Adhesion and Activation of Blood Platelets on Laser-Structured Surfaces of Biomedical Metal Alloys.

The laser surface modification of metallic implants presents a promising alternative to other surface modification techniques. A total of four alloyed metallic biomaterials were used for this study: medical steel (AISI 316L), cobalt-chromium-molybdenum alloy (CoCrMo) and titanium alloys (Ti6Al4V and Ti6Al7Nb). Samples of metallic biomaterials after machining were subjected to polishing or laser modification in two different versions. The results of surface modification were documented using SEM imaging and roughness measurement. After modification, the samples were sterilized with dry hot air, then exposed to citrate blood, washed with PBS buffer, fixed with glutaraldehyde, sputtered with a layer of gold and imaged using SEM to enable the quantification of adhered, activated and aggregated platelets on the surface of biomaterial samples. The average total number, counted in the field of view, of adhered platelets on the surfaces of the four tested biomaterials, regardless of the type of modification, did not differ statistically significantly (66 ± 81, 67 ± 75, 61 ± 70 and 57 ± 61 for AISI 316L, CoCrMo, Ti6Al4V and Ti6Al7Nb, respectively) and the average number of platelet aggregates was statistically significantly higher (p < 0.01) on the surfaces of AISI 316L medical steel (42 ± 53) and of the CoCrMo alloy (42 ± 52) compared to the surfaces of the titanium alloys Ti6Al4V (33 ± 39) and Ti6Al7Nb (32 ± 37). Remaining blood after contact was used to assess spontaneous platelet activation and aggregation in whole blood by flow cytometry. An in-depth analysis conducted on the obtained results as a function of the type of modification indicates small but statistically significant differences in the interaction of platelets with the tested surfaces of metallic biomaterials.

Optimization of Revision Hip Arthroplasty Workflow by Means of Detailed Pre-Surgical Planning Using Computed Tomography Data, Open-Source Software and Three-Dimensional-Printed Models.

BACKGROUND: In revision hip arthroplasty (RHA), establishing the center of rotation (COR) can be technically challenging due to the acetabular bone destruction that is usually present, particularly in severe cases such as Paprosky type II and III defects. The aim of this study was to demonstrate the use of open-source medical image reconstruction software and low-cost 3D anatomical models in pre-surgical planning of RHA. METHODS: A total of 10 patients, underwent RHA and were included in the study. Computed tomography (CT) scans were performed for all cases, before surgery and approximately 1 week after the procedure. The reconstruction of CT data, 3D virtual planning of the COR and positioning of acetabular cups, including their inclination and anteversion angles, was carried out using the free open source software platform 3D Slicer. In addition, anatomical models of the pelvis were built on a desktop 3D printer from polylactic acid (PLA). Preoperative and postoperative reconstructed imaging data were compared for each patient, and the position of the acetabular cups as well as the COR were evaluated for each case. RESULTS: Analysis of the pre- and post-op center of rotation position data indicated statistically insignificant differences for the location of the COR on the X-axis (1.5 mm, t = 0.5741, p = 0.5868) with a fairly strong correlation of the results (r = -0.672, p = 0.0982), whilst for the location of the COR in the Y and Z-axes, there was statistical dependence (Y axis, 4.7 mm, t = 3.168 and p = 0.0194; Z axis, 1.9 mm, t = 1.887 and p = 0.1081). A strong correlation for both axes was also observed (Y and Z) (Y-axis, r = 0.9438 and p = 0.0014; Z-axis, r = 0.8829 and p = 0.0084). Analysis of inclination angle values showed a statistically insignificant difference between mean values (3.9 degrees, t = 1.111, p = 0.3092) and a moderate correlation was found between mean values (r = -0.4042, p = 0.3685). Analysis of the anteversion angle showed a statistically insignificant difference between mean values (1.9 degrees, t = 0.8671, p = 0.4192), while a moderate correlation between mean values was found (r = -0.4782, p = 0.2777). CONCLUSIONS: Three-dimensional reconstruction software, together with low-cost anatomical models, are very effective tools for pre-surgical planning, which have great potential use in orthopedic surgery, particularly RHA. In up and in- and up and out-type defects, it is essential to establish a new COR and to identify three support points within the revision acetabulum in order to correctly position acetabular cups.

