Previously we reported on engineering cell-free vascular grafts that harnessed the potential of the innate immune system to remain patent and regenerate into functional neo-arteries in small and large, preclinical animal models. However, the performance of these grafts in aged hosts—the predominant patient demographic in need of vascular graft replacement—remains elusive. To address this gap, we evaluated graft patency and regenerative potential in aged mice and characterized the cellular phenotypes arising within the grafts using the CX3CR1-confetti mouse model, which we previously developed to trace the fate of cells contributing to graft repopulation. Much like in young mice, these grafts remained patent in aged mice throughout the 4-week implantation period and regenerated endothelial and underlying smooth muscle layers, resembling native arteries. Lineage-tracing analysis revealed that the grafts were repopulated by fluorescently-labeled host monocytes/macrophages that expressed both M1 and M2 macrophage markers, with a bias toward a pro-inflammatory phenotype when implanted in aged hosts. Additionally, cells populating the grafts in aged mice exhibited increased markers of cellular senescence compared with those in young hosts. Nevertheless, the grafts remained patent and developed distinct luminal and medial layers resembling native arteries, underscoring their potential suitability for geriatric recipients.
Intervertebral disc degeneration (IVDD) constitutes the leading cause of chronic low back pain. Oxidative stress and ferroptosis act as critical pathological drivers of IVDD progression. Nevertheless, the cellular heterogeneity of nucleus pulposus cells (NPCs), endogenous repair homeostasis, and the regulatory network linking mechanical signals to ferroptosis remain poorly defined. In the present study, single-cell RNA sequencing was adopted to dissect the dynamic features of human NPC subpopulations, verifying that progressive depletion of progenitor NPCs (P-NPCs) serves as the core trigger for disrupted repair homeostasis during IVDD development. Combined with clinical specimen validation, multi-omics profiling and in vitro cellular models, we demonstrated that aberrant mechanical stress activates the mechanosensitive ion channel PIEZO1 and triggers Ca²⁺ influx. This event downregulates the transcription of GPX4, the master effector against ferroptosis; simultaneously, it upregulates the E3 ubiquitin ligase TRIM21 to accelerate GPX4 ubiquitination and subsequent proteasomal degradation. These two coordinated pathways aggravate oxidative stress, iron overload and lipid peroxidation in NPCs, accompanied by typical mitochondrial morphological damage. Ferroptotic NPCs further shape a pathological microenvironment that suppresses P-NPC viability and triggers P-NPC apoptosis, thereby establishing a cascading and self-amplifying degenerative loop. Both in vitro treatment with a PIEZO1 inhibitor and in vivo mechanical loading assays using nucleus pulposus-specific PIEZO1 knockout mice validated that PIEZO1 blockade restores GPX4 activity, rebalances redox homeostasis and robustly retards IVDD progression.
Traumatic spinal cord injuries (SCIs) exhibit marked differences in severity, yet objective biomarkers reflecting early pathological status remain limited. Here, we identify syntaxin-17 (STX17), a key autophagosomal SNARE protein, as a severity-associated biomarker and functional regulator of autophagic flux following SCI. Proteomic profiling and experimental validation revealed that moderate SCI is characterized by increased autophagic flux accompanied by increased neuronal STX17 expression, whereas severe SCI is characterized by STX17 downregulation, impaired autophagosome–lysosome fusion, and exacerbated neuronal necroptosis. Notably, serum STX17 levels in both SCI patients and mouse models correlated with injury severity, highlighting its translational potential as a biomarker. Mechanistically, restoring STX17 expression reestablished autophagic flux, suppressed necroptosis, and promoted axonal regeneration and functional recovery after severe SCI. We further identified an upstream circRNA–miRNA regulatory mechanism that modulates STX17 expression in neurons. Collectively, our findings establish STX17 as a previously unrecognized biomarker and therapeutic target that links autophagic flux dysregulation to injury severity and neuronal fate after traumatic SCI.
