Growth of Mesenchymal Stem Cells on Poly(3-Hydroxybutyrate) Scaffolds Loaded with Simvastatin.

We studied the effect of porous composite scaffolds based on poly(3-hydroxybutyrate) (PHB) loaded with simvastatin on the growth and differentiation of mesenchymal stem cells. The scaffolds have a suitable microstructure (porosity and pore size) and physicochemical properties to support the growth of mesenchymal stem cells. Scaffold loading with simvastatin suppressed cell growth and increased alkaline phosphatase activity, which can attest to their osteoinductive properties.

Poly(3-hydroxybutyrate)/poly(ethylene glycol) scaffolds with different microstructure: the effect on growth of mesenchymal stem cells.

Development of biocompatible 3D scaffolds is one of the most important challenges in tissue engineering. In this study, we developed polymer scaffolds of different design and microstructure to study cell growth in them. To obtain scaffolds of various microstructure, e.g., size of pores, we used double- and one-stage leaching methods using porogens with selected size of crystals. A composite of poly(3-hydroxybutyrate) (PHB) with poly(ethylene glycol) (PEG) (PHB/PEG) was used as polymer biomaterial for scaffolds. The morphology of scaffolds was analyzed by scanning electron microscopy; the Young modulus of scaffolds was measured by rheometry. The ability to support growth of mesenchymal stem cells (MSCs) in scaffolds was studied using the XTT assay; the phenotype of MSC was preliminarily confirmed by flow cytometry and the activity of alkaline phosphatase and expression level of CD45 marker was studied to test possible MSC osteogenic differentiation. The obtained scaffolds had different microstructure: the scaffolds with uniform pore size of about 125 µm (normal pores) and 45 µm (small pores) and scaffolds with broadly distributed pores size from about 50-100 µm. It was shown that PHB/PEG scaffolds with uniform pores of normal size did not support MSCs growth probably due to their marked spontaneous osteogenic differentiation in these scaffolds, whereas PHB/PEG scaffolds with diverse pore size promoted stem cells growth that was not accompanied by pronounced differentiation. In scaffolds with small pores (about 45 µm), the growth of MSC was the lowest and cell growth suppression was only partially related to stem cells differentiation. Thus, apparently, the broadly distributed pore size of PHB/PEG scaffolds promoted MSC growth in them, whereas uniform size of scaffold pores stimulated MSC osteogenic differentiation.

[Development and preclinical studies of insulating membranes based on poly-3-hydroxybutyrate-co-3-hydroxyvalerate for guided bone regeneration].

Bone tissue damages are one of the dominant causes of temporary disability and developmental disability. Currently, there are some methods of guided bone regeneration employing different osteoplastic materials and insulation membranes used in surgery. In this study, we have developed a method of preparation of porous membranes from the biopolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), produced by a strain of Azotobacter chroococcum 7B. The biocompatibility of the porous membranes was investigated in vitro using mesenchymal stem cells (MSCs) and in vivo on laboratory animals. The cytotoxicity test showed the possibility of cell attachment on membrane and histological studies confirmed good insulating properties the material. The data obtained demonstrate the high biocompatibility and the potential application of insulating membranes based on PHBV in bone tissue engineering.

[Molecular Dynamics of Self-assembling and Rheology of Superhelical Structure of Protofiber of Spider Web].

In this study we suggested a dynamics simulation for the formation of protofiber of spider web nanofiber. It was shown that a bundle of parallel polyalanine ß-strands of sufficient length is arranged through self-assembly into a stable right-handed super helix. By numerical analysis we investigated the rheological properties and provided in nonlinear regime a generalization of the model of Singer for description of the rheological behaviour of super helix.

Culturing of Mouse Mesenchymal Stem Cells on Poly-3-Hydroxybutyrate Scaffolds.