Assessment of the influence of gold nanoparticles stabilized with PAMAM dendrimers on HUVEC barrier cells.

Civilization diseases, cancer, frequent mutations of viruses and other pathogens constitute the need to look for new drugs, as well as systems for their targeted delivery. One of the promising way of using drugs is supplying them by linking to nanostructures. One of the solution for the development of nanobiomedicine are metallic nanoparticles stabilized with various polymer structures. In this report, we present the synthesis of gold nanoparticles, their stabilization with polyamidoamine (PAMAM) dendrimers with ethylenediamine core and the characteristics of the obtained product (AuNPs/PAMAM). The presence, size and morphology of synthesized gold nanoparticles were evaluated by ultraviolet-visible light spectroscopy, transmission electron microscopy and atomic force microscopy. The hydrodynamic radius distribution of the colloids was analyzed by dynamic light scattering technique. Additionally, the cytotoxicity and changes in mechanical properties of human umbilical vein endothelial cell line (HUVEC) cells caused by AuNPs/PAMAM were assessed. The results of studies on the nanomechanical properties of cells suggest a two-step changes in cell elasticity as a response to contact with nanoparticles. When using AuNPs/PAMAM in lower concentrations, no changes in cell viability were observed and the cells were softer than untreated cells. When higher concentrations were used, a decrease in the cells viability to about 80 % were observed, as well as non-physiological stiffening of the cells. The presented results may play a significant role in the development of nanomedicine.

Effects of chronic exposure to cadmium and copper on the proteome profile of hemolymph in false widow spider Steatoda grossa (Theridiidae).

The aim of this study was to evaluate the quantitative and qualitative changes in the proteome of the hemolymph of female Steatoda grossa spiders (Theridiidae) that were chronically exposed to cadmium and copper in food and were additionally immunostimulated (phorbol 12-myristate 13-acetate (PMA); bacterial suspensions: Staphylococcus aureus (G+), Pseudomonas fluorescens (G-). It was found that the expression of nearly 90 proteins was altered in cadmium-intoxicated spiders and more than 60 in copper-exposed individuals. Regardless of the type of metal used, these proteins were mainly overexpressed in the hemolymph of the exposed spiders. On the other hand, immunostimulation did not significantly change the number of proteins with altered expression in metal-intoxicated individuals. Hemocyanin (Hc) was found to be the most abundant of the proteins identified with altered expression. In copper-intoxicated spiders, immunostimulation increased the expression of A-, E-, F-, and G-chain-containing proteins, while in the case of cadmium-intoxicates spiders, it decreased the expression of E- and A-chain-containing Hc and increased the expression of G-chain-containing Hc. Regardless of the type of metal and immunostimulant used, there was an increase in the expression of actin. In addition, cadmium increased the expression of cullin, vimentin, and ceruloplasmin. The changes observed in the expression of hemolymph proteins indicate their protective function in S. grossa (Theridiidae) spiders under conditions of metal exposure.

Investigation of HUVEC response to exposure to PAMAM dendrimers - changes in cell elasticity and vesicles release.

The aim of this study is to assess the effect of PAMAM dendrimers of second, fourth, and seventh generations on human umbilical vein endothelial cells. Primary endothelial cells were exposed to PAMAM dendrimers for 24 h, using concentrations reducing cellular viability to the levels of 90, 75, and 50%. We assumed, that changes in mechanical properties reflect toxicity of PAMAM dendrimers. The mechanical properties were investigated using atomic force spectroscopy (AFS) technique with the use of two approaches for measuring cell elasticity: global, where the tests were performed using a micrometer-hemispherical probe, and local, where a nanometer-sized probe was used. For the sharp probe, a reduction in the elasticity modulus was observed in comparison to untreated control cells, that is related to the depolymerization of the cytoskeleton and the processes leading to cell apoptosis. In the case of the hemispherical probe, cell softening was also observed in comparison to control cells, but with increasing PAMAM concentrations, the modulus of elasticity increases. It is related to the sensing of numerous intracellular vesicles with the use of this probe, e.g. endosomal and empty plasmalemmal which can also alter cell elasticity. The presence of external and intracellular vesicles was confirmed by scanning and transmission electron microscopy. The relationship between the elasticity of HUVEC cells exposed to PAMAM dendrimers of selected generations and their toxic effects was presented herein for the first time. In the transmission electron microscopy images of the cells exposed to PAMAM dendrimers, we have also observed distinctive vesicles with regular multilayer arranged structure.

