Witold Jakubowski

2,7‐DICHLOROFLUORESCIN OXIDATION AND REACTIVE OXYGEN SPECIES: WHAT DOES IT MEASURE?

Intracellular 2,7‐dichlorofluorescin (H2DCF) oxidation is often used to measure the production of reactive oxygen species (ROS) within cells. The rate of H2DCF oxidation depends on the concentration of glutathione, which is an alternative target for ROS. Our results suggest that increased rate of H2DCF oxidation be interpreted as an indication of general oxidative stress due to a variety of reasons, including depletion of antioxidants, rather than as a specific proof of augmented ROS formation.

Estimation of oxidative stress in Saccharomyces cerevisae with fluorescent probes

Oxidation of dichlorofluorescin and dihydrofluorescin and transport of their oxidation products were studied in Saccharomyces cerevisiae cells given diacetates of these compounds. It was found that addition of peroxidase significantly accelerates the formation of dichlorofluorescein and fluorescein, indicating that peroxidase activity may be a limiting factor in studies of cellular oxidation using these probes. A significant leakage of dichlorofluorescin and dihydrofluorescin, and of their oxidized forms from the yeast cells, was found, without any apparent lag period. This phenomenon complicates interpretation of data obtained with the fluorescent precursors used but, on the other hand, points to the possibility of using measurements of dichlorofluorescein or fluorescein in the extracellular medium for studies of cellular oxidation processes.

Adhesion, activation, and aggregation of blood platelets and biofilm formation on the surfaces of titanium alloys Ti6Al4V and Ti6Al7Nb†

Titanium alloys are still on the top list of fundamental materials intended for dental, orthopedics, neurological, and cardiovascular implantations. Recently, a special attention has been paid to vanadium-free titanium alloy, Ti6Al7Nb, that seems to represent higher biocompatibility than traditional Ti6Al4V alloy. Surprisingly, these data are not thoroughly elaborated in the literature; particularly there is a lack of comparative experiments conducted simultaneously and at the same conditions. Our study fills these shortcomings in the field of blood contact and microbiological colonization. To observe platelets adhesion and biofilm formation on the surfaces of compared titanium alloys, fluorescence microscope Olympus GX71 and scanning electron microscope HITACHI S-3000N were used. Additionally, flow cytometry analysis of platelets aggregation and activation in the whole blood after contact with sample surface, as an essential tool for biomaterial thrombocompatibility assessment, was proposed. As a result of our study it was demonstrated that polished surfaces of Ti6Al7Nb and Ti6Al4V alloys after contact with whole citrated blood and E. coli bacterial cells exhibit a considerable difference. Overall, it was established that Ti6Al4V has distinct tendency to higher thrombogenicity, more excessive bacterial biofilm formation and notable cytotoxic properties in comparison to Ti6Al7Nb. However, we suggest these studies should be extended for other types of cells and biological objects.

Surface characterization and biological evaluation of silver-incorporated DLC coatings fabricated by hybrid RF PACVD/MS method

Since the biological response of the body towards an implanted material is mainly governed by its surface properties, biomaterials are improved by various kinds of coatings. Their role is to provide good mechanical and biological characteristics, and exclude some disadvantages like post-implantation infections. This phenomenon may be reduced by introduction of silver as an antibacterial agent. This study evaluates the Ag-DLC films synthesized by the hybrid RF PACVD/MS method according to the patent number PL401955-A1 worked out inter alia by the authors. Such tests as XPS, SEM, EDS, AFM, FTIR, Raman and ICP-TOF-MS were used to determine surface properties of the coatings. The obtained results were correlated with the biological response estimated on the basis of cells viability assay (osteoblast cells line Saos-2) and bacterial colonization test (Escherichia coli strain DH5α). Results showed that the hybrid RF PACVD/MS method allows one to get tight coating preventing the diffusion of harmful elements from the metallic substrate. Ag concentration increases with the growing power density, it occurs in metallic state, does not create chemical bonds and is evenly dispersed within the DLC matrix in the form of nanoscale silver clusters. Increasing silver content above 2at.% improves bactericidal properties, but decreases cell viability.

