The Relevance of Physico-Chemical Properties and Protein Corona for Evaluation of Nanoparticles Immunotoxicity-In Vitro Correlation Analysis on THP-1 Macrophages.

Alongside physiochemical properties (PCP), it has been suggested that the protein corona of nanoparticles (NPs) plays a crucial role in the response of immune cells to NPs. However, due to the great variety of NPs, target cells, and exposure protocols, there is still no clear relationship between PCP, protein corona composition, and the immunotoxicity of NPs. In this study, we correlated PCP and the protein corona composition of NPs to the THP-1 macrophage response, focusing on selected toxicological endpoints: cell viability, reactive oxygen species (ROS), and cytokine secretion. We analyzed seven commonly used engineered NPs (SiO2, silver, and TiO2) and magnetic NPs. We show that with the exception of silver NPs, all of the tested TiO2 types and SiO2 exhibited moderate toxicities and a transient inflammatory response that was observed as an increase in ROS, IL-8, and/or IL-1ß cytokine secretion. We observed a strong correlation between the size of the NPs in media and IL-1ß secretion. The induction of IL-1ß secretion was completely blunted in NLR family pyrin domain containing 3 (NLRP3) knockout THP-1 cells, indicating activation of the inflammasome. The correlations analysis also implicated the association of specific NP corona proteins with the induction of cytokine secretion. This study provides new insights toward a better understanding of the relationships between PCP, protein corona, and the inflammatory response of macrophages for different engineered NPs, to which we are exposed on a daily basis.

Silk fibroin induces chondrogenic differentiation of canine adipose-derived multipotent mesenchymal stromal cells/mesenchymal stem cells.

Under appropriate culture conditions, mesenchymal stem cells (MSC), also called more properly multipotent mesenchymal stromal cells (MMSC), can be induced toward differentiation into different cell lineages. In order to guide stem cell fate within an environment resembling the stem cell niche, different biomaterials are being developed. In the present study, we used silk fibroin (SF) as a biomaterial supporting the growth of MMSC and studied its effect on chondrogenesis of canine adipose-derived MMSC (cADMMSC). Adipose tissue was collected from nine privately owned dogs. MMSC were cultured on SF films and SF scaffolds in a standard cell culture medium. Cell morphology was evaluated by scanning electron microscopy (SEM). Chondrogenic differentiation was evaluated by alcian blue staining and mRNA expression of collagen type 1, collagen type 2, Sox9, and Aggrecan genes. cADMMSC cultured on SF films and SF scaffolds stained positive using alcian blue. SEM images revealed nodule-like structures with matrix vesicles and fibers resembling chondrogenic nodules. Gene expression of chondrogenic markers Sox9 and Aggrecan were statistically significantly upregulated in cADMMSC cultured on SF films in comparison to negative control cADMMSC. This result suggests that chondrogenesis of cADMMSC could occur when cells were grown on SF films in a standard cell culture medium without specific culture conditions, which were previously considered necessary for induction of chondrogenic differentiation.

Viscoelastic behaviour of hydrogel-based composites for tissue engineering under mechanical load.

Along with biocompatibility, bioinductivity and appropriate biodegradation, mechanical properties are also of crucial importance for tissue engineering scaffolds. Hydrogels, such as gellan gum (GG), are usually soft materials, which may benefit from the incorporation of inorganic particles, e.g. bioactive glass, not only due to the acquired bioactivity, but also due to improved mechanical properties. They exhibit complex viscoelastic properties, which can be evaluated in various ways. In this work, to reliably evaluate the effect of the bioactive glass (BAG) addition on viscoelastic properties of the composite hydrogel, we employed and compared the three most commonly used techniques, analyzing their advantages and limitations: monotonic uniaxial unconfined compression, small amplitude oscillatory shear (SAOS) rheology and dynamic mechanical analysis (DMA). Creep and small amplitude dynamic strain-controlled tests in DMA are suggested as the best ways for the characterization of mechanical properties of hydrogel composites, whereas the SAOS rheology is more useful for studying the hydrogel's processing kinetics, as it does not induce volumetric changes even at very high strains. Overall, the results confirmed a beneficial effect of BAG (nano)particles on the elastic modulus of the GG-BAG composite hydrogel. The Young's modulus of 6.6 ± 0.8 kPa for the GG hydrogel increased by two orders of magnitude after the addition of 2 wt.% BAG particles (500-800 kPa).

Characterisation of food contact non-stick coatings containing TiO2 nanoparticles and study of their possible release into food.

