Cystic fibrosis (CF) is a monogenic disease with severe pulmonary manifestations. Nanoparticle carriers of drug and gene therapeutics for CF are being studied, but only few can traverse the highly concentrated, disulfide-crosslinked airway mucus in the CF lung. Here, we developed mucolytic-associated polydisperse micro/nanoscale bubble formulations (MNB) that couple reducing chemistry (tris(2-carboxyethyl)phosphine, TCEP) with an ultrasound-responsive carrier to disrupt mucus. We evaluated the penetration of a model nanocarrier through a CF-mimetic mucus hydrogel following MNB-TCEP treatment. Under the same reducing-equivalent dosing, MNB-TCEP markedly enhanced penetration of 200 nm anionic nanoparticles relative to both PBS and free TCEP. In the more restrictive thick-mucus condition, therapeutic ultrasound increased anionic nanoparticle penetration approximately 3.5-fold in the MNB-TCEP group relative to its no-ultrasound counterpart, without statistically significant effects on PBS or free TCEP controls. MNB-TCEP also retained diagnostic ultrasound visibility in gel phantoms and produced airway-associated echogenicity in ex vivo porcine lungs within fluid-accessible, non-aerated imaging windows. Moreover, MNB-TCEP with or without ultrasound did not reduce acute viability of ex vivo tracheal biopsies under the tested regimens. Introduction of an AAV1 reporter suggested that bubble-ultrasound conditioning may increase localized reporter-positive nuclei in airway epithelium. Together, these data identify mucolytic-associated MNB formulations as a theranostic strategy to enhance nanocarrier access through CF-mimetic mucus, with exploratory tissue-level data supporting further evaluation for gene-delivery applications.
Targeted radioligand therapy (TRT) is an emerging treatment modality that selectively delivers radioisotopes to tumors while sparing healthy tissues. However, optimizing TRT radiopharmaceuticals to concurrently achieve sustained tumor retention and rapid systemic clearance remains a fundamental challenge. Gold nanoclusters with favorable pharmacokinetic properties and tunable blood half-life are advantageous in addressing this issue. Here, we develop a renally clearable GN-based TRT agent (GNFAPI) that efficiently chelates diagnostic 68Ga or therapeutic 177Lu isotopes, conferring precise targeting of fibroblast activation protein (FAP)-enriched and cancer-associated fibroblast (CAF)-dominated tumor stroma for integrated tumor theranostics. PET imaging demonstrates high tumor-specific uptake and renal clearance of 68Ga-GNFAPI in multiple models, with minimal retention in the liver and spleen. For treatment, we propose the Radio-Hook based Crosstalk Cut (RHCC) strategy, synergizing 177Lu-GNFAPI with a TGF-βRI inhibitor. This strategy utilizes β-radiation from 177Lu-GNFAPI like a powerful "hook" to disrupt the stroma and tumor cells, while the inhibitor blocks tumor-stromal crosstalk, thus abrogating the TGF-β-CAF-tumor feedback loop to suppress residual CAF reactivation, inhibit metastasis, and enhance antitumor immunity. Moreover, species-specific transcriptomics in patient-derived xenograft (PDX) models further elucidates the underlying mechanisms. Validation across various tumor models, achieving complete responses in some instances, supports its clinical translational promise.
Bone defect repair confronts a dual challenge of complex immune rejection microenvironments and osteogenic impairment, severely restricting the clinical application of xenogeneic bone graft materials. The dynamic interplay between xenogeneic antigens (α-Gal, Neu5Gc, and SDa) and the host immune system establishes an "antigen-immunity-inflammation" alliance that activates complement cascades, recruits immune cell infiltration, and drives pro-inflammatory macrophage polarization, thereby inducing chronic inflammation and fibrosis. Crucially, this pro-inflammatory microenvironment constitutes the critical determinant of osteogenic failure. Consequently, reshaping the bone repair immunomicroenvironment may fundamentally overturn this paradigm. In this study, we constructed a triple gene-edited and Urist-processed xenogeneic decalcified bone matrix system (3KODBM). Through a synergistic strategy combining genetic editing to knock out major xenogeneic antigen genes (GGTA1, CMAH, and B4GALNT2) with the Urist method to eliminate residual antigenic epitopes, we effectively attenuated immune rejection. Furthermore, we integrated genetic editing with physicochemical modification to synergistically remodel the immunomicroenvironment, promote M1-to-M2 macrophage polarization, and release osteogenic factors such as TGF-β, thereby facilitating bone repair. Our findings demonstrate that 3KODBM significantly downregulates the IL-17A/RORγt inflammatory pathway, reduces fibrosis, and markedly promotes osteogenic markers including Runx2 and Osterix. Mechanistically, by establishing an immune rejection "brake" through antigen gene knockout and an osteogenic "booster" via TGF-β/calcium signaling activation, we modulate the microenvironment to achieve substantial bone defect restoration. This study illuminates the therapeutic prospects of synergistically attenuating xenotransplantation immune rejection, offering an innovative gene-editing bone repair strategy to overcome immune rejection challenges in xenogeneic bone grafts.
