Zuzana Burdikova

Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter

Yeasts, when growing on solid surfaces, form organized multicellular structures, colonies, in which cells differentiate and thus possess different functions and undergo dissimilar fate. Understanding the principles involved in the formation of these structures requires new approaches that allow the study of individual cells directly in situ without needing to remove them from the microbial community. Here we introduced a new approach to the analysis of whole yeast microcolonies either containing specific proteins labelled by fluorescent proteins or stained with specific dyes, by two-photon excitation confocal microscopy. It revealed that the colonies are covered with a thin protective skin-like surface cell layer which blocks penetration of harmful compounds. The cells forming the layer are tightly connected via cell walls, the presence of which is essential for keeping of protective layer function. Viewing the colonies from different angles allowed us to reconstruct a three-dimensional profile of the cells producing ammonium exporter Ato1p within developing microcolonies growing either as individuals or within a group of microcolonies. We show that neighbouring microcolonies coordinate production of Ato1p-GFP. Ato1p itself appears synchronously in cells, which do not originate from the same ancestor, but occupy specific position within the colony.

Enhanced Growth and Osteogenic Differentiation of Human Osteoblast-Like Cells on Boron-Doped Nanocrystalline Diamond Thin Films

Intrinsic nanocrystalline diamond (NCD) films have been proven to be promising substrates for the adhesion, growth and osteogenic differentiation of bone-derived cells. To understand the role of various degrees of doping (semiconducting to metallic-like), the NCD films were deposited on silicon substrates by a microwave plasma-enhanced CVD process and their boron doping was achieved by adding trimethylboron to the CH4:H2 gas mixture, the B∶C ratio was 133, 1000 and 6700 ppm. The room temperature electrical resistivity of the films decreased from >10 MΩ (undoped films) to 55 kΩ, 0.6 kΩ, and 0.3 kΩ (doped films with 133, 1000 and 6700 ppm of B, respectively). The increase in the number of human osteoblast-like MG 63 cells in 7-day-old cultures on NCD films was most apparent on the NCD films doped with 133 and 1000 ppm of B (153,000±14,000 and 152,000±10,000 cells/cm2, respectively, compared to 113,000±10,000 cells/cm2 on undoped NCD films). As measured by ELISA per mg of total protein, the cells on NCD with 133 and 1000 ppm of B also contained the highest concentrations of collagen I and alkaline phosphatase, respectively. On the NCD films with 6700 ppm of B, the cells contained the highest concentration of focal adhesion protein vinculin, and the highest amount of collagen I was adsorbed. The concentration of osteocalcin also increased with increasing level of B doping. The cell viability on all tested NCD films was almost 100%. Measurements of the concentration of ICAM-1, i.e. an immunoglobuline adhesion molecule binding inflammatory cells, suggested that the cells on the NCD films did not undergo significant immune activation. Thus, the potential of NCD films for bone tissue regeneration can be further enhanced and tailored by B doping and that B doping up to metallic-like levels is not detrimental for cells.

Analysis and three-dimensional visualization of collagen in artificial scaffolds using nonlinear microscopy techniques

Extracellularly distributed collagen and chondrocytes seeded in gelatine and poly-ɛ-caprolactone scaffolds are visualized by two-photon excitation microscopy (TPEM) and second-harmonic generation (SHG) imaging in both forward and backward nondescanned modes. Joint application of TPEM and SHG imaging in combination with stereological measurements of collagen enables us not only to take high-resolution 3-D images, but also to quantitatively analyze the collagen volume and a spatial arrangement of cell-collagen-scaffold systems, which was previously impossible. This novel approach represents a powerful tool for the analysis of collagen-containing scaffolds with applications in cartilage tissue engineering.

Osteogenic cell differentiation on H-terminated and O-terminated nanocrystalline diamond films.

Nanocrystalline diamond (NCD) films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination) or oxygen atoms (O-termination). Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers – type I collagen, alkaline phosphatase, and osteocalcin – was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix mineralization, and this is promising for better osteoconductivity of potential bone implant coatings.

A perivascular system releasing sirolimus prevented intimal hyperplasia in a rabbit model in a medium-term study

The main complication of aortocoronary reconstruction with vein grafts is restenosis in the course of time. The aim was to assess the effect of a periadventitial polyester mesh releasing sirolimus on intimal hyperplasia of autologous grafts. We implanted v. jugularis ext. into a. carotis communis in rabbits. The vein graft was either intact, or was wrapped with a pure polyester mesh, or with a sirolimus-releasing mesh. Three and six weeks after surgery, the veins were subjected to standard histological staining and the thicknesses of the tunica intima, the media and the intima-media complex were measured. Wrapping the vein with a mesh releasing sirolimus or with a pure mesh decreased the thickness of the intima in comparison with a vein graft by 73 ± 11% or 73 ± 8% after 3 weeks, and by 73 ± 9% or 59 ± 12% after 6 weeks, respectively. Sirolimus-releasing meshes reduced the thickness of the media by 65 ± 9% and 20 ± 12% after 3 and 6 weeks. The thickness of the intima-media complex in grafts with sirolimus-releasing meshes decreased by 60 ± 6% and 30 ± 13% in comparison with pure PES meshes, after 3 and 6 weeks, respectively. A periadventitial polyester mesh releasing sirolimus has the potential to become an effective device in preventing vein graft restenosis.

Testate amoebae examined by confocal and two-photon microscopy: implications for taxonomy and ecophysiology.

Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiology.

A human pericardium biopolymeric scaffold for autologous heart valve tissue engineering: cellular and extracellular matrix structure and biomechanical properties in comparison with a normal aortic heart valve

Abstract The objective of our study was to compare the cellular and extracellular matrix (ECM) structure and the biomechanical properties of human pericardium (HP) with the normal human aortic heart valve (NAV). HP tissues (from 12 patients) and NAV samples (from 5 patients) were harvested during heart surgery. The main cells in HP were pericardial interstitial cells, which are fibroblast-like cells of mesenchymal origin similar to the valvular interstitial cells in NAV tissue. The ECM of HP had a statistically significantly (p < 0.001) higher collagen I content, a lower collagen III and elastin content, and a similar glycosaminoglycans (GAGs) content, in comparison with the NAV, as measured by ECM integrated density. However, the relative thickness of the main load-bearing structures of the two tissues, the dense part of fibrous HP (49 ± 2%) and the lamina fibrosa of NAV (47 ± 4%), was similar. In both tissues, the secant elastic modulus (Es) was significantly lower in the transversal direction (p < 0.05) than in the longitudinal direction. This proved that both tissues were anisotropic. No statistically significant differences in UTS (ultimate tensile strength) values and in calculated bending stiffness values in the longitudinal or transversal direction were found between HP and NAV. Our study confirms that HP has an advantageous ECM biopolymeric structure and has the biomechanical properties required for a tissue from which an autologous heart valve replacement may be constructed.

Collagen structures in pericardium and aortic heart valves and their significance for tissue engineering

Biological prostheses of human heart valves are prepared from autologous heart valves and from xenogeneic heart valves or pericardium. Xenogenous and allogenous biological prostheses are associated with adverse immune reactions, thrombosis and degeneration, and thus they have a high rate of reoperation. An optimum autologous tissue for heart valve grafts therefore needs to be found. Human pericardium was assessed as a potential source, and was compared with native human heart valves and porcine and bovine pericardium and heart valves. The tissues were evaluated for their mechanical properties, their collagen content and structure, and their histological structure. We observed differences in mechanical properties, collagen fibre orientation, the shape of the collagen bundles, and the collagen content among the pericardium samples and heart valves of different origin.