Biostimulatory dermal fillers are widely used for skin rejuvenation, but how particle physicochemical properties regulate fibroblast–macrophage interactions and collagen remodeling remains unclear. We compared calcium hydroxylapatite (CaHA), polycaprolactone (PCL), spherical poly-L-lactic acid (PLLA-Sp), and irregular PLLA fragments (PLLA-Ir) through comprehensive characterization (SEM, FTIR, zeta potential, AFM, roughness, tapped density, rheology in HA matrices) along with in vitro (human dermal fibroblasts, RAW264.7 macrophages, mono- and co-culture for viability, cytoskeleton, YAP mechanotransduction, macrophage polarization, cytokines, collagen) and in vivo (rat intradermal injection) analyses. CaHA exhibited the highest surface roughness, stiffness, and tapped density with a porous nanosurface and low aggregation, whereas PCL and PLLA-Sp were smoother and more aggregated. CaHA triggered stronger fibroblast cytoskeletal organization, YAP nuclear translocation, and type I collagen-related responses. All materials promoted M2-like macrophage polarization; however, PLLA-Sp induced the greatest total collagen deposition with predominant type III collagen and persistent mixed inflammation, while CaHA led to denser, more organized collagen bundles with a higher type I/III ratio and CD206/CD68 ratio. These findings indicate that particle micro/nanotopography and local cellular microenvironment bias collagen subtype remodeling, providing a physicochemical rationale for selecting biostimulators.
Effective treatment of mature scars remains a clinical challenge due to limited remodeling strategies. This study evaluated the efficacy and mechanisms of hair follicle micro-particle skin grafting in a prospective trial involving 40 scar patients. (Trial registration: Chinese Clinical Trial Registry, PID300363) Participants were allocated to receive either FUE-derived hair follicle micro-graft transplantation combined with punch excision or punch excision alone. After six months, the transplantation group showed significantly greater reductions in scar volume, lower Vancouver Scar Scale scores at 3 and 6 months, and better alleviation of pain and itching compared to controls. Histological analysis revealed that transplantation induced epidermal thickening, enhanced cell proliferation, and increased type IV collagen deposition at the epidermal-dermal junction. Transcriptomic profiling further revealed that these regenerative effects were associated with activation of the PI3K-AKT signaling pathway, suggesting a potential role in scar remodeling. These findings demonstrate that hair follicle micro-particle grafting is a clinically effective approach for scar remodeling, offering a promising therapeutic strategy to improve both aesthetic and functional outcomes.
There is evidence that sorptive clays have been used since prehistory as materials to treat skin abrasions and wounds. Despite this, there is a paucity of studies that systematically address the efficacy of defined clays in improving skin wound healing. In this study, we tested the hypothesis that a well-defined synthetic smectite clay, Laponite, enhances healing in a delayed skin wound healing model. Full-thickness skin wounds were made in the back skin of db/db and wild-type male adult mice (8–10 weeks), and Laponite clay gels or controls of phosphate-buffered saline (PBS) or alginate were applied after 24 h and held in place with a semi-occlusive dressing. Although Laponite treatment did not accelerate macroscopic wound closure, it significantly improved healing quality, including re-epithelialisation, epithelial cell division, epithelial thickness and fibroblast invasion, compared with PBS- or alginate-treated db/db mice. Moreover, hair follicle anagen was stimulated in proximity to Laponite-treated wounds, but was absent in PBS- or alginate-treated wounds. Neutrophil infiltration at day 18 was also reduced. In contrast to alginate, in which VEGF improved wound healing, VEGF in Laponite showed negligible additional benefit. These data indicate a role for synthetic nanoclay gels in wound healing.
