Search Results (22)

Umbilical cord mesenchymal stromal cells (UC-MSCs) are a key element of regenerative medicine due to their ability to secrete growth factors that stimulate proliferation and angiogenesis, and modulate the inflammatory response. Despite their widespread use, the influence of the perinatal microenvironment on their biological properties remains poorly understood. The aim of this study was to assess the influence of pH and blood gas parameters in umbilical cord blood on the global transcriptomic profile of UC-MSCs and to analyze the correlation between the metabolic status of the newborn and the expression of key trophic factors: EGF, FGF2, FGFR1, FGFR3, GDNF, HGF, IGF1, NES, NGF, and PGF. Methods: The study was conducted in two stages. In the first phase, transcriptomic screening was performed using Affymetrix HuGene 2.0 ST microarray on cells isolated from three environmental groups defined by cord blood pH: acidic (pH < 7.35), physiological (7.35–7.39), and alkaline (pH ≥ 7.4). In the second phase, the results were validated using qPCR on an expanded study group (N = 50). Gene expression levels (RQ) were related to blood gas parameters (pH, pCO₂, pO₂, cHCO₃) and the presence of clinical features of threatened neonatal asphyxia. Results: Microarray analysis revealed that environmental pH acts as a molecular phenotypic switch. Under low pH conditions (<7.35), a shift in cell profile from proliferative to structural–migratory was observed. Significant overexpression of genes responsible for extracellular matrix (ECM) organization and adhesion (e.g., COMP, DCN, LUM, FMOD) was observed, while pathways related to cell cycle and cell division (↓CDK1, AURKA, TOP2A) were downregulated. qPCR validation confirmed these observations, demonstrating a strong positive correlation between blood pH and the expression of regenerative mediators: FGFR1 (r = 0.28), EGF (r = 0.30), NGF (r = 0.39), and IGF1 (r = 0.30). A negative correlation was also found between carbon dioxide pressure (pCO₂) and the expression of NGF, FGFR1, and EGF. A significant clinical finding was that in newborns diagnosed with threatened asphyxia, EGF, FGFR1, and NGF gene expression was significantly reduced, indicating impaired trophic potential of the cells in response to metabolic stress. Conclusions: These results indicate that cord blood gas parameters are critical regulators of the genetic activity of UC-MSCs. Metabolic and respiratory acidosis not only inhibit the cells’ proliferative potential but also force them into a matrix remodeling mode, permanently modifying their transcriptomic profile. This suggests that the neonatal acid–base status may serve as an objective indicator of the “biological quality” of isolated stromal cells, which has significant implications for their future applications in cell therapies. Full article

The tumor microenvironment, including extracellular pH (pHe), has emerged as a key regulator of tumor cellular function. Although extracellular acidification sensing and function are well established, the effect of extracellular alkalinization on cellular functioning remains unclear. Here, we report that transient receptor potential ankyrin 1 (TRPA1) functions as an alkaline sensor and mediator of cell death in melanoma cells. Exposure to alkaline pHe (8.1) or allyl isothiocyanate (AITC), a TRPA1 agonist, significantly reduced melanoma cell viability. We found that cell death was propidium iodide-positive and annexin V-negative, suggesting that pHe or AITC treatment induced necrosis rather than apoptosis. TRPA1 activation induced sustained Ca²⁺ influx, which was suppressed by either extracellular Ca²⁺ removal or treatment with the TRPA1 inhibitor, HC-030031, both of which attenuated cell death. Pharmacological screening has identified phosphatidylcholine-specific phospholipase D1 (PLD1) as a positive regulator of cell death. We confirmed that transfection with PLD1 siRNA significantly reduced AITC-induced cell death, whereas PLD2, PLD3, and NAPE-PLD siRNAs had no effect. These observations suggest that the vulnerability of melanoma cells to alkaline pHe is mediated by activation of the TRPA1-PLD1 axis. Thus, TRPA1 and PLD1 are potential targets for therapeutic intervention in melanoma. Full article