We studied the possibility of long-term culturing of mouse mesenchymal stem cells on a porous scaffold made of biocompatible polymer poly-3-hydroxybutyrate. The cells remained viable for at least 2 months and passed more than 65 population doublings in culture. Culturing on the scaffold did not change surface phenotype of cells. 3D poly-3-hydroxybutyrate scaffolds are appropriate substrate for long-term culturing of mesenchymal stem cells.

Distinct impact of targeted actin cytoskeleton reorganization on mechanical properties of normal and malignant cells.

The actin cytoskeleton is substantially modified in cancer cells because of changes in actin-binding protein abundance and functional activity. As a consequence, cancer cells have distinctive motility and mechanical properties, which are important for many processes, including invasion and metastasis. Here, we studied the effects of actin cytoskeleton alterations induced by specific nucleation inhibitors (SMIFH2, CK-666), cytochalasin D, Y-27632 and detachment from the surface by trypsinization on the mechanical properties of normal Vero and prostate cancer cell line DU145. The Young's modulus of Vero cells was 1300±900 Pa, while the prostate cancer cell line DU145 exhibited significantly lower Young's moduli (600±400 Pa). The Young's moduli exhibited a log-normal distribution for both cell lines. Unlike normal cells, cancer cells demonstrated diverse viscoelastic behavior and different responses to actin cytoskeleton reorganization. They were more resistant to specific formin-dependent nucleation inhibition, and reinforced their cortical actin after detachment from the substrate. This article is part of a Special Issue entitled: Mechanobiology.

[Assessment of Binding Properties of Actinomycin and Its Derivatives with DNA Molecule Using Molecular Dynamics Simulation Method].

Molecular dynamics simulation method was used to assess an influence of actinomycins (antibiotics used in chemotherapy for treatment of some oncology diseases) on DNA fragment elasticity. Also the efficiency of binding of actinomycin to DNA fragment was estimated. Energetic contributions of different substitutions of hydroxyl and amino-group to the phenoxazine ring of actinomycin were studied to analyze dynamic behavior and stability of antibiotic-DNA fragment complexes. Young modulus values were calculated for structures: DNA/DNA-actinomycin/DNA-7-hydroxyactinomycin/DNA-7-aminoactinomycin. Free energy calculations were performed for the formation of actinomycin- and two actinomycin analogues-DNA fragment complexes. Our results suggest that the inserted substitutions stabilize the structure of a DNA fragment via the formation of additional hydrogen bonds.

[Biocompatibility of electrospun poly(3-hydroxybutyrate) and its composites scaffolds for tissue engineering].

Development of biodegradable polymers-based scaffolds for tissue engineering is a promising trend in bioengineering. The electrospun scaffolds from poly(3-hydroxybutyrate) (PHB) were produced using different additives that changed the physical and chemical characteristics of the products. As a result, the construct consisting of interwoven threads of different diameter (0.8-3.4 mm) were obtained, the smallest diameter was observed in the threads from the PHB using tetrabutilammonium iodide (TBAI) and titanium oxide II (TiO2) as additives. Mesenchymal stem cells (MSC) were cultivated on the scaffolds for the biocompatibility evaluation of obtained materials. Cells viability was determined by the XTT assay test. It was shown that the scaffold from the interwoven threads of lowest diameter is most favorable for MSC growth in comparison with the polymer film and scaffolds from the threads of larger diameter. Thus, it was shown that the biocompatibility of electrospun PHB scaffolds depended on their microstructure. The obtained data can be used for development of scaffolds for tissue engineering.

Mechanical properties of fibroblasts depend on level of cancer transformation.