Endotracheal intubation is essential in clinical care but often exacerbates airway inflammation, fibrosis, and microbial dysbiosis, contributing to long-term complications. This study investigates how newly developed, coated endotracheal tubes (ETTs) modulate airway healing through localized therapeutic delivery in a swine model of laryngotracheal injury. Animals were intubated with uncoated, dexamethasone-coated, or composite-coated (dexamethasone + PEG hydrogel delivering siRNA against smad3) ETTs for 3, 7, or 14 days. Tissues were analyzed via mechanical testing, immunohistochemistry, ELISA, cytokine profiling, 16S rRNA sequencing, and microscopy. Therapeutic coatings induced region- and time-dependent alterations in airway stiffness and extracellular matrix composition, including remodeling of collagen IV, laminin, and elastin. Reduced nuclear pSMAD3 staining in composite-treated airways relative to uninjured controls provided supportive, indirect evidence of altered SMAD3 pathway activity following smad3-targeting siRNA delivery, although downstream TGF-β1 and collagen I expression did not significantly differ across treatment groups or over the time course. Composite-coated ETTs also promoted a temporal shift from pro-inflammatory (M1) toward reparative (M2) macrophage phenotypes and altered local cytokine profiles. Microbial community composition varied by coating type and duration, with treatment-associated changes in bacterial taxa and predicted metabolic pathways. Mucin composition also shifted over time, reflecting evolving epithelial responses during prolonged intubation. These findings demonstrate that localized therapeutic delivery influences interconnected mechanical, inflammatory, and microbial responses during airway healing and supports the development of multifunctional ETT coatings as a strategy to modulate post-intubation airway remodeling.
Previous cesarean scar defect (PCSD) is the most common complication following cesarean delivery, characterized by myometrial thinning or disruption. Its incidence rises with increasing number of cesarean sections, reaching 76% after three procedures, yet effective clinical interventions remain lacking. Inadequate blood supply and weak regeneration of the lower uterine segment are key pathogenic contributors, suggesting that improving local perfusion could facilitate wound healing. In this study, we developed lipid nanoparticle (LNP)-encapsulated hepatocyte growth factor (HGF) mRNA and applied it locally to uterine incisions in a mouse model. HGF mRNA LNP treatment significantly increased endometrial thickness and gland density, reduced collagen deposition, and promoted smooth muscle regeneration and angiogenesis at days 7 and 30 postoperatively. Favorable trends in pregnancy outcomes were observed. Hematological and serum biochemical parameters remained comparable between groups at all time points examined. Transcriptomic profiling revealed that upregulated genes in the HGF mRNA LNP group were involved in cell proliferation, migration, and survival. In vitro, HGF enhanced endothelial cell migration and tube formation. Collectively, these findings indicate that HGF mRNA LNP delivery at the site of uterine incision injury can promote structural and functional uterine repair in a small‑animal model, and provide a rationale for further preclinical evaluation.
Following radiation injury, intestinal epithelial cells adapt through cellular plasticity to regenerate and repopulate the damaged epithelium by activating Yap, a key effector protein of the Hippo signaling pathway. However, the impact of pharmacologically targeting the Hippo pathway on the regeneration of the irradiated intestinal epithelium remains poorly understood. Here, we investigated this question using NCGC-023, a selective and potent small-molecule inhibitor of Yap’s upstream regulators LATS1/2. NCGC-023 treatment significantly increased active Yap protein expression in human enteroids and protected them against ionizing radiation (IR) in vitro. In mice, transient treatment with NCGC-023 before IR reduced radiation-induced DNA damage, suppressed premitotic apoptosis and aberrant mitosis in intestinal crypt cells, facilitated regenerative reprogramming of the damaged intestinal epithelium, and improved survival of mice subjected to IR doses that precipitate gastrointestinal acute radiation syndrome (GI-ARS). Mechanistically, NCGC-023 treatment before IR upregulated key signaling pathways controlled by LATS1/2, including Yap-dependent regenerative responses involving Il-33 and Yap-independent cellular responses to heavy metals mediated by metallothioneins. Moreover, NCGC-023 treatment did not exacerbate delayed injury in multiple organs of mice that survived 4 months post-irradiation. Together, these results demonstrate that transient NCGC-023 treatment before high-dose IR promotes the regeneration of the damaged intestinal epithelium. This proof-of-concept study supports further development of LATS1/2 inhibitors as prophylactic medical countermeasures for GI-ARS.
