Search Results (1,606)

Articular cartilage is characterized by its avascular, aneural, and alymphatic nature, which confers a limited intrinsic capacity for self-repair. Current regenerative strategies primarily focus on alleviating pain, mitigating symptoms, and restoring joint function. However, their long-term efficacy remains uncertain. Cartilage tissue engineering has emerged as a promising alternative to conventional therapies, offering innovative solutions for articular cartilage regeneration. Central to this approach is the development of functional biomaterials capable of supporting chondrogenic cell adhesion, proliferation, and differentiation, thereby facilitating effective cartilage repair. In this study, we introduce a novel protein-based recombinant spider silk (RSS) as a potential biomaterial for modulating chondrocyte behavior and enabling engineered cartilage formation both in vitro and in vivo. RSS was generated through molecular cloning and processed into silk fibers using biomimetic spinning and acidic coagulation techniques. In micromass cultures of murine chondrocytes, RSS significantly promoted cell aggregation, resulting in increased cell density. Alcian blue and Oil Red O staining demonstrated that RSS-treated cultures produced abundant glycosaminoglycans, a hallmark of chondrogenic activity, while exhibiting minimal lipid accumulation. These findings suggest that RSS supports chondrogenic differentiation and suppresses adipogenic lineage commitment. Real-time PCR analysis revealed upregulation of the chondrogenesis-related gene Sox9 and downregulation of the adipogenic marker PPARγ and the hypertrophic marker Runx2 in RSS-treated micromass cultures. RNA sequencing further corroborated these observations, underscoring the role of RSS in modulating extracellular matrix (ECM) remodeling in chondrocytes. In a subcutaneous transplantation model using severe combined immunodeficiency (SCID) mice, chondrocytes encapsulated in three-dimensional hydrogel scaffolds containing RSS exhibited significantly enhanced ECM accumulation compared to RSS-free controls, indicating that RSS supports the maintenance of the chondrocyte phenotype and promotes cartilage formation in vivo, and underscoring its promising potential as a component of hydrogel composite systems. These findings highlight the potential of RSS as a functional biomaterial to preserve chondrocyte functionality and advance engineered cartilage formation, presenting a promising avenue for cartilage tissue engineering and regeneration. Full article

For testing ocular irritation, 3D corneal models mimicking the corneal epithelium are considered reliable eye irritation tests and are detailed in regulatory guideline OECD Test Guideline (TG) 492. The aim of the present study was to develop and validate a Reconstructed human Cornea-like Epithelium (RhCE) in vitro irritation test method for ophthalmic medical devices according to OECD TG 492. Immortalized Human Primary Corneal Epithelium Cells (IM-HCEpiCs) were cultured on microporous inserts and exposed to an Air–Liquid Interface (ALI). Morphology was examined using standard (immuno-) histological techniques. Viability was quantified with MTT assay. Barrier integrity and function were monitored by trans-epithelial electrical resistance (TEER) and determination of IC₅₀ using MTT assay. Reproducibility was evaluated by calculating the inter-batch coefficient of variation (CV %) of the absorbance values of negative control-treated RhCE model replicates by MTT assay. Technical proficiency was verified using reference chemicals. Irritancy of ophthalmic medical devices was assessed. IM-HCEpiCs developed an epithelium-like barrier under the ALI. TEER increased after ALI introduction, and the obtained IC₅₀ value showed concordance with the guideline’s reference ranges. The developed RhCE test method demonstrated technical proficiency and correctly identified medical devices as non-irritants. A novel RhCE model was developed and validated according to OECD TG 492. Full article

