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Macrophages are the major players and orchestrators of inflammatory response. Expressed proteins and secreted cytokines have been well studied for two polar macrophage phenotypes—pro-inflammatory M1 and anti-inflammatory regenerative M2, but little is known about how the polarization modulates macrophage functions. In this study, we used biochemical and biophysical methods to compare the functional activity and mechanical properties of activated human macrophages differentiated from monocyte with GM-CSF (M0_GM) and M-CSF (M0_M) and polarized into M1 and M2 phenotypes, respectively. Unlike GM-CSF, which generates dormant cells with low activity, M-CSF confers functional activity on macrophages. M0_M and M2 macrophages had very similar functional characteristics—high reactive oxygen species (ROS) production level, and higher phagocytosis and survival compared to M1, while M1 macrophages showed the highest radical-generating activity but the lowest phagocytosis and survival among all phenotypes. All phenotypes decreased their height upon activation, but only M1 and M2 cells increased in stiffness, which can indicate a decrease in the migration ability of these cells and changes in their interactions with other cells. Our results demonstrated that while mechanical properties differ between M0 and polarized cells, all four phenotypes of monocyte-derived macrophages differ in their functional activities, namely in cytokine secretion, ROS production, and phagocytosis. Within the broad continuum of human macrophages obtained in experimental models and existing in vivo, there is a diversity of phenotypes with varying combinations of both markers and functional activities. [ABSTRACT FROM AUTHOR]

The biophysical properties of cells described at the level of whole cells or their membranes have many consequences for their biological behavior. However, our understanding of the relationships between mechanical parameters at the level of cell (stiffness, viscoelasticity) and at the level of the plasma membrane (fluidity) remains quite limited, especially in the context of pathologies, such as cancer. Here, we investigated the correlations between cells' stiffness and viscoelastic parameters, mainly determined via the actin cortex, and plasma membrane microviscosity, mainly determined via its lipid profile, in cancer cells, as these are the keys to their migratory capacity. The mechanical properties of cells were assessed using atomic force microscopy (AFM). The microviscosity of membranes was visualized using fluorescence-lifetime imaging microscopy (FLIM) with the viscosity-sensitive probe BODIPY 2. Measurements were performed for five human colorectal cancer cell lines that have different migratory activity (HT29, Caco-2, HCT116, SW 837, and SW 480) and their chemoresistant counterparts. The actin cytoskeleton and the membrane lipid composition were also analyzed to verify the results. The cell stiffness (Young's modulus), measured via AFM, correlated well (Pearson r = 0.93) with membrane microviscosity, measured via FLIM, and both metrics were elevated in more motile cells. The associations between stiffness and microviscosity were preserved upon acquisition of chemoresistance to one of two chemotherapeutic drugs. These data clearly indicate that mechanical parameters, determined by two different cellular structures, are interconnected in cells and play a role in their intrinsic migratory potential. [ABSTRACT FROM AUTHOR]

Recent developments such as multi-harmonic Atomic Force Microscopy (AFM) techniques have enabled fast, quantitative mapping of nanomechanical properties of living cells. Due to their high spatiotemporal resolution, these methods provide new insights into changes of mechanical properties of subcellular structures due to disease or drug response. Here, we propose three new improvements to significantly improve the resolution, identification, and mechanical property quantification of sub-cellular and sub-nuclear structures using multi-harmonic AFM on living cells. First, microcantilever tips are streamlined using long-carbon tips to minimize long-range hydrodynamic interactions with the cell surface, to enhance the spatial resolution of nanomechanical maps and minimize hydrodynamic artifacts. Second, simultaneous Spinning Disk Confocal Microscopy (SDC) with live-cell fluorescent markers enables the unambiguous correlation between observed heterogeneities in nanomechanical maps with subcellular structures. Third, computational approaches are then used to estimate the mechanical properties of sub-nuclear structures. Results are demonstrated on living NIH 3T3 fibroblasts and breast cancer MDA-MB-231 cells, where properties of nucleoli, a deep intracellular structure, were assessed. The integrated approach opens the door to study the mechanobiology of sub-cellular structures during disease or drug response. [ABSTRACT FROM AUTHOR]

