic intracellularly and induce sonication-dependent expression of green fluorescent protein (GFP). In vivo, CNBs generate contrast in mouse livers for 50 minutes after intravenous administration. Therapeutic ultrasound stimulation over the liver causes a sharp reduction in ultrasound contrast, visualizing localized cavitation in the target organ and inducing a 2.5-fold increase in anti-GFP mean fluorescence intensity relative to the untransfected control. Importantly, no GFP expression is observed without ultrasound stimulation, supporting a mechanism for selective and site-specific gene delivery. This study presents a highly stable CNB capable of efficient DNA loading and ultrasound-dependent gene expression. These results provide a foundation for the future development of CNB platforms to advance image-guided, ultrasound-triggered gene therapy.

Introduction: Advanced biomaterials for regenerative medicine are increasingly expected to combine multifunctionality and compatibility with tissue-specific cellular processes. In this context, hydroxyapatite-based platforms modified through ionic substitution represent promising candidates, as they may integrate structural similarity to bone mineral with additional biological functionality and luminescent properties, enabling diagnostic applications and real-time monitoring. In this study, we evaluated whether silicate-phosphate substituted calcium hydroxyapatite Ca10(PO4)6-x(SiO4)x(OH)2 (where x = 1.5) co-doped with lithium(I), europium(III), and gadolinium(III) ions (Si-HAp-LEG) affects the osteogenic, chondrogenic, and adipogenic differentiation potential of human bone marrow stromal/stem cells (BMSCs). Methods: Human BMSCs were cultured under lineage-specific differentiation conditions in the presence of undoped silicate-substituted phosphate hydroxyapatite (abbr. as Si-HAp), which served as a control, and two distinct Si-HAp-LEG formulations differing in gadolinium(III) (Gd3+) as well as lithium (Li+) and europium(III) (Eu3+) ion concentrations: Si-HAp-LEG-221 (1 mol% Gd3+ ion) and Si-HAp-LEG-222 (2 mol% Gd3+ ion). Differentiation-associated phenotypic outcomes, including extracellular matrix formation and lipid accumulation, were evaluated using Safranin O, Alizarin Red, and Oil Red O staining. In parallel, biomaterial-induced molecular responses were characterized at the transcriptomic and protein levels using RT-qPCR for selected coding and non-coding RNAs and Western blot analysis for representative lineage-associated proteins. Results: Histochemical evaluation confirmed that, across all tested biomaterial groups, BMSCs retained the ability to form mineralized calcium deposits, proteoglycan-rich extracellular matrix, and intracellular lipid accumulation under osteogenic, chondrogenic, and adipogenic conditions, respectively. Quantitative staining analysis revealed no significant Si-HAp-LEG-dependent enhancement of terminal differentiation outcomes compared with undoped Si-HAp. In turn, the molecular response differed between biomaterials modifications. Si-HAp-LEG-222 induced the most prominent changes in transcriptional and post-transcriptional regulators, particularly within BMP/SMAD-associated pathways under osteogenic and chondrogenic conditions, underlying a potential link between gadolinium concentration and osteogenic lineage commitment. However, these transcriptomic responses were not mirrored by consistent changes at the protein level. The results suggest that silicate-phosphate substituted hydroxyapatite co-doped with Li+, Eu3+, and Gd3+ ions primarily affects the early regulatory pathways associated with BMSCs differentiation rather than enhancing their terminal maturation. Discussion: In conclusion, the collective data indicate that Li+, Eu3+, and Gd3+ ions LEG co-doping broadens the multifunctional potential of Si-HAp by introducing imaging-related properties while preserving its underlying pro-regenerative character. Li+, Eu3+, and Gd3+ ions co-doped LEG-substituted Si-HAp may therefore be considered a compatible biomaterial platform that maintains BMSC cellular plasticity and supports balanced, differentiation-dependent modulation of lineage-associated molecular responses.

Lung adenocarcinoma, driven frequently by KRAS and p53 mutations, remains a leading cause of cancer mortality. Current state-of-the-art genetically engineered mouse models often rely on viral delivery of recombinases, such as Cre recombinase, to initiate transformation. However, viral particles can infect and activate innate immune cells, thus potentially impacting studies of tumor-immune dynamics. Here, we develop a layer-by-layer (LbL) polyplex platform using poly({beta}-aminoester) (PBAE) polymers layered with poly-L-aspartic acid (PLD) to deliver Cre mRNA to lungs while avoiding immune cell transfection and activation. PLD-coated nanoparticles (PLD-NPs) exhibit stable mRNA encapsulation and efficient transfection in vitro, even after lyophilization and long-term storage. In KrasLSL-G12D/+;p53flox/flox (KP) mice, PLD-NPs initiate lung adenocarcinomas that mirror human histopathology without infecting or activating dendritic cells and alveolar macrophages, unlike lentiviral (LV) or adenoviral delivery methods. Single-cell transcriptional profiling revealed that LV administration drives long-term upregulation of antigen presentation and costimulatory machinery in lung-resident myeloid populations. This persistent immune activation is avoided by NP delivery. By uncoupling tumor initiation from innate immune activation, this platform enables the high-fidelity interrogation of tumor-immune dynamics, especially for non-inflammation-driven lung cancer.

