Marie Kalbacova

Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes

An emerging concept in cell signalling is the natural role of reactive oxygen species such as hydrogen peroxide (H2O2) as beneficial messengers in redox signalling pathways. The nature of H2O2 signalling is confounded, however, by difficulties in tracking it in living systems, both spatially and temporally, at low concentrations. Here, we develop an array of fluorescent single-walled carbon nanotubes that can selectively record, in real time, the discrete, stochastic quenching events that occur as H2O2 molecules are emitted from individual human epidermal carcinoma cells stimulated by epidermal growth factor. We show mathematically that such arrays can distinguish between molecules originating locally on the cell membrane from other contributions. We find that epidermal growth factor induces 2 nmol H2O2 locally over a period of 50 min. This platform promises a new approach to understanding the signalling of reactive oxygen species at the cellular level.

Dominant Renin Gene Mutations Associated with Early-Onset Hyperuricemia, Anemia, and Chronic Kidney Failure

Through linkage analysis and candidate gene sequencing, we identified three unrelated families with the autosomal-dominant inheritance of early onset anemia, hypouricosuric hyperuricemia, progressive kidney failure, and mutations resulting either in the deletion (p.Leu16del) or the amino acid exchange (p.Leu16Arg) of a single leucine residue in the signal sequence of renin. Both mutations decrease signal sequence hydrophobicity and are predicted by bioinformatic analyses to damage targeting and cotranslational translocation of preprorenin into the endoplasmic reticulum (ER). Transfection and in vitro studies confirmed that both mutations affect ER translocation and processing of nascent preprorenin, resulting either in reduced (p.Leu16del) or abolished (p.Leu16Arg) prorenin and renin biosynthesis and secretion. Expression of renin and other components of the renin-angiotensin system was decreased accordingly in kidney biopsy specimens from affected individuals. Cells stably expressing the p.Leu16del protein showed activated ER stress, unfolded protein response, and reduced growth rate. It is likely that expression of the mutant proteins has a dominant toxic effect gradually reducing the viability of renin-expressing cells. This alters the intrarenal renin-angiotensin system and the juxtaglomerular apparatus functionality and leads to nephron dropout and progressive kidney failure. Our findings provide insight into the functionality of renin-angiotensin system and stress the importance of renin analysis in families and individuals with early onset hyperuricemia, anemia, and progressive kidney failure.

Nanoscale topography of nanocrystalline diamonds promotes differentiation of osteoblasts.

The excellent mechanical, tribological and biochemical properties of diamond coatings are promising for improving orthopedic or stomatology implants. A crucial prerequisite for such applications is an understanding and control of the biological response of the diamond coatings. This study concentrates on the correlation of diamond surface properties with osteoblast behavior. Nanocrystalline diamond (NCD) films (grain size up to 200 nm, surface roughness 20 nm) were deposited on silicon substrates of varying roughnesses (1, 270 and 500 nm) and treated by oxygen plasma to generate a hydrophilic surface. Atomic force microscopy was used for topographical characterization of the films. As a reference surface, tissue culture polystyrene (PS) was used. Scanning electron microscopy and immunofluorescence staining was used to visualize cell morphological features as a function of culture time. Metabolic activity, alkaline phosphatase activity, and calcium and phosphate deposition was also monitored. The results show an enhanced osteoblast adhesion as well as increased differentiation (raised alkaline phosphatase activity and mineral deposition) on NCD surfaces (most significantly on RMS 20 nm) compared to PS. This is attributed mainly to the specific surface topography as well as to the biocompatible properties of diamond. Hence the controlled (topographically structured) diamond coating of various substrates is promising for preparation of better implants, which offer faster colonization by specific cells as well as longer-term stability.

Response of osteoblast-like SAOS-2 cells to zirconia ceramics with different surface topographies

OBJECTIVES Zirconia is a suitable biomaterial for use in medicine (stomatology, orthopaedics) due to its good biocompatibility and outstanding mechanical properties. This study compares the effect of (i) zirconia to the widely used titanium and (ii) zirconia with two different surface topographies (sandblasted and sandblasted/etched) on the adhesion, proliferation and differentiation of SAOS-2 osteoblasts. METHODS SAOS-2 cells were cultured on either sandblasted or sandblasted/etched zirconia and compared with sandblasted/etched titanium. 2 and 24 h after plating, cell morphology was investigated by scanning electron microscope (SEM) and fluorescence imaging. At 24 and 48 h, cell number-relevant parameters were determined. Alkaline phosphatase (ALP) activity and mineral accumulation were measured at days 8, 11, 15 and day 22 of culture, respectively. RESULTS SEM and fluorescence images revealed a faster spreading as well as higher number of adherent cells after 24 h incubation on zirconia compared with titanium. Also, the cellular metabolic activity after 24 h and the proliferation rate after 48 h is higher with zirconia compared with titanium. Zirconia had a more pronounced effect compared with titanium on the differentiation of SAOS-2 cells: ALP activity, an early differentiation marker increased earlier and mineralization, a late differentiation marker was increased. Only minor differences were found between zirconia with two different surface topographies; etched zirconia promoted slightly greater the differentiation of SAOS-2 cells. CONCLUSIONS These data indicate that zirconia mediates a pronounced stronger effect on the adhesion, proliferation and differentiation compared with titanium; and that topographical differences of zirconia have minor effects on osteoblast biology.

