2026-04-02
Valentin von Werz, Aleksander Szarzynski, Matthias Hadrbolec, Gregor Mattert, Sara Zigon-Branc, Bence Kozma, Werner Dammermann, Oliver Spadiut
Natural killer cells are emerging as promising “off-the-shelf” effectors for cancer immunotherapy, yet expansion of the NK-92 cell line in batch cultivation leads to rapid loss of cytotoxicity concomitant with lactate accumulation. In this study we developed and validated a two-phase manufacturing strategy that decouples cell proliferation from functional recovery in order to obtain an improved final product potency. Our 8-day kinetic survey determined declines in viability, metabolite profiles and cytotoxicity during static batch expansion. Guided by these data, in a 32-run full-factorial design-of-experiments approach we varied fresh cultivation medium proportion, temperature, dissolved oxygen, and recovery duration; partial least squares modeling identified fresh-medium ratio and recovery time as the primary drivers of cytotoxicity restoration. Identified optimal conditions (90% fresh medium, 37.2 °C, 3.7 days recovery) recovered cytotoxicity and maximized cytotoxic capacity. These setpoints were then translated to a 2 L stirred-tank bioreactor, where a fed-batch expansion under controlled pH and lactate levels produced 2.0 × 109 cells, followed by recovery that achieved 43% ± 8% cytotoxicity. This scalable, two-phase paradigm minimizes medium usage and obviates continuous perfusion, offering a potential workflow to increase NK-92 potency and a base for manufacturing high-quality advanced therapy medicinal products.
DOI: 10.3389/fbioe.2026.17971292026-04-02
Xiangyong Hu, Liping Du, Hongju Xiang, Yuyu Li, Zhixiong Liao, Jiaqi Yu
IntroductionMyocardial infarction (MI) triggers an excessive inflammatory response that drives adverse cardiac remodeling. Although colchicine has shown clinical promise, its narrow therapeutic window and lack of target specificity limit its efficacy. To address these limitations, we developed a biohybrid nanovesicle (MM‐LP@COL) that encapsulates colchicine, aiming to leverage natural inflammatory tropism for site-specific drug delivery.MethodsMM‐LP@COL nanovesicles were fabricated by fusing macrophage membranes with liposomes. Physicochemical characterization was performed using DLS, TEM, and FRET analysis. Cytokine‐scavenging capacity was evaluated in vitro by ELISA. In a murine MI model, mice were treated with PBS, free colchicine, LP@COL, or MM‐LP@COL. Immune cell populations in blood, bone marrow, and spleen were analyzed by high‐dimensional flow cytometry (UMAP). Cardiac function and tissue remodeling were assessed by echocardiography and histology.ResultsMM‐LP@COL formed uniform spherical nanovesicles (∼200 nm) and demonstrated potent, dose‐dependent neutralization of multiple pro‐inflammatory cytokines in vitro, along with reduced circulating cytokine levels in MI mice. High‐dimensional cytometric analysis revealed that treatment significantly reduced neutrophil infiltration across key immune organs, including blood, bone marrow, and spleen. These effects translated into improved left ventricular function, reduced myocardial inflammation, and attenuated cardiomyocyte hypertrophy.DiscussionThis study demonstrates that the macrophage-inspired nanovesicle system constitutes a potent and targeted combinatorial therapy for post-infarction inflammation and repair.