CRISPR gene editing offers unprecedented genomic and transcriptomic control, positioning it as a powerful tool for cell therapies. Non-viral CRISPR delivery avoids the immunogenicity, genomic integration, and packaging limits of viral vectors, but local or systemic injections of non-viral nanoparticles suffers from transient action and poor biodistribution. Alternatively, biomaterial-based delivery improves nanoparticle localization and sustains delivery, yet current approaches rely on non-specific adsorption of nanoparticles to scaffolds, risking aggregation, destabilization, and unreliable release kinetics. This work establishes a novel strategy to covalently tether nanoparticles to biomaterial substrates through SnoopTag and SpyTag bioconjugation systems for spatiotemporal control of non-viral CRISPR delivery. The RALA cell-penetrating peptide, an efficient and low-cytotoxicity CRISPR delivery system, was functionalized with SnoopTag without altering nanoparticle formation or transfection capacity. SnoopTag-functionalized nanoparticles were then covalently tethered to SpyTag-decorated zwitterionic microgels via a SnoopCatcher-SpyCatcher fusion protein. This platform achieves sustained RALA-mRNA nanoparticle delivery and prolonged CRISPR activation in human mesenchymal stem cells seeded within CRISPR-loaded microgel scaffolds. This work establishes a defined biorthogonal conjugation system that tethers non-viral nanoparticles to biomaterial platforms for sustained and localized CRISPR gene editing.

Disruption of the wound healing cascade can result in pathological outcomes, including fibrosis due to myofibroblast-mediated contraction and collagen deposition. Despite the clinical significance, effective treatments for fibrosis remain limited as current therapies often show inconsistent efficacy, adverse effects, and patient discomfort. Combinatorial therapeutic strategies integrating biomaterial scaffolds with gene delivery have shown promise in regenerative healing. MicroRNAs (miRNAs) are key regulators of fibrotic signalling in cells, including fibroblasts and myofibroblasts. Specifically, miRNA-29b is notable for downregulating pro-fibrotic genes, including collagen type I, reducing ECM accumulation, and limiting fibroblast/myofibroblast overactivation. In this context, the present work develops a collagen-GAG (CG) scaffold platform for delivery of miRNA-29b complexed GET nanoparticles to inhibit fibrosis. Initially, bioinformatic analysis of miRNA-29b validated its involvement in ECM-associated pathways and processes, followed by successful nanoparticle internationalisation in primary dermal fibroblasts. The anti-fibrotic efficacy of the optimised miRNA-29b nanoparticles was subsequently demonstrated by significant reductions in collagen deposition and -SMA expression, both key indicators of myofibroblast differentiation and fibrosis. The optimised miRNA-29b formulation was then incorporated into 3D porous collagen-GAG (CG) scaffolds, which modulated fibrotic gene expression while preserving scaffold structure conducive to fibroblast/myofibroblast infiltration and proliferation. Finally, functional outcomes of seeded TGF-{beta}-stimulated fibroblasts, including reduced matrix contraction, -SMA expression, and ECM deposition, were comparable to those observed in non-fibrotic conditions, thereby confirming the therapeutic potential of scaffold-mediated miRNA-29b delivery. Together, these findings demonstrate that scaffold-mediated miRNA-29b delivery represents a promising anti-fibrotic strategy for wound healing by mitigating myofibroblast activation, limiting matrix contraction, and preventing pathological ECM accumulation.

Developing biomaterials that faithfully recapitulate the complex architecture of native tissues is central to tissue engineering. Conventional hydrogels typically exhibit nanoscale porosity, which limits mass transport and cell migration and thereby constrains tissue development. Here, we exploit liquid-liquid phase separation (LLPS) to generate interconnected macroporous gelatin methacryloyl (GelMA) scaffolds with tunable pore sizes ranging from 2 to 50 m under physiological pH conditions. By mapping the GelMA/dextran phase diagram, we identify a composition range in which LLPS occurs through spinodal decomposition, resulting in a spontaneous bicontinuous, trabecular-like architecture. Pore size can be further tuned by varying polymer concentrations and temperature. We advantageously combined thermal gradients and photopatterning to enable spatial control over pore morphology to fabricate hierarchically structured materials. The biological relevance of these architectures is assessed using fibroblasts cultured within controlled porosity gradients. We show that pore size strongly influences cell infiltration, growth, and spatial organization, with larger pores providing sufficient space for cell migration while reducing the dependence on matrix degradation. Pore size of 7-15 m is identified for maximizing fibroblast growth. Overall, our approach provides a versatile platform for porosity screening, enabling the engineering of biomimetic hydrogels with optimized microarchitectures tailored to specific biological contexts for three-dimensional cell culture.