Seed coating technology is regarded as one of the optimal strategies to promote sustainable agricultural development. It can effectively optimize the physical and physiological characteristics of seeds, improve germplasm quality, and enhance crop resistance to abiotic and biotic stresses. Saline–alkali soils, characterized by high salinity and alkalinity, severely restrict plant growth and development. However, alfalfa, a high-quality leguminous forage, faces substantial challenges in large-scale popularization and cultivation in saline–alkali regions. At present, research on the application of microbial seed coating technology in alfalfa production under saline–alkali conditions remains insufficient, and relevant techniques and formulations still require optimization. Under field conditions, this study used a randomized complete block design with alfalfa as the research material. Different coating treatments combining plant growth-promoting rhizobacteria (PGPR), rhizobia, and extracellular polysaccharides (EPSs) were established to systematically investigate the effects of various coating formulations on alfalfa yield, nutritional quality, root system architecture, and rhizosphere soil properties. Meanwhile, high-throughput sequencing was employed to analyze shifts in rhizosphere soil microbial community structure. The results demonstrated that all microbial coating treatments exerted significant growth-promoting effects on alfalfa grown in saline–alkali soils, among which the T8 treatment (combined coating of rhizobia + PGPR + EPS) performed the best. This treatment not only significantly improved alfalfa yield and nutritional quality but also modified root system architecture and enhanced soil enzyme activities, soil nutrient contents, and soil physical structure, thereby creating a favorable growth environment for plants. Among the single microbial coating treatments, the combined coating of rhizobia and EPS outperformed other single treatments and exhibited favorable application potential. Sequencing results revealed that microbial seed coating treatments significantly increased the relative abundance of beneficial soil bacteria, decreased the abundance of harmful fungi, regulated rhizosphere microbial community structure, and consequently promoted improvements in alfalfa yield and quality by optimizing the plant growth microenvironment. The findings of this study provide important theoretical support for the popularization and application of microbial seed coating technology in crop cultivation in saline–alkali soils, offer a key reference for optimizing alfalfa-specific seed coating formulations for saline–alkali conditions, and are of great significance for promoting the efficient utilization of saline–alkali land resources and the development of ecological agriculture. Full article

Renal cell carcinoma (RCC) is a biologically heterogeneous malignancy accounting for 3% of adult cancers globally. Despite advances in immune checkpoint inhibitors (ICIs) and vascular endothelial growth factor (VEGF)-targeted therapies, durable disease control remains elusive for many patients. Increasing evidence implicates the acidic tumour microenvironment (TME) as a critical mediator of RCC progression, immune evasion, and therapeutic resistance. Solid tumours, including RCC, exhibit reversed pH gradients, characterised by acidic extracellular (pH 6.2–6.9) and alkaline intracellular conditions. This dysregulation arises from enhanced glycolysis, hypoxia-driven lactate accumulation, and the overexpression of pH-regulating enzymes such as carbonic anhydrase (CA9). Acidic TMEs impair cytotoxic T-cell and NK-cell activity, promote tumour-associated macrophage (TAM) polarisation towards an immunosuppressive phenotype, and upregulate alternative immune checkpoints. These mechanisms collectively undermine ICI efficacy and contribute to primary and secondary treatment resistance. Proton-sensing G-protein-coupled receptors (GPCRs), notably GPR65, have emerged as pivotal mediators linking extracellular acidosis to immune dysfunction. Preclinical studies demonstrate that GPR65 antagonists restore anti-tumour immune activity by reversing acidosis-driven immunosuppression and enhancing antigen processing. In RCC models, selective GPR65 inhibitors have shown the ability to reduce immunosuppressive cytokine IL-10 production, induce immunoproteasome activation, and synergise with anti-PD-1 therapy. The first-in-class GPR65 inhibitor, PTT-4256, is now under evaluation in the Phase I/II RAISIC-1 trial (NCT06634849) in solid tumours, including RCC. Targeting acid-sensing pathways represents a novel and promising therapeutic strategy in RCC, aiming to remodel the TME and overcome ICI resistance. Integrating GPR65 inhibition with existing immunotherapies may define the next era of RCC management, warranting continued translational and clinical investigation. Full article