Diclofenac Diminished the Unfolded Protein Response (UPR) Induced by Tunicamycin in Human Endothelial Cells.

Diclofenac belongs to the class of nonsteroidal anti-inflammatory drugs (NSAIDs), which are amongst the most frequently prescribed drugs to treat fever, pain and inflammation. Despite the presence of NSAIDs on the pharmaceutical market for several decades, epidemiological studies have shown new clinical applications of NSAIDs, and new mechanisms of their action were discovered. The unfolded protein response (UPR) activated under endoplasmic reticulum (ER) stress is involved in the pathophysiology of many diseases and may become a drug target, therefore, the study evaluated the effects of diclofenac on the tunicamycin-induced UPR pathways in endothelial cells. RT PCR analysis showed that diclofenac significantly inhibited activation of ER stress-responsive genes, i.e., CHOP/DITT3, GRP78/HSPA5 and DNAJB9. Additionally, the drug diminished the significant upregulation and release of the GRP78 protein, as evaluated using the ELISA assay, which was likely to be involved in the mechanism of the UPR activation resulting in apoptosis induction in endothelial cells. These results suggest the value of diclofenac as a factor capable of restoring the ER homeostasis in endothelial cells by diminishing the UPR.

Interaction of Cationic Carbosilane Dendrimers and Their siRNA Complexes with MCF-7 Cells Cultured in 3D Spheroids.

Cationic dendrimers are effective carriers for the delivery of siRNA into cells; they can penetrate cell membranes and protect nucleic acids against RNase degradation. Two types of dendrimers (CBD-1 and CBD-2) and their complexes with pro-apoptotic siRNA (Mcl-1 and Bcl-2) were tested on MCF-7 cells cultured as spheroids. Cytotoxicity of dendrimers and dendriplexes was measured using the live-dead test and Annexin V-FITC Apoptosis Detection Kit (flow cytometry). Uptake of dendriplexes was examined using flow cytometry and confocal microscopy. The live-dead test showed that for cells in 3D, CBD-2 is more toxic than CBD-1, contrasting with the data for 2D cultures. Attaching siRNA to a dendrimer molecule did not lead to increased cytotoxic effect in cells, either after 24 or 48 h. Measurements of apoptosis did not show a high increase in the level of the apoptosis marker after 24 h exposure of spheroids to CBD-2 and its dendriplexes. Measurements of the internalization of dendriplexes and microscopy images confirmed that the dendriplexes were transported into cells of the spheroids. Flow cytometry analysis of internalization indicated that CBD-2 transported siRNAs more effectively than CBD-1. Cytotoxic effects were visible after incubation with 3 doses of complexes for CBD-1 and both siRNAs.

The Importance of Endoplasmic Reticulum Stress as a Novel Antidepressant Drug Target and Its Potential Impact on CNS Disorders.

Many central nervous system (CNS) diseases, including major depressive disorder (MDD), are underpinned by the unfolded protein response (UPR) activated under endoplasmic reticulum (ER) stress. New, more efficient, therapeutic options for MDD are needed to avoid adverse effects and drug resistance. Therefore, the aim of the work was to determine whether UPR signalling pathway activation in astrocytes may serve as a novel target for antidepressant drugs. Among the tested antidepressants (escitalopram, amitriptyline, S-ketamine and R-ketamine), only S-ketamine, and to a lesser extent R-ketamine, induced the expression of most ER stress-responsive genes in astrocytes. Furthermore, cell viability and apoptosis measuring assays showed that (R-)S-ketamine did not affect cell survival under ER stress. Under normal conditions, S-ketamine played the key role in increasing the release of brain-derived neurotrophic factor (BDNF), indicating that the drug has a complex mechanism of action in astrocytes, which may contribute to its therapeutic effects. Our findings are the first to shed light on the relationship between old astrocyte specifically induced substance (OASIS) stabilized by ER stress and (R-)S-ketamine; however, the possible involvement of OASIS in the mechanism of therapeutic ketamine action requires further study.

