Thermoresponsive electrospun scaffolds based on poly(N-isopropylacrylamide) (PNIPAM) copolymers exhibit morphology-dependent structural disintegration upon cooling to the temperature of the coil-to-globule transition. This behavior does not coincide directly with the classical lower critical solution temperature (LCST) or volume phase transition temperature (VPTT) due to the specific fiber architecture formed by electrospinning. In this study, the Scaffold Disintegration Temperature (SDT) is introduced as an operational descriptor corresponding to the onset of reproducible morphological collapse in fibrous PNIPAM networks. SDT is used as a comparative metric to assess how the macromolecular architecture and processing conditions relate to the scaffold-level stability. Using viscosity-matched statistical and graft copolymers with distinct topologies, architecture-dependent differences in the disintegration behavior were identified. These differences correlate with variations in network morphology and orientational coherence. Scaffolds based on graft copolymers bearing rigid (PLA) or flexible (PCL) side chains exhibited earlier structural disintegration during cooling and reduced network coherence, whereas the statistical P(NIPAM-co-NtBA) system maintained structural integrity over a broader temperature interval and showed greater mechanical robustness under comparable conditions. A variation of the nozzle-to-collector distance further modulated the architecture-dependent differences in the fiber morphology, porosity, and mechanical performance but did not override the dominant influence of macromolecular topology. These results establish SDT as a network-level, morphology-dependent parameter that complements LCST in describing the thermal behavior of electrospun PNIPAM-based materials.

4D printing, a technology that combines additive manufacturing and shape memory polymers, has attracted considerable interest in numerous high-performance fields. However, the majority of existing high-temperature shape memory polymers used in 4D printing suffer from low mechanical strength, which limits their applicability across diverse areas. In this work, we have employed thermally stable cardo polyimide-amide (PIA) to modify the performance characteristics of materials based on photopolymerizable N,N-dimethylacrylamide and bisphenol A ethoxylate diacrylate. It has been demonstrated that the addition of PIA does not affect the ability of the composition to form complex shaped products with high printing resolution using LCD 3D printing technology. In contrast to the conventionally used photopolymerizable composition, the samples obtained with rigid-chain PIA additive have a high tensile strength (up to 108.6 +/- 5.9 MPa) and glass transition temperature (up to 144 degrees C). Furthermore, the 4D-printed structures exhibit excellent high-temperature shape memory performance at recovering temperatures exceeding 150 degrees C.

Olivetol (5-pentylresorcinol) is a naturally occurring alkylresorcinol whose cellular mechanism remains poorly understood. Here, we show that olivetol induces a non-genotoxic nucleolar DNA damage response (n-DDR) in human cells. Although moderately cytotoxic, olivetol did not cause detectable genomic DNA double-strand breaks. Instead, it triggered γH2AX accumulation at ribosomal DNA (rDNA), recruitment of TOPBP1 to Treacle, nucleolar disorganization, and repression of ribosomal RNA synthesis. Mechanistically, olivetol closely phenocopied hypotonic stress, inducing a rapid and reversible n-DDR associated with antisense RNA polymerase II transcription within the rRNA coding region and accumulation of R-loops, consistent with transcriptional interference between RNA polymerases I and II. Both olivetol and hypotonic stress also produced shared membrane-associated phenotypes, including reduced membrane lipid order, calcium redistribution, and plasma membrane blebbing. Importantly, this non-genotoxic nucleolar response depended on cholesterol-sensitive plasma membrane organization.

Poly(N-isopropylacrylamide) (PNIPAM)-based hydrogels are “smart” materials of great interest and demand, primarily in a variety of biomedical applications, due to their LCST close to physiological temperature. The basic biomedical applications of PNIPAM-based injectable hydrogels include controlled drug delivery, tissue engineering, wound healing, and cancer therapy. This review summarizes the thermal phase behavior of PNIPAM-based systems in different conditions, including different scenarios of physical gelation in polymer solutions and volume phase transitions in chemically crosslinked hydrogels. The thermodynamics of coil-to-globule phase transition, its relation to the gel formation, and mechanisms underlying the phase transitions in PNIPAM-based hydrogels are discussed, as well as external parameters which may affect these processes. Different types of phase diagrams observed for PNIPAM-based systems are considered, with particular attention paid to the cases in which gelation occurs separately from the coil-to-globule transition. The rational design of injectable platforms should be based upon a thorough understanding of the PNIPAM-based system’s phase behavior to achieve controllable and reproducible properties.