Biological self-assembly is crucial in the processes of development, tissue regeneration, and maturation of bioprinted tissue-engineered constructions. The cell aggregates—spheroids—have become widely used model objects in the study of this phenomenon. Existing approaches describe the fusion of cell aggregates by analogy with the coalescence of liquid droplets and ignore the complex structural properties of spheroids. Here, we analyzed the fusion process in connection with structure and mechanical properties of the spheroids from human somatic cells of different phenotypes: mesenchymal stem cells from the limbal eye stroma and epithelial cells from retinal pigment epithelium. A nanoindentation protocol was applied for the mechanical measurements. We found a discrepancy with the liquid drop fusion model: the fusion was faster for spheroids from epithelial cells with lower apparent surface tension than for mesenchymal spheroids with higher surface tension. This discrepancy might be caused by biophysical processes such as extracellular matrix remodeling in the case of mesenchymal spheroids and different modes of cell migration. The obtained results will contribute to the development of more realistic models for spheroid fusion that would further provide a helpful tool for constructing cell aggregates with required properties both for fundamental studies and tissue reparation. [ABSTRACT FROM AUTHOR]

Tissue engineering has emerged as an indispensable tool for the reconstruction of organ-specific environments. Organ-derived extracellular matrices (ECM) and, especially, decellularized tissues (DCL) are recognized as the most successful biomaterials in regenerative medicine, as DCL preserves the most essential organ-specific ECM properties such as composition alongside biomechanics characterized by stiffness and porosity. Expansion of the DCL technology to cancer biology research, drug development, and nanomedicine is pending refinement of the existing DCL protocols whose reproducibility remains sub-optimal varying from organ to organ. We introduce a facile decellularization protocol universally applicable to murine organs, including liver, lungs, spleen, kidneys, and ovaries, with demonstrated robustness, reproducibility, high purification from cell debris, and architecture preservation, as confirmed by the histological and SEM analysis. The biomechanical properties of as-produced DCL organs expressed in terms of the local and total stiffness were measured using our facile methodology and were found well preserved in comparison with the intact organs. To demonstrate the utility of the developed DCL model to cancer research, we engineered three-dimensional tissue constructs by recellularization representative decellularized organs and collagenous hydrogel with human breast cancer cells of pronounced mesenchymal (MDA-MB-231) or epithelial (SKBR-3) phenotypes. The biomechanical properties of the DCL organs were found pivotal to determining the cancer cell fate and progression. Our histological and scanning electron microscopy (SEM) study revealed that the larger the ECM mean pore size and the smaller the total stiffness (as in lung and ovary), the more proliferative and invasive the mesenchymal cells became. At the same time, the low local stiffness ECMs (ranged 2.8–3.6 kPa) did support the epithelial-like SKBR-3 cells' viability (as in lung and spleen), while stiff ECMs did not. The total and local stiffness of the collagenous hydrogel was measured too low to sustain the proliferative potential of both cell lines. The observed cell proliferation patterns were easily interpretable in terms of the ECM biomechanical properties, such as binding sites, embedment facilities, and migration space. As such, our three-dimensional tissue engineering model is scalable and adaptable for pharmacological testing and cancer biology research of metastatic and primary tumors, including early metastatic colonization in native organ-specific ECM. [ABSTRACT FROM AUTHOR]

The original article can be found online at https://doi.org/10.1038/s41598-019-42077-1. Correstion to: I Scientific Reports i https://doi.org/10.1038/s41598-019-42077-1, published online 08 April 2019 The original version of this Article contained an error in the Discussion, under the subheading 'Relationship between cytoskeletal organization and mechanical properties', "A positive correlation was found between the density of stress fibers and cell stiffness SP 4,31,32,33 sp and between cell stiffness and cell height SP 34,64 sp ." now reads: "A positive correlation was found between the density of stress fibers and cell stiffness SP 4,31,32,33 sp , while cell stiffness and cell height are inversely correlated SP 34,64 sp .". [Extracted from the article]