Abiotic stress factors, including drought and salinity, severely limit crop productivity worldwide. Furthermore, the extensive use of herbicides, such as glyphosate, disrupts beneficial soil microbiota, further impairing crop growth. Plant growth-promoting bacteria (PGPB) represent a sustainable and efficient strategy to enhance crop yields, particularly under unfavorable environmental and soil conditions. In this study, we characterized Paenibacillus sp. JSM-10, newly isolated from glyphosate-exposed agricultural soil, for its stress tolerance and plant growth-promoting potential, including its morphology examined using complementary microscopy techniques. The strain tolerated up to 0.5 g/L glyphosate, 15 g/L NaCl, and 100 g/L polyethylene glycol (PEG-6000) without significant growth inhibition (p > 0.05), demonstrating robust resilience to such multiple abiotic stresses. Beyond its tolerance, the strain exhibited several beneficial characteristics, including indole-3-acetic acid (IAA) synthesis, siderophore production, and inorganic phosphate solubilization. Furthermore, both living cells and culture filtrates of JSM-10 exhibited a positive trend toward enhancing buckwheat growth under normal and saline conditions, with effect sizes ranging from Hedges’ g = 0.56−0.92. In addition, JSM-10 exhibited antagonistic activity against a range of pathogenic microorganisms, including Nigrospora oryzae, Bipolaris sorokiniana, Alternaria spp., and Escherichia coli. Altogether, these characteristics highlight the Paenibacillus sp. JSM-10 strain and its culture filtrates as promising candidates for application in organic farming aimed at promoting plant growth and improving stress tolerance via plant–microbe interactions. Full article

Background/Objectives: Copper (Cu²⁺) is an essential trace element that participates as a cofactor in key metabolic enzymes such as cytochrome c oxidase and superoxide dismutase. However, excessive copper exposure can be toxic and disturbances in copper homeostasis have been associated with neurodegenerative diseases including Alzheimer’s and Parkinson’s disease. Despite growing evidence linking copper to neuronal dysfunction, the cellular mechanisms by which Cu²⁺ affects neuronal excitability and neurotransmission remain poorly understood. The aim of this study was to investigate the effects of acute Cu²⁺ exposure on ionic currents involved in cellular excitability and neurotransmitter release in bovine chromaffin cells. Methods: Primary cultures of bovine chromaffin cells were used as a neuroendocrine model to study cellular excitability. Voltage-dependent ionic currents were recorded using the whole-cell patch-clamp technique in voltage-clamp configuration. Catecholamine secretion was monitored by amperometry, and cytosolic Ca²⁺ dynamics were measured in fluo-4-loaded cells during depolarization induced by high K⁺ stimulation. Results: Acute Cu²⁺ exposure produced a concentration-dependent enhancement of depolarization-evoked catecholamine release. In parallel, Cu²⁺ inhibited voltage-dependent calcium (ICa), sodium (INa), potassium (IKv), and calcium/voltage-dependent potassium (IKCa-v) currents in a concentration-dependent and partially reversible manner. In addition, Cu²⁺ increased basal cytosolic Ca²⁺ levels while reducing the amplitude of depolarization-evoked Ca²⁺ transients. Conclusions: Acute Cu²⁺ exposure exerts a dual effect in bovine chromaffin cells, inhibiting the ionic currents that support cellular excitability while potentiating catecholamine secretion. This apparent paradox is consistent with a disruption of intracellular Ca²⁺ homeostasis, in which elevated basal cytosolic Ca²⁺ may facilitate exocytosis despite reduced depolarization-evoked Ca²⁺ entry. These findings provide new insight into the mechanisms by which copper may alter neuronal signaling and contribute to neurotoxicity. Full article

