Search Results (508)

Programmed cell death (PCD) is a pivotal biological process that occurs at various stages of plant development, including embryogenesis and seed germination. This study investigated whether the absence of PCD in endosperm cells is connected to the poor germination of Viola odorata seeds. Seeds of poorly germinating V. odorata and well-germinating V. × wittrockiana were either cold-stratified for 10 days or left untreated. Germination tests, tetrazolium viability tests, Western blot analyses for caspase-like proteases, and Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assays for DNA strand break detection were performed. The results revealed that V. odorata seeds did not germinate, regardless of stratification or lack thereof, whereas in V. × wittrockiana, stratification significantly increased their germination capacity (34 ± 6.5% vs. 56.5 ± 9.8% in non-stratified and stratified seeds, respectively). The tetrazolium viability test revealed that V. odorata seeds were nonviable (100% nonviable endosperm and 96% nonviable embryos in total), whereas the seeds of V. × wittrockiana were highly viable (63% and 59% endosperm and embryos in total, respectively). Western blot analysis revealed that in the germinating seeds of V. × wittrockiana, caspase-like activity was detected in the endosperm but not in the embryos, whereas in seeds that failed to germinate, the PCD signal in the endosperm was very weak. In the seeds of V. odorata, caspase-like activity was detected in the embryos and endosperm collected directly after 10 days of stratification, but no signal was detected in the seeds left to germinate for one month after cold stratification. TUNEL assays revealed DNA strand breaks in the peripheral part of the endosperm in V. odorata and in non-germinating V. × wittrockiana, whereas in the germinating seeds of V. × wittrockiana, DNA strand breaks were detected in the endosperm cells adjacent to the embryo. These findings indicate that endosperm-localized PCD facilitates nutrient mobilization to the embryo and seems crucial for successful germination. Overall, these results suggest that PCD contributes to the regulation of seed germination in Viola spp. Full article

Type 2 Diabetes (T2D) is characterized by the toxic aggregation of human islet amyloid polypeptide (hIAPP or amylin) within pancreatic β-cells. IAPP is also a neuropancreatic hormone that plays a significant role in Alzheimer’s disease (AD) by co-depositing with amyloid-beta (Aβ) and Tau, supporting the Type 3 Diabetes (T3D) hypothesis. Soluble IAPP accelerates Aβ aggregation through cross-seeding and causes neurotoxicity by impairing the blood–brain barrier and activating neuroinflammation. Melatonin inhibits these processes by disrupting hydrophobic interactions in both hIAPP and Aβ, preventing the formation of toxic β-sheet structures. Furthermore, melatonin promotes amyloid clearance via the glymphatic and lymphatic systems, protects neurons from oxidative damage, and reduces Tau hyperphosphorylation. This suggests that melatonin serves as a promising multitarget therapeutic agent for both metabolic and neurodegenerative disorders by modulating structural protein transformations. Full article

Background: Nerve conduits are used to bridge peripheral nerve defects caused by trauma, iatrogenic injury, or oncologic disruption. Three-dimensional (3D) biomimetic scaffolds for peripheral nerve regeneration have advanced significantly in recent years, driven by improvements in printing technology and neuronal seeding techniques. We report on published designer conduits that can recreate the epineurium, a critical yet challenging-to-manufacture feature of nerve tissue. Methods: A medical librarian conducted a literature search for our systematic review on EMBASE, Web of Science, and PUBMED, following PRISMA guidelines, for articles from January 2010 to January 2026 for the systematic review. Descriptive statistical analysis was performed using Microsoft 365 Suite software. The literature review was conducted using keywords and search terms describing the history and development of 3DP nerve guidance conduits published prior to January 2026. Results: Our search yielded 273 titles, of which 8 were included after full-text review; these studies used 3D printing to generate nerve conduits for preclinical models. Manual data extraction identified studies reporting successful epineurial recreation. The included scaffold materials were polycaprolactone, poly(l-lactide-co-ε-caprolactone), poly(lactic-co-glycolic acid), acrylate resin, and gelatin methacryloyl. In animal model studies, various terms were used to describe the epineurium outer sheath. Despite this variability in nomenclature, many of these reports indicated successful sciatic functional index (SFI) recovery, favorable g-ratios, good durability, high cell viability, and significant neurite elongation at the time of sacrifice. Conclusions: 3DP nerve conduits targeting the epineurium are promising approaches for treating peripheral nerve defects. The constructs promote oriented growth and myelination. Future research on incorporating the epineurium into nerve scaffolds may consider encapsulating NGF to promote more efficient nerve regeneration, standardizing the definition of epineurial recreation, designing mechanical and permeability reporting benchmarks, and evaluating cell strategies using comparable functional and histologic endpoints. Full article