Background: The nucleus, besides its functions in the gene maintenance and regulation, plays a significant role in the cell mechanosensitivity and mechanotransduction. It is the largest cellular organelle that is often considered as the stiffest cell part as well. Interestingly, the previous studies have revealed that the nucleus might be dispensable for some of the cell properties, like polarization and 1D and 2D migration. Here, we studied how the nanomechanical properties of cells, as measured using nanomechanical mapping by atomic force microscopy (AFM), were affected by the removal of the nucleus. Methods: The mass enucleation procedure was employed to obtain cytoplasts (enucleated cells) and nucleoplasts (nuclei surrounded by plasma membrane) of two cell lines, REF52 fibroblasts and HT1080 fibrosarcoma cells. High-resolution viscoelastic mapping by AFM was performed to compare the mechanical properties of normal cells, cytoplasts, and nucleoplast. The absence or presence of the nucleus was confirmed with fluorescence microscopy, and the actin cytoskeleton structure was assessed with confocal microscopy. Results: Surprisingly, we did not find the softening of cytoplasts relative to normal cells, and even some degree of stiffening was discovered. Nucleoplasts, as well as the nuclei isolated from cells using a detergent, were substantially softer than both the cytoplasts and normal cells. Conclusions: The cell can maintain its mechanical properties without the nucleus. Together, the obtained data indicate the dominating role of the actomyosin cytoskeleton over the nucleus in the cell mechanics at small deformations inflicted by AFM. [ABSTRACT FROM AUTHOR]

The measurement of local mechanical properties of living cells by nano/micro indentation relies on the foundational assumption of locally isotropic cellular deformation. As a consequence of assumed isotropy, the cell membrane and underlying cytoskeleton are expected to locally deform axisymmetrically when indented by a spherical tip. Here, we directly observe the local geometry of deformation of membrane and cytoskeleton of different living adherent cells during nanoindentation with the integrated Atomic Force (AFM) and spinning disk confocal (SDC) microscope. We show that the presence of the perinuclear actin cap (apical stress fibers), such as those encountered in cells subject to physiological forces, causes a strongly non-axisymmetric membrane deformation during indentation reflecting local mechanical anisotropy. In contrast, axisymmetric membrane deformation reflecting mechanical isotropy was found in cells without actin cap: cancerous cells MDA-MB-231, which naturally lack the actin cap, and NIH 3T3 cells in which the actin cap is disrupted by latrunculin A. Careful studies were undertaken to quantify the effect of the live cell fluorescent stains on the measured mechanical properties. Using finite element computations and the numerical analysis, we explored the capability of one of the simplest anisotropic models – transverse isotropy model with three local mechanical parameters (longitudinal and transverse modulus and planar shear modulus) – to capture the observed non-axisymmetric deformation. These results help identifying which cell types are likely to exhibit non-isotropic properties, how to measure and quantify cellular deformation during AFM indentation using live cell stains and SDC, and suggest modelling guidelines to recover quantitative estimates of the mechanical properties of living cells. [ABSTRACT FROM AUTHOR]

Biodegradable polymeric fibrous non-woven materials are widely used type of scaffolds for tissue engineering. Their morphology and properties could be controlled by composition and fabrication technology. This work is aimed at development of fibrous scaffolds from a multicomponent polymeric system containing biodegradable synthetic (polylactide, polycaprolactone) and natural (gelatin, chitosan) components using different methods of non-woven mats fabrication: electrospinning and electro-assisted solution blow spinning. The effect of the fabrication technique of the fibrous materials onto their morphology and properties, including the ability to support adhesion and growth of cells, was evaluated. The mats fabricated using electrospinning technology consist of randomly oriented monofilament fibers, while application of solution blow spinning gave a rise to chaotically arranged multifilament fibers. Cytocompatibility of all fabricated fibrous mats was confirmed using in vitro analysis of metabolic activity, proliferative capacity and morphology of NIH 3T3 cell line. Live/Dead assay revealed the formation of the highest number of cell–cell contacts in the case of multifilament sample formed by electro-assisted solution blow spinning technology. [ABSTRACT FROM AUTHOR]