Nanogrooves provide instructive cues to cells in culture. Several nanofabrication techniques have been developed to create biomimetic substrates, advancing our understanding of cell adhesion. Their integration into nervous system models highlights the critical role of the extracellular matrix (ECM) in developing functional tissue constructs for in vitro platforms such as Brain-on-Chip (BoC) and Nervous System-on-Chip (NoC). This study presents a nanofabrication approach that integrates photolithography and microtransfer molding (μTM) to pattern nanogrooves using photocurable polymer NOA81 onto microelectrode array (MEA) plates. The resulting nanogrooves exhibited a pattern periodicity of 976 nm and a ridge width of 232 nm, as confirmed by scanning electron microscopy and atomic force microscopy. We assessed the biocompatibility and functional impact of these modified substrates using human induced pluripotent stem cell (hiPSC)-derived neuronal cultures. Neurons cultured on nanogroove-modified MEAs exhibited aligned neural processes due to the anisotropic surface features and expressed vivid spiking behavior and higher burst frequency compared to randomly cultured neuronal networks. In conclusion, the proposed fabrication technique integrates nanogrooves with commercial MEAs using a combination of microtransfer molding and photolithography, resulting in modified culture substrates that enhance spike activity and network organization, aiding in the development of more in vivo-like neural models. Full article

Pulmonary bioengineering holds significant promise for the development of functional lungs suitable for transplantation in patients with terminal lung diseases; however, it encounters considerable challenges. The inherent structural complexity, diverse cellular composition, and the intricate process of re-endothelialization the pulmonary vasculature complicate efforts to reconstruct viable lungs for transplantation. This study aimed to establish an innovative re-endothelialization technique utilizing decellularized scaffolds, integrating canine yolk sac-derived endothelial precursor cells with mechanical respiratory stimuli within a bioreactor framework. Wistar rat lungs were subjected to a decellularization protocol employing SDS + Triton X-100 0.5% and subsequently assessed for cytocompatibility with murine fibroblasts (3T3) and yolk sac (YS) cells in fragments. Following this, the recellularization of the whole-lung scaffold was evaluated under constant mechanical respiratory stimulation with YS cells. Each stage of the process was rigorously analyzed using histological staining, DAPI, scanning electron microscopy (SEM), and genomic DNA quantification. The findings reveal that the implemented alternating decellularization protocol resulted in a structured scaffold conducive to the culture of various cell types in fragments. When subjected to the complete scaffold recellularization model, the results indicated that YS cells are advantageous for the re-endothelialization process. Moreover, when employed in conjunction with the bioreactor model incorporating respiratory stimulation, these cells demonstrated enhanced cellular diffusion capacity and facilitated more homogeneous recellularization of the entire organ. These results signify a notable advancement in the reconstruction of new tissues for pulmonary transplantation. Full article

Microorganisms represent the Earth’s most abundant biomass and a vast reservoir of genetic diversity. However, traditional agar plate methods fail to recover the vast majority of these species, leaving a “microbial dark matter” that holds immense potential for the discovery of novel antibiotics and bioactive compounds. While conventional techniques such as selective media and enrichment culture remain foundational, they are inherently limited by community biases and the inability to support low-abundance, oligotrophic species. To address these bottlenecks, a diverse array of innovative isolation strategies has emerged. This review systematically categorizes and evaluates these methodologies, ranging from in situ cultivation to high-resolution single-cell manipulation. We first examine membrane diffusion-based cultivation (e.g., iChip), which mimics natural microenvironments to resuscitate recalcitrant microbes. Subsequently, we explore high-throughput single-cell technologies, including microfluidics for physicochemical separation, optical tweezers for precise manipulation, and fluorescence-activated cell sorting (FACS). Special attention is given to Raman-activated cell sorting (RACS) as a label-free functional screening tool and reverse genomics for targeted capture. By synthesizing the strengths and limitations of these approaches, we propose integrated workflows designed to accelerate the mining of untapped microbial resources. Full article