For regenerative medicine applications, transplanted cells are difficult to retain in a specific place to maximize the localized release of cytokines, which can positively modulate immune responses, angiogenesis and tissue regeneration. Here, we developed a hollow electrospun (ESP) scaffold with one open and one closed end to enclose a hydrogel laden with human mesenchymal stem cells (hMSCs); we termed this combination the hydrocup. hMSCs remained viable for 28 days in vitro and released functional cytokines similar to hMSCs in transplanted but non-enclosed hydrogels. The hydrocups remained intact and fixed in place for 6 weeks after subcutaneous implantation in rats, demonstrating in vivo compatibility. These results demonstrate that the hydrocup is the first, to our knowledge, cell-laden hydrogel delivery scaffold. This innovation could be developed for a wide variety of cell-based cytokine secretion or drug-laden hydrogel applications.

Osteoarthritis, characterized by cartilage degradation and synovial inflammation, has spurred interest in mechano-piezoelectric bio-hydrogel therapies that can both relieve symptoms and reverse progression. However, current approaches lack sufficient piezoelectric output and dual cartilage/inflammation targeting. To address this, we demonstrated a mechano-piezoelectric peptide hydrogel composed of nanofibers integrating piezoelectric cues with mesenchymal stromal cells (MSCs) recruitment and PIEZO2 mechanosignaling. Molecularly, the hydrogel's seed peptide incorporated four functions: COL2A1 targeting, MMP-13 responsiveness, MSCs homing, and self-assembly. Overexpressed MMP-13 in the osteoarthritis niche triggers gelation, promoting MSCs recruitment and drug retention. Fluorination modulates hierarchical nanofiber assembly, enhancing mechanical and piezoelectric properties, as confirmed by morphological, biophysical, and computational analysis. The trifluoromethyl-modified, 4-octyl itaconate (4-OI) loaded formulation reverses osteoarthritis via PI3K/AKT activation and Wnt/{beta}-catenin suppression, as shown by improved Osteoarthritis Research Society International (OARSI) scores, bone microarchitecture, and cartilage matrix. This synergy of mechano-piezoelectric cues and 4-OI offers a clinically promising strategy for osteoarthritis.

Transcatheter embolization requires materials that can be steered through tortuous vessels, solidify rapidly in situ, remain clearly visible under fluoroscopy, and, ideally, carry therapeutic cargo without harming tissue. To meet these requirements, we present a fully water-based, two-component PEI-PEG hydrogel delivered through a soft-robotic, microfluidic catheter that keeps the precursors separate until they meet in a millimetre-scale mixing chamber at the tip. Fast amide cross-linking converts the liquid pair into a self-supporting gel within seconds, eliminating organic solvents and preventing catheter blockage. By adjusting precursor ratio and flow regime, the gel's stiffness and viscosity can be tuned over orders of magnitude, with the same chemistry allowing to occlude both high-flow arteries and fragile micro-vessels. The platform was validated in three escalating models. First, in ex-vivo perfused human placenta, the hydrogel filled targeted branches without reflux or fragmentation, demonstrating controlled delivery in clinically relevant vasculature. Next, in three porcine embolizations, splenic, hepatic and ascending pharyngeal arteries, the material achieved stable, selective occlusion with no migration, vasospasm or recanalization, showing seamless compatibility with standard interventional workflows. Finally, in rats bearing orthotopic liver tumours, drug-loaded hydrogel delivered through the hepatic artery concentrated doxorubicin inside tumours while sparing healthy tissue, confirming its potential for precision chemoembolization. These results position the PEI-PEG hydrogel and microfluidic catheter as a unified, image-guided platform that couples robust mechanical occlusion with site-specific drug delivery, offering a biocompatible alternative to current liquid embolics and expanding the therapeutic reach of minimally invasive embolization procedures.