DOI: 10.3389/fbioe.2026.17516402026-04-01
Qi Yan, Yu Zheng, Long Chen, Hongmin Ma, Chao Ding, Xiaoxiao Pang, Tingting Xia, Jingyan Wei, Yinlong Zhang, Guoxin Xu
Reactive oxygen species (ROS) are inevitable by-products of aerobic metabolism and play a dual role in skin physiology and pathology. At physiological levels, ROS act as essential second messengers regulating cellular signaling and maintaining skin homeostasis. However, excessive ROS accumulation disrupts redox balance, leading to oxidative stress, inflammation, barrier dysfunction, and macromolecular damage, which are closely associated with the pathogenesis of various skin diseases, including psoriasis, atopic dermatitis, pigmentary disorders, photoaging, and skin cancers. In recent years, increasing attention has been directed toward nanomaterial-based strategies for precise ROS regulation, owing to their unique physicochemical properties, such as high surface area, tunable antioxidant activity, and enhanced skin permeability. Compared with conventional antioxidants, nanomaterials, including nanozymes, metal-based nanoparticles, biomacromolecular nanomaterials, and ROS-responsive nanocarriers, exhibit superior stability, targeted delivery capability, and sustained therapeutic efficacy. These nanoplatforms can not only efficiently scavenge excessive ROS but also modulate redox-sensitive signaling pathways, inflammatory responses, and skin barrier repair in a disease-specific manner. This review systematically summarizes the core mechanisms by which ROS contribute to the development of skin diseases, with an emphasis on oxidative stress mediated inflammation, macromolecular damage, and barrier impairment. Furthermore, we comprehensively discuss recent advances in nanomaterial-based therapeutic approaches for ROS regulation, highlighting self-therapeutic nanozymes, biomacromolecular antioxidant materials, and antioxidant-loaded nanodelivery systems. Finally, current challenges and future perspectives for the clinical translation of ROS-targeted nanotherapies in dermatology are discussed, aiming to provide a theoretical basis for the rational design of next-generation nanomedicines for skin disease treatment.
DOI: 10.3389/fbioe.2026.17970442026-04-01
Priya Das, Matthew McGrath, Noof Sulaiman, Martin Maresch, Nigamananda Dey, Melvin Varghese Jacob, Mohammed Al Muharraqi, Shane Browne, Fergal J. O’Brien, Michael B. Keogh
IntroductionThe treatment of chronic wound is extremely challenging and is often exacerbated by inflammation, poor angiogenesis and recurrent bacterial infections. To address this, we have developed a novel biomimetic bilayer three-dimensional scaffold with a chitosan-collagen upper epidermal layer, on top of a porous collagen-glycosaminoglycan dermal layer.MethodsIn this study we assess this scaffold’s efficacy in a preclinical wound model. In addition, we examined the scaffold with the addition of plasmid DNA encoding pro-angiogenic stromal derived factor-1α (SDF-1α) and anti-fibrotic β-klotho in a splinted full-thickness skin wound model on young Sprague Dawley rats for 14 days.ResultsAll the scaffold groups showed uniform deposition of extracellular matrix and showed no signs of wound contraction unlike our ‘empty’ defect group. Both the ‘bilayer chitosan- collagen’ group and ‘gene activated group’ showed that the upper chitosan layer was filled with exudate, which dried over time and formed a protective scab that delaminated easily at day 14. Our Chitosan- collagen scaffolds showed a decrease in pro-inflammatory IL-1β, an increase in the pro-angiogenic CD31 and a decrease in pro-fibrotic α-SMA protein expression. We showed enhanced pro-angiogenic and reduced pro-fibrotic expression with the addition of SDF and Klotho plasmids respectively (p < 0.01); however, the rate of wound healing was reduced with gene activation.DiscussionWhile the chitosan layer of the bilayer scaffold does not integrate into the wound bed it does form a protective covering with enhanced anti-inflammatory cues that support the lower integrating dermal collagen layer yielding optimal anti-fibrotic wound healing. These properties highlight the potential of this chitosan-collagen bi-layered scaffold, suggesting its suitability for promoting enhanced healing of chronic wounds in clinical settings.