Adipose tissue engineering (ATE) is an interdisciplinary field integrating materials science, cell biology, and engineering, aiming to construct functional artificial adipose tissue for addressing adipose tissue deficiency, metabolic disorders, and related clinical challenges. This review systematically summarizes the core advances, critical limitations, and translational potential of ATE. First, we elaborate on the three fundamental elements of ATE: scaffold materials (hydrogels, porous materials, microspheres, fibrous materials, decellularized extracellular matrix, 3D-printed/bioprinted scaffolds, and prevascularized constructs), seed cells (adipose-derived stem cells, mesenchymal stem cells, etc.), and growth factors (vascular endothelial growth factor, fibroblast growth factor, etc.), as well as their synergistic regulatory roles in adipose tissue regeneration. We then discuss the key factors influencing adipogenic differentiation and vascularization, which are pivotal for the formation of functional ATE constructs. Furthermore, we detail the construction and evaluation of in vitro and in vivo ATE models, highlighting the value of large animal models in bridging preclinical and clinical gaps. The applications of ATE in soft tissue repair and reconstruction, drug screening and disease modeling, and cultured meat manufacturing are comprehensively analyzed, with emphasis on technical challenge across different directions. Finally, we discuss the core challenges hindering ATE clinical translation, including lack of standardization of adipose-derived stem cells, immunogenicity issues, regulatory barriers, and technical limitations, and propose targeted future perspectives. This review provides a comprehensive and critical overview of ATE, offering guidance for promoting its translation from preclinical research to clinical practice and industrial application. Full article

Despite advances in the treatment of cutaneous melanoma, there is still a high percentage of patients who fail to respond or develop resistance to treatment. Establishing robust in vitro melanoma models will enable mechanism-based drug screening while reducing animal testing. In this work, a three-dimensional (3D) melanoma skin model (3DMSM) was developed on a porous scaffold. The culture of three melanoma cell lines (SKMEL-1, A375, and G361) in co-culture with human fibroblasts, melanocytes, and keratinocytes allowed the formation of the dermis, and stratified epidermis. Tumors were established in this model using two methodologies: adding previously formed melanoma cell aggregates (CA) or seeding melanoma cells directly into the dermis (CD). In this model, melanoma cells remain in their original microenvironment and, after proliferation, invade the basal layer. The model recapitulates correct melanocyte localization, epidermal disruption, extracellular matrix (ECM) remodeling, including collagen deposition, and epithelial-to-mesenchymal transition (EMT). Additionally, the cytokine profiles studied indicate that the model could mirror the inflammatory and immune-evasive traits of melanoma. Overall, 3DMSM provides a useful tool for understanding the mechanisms of melanoma progression and invasion, and for developing personalized medicine strategies through the implementation of a patient-derived model. Full article

Background: Embryoid bodies (EBs) play a central role in organoid engineering, where their formation fidelity and size critically influence downstream differentiation outcomes. Current EB production workflows primarily rely on retrospective quality assessment, which limits reproducibility in high-throughput culture systems. Objective: This study aimed to develop a prospective, non-invasive framework that integrates early-phase bright-field time-lapse imaging with a three-dimensional convolutional neural network to predict EB formation outcomes and final EB diameter within the microwell platform. Methods: Time-lapse image sequences collected during the first hours after cell seeding on the microwell array were used to train 3D-CNN models for classification (formation vs. non-formation) and regression (final diameter). A balanced dataset was constructed through under-sampling, and five-fold cross-validation with data augmentation was applied to evaluate model performance. Results: The classification model achieved an accuracy of 96.5%, reliably distinguishing between successful and failed EB formation using short-duration image sequences. The regression model predicted the final EB diameter with a mean absolute error of ±7.1 µm, reflecting strong agreement with measured values and capturing seeding-density-dependent size variations. Conclusions: Early aggregation dynamics captured by bright-field time-lapse imaging contain sufficient spatiotemporal information to enable accurate, prospective EB quality prediction. The proposed framework provides a label-free and automation-compatible strategy for improving reproducibility in large-scale EB manufacturing and supports the future development of adaptive and closed-loop organoid culture systems for clinical applications. Full article

