Petr Humpolíček

Cell Proliferation of HaCaT Keratinocytes on Collagen Films Modified by Argon Plasma Treatment

Argon plasma treatment was used to modify the surface of atelocollagen films using a plasmochemical reactor. To evaluate the effects of the treatment, the untreated and treated samples were characterized by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM) imaging, and X-ray Photoelectron Spectroscopy (XPS) techniques. Cell growth was carried out by culturing human immortalized keratinocyte (HaCaT) cells and proliferation was measured via MTT assay. It was observed that argon plasma treatment significantly enhanced the extent of cell proliferation, which was ascribed to the favourable role of plasma treatment in inducing surface oxygen-containing entities together with increasing surface roughness. This can be considered as a potentially promising approach for tissue regeneration purposes.

Colloidal polyaniline dispersions: Antibacterial activity, cytotoxicity and neutrophil oxidative burst

Polyaniline colloids rank among promising application forms of this conducting polymer. Cytotoxicity, antibacterial activity, and neutrophil oxidative burst tests were performed on cells treated with colloidal polyaniline dispersions. The antibacterial effect of colloidal polyaniline against gram-positive and gram-negative bacteria was most pronounced for Bacillus cereus and Escherichia coli, with a minimum inhibitory concentration of 3,500 μg mL(-1). The data recorded on human keratinocyte (HaCaT) and a mouse embryonic fibroblast (NIH/3T3) cell lines using an MTT assay and flow cytometry indicated a concentration-dependent cytotoxicity of colloid, with the absence of cytotoxic effect at around 150 μg mL(-1). The neutrophil oxidative burst test then showed that colloidal polyaniline, in concentrations <150 μg mL(-1), was not able to stimulate the production of reactive oxygen species in neutrophils and whole human blood. However, it worked efficiently as a scavenger of those already formed.

Antibacterial properties of polyaniline-silver films

In situ polymerised thin polyaniline (PANI) films produced on polystyrene dishes were tested for their antibacterial activity with respect to Escherichia coli and Staphylococcus aureus, representing both gram-positive and gram-negative bacteria. PANI films were subsequently used for the reduction of silver ions to metallic Ag. PANI salt and base in original forms and after the deposition of Ag were studied. PANI salt showed a significant antibacterial effect against both bacteria strains while the efficacy of neat PANI base was only marginal. After the Ag deposition, the PANI base exhibited different levels of antibacterial effect depending on the type of the bacterial strain; the growth of gram-positive Staphylococcus aureus was inhibited depending on the Ag concentration on the film, while Escherichia coli remained uninfluenced. Efficacy of the PANI salt with deposited Ag against both bacteria strains was comparable with that of PANI alone and was not affected by the Ag concentration. The results show that Ag deposition can be a suitable method for the preparation of PANI base films with improved antibacterial properties.

HaCaT Keratinocytes Response on Antimicrobial Atelocollagen Substrates: Extent of Cytotoxicity, Cell Viability and Proliferation

The effective and widely tested biocides: Benzalkonium chloride, bronopol, chitosan, chlorhexidine and irgasan were added in different concentrations to atelocollagen matrices. In order to assess how these antibacterial agents influence keratinocytes cell growth, cell viability and proliferation were determined by using MTT assay. Acquired data indicated a low toxicity by employing any of these chemical substances. Furthermore, cell viability and proliferation were comparatively similar to the samples where there were no biocides. It means that regardless of the agent, collagen-cell-attachment properties are not drastically affected by the incorporation of those biocides into the substrate. Therefore, these findings suggest that these atelocollagen substrates enhanced by the addition of one or more of these agents may render effectiveness against bacterial stains and biofilm formation, being the samples referred to herein as “antimicrobial substrates” a promising view in the design of novel antimicrobial biomaterials potentially suitable for tissue engineering applications.

Phenolic compounds from Allium schoenoprasum, Tragopogon pratensis and Rumex acetosa and their antiproliferative effects.

Experimental studies have shown that phenolic compounds have antiproliferative and tumour arresting effects. The aim of this original study was to investigate the content of phenolic compounds (PhC) in flowers of Allium schoenoprasum (chive), Tragopogon pratensis (meadow salsify) and Rumex acetosa (common sorrel) and their effect on proliferation of HaCaT cells. Antiproliferative effects were evaluated in vitro using the following concentrations of phenolic compounds in cultivation medium: 100, 75, 50 and 25 µg/mL. Phenolic composition was also determined by HPLC. The results indicate that even low concentrations of these flowers’ phenolic compounds inhibited cell proliferation significantly and the possible use of the studied herb’s flowers as sources of active phenolic compounds for human nutrition.