Novel nanoparticles containing non-stick coatings have been developed for food contact applications such as frying pans. Possible release of nanoparticles from such coatings into food is not known. In this paper, the characterisation of commercially available non-stick coatings was performed by use of FTIR, electron and optical microscopy, EDXS and XRD analysis. Characterisation revealed that the coatings contained micron- and nanosized rutile TiO2 particles, and quartz SiO2 embedded in a silicone polymer matrix. In order to estimate possible migration of TiO2 nanoparticles from coatings into food, migration tests into simulants (deionised water, 3% acetic acid and 5 g l-1 citric acid) were performed (2 h at 100°C), and thermal and mechanical degradation of the matrix was studied. Simulants were analysed by ICP-MS after ultrafiltration and by microwave-assisted digestion. The concentration of titanium-containing particles that migrated into simulants was up to 861 µg l-1 (147 µg dm-2). Titanium was present in simulants in ionic form as well. The presence of TiO2 nanoparticles in 3% acetic acid was confirmed by SEM-EDXS analysis. Thermal stability study (TG/DSC MS analysis) did not show degradation of the matrix under foreseeable conditions of use, but mechanical degradation studies (scratch and tribological testing) showed possible release (microgram quantities per punched sample) of titanium-containing nanoparticles. The matrix degradation results were confirmed by observations of the morphology of the same type of coatings actually used for food preparation. Dissolution from the surface and matrix degradation can both contribute to nanoparticles release from this type of non-stick food contact coatings.

Electrokinetic behaviour of porous TiO2-coated implants.

It is known that the "race for the surface" determining the in vivo response is strictly connected to the physico-chemical properties of the material, especially at its surface. Accordingly, the study of surface roughness, charge and wettability is fundamental to predict the bio-response to the implant. In this work, streaming potential was chosen as a reliable method to quantify the solid surface charge of hydrothermally treated (HT) TiO2-anatase nano-crystalline coatings, grown on titanium substrates. The influence of metal and ionic conductance on the zeta potential values was taken into account, allowing for the correlation of the surface charge with the coating porosity, the semiconductor character of the TiO2 nano-crystals and the metallic nature of the bulk titanium.

Enhanced osteogenesis on titanium implants by UVB photofunctionalization of hydrothermally grown TiO2 coatings.

Even though Ti-based implants are the most used materials for hard tissue replacement, they may present lack of osseointegration on the long term, due to their inertness. Hydrothermal treatment (HT) is a useful technique for the synthesis of firmly attached, highly crystalline coatings made of anatase titanium dioxide (TiO2), providing favorable nanoroughness and higher exposed surface area, as well as greater hydrophilicity, compared to the native amorphous oxide on pristine titanium. The hydrophilicity drops even more by photofunctionalization of the nanostructured TiO2-anatase coatings under UV light. Human mesenchymal stem cells exhibited a good response to the combination of the positive surface characteristics, especially in respect to the UVB pre-irradiation. The results showed that the cells were not harmed in terms of viability; even more, they were encouraged to differentiate in osteoblasts and to become osteogenically active, as confirmed by the calcium ion uptake and the formation of well-mineralized, bone-like nodule structures. In addition, the enrichment of hydroxyl groups on the HT-surfaces by UVB photofunctionalization accelerated the cell differentiation process and greatly improved the osteogenesis in comparison with the nonirradiated samples. The optimal surface characteristics of the HT-anatase coatings as well as the high potentiality of the photo-induced hydrophilicity, which was reached during a relatively short pre-irradiation time (5 h) with UVB light, can be correlated with better osseointegration ability in vivo; among the samples, the superior biological behavior of the roughest and most hydrophilic HT coating makes it a good candidate for further studies and applications.

The influence of surface modification on bacterial adhesion to titanium-based substrates.

This study examines bacterial adhesion on titanium-substrates used for bone implants. Adhesion is the most critical phase of bacterial colonization on medical devices. The surface of titanium was modified by hydrothermal treatment (HT) to synthesize nanostructured TiO2-anatase coatings, which were previously proven to improve corrosion resistance, affect the plasma protein adsorption, and enhance osteogenesis. The affinity of the anatase coatings toward bacterial attachment was studied by using a green fluorescent protein-expressing Escherichia coli (gfp-E. coli) strain in connection with surface photoactivation by UV irradiation. We also analyzed the effects of surface topography, roughness, charge, and wettability. The results suggested the dominant effects of the macroscopic surface topography, as well as microasperity at the surface roughness scale, which were produced during titanium machining, HT treatment, or both. Macroscopic grooves provided a preferential site for bacteria deposit within the valleys, while the microscopic roughness of the valleys determined the actual interaction surface between bacterium and substrate, resulting in an "interlocking" effect and undesired high bacterial adhesion on nontreated titanium. In the case of TiO2-coated samples, the nanocrystals reduced the width between the microasperities and thus added nanoroughness features. These factors decreased the contact area between the bacterium and the coating, with consequent lower bacterial adhesion (up to 50% less) in comparison to the nontreated titanium. On the other hand, the pronounced hydrophilicity of one of the HT-coated discs after pre-irradiation seemed to enhance the attachment of bacteria, although the increase was not statistically significant (p > 0.05). This observation may be explained by the acquired similar degree of wetting between gfp-E. coli and the coating. No correlation was found between the bacterial adhesion and the ζ-values of the samples in PBS, so the effect of surface charge was considered negligible in this study.