Spinal cord injury (SCI) is a severe neurological disease resulting in the formation of a harsh microenvironment that hinders neural regeneration. By preserving the components and structure of natural tissues, the decellularized extracellular matrix (dECM) mimics the neural regeneration microenvironment and provides the biochemical signals and structural support for neural regeneration. However, the regenerated neurons lack the ability to autonomously restore neural communication. In this study, we assemble the conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) within spinal cord-derived decellularized extracellular matrix (pdECM) to construct a novel pdECM-based conductive hydrogel for enhancing neural communication among regenerated neurons via biochemical and bioelectrical signaling. This conductive hydrogel induces the differentiation of neural stem cells into neurons, modulates cellular membrane potential, enhances 4-fold cellular calcium activity in neural cells, upregulates expression of calcium-dependent signaling genes, and stimulates BDNF expression. In SCI mice model, the conductive hydrogel promotes endogenous neural regeneration, and sustained activates the secretion of neurotrophic factors and calcium-dependent signaling pathways for enhancing neural communication, thereby accelerating motor function recovery. This study addresses the challenge of neural communication among regenerated neurons by activating calcium-dependent signals, providing a novel strategy for the treatment of SCI through integrated biochemical and bioelectrical signaling.
Effective T-cell activation plays the critical role in cancer immunotherapy; however, strategies to precisely and sustainably stimulate antitumor T-cell responses while minimizing systemic toxicity remain limited. Here, we develop a sonogenetic artificial antigen-presenting cell (aAPC) platform for remote, spatiotemporally controlled Ca2+ influx in tumor-infiltrating T cells. The platform consists of poly (lactic-co-glycolic acid) nanoparticles loaded with the plasmid encoding the Mechanosensitive channel of large conductance (MSCL), coated with tumor-pulsed dendritic cell membranes to provide costimulatory signals, and conjugated with PD-1-targeting aptamers for T-cell-specific delivery. The nanoplatform exhibited high gene transfection efficiency within T cells and realize the expression of US (US)-responsive MSCL on the surface of T cells. Upon US irradiation, the engineered MSCL open, triggering controlled Ca2+ influx and activating downstream NFAT signalling. Therefore, sonogenetic aAPC results in the efficient and specific synergistic activation of T cells, improves the killing effects of T cells and ameliorated the dysfunction of exhausted T cells. In B16-F10 melanoma-bearing mice, sonogenetic aAPC promotes tumor infiltration and proliferation of CD8+ T cells, reduces immunosuppressive cells, and significantly suppresses tumor growth without eliciting adverse systemic immune-related events. This work demonstrates a safe and effective strategy for remotely potentiating T-cell-mediated antitumor immunity through precise sonogenetic control of calcium signalling.
Macrophages serve as central units in immune regulation, with their polarization influencing distinct stages of the bone healing process. Here, we report an immune-adaptive 3D-printed scaffold that integrates engineered macrophage capture traps, an HA network-mediated macrophage anchoring interface, onto a poly(lactic-co-glycolic acid)/hydroxyapatite microsphere composite framework. These traps selectively enrich macrophages via CD44 receptor-mediated recognition and dynamically guide their polarization. Intramuscular implantation experiments demonstrated a sequential shift in polarization of scaffold-enriched macrophages from M1 at 3 days to M2 at 7 days. Omics analysis of the short-term rabbit calvarial defect model revealed that HA released from this scaffold is phagocytosed by macrophages, gradually enters lysosomes, and activates antioxidant pathways, including negative regulator of reactive oxygen species (NRROS), sestrin 2 (SESN2), and glucose-6-phosphate dehydrogenase (G6PD), thereby reducing intracellular levels of reactive oxygen and nitrogen species. These processes collectively drive the shift in macrophage polarization from M1 to M2, and accelerate angiogenesis and bone matrix deposition. These findings provide new insights for the design of immunomodulatory bone regenerative materials.