Osteosarcoma cells in early and late stages as cancer in vitro progression model for assessing the responsiveness of cells to silver nanoparticles.

Understanding of biology of osteosarcoma malignant progression is indispensable for enhancement of conventional chemotherapy by the use of silver nanoparticles (AgNPs). We presented an in vitro model of cancer progression closely resembling processes occurring in vivo in terms of protein profile. A comparison of cytotoxic and genotoxic potential of AgNPs in Saos-2 cells in early stages of cancerous progression (early passages) with the cells in advanced stages (late passages) demonstrated significantly reduced responsiveness of the late passage cells to nanoparticles toxicity. It was also confirmed by proteome analysis as we identified considerably higher number of differentially expressed proteins in Saos-2 cells in early passages compared to the late passage cells. Our studies showed that the ability of AgNPs as potential drug carriers to deliver a medication and/or to evoke toxic effects might be significantly diminished in advanced stages of cancer progression.

Isolation, Biochemical Characterisation and Identification of Thermotolerant and Cellulolytic Paenibacillus lactis and Bacillus licheniformis.

RESEARCH BACKGROUND: Cellulose is an ingredient of waste materials that can be converted to other valuable substances. This is possible provided that the polymer molecule is degraded to smaller particles and used as a carbon source by microorganisms. Because of the frequently applied methods of pretreatment of lignocellulosic materials, the cellulases derived from thermophilic microorganisms are particularly desirable. EXPERIMENTAL APPROACH: We were looking for cellulolytic microorganisms able to grow at 50 °C and we described their morphological features and biochemical characteristics based on carboxymethyl cellulase (CMCase) activity and the API® ZYM system. The growth curves during incubation at 50 °C were examined using the BioLector® microbioreactor. RESULTS AND CONCLUSIONS: Forty bacterial strains were isolated from fermenting hay, geothermal karst spring, hot spring and geothermal pond at 50 °C. The vast majority of the bacteria were Gram-positive and rod-shaped with the maximum growth temperature of at least 50 °C. We also demonstrated a large diversity of biochemical characteristics among the microorganisms. The CMCase activity was confirmed in 27 strains. Hydrolysis capacities were significant in bacterial strains: BBLN1, BSO6, BSO10, BSO13 and BSO14, and reached 2.74, 1.62, 1.30, 1.38 and 8.02 respectively. Rapid and stable growth was observed, among others, for BBLN1, BSO10, BSO13 and BSO14. The strains fulfilled the selection conditions and were identified based on the 16S rDNA sequences. BBLN1, BSO10, BSO13 were classified as Bacillus licheniformis, whereas BSO14 as Paenibacillus lactis. NOVELTY AND SCIENTIFIC CONTRIBUTION: We described cellulolytic activity and biochemical characteristics of many bacteria isolated from hot environments. We are also the first to report the cellulolytic activity of thermotolerant P. lactis. Described strains can be a source of new thermostable cellulases, which are extremely desirable in various branches of circular bioeconomy.

Interaction of Cationic Carbosilane Dendrimers and Their siRNA Complexes with MCF-7 Cells.

The application of siRNA in gene therapy is mainly limited because of the problems with its transport into cells. Utilization of cationic dendrimers as siRNA carriers seems to be a promising solution in overcoming these issues, due to their positive charge and ability to penetrate cell membranes. The following two types of carbosilane dendrimers were examined: CBD-1 and CBD-2. Dendrimers were complexed with pro-apoptotic siRNA (Mcl-1 and Bcl-2) and the complexes were characterized by measuring their zeta potential, circular dichroism and fluorescence of ethidium bromide associated with dendrimers. CBD-2/siRNA complexes were also examined by agarose gel electrophoresis. Both dendrimers form complexes with siRNA. Moreover, the cellular uptake and influence on the cell viability of the dendrimers and dendriplexes were evaluated using microscopic methods and XTT assay on MCF-7 cells. Microscopy showed that both dendrimers can transport siRNA into cells; however, a cytotoxicity assay showed differences in the toxicity of these dendrimers.

Dysfunction of endothelial cells exposed to nanomaterials assessed by atomic force spectroscopy.