Nanoparticulate bioactive-glass-reinforced gellan-gum hydrogels for bone-tissue engineering.

This work presents bioactive-glass-reinforced gellan-gum spongy-like hydrogels (GG-BAG) as novel hydrophilic materials for use as the scaffolding in bone-tissue engineering. The reinforcement with bioactive-glass particles resulted in an improvement to the microstructure and to the mechanical properties of the material. These mechanical properties were found to be dependent on the composition and improved with the amount of bioactive glass; however, values necessary to accommodate biomechanical loading were not achieved in this study. Nevertheless, by incorporating the bioactive-glass particles, the composite material acquired the ability to form an apatite layer when soaked in simulated body fluid. Furthermore, human-adipose-derived stem cells were able to adhere and spread within the gellan-gum, spongy-like hydrogels reinforced with the bioactive glass, and remain viable, which is an important result when considering their use in bone-tissue engineering. Thus, hydrogels based on gellan gum and bioactive glass are promising biomaterials for use either alone or with cells, and with the potential for use in osteogenic differentiation.

Photoinduced properties of nanocrystalline TiO2-anatase coating on Ti-based bone implants.

The paper reports on the photoinduced properties of hydrothermally treated (HT) titanium used for bone implants. The anatase coatings composed of 30-100nm anatase crystals exhibited high photocatalytic activity and good photo-induced wettability, reaching a superhydrophilic state, despite the larger crystal dimensions than the previously reported optimal ones. These properties are due to a suitable combination of surface texture, roughness, thickness, crystal morphology and particle size, which allowed the two independent photo-induced phenomena to occur simultaneously. The results on caffeine degradation by photocatalysis and the prolonged effect (up to two weeks) of photo-induced wettability in dark suggested a possible applicability of the HT anatase coatings as bacteria-repelling surfaces for body implants, in favor of a better osseointegration in vivo.

Improvement to the Corrosion Resistance of Ti-Based Implants Using Hydrothermally Synthesized Nanostructured Anatase Coatings.

The electrochemical behavior of polycrystalline TiO2 anatase coatings prepared by a one-step hydrothermal synthesis on commercially pure (CP) Ti grade 2 and a Ti13Nb13Zr alloy for bone implants was investigated in Hank's solution at 37.5 °C. The aim was to verify to what extent the in-situ-grown anatase improved the behavior of the substrate in comparison to the bare substrates. Tafel-plot extrapolations from the potentiodynamic curves revealed a substantial improvement in the corrosion potentials for the anatase coatings. Moreover, the coatings grown on titanium also exhibited lower corrosion-current densities, indicating a longer survival of the implant. The results were explained by considering the effects of crystal morphology, coating thickness and porosity. Evidence for the existing porosity was obtained from corrosion and nano-indentation tests. The overall results indicated that the hydrothermally prepared anatase coatings, with the appropriate morphology and surface properties, have attractive prospects for use in medical devices, since better corrosion protection of the implant can be expected.

Analytical electron microscopy of W-core ß-SiC fibers for use in an SiC-based composite material for fusion applications.

In this work, the interactions between tungsten (W) and silicon carbide (SiC) in Sigma SiC fibers at high temperatures were characterized using scanning and transmission electron microscopy. These fibers could have the potential for use in fusion-related applications owing to their high thermal conductivity compared with pure SiC-based fibers. The as-received fibers were composed of a 100-µm-thick shell of radially textured ß-SiC grains and a 15-µm-thick tungsten core, composed of a few hundreds of nm-sized elongated tungsten grains. The interfaces between the tungsten and the SiC and the SiC and the outer coatings were sharp and smooth. After heat treatment at 1,600°C for 3 h in Ar, the tungsten core reacted with SiC to form a rough interface surface. Inside the core, W5Si3, W3Si, and W2C phases were detected using energy-dispersive X-ray spectroscopy and electron-diffraction techniques. The mechanical properties of the fibers deteriorate after the heat treatment.