Cell–cell communication and extracellular matrix (ECM) organization in a bone microenvironment are essential to replicate the bone microenvironment accurately. In this study, the extracellular matrix (ECM) was emulated by incorporating M13 phages, selected through phage display for displaying engineered peptides that mimic bone matrix proteins, into human osteoblast cultures to develop a three-dimensional bone model (3D BMP-Phage). Comprehensive analysis was performed to investigate: (i) the morphological development of spheroids, assessed by optical microscopy and quantified via fractal dimension analysis using box-counting algorithms; (ii) the biochemical composition of the extracellular matrix, evaluated by Raman spectroscopy; (iii) ECM protein deposition, analyzed through immunofluorescence staining; (iv) matrix mineralization, assessed by Alizarin Red staining and alkaline phosphatase (ALP) activity assay; and (v) osteogenic gene expression, measured by quantitative RT-PCR. The findings demonstrate that the 3D BMP-Phage model, facilitated by a cocktail of bone-mimicking peptides, enhances structural integrity, ECM complexity, mineralization, and osteogenic pathways compared to the control. This novel approach replicates key aspects of the bone microenvironment, providing a valuable platform for advanced physiological and regenerative medicine research under controlled conditions. Full article

To address the dual clinical challenges of poor osseointegration and inadequate analgesia caused by postoperative infections in traditional titanium implants, this study proposes a multifunctional synergistic strategy based on metal—organic frameworks (MOFs). By integrating drug-controlled release and ionic microenvironment regulation, it constructs a titanium-based implant coating system with antibacterial and bone-regenerative properties. Ketoprofen, a drug with excellent analgesic properties, was loaded into MgCu-MOF74 powder, and the Ket@MgCu-MOF74 powder was successfully anchored onto the surface of the titanium alloy through dopamine-mediated adhesion. The maximum load of ketoprofen to MgCu-MOF74 is 18.55%, and it has a good controllable release effect. The results showed that MgCu-MOF74/Ti and Ket@MgCu-MOF74/Ti coatings enhanced osteogenic performance by promoting alkaline phosphatase activity, collagen secretion, and extracellular matrix mineralization. Additionally, the release of Mg²⁺ and Cu²⁺ created an alkaline environment, providing antibacterial properties. In summary, the MOF enabled the controlled release of ketoprofen, and the composite coating can improve osteogenic differentiation of osteoblasts and enhance the antibacterial properties of titanium alloy implants. Full article

Hair loss is often associated with oxidative stress and mitochondrial dysfunction in human follicle dermal papilla cells (HFDPCs), resulting in impaired cellular function and follicle degeneration. Thus, many studies have been conducted on natural plants aimed at inhibiting hair loss. This study investigated the therapeutic potential of exosomes derived from the rhizomes of Iris germanica L. (Iris-exosomes) in HFDPCs damaged by dihydrotestosterone (DHT). Iris-exosomes significantly reduced reactive oxygen species (ROS) levels, restoring mitochondrial membrane potential and ATP production, thereby mitigating oxidative stress and improving mitochondrial function. These effects occurred alongside enhanced cellular processes critical for hair follicle regeneration, including increased cell migration, alkaline phosphatase (ALP) activity, and three-dimensional (3D) spheroid formation, which replicates the follicle-like microenvironment and promotes inductive potential. Furthermore, Iris-exosomes stimulated the Wnt/β-catenin signaling pathway by enhancing glycogen synthase kinase-3β (GSK-3β), AKT, and extracellular signal-regulated kinase (ERK), leading to β-catenin stabilization and nuclear translocation, thereby supporting the expression of genes essential for hair growth. Taken together, these findings suggest that Iris-exosomes can be promising ingredients for alleviating hair loss. Full article

