Advanced Healthcare Materials

Modeling neural-immune interactions in neurodegenerative and immune-mediated central nervous system (CNS) diseases requires human 3D models that capture cellular diversity and long-term tissue maturation. Here, we present an enhanced human induced pluripotent stem cell (hiPSC)-derived cerebral organoid (CO) platform optimized to mitigate core hypoxia for over 200 days. Timed pro-myelinating cues established organized neuronal layering and progressive axonal myelination through day 140, while vascular fusion yielded assembloids incorporating endothelial structures and microglia. Extended culture (>500-750 days) spontaneously reproduced hallmark features of human CNS aging, including cellular senescence signatures, neuroaxonal loss, hypomyelination, and the autonomous emergence of a neurotoxic astrocyte transcriptional profile in the complete absence of microglia or immune cells. Co-culture with autologous activated peripheral blood mononuclear cells (PBMC) resulted in transient immune infiltration and a pronounced type II interferon response across CNS lineages. High-plex spatial transcriptomics revealed that immune cell infiltration was associated with oligodendrocyte loss and in aged organoids also with downregulated oligodendrocyte myelin gene transcription. While not fully reproducing adult tissue stoichiometry, this platform enables longitudinal modeling of neural-immune crosstalk in age-related and neuroinflammatory CNS disorders.

Osteochondral repair requires restoration of zonally organised articular cartilage and subchondral bone, yet translatable implants rarely reproduce this spatial complexity. Here, we developed an off-the-shelf, cell-free multilayer scaffold comprising a superficial 2% (w/v) articular cartilage extracellular matrix (AC-ECM) phase, an intermediate 5% AC-ECM phase and a basal 6% bone ECM (BN-ECM) phase. The scaffold formed continuous interfaces, displayed regionally distinct pore sizes and resisted permanent deformation during cyclic compression. In vitro, constructs seeded with caprine mesenchymal stromal and articular cartilage progenitor cells supported cell expansion and the accumulation of sulfated glycosaminoglycan- and collagen-rich matrix, with regional differences in collagen I, II and X deposition. Following eight weeks of subcutaneous implantation, cell-seeded scaffolds contained more collagenous matrix than unseeded controls, while vascularisation preferentially localised to the BN-ECM phase. The scaffold was then evaluated against empty defects in a caprine osteochondral model for six months. Scaffold treatment significantly improved macroscopic and histological repair, increased chondral tissue fill (~60% versus ~40%), limited cartilage-like tissue extension into the subchondral region and generated a more native-like superficial collagen organisation. Repair tissue further exhibited greater collagen II immunoreactivity, increased ACAN and COL2A1 expression and reduced COL1A2 expression relative to empty defects, although deeper bone repair was not significantly improved. These findings demonstrate that tissue-specific ECM layering can spatially guide endogenous repair and substantially improve cartilage restoration without exogenous cells or growth factors in a clinically relevant large-animal model, while identifying subchondral bone regeneration as the remaining design challenge for complete osteochondral repair.

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.

Decellularized extracellular matrix (dECM) scaffolds are increasingly used in regenerative medicine, yet the extent to which processed placental dECM retains properties capable of influencing cellular responses remains unclear. This study combines functional cell assays with deep learning-enabled quantitative imaging to determine how dehydrated placental ECM regulates cellular behavior across multiple human cell lineages. Human dermal fibroblasts, cardiac fibroblasts, and osteoblasts were cultured on dehydrated placental ECM or standard cell culture surfaces and assessed for cell attachment, viability, extracellular matrix production, and nuclear morphology. Placental dECM supported attachment and survival across all three cell types, while Pro-Collagen I Alpha 1 secretion varied by cell lineage relative to negative controls. To identify structural responses associated with scaffold culture, an automated imaging pipeline combining Cellpose-based nuclear segmentation with nuclear morphometric analysis was used to quantify nuclear area, eccentricity, and circularity. Quantitative profiling of hundreds of nuclei revealed scaffold-dependent remodeling of nuclear morphology that was not apparent by conventional microscopy. Cells cultured on placental dECM exhibited reduced nuclear area and increased nuclear eccentricity, while cardiac fibroblasts and osteoblasts showed alterations in nuclear circularity. These lineage-dependent morphological responses demonstrate that placental dECM provides more than a permissive substrate for cell attachment and is associated with measurable changes in cellular architecture following processing. Together, these findings support the biological relevance of processed placental dECM as a regenerative biomaterial and demonstrate the utility of quantitative single-cell morphometric analysis for detecting cell-material interactions that may not be apparent through qualitative imaging alone, guiding the rational design of regenerative therapies.

