Excessive macrophage pyroptosis is a central driver of inflammatory microenvironment deterioration and alveolar bone loss in diabetic periodontitis (DP), posing significant challenges for precise immune modulation and tissue preservation. Given that cellular bioelectric cues have emerged as critical regulators of immune and inflammatory responses, mimicking endogenous bioelectric signals represents a promising strategy to restore immune homeostasis. Here, we report a polarized, ball-milled K0.5Na0.5NbO3 (PMKNN) ferroelectric nanoplatform capable of establishing a sustained nanoscale intracellular electric field (NIEF) upon endocytosis by macrophages. Internalized PMKNN nanoparticles (NPs) predominantly localize within lysosomes, where their NIEF modulates TRPML1-dependent lysosomal Ca2+ signaling, leading to TFEB nuclear translocation, thereby promoting lysosomal biogenesis and mitophagy. This coordinated lysosome-mitochondria crosstalk ameliorates mitochondrial dysfunction, mitigates reactive oxygen species (ROS) accumulation, suppresses NLRP3 inflammasome activation, and attenuates macrophage pyroptosis. In a DP rat model, local administration of PMKNN NPs reduces inflammatory infiltration and alveolar bone loss. This study unveils a nanoscale electric-field-mediated immunomodulatory paradigm, providing novel insights for the development of wireless, noninvasive electroceutical approaches in chronic inflammatory diseases. (c) 2026 Published by Elsevier Ltd on behalf of The editorial office of Journal of Materials Science & Technology.

Study DesignRetrospective cohort study.ObjectiveTo evaluate the therapeutic effects of postoperative repetitive transcranial magnetic stimulation (rTMS) on neuropathic pain (NP) and neurological recovery in patients with degenerative cervical myelopathy (DCM).MethodsFifty-nine DCM patients who underwent cervical decompression between 2017 and 2024. Twenty-seven received postoperative rTMS (20 Hz, 20 trains of 40 pulses at 90% resting motor threshold over the biceps brachii for 5 consecutive days) in addition to routine care, while 32 received routine care only. Pain intensity at the neck, upper limbs, and below-neck regions was assessed using the 10-cm Visual Analog Scale (VAS). NP was identified by a Douleur Neuropathique 4 (DN-4) score ≥4. Hand dexterity, myelopathy severity, and conduction function were evaluated by the 10-second grip-and-release test, modified Japanese Orthopedic Association (mJOA) scale, and somatosensory/motor evoked potentials (SSEP/MEP) respectively.ResultsAmong patients with NP (n = 25), rTMS significantly reduced VAS pain scores in the upper limbs and below-neck regions (P < 0.05), but not in the neck. No effect was observed in non-NP patients (n = 34). rTMS also improved hand dexterity, mJOA scores, and recovery rates compared with controls, particularly in patients with preoperative mJOA ≤14 (P < 0.05). These benefits persisted for 6 months. However, SSEP and MEP results were comparable between groups, suggesting that rTMS did not alter cervical conduction.ConclusionFive-day postoperative rTMS was associated with NP and functional recovery in DCM, especially in severe cases, possibly by modulating higher central pathways.

Introduction:Abnormal mechanical loading is a significant pathogenic factor in intervertebral disc degeneration (IVDD), yet the underlying mechanotransduction mechanisms remain incompletely elucidated. This study aimed to investigate the role of integrin α5β1 as a key mechanosensor in regulating the autophagy-apoptosis balance under mechanically induced IVDD. Methods:Bovine intervertebral discs (IVDs) with intact endplates were cultured in a bioreactor and subjected to dynamic mechanical loading, including physiological loading (PL: 0.02-0.2 MPa, 0.2 Hz) and degenerative loading (DL: 0.32-0.5 MPa, 5 Hz) for 3 and 7 days. Interventions involved the autophagy inhibitor 3-Methyladenine (3-MA), integrin α5β1-specific inhibitory peptide RGD (Arg-Gly-Asp), and the autophagy activator rapamycin. A systematic evaluation was performed, assessing disc height, histomorphology, cell viability, gene/protein expression, autophagy levels, and apoptosis. Results:Degenerative loading induced progressive IVD degeneration, characterized by irreversible disc height loss, structural disruption, decreased cell viability, and extracellular matrix (ECM) metabolic imbalance. Treatment with 3-MA exacerbated these degenerative changes, confirming the protective role of autophagy. Integrin α5β1 exhibited distinct spatial distribution patterns: its expression was significantly upregulated in the nucleus pulposus (NP) and inner annulus fibrosus (IAF) under degenerative loading, whereas only the β1 subunit was increased in the outer annulus fibrosus (OAF). Functional experiments demonstrated that competitive inhibition of integrin α5β1 by RGD peptide significantly suppressed autophagy activity, exacerbated apoptosis, and promoted ECM degradation. Conversely, rapamycin alleviated degeneration by restoring autophagic flux. Mechanistically, degenerative loading suppressed the FAK/PI3K/AKT/mTOR pathway while upregulating ULK1, and these effects were partially reversed by RGD inhibition. Discussion:The autophagy-apoptosis balance plays a critical regulatory role in IVDD progression, with integrin α5β1 serving as a crucial upstream mechanosensor that may exert its protective function through modulating the FAK/PI3K/AKT/mTOR pathway. The region-specific distribution of integrin subtypes determines the specificity of mechanotransduction across different disc areas. Targeting the integrin-autophagy axis and its associated signaling pathways may represent a potential therapeutic strategy for mitigating mechanically induced IVDD.