Dermal fibroblasts exhibit spatiotemporal heterogeneity, transitioning from coordinating immune signaling and depositing a provisional extracellular matrix during the early inflammatory phase to driving directional migration and mature matrix synthesis in the proliferative phase of wound healing. However, the molecular mechanisms underlying these fibroblast state transitions remain unclear. Here, by integrating single-cell RNA-sequencing, spatial transcriptomics, and in vivo and in vitro analyses in both mouse and skin organoid models for functional validation, we identify a previously underappreciated Gli2-Serpinh1 regulatory axis that plays a key role in fibroblast state transitions during skin wound healing. Serpinh1⁺ fibroblast subsets drive wound closure through re-epithelialization, vascular regeneration, and actin-mediated filopodia formation. Gli2, as a key upstream regulator of Serpinh1, modulates fibroblast function in a level-dependent manner across different healing states. Leveraging organoid technology as a drug discovery platform, we further identify three traditional Chinese medicine candidates that enhance wound healing by activating the Gli2-Serpinh1 axis. Our study reveals the Gli2-Serpinih1 axis as a pivotal regulator of fibroblast state transitions, providing mechanistic insight into fibroblast heterogeneity during wound healing and opening new avenues for precision regenerative therapies.
Regenerative capacity varies widely across tissues and species. Most regeneration studies have focused on identifying genes and pathways that promote regeneration, whereas mechanisms that inhibit regeneration to enforce organ size control and prevent excessive proliferation remain less well defined. Here, we identify isl1a as a dynamically expressed negative regulator of lateral line hair cell regeneration. isl1a is highly expressed during homeostasis but becomes transiently downregulated following hair cell loss. Loss of isl1a leads to proliferative expansion of support cells, producing enlarged sensory organs with increased hair cell numbers after injury. Mechanistically, isl1a downregulation increases chromatin accessibility at pro-proliferative loci, including AP-1 transcription factors, accompanied by sustained expression of injury- and regeneration-responsive genes. Together, these findings demonstrate that injury-induced downregulation of isl1a promotes the initiation of regeneration, while its subsequent re-expression restricts the duration of the response, thereby fine-tuning hair cell regeneration and maintaining appropriate support cell numbers.
Danio rerio (zebrafish) can regenerate their heart upon loss of over 20% of the total myocardium. A second teleost species, Oryzias latipes (medaka), however, has been described to lack this capacity and respond to a lesion forming a permanent fibrotic scar after heart resection. An altered immune response has been suggested to underlie the differences in heart regeneration. However, a thorough comparative analysis between zebrafish and medaka through the different steps of regeneration was lacking. Here, we provide an in-depth characterization of the injury response and heart regeneration in both teleost species (AB and Cab strains, respectively) after ventricular cryoinjury (CI). We observed an exacerbated neutrophil activation with little evidence of macrophage recruitment in medaka compared to zebrafish. However, cardiomyocyte proliferation significantly increased in both teleosts upon CI. We also observed transient blood vessel formation in medaka and a significant reduction of fibrotic tissue at 30 and 60 dpi compared to 7 dpi. However, some animals presented signs of an irreversible scar at 60 dpi and ventricular indentations suggestive of impaired regeneration. Nonetheless, echocardiographic assessment revealed cardiac function recovery in a subset of medaka, independent of the fibrotic tissue resolution. Overall, we conclude that the cardiac regenerative ability is broader in medaka than anticipated.
Cellular senescence is a key driver of kidney aging, leading to functional decline and increased susceptibility to chronic kidney disease. While the senolytic combination of dasatinib and quercetin (D + Q) has shown promise in mitigating age-related pathologies, its long-term effects and underlying multi-level systemic mechanisms in the aging kidney remain poorly defined. Here, we systematically evaluated the long-term effects of D + Q in naturally aged mice using multi-omics approaches. We show that D + Q treatment reduces senescence markers (p16, p21, SA-β-gal), restores the anti-aging protein Klotho, and attenuates renal fibrosis and inflammation. Proteomic profiling reveals that D + Q enhances apoptotic clearance of senescent cells and promotes proliferative and regenerative pathways. Moreover, D + Q reactivates PPARα signaling, improves fatty acid oxidation, and reduces lipid accumulation in aged kidneys. Single-cell transcriptomics further demonstrates that D + Q reverses transcriptional aging signatures across multiple renal cell types and remodels cell-type-specific pathways associated with metabolism, inflammation, and fibrosis. Cell-cell communication analysis reveals that D + Q normalizes the hyperconnected intercellular network in aged kidneys, particularly by modulating inflammation-related signaling. Our findings offer a comprehensive, systems-level understanding of how senolytic therapy restores renal homeostasis, emphasizing its potential as a multifaceted intervention to combat kidney aging.
