Interfacial blending in co-axially electrospun polymer core-shell fibers and their interaction with cells via focal adhesion point analysis.

Electrospun polymer scaffolds have gained prominence in biomedical applications, including tissue engineering, drug delivery, and wound dressings, due to their customizable properties. As the interplay between cells and materials assumes fundamental significance in biomaterials research, understanding the relationship between fiber properties and cell behaviour is imperative. Nevertheless, altering fiber properties introduces complexity by intertwining mechanical and surface chemistry effects, challenging the differentiation of their individual impacts on cell behaviour. Core-shell fibers present an appealing solution, enabling the control of mechanical properties of scaffolds, flexibility in material and drug selection, efficient encapsulation, strong protection of bioactive drugs against harsh environments, and controlled, prolonged drug release. This study addresses a key challenge in core-shell fiber design related to the blending effect between core and shell polymers. Two types of fibers, PMMA and core-shell PC-PMMA, were electrospun, and thorough analyses confirmed the desired core-shell structure in PC-PMMA fibers. Surface chemistry analysis revealed PC diffusion to the PMMA shell of the core-shell fiber during electrospinning, subsequently prompting an investigation of the fiber's surface potential. Conducting cellular studies on osteoblasts by super-resolution confocal microscopy provided insights into the direct influence of interfacial polymer blending and, consequently, altered fiber surface and mechanical properties on cell focal adhesion points, bridging the gap between material attributes and cell responses in core-shell fibers.

Cell-Material Interplay in Focal Adhesion Points.

The complex interplay between cells and materials is a key focus of this research, aiming to develop optimal scaffolds for regenerative medicine. The need for tissue regeneration underscores understanding cellular behavior on scaffolds, especially cell adhesion to polymer fibers forming focal adhesions. Key proteins, paxillin and vinculin, regulate cell signaling, migration, and mechanotransduction in response to the extracellular environment. This study utilizes advanced microscopy, specifically the AiryScan technique, along with advanced image analysis employing the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) cluster algorithm, to investigate protein distribution during osteoblast cell adhesion to polymer fibers and glass substrates. During cell attachment to both glass and polymer fibers, a noticeable shift in the local maxima of paxillin and vinculin signals is observed at the adhesion sites. The focal adhesion sites on polymer fibers are smaller and elliptical but exhibit higher protein density than on the typical glass surface. The characteristics of focal adhesions, influenced by paxillin and vinculin, such as size and density, can potentially reflect the strength and stability of cell adhesion. Efficient adhesion correlates with well-organized, larger focal adhesions characterized by increased accumulation of paxillin and vinculin. These findings offer promising implications for enhancing scaffold design, evaluating adhesion to various substrates, and refining cellular interactions in biomedical applications.

Mimicking natural electrical environment with cellulose acetate scaffolds enhances collagen formation of osteoblasts.

The medical field is continuously seeking new solutions and materials, where cellulose materials due to their high biocompatibility have great potential. Here we investigate the applicability of cellulose acetate (CA) electrospun fibers for bone tissue regeneration. For the first time we show the piezoelectric properties of electrospun CA fibers via high voltage switching spectroscopy piezoresponse force microscopy (HVSS-PFM) tests, which are followed by surface potential studies using Kelvin probe force microscopy (KPFM) and zeta potential measurements. Piezoelectric coefficient for CA fibers of 6.68 ± 1.70 pmV-1 along with high surface (718 mV) and zeta (-12.2 mV) potentials allowed us to mimic natural electrical environment favoring bone cell attachment and growth. Importantly, the synergy between increased surface potential and highly developed structure of the fibrous scaffold led to the formation of a vast 3D network of collagen produced by osteoblasts only after 7 days of in vitro culture. We clearly show the advantages of CA scaffolds as a bone replacement material, when long-lasting structural support is needed.

Stretchable skin hydrating PVB patches with controlled pores' size and shape for deliberate evening primrose oil spreading, transport and release.

With the increasing number of skin problems such as atopic dermatitis and the number of affected people, scientists are looking for alternative treatments to standard ointment or cream applications. Electrospun membranes are known for their high porosity and surface to volume area, which leads to a great loading capacity and their applications as skin patches. Polymer fibers are widely used for biomedical applications such as drug delivery systems or regenerative medicine. Importantly, fibrous meshes are used as oil reservoirs due to their excellent absorption properties. In our study, nano- and microfibers of poly (vinyl butyral-co-vinyl alcohol-co-vinyl acetate) (PVB) were electrospun. The biocompatibility of PVB fibers was confirmed with the keratinocytes culture studies, including cells' proliferation and replication tests. To verify the usability and stretchability of electrospun membranes, they were tested in two forms as-spun and elongated after uniaxially stretched. We examine oil transport through the patches for as-spun fibers and compare it with the numerical simulation of oil flow in the 3D reconstruction of nano- and microfiber networks. Evening primrose oil spreading and water vapor transmission rate (WVTR) tests were performed too. Finally, for skin hydration tests, manufactured materials loaded with evening primrose oil were applied to the forearm of volunteers for 6 h, showing increased skin moisture after using patches. This study clearly demonstrates that pore size and shape, together with fiber diameter, influence oil transport in the electrospun patches allowing to understand the key driving process of electrospun PVB patches for skin hydration applications. The oil release improves skin moisture and can be designed regarding the needs, by manufacturing different fibers' sizes and arrangements. The fibrous based patches loaded with oils are easy to handle and could remain on the altered skin for a long time and deliver the oil, therefore they are an ideal material for overnight bandages for skin treatment.