Vessel-microenvironment interactions are pivotal in tissue development and disease. Existing experimental platforms fail to recapitulate the seamless extracellular matrix (ECM) continuity required for physiological vessel-stromal communications, often creating disruptive material boundaries. In this study, we developed a consubstantial bioink-supporting bath system using collagen modified with nanoscale PEGylated polyhedral oligomeric silsesquioxane, enabling interface-free vessel-microenvironment modeling. This suspension matrix approach maintains uninterrupted, biomimetic ECM to preserve spatial complexity and multicellular crosstalk. We validated this platform through complementary models: a regenerative vessel-stromal model demonstrating the dominance of contact-dependent signaling over paracrine effects and a pathological vessel-tumor model. Single-cell RNA sequencing and cell-cell communication analysis revealed cancer-associated fibroblast-mediated networks driving pathological angiogenesis. This study advances vessel-microenvironment biology and provides a platform to investigate vascular mechanisms and therapies.

Currently, targeting the Wnt/β-catenin pathway to promote bone formation is a primary strategy for the development of osteoporosis drugs. Here, we demonstrate that vinculin promotes bone mass increase and fracture repair by elevating the β-catenin protein levels in mesenchymal stem cells (MSCs). Furthermore, it is revealed that vinculin is required for sclerostin-neutralizing antibody (Scl-Ab) to increase the bone mass in mice. We find that promoter accessibility and the expression of the Vcl gene, which encodes vinculin, are reduced in the MSCs from elderly human individuals, and vinculin knockdown impairs osteoblast differentiation in vitro. Genetic deletion of Vcl in Prx1-expressing cells in mice leads to pronounced bone loss in weight-bearing long bones, but not in the non-weight-bearing skull, primarily attributed to severely impaired bone formation, characterized by reduced osteoblastic and increased adipogenic differentiation. Unexpectedly, vinculin loss decreases the β-catenin protein levels by approximately 80

The anti-osteoporotic effects of parathyroid hormone (PTH) analogs for patients with severe osteoporosis decrease with age; however, the underlying mechanism(s) are poorly understood. Here we provide evidence that enhanced TNF-mediated chronic inflammation during aging may play a critical role in promoting bone loss and attenuating the anabolic effects of PTH on bone in aged individuals. Specifically, we find that abolishment of TNF signaling activation by global deletion of the Tnfr1/2 genes, which encode the TNF receptors, prevents aging-related bone mass loss and improves the anabolic effects of PTH (1-34) analog to increase bone mass and bone mineral density in aged, but not young adult, mice. These effects are achieved mainly by enhancing PTH signaling in osteoblastic cells, leading to accelerated bone formation. TNF-α inhibition of PTH signaling is mediated via TRAF2, an E3 ligase. TRAF2 promotes the non-canonical K27- and K33-linked ubiquitination of Gαs, which increases Gαs protein stability but blocks Gαs activation and downstream signaling. Of clinical significance, infliximab, a TNF-α neutralizing antibody, reduces the bone loss in aged mice and improves the skeletal response to PTH. Collectively, we demonstrate a novel mechanism whereby TNF/TNFR/TRAF2 promotes aging-related bone loss and impairs the anabolic effects of PTH on bone in aged animals by inhibiting Gαs activation, and our findings suggest that modulating the TNF/TNFR signaling pathway may improve the effectiveness of PTH treatment in senile osteoporosis patients.

