I. I. Selezneva

Bactericidal effects of non-thermal argon plasma in vitro, in biofilms and in the animal model of infected wounds.

Non-thermal (low-temperature) physical plasma is under intensive study as an alternative approach to control superficial wound and skin infections when the effectiveness of chemical agents is weak due to natural pathogen or biofilm resistance. The purpose of this study was to test the individual susceptibility of pathogenic bacteria to non-thermal argon plasma and to measure the effectiveness of plasma treatments against bacteria in biofilms and on wound surfaces. Overall, Gram-negative bacteria were more susceptible to plasma treatment than Gram-positive bacteria. For the Gram-negative bacteria Pseudomonas aeruginosa, Burkholderia cenocepacia and Escherichia coli, there were no survivors among the initial 10(5) c.f.u. after a 5 min plasma treatment. The susceptibility of Gram-positive bacteria was species- and strain-specific. Streptococcus pyogenes was the most resistant with 17 % survival of the initial 10(5) c.f.u. after a 5 min plasma treatment. Staphylococcus aureus had a strain-dependent resistance with 0 and 10 % survival from 10(5) c.f.u. of the Sa 78 and ATCC 6538 strains, respectively. Staphylococcus epidermidis and Enterococcus faecium had medium resistance. Non-ionized argon gas was not bactericidal. Biofilms partly protected bacteria, with the efficiency of protection dependent on biofilm thickness. Bacteria in deeper biofilm layers survived better after the plasma treatment. A rat model of a superficial slash wound infected with P. aeruginosa and the plasma-sensitive Staphylococcus aureus strain Sa 78 was used to assess the efficiency of argon plasma treatment. A 10 min treatment significantly reduced bacterial loads on the wound surface. A 5-day course of daily plasma treatments eliminated P. aeruginosa from the plasma-treated animals 2 days earlier than from the control ones. A statistically significant increase in the rate of wound closure was observed in plasma-treated animals after the third day of the course. Wound healing in plasma-treated animals slowed down after the course had been completed. Overall, the results show considerable potential for non-thermal argon plasma in eliminating pathogenic bacteria from biofilms and wound surfaces.

Acceleration of fibril formation and thermal stabilization of collagen fibrils in the presence of taxifolin (dihydroquercetin).

We studied the effect of flavonoid taxifolin (dihydriquercetin) on the structure and thermal stability of collagen I fibrils. Taxifolin accelerated fibril formation with reconstruction of periodical cross-striation characteristic of these fibrils. Differential scanning calorimetry showed elevation of melting temperature of collagen fibrils formed in neutral or weakly alkaline media, but not of individual tropocollagen molecules in acid medium. Taxifolin capacity to stimulate fibril formation and promote stabilization of fibrillar forms of collagen can be used in medicine.

Cerium oxide nanoparticles stimulate proliferation of primary mouse embryonic fibroblasts in vitro

The increasing application of cell therapy technologies in the treatment of various diseases requires the development of new effective methods for culturing primary cells. The major limitation for the efficient use of autologous cell material is the low rate of cell proliferation. Successful cell therapy requires sufficient amounts of cell material over a short period of time with the preservation of their differentiation and proliferative potential. In this regard, the development of novel, highly efficient stimulators of proliferative activity in stem cells is a truly urgent task. In this paper we have demonstrated that citrate-stabilized cerium oxide nanoparticles (nanoceria) enhance the proliferative activity of primary mouse embryonic fibroblasts in vitro. Cerium oxide nanoparticles stimulate cell proliferation in a wide range of concentrations (10(-3)М-10(-9)M) through reduction of intracellular levels of reactive oxygen species (ROS) during the lag phase of cell growth and by modulating the expression level of the major antioxidant enzymes. We found the optimal concentration of nanoceria, which provides the greatest acceleration of cell proliferation in vitro, while maintaining the levels of intracellular ROS and mRNA of antioxidant enzymes in the physiological range. Our results confirm that nanocrystalline ceria can be considered as a basis for effective and inexpensive supplements in cell culturing.

Mesenchymal Stem Cells from Human Dental Pulp: Isolation, Characteristics, and Potencies of Targeted Differentiation

We studied cell cultures isolated from the pulp of third molar germ of an adult human and from the skin of a human fetus on gestation day 10. Both cultures expressed similar repertoire of surface markers typical of multipotent mesenchymal cells (CD44, CD90, and CD105). Under in vitro conditions, dental pulp cells were more susceptible to factors inducing their differentiation into adipogenic, chondrogenic, and osteogenic lineage cells.

Immobilization and long-term culturing of mouse embryonic stem cells in collagen-chitosan gel matrix.

We propose a method of creation of a 3D matrix consisting of native collagen fibers and natural polysaccharide chitosan. The collagen-chitosan hydrogels maintain viability and prolipherative activity of embryonic stem cells obtained from internal cells of mouse blastocyst. The proposed system forming hydrogels in situ can be used in cell therapy for immobilization and targeted delivery of stem cells.