Astrocyte border formation is a conserved neuroprotective response to neural tissue disruption, yet astrocyte border states at implanted biomaterials remain less well characterized than injury responses. Here, we developed the Astrocyte Border Characterization (ABC) Tool, which leverages a shear-thinning, injectable biomaterial to locally deliver astrocyte-specific RiboTag AAVs and small molecule regulators in the mouse striatum, enabling molecular profiling and phenotypic modulation of astrocyte border (AB) cells. Spatially precise delivery of AAV using the ABC Tool yielded enhanced specificity and robust RiboTag expression in AB cells from 7-70 days post injection. Temporal transcriptomic profiling of AB cells revealed predominantly acute, transient changes in genes governing dedifferentiation, proliferation, metabolic reprogramming, and inflammation regulation. Persistent changes accounted for only 14% of regulated genes but involved critical gain of functions in immune regulation and host defense that mirrored astrocyte border responses at chronic CNS injuries. Local delivery of indiscriminate or astrocyte-selective ablation molecules delayed, rather than prevented, border formation, ultimately yielding thicker astrocytes borders with increased inflammation and fibrosis at the biomaterial-tissue interface. Conversely, local delivery of {beta}-hydroxybutyrate (BHB) from the ABC Tool altered key aspects of the transcriptional reprogramming to attenuate chronic astrocyte reactivity and prevent biomaterial contraction without exacerbating inflammation or fibrosis. Our findings establish the ABC Tool as a bioassay for studying and manipulating astrocyte borders at implanted biomaterials and identify focal metabolic regulation as a strategy to modulate AB cell phenotypes and enhance the CNS biocompatibility of biomaterials.

We present an active mixing toolhead for extrusion bioprinting. The tool enables the programmable fabrication of tumor microenvironment gradient arrays, through controlled deposition of mixed hydrogel precursor formulations into 384-well plates, pre-seeded with tumor cells. It operates on an open-source bioprinter and can actively mix arbitrary ratios of two hydrogel precursors prior to extrusion. These concentration gradient arrays are compatible with quantitative image analysis of cell viability and morphological responses to hydrogels conditioned with drug or extracellular matrix (ECM) proteins. The tools capacity to mix and print hydrogel precursor gradients was demonstrated using alginate and highly concentrated mCherry-conjugated mini-spidroin solutions. Hydrogel precursor stocks contained fluorescent reporters to facilitate quantifications of mixing efficiency, and as proxies for drug and ECM protein concentrations. The tool was applied to generate hydrogel-based gradients of the apoptosis-inducer staurosporine, from which concentration-dependent MDA-MB-231 breast cancer cell death responses were quantified. Gradient arrays of the ECM protein laminin-511, implicated in breast cancer tumorigenesis, were generated and revealed that increasing laminin-511 concentrations potentiated staurosporine-induced cell death. The study demonstrates the utility of this active mixing toolhead for producing hydrogel gradient arrays, and demonstrates the relevance of studying drug-responses in tumor microenvironment models that account for disease-specific ECM components.

Precise control of growth factor delivery remains a challenge for directing stem cell differentiation in three-dimensional (3D) engineered tissues. In this study, engineered cell-like vesicles are used as programmable microenvironments to enable sustained and localized delivery of growth factors within visible-light-crosslinked GelMA hydrogels. Giant unilamellar vesicles (GUV) loaded with FGF-2 and TGF-{beta}3 were incorporated into bioinks with BM-MSC to drive keratocyte differentiation without repeated soluble growth factor supplementation. ELISA measurements confirmed the removal of non-encapsulated growth factors and the release of the vesicle cargo following induced vesicle rupture. Fluorescence monitoring showed a progressive reduction in detectable FGF-2- and TGF-{beta}3-loaded GUV during culture, while droplet-scale analysis demonstrated the co-deposition of cells and vesicles after printing. After 14 days of differentiation, differentiated cells expressed ALDH1A1, ALDH3A1, lumican, keratocan, and collagen I without induction of -SMA. Interestingly, keratocyte-associated differentiation was retained after drop-on-demand bioprinting, confirmed by qPCR analysis. These findings establish growth factor-loaded vesicles as bioprintable instructive niches capable of supporting localized keratocyte differentiation within 3D corneal constructs.