As the most prevalent subtype of colorectal cancer, microsatellite-stable colorectal cancer (MSS CRC) is resistant to T cell-focused immune checkpoint blockade due to its low mutational burden and immunosuppressive tumor microenvironment. Despite reprogramming tumor-associated macrophages toward a tumoricidal M1-like state being a promising alternative, its efficacy is limited by paradoxical upregulation of programmed cell death-ligand 1 (PD-L1) on M1-like macrophages and their inherently poor antigen cross-presentation capacity. Here, we report that poly-metformin (PMet) can mimic the intracellular domain of PD-L1, competitively inhibit the membrane anchoring of PD-L1, and thereby effectively downregulate cell-surface PD-L1. To evaluate the therapeutic efficacy of PMet in immunosuppressive MSS CRC, we further developed an oral probiotic outer membrane vesicle (OMV) gene delivery system, siYthdf2/PMet@Akk-OMV, and demonstrated its ability to reduce PD-L1 levels on both macrophages and tumor cells while synergistically enhancing macrophage cross-presentation of tumor antigens, leading to potent activation of specific antitumor immunity and significant tumor growth inhibition. This work thus provides both theoretical and experimental foundations for treating cancers resistant to traditional immune checkpoint blockade therapy.
Cranial bone defects remain a significant clinical challenge due to limited intrinsic regenerative capacity and an adverse oxidative microenvironment that impairs osteogenesis. Demineralized bone matrix (DBM), a clinically used bone graft substitute, exhibits osteoinductive potential but suffers from inconsistent performance and poor retention at defect sites. Here, we report a DBM-loaded injectable dynamic hydrogel that enhances bone regeneration through coordinated redox modulation, reactive oxygen species (ROS)-triggered biomineralization, and Tribbles homolog 3 (Trb3)-mediated osteogenic signaling. Black phosphorus (BP) nanosheets were functionalized with nuclear localization signal (NLS) peptides through a branched NLS-PEG construct formed by conjugating NLS to multi-armed PEG, followed by electrostatic assembly onto BP nanosheets and subsequent incorporation into a self-healing hydrogel network via dynamic Schiff base crosslinking. The hydrogel effectively scavenges excessive ROS and restores redox balance, while BP degradation releases phosphate to induce ROS-triggered biomineralization and promote a pro-osteogenic microenvironment. In parallel, the BP/NLS system enables gene delivery and nuclear localization of Trb3 plasmid DNA, leading to enhanced Trb3 expression and promotion of BMP/Smad-mediated osteogenic signaling. These combined effects significantly improve the osteoinductive capacity of DBM and promote enhanced bone regeneration in cranial defects. This study provides a strategy to enhance the therapeutic performance of clinically relevant bone graft materials through integrated microenvironment regulation and gene activation.
Diabetes mellitus severely impair fracture healing due to a pathological microenvironment characterized by persistent hyperglycemia, excessive reactive oxygen species (ROS), tissue hypoxia, and chronic inflammation, which together suppress angiogenesis and osteogenesis. Most current biomaterial strategies target only a single pathological factor and therefore fail to disrupt the interconnected metabolic cascade that perpetuates tissue dysfunction. Here, we developed a pathology-driven catalytic nanozyme-integrated adhesive hydrogel (Gel-FQG) to simultaneously regulate hyperglycemia, oxidative stress, hypoxia, and immune imbalance in diabetic fractures. Gel-FQG incorporates glucose oxidase (GOx)-loaded Fe3+-quercetin nanozymes into a dynamic Schiff-base hydrogel network, providing strong tissue adhesion, injectability, and sustained catalytic activity. Through sequential glucose oxidation and H2O2 decomposition, Gel-FQG consumes excess glucose, scavenges ROS, and generates oxygen, thereby disrupting the hyperglycemia-ROS-hypoxia feedback loop. In vitro, Gel-FQG alleviates oxidative stress and hypoxia, promotes macrophage polarization toward the pro-regenerative M2 phenotype via suppression of the TNF-α/NF-κB/HIF-1α signaling, and enhances osteogenic and angiogenic responses under hyperglycemic conditions. In a diabetic rat femoral fracture model, local implantation of Gel-FQG significantly accelerates vascularized bone regeneration and fracture healing. These findings demonstrate that Gel-FQG actively remodels the diabetic fracture microenvironment and offers a promising strategy for diabetic bone repair.