Lung repair involves coordination of multicellular processes, including endothelial angiogenesis and epithelial repopulation. Retinoic acid (RA) signalling is crucial for lung development, homeostasis, and repair; however, the mechanisms through which RA drives repair are still unknown. It has previously been shown that RA has no direct effects on the repair of alveolar epithelium, yet in animal studies, RA induces alveolar regeneration. Here we show that RA induces endothelial angiogenesis, which is associated with enhanced paracrine signalling to alveolar epithelial cells. Transcriptomic profiling of RA-treated HPMECs undergoing angiogenesis revealed enrichment of wound healing pathways, and subsequent in silico analysis identified several candidate mediators of endothelial-epithelium crosstalk. Scratch assays demonstrated that of these candidates, only TGFα promoted wound healing in alveolar epithelial A549 and primary human alveolar type 2 (hAT2) cells. Mechanistically, TGFα was associated with increased epithelial cell migration and activation of EGFR signalling, without detectable effects on proliferation or apoptosis. Our findings indicate that RA-induced endothelial angiogenesis promotes epithelial EGFR signalling through paracrine mediators, including TGFα. This study highlights the importance of endothelial-epithelial interactions in lung repair and provides insight into pathways that may be targeted to enhance alveolar regeneration.
This study investigated xenoreduced generation of oral keratinocytes from induced pluripotent stem cells (iPSCs) derived from adult oral and skin fibroblasts for oral mucosal regeneration. Using an extracellular matrix-based protocol, iPSCs were differentiated into cells with a phenotype and molecular profile of oral epithelium, in both serum-containing and xenoreduced conditions. In vivo tests validated the viability of multilayered squamous epithelial-like tissues formed by cells derived from gingival fibroblast iPSCs.
This study evaluated a chitosan-fibrinogen-thrombin (CFT) matrix enriched with autologous skin cells and a fatty acid sodium salt (FASS) to enhance wound closure and regeneration. A rat excisional wound model compared six treatments: CFT with or without autologous cells and/or FASS, lubricating jelly, and cell suspension alone. Wounds were splinted and assessed at day 14. Tissue thickness and force at 10% strain were measured by indentation, followed by histological staining (Hematoxylin and Eosin, Masson’s Trichrome, Picrosirius Red) and immunostaining (CD31, CD68, Neutrophil elastase, Arginase-1). CFT-treated wounds formed a compact crust that supported keratinocyte migration, unlike Jelly and Cells groups which had delayed closure. All wounds remained in active repair, with CFT groups showing more advanced epithelialization and cornification, but no significant benefit from adding cells or FASS. There was greater angiogenesis and presence of macrophages in all wounds versus native skin, especially in Jelly and Cells groups, indicating earlier repair phases. Picrosirius red revealed immature collagen in all treated wounds. Mechanical testing showed wound thickness and resistance similar to native skin, suggesting near-functional recovery despite incomplete maturation. The main effect came from the intrinsic properties of the CFT matrix, which provided a biocompatible barrier promoting granulation and keratinocyte migration.
Axon regeneration is the key to repairing spinal cord injury. Circadian rhythm plays regulatory roles in axonal regeneration. The endogenous molecular clock serves as the molecular basis for the generation and precise maintenance of circadian rhythms. CLOCK is one of the most core transcription factors in the endogenous molecular clock. However, the role of CLOCK in axon regeneration has so far remained elusive. Therefore, this study explored how the circadian gene clocka regulates the zebrafish central neuronal Mauthner cells (M-cells) axon regeneration. Using the M-cell axonal regeneration model, we found that M-cell axons exhibited circadian-phase-dependent regeneration after two-photon laser ablation, while in the clocka mutant, the circadian-phase-dependent regeneration of M-cells was deprived and their regenerative ability was inhibited. Subsequently, a combination of single-cell electroporation, single-cell capture, transcriptome sequencing, and Rolipram treatment demonstrated that clocka is required for M-cell axonal regeneration and regulates axonal regeneration through phosphodiesterase pde4a-cAMP axis. Together, these findings demonstrate that endogenous molecular clock gene clocka regulates zebrafish central M-cell axonal regeneration via pde4a-cAMP pathway and provide new insights into rhythmic regulation for central nerve repair.