Thermally responsive shape memory polyimides (PIs) have attracted significant attention in the high-tech fields like aerospace industry. At the same time creating of materials and approaches to the formation of three-dimensional (3D) shape memory structures containing PIs remains an urgent task. In this work we have developed new photopolymeric compositions (PPCs) based on high-performance readily soluble PI, reactive solvent (N,N-dimethylacrylamide) and photoactive cross-linker (bisphenol A ethoxylate diacrylate (BAEDA) or tris [2-(acryloyloxy)ethyl] isocyanurate (TAI)). The resulting PPCs were used to form 3D-structures using LCD 3D-printing. It has been found that the nature of the cross-linker and post-processing temperature have a significant impact on the thermal and mechanical properties of the materials. The tensile strength has the maximum value after the thermal post-curing at 250°С for 1 h (83.3 ± 7.4 MPa and 60.8 ± 4.8 MPa for PPCs with TAI and BAEDA, respectively). Moreover, 4D-printed PI-containing materials exhibit an excellent shape memory performance at recovery temperatures >150 °C, with shape fixity ratio >99% and shape recovery ratio up to 95.6%. [Display omitted] • A composition based on PI, TAI or BAEDA and DMAA can be used for LCD printing. • The materials are characterized by a high tensile strength (up to 83.3 ± 7.4 MPa). • The 4D-printed structures possess a good shape memory performance at T rec = 194 °C. [ABSTRACT FROM AUTHOR]

Most of the presently known thermosensitive shape‐memory polymers suitable for 4D printing have insufficient mechanical strength and thermal stability that restricts their potential areas of application. Here, new photosensitive compositions (PSCs) based on aromatic heterochain polymers – poly‐N,N′‐(m‐phenylene)isophthalamide (MPA) or poly‐2,2′‐(p‐oxydiphenylene)‐5,5′‐dibenzimidazole (OPBI) – for DLP printing are proposed. Thermal post‐curing and supercritical carbon dioxide (scCO2) are used for post‐processing of the structures. During the scCO2 treatment the removal of unreacted monomeric component (N,N‐dimethylacrylamide) and its uncrosslinked oligomers is accompanied by the preservation of the initial degree of crosslinking. The more stable shrinkage is observed for the combined post‐processing method (T°+scCO2) in the case of OPBI‐PSC and after the heat treatment for MPA‐PSC specimens. The method of post‐processing and the nature of the heterochain polymer strongly affect the mechanical properties and thermal resistance of the structures. The tensile strength has the maximum value after the thermal post‐treatment (101.1 ± 7.1 and 78.4 ± 5.1 MPa of OPBI‐PSC and MPA‐PSC, respectively). The intense destruction of the materials is observed at 393 and 408 °C for MPA‐PSC and OPBI‐PSC, respectively. Moreover, the 4D‐printed structures exhibit excellent shape memory performance at transition temperatures >100 °C, thus have a great potential for the use in aerospace, robotics, sensorics. [ABSTRACT FROM AUTHOR]

The self‐assembly of peptides is a key direction for fabrication of advanced materials. Novel approaches for fine tuning of macroscopic and microscopic properties of peptide self‐assemblies are of a high demand for constructing biomaterials with desired properties. In this work, while studying the kinetics of the Fmoc‐Diphenylalanine (Fmoc‐FF) dipeptide self‐assembly using the Thioflavin T (ThT) dye, we observed that the presence of ThT strongly modifies structural and mechanical properties of the Fmoc‐FF hydrogel. Notably, the presence of ThT resulted in a tenfold increase of the gelation time and in the formation of short and dense fibers in the hydrogel. As a result of these morphological alteration higher thermal stability, and most important, tenfold increase of the hydrogel rigidity was achieved. Hence, ThT not only slowed the kinetics of the Fmoc‐FF hydrogel formation, but also strongly enhanced its mechanical properties. In this study, we provide a detailed description of the ThT effect on the hydrogel properties and suggest the mechanisms for this phenomenon, paving the way for the novel approach to the control of the peptide hydrogels' micro‐ and macroscale properties. [ABSTRACT FROM AUTHOR]