[This corrects the article DOI: 10.3389/fbioe.2026.1741808.].

Immunomodulatory bioactivity is crucial for biomaterials to effectively promote bone regeneration, yet this remains a significant clinical challenge.Here, we develop a bioactive 3D-printed scaffold by encapsulating transfected bone marrow-derived mesenchymal stem cells (BMSCs) within a hybrid hydrogel, followed by UV crosslinking. While the hybrid hydrogel ColMA/CSMA/HA is composed of methacrylated type-I collagen, methacrylated chitosan, and nano-hydroxyapatite, encapsulating insulin-like growth factor-1 (IGF-1)-transfected BMSCs leads to the 3D scaffold ColMA/CSMA/HA/BMSCs@IGF-1. The in vitro evaluation demonstrates excellent biocompatibility and the capability of the scaffold to promote M2 macrophage polarization within an immunomodulatory microenvironment and thereby facilitate the osteogenic differentiation of BMSCs. Transcriptomic analyses reveal that the scaffold ColMA/CSMA/HA/BMSCs@IGF-1 regulates osteogenic differentiation and promote bone regeneration by modulating immune responses and suppressing inflammatory cytokines. Through the synergistic effects of immunomodulation, inflammation suppression, and osteogenic differentiation, the 3D-printed scaffold efficiently promotes the repair of bone defects. Overall, the 3D-printed hybrid scaffold exhibits synergistic effects under the interplay of the bone immune microenvironment to enhance bone regeneration. This study provides a new strategy for the development of bone biomaterials capable of mediating immunomodulation for bone tissue engineering and regeneration.

Background Osteoporosis (OP) combined with Intervertebral disc degeneration (IDD) is a chronic degenerative disease. There are no drugs that simultaneously treat both IDD and OP. Panax notoginseng saponins (PNS) can treat OP or ameliorate IDD, respectively, indicating its potential for simultaneous treatment of both conditions. The specific biological mechanism by which PNS treats OP combined with IDD and the key bioactive compounds involved require further investigation. Methods The effects of oral administration of PNS on OP and IDD were evaluated in mice using micro-CT scanning and immunofluorescence. In vitro experiments involved the preparation of PNS serum for cell culture, performing tube formation assays on HUVECs, RNA sequencing followed by KEGG, GO, and DEGs analysis, as well as Western blotting. Furthermore, HPLC-MS analysis and molecular docking were conducted to identify key bioactive biomolecules in PNS serum. Results The animal model confirmed that the OP condition can exacerbate IDD and that PNS treatment simultaneously treats OP combined with IDD. Additionally, PNS promotes the coupling of osteogenesis and angiogenesis in the lumbar vertebrae. Moreover, PNS serum enhances the tube formation ability of HUVECs and promotes the expression of angiogenesis-related genes in bEnd.3 cells, increases the expression of Slit3 in BMSCs and lumbar vertebrae, promoting the phosphorylation of the ERK signalling pathway. Finally, through HPLC-MS analysis and molecular docking predictions, the key bioactive molecule NG-R4 was identified in PNS serum. Conclusion PNS enhances the ERK phosphorylation in BMSCs through its metabolic component NG-R4, stimulating Slit3 secretion to promote the coupling of osteogenesis and angiogenesis.

Periodontitis, a prevalent microbe-driven disease, is characterized by the progressive destruction of tooth-supporting tissues. Porphyromonas gingivalis ( P. gingivalis), a keystone pathogen in chronic periodontitis, evades clearance by periodontal immune cells, leading to microbial dysbiosis and alveolar bone resorption. Effective treatment requires the elimination of periodontopathogens and restoration of alveolar bone. Macrophages serve as the primary defense against periodontal pathogens. Cell stiffness, an intrinsic mechanical property of macrophage, is closely linked to their motility, deformability, and phagocytic function, presenting a potential target for enhancing macrophage antibacterial activities. Black phosphorus nanosheets (BPNS), a novel two-dimensional material that degrades into nontoxic phosphate in physiological environments, offer distinct advantages in bone regeneration. In this study, BPNS was functionalized with spermidine (Spd), a natural polyamine with immunomodulatory effects, to improve periodontal infection control. In addition to preserving BPNS's osteogenic activity, BPNS@Spd further enhanced macrophage antibacterial function by targeting cell stiffness. Reduced cell stiffness enhanced macrophage deformability and membrane fluidity, facilitating more efficient phagocytosis and clearance of P. gingivalis. Mechanistically, BPNS@Spd modulated macrophage stiffness and function by decreasing membrane cholesterol content, in which autophagy-mediated cholesterol efflux plays a critical role. This work underscores the potential of modulating membrane cholesterol and cell stiffness to boost macrophage-mediated bacterial clearance, offering an innovative immunomodulatory approach for managing microbe-driven inflammatory diseases beyond periodontitis. (c) 2025 Published by Elsevier Ltd on behalf of The editorial office of Journal of Materials Science & Technology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