Thermoresponsive polymers have attracted significant attention in the design of injectable hydrogels for biomedical applications. Their gelation is conventionally attributed to the coil-to-globule transition, and other possible gelation mechanisms are frequently overlooked. Here, we present a graft copolymer P(NIPAM-g-PLA), representative of a distinct type of thermoresponsive polymers with gelation and coil-to-globule transition being separate processes. Its temperatures of gelation (Tgel) are consistently lower than the binodal temperatures (Tb); it forms a transparent gel at Tgel < T < Tb, and Tgel significantly depends on the polymer concentration, in contrast to Tb. At Tb, the polymer becomes opaque. Thus, the P(NIPAM-g-PLA)-water system exists in four phases: transparent sol, opaque sol, transparent gel, and opaque gel. P(NIPAM-g-PLA) gelation may be modeled by the formation of physical cross-links between hydrophobic "pearls" in the "pearl-necklace" polymer chains. P(NIPAM-g-PLA) forms viscous solutions at T < Tgel, with the viscosity dramatically depending on the polymer concentration. Its injectability appears restricted by approximately 10 wt %, above which the solutions become too viscous. Upon gelation at Tgel < Tb, transparent gels with stable mechanical properties and storage moduli of up to megapascals are formed. The rheological properties of P(NIPAM-g-PLA) were essentially modified by the presence of a biological buffer (PBS) as compared with pure water. An important finding of the study was the complete loss of the copolymer's ability to form gels due to aging (after a year-long storage). The potential influence of salts in biological buffers and the adverse effects of polymer aging should be considered when creating injectable hydrogels for biomedical applications.

Today, organ building blocks (OBBs) serve as important tools for in vitro tissue modeling, personalized medicine, and regenerative approaches. Despite substantial advances in dental reconstruction methods, tissues of the oral cavity remain challenging to regenerate due to their complex structure and microenvironment. However, effective regeneration of the periodontal complex, e.g., in diseases such as periodontitis, persists, as current methods do not achieve complete tissue restoration. Cells from the gingiva and dental pulp are accessible sources of mesenchymal stem cells with high regenerative potential, making them promising materials for creating OBBs. These cells can serve as fundamental units for restoring the periodontal complex using techniques such as 3D bioprinting. This study aims to characterize and compare OBBs derived from gingival cells, pulp cells, and their combinations by assessing key parameters, including morphology, extracellular matrix composition, biomechanical properties, histology, and metabolic activity. Combining the two cell types improved the structural, mechanical, and functional properties of OBBs, making them more suitable for bioprinting than those derived from a single cell type. Moreover, all types of OBBs from the two cell cultures may be suitable as components of bioinks, depending on the specific purposes. The results provide insights into the potential use of these cell sources for tissue engineering and the development of personalized periodontal bio-constructs that may significantly improve treatment approaches for oral diseases.

The ability of tumor cells to migrate and invade adjacent tissue is a key property underlying the metastatic process. To ensure greater deformability and to facilitate movement, migratory cells undergo multiple changes in biophysical parameters, including those of stiffness and membrane viscosity. However, reports on correlations between cell motility and stiffness, or between cell motility and membrane microviscosity are rather limited and conflicting. Here, using atomic force microscopy (AFM) and fluorescence lifetime imaging (FLIM), we have investigated alterations in the mechanical properties of cancer cells and in the microviscosity of their plasma membranes that are associated with the migration process. It was found that upon activation of migration either through a "wound healing" test or by inducing epithelial-mesenchymal transition, human colorectal cancer cells undergo profound biomechanical remodeling characterized by simultaneous decreases in cell stiffness and in plasma membrane microviscosity. Our findings, therefore, support the results of previous studies that have shown cell softening and membrane fluidization to be critical adaptive responses enabling cell movement and that these can be regarded as potential biomarkers of tumor cell motility, offering scope for identifying new therapeutic targets.