The self‐assembly of peptides is a key direction for fabrication of advanced materials. Novel approaches for fine tuning of macroscopic and microscopic properties of peptide self‐assemblies are of a high demand for constructing biomaterials with desired properties. In this work, while studying the kinetics of the Fmoc‐Diphenylalanine (Fmoc‐FF) dipeptide self‐assembly using the Thioflavin T (ThT) dye, we observed that the presence of ThT strongly modifies structural and mechanical properties of the Fmoc‐FF hydrogel. Notably, the presence of ThT resulted in a tenfold increase of the gelation time and in the formation of short and dense fibers in the hydrogel. As a result of these morphological alteration higher thermal stability, and most important, tenfold increase of the hydrogel rigidity was achieved. Hence, ThT not only slowed the kinetics of the Fmoc‐FF hydrogel formation, but also strongly enhanced its mechanical properties. In this study, we provide a detailed description of the ThT effect on the hydrogel properties and suggest the mechanisms for this phenomenon, paving the way for the novel approach to the control of the peptide hydrogels' micro‐ and macroscale properties. [ABSTRACT FROM AUTHOR]

Poly(3-hydroxybutyrate) and chitosan are among the most widely used polymers for biomedical applications due to their biocompatibility, renewability and low toxicity. The creation of composite materials based on biopolymers belonging to different classes makes it possible to overcome the disadvantages of each of the components and to obtain a material with specific properties. Solving this problem is associated with difficulties in the selection of conditions and solvents for obtaining the composite material. In our study, acetic acid was used as a common solvent for hydrophobic poly(3-hydroxybutyrate) and chitosan. Mechanical, thermal, physicochemical and surface properties of the composites and homopolymers were investigated. The composite films had less crystallinity and hydrophobicity than poly(3-hydroxybutyrate), and the addition of chitosan caused an increase in moisture absorption, a decrease in contact angle and changes in mechanical properties of the poly(3-hydroxybutyrate). The inclusion of varying amounts of chitosan controlled the properties of the composite, which will be important in the future for its specific biomedical applications. [ABSTRACT FROM AUTHOR]

Cytotoxicity assays are essential tests in studies on the safety and biocompatibility of various substances and on the efficiency of anticancer drugs. The most frequently used assays commonly require application of externally added labels and read only collective response of cells. Recent studies show that the internal biophysical parameters of cells can be associated with the cellular damage. Therefore, using atomic force microscopy, we assessed the changes in the viscoelastic parameters of cells treated with eight different common cytotoxic agents to gain a more systematic view of the occurring mechanical changes. With the robust statistical analysis to account for both the cell-level variability and the experimental reproducibility, we have found that cell softening is a common response after each treatment. More precisely, the combined changes in the viscoelastic parameters of power-law rheology model led to a significant decrease of the apparent elastic modulus. The comparison with the morphological parameters (cytoskeleton and cell shape) demonstrated a higher sensitivity of the mechanical parameters versus the morphological ones. The obtained results support the idea of cell mechanics-based cytotoxicity tests and suggest a common way of a cell responding to damaging actions by softening. [Display omitted] • Cell mechanical properties change during different cytotoxic effects. • Cell softening was observed after treatment with a range of different cytotoxic agents. • Softening might be a universal and sensitive cell response to cytotoxic effects. [ABSTRACT FROM AUTHOR]

Significance: Terahertz (THz) radiation has demonstrated a great potential in biomedical applications over the past three decades, mainly due to its non-invasive and label-free nature. Among all biological specimens, skin tissue is an optimal sample for the application of THz-based methods because it allows for overcoming some intrinsic limitations of the technique, such as a small penetration depth (0.1 to 0.3 mm for the skin, on average). Aim: We summarize the modern research results achieved when THz technology was applied to the skin, considering applications in both imaging/detection and treatment/modulation of the skin constituents. Approach: We perform a review of literature and analyze the recent research achievements in THz applications for skin diagnosis and investigation. Results: The reviewed results demonstrate the possibilities of THz spectroscopy and imaging, both pulsed and continuous, for diagnosis of skin melanoma and non-melanoma cancer, dysplasia, scars, and diabetic condition, mainly based on the analysis of THz optical properties. The possibility of modulating cell activity and treatment of various diseases by THz-wave exposure is shown as well. Conclusions: The rapid development of THz technologies and the obtained research results for skin tissue highlight the potential of THz waves as a research and therapeutic instrument. The perspectives on the use of THz radiation are related to both non-invasive diagnostics and stimulation and control of different processes in a living skin tissue for regeneration and cancer treatment. [ABSTRACT FROM AUTHOR]