Vascularization, which is critical for most engineered tissue constructs, is dependent on interactions between endothelial cells and spatiotemporally presented biochemical and biophysical cues. Yet, most hydrogels define these cues at the time of fabrication, thus precluding adjustments to actively drive vascular formation. We developed acoustically responsive scaffolds (ARSs) that use focused ultrasound to trigger growth factor release and localized matrix remodeling within fibrin hydrogels. ARSs were formed by incorporating phase-shift emulsions containing basic fibroblast growth factor (bFGF) along with perfluorohexane (C6) or perfluorooctane (C8). Upon ultrasound exposure, stable bubbles were generated in C6-ARSs that locally compacted the matrix and increased macroscale stiffness. Comparatively, in C8-ARSs, ultrasound generated macropores without impacting viscoelastic properties. Ultrasound increased bFGF release from both ARS types, which enhanced in vitro and in vivo vasculogenic assembly in ARSs with co-encapsulated endothelial cells and fibroblasts. Our data also show that ultrasound-driven matrix remodeling without bFGF release increased endothelial sprouting. In C6-ARSs, elevated levels of F-actin were observed in both cell types adjacent to bubbles as well as increased YAP intensity and nuclear asymmetry. Together, these results establish ARSs as reconfigurable hydrogels that couple on-demand release of biochemical cues with programmable matrix restructuring to direct three-dimensional microvascular assembly.

Graphene oxide (GO) has primarily been investigated as a carrier for intracellular delivery of therapeutic molecules. In previous work, we identified a cell type-dependent interaction pattern in which GO remained predominantly associated with the plasma membrane of cancer cells but was internalised by non-cancerous epithelial cells. Here, we explored whether plasma membrane-associated GO can be used as a platform to present bioactive ligands and influence cell-surface receptor signalling in cancer cells. To test this hypothesis, we targeted integrin receptors at the plasma membrane in glioblastoma cell models using an RGD-containing peptide non-covalently complexed with GO. We assessed GO-peptide interactions, cellular interactions/uptake, motility, and focal adhesion signalling readouts. Peptide association was quantified using a 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay, and GO was characterised by atomic force microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and colloidal measurements. Immediately after complexation, ~70% of RGD was associated with GO. Peptide association increased the nitrogen signal and shifted the principal GO XRD peak while retaining nanosheet morphology. Biological responses were examined in U87 and U251 glioblastoma cells with different integrin-positive fractions, and in non-cancerous BEAS-2B bronchial epithelial cells. Confocal microscopy showed that GO and GO:RGD remained predominantly localised on the plasma membrane in U87 and U251 cells, whereas greater intracellular localisation was observed in BEAS-2B cells. Importantly, GO:RGD significantly reduced key indicators of cell motility: cell velocity in U87 and U251 cells, with trajectory and mean-square-displacement analyses supporting restricted cellular movement. Free RGD had no significant effect, while GO alone produced a smaller reduction in motility only in U251 cells. No treatment significantly altered BEAS-2B motility. Flow cytometry also showed a reduced pFAK-associated signal in GO:RGD-treated U87 cells. These findings establish a proof of concept that the cell-line-dependent plasma membrane localisation of GO can be exploited as a membrane-associated nano-bio interface for cell-surface-active ligands, opening the way for the development of GO-based platforms that modulate receptor-mediated signalling and cell behaviour.

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.

Embedded extrusion printing can process collagen-rich bioinks, but their low viscosity and slow fibrillogenesis compromise print fidelity and post-deposition stability. Here, we developed a collagen fibril-inducing support bath (FIB) that combines mechanical support for embedded printing with biochemical induction of collagen assembly. Microfibrillated or nanofibrillated cellulose was incorporated into a fibril-inducing buffer, and formulations were screened at 37 degrees Celsius for rheological behaviour and optical transparency. The selected FIB was evaluated by printing 1% and 5% (w/v) articular cartilage-derived extracellular matrix (ECM) inks at 10-20 mm/s and compared with a cellulose-only control bath. FIB exhibited yield-stress, shear-thinning and rapid recovery behaviour that supported reproducible filament deposition. Unlike the control bath, FIB enabled intact construct retrieval following stabilisation and promoted the formation of fibrillar collagen within the printed strands. Scanning electron microscopy revealed D-banded collagen fibrils preferentially oriented along the deposition direction, with dominant orientation peaks within +/- 10-15 degrees. The platform supported the fabrication of 15 x 15 x 1.5 mm sheets and 6 x 6 x 6 mm scaffolds whose macroscopic dimensions were retained after processing. Constructs produced from 5% ECM inks exhibited approximately fourfold higher ramp and relaxation moduli than those produced from 1% ECM inks. Extracts from both formulations caused no detectable reduction in cell metabolic activity after 24 h or 72 h. Mesenchymal stem/stromal cells (MSCs) seeded onto printed sheets became markedly elongated and aligned by day 3, with approximately 80% of cells having an aspect ratio exceeding 1.5, significantly greater than cells seeded onto casted ECM controls, with a mean deviation of ~9 degrees from the filament print direction. These findings establish FIB as a bioactive support bath that couples embedded printability with collagen fibrillogenesis, enabling recoverable collagen-rich constructs with aligned fibrillar architecture that directs early cellular organisation.