Three-dimensional (3D) cell spheroids are widely used asin vitrotissue models, yet quantitative understanding of their morphogenesis remains limited. We present an integrated experimental-computational framework to analyze, model, and modulate the compaction of cell aggregates in agarose microwells of defined geometries. Custom 3D-printed stamps produced circular, square, and triangular microwells of equal cross-sectional area. Time-lapse imaging combined with AI-based segmentation enabled tracking of spheroid morphology, with circularity and projected area serving as quantitative descriptors of compaction. The process followed predictable exponential kinetics, with mesenchymal (HDF) spheroids compacting faster than epithelial (ARPE-19) ones. Computational fluid dynamics (CFD) simulations modeled spheroid rounding as a visco-capillary-driven process, where the extracted visco-capillary velocity unified experimental and simulated dynamics. Mechanical measurements by atomic force microscopy and compression confirmed that differences in surface tension predominantly governed the observed kinetics. Pharmacological modulation of cytoskeletal tension revealed that inhibition of contractility markedly altered spheroid formation dynamics, enabling the generation of stable, non-spherical aggregates. Using this principle as a shape-engineering strategy, we produced aggregates with distinct geometries (brick-like, prismatic, and star-shaped), characterized by an increased surface-to-volume ratio compared to conventional spheroids. Limitations of the approach include the use of pharmacological cytoskeletal modulation and constraints in geometric fidelity arising from printing resolution, agarose casting, cell filling, and intrinsic smoothing of sharp features during cell aggregation. Collectively, this work establishes a geometry-controlled platform for quantitative analysis of spheroid formation and mechanical behavior, and provides a versatile framework for designing cell aggregates with defined shapes.

Three-dimensional (3D) cultivation of cells and tissues is currently of great interest for testing various drugs. Unlike standard cultivation, 3D cultures demonstrate increased resistance to anticancer drugs. In this work, we first developed a hydrogel based on bacterial cellulose (BC), polyvinyl alcohol (PVA) and polyethylene glycol (PEG) for 3D cultivation of tumor cells. The addition of 1.5

The morphological characterisation is crucial for analysing cell states, especially for red blood cells (RBCs), which are used in transfusions. This study compared the applicability of atomic force microscopy (AFM) and confocal optical profilometry in the accurate characterisation of the RBC morphological parameters. The imaging of RBCs thawed after cryopreservation with immediate and delayed washing steps (deglycerolisation) was performed, and the morphological data obtained with AFM and optical profilometry were compared with the clinical laboratory studies. Both techniques provided close data on the morphological parameters, but optical profilometry allowed a faster and more convenient data acquisition. However, the membrane roughness analysis on discocytes and the submembrane cytoskeleton analysis on RBC ghosts was only possible with AFM due to its higher spatial resolution. Both techniques confirmed that delayed washing did not have negative effects on cells compared to immediate washing. Additional 3-day storage of both types of RBCs resulted in increased haemolysis. A decrease in the fraction of area occupied by pores in the submembrane cytoskeleton with the storage time was observed, possibly associated with the cytoskeleton deterioration. The studied conditions model the transportation of thawed RBCs in a cryoprotectant solution to medical facilities that have technical conditions to wash thawed RBCs and confirm its feasibility.

Glycation, or non-enzymatic glycosylation, has recently attracted increasing interest in the context of its impact on aging. Advanced glycation end products (AGEs) contribute to various age-related pathological conditions such as inflammation, fibrosis, and vascular calcification. However, the molecular mechanisms underlying glycation-induced disruption of cell-matrix interactions during cellular senescence are not fully understood. The aim of this study was to investigate transcriptomic changes in young and senescent dermal fibroblasts (HdFbs) cultured in 3D post-glycated collagen type I matrices after 10 and 17 days. Our findings indicate that D-ribose-mediated glycation increases the accumulation of fluorescent AGEs and the stiffness of matrices in a dose-dependent manner. The transcriptome alterations in cells encompassed the modulation of age-related genes and signaling pathways, including activation of genes related to senescence-associated secretory phenotype (SASP). Notably, the alterations in the transcriptome profiles due to glycation were more pronounced (in terms of both the number of genes and their fold changes) after 10 days of culture compared to day 17 in both passages. These findings suggest that cellular responses to glycation and resulting stiffness depend on both the concentration of reducing sugar and the time spent under those conditions.