DOI: 10.3389/fbioe.2026.18023082026-04-01
Zhichao Liu, Haiyan Fan, Yun Yang
Functional regeneration of bone and cartilage remains an urgent clinical challenge in orthopedics, as its repair process involves the synergistic participation of multiple systems and cell types. Traditional studies have mostly focused on the regulatory roles of individual cells or signaling pathways, while recent research has confirmed that bone/cartilage regeneration is governed by a regulatory mechanism centered on the neuro-immune-vascular axis. In this mechanism, mesenchymal stem cells (MSCs), bone marrow mesenchymal stem cells (BMSCs), adipose-derived mesenchymal stem cells (ADSCs), and cartilage progenitor cells (CPCs) serve as key functional cells, interacting sequentially and transcellularly with immune cells and endothelial cells through multiple core signaling pathways. This review systematically summarizes these core signaling pathways, including neurosignal-mediated pathways (CGRP/CRLR, NGF/TrkA, SP/NK1R), immune signal-mediated pathways (IL-4/IL-4R, TGF-β/Smad, TNF-α/NF-κB), endothelial cell-mediated pathways (VEGF/VEGFR, Notch, PDGF/PDGFR), and cross-regulatory pathways (PI3K/Akt, MAPK). These pathways collectively mediate the sequential crosstalk and functional coordination among the four cellular components. Additionally, the review highlights the application achievements of cutting-edge technologies in this field, such as single-cell omics, organoid models, in vivo imaging, new approach methodologies (NAM), microphysiological systems (MPSs), and biosensor-integrated platforms. It thoroughly analyzes the current bottlenecks in network mechanism research and clinical translation, including the spatiotemporal specificity of regulatory targets and the difficulty in simulating complex microenvironments, while proposing breAkthrough directions such as optimizing targeted regulatory strategies, developing intelligent biomaterials, and integrating multi-disciplinary technologies. Notably, the traditional M1/M2 macrophage dichotomy can no longer capture the high heterogeneity of immune cells. Recent single-cell omics studies have identified multiple functionally distinct macrophage subsets in the bone/cartilage regeneration microenvironment. This discovery provides a new perspective for precise immune regulation strategies and also underscores the limitations of the traditional classification framework. Overall, this review aims to establish a systematic framework for understanding the complex regulatory mechanisms of bone/cartilage regeneration and offer theoretical support and research insights for the development of efficient repair strategies.
DOI: 10.3389/fbioe.2026.17785782026-04-01
Tianfei Zheng, Dongfeng Guo, Yaqi Shi, Jinlong Zhou, Kun Zong, Naihong Ding, Xingjiang Li
Microbial communities play pivotal roles in the fermentation of cigar tobacco leaves. Although high-throughput sequencing technology has facilitated the exploration of these communities, a comprehensive understanding of their assembly mechanisms remains elusive. This review integrates the current knowledge regarding microbial sources, ecological dynamics, and evolutionary processes during cigar tobacco fermentation. We systematically assess the abiotic factors (temperature, humidity, nutrients) and biotic interactions (quorum sensing, metabolic coordination) which influence the microbial community. Moreover, we put forward strategies for synthetic community engineering and discuss the emerging applications of artificial intelligence in fermentation optimization. These insights deepen the understanding of microbial communities in cigar tobacco leaves, and provide new perspectives on regulating microbial communities to enhance the fermentation quality of cigar tobacco leaves.
DOI: 10.3389/fbioe.2026.17791822026-04-01
Xuekun Fu, Shaochuan Huo, Chunhao Cao, Hanson Hsu, Jie Huang, Yuhe Lei, Jie Li, Peng Liu, Jincheng Zeng, Songqing Lin, Chao Liang
BackgroundMesenchymal stem cell (MSC) dysfunction contributes to impaired bone regeneration in osteoporosis. Lamin B1, a nuclear lamina protein implicated in stem cell aging, has an unclear role in osteogenesis.MethodsWe examined Lamin B1 expression during osteogenic differentiation, assessed its pathological alterations in OVX-derived BMSCs, and generated Lamin B1–knockout MSCs to evaluate effects on osteogenesis, migration, and KAT2A regulation. We performed transcriptomic profiling and intra–bone marrow transplantation in OVX mice to determine functional relevance.ResultsLamin B1 expression progressively decreased during osteogenic induction but was markedly upregulated in OVX BMSCs, where it correlated with impaired migration. Lamin B1 deletion enhanced alkaline phosphatase activity, mineralization, and migration. Mechanistically, Lamin B1 interacted with KAT2A and promoted its ubiquitin-dependent degradation, thereby reducing KAT2A protein stability. Knockdown-induced transcriptional changes indicated activation of osteogenic and migration-related pathways. In vivo, Lamin B1-deficient MSCs showed improved engraftment and substantially enhanced bone regeneration, reflected by increased BMD, BV/TV, MAR, and BFR in OVX mice.ConclusionLamin B1 depletion enhances BMSC osteogenesis by preventing KAT2A degradation. Lamin B1-deficient BMSCs provide a promising gene-enhanced cell therapy strategy for osteoporosis.