Sensory and motor neurons differ significantly in their morphology, structural organization, functional properties, and mode of action. However, despite this heterogeneity, many in vitro studies focus only on a single neuronal subtype, mainly being sensory neurons, limiting the translational potential and relevance of these studies for the evaluation of therapeutic options for spinal cord injury. In this study, we investigate the differentiation, maturation, and neuronal outgrowth of human induced pluripotent stem cell (iPSC)-derived motor and sensory neurospheres using polyethylene glycol (PEG)-microgels with various bioactive coatings. Our results show subtype-specific responses to the PEG-microgel scaffolds, with respect to motor and sensory neurosphere morphology and size. Furthermore, we compare the formation of the PEG-microgel/scaffolds when starting from iPSCs-derived precursor neuron spheres versus undifferentiated iPSCs. We observe notable differences in structural organization, maturity, and neuronal outgrowth between the two approaches, as well as between motor and sensory neurospheres. Together, these results underline the importance of studying motor and sensory neurons separately and highlight the need for a controlled, tunable culture platform to assess the impact of the microenvironment and to improve the physiological relevance of in vitro platforms for neuron-based research.

Decellularized extracellular matrices (ECMs) are widely used in regenerative medicine, yet current evaluation criteria prioritize cellular removal rather than preservation of the ECM characteristics that govern tissue behavior. Here, we demonstrate that efficient decellularization is achieved across a broad range of chemical conditions, whereas preservation of structurally and biologically relevant ECM components is confined to narrow, tissue-specific windows defined by coupled detergent interactions. Quantitative proteomics revealed that intrinsic ECM composition is strongly associated with tissue-specific susceptibility to decellularization-induced damage and provided molecular context for the distinct preservation responses between tissues. Optimized matrices retained major structural ECM components and supported tissue-specific cellular organization and cell-mediated mechanical reinforcement following cellular repopulation despite uniformly low residual DNA across protocols. Together, these findings support a shift in decellularization quality assessment from DNA-based evaluation toward preservation of biologically relevant ECM and establish a composition-driven strategy for the rational design of regenerative biomaterials with tissue-relevant biological and mechanical properties.

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.

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.

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.

Ulcerative colitis (UC) is a global health challenge driven by immune dysregulation and gut microbiota imbalance.1 Current treatments, limited by insufficient efficacy and systemic toxicity during prolonged use, fail to resolve the intertwined immune-microbial pathology.2 Here, we report an orally administered self-assembled hydrogel C2-(IIRR)2I-NH2 (CIR), engineered from host defense peptides, which disrupts the immune-microbiota entanglement. The CIR hydrogel exhibits structural transformation at the inflamed sites rich in liposaccharide (LPS), a pro-inflammatory molecule derived from pathogenic bacteria. Stable {beta}-sheet nanofibers can transfer to bioactive -helix conformations, enabling localized therapeutic action with minimal off-target toxicity. In murine colitis models, CIR restores mucosal integrity and suppresses disease severity, outperforming the first-line drug 5-aminosalicylic acid (5-ASA). Microbiome profiling reveals its capacity to rebalance gut microbiota, depleting LPS produced pathogenic bacteria like Prevotellaceae. Transcriptomic analyses further indicate that CIR silences TLR4-mediated signaling pathway. By synergistically targeting immune dysregulation and microbial dysbiosis, this self-assembled peptide hydrogel establishes a paradigm-shifting strategy for UC, offering clinically translatable potential for multifactorial gastrointestinal disorders.