The testicular spermatogenic epithelium, the fundamental functional unit of spermatogenesis, comprises Sertoli cells and a sequence of spermatogenic cells, with the Leydig cells (LCs) playing a pivotal supporting role in sperm development. In this study, we developed a microfluidic testicular organ-on-a-chip (OoC) composed of spermatogonial stem cells, Sertoli cells, and LCs. After 28 d of culture, the testicular OoC demonstrated the formation of a spermatogenic epithelial structure, with observed proliferation and differentiation of spermatogonial stem cells. Both Sertoli and LCs were noted to perform their fundamental cellular functions and engage in intercellular communication. Applying reproductive toxicity factors to testicular OoC reduced the proliferation of spermatogonia stem cell in the chip. This testicular OoC model revealed its potential for exploring physiological functions of the testicular spermatogenic epithelium and serving as a platform for pharmacological and toxicological screening.

Craniofacial bone defect healing in periodontitis patients with diabetes background has long been difficult due to increased blood glucose levels which cause overproduction of reactive oxygen species (ROS) and a low pH environment. These conditions negatively affect the function of macrophages, worsen inflammation and oxidative stress, and ultimately, hinder osteoblasts' bone repair potential. In this study, we for the first time found that annexin A1 (ANXA1) expression in macrophages was reduced in a diabetic periodontitis (DP) environment, with the activation of the NLRP3/Caspase-1/GSDMD signaling pathway, and, eventually, increased macrophage pyroptosis. Next, we have developed a new GPPG intelligent hydrogel system which was ROS and pH responsive, and loaded with Ac2-26, an ANXA1 bioactive peptide, and osteogenic peptide OGP as well. We found that Ac2-26/OGP/GPPG can effectively reduce ROS, mitigates macrophage pyroptosis via the ANXA1/NLRP3/Caspase-1/GSDMD pathway and enhanced osteogenic differentiation. The effect of Ac2-26/OGP/GPPG in regulation of pyroptosis and bone defect repair was also further validated by animal experiments on periodontitis-induced tooth loss model in diabetic rats. To conclude, our study unveils the effect of ANXA1 on macrophage pyroptosis in periodontitis patients with diabetes, based on which we introduced a promising innovative hydrogel system for improvement of bone defects repair in DP patients via targeting macrophage pyroptosis and enhancing osteogenic potential.

The regeneration of critical-sized osteochondral defects remains a significant challenge due to the limited self-healing capacity of cartilage. Traditional approaches, such as autologous chondrocyte implantation (ACI) and matrix-induced autologous chondrocyte implantation (MACI), have shown promise but are limited by issues like insufficient cell availability, dedifferentiation of chondrocytes during expansion, and the formation of fibrocartilage rather than functional hyaline cartilage. This study presents a promising approach utilizing transcript-activated matrices (TAMs) with mRNA to enhance the therapeutic potential of bone marrow mesenchymal stem cells (BMSCs) in situ. Chemically modified mRNA (cmRNA) encoding transforming growth factor β3 (TGF-β3) was encapsulated in a collagen hydrogel to provide localized, sustained delivery of chondrogenic signals. In a rat model of critical-sized osteochondral defects, this strategy significantly promoted cartilage regeneration, achieving structural and molecular restoration within six weeks. Histological and biochemical analyses revealed robust chondrogenesis, enhanced extracellular matrix deposition, and superior mechanical properties. Moreover, TAM therapy maintained subchondral bone integrity This work highlights the transformative potential of mRNA-activated matrices as a platform technology that not only addresses key limitations of existing cartilage repair strategies but also provides a biomimetic microenvironment that guides stem cell differentiation and tissue regeneration.