Sensitivity of antioxidant-deficient yeast Saccharomyces cerevisiae to peroxynitrite and nitric oxide

Sensitivity of Saccharomyces cerevisiae strains deficient in superoxide dismutases and catalases and of decreased level of glutathione to peroxynitrite and a nitric oxide donor, S-nitrosoglutathione was compared. Moderate but significant differences observed point to increased sensitivity to both agents of yeast deficient in antioxidant defense, the superoxide dismutase-deficient strain showing the highest sensitivity, The sequence of sensitivity of various strains to peroxynitrite and nitric oxide was the same. The results are compatible with the view that cytotoxic effects of peroxynitrite involve formation of secondary reactive oxygen species.

Bacterial colonisation of bioceramic surfaces

Abstract Hydroxyapatite (HAp – Ca10(PO4)6(OH)2) and tricalcium phosphate (TCP – Ca3(PO4)2) are bioceramic materials of special interest with regards to bone surgery, in particular the repair of bone tissue defects. These materials are highly biocompatible with bone and soft tissue; they are bioactive, osteoconductive and resistant to sterilisation processes. In comparison with other biomaterials, particularly metallic materials, bioceramic surfaces exhibit high resistance to bacterial colonisation. This is currently considered to be one of the most important issues concerning materials used in medicine, due to the fact that bacterial biofilm is difficult to combat or remove and can be responsible for recurrent infections. The aim of the present study was to evaluate bacterial colonisation on the surface of different calcium phosphate based materials.

Resistance of oxidative stress in biofilm and planktonic cells

This work studied the susceptibility of biofilm produced by E. coli to oxidative stress, and compared the components of free radicals defences: level of glutathione, catalase and dismutase activities in planktonic and biofilm located cells. Results showed the diversity of responses to oxidative stress in bacterial cells in log or stationary phases in both planktonic and biofilm forms. The bacteria were exposed to free-radical donors (H2O2, tBOOH, menadione, SIN-1 or peroxynitrite) in a wide range of final concentrations, from 0.5 to 10mM. Different level of toxicity of individual donors, independence of cell type (planktonic forms or biofilm) and phases of growth were observed. The highest oxidative stress resistance was observed for the cells in logarithmic phase of growth treated with H2O2, both in planktonic and biofilm forms, whereas for the cells in stationary phase, the highest resistance was observed for menadione. These results showed higher efficiency of agents based on superoxide anion donors in combating bacteria colonizing abiotic surfaces stainless steel (AISI 316L).

The effect of carbon nanoparticles on biological properties of polyester nanocomposites

The aim of present study was to determine the hemocompatibility, cellular response of endothelial cells and bacterial adhesion to a new polyester nanocomposite. The carbon nanoparticle nanocomposite was prepared via in situ polymerization of monomers to obtain material of hardness 55 Sh D similar to polyurethanes used in medical applications, for example, in heart-assisting devices. The carbon nanoparticle-containing polyester exhibits markedly reduced bacterial colonization, as compared to commercially available polyurethanes. Further the nanocomposite possesses markedly improved hemocompatibility, as determined by flow cytometry, and robust endothelialization. Possible explanations for these beneficial properties include surface nanoroughness of carbon nanoparticle-containing nanocomposites and presence of fatty acid sequences within polymer structure.

Physicochemical and Biological Investigation of Different Structures of Carbon Coatings Deposited onto Polyurethane

The aim of this study was to examine the thrombogenic properties of polyurethane that was surface modified with carbon coatings. Physicochemical properties of manufactured coatings were investigated using transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray Photoelectron Spectroscopy (XPS), Raman spectroscopy and contact angle measurement methods. Samples were examined by the Impact-R method evaluating the level of platelets activation and adhesion of particular blood cell elements. The analysis of antimicrobial resistance against E. coli colonization and viability of endothelial cells showed that polyurethane modified with use of carbon layers constituted an interesting solution for biomedical application.

Interaction of body fluids with carbon surfaces

The use of medical implants allows one to improve patients lives, and quite often it can return patients back to normal activity in their personal and professional lives. One of the most difficult problems, which is necessary to solve, is a proper selection of the materials to be used for implant construction and/or implant coating. The surface of an implant is exposed to continuous contact with body fluids and several unwanted processes may occur there. Titanium and its alloys are generally accepted as the best tolerated materials for implants. But currently many efforts are focused on thin layers of crystalline carbon, i.e. diamond like carbon (DLC) and nanocrystalline diamond (NCD), used for coating of metal implants. This technology was successfully applied in bone surgery (screws), and more recently in heart surgery (stents). We found, with the fluorescence microscopy technique, that bacterial growth was possible on stainless steel, to a lesser degree on titanium, but NCD was almost totally resistant to bacterial colonization.