Collagen, a major component of the extracellular matrix, regulates cellular behaviors, such as adhesion, differentiation, and angiogenesis. These functions are mediated by interactions between specific amino acid motifs within the collagen triple-helical structure and collagen-binding biomolecules. These include cell-surface receptors, such as integrins, discoidin domain receptors (DDRs), and syndecans, a family of transmembrane heparan sulfate proteoglycans (HSPGs). Signals mediated by these receptors are integrated to regulate cell fate. However, native collagen simultaneously presents multiple receptor-binding motifs, making it difficult to isolate receptor-specific functions and to evaluate receptor crosstalk. Here, we introduce a composition-controlled artificial collagen matrix platform that enables independent tuning of multiple receptor-binding motifs within a constant triple-helical scaffold. This material was produced by disulfide crosslinking of chemically synthesized collagen-like triple-helical peptides, each bearing a single defined receptor-binding sequence. By varying the mixing ratios of these peptides before crosslinking, we systematically controlled the composition of receptor-binding motifs within the matrices. We applied this platform to nerve growth factor-dependent neuronal differentiation of PC12 cells, a process supported by collagen. Matrices containing only integrin-binding sequences were sufficient to support this differentiation. Incorporation of an HSPG-binding sequence had little additional effect, whereas incorporation of a DDR-binding sequence suppressed integrin-mediated differentiation and coincided with DDR phosphorylation. These results reveal opposing roles of collagen-binding integrins and DDRs in regulating PC12 cell differentiation. Composition-controlled artificial collagen provides a versatile matrix platform for dissecting functional crosstalk among collagen receptors.

Tympanic membrane perforations remain a common clinical problem, and although surgical intervention through tympanoplasty achieves high success rates, it is associated with donor-site morbidity, surgical complexity and limited restoration of the native radial and circumferential collagen architecture. In this study, 3D extrusion printing was utilized to create an active scaffold and attempt to promote collagen organization through shear-mediated structural alignment. An alginate-carboxymethyl cellulose (CMC) hydrogel with bovine SIS-dECM was prepared and investigated for its suitability as a bioink alternative to tympanoplasty grafts. The physiochemical, rheological and printability characteristics of the hydrogel were assessed. Successful decellularization was confirmed by histological analysis. The incorporation of the SIS-dECM into the hydrogel led to increased swelling, lower apparent viscosity, yield stress and flow stress while maintaining favourable printability and filament stability. Polarized optical microscopy was also used to study the influence of printing speed on the alignment of collagen to mimic the native tympanic membrane radial collagen architecture. Compared with the cast controls, the printed samples presented stronger birefringence signals. Biological evaluation demonstrated that the 15% dECM hydrogel exhibited the highest live cell area percentage and live/dead ratio after 48 h. In addition, the chick chorioallantoic membrane assay demonstrated that the dECM-containing hydrogels improved vascular density. The findings establish a printable, biologically active dECM bioink capable of generating bulk collagen organization through extrusion printing as a platform for tympanic membrane regeneration.

Astrocytes are essential regulators of synaptic transmission, brain homeostasis and behavior, and direct astrocyte activation has emerged as a promising therapeutic modality for a broad range of CNS disorders. However, selective activation of astrocytes in vivo currently requires viral transgene delivery, tethered fiber implants, or MRI-scale magnetic fields, limiting deployment across laboratories and translational settings. Here we introduce anti-GLAST-conjugated magnetite vortex nanodiscs (GLAST-MND) that, together with a low-amplitude alternating magnetic field, enable wireless, transgene-free, cell-type-specific astrocyte stimulation. The torque generated by MND is sufficient to trigger mechanosensors without the strong spatial gradient required by prior magnetomechanical glial stimulation approaches. GLAST-MND injected into the mouse dentate gyrus co-localized with the astrocyte membrane marker GLAST in vivo. Fiber-photometry in paired astrocyte (gfaABC1D-GCaMP6f) and neuron (Thy1-GCaMP6s) reporter cohorts showed that stimulation at 25 to 28 mT and 10 Hz evoked astrocyte Calcium responses 2- to 3-fold larger than non-magnetic hematite controls, while the neuron cohort showed no material-specific response. A single intracranial injection sustained stable astrocyte responses across at least five weeks and extended to seven weeks in a smaller follow-up cohort. Immunohistochemistry using the contralateral hemisphere as an internal reference confirmed that GLAST-MND stimulation did not induce local c-Fos, consistent with the absence of downstream neuronal excitation. This approach uses a scalable and affordable magnetic system compatible with freely-behaving animals, opening opportunities for astrocyte-focused neuroscience research and for the future development of astrocyte-targeted therapeutics.