This study proposes a novel therapeutic strategy for cancer management by combining the antitumor effects of hydrogen sulfide (H₂S) and inhibition of carbonic anhydrases (CAs; EC 4.2.1.1), specifically isoforms IV, IX, and XII. H₂S has demonstrated cytotoxicity against various cancers at high concentrations. The inhibition of tumor-associated CAs leads to lethal intracellular alkalinization and acidification of the extracellular tumor microenvironment and restores tumor responsiveness to the immune system, chemotherapy, and radiotherapy. The study proposes H₂S donor–CA inhibitor (CAI) hybrids for tumor management. These compounds effectively inhibit the target CAs, release H₂S consistently, and exhibit potent antitumor effects against MDA-MB-231, HCT-116, and A549 cancer cell lines. Notably, some compounds display high cytotoxicity across all investigated cell lines. Derivative 30 shows a 2-fold increase in cytotoxicity (0.93 ± 0.02 µM) under chemically induced hypoxia in HCT-116 cells. These compounds also disturb the cell cycle, leading to a reduction in cell populations in G₀/G₁ and S phases, with a notable increase in G₂/M and Sub-G₁. This disruption is correlated with induced apoptosis, with fold increases of 37.2, 24.5, and 32.9 against HCT-116 cells and 14.2, 13.1, and 19.9 against A549 cells compared to untreated cells. These findings suggest the potential of H₂S releaser–CAI hybrids as effective and versatile tools in cancer treatment. Full article

Polymer microspheres have recently shown outstanding potential for bone tissue engineering due to their large specific surface area, good porosity, injectable property, good biocompatibility, and biodegradability. Their good load-release function and surface modifiability make them useful as a carrier of drugs or growth factors for the repair of bone defects in irregularly injured or complex microenvironments, such as skull defects. In this study, berberine (BBR)-encapsulated poly(lactic-co-glycolic acid) (PLGA)/hydroxyapatite (HA) microspheres were fabricated using electrified liquid jets and a phase-separation technique, followed by modification with the 3,4-hydroxyphenalyalanine-containing recombinant insulin-like growth–factor-1 (DOPA-IGF-1). Both the BBR and the IGF-1 exhibited sustained release from the IGF-1@PLGA/HA-BBR microspheres, and the composite microspheres exhibited good biocompatibility. The results of the alkaline phosphatase (ALP) activity assays showed that the BBR and IGF-1 in the composite microspheres synergistically promoted the osteogenic differentiation of MC3T3-E1 cells. Furthermore, it was confirmed that immobilized IGF-1 enhances the mRNA expression of an osteogenic-related extracellular matrix and that BBR accelerates the mRNA expression of IGF-1-mediated osteogenic differentiation and cell mineralization. Further cellular studies demonstrate that IGF-1 could further synergistically activate the IGF-1R/PI3K/AKT/mTOR pathway using BBR, thereby enhancing IGF-1-mediated osteogenesis. Rat calvarial defect repair experiments show that IGF-1@PLGA/HA-BBR microspheres can effectively promote the complete bony connection required to cover the defect site and enhance bone defect repair. These findings suggest that IGF-1@PLGA/HA-BBR composite microspheres show a great potential for bone regeneration. Full article

Tumor necrosis is a recurrent characteristic of head and neck squamous cell carcinomas (HNSCCs). There is a need for more investigations on the influence of biomolecules released by these necrotic foci in the HNSCC tumor microenvironment. It is suspected that a fraction of the biomolecules released by necrotic cells are damage-associated molecular patterns (DAMPs), which are known to be natural endogenous ligands of Toll-like receptors (TLRs), including, among others, proteins and nucleic acids. However, there has been no direct demonstration that biomolecules released by HNSCC necrotic cells can activate TLRs. Our aim was to investigate whether some of these molecules could behave as agonists of the TLR3, either in vitro or in vivo. We chose a functional approach based on reporter cell exhibiting artificial TLR3 expression and downstream release of secreted alkaline phosphatase. The production of biomolecules activating TLR3 was first investigated in vitro using three HNSCC cell lines subjected to various pronecrotic stimuli (external irradiation, serum starvation, hypoxia and oxidative stress). TLR3 agonists were also investigated in necrotic tumor fluids from five oral cancer patients and three mouse tumor grafts. The release of biomolecules activating TLR3 was demonstrated for all three HNSCC cell lines. External irradiation was the most consistently efficient stimulus, and corresponding TLR3 agonists were conveyed in extracellular vesicles. TLR3-stimulating activity was detected in the fluids from all five patients and three mouse tumor grafts. In most cases, this activity was greatly reduced by RNAse pretreatment or TLR3 blocking antibodies. Our data indicate that TLR3 agonists are consistently present in necrotic fluids from HNSCC cells and mainly made of dsRNA fragments. These endogenous agonists may induce TLR3, which might lead to a protumorigenic effect. Regarding methodological aspects, our study demonstrates that direct investigations—including functional testing—can be performed on necrotic fluids from patient tumors. Full article