Preparation of active antibacterial LDPE surface through multistep physicochemical approach II: Graft type effect on antibacterial properties

Three monomers (allylamine, N-allylmethylamine and N,N-dimethylallylamine) were used for grafting onto air plasma activated LDPE surface. Antibacterial agent triclosan was anchored on such substrates. Influence of graft type on the antibacterial properties was determined. Increase of antibacterial activity and amount of deposited antibacterial agent for N-allylmethylamine and N,N-dimethylallylamine monomers were examined. Surface characteristics were measured by means of static contact angle measurement with surface energy evaluation, ATR-FTIR spectroscopy, XPS and SEM characterization analysis. Antibacterial properties were tested in vitro by inhibition zone method on agar plates for Staphylococcus aureus and Escherichia coli strains.

Influence of various sterilization procedures on TiO2 nanotubes used for biomedical devices

Sterilization is the final surface treatment procedure of all implantable devices and is one of the key factors which have to be considered before implementation. Since different sterilization procedures for all implantable devices influence mechanical properties as well as biological response, the influence of different sterilization techniques on titanium nanotubes was studied. Commonly used sterilization techniques such as autoclaving, ultra-violet light sterilization, hydrogen peroxide plasma sterilization as well as the not so frequently used gaseous oxygen plasma sterilization were used. Three different nanotube diameters; 15 nm, 50 nm and 100 nm were employed to study the effects of various sterilization techniques. It was observed that autoclave sterilization resulted in destruction of nanotubular features on all three studied nanotube diameters, while UV-light and both kinds of plasma sterilization did not cause any significant morphological changes on the surfaces. Differences between the sterilization techniques employed influenced cytocompatibility, especially in the case of nanotubes with 100 nm diameter.

Conducting polyaniline based cell culture substrate for embryonic stem cells and embryoid bodies

In this work, thin films consisting of electrically conducting polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanesulfonate) (PAMPSA) have been used as cell culture substrates for embryonic stem cells (ESC) and embryoid bodies (EMB). The PANI–PAMPSA films having fibrillar morphology were electrochemically polymerized in a single-step by cyclic voltammetry from an aqueous solution containing aniline and PAMPSA. UV-visible spectroscopy showed that the PANI films were electrically conducting still at pH 10. This makes them suitable for tissue engineering applications operating at physiological pH, in contrast to the commonly used PANI hydrochloride films which loose their electrical conductivity at pH ≥ 4. Our results reveal that the PANI–PAMPSA films allow only for limited ESC adhesion and growth. The inhibition of the EMB growth and adhesion on the PANI–PAMPSA surface in serum-free medium indicates that it can be used as a cell-selective substrate for the growth of only some specific differentiated EMB cell types.

Edible flowers — antioxidant activity and impact on cell viability

The phenolic compound composition, antioxidant activity and impact on cell viability of edible flower extracts of Allium schoenoprasum; Bellis perennis; Cichorium intybus; Rumex acetosa; Salvia pratensis; Sambucus nigra; Taraxacum officinale; Tragopogon pratensis; Trifolium repens and Viola arvensis was examined for the first time. Total phenolic content of the flowers of these plants fell between 11.72 and 42.74 mg of tannin equivalents/kg of dry matter. Antioxidant activity ranged from 35.56 to 71.62 g of ascorbic acid equivalents/kg of dry matter. Using the Human Hepatocellular Carcinoma cell-line (HepG2) and the Human Immortalized Non-tumorigenic Keratinocyte cell line (HaCaT), we assessed cell viability following a 3 day incubation period in media containing 25, 50, 75 and 100 μg/ml of total phenolic compounds using a colorimetric MTT assay. These three properties could make the herbs useful in treatment of various diseases like cancer. The tested extracts had significant effects on cell viability, but the effects were dependent not only on the phenolic compound concentration and the edible flowers species, but also on the phenolic compound and antioxidant profiles. In addition, responses differed between cell lines.

Stem cell differentiation on conducting polyaniline

Polyaniline is a promising conducting polymer with broad application potential in biomedicine. Its medical use, however, requires both biocompatibility and suitable physico-chemical and surface properties. The microstructure, electrical properties, and surface characteristics of polyaniline salt, polyaniline base, and polyaniline deposited with biologically active poly(2-acrylamido-2-methyl-1-propanesulfonic acid) were revealed using atomic force microscopy, contact angle measurements, and Raman spectroscopy. As conducting polymers can be preferentially applied in tissue engineering of heart and nervous tissues, the cardiomyogenesis in pure cardiomyocytes derived from embryonic stem cells and neurogenesis in neural progenitors isolated from embryonal 13 dpc brain were further investigated. The results show that neither cardiomyogenesis nor neurogenesis were influenced by any of the tested polyaniline films. However, the most favorable cell behaviour was observed on pristine polyaniline base; therefore, polyaniline in pristine forms without any further modification can be applied in a variety of biomedical fields.