The study of the impact of nanomaterials on endothelial cell elasticity with the atomic force spectroscopy (AFS) can be a significant model for assessing nanomaterials toxic effects in vitro. The mechanical properties of cells exposed to nanostructures can provide information not only about cellular nano and micro-structure, but also about cell physiology. The toxicity of nanostructures is an important issue which must be carefully considered when the optimal nanomaterial is defined. There are no universal properties characterizing such a nanomaterial, i.e. depending on the intended use, the requirements can be diverse. For example, for biomedical use a nanomaterial should not negatively affect the cells or should cause the expected therapeutic or diagnostic effects in justified cases. The present study was devoted to the effects of silver nanoparticles (SNPs), multi-walled carbon nanotubes (MWCNTs) and poly(amidoamine) (PAMAM) dendrimers of 4th generation on functioning of endothelial cells. Immortalized endothelial cells were exposed for 24 h to the tested nanomaterials used in concentrations reducing cellular viability to the levels of 90 % and 75 %. The innovative nature of our work is the comparison of cell elasticity performed with various AFS probes, which enabled detection of local and global elasticity alteration caused by the nanostructures. The obtained results demonstrated changes in elasticity of endothelial cell induced by the nanostructures, which were closely correlated with the level of cellular viability, forming of actin stress fibres and elevated levels of reactive oxygen species. Trend of changes in local and global elasticity of cells exposed to nanostructures was similar, but the magnitude of the response was dependent on the selected probe. SNPs and MWCNTs evoked cells stiffening, which was correlated with changes in production levels of reactive oxygen species (ROS) and the cytoskeletal alteration. Softening of cells exposed to PAMAM dendrimers correlated with increased number of apoptotic cells and ROS production levels. Based on the obtained results we conclude, that the structure and the type of nanostructure (nanoparticle) is essential for their localization inside the cells and for the toxic effect on the endothelial cells.

Comprehensive Biological Evaluation of Biomaterials Used in Spinal and Orthopedic Surgery.

Biological acceptance is one of the most important aspects of a biomaterial and forms the basis for its clinical use. The aim of this study was a comprehensive biological evaluation (cytotoxicity test, bacterial colonization test, blood platelets adhesion test and transcriptome and proteome analysis of Saos-2 cells after contact with surface of the biomaterial) of biomaterials used in spinal and orthopedic surgery, namely, Ti6Al4V ELI (Extra Low Interstitials), its modified version obtained as a result of melting by electron beam technology (Ti6Al4V ELI-EBT), polyether ether ketone (PEEK) and polished medical steel American Iron and Steel Institute (AISI) 316L (the reference material). Biological tests were carried out using the osteoblasts-like cells (Saos-2, ATCC HTB-85) and bacteria Escherichia coli (DH5α). Results showed lack of cytotoxicity of all materials and the surfaces of both Ti6Al4V ELI and PEEK exhibit a significantly higher resistance to colonization with E. coli cells, while the more porous surface of the same titanium alloy produced by electron beam technology (EBT) is more susceptible to microbial colonization than the control surface of polished medical steel. None of the tested materials showed high toxicity in relation to E. coli cells. Susceptibility to platelet adhesion was very high for polished medical steel AISI 316L, whilst much lower for the other biomaterials and can be ranked from the lowest to the highest as follows: PEEK < Ti6Al4V ELI < Ti6Al4V ELI-EBT. The number of expressed genes in Saos-2 cells exposed to contact with the examined biomaterials reached 9463 genes in total (ranging from 8455 genes expressed in cells exposed to ELI to 9160 genes in cells exposed to PEEK). Whereas the number of differentially expressed proteins detected on two-dimensional electrophoresis gels in Saos-2 cells after contact with the examined biomaterials was 141 for PEEK, 223 for Ti6Al4V ELI and 133 for Ti6Al4V ELI-EBT. Finally, 14 proteins with altered expression were identified by mass spectrometry. In conclusion, none of the tested biomaterials showed unsatisfactory levels of cytotoxicity. The gene and protein expression analysis, that represents a completely new approach towards characterization of these biomaterials, showed that the polymer PEEK causes much more intense changes in gene and protein expression and thus influences cell metabolism.

Spheroids as a Type of Three-Dimensional Cell Cultures-Examples of Methods of Preparation and the Most Important Application.

