(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (γH2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The γH2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for γH2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in γH2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of γH2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.
Mikrofluidika je inovativní obor, který se zabývá zpracováním malého množství kapaliny v mikrokanálech. V kombinaci s pokročilými analytickými technikami, jako je např. mikrofluidní PCR, nabízí významné výhody nejen pro analýzu genové exprese. Tato metoda využívá mikrokanály a mikroventily k přesnému dávkování a míchání činidel, čímž se minimalizuje spotřeba vzorku a činidla a také čas stráve‐ ný pipetováním. Tyto vlastnosti činí mikrofluidní PCR ideální pro analýzu genové exprese, kde je vyžadováno podrobné monitorování a kvantifikace mRNA. Jedním z přístrojů umožňujícím mikrofluidní PCR je Biomark X. Díky své schopnosti multiplexování a také díky své‐ mu mikrofluidnímu designu umožňuje analýzu mnoha vzorků současně. Tato pokročilá technologie má široké uplatnění v biologickém výzkumu, diagnostice a personalizované medicíně a nabízí nové příležitosti k objevování a pochopení genetických procesů.
Microfluidics is an innovative science that deals with the manipulation of small volumes of fluid in microchannels. In combination with advanced analytical techniques such as microfluidic PCR, it offers significant advantages not only for gene expression analysis. Microflui‐ dic PCR enables PCR reactions to be performed using very small sample volumes, as it utilizes microchannels and microvalves for precise reagent dispensing and mixing. This fact increases both sensitivity and accuracy of the analysis. The Biomark X instrument utilizes micro‐ fluidic PCR for gene expression analysis, as it is ideal for mRNA quantification. With its multiplexing capability and microfluidic design, it enables the analysis of multiple samples simultaneously. This advanced technology finds broad applications in biological research, diagnostics, and provides new opportunities for the discovery and understanding of genetic processes.
This research work is focused on the investigation of newly developed titania sol-gel coatings containing silver, calcium and phosphate with appropriate abilities to be implanted into the human body. These abilities include adhesion, bioactivity, antibacterial property and cytocompatibility of prepared coatings. Four types of coatings were applied on a titanium substrate by dip-coating technique under different conditions (TCP1, TCP2, TCPA1 and TCPA2). Surfaces of coatings after the firing without silver featured different distribution of circular areas containing Ca. The coatings TCPA1 and TCPA2 were made up of unhomogeneously situated silver. Adhesion of the coatings to the substrates was measured by a tape test. All types of the coatings demonstrated very good adhesion. Isolated cracks that appeared during the firing did not have a negative influence on the adhesion properties. Bioactivity of the coatings was tested in vitro using a simulated body fluid. Three of the four types demonstrated bioactive properties (TCP1, TCP2 and TCPA2), that is, precipitation of crystalline hydroxyapatite as was confirmed by X-ray diffraction. The antibacterial effect (against Escherichia coli and Staphylococcus epidermidis) and cytotoxicity (toward L929 and U-2 OS cell lines, direct and indirect test) were then tested. All the coatings demonstrated very good antibacterial effect against both bacteria after 4- and 24-hr interaction. All the coating types were evaluated as cytocompatible in the indirect test. Cells were able to grow even directly on the coatings.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- biokompatibilní potahované materiály chemie farmakologie MeSH
- Escherichia coli MeSH
- hydroxyapatit farmakologie MeSH
- lidé MeSH
- stříbro * farmakologie MeSH
- titan * chemie farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Polymerase chain reaction (PCR) is one of the main techniques of molecular biology. Currently, there is a boom of digital PCR which allows precise, sensitive and reproducible quantification without using a calibration curve. Due to its features, digital PCR has a wide analyti-cal application in many areas (e.g. biomedical applications or food analysis). Presently, there are several types of digi-tal PCR that differ from each other particularly in the way of dispersing the sample into many aliquots of very small volumes. All platforms allow multiplex analyses based on the use of differently labelled probes and/or different con-centrations of probes labelled with one fluorescent color. This can reduce the financial and time requirements for a single sample analysis. The advantages and disadvantages of each type of digital PCR are summarized in this article.
Titanium biomaterials are widely used in the medical field due to their biocompatibility and excellent corrosion and mechanical resistance. However, these materials have no antibacterial properties. To obtain an antibacterial active surface, a nanostructure of Ti6Al4V alloy was created. This specific nanostructure contained nanotubes and micro-cavities and was used as a substrate for silver anchoring. The electrochemical approach to silver reduction was studied. It is a common approach for silver deposition and in this work, inhomogeneities in the nanostructure were used as a preferential area for silver localisation. The galvanostatic regimen of deposition allowed for a technically quantitative process and the required silver placement. The experimental conditions used enabled testing and silver dissolution rate evaluation within a reasonable time span. Based on the corrosion and analytical results (EDS, XPS and ICP-MS), a two-phase silver release mechanism was confirmed. The openings of the individual nanotubes were filled with silver nanoparticles, whose release was relatively fast. By contrast, the silver anchored inside the cavities allowed the silver to release gradually. Antibacterial efficiency against Staphylococcus aureus and Escherichia coli was successfully demonstrated. Cytotoxicity testing with murine fibroblasts showed cell metabolic activity far above the normative limit of 70%.