The mimicking of the architectonics of native tissue, biodegradable non-woven fibrous mats is one of the most promising forms of scaffolding for tissue engineering. The key properties needed for their successful application in vivo, such as biodegradability, biocompatibility, morphology, mechanical properties, etc., rely on their composition and appropriate 3D structure. A multicomponent system based on biodegradable synthetic (polycaprolactone, oligo-/polylactide) and natural (chitosan, gelatin) polymers, providing the desired processing characteristics and functionality to non-woven mats fabricated via the electrospinning technique, was developed. The solid-state reactive blending of these components provided a one-step synthesis of amphiphilic graft copolymer with an ability to form stable ultra-fine dispersions in chlorinated solvents, which could be successfully used as casting solvents for the electrospinning technique. The synthesized graft copolymer was analyzed with the aim of fractional analysis, dynamic laser scattering, FTIR-spectroscopy and DSC. Casting solution characteristics, namely viscosity, surface tension, and electroconductivity, as well as electrospinning parameters, were studied and optimized. The morphology, chemical structure of the surface layer, mechanical properties and cytocompatibility were analyzed to confirm the appropriate functionality of the formed fibrous materials as scaffolds for tissue engineering. [ABSTRACT FROM AUTHOR]

Renal cancer would greatly benefit from new therapeutic strategies since, in advanced stages, it is refractory to classical chemotherapeutic approaches. In this context, lysosomal protease cysteine cathepsins may represent new pharmacological targets. In renal cancer, they are characterized by a higher expression, and they were shown to play a role in its aggressiveness and spreading. Traditional studies in the field were focused on understanding the therapeutic potentialities of cysteine cathepsin inhibition, while the direct impact of such therapeutics on the expression of these enzymes was often overlooked. In this work, we engineered two fluoromethyl ketone-based peptides with inhibitory activity against cathepsins to evaluate their potential anticancer activity and impact on the lysosomal compartment in human renal cancer. Molecular modeling and biochemical assays confirmed the inhibitory properties of the peptides against cysteine cathepsin B and L. Different cell biology experiments demonstrated that the peptides could affect renal cancer cell migration and organization in colonies and spheroids, while increasing their adhesion to biological substrates. Finally, these peptide inhibitors modulated the expression of LAMP1, enhanced the expression of E-cadherin, and altered cathepsin expression. In conclusion, the inhibition of cysteine cathepsins by the peptides was beneficial in terms of cancer aggressiveness; however, they could affect the overall expression of these proteases. [ABSTRACT FROM AUTHOR]

Significance: Currently, various scaffolds with immobilized cells are widely used in tissue engineering and regenerative medicine. However, the physiological activity and cell viability in such constructs might be impaired due to a lack of oxygen and nutrients. Photobiomodulation (PBM) is a promising method of preconditioning cells to increase their metabolic activity and to activate proliferation or differentiation. Aim: Investigation of the potential of PBM for stimulation of cell activities in hydrogels. Approach: Mesenchymal stromal cells (MSCs) isolated from human gingival mucosa were encapsulated in modified fibrin hydrogels with different thicknesses and concentrations. Constructs with cells were subjected to a single-time exposure to red (630 nm) and near-infrared (IR) (840 nm) low-intensity irradiation. After 3 days of cultivation, the viability and physiological activity of the cells were analyzed using confocal microscopy and a set of classical tests for cytotoxicity. Results: The cell viability in fibrin hydrogels depended both on the thickness of the hydrogels and the concentration of gel-forming proteins. The PBM was able to improve cell viability in hydrogels. The most pronounced effect was achieved with near-IR irradiation at the 840-nm wavelength. Conclusions: PBM using near-IR light can be applied for stimulation of MSCs metabolism and proliferation in hydrogel-based constructs with thicknesses up to 3 mm. [ABSTRACT FROM AUTHOR]