Cell cultures are very important for testing materials and drugs, and in the examination of cell biology and special cell mechanisms. The most popular models of cell culture are two-dimensional (2D) as monolayers, but this does not mimic the natural cell environment. Cells are mostly deprived of cell-cell and cell-extracellular matrix interactions. A much better in vitro model is three-dimensional (3D) culture. Because many cell lines have the ability to self-assemble, one 3D culturing method is to produce spheroids. There are several systems for culturing cells in spheroids, e.g., hanging drop, scaffolds and hydrogels, and these cultures have their applications in drug and nanoparticles testing, and disease modeling. In this paper we would like to present methods of preparation of spheroids in general and emphasize the most important applications.

Endothelial cell aging detection by means of atomic force spectroscopy.

Endothelial cell aging is related to changes not only in cell phenotype, such as luminal changes, intimal and medial thickening, and increased vascular stiffness, but encompasses different cell responses to various substances including drugs or nanomaterials. In the present work, time- and dose-dependent elasticity changes evoked by silver nanoparticles in endothelial cells in early (below 15) passages were analyzed. Silver nanoparticle concentrations of 3, 3.6, and 16 µg/mL were selected for elasticity measurements for long incubation (24 hours) and of 1 and 3 µg/mL for monitoring dynamic elasticity changes of 1-, 3-, and 6-hour incubations. Surprisingly, a significant reduction in the cells elasticity modulus at lower number of passages exposed to silver nanoparticles used at 3 µg/mL for 24 hours was demonstrated. These results are in contrast to those obtained for endothelial cells in late (33-43) passages that may result from cellular aging in response to nanosilver. Furthermore, for short incubation times (1 and 3 hours), SNP-induced significant increase in the cell elasticity modulus was detected. In current work, we also attempted to answer the question whether the changes in cell elasticity were induced by the silver nanoparticles stabilized with polyvinyl pyrrolidone or by stabilizer itself. Elasticity measurements were supplemented by observations made with transmission electron microscopy and scanning electron microscopy, which confirmed the presence of silver nanoparticles inside the cells and on the cell membrane. Additionally, activation of reactive oxygen species was detected for cells exposed to SNPs for 1 and 3 hours, which was accompanied by increased cell elasticity modulus suggesting a possible mechanism of observed phenomenon.

Designing laser-modified surface structures on titanium alloy custom medical implants using a hybrid manufacturing technology.

The hybrid technology combines an efficient material-removal process and implant surface treatment by the laser reducing time of manufacture process compared to currently used machining technologies. It also permits precise structuring of the implant material surface. Six structures of the Ti6Al4V ELI surface were designed and studied how the structure topography prepared with the hybrid technology affected the Escherichia coli adhesion to the surface and viability, as well as the growth, adhesion, and viability of human osteogenic Saos-2 cells cultured on the investigated surfaces. Results have confirmed that the microtopography of medical titanium alloy plays a beneficial role in bacterial adhesion and viability (number of bacteria found on reference surface: [5.9 ± 0.44] × 106 CFU/ml, sample no. 3: [8.8 ± 0.93] × 104 CFU/ml, and sample no. 5: [1.2 ± 0.23] × 107 CFU/ml; CFU - Colony Forming Unit). All tested structured surfaces enabled good cell attachment and proliferation of Saos-2 cells (viability of Saos-2 cells [% of control] for reference surface: 81.93%; sample no. 3: 75% and sample no. 5: 100%). Transcriptome analysis of genes commonly expressed in the process of osseointegration demonstrated that the use of hybrid technology allows designing structures that enhance osseointegration but it should be coupled with other methods of preventing bacterial growth, or with a different strategy to limit microbial colonization with the satisfactory osseointegration potential.

Surface Characteristics and Biological Evaluation of Si-DLC Coatings Fabricated Using Magnetron Sputtering Method on Ti6Al7Nb Substrate.