- MeSH
- antibakteriální látky aplikace a dávkování chemie farmakologie MeSH
- biokompatibilní materiály chemie MeSH
- buněčné linie MeSH
- Escherichia coli účinky léků MeSH
- infekce vyvolané Escherichia coli farmakoterapie MeSH
- lékové transportní systémy metody MeSH
- lidé MeSH
- myši MeSH
- nanostruktury chemie MeSH
- protézy a implantáty MeSH
- stafylokokové infekce farmakoterapie MeSH
- Staphylococcus aureus účinky léků MeSH
- stříbro aplikace a dávkování chemie farmakologie MeSH
- titan chemie MeSH
- uvolňování léčiv MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
3D printing seems to be the technology of the future for the preparation of metallic implants. For such applications, corrosion behaviour is pivotal. However, little is published on this topic and with inconsistent results. Therefore, we carried out a complex study in which we compared two techniques of the 3D printing technology - selective laser melting and electron beam melting. The corrosion behaviour was studied in physiological solution by standard electrochemical techniques and susceptibility to localised corrosion was estimated too. All samples showed typical passive behaviour. Localised corrosion was shown to be possible on the original as-printed surfaces. Corrosion experiments were repeated tree times. To reveal possible negative effects of 3D printing on cytocompatibility, direct in vitro tests were performed with U-2 OS cells. The cells showed good viability and proliferation, but their growth was impeded by surface unevenness. Our results suggest that both techniques are suitable for implants production. Statistical evaluation was performed by ANOVA followed by Tukey's test.
- MeSH
- 3D tisk * MeSH
- koroze MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- proliferace buněk účinky léků MeSH
- testování materiálů * MeSH
- titan * chemie farmakologie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Effect of processing by equal channel angular pressing (ECAP) on the degradation behaviour of extruded LAE442 magnesium alloy was investigated in a 0.1M NaCl solution, Kirkland's biocorrosion medium (KBM) and Minimum Essential Medium (MEM), both with and without 10% of foetal bovine serum (FBS). Uniform degradation of as extruded and ECAP processed samples in NaCl solution was observed, nevertheless higher corrosion resistance was found in the latter material. The increase of corrosion resistance due to ECAP was observed also after 14-days immersion in all media used. Higher compactness of the corrosion layer formed on the samples after ECAP was responsible for the observed decrease of corrosion resistance, which was proven by scanning electron microscope investigation. Lower corrosion rate in media with FBS was observed and was explained by additional effect of protein incorporation on the corrosion layer stability. A cytotoxicity test using L929 cells was carried out to investigate possible effect of processing on the cell viability. Sufficient cytocompatibility of the extruded samples was observed with no adverse effects of the subsequent ECAP processing. In conclusion, this in vitro study proved that the degradation behaviour of the LAE442 alloy could be improved by subsequent ECAP processing and this material is a good candidate for future in vivo investigation.
- MeSH
- buněčná smrt účinky léků MeSH
- buněčné linie MeSH
- chlorid sodný farmakologie MeSH
- fibroblasty cytologie účinky léků metabolismus MeSH
- hořčík chemie MeSH
- ionty MeSH
- koroze MeSH
- myši MeSH
- roztoky MeSH
- slitiny chemie MeSH
- testování materiálů metody MeSH
- vodík analýza MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Vydání první 133 stran : ilustrace, tabulky ; 28 cm
Recently, iron-based materials have been considered as candidates for the fabrication of biodegradable load-bearing implants. Alloying with palladium has been found to be a suitable approach to enhance the insufficient corrosion rate of iron-based alloys. In this work, we have extensively compared the microstructure, the mechanical and corrosion properties, and the cytotoxicity of an FePd2 (wt%) alloy prepared by three different routes - casting, mechanical alloying and spark plasma sintering (SPS), and mechanical alloying and the space holder technique (SHT). The properties of the FePd2 (wt%) were compared with pure Fe prepared in the same processes. The preparation route significantly influenced the material properties. Materials prepared by SPS possessed the highest values of mechanical properties (CYS~750-850MPa) and higher corrosion rates than the casted materials. Materials prepared by SHT contained approximately 60% porosity; therefore, their mechanical properties reached the lowest values, and they had the highest corrosion rates, approximately 0.7-1.2mm/a. Highly porous FePd2 was tested in vitro according to the ISO 10993-5 standard using L929 cells, and two-fold diluted extracts showed acceptable cytocompatibility. In general, alloying with Pd enhanced both mechanical properties and corrosion rates and did not decrease the cytocompatibility of the studied materials.
New materials with appropriate mechanical properties and an antibacterial effect are constantly being sought for orthopedic and dental applications. The aim of this study was to investigate newly developed TiSi alloys coated with titania sol-gel containing silver. Titanium alloys with 5 or 10wt% of silicon were prepared by vacuum arc remelting and dip-coated with titania sol containing either AgNO3or Ag3PO4in two concentrations. The size and distribution of the particles in the layer were evaluated, as well as layer compactness (SEM). The antibacterial effect (against E. coli and S. epidermidis) and cytotoxicity (towards L929 and U-2 OS cell lines) of these materials were then tested. Despite cracking of the coatings after firing, the coatings demonstrated very good antibacterial effects against both E. coli and S. epidermidis after 24h of interaction. None of the tested materials were toxic to both cell lines. Collectively, our results suggest that these materials are promising candidates for orthopedic applications.