Background/Objectives: Sepsis is a leading cause of hospital mortality and represents a time-sensitive medical emergency. Current diagnostic strategies rely on clinical assessment, severity scores, biomarkers, and blood cultures. However, blood cultures require 24–72 h for pathogen identification and demonstrate limited sensitivity, while biomarkers such as procalcitonin and C-reactive protein lack optimal specificity. These limitations support the widespread empirical use of broad-spectrum antibiotics and highlight the need for rapid, sensitive, and culture-independent diagnostic tools. Methods: A narrative literature review was conducted using PubMed and Google Scholar, including 28 studies published over the past 10 years, encompassing observational and preclinical investigations. Current evidence on the application of Raman spectroscopy in sepsis was summarized, with a dual focus on pathogen identification and the assessment of the host response. Results: Raman spectroscopy has demonstrated the ability to detect early molecular alterations in circulating immune cells and mitochondrial redox status, potentially preceding conventional biomarkers. For pathogen identification, Raman techniques have achieved diagnostic accuracies comparable to automated systems, but with significantly shorter turnaround times. Integration with microfluidics, optical tweezers, and deep learning algorithms has further enhanced performance, although these applications remain largely experimental. Conclusions: Despite these promising results, the lack of methodological standardization, spectral overlap among phylogenetically related species, limited large-scale validation, and challenges in interpreting certain spectral signatures remain unresolved. Most available evidence originates from preclinical, single-center, and controlled studies, underscoring the need for prospective multicenter trials and harmonized protocols. Full article

In vitro cell barrier models have been increasingly integrated into pharmaceutical and academic research pipelines to evaluate drug safety and drug delivery due to a shift towards New Approach Methodologies (NAMs) in research and regulatory safety assessment. Such models require reliable and interpretable functional readouts. Bioimpedance-based monitoring, particularly transepithelial/endothelial electrical resistance (TEER), is a widely adopted readout due to its non-invasive and real-time capabilities. However, substantial variability arises from differences in measurement settings, frequency selection, electrode configuration, impedance measuring techniques, and data analysis strategies. In numerous studies, TEER is approximated from single-frequency impedance magnitude measurements, which do not isolate the resistive component associated with tight junction-mediated paracellular transport but instead reflect the combined response of a coupled electrochemical system. This review clarifies impedance measuring techniques and systematically analyzes impedance-based measurement and analysis strategies for in vitro biological cell barrier integrity. We compare mono-frequency and broadband acquisition approaches, examine the influence of electrode–electrolyte interfaces, electrode geometry, and culture configuration, and evaluate equivalent circuit modeling and phase-resolved electrical impedance spectroscopy (EIS). Based on this comparison, we propose a three-level analytical hierarchy adapted to experimental objectives and instrumentation constraints. We conclude that phase-informed impedance analysis and harmonized reporting are essential to improve measurement reproducibility, inter-platform comparability, and integration of impedance-derived cell barrier assessment within NAMs-oriented research workflows. Full article

Background: Influenza causes substantial morbidity in young children, particularly those aged 6–35 months. In this age group, optimisation of vaccine dose regimens remains important to ensure adequate immunogenicity while maintaining acceptable safety. This study evaluated the immunogenicity and safety of a full 0.5 mL dose of quadrivalent inactivated influenza vaccine (NBP607-QIV) in young children. Methods: This Phase 3, randomised, double-blind, active-controlled, multicentre study was conducted in Korea, Thailand, and Malaysia. Healthy children aged 6–35 months were randomised 2:1 to receive NBP607-QIV (0.5 mL) or control vaccine (0.25 mL). Immunogenicity was assessed using the haemagglutination inhibition assay. Primary endpoints were non-inferiority of NBP607-QIV versus Agrippal for seroconversion rate (SCR) and adjusted post-vaccination geometric mean titre (GMT) ratio against three shared strains. Immunogenicity against the additional B/Yamagata strain was evaluated according to Committee for Medicinal Products for Human Use (CHMP) criteria. Safety was assessed based on adverse events. Results: A total of 676 participants were randomised, and 675 were included in the safety set. Non-inferiority of NBP607-QIV versus control vaccine was demonstrated for SCR for all shared strains and for the adjusted GMT ratio for A/H1N1 and B/Victoria, but not for A/H3N2. Immunogenicity against the B/Yamagata strain met CHMP criteria for SCR and geometric mean ratio (GMR). Immunogenicity was consistent across prespecified subgroups, and the incidence of adverse events was comparable between groups, with no clinically meaningful safety concerns. Conclusions: NBP607-QIV administered at a 0.5 mL dose demonstrated acceptable immunogenicity and a safety profile comparable to that of a licensed trivalent influenza vaccine in children aged 6–35 months, supporting its use in this paediatric population. Full article