The modifications in biomechanical properties of cells and tissue are important in cancer progression, including its different aspects, e.g. invasion, migration, adhesion, signaling, interactions with microenvironment, and immune response. However, our understanding of the changes in physical characteristics of cells, and especially the molecular basis for these changes, caused by malignant transformation remains rather limited. While the differences in stiffness and viscoelasticity of tumor and normal cells have been well documented at the cellular scale, subcellular and molecular alterations have been poorly characterised. In our work, we investigated the stiffness and viscoelastic parameters, mainly determined by the actin cortex, and the microviscosity of the plasma membrane, mainly determined by its lipid profile, in normal and cancer cells. The mechanical properties of the cells were assessed using atomic force microscopy (AFM). The microviscosity of the membrane was visualized by fluorescence lifetime imaging microscopy (FLIM) with the viscosity-sensitive probe BODIPY2. Chemical analysis of cell membranes was performed by secondary ion time-of-flight mass spectrometry (ToF-SIMS). The MCF-10 A (normal epithelial cells) and MCF-7 (human breast cancer) cell lines were used in the study. It was shown that cancer cells were more deformable due to a less organized and more isotropic filamentous structure of the actin cytoskeleton. At the same time they had more viscous plasma membranes, compared to normal cells, in both in vitro and tissue conditions. In the membrane lipid profile, increased signals of sphingomyelin and saturated fatty acids and decreased signals of polyunsaturated fatty acids were detected in cancer cells, which explain the higher microviscosity of their membranes. The obtained data indicate a complex reorganization of cell biomechanics at the cellular, subcellular and molecular levels during malignant transformation, which is important for better understanding of fundamental mechanisms of tumor development.

Three-dimensional (3D) cell spheroids are widely used in biomedical research as in vitro tissue models, yet quantitative understanding of their morphogenesis remains limited. Here, we present an integrated experimental and computational approach to analyze and model the compaction of cell aggregates in agarose microwells with defined cross-sectional geometries. Custom 3D-printed stamps were designed to produce circular, square, and triangular microwells with equal cross-sectional area. Time-lapse imaging and AI-based segmentation were employed to track the evolution of spheroid morphology, with circularity and projected area used as quantitative indicators of compaction dynamics. We show that the compaction process follows predictable exponential trends in both parameters, with mesenchymal spheroids (from human dermal fibroblasts line HDF) compacting faster than epithelial spheroids (from ARPE-19 cells). Spheroid rounding was simulated as a visco-capillary-driven process with a computational fluid dynamics (CFD) model using the Volume of Fluid (VoF) method in OpenFOAM. The visco-capillary velocity extracted from both experimental and simulation data served as a unifying parameter that explained differences in compaction kinetics. Using additional mechanical measurements (AFM and compression), we estimated surface tension and effective viscosity, confirming that surface tension differences predominantly drive the observed kinetics. Pharmacological treatments modulating cytoskeletal tension revealed that contractility inhibition significantly modified spheroid formation dynamics, allowing acquisition of non-spherical cell aggregates. Taken together, our study establishes a robust, geometry-controlled platform for analyzing spheroid formation and quantifying their mechanical properties, as well as provides a framework for creating cellular aggregates of defined shapes. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Russian Science Foundation, grant No. 23-74-10113, https://rscf.ru/en/project/23-74-10113/ Ministry of Health of the Russian Federation, Theme No. NZAF-2024-0006 [1]: pending:yes

Multicellular structures, including cell sheets, are actively used as model systems to study intercellular interactions and can be applied in different areas of regenerative medicine. In this paper, we present a novel approach for measuring mechanical properties of cell sheets based on a simple experimental setup and numerical simulations. The advantage of the present approach is the relative ease of the sample preparation, while previous systems for the tensile tests required specialized and sensitive equipment. With the developed approach, the cell sheet on a polymer membrane is mounted in the holder on one side, and then deflection of the free end of the membrane is measured. The deflection of this cantilever-like construction depends on the elastic modulus of the membrane (which is known) and cell sheet, and also from the traction force generated by the cell sheet. By involving an experimental step with relaxing the traction force and by conducting finite element simulations, both traction force and elastic properties of the cell sheet can be estimated. We performed such measurements on cell sheets from keratocytes from corneal explants and confirmed that the developed approach is applicable for measurement of both traction force and elastic modulus of cell sheets.