Globular proteins are difficult to convert into robust hydrogels, as their compact, folded structures bury reactive residues, forcing conventional strategies to rely on denaturation or synthetic-polymer reinforcement that compromise the native protein. Inspired by the beta-ketoenamine bond-forming chemistry of covalent organic frameworks (COFs), we report the crosslinking of native bovine serum albumin (BSA) with 1,3,5-triformylphloroglucinol (TFP), a C3-symmetric trialdehyde, into a chemically defined hydrogel. TFP reacts with surface-exposed lysine residues through an irreversible enol-to-keto tautomerization, confirmed by FTIR and NMR spectroscopy, generating stable {beta}-ketoenamine crosslinks under mild aqueous conditions without denaturing the protein, as verified by intrinsic tryptophan fluorescence. The resulting hydrogels are mechanically robust compared to a reversible-imine control, injectable and self-recovering, exhibit reversible shape memory and substantial load-bearing capacity, and remain stable across a broad pH range over extended periods. The network shows consistent swelling behavior at physiological and mildly acidic pH, with modest compaction under strongly basic conditions; scanning electron microscopy reveals a dense, nodular network for the TFP hydrogel versus an open, sheet-like lamellar morphology for the reversible-imine control. The hydrogel efficiently encapsulates doxorubicin and displays pH-triggered, acid-selective release, which comparative kinetic analysis attributes principally to pH-dependent weakening of DOX-BSA binding affinity (linked to the N-to-F conformational transition of BSA near its isoelectric point) rather than to bulk network swelling or degradation. Doxorubicin-loaded hydrogels show enhanced killing of MCF-7 breast cancer cells relative to the free drug while remaining cytocompatible toward normal mammalian cells, and a ciprofloxacin-loaded variant exhibits potent antibacterial activity against both Gram-positive (M. luteus) and Gram-negative (E. coli) bacteria. This work translates reticular {beta}ketoenamine chemistry into a general platform for robust, stimuli-responsive protein biomaterials.

Engineering a transplantable artificial liver requires reconstituting the hepatic lobule, whose densely cellular parenchyma is organized around a hierarchically branched vascular network. Achieving high cellular density and hierarchical vascularization within a single fabrication step remains a major challenge. Here, we present an ultrasonic standing wave (USW)-based strategy that concurrently organizes vascular and parenchymal compartments during a single extrusion. HepG2-endothelial cell aggregates were fabricated as building blocks and suspended in a liver-derived dECM-alginate hydrogel for rapid, ultraviolet-free crosslinking. They were then co-extruded with single endothelial cells through a glass capillary under USW actuation. Due to their size difference, the single cells were focused at the pressure nodes, forming patterns that transitioned from a single supplying line to four microvascular lines. The aggregates instead accumulated at the pressure antinodes, reconstituting the basic microvascular-parenchymal unit of the lobule. Aggregate size increased with seeding density while maintaining uniformity and over 90% viability. The patterned endothelial cells formed perfusable lumina through which 5 um microparticles flowed freely. Compared with hepatocyte-only cultures, the vascularized constructs showed a 1.7-fold increase in urea production, and 2.7- and 6.2-fold increases in CYP1A2 and CYP3A4 expression, respectively. Following implantation into the mouse liver, the scaffolds showed favorable biocompatibility, sustained proliferation over 21 days, and progressive host integration. The smallest blood-containing lumina matured from 26.1 um to 7.9 um in diameter, accompanied by increasing collagen deposition at the graft-host interface. This approach provides a template-free route to hierarchically vascularized hepatic units and a promising step towards implantable liver tissue.

Myocardial infarction highlights an urgent need for strategies to regenerate functional cardiac tissue. Cardiac tissue engineering offers a promising approach; however, fabricating scaffolds that simultaneously integrate precise architectural anisotropy, mechanical compliance, and electrical conductivity remains an open challenge. In this work, we utilized melt electrowriting (MEW) to construct well-defined, 20-layer anisotropic rhomboidal polycaprolactone (PCL) scaffolds. We characterised them by tensile testing and by micro- and nanoscale microscopy. While introducing electrical conductivity via bulk blending with fillers (polypyrrole (PPy), polyaniline, or graphene oxide) compromised MEW print fidelity and failed to achieve physiological conductivity, surface coating strategies effectively combined conductivity from structural mechanics. Electrical and mechanical testing revealed that gold sputter coating and in situ PPy polymerization both imparted robust electrical conductivity while preserving the microfibrous architecture. However, when seeded with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in fibrin hydrogels, only the gold-coated scaffolds supported synchronized, robust, and sustained contractile activity. PPy-coating resulted in functionally restricted constructs, suggesting that excessive structural rigidity limited tissue deformability. Gene expression analysis further revealed that elevated electrical conductivity alone does not drive hiPSC-CM maturation. Our data indicates that successful cardiac patch design relies on the integrated optimization of mechanics and architecture rather than treating conductivity as an isolated parameter, offering foundational guidelines for developing translational bioengineered heart patches.