Recently, it was revealed that tumor cells are significantly softer than normal cells. Although this phenomenon is well known, it is connected with many questions which are still unanswered. Among these questions are the molecular mechanisms which cause the change in stiffness and the correlation between cell mechanical properties and their metastatic potential. We studied mechanical properties of cells with different levels of cancer transformation. Transformed cells in three systems with different transformation types (monooncogenic N-RAS, viral and cells of tumor origin) were characterized according to their morphology, actin cytoskeleton and focal adhesion organization. Transformation led to reduction of cell spreading and thus decreasing the cell area, disorganization of actin cytoskeleton, lack of actin stress fibers and decline in the number and size of focal adhesions. These alterations manifested in a varying degree depending on type of transformation. Force spectroscopy by atomic force microscopy with spherical probes was carried out to measure the Young's modulus of cells. In all cases the Young's moduli were fitted well by log-normal distribution. All the transformed cell lines were found to be 40-80% softer than the corresponding normal ones. For the cell system with a low level of transformation the difference in stiffness was less pronounced than for the two other systems. This suggests that cell mechanical properties change upon transformation, and acquisition of invasive capabilities is accompanied by significant softening.

[application of the analytical transmission electron microscopy techniques for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in mammalian cells].

This work represents the results of the study on applicability of the modern methods of analytical transmission electron microscopy for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in A549 cell, human lung adenocarcinoma cell line. A comparative analysis of images of the nanoparticles in the cells obtained in the bright field mode of transmission electron microscopy, under dark-field scanning transmission electron microscopy and high-angle annular dark field scanning transmission electron was performed. For identification of nanoparticles in the cells the analytical techniques, energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy, were compared when used in the mode of obtaining energy spectrum from different particles and element mapping. It was shown that the method for electron tomography is applicable to confirm that nanoparticles are localized in the sample but not coated by contamination. The possibilities and fields of utilizing different techniques for analytical transmission electron microscopy for detection, visualization and identification of nanoparticles in the biological samples are discussed.

[Investigation of the conformational dynamics of the adenosine A2A receptor by means of molecular dynamics simulation].

In this work structural behavior of apo form of the adenosine A2A receptor in the implicit membrane-mimicking environment was investigated by means of molecular dynamics (MD) technique. For better interpretation of the obtained data they were analyzed using principal components analysis. The principal components analysis technique was applied to both MD snapshots as well as X-ray structures of the adenosine receptor. As the result the charts were obtained which reflected an interconnection interdependence between dynamic behavior of the receptor observed on the MD trajectories as well as experimental dataset of investigated protein. The calculated MD trajectories allow to observe represent pronounced structural dynamics of the A2A receptor especially in the intracellular part loop connecting TM 5 and 6 of that protein. This observation generally corresponds to the dynamic behavior of the investigated protein seen on the experimental dataset. Therefore the pattern of the intramolecular motions might be following directly from the spatial architecture (fold) of the receptor under study.

[Three-dimensional structure of human Kv10.2 ion channel studied by single particle electron microscopy and molecular modeling].

Here we present a three-dimensional structure of human voltage gated Kv10.2 ion channel solved at 2.5 nm resolution. We demonstrated that Kv10.2 channel structure is subdivided into two layers. For interpretation of the structure we used the homology modeling, using the transmembrane regions of MlotiK1 channel (C subunit), and cytoplasmic PAS-PAC and cNBD domains of the N-terminal tail of hERG (A subunit) and the bacterial cyclic nucleotide-activated K+ channel binding domain as the templates. The homologous transmembrane part can be fitted into the upper part of the reconstruction. The cytoplasmic domains form the structure, similar to a "hanging gondola", which is connected to the membrane-embedded domain with linkers. The length of linkers allow contacts between C-terminal cNBD domains and N-terminal PAS domains.

Correlation between biological activity and conformational dynamics properties of tetra- and pentapeptides derived from fetoplacental proteins.