Micro-Pattern Guided Adhesion of Osteoblasts on Diamond Surfaces

Microscopic chemical patterning of diamond surfaces by hydrogen and oxygen surface atoms is used for self-assembly of human osteoblastic cells into micro-arrays. The cell adhesion and assembly is further controlled by concentration of cells (2,500-10,000 cells/cm2) and fetal bovine serum (0-15%). The cells are characterized by fluorescence microscopy of actin fibers and nuclei. The serum protein adsorption is studied by atomic force microscopy (AFM). The cells are arranged selectively on O-terminated patterns into 30-200 μm wide arrays. Higher cell concentrations allow colonization of unfavorable H-terminated regions due to mutual cell communication. There is no cell selectivity without the proteins in the medium. Based on the AFM, the proteins are present on both H- and O-terminated surfaces. Pronounced differences in their thickness, surface roughness, morphology, and phase images indicate different conformation of the proteins and explain the cell selectivity.

Comparison of the Effect of Mitochondrial Inhibitors on Mitochondrial Membrane Potential in Two Different Cell Lines Using Flow Cytometry and Spectrofluorometry

Determination of mitochondrial membrane potential (ΔΨm) is widely used to characterize cellular metabolism, viability, and apoptosis. Changes of ΔΨm induced by inhibitors of oxidative phosphorylation characterize respective contributions of mitochondria and glycolysis to adenosine triphosphate (ATP) synthesis.

Effects of protein inter-layers on cell–diamond FET characteristics

Diamond is recognized as an attractive material for merging solid-state and biological systems. The advantage of diamond field-effect transistors (FET) is that they are chemically resistant, bio-compatible, and can operate without gate oxides. Solution-gated FETs based on H-terminated nanocrystalline diamond films exhibiting surface conductivity are employed here for studying effects of fetal bovine serum (FBS) proteins and osteoblastic SAOS-2 cells on diamond electronic properties. FBS proteins adsorbed on the diamond FETs permanently decrease diamond conductivity as reflected by the -45 mV shift of the FET transfer characteristics. Cell cultivation for 2 days results in a further shift by another -78 mV. We attribute it to a change of diamond material properties rather than purely to the field-effect. Increase in gate leakage currents (by a factor of 4) indicates that the FBS proteins also decrease the diamond-electrolyte electronic barrier induced by C-H surface dipoles. We propose a model where the proteins replace ions in the very vicinity of the H-terminated diamond surface.

Influence of the fetal bovine serum proteins on the growth of human osteoblast cells on graphene

The influence of single-layer graphene produced by chemical vapor deposition on human osteoblast cells under different conditions was studied. Measurements probed the ability of cells to adhere and proliferate on graphene compared with SiO(2)/Si substrates and standard tissue culture plastic when cells were incubated for the first 2 h in the presence or the absence of fetal bovine serum (FBS), thus influencing the initial, direct interaction of cells with the substrate. It was found that after 48 h of human osteoblast incubation on graphene films, there were a comparable number of cells of a similar size irrespective of the presence or the absence of serum proteins. On the other hand, a strong initial influence through the presence of FBS proteins on cell number and cell size was observed in the case of the SiO(2)/Si substrate and control plastic. Thus, our study showed that the initial presence/absence of FBS in the medium does not determine cell fate in the case of a graphene substrate, which is very unusual and different from the behavior of cells on other materials.

Evaluation of Sericin as a Fetal Bovine Serum-Replacing Cryoprotectant During Freezing of Human Mesenchymal Stromal Cells and Human Osteoblast-Like Cells

A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate or reduce the presence of dimethyl sulfoxide (DMSO) in the medium. Sericin, a sticky protein derived from the silkworm cocoon, was investigated as a substitute for FBS and DMSO in the freezing medium. Cell viability (24 hours after thawing, both hMSC and SAOS-2) and colony-forming ability (2 weeks after thawing, only for hMSCs) were both determined. The FBS-free medium with 1% sericin in 10% DMSO was found to be a suitable freezing medium for primary hMSCs, in contrast to immortalized human osteoblasts. Surprisingly, the storage of hMSCs in a cultivation medium with only 10% DMSO also provided satisfactory results. Any drop in DMSO concentration led to significantly worse survival of cells, with little improvement in hMSC survival in the presence of sericin. Thus, sericin may substitute for FBS in the freezing medium for primary hMSCs, but cannot substitute for DMSO.

The impact of doped silicon quantum dots on human osteoblasts

Silicon (Si) nanostructures allow for the expansion of the application spectrum of this important semiconductor material with respect to the fields of optoelectronics and photonics. At the same time, the significant potential of Si quantum dots (SiQDs) has been revealed in terms of their potential application in the areas of biology and medicine due to their biocompatibility, low toxicity and natural biodegradability, unlike currently used semiconductor quantum dots. As far as this study is concerned, SiQDs co-doped with boron and phosphorus were used for the in vitro evaluation of their cytotoxicity in human osteoblasts. Two chemically identical types of SiQD differing in terms of their size and photoluminescence (PL) were studied. They both display long-lasting dispersion in methanol and even in aqueous media as well as PL which is not sensitive either to changes in the environment or surface modifications. Our experiments revealed significant differences between the two types of SiQD tested in regard to their behavior in a cell culture environment depending on increasing concentration (25–125 μg ml−1) and cultivation conditions (the presence or absence of proteins from the fetal bovine serum – a component of the cultivation medium). A detailed description of their optical parameters and the evaluation of zeta potential enhance the understanding of the complexities of the in vitro results obtained.