SiC-CNT composite prepared by electrophoretic codeposition and the polymer infiltration and pyrolysis process.

The paper reports on the successful anodic codeposition of submicrometer SiC powder and multiwalled carbon nanotubes from aqueous suspensions to form SiC-CNT composites. On the basis of the comprehensive analysis of the aqueous suspensions with different pHs, solids contents, and CNT contents, optimal conditions for deposition were determined. Besides having the necessary high absolute value of the ζ-potential, the suspensions that resulted in firm deposits were characterized by limited conductivity (<1 mS/cm). Lowering of suspension conductivity was achieved either by dilution of the suspension or by dialysis of the as-received CNT suspension with high intrinsic conductivity. Selected SiC-CNT deposits were further densified by use of the polymer infiltration and pyrolysis process.

Bioactive glass enhances bone ingrowth into the porous titanium coating on orthopaedic implants.

PURPOSE: The aim of the study was to verify the ability of nanoparticulate bioactive glass (BAG) to infiltrate into the porous titanium (Ti) layer on Ti-based implants to promote osseointegration. METHODS: The porous titanium layer on Ti-based implants was impregnated with nanoparticulate BAG. The implants without or with BAG were implanted bilaterally in tibial holes of ten New Zealand white rabbits. The rabbits were sacrificed after ten weeks for examinations. Beside histological examination, EDXS analysis of polished cross-sections of explanted implants was also performed with the aim to quantitatively evaluate the bone-to-pore contact and bone-in-pore ratio. RESULTS: After ten weeks, EDXS analyses of cross-sections of the explanted implants confirmed that bioactive glass was fully resorbed and that the pores throughout the thickness of the porous titanium layer were to a large extent filled with a new bone. In the absence of bioactive glass, only the outer part of the porous layer was filled with bone. The implants without BAG in the porous Ti-layer exhibited similar bone-to-pore contact, while significant improvement of bone ingrowth into the pores was observed for the implants with BAG (38%), as opposed to those without it (22%). CONCLUSION: This study confirmed that the nanoparticulate bioactive glass within the porous titanium surface layer on implants promotes osseointegration and stimulates the formation of bone within the pores.

Pre-irradiation of anatase TiO2 particles with UV enhances their cytotoxic and genotoxic potential in human hepatoma HepG2 cells.

Titanium dioxide (TiO(2)) is active in the UV region of the light spectra and is used as a photocatalyst in numerous applications. Photo-activated anatase TiO(2) particles promote increased production of free radicals. This is a desirable property, although the potential toxicity of such photo-activated TiO(2) particles on exposure of humans and the environment remains unknown. Therefore, we studied whether pre-irradiation of TiO(2) particles with UV influences their cytotoxic and genotoxic potential. The TiO(2) particles, as TiO(2)-A (<25 nm) and TiO(2)-B (>100 nm), were UV pre-irradiated (24h) and tested for cytotoxic and genotoxic activities in human hepatoma HepG2 cells. Non-irradiated TiO(2)-A/B at 1.0-250 µg/ml did not reduce viability of HepG2 cells, nor induce significant increases in DNA strand breaks; only TiO(2)-A induced significant increases in oxidative DNA damage. After UV pre-irradiation, both TiO(2)-A and TiO(2)-B reduced cell viability and induced significant increases in DNA strand breaks and oxidative DNA damage. This is the first study that shows that UV pre-irradiation of anatase TiO(2) particles results in increased cytotoxic and genotoxic potential. This warrants further studies as it has important implications for environmental and human health risk assessment and preventive actions to limit human exposure.

DNA damage and alterations in expression of DNA damage responsive genes induced by TiO2 nanoparticles in human hepatoma HepG2 cells.

We investigated the genotoxic responses to two types of TiO2 nanoparticles (<25 nm anatase: TiO(2)-An, and <100 nm rutile: TiO2-Ru) in human hepatoma HepG2 cells. Under the applied exposure conditions the particles were agglomerated or aggregated with the size of agglomerates and aggregates in the micrometer range, and were not cytotoxic. TiO2-An, but not TiO2-Ru, caused a persistent increase in DNA strand breaks (comet assay) and oxidized purines (Fpg-comet). TiO2-An was a stronger inducer of intracellular reactive oxygen species (ROS) than TiO2-Ru. Both types of TiO2 nanoparticles transiently upregulated mRNA expression of p53 and its downstream regulated DNA damage responsive genes (mdm2, gadd45α, p21), providing additional evidence that TiO2 nanoparticles are genotoxic. The observed differences in responses of HepG2 cells to exposure to anatase and rutile TiO2 nanoparticles support the evidence that the toxic potential of TiO2 nanoparticles varies not only with particle size but also with crystalline structure.