The 4D printing of high-temperature shape memory polymers (SMPs) has attracted tremendous interest because of its potential application in smart devices in the aerospace field. The development of SMPs with high thermal and mechanical properties as well as radiation resistance is challenging. In this work, we developed UV-curable resins based on rigid thermally stable poly-2,2 '-(p-oxydiphenylene)-5,5 '-dibenzimidazole (OPBI), a photosensitive solvent (N,N-dimethylacrylamide), and a crosslinker (bisphenol A ethoxylate diacrylate or tris[2-(acryloyloxy)ethyl] isocyanurate). 4D-printed high-performance SMPs were obtained through a UV-thermal dualcuring process, which resulted in materials with high glass transition temperatures (155-185 degrees C) and increased tensile strengths (115-144 MPa). The combination of hard segments from OPBI and crosslinkers, as well as soft segments from N,N-dimethylacrylamide, results in excellent shape memory performance of the materials (the average shape fixity and shape recovery ratios both exceed 94 %). In addition, we found that 4Dprinted OPBI-based SMPs are resistant to gamma-radiation at doses up to 10<^>6 Gy. After gamma-irradiation, the materials maintain high tensile strengths (up to 124 MPa), glass transition temperatures (up to 179 degrees C) and shape memory behavior. Thus, these new 4D-printed OPBI-based SMPs offer great potential for the aerospace industry.

AIM:This study establishes the mechanism of stable emulsion capsules formation using sulfhydryl-free polysaccharides (xanthan gum, chitosan, and their mixtures), comparing ultrasonic versus conventional mechanical preparation methods. METHODS:Capsules were fabricated using both mechanical and ultrasonic processing, followed by comprehensive characterization through DLS, CLSM, CRYO electron microscopies, XPS, FTIR, AFM, XRD, and TGA. RESULTS:Ultrasonically processed xanthan gum/chitosan capsules exhibit a well-defined morphology (1 μm average size), stable surface characteristics (-19 mV zeta potential), and enhanced resistance to aggregation and coalescence. The results demonstrate not only formation of polyelectrolyte complexes in the mixed shells (with xanthan gum/chitosan ratios of 1 and 1.17 for mechanically and ultrasonically prepared capsules, respectively, with an initial 1:1 polymer solution ratio), but also reveals molecular scissoring effects. Structural characterization reveals semi-crystalline shell organization with significantly improved mechanical strength, as evidenced by the 48 kPa Young's modulus. The capsules exhibit excellent hemocompatibility (hemolysis rate < 0.02 μL/mL) for intravenous delivery applications. CONCLUSION:Our findings reveal fundamental insights into polysaccharide behavior on the phase interface under ultrasonication, demonstrating how acoustic energy drives molecular reorganization to create structurally superior capsules. This work provides a new paradigm for polysaccharide-based drug carrier design to create high-performance delivery systems with enhanced stability.

Soft tissues exhibit predominantly time-dependent mechanical behavior critical for their biological function in organs like the lungs and aorta, as they can deform and stretch at varying rates depending on their function. Collagen type I serves as the primary structural component in these tissues. The viscoelastic characteristics of such tissues, stemming from diverse energy dissipation mechanisms across various length scales, remains poorly characterized at the nanoscale. Prior experimental investigations have predominantly centered on analyzing tissue responses largely attributed to interactions between cells and fibers. Despite many studies on tissue viscoelasticity from scaffolds to single collagen fibrils, the time-dependent mechanics of collagen fibrils at the sub-fibrillar level remain poorly understood. This pioneering study employs atomic force microscopy (AFM) nano-rheometry and indentation testing to examine the viscoelastic characteristics of individual collagen type I fibrils at the ultrastructural level within distinct topographical zones, specifically focusing on gap and overlap regions. Our investigation has unveiled that collagen fibrils display a viscoelastic response that replicates the mechanical behavior of the tissue at the macroscale. Further, our findings suggest a distinct viscoelastic behavior between the gap and overlap regions, likely stemming from variances in molecular organization and cross-linking modalities within these specific sites. The results of our investigation provide unequivocal proof of the temporal dependence of mechanical properties and provides unique data to be compared to atomistic models, laying a foundation for refining the precision of macroscale models that strive to capture tissue viscoelasticity across varying length scales. Statement of Significance Soft tissues such as the lungs and aorta depend on collagen to stretch and perform their functions, which involve continuous and dynamic deformation. Although these tissues are known to exhibit viscoelastic behavior, the mechanisms behind this at the fine scale of individual collagen fibril ultrastructure are not well understood. In this study, we used AFM nano-rheometry and direct indentation to be the first to directly measure the viscoelastic properties of collagen at the ultrastructural level. We discovered that single fibrils show time-dependent behavior similar to that of whole tissues, with distinct mechanical differences between regions likely due to variations in molecular organization and bonding. These insights advance our understanding of tissue mechanics and contribute to more accurate multi-scale modeling.