Large bone defects remain a major clinical challenge, as current treatments primarily provide mechanical stability while often insufficiently addressing the biological microenvironment. The cell-deposited extracellular matrix (CD-ECM) represents a promising strategy to improve implant bioactivity by mimicking key features of the native tissue. In this study, we compared CD-ECMs from adipose tissue-derived mesenchymal stromal cells (ASCs), ASC-derived osteoprogenitor cells, and dermal fibroblasts. ECM composition was analyzed, and its ability to support the osteogenesis of reseeded skeletal stem cells (SSCs) was assessed. Subsequently, the best performing cells were used to produce CD-ECM on a 3D scaffold. Furthermore, we improved the ECM by treating the ECM-producing cells with dextran sulfate (Dx-S). Fibroblast-derived ECM showed higher collagen and glycosaminoglycan contents compared to ASC-ECM or osteoprogenitor-ECM. Furthermore, only the fibroblast-derived ECM (Fibro-ECM) exerted a supportive effect on the osteogenesis of SSCs. SSCs seeded on ECM showed a higher proliferation rate and enhanced osteogenesis. Supplementation with dextran sulfate further increased ECM deposition and osteogenic potential. We showed that fibroblasts produced substantially more ECM with a stronger pro-osteogenic effect than ASCs or osteoprogenitor cells. The ECM and its pro-osteogenic effect could further be increased when fibroblasts were treated with Dx-S. Together, these results highlight Fibro-ECM as a promising and easily accessible cell-derived ECM deposition strategy to improve the biological performance of implants in bone regeneration. Full article

Bioreactors used for the maturation of cell-seeded tissue-engineered scaffolds should essentially mimic the dynamic in vivo environments experienced by the native tissues they intend to substitute. In addition to perfusion of growth medium to facilitate continuous mass transfer, application of appropriate mechanical stimulation is important to enhance cellular responses in scaffolds for tissues such as tendons, skin, and cardiac muscle that experience dynamic loading. This study focuses on the development of a multi-modal custom bioreactor capable of applying cyclic tensile stimulation and perfusion within physiologically relevant ranges while minimizing shear stress detrimental to cells seeded on scaffolds. To validate the bioreactor design and operation, we assessed the effects of tensile stimulation (0.1 Hz, 2000 cycles/day) and perfusion (media flow rate = 0.15 mL/min) over 21 days on the biofunctional performance of 3D-bioplotted polycaprolactone (PCL) auxetic scaffolds with a representative design (missing-rib pattern) characterized by negative Poisson’s ratio similar to the aforementioned soft tissues. The scaffold had a tensile yield strain of 9.14%, yield strength of 0.25 MPa, elastic modulus of 2.85 MPa, and ultimate tensile strength (UTS) of 1.32 MPa. The application of perfusion and tensile stimulation (0–5% cyclic strain) for 21 days did not adversely affect the yield strength and elastic modulus of the scaffold but affected its UTS (22.5% decrease) compared to the control cultured without perfusion or stimulation. Notably, it resulted in significantly improved fibroblast cellular responses (DNA = 29 µg/g sample and collagen = 371.78 µg/g sample) compared to the control (7.52 µg/g sample and 163.51 µg/g sample, respectively). These results validate the bioreactor system operation and the ability of multi-modal stimulation to control biofunctional responses of auxetic scaffolds, which will serve as the basis for future studies that will optimize auxetic scaffold design and dynamic culture parameters for NPR tissue-specific applications. Full article

Background/Objectives: A stem cell therapy for type 1 diabetes (T1D) is experimentally available but only to those few humans in whom the use of systemic immunosuppression can be justified. For others with T1D, a means to deliver the islets needs to be perfected. We have previously bioengineered a removable device for this purpose and now wish to test the effect of adding extracellular matrix (ECM) derived from decellularised human pancreas to it. Methods: The complete device consists of encapsulated pluripotent stem cell differentiated islets seeded into tubular scaffolds of polycaprolactone made by melt electrospin writing and to which ECM was added. The seeded device was implanted either subcutaneously (SC) or intraperitoneally (IP) into streptozotocin diabetic immunodeficient mice. The outcome over the next few months was compared with that achieved in diabetic mice implanted IP with encapsulated islets alone. Results: The device seeded with encapsulated islets but not containing ECM functioned less well than encapsulated islets implanted alone, with lower human C-peptide production. However, when ECM was added to the seeded device and whether implanted SC or IP, islets functioned as efficiently as those implanted without use of a scaffold. Conclusions: These data provide optimism for the use of seeded scaffolds in diabetic humans in whom a single scaffold seeded with multiple encapsulated islets can more readily be removed if needed for safety reasons than can multiple encapsulated islets not seeded into a scaffold. Full article