DOI: 10.3389/fbioe.2026.18035142026-04-01
Ming Zhang, Yinan Cui, Su Yang, Xin Liu, Baiyan Sui, Jiao Sun
ObjectivesResin-based sealants (RBSs) are widely used to prevent pit and fissure caries in clinical dentistry. However, their high density of C=C double bonds leads to volumetric shrinkage during photo-polymerization, which may compromise enamel bonding. Additionally, the biosafety of commonly used monomers such as bisphenol A glycerolate dimethacrylate (bis-GMA) and triethylene glycol dimethacrylate (TEGDMA) remains a concern due to potential health risks in adolescent patients. This study aimed to develop a bis-GMA- and TEGDMA-free pit and fissure sealant to address these limitations.MethodsA novel family of isosorbide-derived dental sealants (ISDSs) was developed using a synthesized isosorbide-based matrix, bis(2-(methacryloyloxy)ethyl) disuccinate isosorbide (IBMEDS), and a diluent, bis(2-methylacrylate) isosorbide (IBM). The performance of ISDS was evaluated against the commercial Clinpro™ sealant in terms of polymerization shrinkage, cytotoxicity, physicochemical properties, microleakage resistance, and stability of the sealed area under artificial aging conditions.ResultsCompared with Clinpro, ISDS exhibited a 34% reduction in polymerization shrinkage and a more than sevenfold increase in L929 cell viability, attributed to its bio-based structure and low double-bond density. Moreover, ISDS demonstrated superior physicochemical properties, including higher shear bond strength, more effective marginal adaptation to enamel, lower water absorption and solubility, and more sustained and stable long-term fluoride release, while maintaining comparable microleakage resistance.ConclusionA fully bio-based, fluoride-containing isosorbide-derived dental sealant (ISDS) was developed to overcome the polymerization shrinkage and biocompatibility limitations of conventional resin-based sealants. This novel sealant offers enhanced long-term stability, excellent biocompatibility, improved marginal adaptation, and sustained fluoride release, representing a safer and more effective alternative for caries prevention in children and adolescents.
DOI: 10.3389/fbioe.2026.17307492026-04-01
Yi-Chen Chen, Shuo-Wen Hsu, Eisuke Shimokita, Tatsuya Takemoto
The systematic culling of male layer chicks raises ethical concerns, leading to bans in Germany and other European countries and spurring the search for reliable in-ovo sexing methods. Most existing strategies rely on integration of exogenous DNA or are limited to specific chicken strains, and none meet commercial requirements. Here, we present a broadly applicable in-ovo sexing method that avoids exogenous DNA integration. We developed precision-bred chickens with targeted disruption of the Z-linked SLC45A2 gene, which encodes a transporter essential for pigmentation. Hemizygous knockout females (ZW; SLC45A2KO/W) exhibited eye depigmentation at embryonic day 7 (E7), whereas heterozygous knockout males (ZZ; SLC45A2KO/+) retained normal pigmentation. This clear visual dimorphism enables accurate sexing by routine egg candling. Fertility and reproductive performance of knockouts were comparable to wild-type chickens, and genotyping confirmed 100% prediction accuracy. Unlike many current technologies, our approach requires no complex instrumentation and allows early detection during incubation. This work provides a practical and ethical solution to chick sexing, with significant advantages for commercial hatcheries. More broadly, this study illustrates the potential of precision breeding to address pressing animal welfare concerns in the modern poultry industry.