In this work, using molecular dynamics simulation, we study conformational and dynamic properties of biologically active penta- and tetrapeptides derived from fetoplacental proteins such as alpha-fetoprotein, pregnancy specific ß1-glycoprotein, and carcinoembryonic antigen. Existence of correlation between flexibility of peptide backbone and biological activity of the investigated peptides was shown. It was also demonstrated that flexibility of peptide backbone depends not only on its length, but also on the presence of reactive functional groups in amino acid side chains that participate in intramolecular interactions. Peptides that demonstrate similar biological effects in regulation of proliferation of lymphocytes and expression of differentiation antigens on their surface (LDSYQCT, PYECE, YECE, and YVCE) are characterized by rigidity of their peptide backbone. Increased backbone flexibility in peptides PYQCE, YQCE, SYKCE, YQCT, YQCS, YVCS, YACS, and YACE is correlated with decreased biological activity. Conformational mobility of amino acid residues does not depend on physicochemical properties only, but also on intramolecular interactions. So, evolutionary restrictions should exist to maintain such interactions in the environment of functionally important sites.

[The effect of poly(3-hydroxybutyrate) modification by poly(ethylene glycol) on the viability of cells grown on the polymer films].

A biodegradable polymer of bacterial origin, poly(3-hydroxybutyrate) (PHB), is intensively studied as biomaterial for tissue engineering. However, factors determining its biocompatibility still require better understanding. To analyze the PHB films biocompatibility, the polymer material was modified by hydrophilic polymer, poly(ethylene glycol) 300 (PEG). The blends PHB/PEG with different PEG content (10, 20, 30 and 50%) were produced by subsequent incubation in water resulted in removal of 95% PEG. The surface roughness and hydrophilicity were studied by atomic force microscopy (AFM) and contact angle "water-polymer" measurement, respectively. The film biocompatibility on cell culture of COS-1 fibroblasts was studied in vitro. It was shown that both roughness and hydrophobicity are directly proportional to initial PEG content in the PHB/PEG blends. The growth rate of COS-1 fibroblasts on polymer films is determined by combination of two basic physicochemical properties of the polymer surface: the roughness and hydrophilicity. The optimal roughness requred for COS-1 cells growth is the average roughness more than 25 nm, whereas the limit values of the contact angle "water-polymer" that was responsible for relatively high cell viability were not found. These data indicate that the film surface roughness had the greatest effect on the cell growth, whereas the increase in the polymer surface hydrophilicity caused the additional positive effect on viability of attached cells. Thus, the modification of PHB polymer material by PEG resulted in the improved viability of cells cultivated on the polymer films in vitro. The obtained data can be used for development of such medical devices as surgeon patches and periodontal membranes.

[Sustained release of the antitumor drug paclitaxel from poly(3-hydroxybutyrate)-based microspheres].

Development of systems of medicines with sustained action on the basis of biodegradable polymers is a promising trend in modem pharmacology. Polyhydroxyalkanoates (POA) attract increasing attention due to their biodegradability and high biocompatibility, which make them suitable for development of novel drug dosage forms. We obtained microspheres on the basis of poly(3-hydroxybutyrate) (PHB) loaded with the antitumor drug paclitaxel. Morphology, drug release kinetics and effect on tumor cells in vitro of microspheres were studied. The data on the kinetics of drug release, biocompatibility and biological activity of the biopolymer microspheres in vitro showed that the studied system of prolonged drug release had lower toxicity and higher efficiency compared to the traditional dosage forms of paclitaxel.

[Atomic force microscopy of living cells: advances and future outlooks].

The advances of the method of atomic force microscopy for investigating the animal cells and an analysis of its development have been reviewed, with much attention being given to studies of living cells. The features and problems of the method have been considered, and a number of special methods based on the use of atomic force microscopy have been analyzed. The problems of choosing the geometry of probes for studies of animal cells, determination of cell adhesion on substrate, mapping of the cell surface using chemically modified cantilevers, and the distribution of molecular components inside the cell with the use of micro- and nanosurgical approaches have been discussed. The problems of combining the atomic force microscopy with optical and laser scanning confocal microscopy have been considered. Possible applications of the method in biotechnology and medicine are discussed.

[Comparative studies of structural properties of melittin in water and trifluroethanol/water mixture by the molecular dynamics method].