The main factors that determine the low effectiveness of chemotherapy are the low target bioavailability of antitumor drugs and the efflux process. In attempts to overcome this problem, several approaches are proposed here. Firstly, the development of polymeric micellar systems based on chitosan grafted by fatty acids (different types to optimize their properties), which, on the one hand, increase the solubility and bioavailability of cytostatics and, on the other hand, effectively interact with tumor cells due to the polycationic properties of chitosan, allowing for more effective penetration of cytostatic drugs into the cells. Secondly, the use of adjuvants—synergists of cytostatics (such as eugenol) included in the same micellar formulation—that selectively enhance the accumulation and retention of cytostatics in the tumor cells. pH- and temperature-sensitive polymeric micelles developed show high entrapment efficiency for both cytostatics and eugenol (EG) >60% and release the drug in a prolonged manner for 40 h in a weakly acidic medium corresponding to the microenvironment of tumors. In a slightly alkaline environment, the drug circulates longer (more than 60 h). The thermal sensitivity of micelles is realized due to an increase in the molecular mobility of chitosan, which undergoes a phase transition at 32–37 °C. The effect of the cytostatic drug doxorubicin (Dox) on cancerous A549 cells and model healthy cells of human embryonic renal epithelium (HEK293T) was studied by FTIR spectroscopy and fluorescence microscopy. Micellar Dox penetrates into cancer cells 2–3 times more efficiently when using EG adjuvant, which inhibits efflux, as demonstrated by a significant increase in the ratio of intra- and extracellular concentrations of the cytostatic. However, here it is worth remembering about healthy cells that they should not be damaged: according to changes in the FTIR and fluorescence spectra, the penetration of Dox into HEK293T when using micelles in combination with EG is reduced by 20–30% compared to a simple cytostatic. Thus, experimental developments of combined micellar cytostatic drugs have been proposed to increase the effectiveness of cancer treatment and overcome multiple drug resistance. Full article

Glioblastoma multiforme (GBM) is the most common and malignant brain tumour. It is characterised by transcriptionally distinct cell populations. In tumour cells, physiological pH gradients between the intracellular and extracellular compartments are reversed, compared to non-cancer cells. Intracellular pH in tumour cells is alkaline, whereas extracellular pH is acidic. Consequently, the function and/or expression of pH regulating transporters might be altered. Here, we investigated protein expression and regulation of the electrogenic sodium/bicarbonate cotransporter 1 (NBCe1) in mesenchymal (MES)-like hypoxia-dependent and -independent cells, as well as in astrocyte-like glioblastoma cells following chemical hypoxia, acidosis and elucidated putative underlying molecular pathways. Immunoblotting, immunocytochemistry, and intracellular pH recording with the H⁺-sensitive dye 2′,7′-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein were applied. The results show NBCe1 protein abundance and active NBCe1 transport. Hypoxia upregulated NBCe1 protein and activity in MES-like hypoxia-dependent GBM cells. This effect was positively correlated with HIF-1α protein levels, was mediated by TGF-β signalling, and was prevented by extracellular acidosis. In MES-like hypoxia-independent GBM cells, acidosis (but not hypoxia) regulated NBCe1 activity in an HIF-1α-independent manner. These results demonstrate a cell-specific adaptation of NBCe1 expression and activity to the microenvironment challenge of hypoxia and acidosis that depends on their transcriptional signature in GBM. Full article