Diamond-like carbon (DLC) coatings are well known as protective coatings for biomedical applications. Furthermore, the incorporation of different elements, such as silicon (Si), in the carbon matrix changes the bio-functionality of the DLC coatings. This has also been proven by the results obtained in this work. The Si-DLC coatings were deposited on the Ti6Al7Nb alloy, which is commonly used in clinical practice, using the magnetron sputtering method. According to the X-ray photoelectron spectroscopy (XPS) analysis, the content of silicon in the examined coatings varied from ~2 at.% up to ~22 at.%. Since the surface characteristics are key factors influencing the cell response, the results of the cells' proliferation and viability assays (live/dead and XTT (colorimetric assays using tetrazolium salt)) were correlated with the surface properties. The surface free energy (SFE) measurements, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS) analysis demonstrated that the polarity and wettability of the surfaces examined increase with increasing Si concentration, and therefore the adhesion and proliferation of cells was enhanced. The results obtained revealed that the biocompatibility of Si-doped DLC coatings, regardless of the Si content, remains at a very high level (the observed viability of endothelial cells is above 70%).

Simultaneous transcriptome and proteome analysis of EA.hy926 cells under stress conditions induced by nanomaterials.

Today, the extensive and constantly growing number of applications in the field of nanotechnology poses a lot of questions about the potential toxicity of nanomaterials (NMs) toward cells of different origins. In our work we employed the tools of molecular biology to evaluate changes that occur in human endothelial cells at the transcriptomic and proteomic level, following 24 h of exposure to three different classes of NMs. Using microarray technology, we demonstrated that 24 h of exposure to silver nanoparticles (SNPs), multiwalled carbon nanotubes (MWCNTs) and polyamidoamine dendrimers (PAMAMs) leads to changes in 299, 1271, and 431 genes, respectively, influencing specific molecular pathways. The 2D-DIGE and mass spectrometry analysis revealed that differentially expressed proteins were involved in numerous cellular processes, for example, cytoskeletal reorganization, cell growth and proliferation, or response to stress. Both, transcriptome and proteome alterations indicate reorganization of mechanism regulating cell functioning. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1024-1034, 2019.

Sensing of silver nanoparticles on/in endothelial cells using atomic force spectroscopy.

Endothelial cells, due to their location, are interesting objects for atomic force spectroscopy study. They constitute a barrier between blood and vessel tissues located deeper, and therefore they are the first line of contact with various substances present in blood, eg, drugs or nanoparticles. This work intends to verify whether the mechanical response of immortalized human umbilical vein endothelial cells (EA.hy926), when exposed to silver nanoparticles, as measured using force spectroscopy, could be effectively used as a bio-indicator of the physiological state of the cells. Silver nanoparticles were characterized with transmission electron microscopy and dynamic light scattering techniques. Tetrazolium salt reduction test was used to determine cell viability after treatment with silver nanoparticles. An elasticity of native cells was examined in the Hanks' buffer whereas fixed cells were softly fixed with formaldehyde. Additional aspect of the work is the comparative force spectroscopy utilizing AFM probes of ball-shape and conical geometries, in order to understand what changes in cell elasticity, caused by SNPs, were detectable with each probe. As a supplement to elasticity studies, cell morphology observation by atomic force microscopy and detection of silver nanoparticles inside cells using transmission electron microscopy were also performed. Cells exposed to silver nanoparticles at the highest selected concentrations (3.6 µg/mL, 16 µg/mL) are less elastic. It may be associated with the reorganization of the cellular cytoskeleton and the "strengthening" of the cell cortex caused by presence of silver nanoparticles. This observation does not depend on cell fixation. Agglomerates of silver nanoparticles were observed on the cell membrane as well as inside the cells.

Toxicity of silver nanoparticles, multiwalled carbon nanotubes, and dendrimers assessed with multicellular organism Caenorhabditis elegans.

Nematode Caenorhabditis elegans (C. elegans) was used to investigate the impact of silver nanoparticles (SNP), multiwalled carbon nanotubes (MWCNT), and polyamidoamine dendrimers (PAMAM) used in concentration of 1010 particle/mL. Population-based observations and gene expression analysis were employed in this study. SNP and PAMAM caused decrease in the number of live nematodes and their body length, but MWCNT did not affect the population of nematodes. Gene expression analysis revealed significant changes caused by the presence of all studied nanomaterials, and the results strongly suggest a specific metabolic response of the nematode organism to exposure to various nanomaterials. It was shown that C. elegans is a very sensitive organism capable to respond specifically to the exposure to some nanomaterials and therefore could be considered as a possible biosensor for early warning of presence of some nanoparticles.