The focal adhesion (FA) is the structural basis of the cell-extracellular matrix crosstalk and plays important roles in control of organ formation and function. Here we show that expression of FA protein vinculin is dramatically reduced in osteocytes in patients with aging-related osteoporosis. Vinculin loss severely impaired osteocyte adhesion and dendrite formation. Deleting vinculin using the mouse 10-kb Dmp1-Cre transgenic mice causes dramatic bone loss in the weight-bearing long bones and spine, but not in the skull, in both young and aged mice by impairing osteoblast formation and function without markedly affecting bone resorption. Vinculin loss impairs the anabolic response of skeleton to mechanical loading in mice. Vinculin knockdown increases, while vinculin overexpression decreases, sclerostin expression in osteocytes without impacting expression of Mef2c, a major transcriptional regulator of the Sost gene, which encodes sclerostin. Vinculin interacts with Mef2c and retains the latter in the cytoplasm. Thus, vinculin loss enhances Mef2c nuclear translocation and binding to the Sost enhancer ECR5 to promote sclerostin expression in osteocytes and reduces bone formation. Consistent with this notion, deleting Sost expression in osteocytes reverses the osteopenic phenotypes caused by vinculin loss in mice. Finally, we find that estrogen is a novel regulator of vinculin expression in osteocytes and that vinculin-deficient mice are resistant to ovariectomy-induced bone loss. Thus, we demonstrate a novel mechanism through which vinculin inhibits the Mef2c-driven sclerostin expression in osteocytes to promote bone formation.

We previously found that oral administration of Panax notoginseng saponins (PNS) alleviated bone loss in ovariectomy(OVX)-induced osteoporotic mice. However, the specific active component responsible for this effect and its underlying mechanism remained unclear. Ginsenoside compound K (CK), one of the main active components of PNS, may serve as a promising therapeutic agent for osteoporosis. This study demonstrated that CK inhibited osteoclastogenesis and promoted type H vessel formation to alleviate bone loss in OVX mice. In vitro, CK concentration-dependently inhibited RANKL-induced osteoclastogenesis. In addition, high concentration CK inhibited the migration and tubule formation of HUVECs. However, treating HUVECs with CK + RANKL-stimulated RAW264.7 conditional medium showed enhancement of migration and tubule formation ability, which was blocked by adding PDGF-BB neutralising-antibody. Proteomics and network pharmacological analysis revealed CK may directly target CSF1R and inhibit osteoclast differentiation via PI3K/AKT/NFκB pathway, which subsequently conformed by drug affinity responsive target stability, cellular thermal shift, surface plasmon resonance and western blot assays. Furthermore, adding macrophage colony-stimulating factor (M-CSF) mitigated the inhibitory effect of CK on osteoclast differentiation and PI3K/AKT/NFκB pathway activation. Taken together, we demonstrated that CK exerts osteoprotective effects by targeting CSF1R to inhibit PI3K/AKT/NFκB pathway, thereby suppressing osteoclastogenesis and promoting preosteoclast PDGF-BB-induced angiogenesis.

Prostate cancer bone metastases often harbor a rare subset of tumor cells with suppressed proliferation, contributing to therapy resistance and disease relapse. However, the lack of physiologically relevant in vitro models has hindered mechanistic and therapeutic advances. Here, we engineered a 3D bone-like microenvironment by integrating calcium phosphate scaffolds, decellularized extracellular matrix (dECM), mesenchymal stem cells (MSCs), and osteoblasts. This biomimetic niche induced a proliferation-inhibited state in prostate cancer cells, closely mirroring transcriptomic signatures identified from patient-derived single-cell RNA sequencing datasets. Tumor cells in this niche also displayed enzalutamide resistance, accompanied by metabolic reprogramming and activation of pro-survival signaling. This platform provides a clinically relevant tool for modeling bone metastatic prostate cancer and accelerating the development of therapies targeting resistant tumor states.