Human umbilical cord mesenchymal stem cells (hUCMSCs) are promising for bone tissue engineering, which have a non-invasive harvesting process, high cell yield, favorable proliferation capacity, and low immunogenicity. However, the osteogenic efficacy of hUCMSCs is relatively lower than that of bone marrow mesenchymal stem cells (BMSCs). Hydrogels from decellularized extracellular matrix (dECM) preserve the biological compositions and functions of natural ECM, which can provide tissue-specific cues to regulate phenotypic expression and cell fate. It is unknown, however, whether hydrogels from periosteum can serve as pro-osteogenic carriers of hUCMSCs. Herein, a decellularized periosteum-derived hydrogel (dPH) was fabricated to reveal the effects of periosteum-specific cues on the bioactivities of hUCMSCs. A widely used non-bone/periosteum-derived ECM hydrogel product, Matrigel, was used as the control group. After decellularization, the absence of nuclei in the histological analysis indicated a successful removal of cellular components, which was also confirmed by DNA content quantification. The storage modulus of dPH increased (from 164.49 ± 29.92 Pa to 855.20 ± 20.67 Pa) with increasing concentration (from 0.5% to 1%). With a highly porous, fibrous microstructure, dPH had a more hydrophilic surface than Matrigel, of which the water contact angle reduced 62.62 ± 0.04%. Furthermore, dPH prominently promoted the initial cellular spreading with a significantly higher cell surface area (1.47-fold), cell spreading length (1.45-fold) and proliferation (approximately 1.05–1.13-fold) of hUCMSCs than those of Matrigel. Additionally, dPH was conducive to cell migration, whereas no cells migrated to Matrigel in the Transwell model. Compared with those of the Matrigel group, the osteogenesis-related genes expression levels (runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN)) and mineralized matrix formation (9.74-fold) of the hUCMSCs significantly increased in the dPH group. Our study indicated that dPH could provide a pro-osteogenic microenvironment for hUCMSCs, thereby revealing a promising application potential to repair bone defects. Full article

Epigenetic approaches using the histone deacetylase 2 and 3 inhibitor-MI192 have been reported to accelerate stem cells to form mineralised tissues. Gelatine methacryloyl (GelMA) hydrogels provide a favourable microenvironment to facilitate cell delivery and support tissue formation. However, their application for bone repair is limited due to their low mechanical strength. This study aimed to investigate a GelMA hydrogel reinforced with a 3D printed scaffold to support MI192-induced human bone marrow stromal cells (hBMSCs) for bone formation. Cell culture: The GelMA (5 wt%) hydrogel supported the proliferation of MI192-pre-treated hBMSCs. MI192-pre-treated hBMSCs within the GelMA in osteogenic culture significantly increased alkaline phosphatase activity (p ≤ 0.001) compared to control. Histology: The MI192-pre-treated group enhanced osteoblast-related extracellular matrix deposition and mineralisation (p ≤ 0.001) compared to control. Mechanical testing: GelMA hydrogels reinforced with 3D printed poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) scaffolds exhibited a 1000-fold increase in the compressive modulus compared to the GelMA alone. MI192-pre-treated hBMSCs within the GelMA–PEGT/PBT constructs significantly enhanced extracellular matrix collagen production and mineralisation compared to control (p ≤ 0.001). These findings demonstrate that the GelMA–PEGT/PBT construct provides enhanced mechanical strength and facilitates the delivery of epigenetically-activated MSCs for bone augmentation strategies. Full article

Tumour cells maintain a local hypoxic and acidic microenvironment which plays a crucial role in cancer progression and drug resistance. Urease is a metallohydrolases that catalyses the hydrolysis of urea into ammonia and carbon dioxide, causing an abrupt increase of pH. This enzymatic activity can be employed to target the acidic tumour microenvironment. In this study, we present the anticancer activities of urease mimetic cobalt (III) complexes on A549 cells. The cells were treated with different doses of cobalt (III) complexes to observe the cytotoxicity. The change in cellular morphology was observed using an inverted microscope. The cell death induced by these complexes was analysed through ATP proliferation, LDH release and caspase 3/7 activity. The effect of extracellular alkalinization by the cobalt (III) complexes on the efficacy of the weakly basic drug, doxorubicin (dox) was also evaluated. This combination therapy of dox with cobalt (III) complexes resulted in enhanced apoptosis in A549 cells, as evidenced by elevated caspase 3/7 activity in treated groups. The study confirms the urease mimicking anticancer activity of cobalt (III) complexes by neutralizing the tumour microenvironment. This study will motivate the applications of transition metal-based enzyme mimics in targeting the tumour microenvironment for effective anticancer treatments. Full article