Introduction: A 3D endothelial spheroid model expressing mosaic gain-of-function KRAS mutations was established to further understand the molecular changes associated with sporadic brain arteriovenous malformations (AVMs). Methods: Repellent 96-well U-bottom plates were seeded with human cerebral microvascular endothelial cells and resultant spheroids transduced with recombinant adeno-associated virus expressing KRAS^G12V. Spheroids were monitored using live-cell imaging for extended culture periods. Results: In the early growth period, KRAS^G12V expression increased spheroid growth rates and enhanced spheroid sprouting on gel matrices consistent with known AVM characteristics. With extended culture, novel endothelial characteristics were observed. KRAS^G12V-expressing spheroids displayed dynamic blebbing associated with the formation of rounded, hypertrophic cells disposed to engage in spheroid escape. These cells displayed reduced cell–cell adherence with rapid plasma membrane blebbing characteristic of amoeboid-like migration and mesenchymal-to-amoeboid transition. Spheroid growth and blebbing were reversed with MEK and mTOR inhibitors; Rho/ROCK inhibition specifically targeted the blebbing phenotype. Conclusions: Endothelial spheroids expressing KRAS^G12V exhibit characteristic features associated with abnormal vessel development in brain AVMs as well as novel phenotypes not previously observed in 2D monolayers. The ability to extend culture periods in this simple 3D model may allow further phenotypic exploration of important AVM driver mutations. Full article

The genus Plukenetia includes lianas or vines with oleaginous seeds rich in omega-3 and omega-6 fatty acids, proteins, and vitamin E, and the presence of flavonoids, steroids, and terpenoids has also been reported in leaves. Several species of Plukenetia have traditionally been cultivated in their native distribution areas, and their propagation is usually by seed. The aim of this work was to establish callus and cell suspension cultures of P. carabiaseae, an endemic species of Mexico, for the evaluation of the in vitro antioxidant and anti-inflammatory potential of its extracts. Three light conditions were evaluated for the establishment of P. carabiaseae callus lines from leaf explants. Friable calluses obtained under constant light were used to initiate a cell suspension cultures in Gamborg basal (B5) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (CIN), as growth regulators. After 35 days of cultivation, different polarity extracts from biomass were obtained, showing that the acetone extract had the highest antioxidant activity and a high total phenolic content (30.57 mg of gallic acid equivalent (GAE)/g dry weight). The anti-inflammatory activity of the methanolic extract, evaluated in murine macrophages induced with bacterial lipopolysaccharides, was dose-dependent, without cytotoxic effects. This is the first report of the establishment of P. carabiasiae cell suspension culture and demonstrates its potential as a biotechnological source of antioxidant and anti-inflammatory metabolites. Full article

Calcium phosphate cements (CPCs) and biomaterials, such as mesoporous bioactive glass (MBG), are critical for bone tissue engineering. This study aimed to 3D-print CPC scaffolds modified with MBG to enhance their osteogenic potential and regenerative ability. MBG powder was synthesized and characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), and nitrogen adsorption–desorption techniques. A commercial CPC ink (hydroxyapatite/α-tricalcium phosphate) was mixed with 5% MBG (w/w; CPC/MBG), and, after rheological assessment, the mixture was used to obtain scaffolds via 3D printing. These scaffolds were then tested for chemical, morphological, and mechanical properties, as well as ion release analysis. Unmodified CPC 3D-printed scaffolds served as controls. Biological experiments, including cell viability, DNA content, cell adhesion/spreading, and osteogenic gene expression, were performed by seeding alveolar bone-derived mesenchymal stem cells onto the scaffolds. Statistics were performed using Student’s t-test and ANOVA with post hoc tests (α = 5%). MBG characterization showed a typical mesoporous structure with aligned microchannels and an amorphous structure. Both formulations released calcium and phosphate ions; however, CPC/MBG also released silicon. Cell viability, adhesion/spreading, and DNA content were significantly greater in CPC/MBG scaffolds compared to CPC (p < 0.05) after 3 and 7 days of culture. Furthermore, CPC/MBG supported increased expression of key osteogenic genes, including collagen (COL1A1), osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2), after 14 days (p < 0.05). The combination of CPC ink with MBG particles effectively enhances the biocompatibility and osteogenic potential of the scaffold, making it an innovative bioceramic ink formulation for 3D printing personalized scaffolds for bone regeneration. Full article