DOI: 10.3389/fbioe.2026.17858932026-04-01
Nahidah Ibrahim Hammadi, Ahmed J. Alfahdawi, Athraa Basheer Radhi, Alaa Imad Abdulrazzaq
BackgroundHealing of cutaneous wound is a complex biological process that requires the migration of fibroblasts, production of extracellular matrix (ECM) component, angiogenesis and a highly regulated expression pattern of inflammatory mediators. Owing to the interest in wound dressings based on renewable biomaterials, much attention has been paid to their biocompatibility and sustainability. Within these materials, nanocellulose features the physical and chemical properties to promote important cellular processes for tissue regeneration.MethodsWheat straw–derived nanocellulose was isolated and its nanostructure and physicochemical properties were characterised using FESEM, XRD, EDX, and FTIR analyses. In vitro, its biological activity was evaluated in NIH-3T3 fibroblasts through cell viability, migration, and expression of wound-healing–related genes and proteins (n = 3). The effects of nanocellulose on wound healing were further evaluated using a rat full-thickness skin wound model (n = 10 per group), with macroscopic and histological assessments conducted at 3, 7, and 14 days after wounding.ResultsThe extracted nanocellulose exhibited a cellulose I crystalline structure and showed no cytotoxic effects on fibroblasts (p > 0.05). Nanocellulose-conditioned medium significantly enhanced fibroblast migration compared with control conditions (p 0.05). In the rat wound model, nanocellulose treatment significantly accelerated wound closure at days 3, 7, and 14 (p < 0.01–0.0001) and improved histological features of healing, including reduced inflammatory cell infiltration, more organised granulation tissue formation, enhanced angiogenesis, and earlier re-epithelialisation.ConclusionThese results suggest that nanocellulose derived from wheat straw can promote cutaneous wound healing by stimulating fibroblast-mediated matrix synthesis and angiogenesis without resulting in excess inflammatory stimulation. Taken together, the in vitro and in vivo findings demonstrate that nanocellulose is not only a renewable biomaterial but also an active biological material for use as functional wound-healing dressings.
DOI: 10.3389/fbioe.2026.18004162026-03-27
Xiong Shen, Hanlin Li, Tingting Liu, Yuanyuan Wang, Chenxing Liu, Yao Chen, Maosen Hong, Yusheng Lin, Zhijie Guo, Zhenzhou Li, Fei Yan
PurposeTo achieve ultrasound molecular imaging (UMI) of various tumor types, this study developed a novel integrin-targeted probe, RGD-hGVs, based on gas vesicles (GVs) derived from Halobacterium salinarum NRC-1 (Halo) and cyclic RGDfK peptides. The application potentials of UMI were evaluated in osteosarcoma and melanoma.Materials and MethodsRGD-hGV acoustic nanoprobes were constructed by conjugating Halo-derived GVs with cyclic RGD peptides. Morphology was characterized by PCM and TEM, and hydrodynamic size and zeta potential were measured. In vitro targeting to bEnd.3, K7M2, and B16-F10 cells was evaluated by flow cytometry and confocal microscopy. Ultrasound imaging was performed in osteosarcoma and melanoma models, followed by immunofluorescence staining for αvβ3 and probe distribution. Biocompatibility was assessed by hemolysis, CCK-8 assays, serum biochemistry, and H&E staining of major organs.ResultsRGD-hGVs (∼240 nm) demonstrated targeted binding to tumor cells (bEnd.3, K7M2, B16-F10) in vitro and in vivo. They produced stronger, longer-lasting tumor ultrasound signals than non-targeted controls, with immunofluorescence confirming localization to tumor vasculature and cells, and showed no significant toxicity.ConclusionRGD-hGVs may serve as a novel UMI probe, suitable for diagnosing multiple tumors.
DOI: 10.3389/fbioe.2026.1808539