Cardiac extracellular vesicles (EVs) hold promise as cell-free therapeutics for heart repair. However, robust methods to produce scalable, functional and cardiac-specific EVs at high yield remain a limiting factor in their exploitation. Here, we report an engineered platform that combines 3D cardiac microtissues with hydrodynamic stimulation to address these hurdles. We exploited differential cell mechanics, quantified via surface tension measurements, to establish a unique 3D spheroid architecture showing cardiomyocytes preferentially localizing at the spheroid periphery. A controlled-flow bioreactor then enabled high-yield EV production, at a 10-fold increase compared to non-stimulated spheroids, all while maintaining cell viability comparable to standard 2D production conditions while preserving vesicle structural integrity. Proteomic profiling revealed that EVs generated under these conditions carry a cardiac-specific signature, enriched in sarcomeric, mitochondrial, ribosomal and heat shock proteins, all while retaining core EV markers. Functionally, these EVs enhanced wound closure and reduced fibroblast activation more effectively than EVs derived from standard 2D fibroblast cultures, at 1.3 and 1.5-fold, respectively. Our findings establish a scalable, physiologically relevant strategy for generating cardiac EVs and demonstrate that combining 3D microenvironment engineering with hydrodynamic cues can yield therapeutically potent vesicles suitable for regenerative medicine.
Severe burn wounds with excessive exudation and high infection risk disrupt all four healing phases, requiring antibacterial, exudate-managing, and phase-specific strategies to drive wound contraction and tissue regeneration. Herein, we develop a spatially programmed hydrogel dressing (SPHD) via confined layer-by-layer photopolymerization, with Ga3+ and Fe3+ ions layer-specifically immobilized to enable chelation- and spatially guided programmable release with phase-matched kinetics. Abundant hydrophilic groups and dynamic catechol-boronate ester network render the hydrogel high swelling capacity, resilience, self-healing, and strong tissue adhesion. Its pH/temperature dual responsiveness enables adaptation to wound microenvironmental changes through reversible metal coordination and polymer conformational transitions. Hierarchically designed layers yield release kinetics aligned with healing phases, the inner layer rapidly releases Ga3+ within 16 h to suppress infection during the inflammatory phase, whereas the outer layer sustains Fe3+ release over 96 h, activating prolyl hydroxylase and promoting collagen synthesis and angiogenesis in the proliferative phase. Molecular dynamics simulations elucidate ion-specific diffusion barriers and mechanisms underlying phase-matched release. In murine deep second-degree burn model, SPHD accelerates wound closure to 96% in 12 days, enhancing epidermal barrier recovery and neovascularization. This work defines a paradigm for programmable wound management, offering a versatile platform extendable to regenerative medicine.
Ferroptosis holds great promise for cancer immunotherapy, yet elevated cholesterol levels in tumor cells impose substantial structural and functional barriers to ferroptosis. Here, a cascade-catalytic nanocomposite microneedle platform (CSMZC MNs) is developed to convert this tumor-intrinsic cholesterol shield into an oxidative spear for ferroptosis-amplified cancer immunotherapy. By integrating superoxide dismutase (SOD) and cholesterol oxidase (COD) within Mn-doped zeolitic imidazolate framework nanoparticles and embedding them into dissolvable poly(γ-glutamic acid) microneedles, the platform enables localized intratumoral delivery and coordinated catalytic activation. After tumor cell internalization, the SOD-COD-Mn2+ cascade rewires redox metabolism and membrane lipid homeostasis by depleting cholesterol and 7-dehydrocholesterol, thereby dismantling tumor resistance to ferroptosis while amplifying reactive oxygen species generation and lipid peroxidation. This self-reinforcing oxidative amplification induces ferroptosis-associated immunogenic cell death and promotes cytosolic accumulation of nuclear and mitochondrial DNA, resulting in endogenous cGAS-STING activation. Meanwhile, Mn2+ enhances cGAS sensitivity to cytosolic DNA and amplifies type I interferon-mediated innate immune signaling. Through catalytic amplification and immune remodeling, CSMZC MNs reshape the immunosuppressive tumor microenvironment and elicit systemic T cell-mediated antitumor immunity against both primary and distant tumors. This work establishes an agonist-free immunometabolic strategy for converting tumor-protective cholesterol metabolism into a therapeutic vulnerability for tumor immunotherapy.