Objective: This study investigates the efficacy of indocyanine green-mediated photodynamic therapy (PDT) in targeting MCF-7 breast cancer cells, a representative model of luminal A subtype, compared to healthy breast epithelial cells. Methods: MCF-7 cells and healthy breast cells were cultured in a three-dimensional (3D) hollow fiber bioreactor to mimic the tumor microenvironment in vivo. Cells were treated with ICG at concentrations ranging from 1 to 1000 μM and then photoactivated using a diode laser. Cell viability was assessed by trypan blue staining, and the production of reactive oxygen species (ROS), including singlet oxygen (¹O₂) was measured. Results: Cell viability, assessed via trypan blue exclusion, decreased dose-dependently with ICG concentrations (1–1000 μM), with MCF-7 viability dropping from 94.5% ± 0.8% at 0.1 μM to 15.83% ± 0.66% at 1000 μM, compared to healthy cells retaining >50% viability up to 500 μM (55.2% ± 2.0% at 1000 μM). Student’s t-tests confirmed significant differences (p < 0.05) between MCF-7 and control (0 μM) at all concentrations, and between MCF-7 and healthy cells, indicating selective cytotoxicity (IC₅₀: ~75 μM for MCF-7). Flow cytometry revealed MCF-7 cell concentrations were significantly lower than healthy cells’ across all ICG doses and seeding densities (p < 0.05). Spectroscopic analyses showed ICG absorption peaks at 800–900 nm, fluorescence at 800–820 nm, and singlet oxygen phosphorescence at 1270 nm, confirming effective ROS generation. Conclusions: Cell concentrations confirmed selective MCF-7 cytotoxicity (p < 0.05). Spectroscopic data validated ROS generation, supporting ICG-PDT’s potential as a selective therapy for early-stage breast cancer within a 50–500 μM therapeutic window. Full article

Background/Objectives: Pulmonary fibrosis (PF) is a progressive interstitial lung disease with limited therapeutic options. Lepidium sativum (cress seeds) possess recognized antioxidant and anti-inflammatory properties, yet its potential antifibrotic activity has not been investigated. This study evaluated the phytochemical composition and antifibrotic efficacy of cress seed extract (CSE) and examined whether its effects are associated with modulation of the ncNRFR/let-7d pathway in methotrexate (Mtx)-induced PF. Methods: Comprehensive metabolite profiling was performed using GC–MS, HPLC, and UPLC–T-TOF–MS/MS. Antioxidant capacity and antiproliferative effects were assessed in vitro. Network pharmacology was used to identify CSE-related PF targets and regulatory pathways. In vivo, PF was induced in adult male Wistar rats by Mtx, followed by oral CSE administration (50–150 mg/kg). Biochemical markers of inflammation, oxidative stress, extracellular matrix deposition, EMT, and ncRNA expression (ncNRFR and let-7d) were quantified alongside histopathology and immunohistochemistry. Results: CSE contained diverse terpenes, phenolics, flavonoids, glucosinolates, and amino acid derivatives. It exhibited potent antioxidant activity and antiproliferative effects against A549 and Hep2 lung cancer cells. Network analysis identified 997 overlapping CSE–PF targets and highlighted IL6 and MMP1 as relevant miR-let-7d–associated nodes. In vivo, Mtx-induced marked fibrosis characterized by increased ncNRFR, reduced let-7d, elevated IL6, HMGB1, TGF-β, MMP1, collagen, and hydroxyproline, and reduced antioxidant enzyme activity. CSE treatment dose-dependently mitigated these alterations, improved histoarchitecture, and reduced collagen deposition. Conclusions: CSE showed antifibrotic, antioxidant, and anti-inflammatory activity in MTX-induced PF in rats and modulated the reciprocal expression patterns of ncNRFR and let-7d. These findings support CSE as a potential source of bioactive constituents for PF management and identify the putative ncNRFR–let-7d regulatory relationship as a novel pathway in fibrotic lung disease, warranting further mechanistic investigation. Full article

Natural or synthetic scaffolds are essential for developing three-dimensional (3D) cell culture models, as they provide structural stability and accurately replicate the cellular microenvironment. When integrated into optimized setups, scaffold-supported cellular aggregates, such as spheroids, can be non-destructively characterized and monitored using 3T Magnetic Resonance Imaging (MRI). However, a significant technical limitation is the presence of MR artifacts generated by scaffolds, which can severely obscure the visualization of the embedded spheroids. This study systematically evaluated the suitability of various scaffolds and matrices (including Matrigel^®, fibrin glue, and several hydrogels) for MRI and MR spectroscopy (MRS). The materials were investigated both native and seeded with chondrosarcoma cells (SW1353). Our findings revealed considerable variability in MR compatibility across different materials. Specifically, fibrin glue proved unsuitable for MR applications due to substantial artifact generation that interfered with the visualization of cellular components. Furthermore, the results emphasize the importance of the observation period, as material degradation processes can introduce confounding factors in longitudinal MR studies. The choice of scaffold material is paramount for the successful analysis of 3D cell models via MRI. Careful selection is required, as the materials’ properties and temporal stability directly impact the interpretability of the acquired data. Full article