Neurofilament light chain (NfL) is a promising biomarker of axonal injury across acute and chronic neurodegeneration, which can improve drug discovery and disease monitoring models. Traditional in vivo animal models cannot fully mimic human pathophysiology of neurodegenerative diseases (NDDs), but in vitro models based on human cells solve this problem, reducing the time and cost of drug testing. We developed an electrochemical immunosensor for NfL detection in cell culture media to monitor acute neuronal injury in in vitro models. The biosensor was designed in two configurations: the label-free system, which directly detects NfL in the sample via the antibody–antigen interaction, and the sandwich configuration, which incorporates two additional antibodies. Detection was examined using electrochemical techniques, including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronoamperometry (CA). The sensor demonstrated a detection limit of 3–9 pg mL⁻¹, and a dynamic working range spanning from 10 up to 10⁷ pg mL⁻¹. Importantly, NfL was successfully detected in physiological media collected from cultured neurons that were differentiated from the long-term human neuroepithelial-like stem cells. This discovery highlights the platform’s applicability for in vitro neurodegenerative models. The immunosensor offers a sensitive, scalable, and cost-effective alternative for neurodegeneration detection in drug testing applications. Full article

Background: Konjac (Amorphophallus spp.) is an economically important crop valued for the glucomannan content in its corms. Currently, the konjac industry faces germplasm degeneration due to long-term asexual propagation. Developing tissue culture and genetic transformation techniques is essential for its genetic improvement. The WUSCHEL-related homeobox (WOX) transcription factors are critical regulators of somatic embryogenesis and stem cell maintenance in plants. Methods: In this study, we performed genome-wide identification and characterization of WOX genes in the A. konjac reference genome. Furthermore, comparative transcriptomic analyses and functional verification were conducted in A. bulbifer. Results: A total of 12 AkWOX genes were identified in A. konjac, and their structural features were documented. Comparative transcriptomic analysis of A. bulbifer revealed that AbWOX genes were differentially expressed between embryogenic calli (EC) and non-embryogenic calli (nEC). Notably, AbWOX2 was significantly upregulated in EC. Overexpression of AbWOX2 significantly promoted callus proliferation and shoot regeneration in A. bulbifer. Furthermore, AbWOX2-overexpressing lines exhibited a 5.3-fold increase in genetic transformation efficiency (from 5.12% to 27.31%) compared to the control. Conclusions: We characterized the diverse expression patterns of the WOX gene family in Amorphophallus. Crucially, we identified specific individual members—most notably the markedly upregulated AbWOX2—that function as pivotal drivers of somatic embryogenesis and serve as promising candidates for enhancing regeneration and genetic engineering efficiency in Amorphophallus species. Full article

Accurate biomass quantification is important for evaluating growth kinetics and performance of microalgal and microalgal–bacterial wastewater treatment systems. However, small-scale studies frequently encounter methodological limitations due to low biomass concentrations, limited sampling volumes, and/or interference from non-biotic solids in complex wastewaters. This work adopts a two-fold approach: (i) a concise review of current biomass quantification methods for bench-scale systems, and (ii) an experimental evaluation of a gravimetric protocol for complex wastewaters. The review discusses commonly applied techniques, highlights their strengths and weaknesses, and identifies research gaps in data comparability and reproducibility. The laboratory investigations evaluated the effects of key factors, namely culture volume (250 mL to 1 L), test aliquots (2.5 mL to 10 mL), and the absolute weight of total suspended solids (3.43 g to 14.5 g) on total suspended solids measurements. Aliquots containing <5 mg total suspended solids produced statistically significant variability, whereas reliable and reproducible results were obtained when >8–10 mg absolute total suspended solids per aliquot was present. In complex wastewater matrices, approximately 18% of total suspended solids consisted of non-volatile solids, demonstrating that the method can systematically over-estimate true dry cell weight in microalgal–bacterial systems. The findings emphasized the need for procedural standardization. Finally, a practical gravimetric protocol is proposed for both axenic and consortium-based small-scale studies dealing with complex wastewater, providing an evidence-based roadmap for obtaining more reliable biomass estimations. Full article