Antibiotic-resistant wound infections require dressings that rapidly remove protein-rich exudate, resist fouling and bacterial adhesion, and provide drug-sparing antimicrobial activity. Here we develop asymmetric-interfacial Janus nanofibrous membranes that integrate diode-like exudate transport with infection-microenvironment-responsive nanozyme catalysis for antibacterial wound healing. The membrane couples a multiscale-rough superhydrophobic outer layer with a hydrophilic inner layer, establishing a through-thickness wettability gradient and capillary-pressure asymmetry that preferentially pumps liquids from the hydrophilic side to the superhydrophobic side while suppressing reverse wetting. An asymmetric Janus nanofibrous membrane (Janus-ZnCu-BIF NFM), composed of a hydrophilic nanofiber layer selectively loaded with copper-doped zinc boron imidazolate framework (ZnCu-BIF) nanocubes and an integrated hydrophobic polylactic acid layer, exhibits catalytic antibacterial activity by promoting reactive oxygen species generation and disrupting bacterial membrane integrity. The resulting membrane nearly eliminated viable multidrug-resistant bacteria in vitro. In a murine full-thickness wound model infected with methicillin-resistant Staphylococcus aureus, the dressing significantly reduced bacterial burden and accelerated healing, reaching 99.8% wound closure by day 12. Histological analyses indicated attenuated inflammation, enhanced angiogenesis, and improved dermal regeneration, while transcriptomic profiling revealed enrichment of pathways associated with extracellular-matrix remodeling and vascular development. Overall, this work establishes a scalable, biomimetic dressing that couples rectified exudate management with robust catalytic antibacterial efficacy, providing a practical route toward next-generation wound care materials for drug-resistant infections.
In this work, we explore the potency of poly(D-peptide) (D-PP) in combination with diverse classes of antibiotics to address multidrug-resistant (MDR) Gram-negative superbugs, including their mature biofilms, polymicrobial assemblies, and dormant subpopulations, which are among complex challenging targets. The polypeptide adjuvant displayed significant antibiotic potentiation, resulting in a moderate to rapid bactericidal activity against the Gram-negative pathogens in their planktonic stage. The adjuvant and its combination restored susceptibility in dormant bacterial subpopulations. In polymicrobial settings, these combinations were able to reduce both Gram-negative and Gram-positive bacterial species, unlike individual antibiotic exposure. Importantly, one of the lead combinations (D-PP-rifampicin) resulted in moderate biofilm eradication of P. aeruginosa. Transcriptomic analysis demonstrated the differentially expressed genes (DEGs) involved in membrane integrity, stress response, and transport systems. Together with our previous mechanistic findings, this further confirmed that bacterial membrane perturbation is caused by the adjuvant. Such perturbation facilitated antibiotic accumulation within microbial cells. Furthermore, polypeptide reduces the bacterial resistance development towards doxycycline and rifampicin, unlike polymyxin B. Taken together, these findings exhibit that membrane-active polypeptide adjuvants can enhance antibiotic efficacy across diverse bacterial states and environments, providing a promising strategy to combat persistent and multidrug-resistant infections.