INTRODUCTION:Notoginsenoside R1 (NGR1), a bioactive compound, exhibits significant pro-angiogenic potential, making it a promising candidate for treating various diseases. Since angiogenesis and osteogenesis are synergistically coupled processes, NGR1's capacity to stimulate blood vessel formation may critically promote bone regeneration. However, the underlying molecular mechanisms through which NGR1 promotes angiogenesis in bone repair remain to be fully elucidated. OBJECTIVES:To investigate the potential mechanism by which NGR1 promotes angiogenesis and to validate the therapeutic effect of NGR1-loaded biomaterials on bone defect regeneration. METHODS:Human umbilical vein endothelial cells (HUVECs) were cultured in complete medium containing the screened concentration of NGR1 to investigate its pro-angiogenic phenotype and potential mechanism in vitro. Subsequently, an injectable nano-hydroxyapatite-incorporated GelMA hydrogel was synthesized as an active drug-delivery delivery system for NGR1. The therapeutic effect of this fabricated NGR1-loaded biomaterial on bone defect regeneration was further evaluated in a rat cranial bone defect model. The key molecules in relevant signaling pathways was analyzed by immunohistochemistry. RESULTS:In vitro experiments demonstrated that NGR1 exhibits good biocompatibility and angiogenic capacity, as it promoted cell proliferation, enhanced cell migration, upregulated the angiogenic-related gene expression, and increased the protein expression of VEGF and VEGFR-2. Furthermore, the implantation of the injectable nano-hydroxyapatite-incorporated GelMA hydrogel loaded with NGR1 significantly enhanced bone defect regeneration in a rat cranial bone defect model compared to hydrogel-only group. Additionally, NGR1 supplementation markedly upregulated CD31 expression during bone formation, suggesting its role in coupling of angiogenesis and osteogenesis. Mechanistically, both in vivo and in vitro experiments indicated that NGR1 likely promote angiogenesis via activating Notch1/Akt singling pathway during bone regeneration. CONCLUSIONS:These findings indicate that NGR1 promotes angiogenesis through Notch1/Akt signaling activation during bone regeneration, which might offer potential therapeutic targets for bone-related diseases. Moreover, the application of NGR1-loaded biomaterials could represent a promising strategy to enhance bone regeneration.

The death of spermatogonia leads to decreased spermatogenesis and male infertility. Spermatogonia are vulnerable to various external damaging factors, which can cause cell death. However, the mechanism is still unclear. In this study, we found that the actin-related protein T2 (ACTRT2) is specifically expressed in testicular tissue and is associated with spermatogenesis. In vitro, when GC-1 cells (spermatogonial cell line) were treated with busulfan, the proportion of cell death in the low-ACTRT2 group increased significantly. Reactive oxygen species accumulation and typical mitochondrial changes associated with ferroptosis occurred. In vivo, the seminiferous tubules in ACTRT2-/- mice were significantly shrunken. In addition, after being treated with busulfan, spermatogenesis in ACTRT2+/- mice decreased significantly compared to that in wild-type mice. In ACTRT2+/- testes, the expression levels of acyl-CoA synthetase long-chain family member 4 and arachidonic acid 15-lipoxygenase-1 (ALOX15) were upregulated, while the expression levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were downregulated. Finally, we found that the expression of solute carrier family 11 member 2 (SLC11A2), iron responsive element binding protein 2 (IREB2), and transferrin receptor protein 1 (TFRC) increased significantly in the low-ACTRT2 group, which transports iron into the cell to increase the intracellular unstable iron pool. In conclusion, ACTRT2 deficiency leads to intracellular iron overload and damage to mitochondria, ultimately increasing spermatogonia vulnerability to ferroptosis.

Extracellular matrix (ECM) assembly/disassembly is a critical regulator for airway epithelial development and remodeling. Airway organoid is widely used in respiratory research, yet there is limited study to indicate the roles and mechanisms of ECM organization in epithelial growth and differentiation by using in vitro organoid system. Moreover, most of current Matrigel-based airway organoids are in basal-out orientation where accessing the apical surface is challenging. We present a human apical-out airway organoid using a biochemically defined hybrid hydrogel system. During human nasal epithelial progenitor cells (hNEPCs) differentiation, the gel gradually degrade, leading to the organoid apical surfaces facing outward. The expression and activity of ECM-degrading enzymes, matrix metalloproteinases (MMP7, MMP9, MMP10 and MMP13) increases during organoid differentiation, where inhibition of MMPs significantly suppresses the normal ciliation, resulting in increased goblet cell proportion. Moreover, a decrease of MMPs is found in goblet cell hyperplastic epithelium in inflammatory mucosa. This system reveals essential roles of epithelial-derived MMPs on epithelial cell fate determination, and provides an applicable platform enabling further study for ECM in regulating airway development in health and diseases.