This study presents a comprehensive characterization of Argopecten purpuratus (AP) shells—a marine-derived natural bioceramic composed predominantly of calcium carbonate (CaCO₃)—to evaluate their potential as biomaterials for regenerative medicine. Structural and compositional analyses were performed using micro-computed tomography (MicroCT), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and X-ray diffraction (XRD). These techniques confirmed a high CaCO₃ content (>96 wt%) and revealed distinct microstructural features: the outer surface showed irregular grooves and rough textures, while the inner surface exhibited smoother, foliated morphologies with mixed calcite and aragonite phases. To assess biocompatibility, human gingival mesenchymal stem cells (hGMSCs) were cultured on both shell surfaces. Viability and adhesion were evaluated via MTS assays and fluorescence microscopy at time points ranging from 30 min to four weeks. Both surfaces supported robust early metabolic activity and long-term proliferation, with cells covering the entire surface area after four weeks. Morphometric analysis indicated time-dependent changes in cell shape, transitioning from rounded to elongated morphologies, with minor differences linked to surface topography. The integration of structural, compositional, and biological data demonstrates that AP shells provide a cytocompatible and sustainable natural material platform capable of supporting cell adhesion and proliferation. Their inherent micro- and nanoscale surface features may facilitate protein adsorption and cell–material interactions. These findings highlight the importance of correlating microstructural material properties with cellular responses and support the future exploration of marine-derived bioceramics for regenerative medicine applications. Full article

Background/Objectives: In the last decade, non-invasive methods for aneuploidy detection have been explored. The most successful approach involves analyzing the cell-free DNA (cfDNA) released by the embryo into the culture medium. The main objective of this study is to examine the technical feasibility of this new approach called non-invasive PGT-A or niPGT-A. In addition, as an exploratory objective, the impact of the niPGT-A results on clinic outcomes will be assessed. Methods: This was a multicenter, international study that included 716 patients and 2539 blastocysts (ClinicalTrials.gov: NCT03520933). Each embryo was cultured following a specific protocol for niPGT-A. Individual spent blastocyst medium (SBM) and trophectoderm (TE) biopsy were obtained, analyzed, and compared to assess concordance. In a subset of embryos, the comparison also included an inner cell mass (ICM) biopsy. Clinical outcomes from the embryo transfers performed (all based on the TE result) were registered, and results were analyzed blindly regarding the impact of aneuploidies in the culture medium. Results: The concordance rate between SBM and TE was 79.1% (range: 74.1–82.1; cycles with autologous oocytes). This value increased to 87.0% when comparing SBM and ICM. Applying an adapted embryo culture protocol to collect the SBM for niPGT-A did not affect blastocyst quality. Analysis of the embryo transfers performed (n = 265) revealed a trend towards lower miscarriage rate in blastocysts where both TE and SBM were concordant and euploid (13.0%), compared to blastocysts with a euploid TE and an aneuploid SBM (22.2%). Conclusions: The results obtained show a high concordance between the SBM and TE biopsies. Although additional refinement of the technique would further increase niPGT-A’s performance, the results obtained support the potential use of this non-invasive approach for aneuploidy detection. The high concordance of the cfDNA present in the SBM with the corresponding ICM biopsy and the miscarriage rate observed in cases with an aneuploid SBM, despite the euploid TE results, also support niPGT-A’s capacity to assess embryo aneuploidies and its potential as a prioritization system for selecting blastocysts to transfer. This approach could hold special interest in patients with no PGT-A indications, couples that prefer not to biopsy their embryos or those who do not have access to invasive PGT-A. Full article