Obesity represents a complex systemic disease resulting in the global metabolic syndrome epidemic, yet our understanding of the underlying mechanisms in adipose tissue driving obesity remains limited. As the primary cellular targets of increased caloric intake, adipocytes undergo dramatic morphological and functional changes that trigger systemic metabolic dysfunction. To study the dysfunctional obese adipocyte phenotype in vitro, revolutionary three-dimensional (3D) adipocyte models have shown to be uniquely capable of recapitulating the lipid-laden, hypertrophic adipocyte characteristic of obesity. This feature makes 3D in vitro platforms powerful alternatives to traditional two-dimensional monolayer systems where the large, spherical and buoyant adipocytes simply detach. These sophisticated 3D in vitro New Approach Methodologies (NAMs) now enable researchers to dissect the multifactorial nature of obesity through diverse functional assays that validate individual disease hallmarks and assess therapeutic interventions. However, the field faces a critical challenge: integrating multiple disease-relevant readouts into holistic interpretations that can unlock novel adipocyte-targeting mechanisms. This review provides a comprehensive roadmap of cutting-edge 3D adipose tissue systems, showcasing how these NAMs can revolutionize biological and drug target discovery in obesity research. We examine the functional assays that define these models' capabilities, address key interpretative challenges, and explore transformative opportunities to amplify their impact through high-throughput approaches and scalable functional platforms that will accelerate the next generation of obesity therapies.
Scaffold-based retinal pigment epithelium (RPE) transplantation is a candidate approach to preserve or recover vision for macular degeneration. Electrospun poly(ε-caprolactone) (PCL)/Gelatin (Gel) scaffolds have demonstrated support for cellular proliferation and function, yet their inadequate mechanical performance compromises surgical handling and deliverability. In this study, we incorporated low-oxidation graphene oxide (GO) into electrospun PCL/Gel scaffolds and evaluated their performance for human embryonic stem cell (hESC)-derived RPE cell transplantation. We found that the incorporation of low-oxidation GO improved scaffolds’ WCA from 45.47 ± 8.44° to 27.48 ± 1.42° and tensile strength from 1.60 ± 0.06 MPa with an ultimate strain of 16% to 1.75 ± 0.07 MPa with an elongation at break of 44%, while maintaining fibrous formability and biocompatibility. The PCL/Gel/GO scaffolds supported RPE-specific protein expression (MITF, PAX6, CRALBP, ZO-1) and established favorable cell polarity, as indicated by apical Na+/K+-ATPase. Transmission electron microscopy assay also showed that the hESC-RPE cells on PCL/Gel/GO scaffolds formed apical microvilli. RNA-seq revealed the preservation of trophic factors and phagocytic function on PCL/Gel/GO scaffolds, which was further validated by augmented expression levels of CNTF, IGF1, and VEGFA, as well as improved fluorescent microsphere uptake capacity. Furthermore, the enhanced mechanical properties of the PCL/Gel/GO scaffold eliminated the need for additional treatment before implantation and facilitated surgical handling during implantation. In addition, the scaffold did not cause any observable short-term retinal dysfunction, indicating good short-term feasibility. Collectively, the scaffold structural features, RPE functional profiles, and surgical findings establish PCL/Gel/GO scaffolds as a versatile platform, warranting further investigation into their application in scaffold-based cell therapy for the treatment of macular degeneration.
Avascular meniscus tears exhibit minimal intrinsic healing and frequently progress to joint degeneration because of limited vascularity, poor biological repair capacity, and inadequate restoration of tissue-level mechanical continuity. Here, we report a hydrophilic polydopamine (hPDA) fueled bioglue platform that overcomes the aqueous insolubility limitations of conventional polydopamine (PDA) and enables functional repair of avascular meniscus injuries. Water soluble hPDA was synthesized through controlled depolymerization and recrystallization, yielding monomeric and oligomeric species rich in catechol, amine, and hydroxyl functionalities. Incorporation of hPDA into fibrin bioglues significantly improved both bulk mechanical and interfacial adhesive properties, producing 515-525% increases in lap-shear modulus, 165-190% increases in adhesive strength, up to 175% increases in compressive modulus, and approximately 176% increases in compressive strength relative to fibrin controls, while also improving degradation stability over 14 days. In contrast to genipin-mediated covalent crosslinking, hPDA primarily enhanced mechanical performance through catechol-mediated intermolecular interactions, improved energy dissipation, and reinforced interfacial load transfer. hPDA exhibited excellent cytocompatibility in both 2D and 3D cultures. In a bovine avascular meniscus explant model, hPDA fueled bioglues promoted tissue integration and aligned collagen remodeling, restoring interfacial mechanics with a 482-582% increase in tensile (pull-out) modulus and up to 152-285% higher pull-out strength after 6 weeks. These findings establish hPDA as a versatile bioadhesive building block with strong potential for repairing avascular meniscus tears and other mechanically demanding connective tissues.
