Verhalten von toxikologisch relevanten Silberverbindungen und Bariumsulfat-Partikeln in komplexen Medien: von der Synthese zu biologischen Untersuchungen

This work comprises the synthesis procedures of toxicologically relevant particles, and their behavior in complex biological media in terms of stability and dissolution, with specific biological responses to the synthesized materials. Two toxicologically relevant particulate systems were studied. As a model of a biologically active material, silver compounds, especially silver nanoparticles, were investigated. In contrast, barium sulfate was examined as a bioinert system. The formation of silver nanoparticles during the reduction with glucose in the presence of poly(vinylpyrrolidone) as a capping agent was followed for more than 3000 min. It was shown that spherical silver nanoparticles were formed first, but in later stages, an increasing fraction of nano-triangles and also a few nanorods developed. By electron microscopy, the growth of spherical and trigonal nanoparticles with time was described. From X-ray powder diffractometry, it was concluded that the domain size in the spherical nanoparticles increased proportionally to the particle diameter and was always about ¼ of the diameter, indicating that twinned seeds were formed very early in the process and then simply grew by extending their size. The lattice constant of the nanoparticles was systematically increased in comparison to microcrystalline silver, but did not change as a function of particle diameter. The behavior of silver ions was investigated in biologically relevant concentrations in different media, from physiological salt solution over phosphate-buffered saline solution to protein-containing cell culture media. The results showed that silver ions that were initially present were bound as silver chloride due to the presence of chloride. Only in the absence of chloride, glucose was able to reduce Ag+ to Ag0. The precipitation of silver phosphate was never observed in any case. It was concluded that the predominant silver species in biological media is dispersed nanoscopic silver chloride, surrounded by a protein corona which prevents the growth of crystals and leads to colloidal stabilization. Using bacteria (S. aureus) and cell culture experiments (human mesenchymal stem cells; T-cells; monocytes), it was possible to show that a toxic effect occurred at the same silver concentration of ionic silver and synthetically prepared silver chloride nanoparticles. Furthermore, the dissolution of silver nanoparticles and the solubility products of silver chloride and silver nitrate were quantified in protein-containing cell culture media. Barium sulfate micro-, sub-micro-, and nanoparticles were synthesized for evaluation of inflammatory potential. The synthesis of fluorescent labeled BaSO4 particles with a narrow size distribution was achieved. The resultant powders were uniform in form, chemical composition and surface modification by the fluorescent dye. All of the particles were colloidally dispersed in cell culture medium with serum. From the cellular uptake studies on rat alveolar macrophages, it was concluded that a significant difference occurred in the case of barium sulfate microparticles for suspended and adherent cell fractions. This was explained by the sedimentation of synthesized particles over time, and confirmed by stability studies of dispersed particles in biological medium by dynamic light scattering. However, in a particle-induced cell migration assay for evaluation of the inflammation potential of the system, no dose- and size-dependency was found

3D biodegradable scaffolds of polycaprolactone with silicate-containing hydroxyapatite microparticles for bone tissue engineering: high-resolution tomography and in vitro study

To date, special interest has been paid to composite scaffolds based on polymers enriched with hydroxyapatite (HA). However, the role of HA containing different trace elements such as silicate in the structure of a polymer scaffold has not yet been fully explored. Here, we report the potential use of silicate-containing hydroxyapatite (SiHA) microparticles and microparticle aggregates in the predominant range from 2.23 to 12.40 µm in combination with polycaprolactone (PCL) as a hybrid scaffold with randomly oriented and well-aligned microfibers for regeneration of bone tissue. Chemical and mechanical properties of the developed 3D scaffolds were investigated with XRD, FTIR, EDX and tensile testing. Furthermore, the internal structure and surface morphology of the scaffolds were analyzed using synchrotron X-ray µCT and SEM. Upon culturing human mesenchymal stem cells (hMSC) on PCL-SiHA scaffolds, we found that both SiHA inclusion and microfiber orientation affected cell adhesion. The best hMSCs viability was revealed at 10 day for the PCL-SiHA scaffolds with well-aligned structure (~82%). It is expected that novel hybrid scaffolds of PCL will improve tissue ingrowth in vivo due to hydrophilic SiHA microparticles in combination with randomly oriented and well-aligned PCL microfibers, which mimic the structure of extracellular matrix of bone tissue

Author Correction: 3D biodegradable scaffolds of polycaprolactone with silicate-containing hydroxyapatite microparticles for bone tissue engineering: high-resolution tomography and in vitro study

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A multi-center study of their physicochemical characteristics, cell culture and in vivo experiments