The structural properties and dynamic behavior of the antimicrobial peptide melittin in hydrophobic and polar environments have been investigated. The main characteristics of the secondary structure of melittin in different media have been analyzed, and compared with the data on the ideal alpha-helix. It has been shown that melittin is an alpha-helix bent in the region of residue Pro14; in this case, the N-terminus of the peptide tends to unfold, while the C-terminal segment (residues 14-23) retains the helical structure for 20 ns of the simulation. 2,2,2-Trifluoroethanol molecules stabilize the helical structure of the peptide through lowering the dielectric constant of the environment and preferential accumulation nearby particular segments of the polypeptide chain.

Steered molecular dynamics simulations of cobra cytotoxin interaction with zwitterionic lipid bilayer: no penetration of loop tips into membranes.

Cobra cytotoxins, small proteins of three-fingered toxin family, unspecifically damage membranes in different cells and artificial vesicles. However, the molecular mechanism of this damage is not yet completely understood. We used steered molecular dynamics simulations to study the interaction of cardiotoxin A3 from Naja atra cobra venom with hydrated 1-palmitoyl-2-oleoyl-1-sn-3-phosphatidylcholine (POPC) bilayer. The studied system included one cytotoxin molecule, 64 lipid molecules (32 molecules in each monolayer) and 2500 water molecules. It was found that the toxin interacted with zwitterionic bilayer formed by POPC. During first nanosecond of simulation the toxin molecule was oriented toward membrane surface by loops' basement including cytotoxin regions Cys14-Asn19 and Cys38-Ser46. This orientation was stable enough and was not changed during next 6 ns of simulation. The obtained data suggest that cytotoxin molecule cannot penetrate into membrane composed of zwitterionic lipids without some auxiliary interaction.

Conformational dynamics of human alpha-fetoprotein-derived heptapeptide LDSYQCT analogs.

Conformational dynamics of a biologically active fragment of alpha-fetoprotein, the heptapeptide LDSYQCT, and its analogs obtained by site-directed substitutions of amino acid residues were studied. The conformational dynamics of the peptide were conservative under the substitutions Y17F, Y17S, and D15E. Substitutions C19A and S16V resulted only in local changes in the dynamic behavior of the peptide. Chemical modification of cysteine (C19) or dimerization of the peptide by producing a disulfide bond between cysteine residues of two parallel peptide chains, as well as the substitutions C19G, C19S, Q18E, and D15N changed a set of possible conformations and dynamic behavior of all amino acid residues. The most significant changes were caused by substitution of uncharged amino acid residues by charged ones, and vice versa.

[Relationship between structure and function of alpha-fetoprotein: conformational status and biological activity].

Alpha-fetoprotein (AFP) is the major mammalian fetal protein and the recognized tumor marker. This review summarizes data on structure and function of AFP with emphasis on human AFP, which is intensively investigated. During the last decade multiple functionally important sites of human AFP have been revealed or predicted by searching of similarity between primary structures of AFP and other proteins or their DNA sequences. A number of peptides derived from human AFP have been studied by different teams of investigators. These peptides were obtained by limited proteolysis of AFP or synthesized using solid phase chemistry. Study of biological (physiological) activities of these peptides allows determining biologically active sites of alpha-fetoprotein and constructing its structural and functional map. Biomodulating properties of these peptides make them a potential basis for design of drugs for different purposes including using in anticancer therapy. Conformational changes in AFP molecule have been intensively studied for the last few years and sufficient conformational mobility of AFP with the ability to form molten globule form (MGF) despite its stability in solution has been demonstrated. Native molecule of AFP may contain cryptic biologically active sites, which are not available for ligand binding. These sites become open and available for interaction after changes in conformation of AFP molecule. Study of conformational changes of AFP under different conditions allows understanding molecular mechanisms of its functioning. This review describes and analyses data obtained, mainly, during the last few years on study of conformational states of alpha-fetoprotein and relationship between conformational changes of AFP and its biological activity. Biochemical, biophysical and functional characterislics of some well-studied peptide fragments of AFP and their structural and functional mapping are presented.