Matrix stiffness is a critical biophysical cue that governs mesenchymal stromal cell (MSC) fate, yet the molecular mechanisms by which soft extracellular environments impair osteogenesis remain poorly defined. In this study, we utilized a stiffness-tunable in vitro model to dissect focal adhesion (FA)-mediated mechanotransduction and identified the small heat shock protein Hsp27 as a mechanosensitive regulator that selectively localizes to the FAs of MSCs on soft matrices. Proteomic profiling revealed that soft substrates reprogram FA composition to recruit chaperones and ubiquitin-proteasome system (UPS) components, with Hsp27 acting as a central effector. Mechanistically, Hsp27 facilitates the localized, proteasome-mediated degradation of the small GTPase Rac1, thereby attenuating lamellipodia formation, cytoskeletal tension, and pro-osteogenic signaling. Genetic deletion or pharmacological inhibition of Hsp27 successfully restored FA maturation, actin stress fiber architecture, and intracellular traction forces, effectively rescuing osteogenic differentiation on compliant substrates. In a murine model of age-related osteoporosis, we observed that femoral bone tissues exhibit hallmarks of matrix softening, including collagen fiber misalignment and increased marrow adiposity, accompanied by Hsp27-mediated Rac1 depletion. These findings establish Hsp27 as a mechanically responsive regulator of FA proteostasis and identify a stiffness-sensitive degradation pathway as a potential therapeutic target to restore bone formation in mechanically compromised environments.
Providing transport routes for oxygen and nutrient supply in macroscopic tissue remains a major challenge in tissue engineering. Among fabrication methods for perfusable microtubular structures, two-photon stereolithography (TPS) stands out due to its superior resolution, achieving features at the capillary scale. This study uses TPS to fabricate hollow tubes and investigates their feasibility as microtubular supply structures in in vitro tissue models. With optimized parameters, porous hollow tubes of different sizes (8 μm to 2 mm in diameter) with pore diameters of 1 to 50 μm were printed with the two photoresins IP-S and IP-Visio. IP-S demonstrated superior printability and higher shape accuracy compared to IP-Visio. IP-Visio prints were compatible with fluorescence microscopy, whereas IP-S exhibited high autofluorescence, which interfered with fluorescence imaging. The prints displayed partial wettability with similar contact angles between 71° and 76° on both flat and porous curved surfaces, with none of the liquids passing through the pores. Printed hollow tubes with pores were integrated into a perfusion system, and the permeability of molecules of 4, 70 and 150 kDa into the surrounding hydrogel matrix was assessed under physiological flow conditions. Structures printed with both photoresins facilitated the attachment and survival of primary human endothelial cells on top of flat prints (viability ≥97 %) as well as inside printed tubes. Finally, flat prints were tested in cytokine release experiments and provoked neither pro- nor anti-inflammatory responses of human macrophages and monocytes.
Overcoming short intra-articular retention and poor chondrocyte uptake of drugs remains a major challenge for osteoarthritis (OA) therapy. Thereby, we developed a tetrahedral DNA (TDN)-based nano-delivery system (TLM@CAP) via a two-step engineering strategy, involving the introduction of tunable numbers of specific DNA loops to significantly enhance metformin loading capacity, and conjugation with a chondrocyte-affinity peptide (CAP) to confer chondrocyte-targeting capability. The TLM@CAP complex demonstrated improved chondrocyte uptake, prolonged joint retention exceeding 72 h, and deep cartilage penetration. In vivo, TLM@CAP outperformed free metformin in restoring mitochondrial function, reducing cellular senescence, and rebalancing extracellular matrix metabolism via AMPK/mTOR pathway activation in senescent chondrocytes. In an OA rat model, intra-articular TLM@CAP administration effectively alleviated pain, improved gait, and protected against cartilage and subchondral bone destruction. This work not only presents a potent targeted therapeutic strategy for OA but also advances the rational design of TDN-based nanoplatforms for drug delivery.