Oral ulcers represent a prevalent mucosal disease with incompletely elucidated pathogenesis and a clinical deficiency in multifunctional therapeutics. This study investigates a food-derived peptide, RDP3, with tissue-penetrating capability, which significantly promotes repair of oral ulcer mucosal in vivo and in vitro in a low concentration (1 nM). This is the first time to report a food-origin peptide capable of accelerating oral ulcer mucosal repair. RDP3 can not only accelerate wound healing but also restore microbiome homeostasis. Mechanistically, RDP3 functions as a novel peptide-antagonist of the interleukin-2 receptor β subunit (IL-2Rβ; binding affinity KD = 0.99 μM). This interaction suppresses pathological PI3K signaling, thereby inhibiting NLRP3/GSDMD-mediated pyroptosis, reducing inflammation, and promoting mucosal regeneration. These findings position RDP3 as a promising multifunctional therapeutic candidate for oral ulcer treatment.
Chronic nonhealing wounds remain a major clinical challenge, driven by persistent inflammation, impaired angiogenesis, defective extracellular matrix remodeling, and incomplete functional restoration. While stem cell therapies can modulate these processes, their clinical use is limited by low engraftment, variability, and safety concerns. Stem cell-derived extracellular vesicles (SC-EVs) and artificial nanovesicles (SC-ANVs) have emerged as cell-free alternatives that aim to capture key paracrine functions of stem cells. In this review, we evaluate these vesicle-based approaches within a problem-oriented framework linking molecular and cellular effects to clinically meaningful outcomes. SC-EVs and SC-ANVs influence inflammation, angiogenesis, and tissue regeneration in preclinical wound models; however, current evidence is largely limited to improvements in wound closure and histological parameters. We highlight that proof of concept for clinically relevant endpoints, such as durable function, reduced scarring, and recurrence prevention, remains insufficient. Key translational challenges, including delivery, dosing, and endpoint selection, are discussed, along with a framework for future studies required to establish therapeutic efficacy. Collectively, SC-EVs and SC-ANVs represent emerging platforms whose clinical potential depends on rigorous validation against defined clinical benchmarks.
Exosome-based therapies are emerging as promising tools in regenerative medicine and tissue protection. In this study, we engineered exosomes by mimicking paracrine signaling between umbilical cord-derived mesenchymal stem cells (UC-MSCs) and granulosa cells to mitigate chemotherapy-induced ovarian toxicity. Enhanced exosomes were evaluated in vitro, in vivo, and ex vivo using human granulosa cells, cyclophosphamide-treated mice, and cultured ovarian tissues from humans and rats. Enhanced exosomes demonstrated protective effects compared with chemotherapy-only controls. Molecular analyses revealed increased pro-proliferative and anti-apoptotic gene expression and reduced apoptotic markers following treatment. In vivo, enhanced exosome administration was associated with 70% higher primordial follicle counts and a two-fold increase in combined primordial and primary follicles relative to CTX controls. Primary follicle numbers were markedly elevated ( > 20-fold vs CTX). In breeding studies, pup numbers increased from 1 in CTX-treated mice to 7 in enhanced exosome-treated mice in the first mating, and pregnancies were observed in later breeding rounds only in the enhanced group. Enhanced exosomes also modulated ATP-binding cassette transporter expression. In human ovarian tissue ex vivo, treatment resulted in a two-fold increase in AMHR2 expression. These findings support engineered exosomes as a potential platform for oncofertility preservation and mitigation of chemotherapy-associated gonadotoxicity.