Titanium dioxide in our everyday life; is it safe?

BACKGROUND: Titanium dioxide (TiO(2)) is considered as an inert and safe material and has been used in many applications for decades. However, with the development of nanotechnologies TiO(2) nanoparticles, with numerous novel and useful properties, are increasingly manufactured and used. Therefore increased human and environmental exposure can be expected, which has put TiO(2) nanoparticles under toxicological scrutiny. Mechanistic toxicological studies show that TiO(2) nanoparticles predominantly cause adverse effects via induction of oxidative stress resulting in cell damage, genotoxicity, inflammation, immune response etc. The extent and type of damage strongly depends on physical and chemical characteristics of TiO(2) nanoparticles, which govern their bioavailability and reactivity. Based on the experimental evidence from animal inhalation studies TiO(2) nanoparticles are classified as "possible carcinogenic to humans" by the International Agency for Research on Cancer and as occupational carcinogen by the National Institute for Occupational Safety and Health. The studies on dermal exposure to TiO(2) nanoparticles, which is in humans substantial through the use of sunscreens, generally indicate negligible transdermal penetration; however data are needed on long-term exposure and potential adverse effects of photo-oxidation products. Although TiO(2) is permitted as an additive (E171) in food and pharmaceutical products we do not have reliable data on its absorption, distribution, excretion and toxicity on oral exposure. TiO(2) may also enter environment, and while it exerts low acute toxicity to aquatic organisms, upon long-term exposure it induces a range of sub-lethal effects. CONCLUSIONS: Until relevant toxicological and human exposure data that would enable reliable risk assessment are obtained, TiO(2) nanoparticles should be used with great care.

[Topographic localization of an intraocular foreign body by B-scan echography].

BACKGROUND/AIM: In cases of blurred optic media the ultrasound diagnostics offers useful data about eventual presence of intraocular foreign body as well as about its precise localization in the eye. The aim of this study was to retrospectively analyze echographic findings in patients with the diagnosis of intraocular foreign body with a special interest in localizations of a intraocular foreign body in the eye and the presence of an eventual infection - endophthalmitis. The aim of this study was also to confirm the localization of intraocular foreign body by echography and to test the precision of this method. METHODS: We performed analysis of all cases that had been referred to the ultrasound diagnostices, in which the presence of intraocular foreign body had been confirmed in the period of one year. All examinations were done with B-scan and were confirmed during the surgery - vitrectomy. RESULTS: In the one-year period we were contacted by 27 patients with intraocular foreign body. In one injured eye the intraocular foreign body was in the lens (3.70%), in 10 injured eyes (37.03%) intraocular foreign body was in the vitreal body. In 15 patients (55.5%) intraocular foreign body was fixed in the retina. In one patient (3.70%) there was a perforating injury, intraocular foreign body was found in the retrobulbar part of the orbit. In 7 injured eyes (25.9%), with the presence of intraocular foreign body, we found signs of endophthalmitis (organized blurring in vitreal space, thickened choroid). Other accompanying echographic findings were: blood in vitreal space, haemophthalmus in 12 cases (44.4%), retinal detachment in 5 cases (18.5%) and subretinal hemorrhagies in 4 cases (14.8%). CONCLUSION: Ultrasound diagnostics can very precisely show the localization of intraocular foreign body in the eye that is very important in the choice of approach and timing of surgical treatment. Also the echographic diagnostics may find an accompanying endophthalmitis in the posterior segment of the eye, that is very important for an urgent therapeutic approach.

Electron microscopy and microanalysis of the fiber-matrix interface in monolithic silicone carbide-based ceramic composite material for use in a fusion reactor application.

A composite material made from continuous monolithic silicone carbide (SiC) fibers and a SiC-based matrix (SiC(f)/SiC), was prepared using a novel technique, i.e. adapted dip coating and infiltration of SiC fibers with a water suspension containing SiC particles and a sintering additive. This kind of material could be used in the first-wall blanket of a future fusion reactor. Using magnetron sputtering, the SiC fibers were coated with various thin layers (TiC, CrN, CrC, WC, DLC-diamond-like carbon) of the interface material by physical vapor deposition (PVD). Using scanning and transmission electron microscopy and microanalysis, detailed microstructural studies of the fiber-matrix interface were performed. Both samples, with coated and uncoated fibers, were examined under a load. The microcracks introduced by the Vickers indenter continued their path through the fibers, and thus caused the failure of the composite material, in the case of the uncoated fibers or deviated from their primary direction at the fiber-matrix interface in the case of the coated fibers.