PVP-capped silver nanoparticles with a diameter of the metallic core of 70 nm, a hydrodynamic diameter of 120 nm and a zeta potential of −20 mV were prepared and investigated with regard to their biological activity. This review summarizes the physicochemical properties (dissolution, protein adsorption, dispersability) of these nanoparticles and the cellular consequences of the exposure of a broad range of biological test systems to this defined type of silver nanoparticles. Silver nanoparticles dissolve in water in the presence of oxygen. In addition, in biological media (i.e., in the presence of proteins) the surface of silver nanoparticles is rapidly coated by a protein corona that influences their physicochemical and biological properties including cellular uptake. Silver nanoparticles are taken up by cell-type specific endocytosis pathways as demonstrated for hMSC, primary T-cells, primary monocytes, and astrocytes. A visualization of particles inside cells is possible by X-ray microscopy, fluorescence microscopy, and combined FIB/SEM analysis. By staining organelles, their localization inside the cell can be additionally determined. While primary brain astrocytes are shown to be fairly tolerant toward silver nanoparticles, silver nanoparticles induce the formation of DNA double-strand-breaks (DSB) and lead to chromosomal aberrations and sister-chromatid exchanges in Chinese hamster fibroblast cell lines (CHO9, K1, V79B). An exposure of rats to silver nanoparticles in vivo induced a moderate pulmonary toxicity, however, only at rather high concentrations. The same was found in precision-cut lung slices of rats in which silver nanoparticles remained mainly at the tissue surface. In a human 3D triple-cell culture model consisting of three cell types (alveolar epithelial cells, macrophages, and dendritic cells), adverse effects were also only found at high silver concentrations. The silver ions that are released from silver nanoparticles may be harmful to skin with disrupted barrier (e.g., wounds) and induce oxidative stress in skin cells (HaCaT). In conclusion, the data obtained on the effects of this well-defined type of silver nanoparticles on various biological systems clearly demonstrate that cell-type specific properties as well as experimental conditions determine the biocompatibility of and the cellular responses to an exposure with silver nanoparticles

Incorporation of Fluoride into Human Teeth after Immersion in Fluoride-Containing Solutions

Toothpastes and mouth rinses contain fluoride as a protective agent against caries. The aim of this study was to determine the degree of fluoride-uptake by human tooth mineral during immersion into fluoride-containing aqueous solutions as different pH. Human teeth were immersed in fluoride-containing solutions to assess the extent of fluoride incorporation into tooth enamel. A total of 16 extracted teeth from 11 patients were immersed at 37 °C for one minute into aqueous fluoride solutions (potassium fluoride; KF) containing either 250 ppm or 18,998 ppm fluoride (1-molar). Fluoride was dissolved either in pure water (neutral pH) or in a citrate buffer (pH 4.6 to 4.7). The elemental surface composition of each tooth was studied by energy-dispersive X-ray spectroscopy in combination with scanning electron microscopy and X-ray powder diffraction. The as-received teeth contained 0.17 ± 0.16 wt% fluoride on average. There was no significant increase in the fluoride content after immersion in 250 ppm fluoride solution at neutral or acidic pH values. In contrast, a treatment with a 1-molar fluoride solution led to significantly increased fluoride concentrations by 0.68 wt% in water and 9.06 wt% at pH 4.7. Although such fluoride concentrations are far above those used in mouth rinses or toothpastes, this indicates that fluoride can indeed enter the tooth surface, especially at a low pH where a dynamic dissolution-reprecipitation process may occur. However, precipitations of calcium fluoride (globuli) were detected in no cases

Automated analysis of transmission electron micrographs of metallic nanoparticles by machine learning

Metallic nanoparticles were analysed with respect to size and shape by a machine learning approach. This involved a separation of particles from the background (segmentation), a separation of overlapping particles, and the identification of individual particles. An algorithm to separate overlapping particles, based on ultimate erosion of convex shapes (UECS), was implemented. Finally, particle properties like size, circularity, equivalent diameter, and Feret diameter were computed for each particle of the whole particle population. Thus, particle size distributions can be easily created based on the various parameters. However, strongly overlapping particles are difficult and sometimes impossible to separate because of an a priori unknown shape of a particle that is partially lying in the shadow of another particle. The program is able to extract information from a sequence of images of the same sample, thereby increasing the number of analysed nanoparticles to several thousands. The machine learning approach is well-suited to identify particles at only limited particle-to-background contrast as is demonstrated for ultrasmall gold nanoparticles (2 nm)

Determination of the Ca/P ratio in calcium phosphates during the precipitation of hydroxyapatite using X-ray diffractometry

The applicability of the X-ray powder diffraction method to the determination of phase composition and Ca/P ratio in precipitates during the nitrous wet synthesis of hydroxyapatite (HA) has been shown. The plotted dependences of the phase composition and the Ca/P ratio on the synthesis time can be used as initial data for the development of new and simple processing routes of calcium phosphate ceramics based on HA of any desired composition