Calcium is a key macroelement involved in a range of physiological processes in the body, and its concentration in blood is an important diagnostic indicator in various diseases. This work presents a novel rapid method for the point-of-care determination of total calcium content in patient blood, by applying a drop of capillary blood from a finger onto a hydrogel. Gelatin hydrogel, modified with an optical sensor for calcium, Arsenazo III, was used as a platform for the separation of blood into plasma and erythrocytes. A comparative analysis of various types of hydrogel materials (polyacrylamide, PVA, Fmoc-FF, carbomer, carbopol, gelatin) was performed, demonstrating that among the studied systems, only gelatin hydrogel is suitable as a platform for the determination of calcium in blood plasma. The binding of calcium ions from blood plasma with the calcium sensor embedded in the hydrogel leads to a change in the absorption spectrum of the system, enabling photometric determination of calcium concentrations below and above the normal range in blood plasma. Therefore, this rapid assay allows monitoring of calcium metabolism disorders in the human organism. The method is characterized by its speed, simplicity of sample preparation, and potential for integration into clinical practice.

Wound care remains a significant healthcare challenge, with associated costs continuing to rise over the past decade. The advancement of innovative wound dressings that actively facilitate healing has emerged as a pivotal research focus. This study introduces a novel design for functional microstructured wound dressings, utilising microencapsulation technology to enhance wound healing. This methodology allows for precise tuning of the chemical microenvironment within the wound bed. An original solid-powder encapsulation methodology is employed, facilitating the sustained and controlled release of bioactive compounds. The effects of sustained release are demonstrated by modulating oxidative stress through the administration of antioxidants and hydrogen peroxide. Sustained release of active compounds within the wound is shown to modulate angiogenesis, exert antiseptic and anti-inflammatory effects, and promote scar-free tissue formation. The fundamental role of ultralow doses and sustained-release of hydrogen peroxide and antioxidants in tissue regeneration was simultaneously investigated. The observed bioeffects of microchamber-based wound dressings highlight the potential of advanced materials and encapsulation strategies to actively regulate the wound milieu and provide sustained, tailored therapeutic effects by a slight shift of the chemical profile. The proposed technique establishes a foundation for the development of wound healing systems characterised by hierarchical multifunctionality. Our approach presents a variety of unique features and potential applications, including anisotropic manipulations, site specific and on-demand release, integration with cell therapy, and more. Further research is warranted to explore the clinical translation and broader applications of this technology for wound management.

Biomimetic hydrogels have garnered increased interest due to their considerable potential for use in various fields, such as tissue engineering, 3D cell cultivation, and drug delivery. The primary challenge for applying hydrogels in tissue engineering is accurately evaluating their mechanical characteristics. In this context, we propose a method using scanning ion conductance microscopy (SICM) to determine the rigidity of living human breast cancer cells MCF-7 cells grown on a soft, self-assembled Fmoc-FF peptide hydrogel. Moreover, it is demonstrated that the map of Young’s modulus distribution obtained by the SICM method allows for determining the core location. The Young’s modules for MCF-7 cells decrease with the substrate stiffening, with values of 1050 Pa, 835 Pa, and 600 Pa measured on a Petri dish, Fmoc-FF hydrogel, and Fmoc-FF/chitosan hydrogel, respectively. A comparative analysis of the SICM results and the data obtained by atomic force microscopy was in good agreement, allowing for the use of a composite cell–substrate model (CoCS) to evaluate the ‘soft substrate effect’. Using the CoCS model allowed us to conclude that the MCF-7 softening was due to the cells’ mechanical properties variations due to cytoskeletal changes. This research provides immediate insights into changes in cell mechanical properties resulting from different soft scaffold substrates.