Tackling Tumour Cell Heterogeneity at the Super-Resolution Level in Human Colorectal Cancer Tissue.

Tumour cell heterogeneity, and its early individual diagnosis, is one of the most fundamental problems in cancer diagnosis and therapy. Single molecule localisation microscopy (SMLM) resolves subcellular features but has been limited to cultured cell lines only. Since nuclear chromatin architecture and microRNAs are critical in metastasis, we introduce a first-in-field approach for quantitative SMLM-analysis of chromatin nanostructure in individual cells in resected, routine-pathology colorectal carcinoma (CRC) patient tissue sections. Chromatin density profiles proved to differ for cells in normal and carcinoma colorectal tissues. In tumour sections, nuclear size and chromatin compaction percentages were significantly different in carcinoma versus normal epithelial and other cells of colorectal tissue. SMLM analysis in nuclei from normal colorectal tissue revealed abrupt changes in chromatin density profiles at the nanoscale, features not detected by conventional widefield microscopy. SMLM for microRNAs relevant for metastasis was achieved in colorectal cancer tissue at the nuclear level. Super-resolution microscopy with quantitative image evaluation algorithms provide powerful tools to analyse chromatin nanostructure and microRNAs of individual cells from normal and tumour tissue at the nanoscale. Our new perspectives improve the differential diagnosis of normal and (metastatically relevant) tumour cells at the single-cell level within the heterogeneity of primary tumours of patients.

Enhanced Cells Anchoring to Electrospun Hybrid Scaffolds With PHBV and HA Particles for Bone Tissue Regeneration.

Hybrid materials combining organic and inorganic compounds used as scaffolds are highly beneficial in bone regeneration. In this study, we successfully produced by blend electrospinning poly(3-hydroxybutyric acid-co-3-hydrovaleric acid) (PHBV) scaffolds enriched with hydroxyapatite (HA) particles to biomimic bone tissue for improved and faster regeneration processes. The morphology, fiber diameters, and composition of the scaffolds were investigated by scanning electron microscopy (SEM) techniques followed by focused ion beam (FIB) sectioning to verify HA particles integration with PHBV fibers. In vitro cell culture was performed for 7 days and followed with the cell proliferation test (CellTiter-Blue® Assay). Additionally, cell integration with the scaffold was visualized by confocal and SEM imaging. We developed a simple way of obtaining hybrid scaffolds by electrospinning PHBV solution with HA particles without any post-processing. The PHBV + HA scaffold enhanced cell proliferation and filopodia formation responsible for cell anchoring within the created 3D environment. The obtained results show the great potential in the development of hybrid scaffolds stimulating bone tissue regeneration.

PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks.

DNA lesions induce recruitment and accumulation of various repair factors, resulting in formation of discrete nuclear foci. Using superresolution fluorescence microscopy as well as live cell and quantitative imaging, we demonstrate that X-ray repair cross-complementing protein 1 (XRCC1), a key factor in single-strand break and base excision repair, is recruited into nuclear bodies formed in response to replication-related single-strand breaks. Intriguingly, these bodies are assembled immediately in the vicinity of these breaks and never fully colocalize with replication foci. They are structurally organized, containing canonical promyelocytic leukemia (PML) nuclear body protein SP100 concentrated in a peripheral layer, and XRCC1 in the center. They also contain other factors, including PML, poly(ADP-ribose) polymerase 1 (PARP1), ligase IIIα, and origin recognition complex subunit 5. The breast cancer 1 and -2 C terminus domains of XRCC1 are essential for formation of these repair foci. These results reveal that XRCC1-contaning foci constitute newly recognized PML-like nuclear bodies that accrete and locally deliver essential factors for repair of single-strand DNA breaks in replication regions.-Kordon, M. M., Szczurek, A., Berniak, K., Szelest, O., Solarczyk, K., Tworzydlo, M., Wachsmann-Hogiu, S., Vaahtokari, A., Cremer, C., Pederson, T., Dobrucki, J. W. PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks.

Entry of Human Coronavirus NL63 into the Cell.

The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into the LLC-Mk2 cell line and ex vivo three-dimensional (3D) tracheobronchial tissue. Using a variety of techniques, we have shown that HCoV-NL63 virions require endocytosis for successful entry into the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. For 3D tracheobronchial tissue cultures, we also observed that the virus enters the cell by clathrin-mediated endocytosis, but we obtained results suggesting that this pathway may be bypassed.IMPORTANCE Available data on coronavirus entry frequently originate from studies employing immortalized cell lines or undifferentiated cells. Here, using the most advanced 3D tissue culture system mimicking the epithelium of conductive airways, we systematically mapped HCoV-NL63 entry into susceptible cells. The data obtained allow for a better understanding of the infection process and may support development of novel treatment strategies.

Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks.

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 µM camptothecin, 10 µM etoposide or 0.2 µM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.

Two stages of XRCC1 recruitment and two classes of XRCC1 foci formed in response to low level DNA damage induced by visible light, or stress triggered by heat shock.

Induction of local photosensitised DNA damage has been used to study recruitment of repair factors, spatial organisation and subsequent stages of the repair processes. However, the damage induced by a focused laser beam interacting with a photosensitiser may not fully reflect the types of damage and repair encountered in cells of an animal under typical conditions in vivo. We report on two characteristic stages of recruitment of XRCC1 (a protein engaged in BER and SSB repair pathways), in response to low level DNA damage induced by visible light. We demonstrate that, when just a few DNA breaks are induced in a small region of the nucleus, the recruited XRCC1 is initially distributed uniformly throughout this region, and rearranges into several small stationary foci within minutes. In contrast, when heavy damage of various types (including oxidative damage) is induced in cells pre-sensitized with a DNA-binding drug ethidium bromide, XRCC1 is also recruited but fails to rearrange from the stage of the uniform distribution to the stage of several small foci, indicating that this heavy damage interferes with the progress and completion of the repair processes. We hypothesize that that first stage may reflect recruitment of XRCC1 to poly(ADP-ribose) moieties in the region surrounding the single-strand break, while the second-binding directly to the DNA lesions. We also show that moderate damage or stress induces formation of two types of XRCC1-containing foci differing in their mobility. A large subset of DNA damage-induced XRCC1 foci is associated with a major component of PML nuclear bodies--the Sp100 protein.

DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5-ethynyl-2'-deoxyuridine incorporated into DNA.

The "click chemistry" approach utilizing 5-ethynyl-2'-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication and adapted to flow- and imaging-cytometry. In the present study, we observed that EdU, once incorporated into DNA, induces DNA damage signaling (DDS) such as phosphorylation of ATM on Ser1981, of histone H2AX on Ser139, of p53 on Ser15, and of Chk2 on Thr68. It also perturbs progression of cells through the cell cycle and subsequently induces apoptosis. These effects were observed in non-small cell lung adenocarcinoma A549 as well as in B-cell human lymphoblastoid TK6 and WTK1 cells, differing in the status of p53 (wt versus mutated). After 1 h EdU pulse-labeling, the most affected was cells progression through the S phase subsequent to that at which they had incorporated EdU. This indicates that DNA replication using the template containing incorporated EdU is protracted and triggers DDS. Furthermore, progression of cells having DNA pulse-labeled with EdU led to accumulation of cells in G2 , likely by activating G2 checkpoint. Consistent with the latter was activation of p53 and Chk2. Although a correlation was observed in A549 cells between the degree of EdU incorporation and the extent of γH2AX induction, such correlation was weak in TK6 and WTK1 cells. The degree of perturbation of the cell cycle kinetics by the incorporated EdU was different in the wt p53 TK6 cells as compared to their sister WTK1 cell line having mutated p53. The data are thus consistent with the role of p53 in modulating activation of cell cycle checkpoints in response to impaired DNA replication. The confocal microscopy analysis of the 3D images of cells exposed to EdU for 1 h pulse and then grown for 24 or 48 h revealed an increased number of colocalized γH2AX and p53BP1 foci considered to be markers of DNA double-strand breaks and enlarged nuclei.

Analysis of spatial correlations between patterns of DNA damage response and DNA replication in nuclei of cells subjected to replication stress or oxidative damage.

Sites of DNA replication (EdU incorporation) and DNA damage signaling (γH2AX) induced by camptothecin (Cpt) or hydrogen peroxide (H2O2) form characteristic patterns of foci in cell nuclei. The overlap between these patterns is a function of the number of DNA double strand breaks (DSBs) formed in replication sites. The goal of this study was to optimize a method of quantitative assessment of a degree of correlation between these two patterns. Such a correlation can be used to estimate a probability of inducing damage in sections of replicating DNA. The damage and replication foci are imaged in 3D with confocal microscopy and their respective positions within nuclei are determined with adaptive image segmentation. Using correlation functions spatial proximity of the resultant point patterns is quantified over the range of distances in cells in early-, mid- and late S-phase. As the numbers (and nuclear densities) of γH2AX and replication foci differ significantly in the subsequent substages of S phase, the detected association values were corrected for the expected random overlap between both classes of foci. Thus, the probability of their nonrandom association was estimated. Moreover, self association (clustering) of DNA replication sites in different stages of S-phase of the cell cycle was detected and accounted for. While the analysis revealed a strong correlation between the γH2AX foci and the sites of DNA replication in cells treated with Cpt, only a low correlation was apparent in cells exposed to H2O2. © 2013 International Society for Advancement of Cytometry.