Evaluation of Collagen Gel Microstructure by Scanning Electron Microscopy

We performed qualitative comparison of freeze drying and chemical drying as methods of preparing 3D wet specimens for scanning electron microscopy. Human fibroblasts immobilized in collagen gel were used as a model system. Specimens fixed with glutaraldehyde were frozen in liquid nitrogen and freeze-dried at low temperature in high vacuum. In parallel experiments, glutaraldehyde-fixed samples were dehydrated in ascending ethanol solutions, absolute ethanol, and 100% hexamethyldisilazane and then dried at room temperature. Scanning electron microscopy microphotographs of collagen fibers and cells were characterized by high resolution and the absence of collapsed or deformed structures even at high magnification (×50,000) for both chemical drying and high-vacuum freeze drying. However, high-vacuum freeze drying is superior to chemical drying for the investigation of the internal space of 3D scaffolds, because sample fracture can be prepared directly in liquid nitrogen. These techniques are a part of the sample preparation process for scanning electron microscopy and can also be used for studies of cell adhesion, morphology, and arrangement in wet specimens (3D gels and flexible tissue engineering scaffolds).

Materials from Titanium—Cobalt Alloys for Hybrid Implants

We proposed a new method to increase the biocompatibility of porous materials that were synthesized from titanium and cobalt allows by the method of self-propagating high-temperature synthesis. This method suggested the introduction of calcium hydroxyapatite into the reaction mixture. Administration of calcium hydroxyapatite into the reaction mixture had a modifying effect on the structure and surface of the pore space and biocompatibility of composite materials. Administration of calcium hydroxyapatite crystals was followed by a significant decrease in the size of pores and appearance of water-soluble fractions, which inhibited the activity of cells. However, treatment with amorphous nanodispersed calcium hydroxyapatite increased the biocompatibility and adhesiveness of materials for mesenchymal stem cells. The pore space and mechanical characteristics of materials obtained with amorphous nanodispersed calcium hydroxyapatite were similar to the properties of natural bone. Moreover, these materials surpassed titanium—cobalt allows in biocompatibility. Our results indicate that the introduction of amorphous nanodispersed calcium hydroxyapatite into the reaction mixture during self-propagating high-temperature synthesis has a modifying effect on the pore space of composite materials and increases their biocompatibility and adhesiveness for cells. We conclude that these materials may be used as a carrier of stem cells and progenitor cells in hybrid implants.

Radioprotective effects of ultra-small citrate-stabilized cerium oxide nanoparticles in vitro and in vivo

A comprehensive study of the radioprotective action mechanisms of ultra-small, citrate-stabilized, nanocrystalline cerium oxide (CeO2 nanoparticles, nanoceria) in vitro and in vivo was carried out. CeO2 nanoparticles were shown to significantly reduce the levels of hydrogen peroxide and hydroxyl radicals in aqueous solutions under X-ray irradiation. Pretreatment of primary mouse fibroblasts by CeO2 nanoparticles in a wide range of concentrations (10−5 to 10−9 M) protects cells from radiation-induced oxidative stress, maintaining a high level of dehydrogenase activity. A protective effect of CeO2 nanoparticles is also manifested in a decrease in the number of apoptotic cells and the level of reactive oxygen species (ROS) in a culture of primary mouse fibroblasts in vitro, as well as in a significant reduction (up to 50% of the control) of cytogenetic damage of mouse bone marrow in vivo. The intraperitoneal administration of CeO2 nanoparticles (200 nM) increased the rate of survival of mice exposed to X-ray radiation at a lethal dose, both in the prophylactic and the therapeutic schemes of administration, testifying the protection at the molecular, cellular and organism levels. The physical, chemical and biological origins of the protective action of CeO2 nanoparticles upon exposure of living systems to ionizing radiation in vitro and in vivo are discussed.

Antiproliferative Effects of Mesenchymal Stem Cells and Epithelial Cells on Lymphocytes

We analyzed the interactions between peripheral blood lymphocytes from heterologous donors with mesenchymal stem cells obtained from the tooth pulp and trophoblast. In mixed cultures, proliferation of both lymphocytes and mesenchymal stem cells was suppressed. Similar suppressive effects were observed in lymphocyte cultures mixed with epithelial cells (hepatocytes HeG2 and renal epithelial cells HEK293). This suppression can be determined by impairment of normal adhesion contacts between cells of different origin.

Interaction of Lymphocytes with Mesenchymal Stem Cells

We studied the interaction of neural stem cells and dental pulp-derived mesenchymal stem cells with lymphocytes from autologous and heterologous donors. Flow cytometry analysis with the use of CFSE-labeled lymphocytes demonstrated an increase in the content of proliferating CD8, CD16 and CD56 cells, but not CD4 cells in cultures of HLA-DR-negative mesenchymal stromal cells from the dental pulp co-cultured with lymphocytes. In neural cultures expressing HLA-DR, all subpopulations of T cells and NK cells were activated. No differences between the autologous and heterologous cultures were revealed.