V. G. Debabov

Tissue regeneration in vivo within recombinant spidroin 1 scaffolds

One of the major tasks of tissue engineering is to produce tissue grafts for the replacement or regeneration of damaged tissue, and natural and recombinant silk-based polymer scaffolds are promising candidates for such grafts. Here, we compared two porous scaffolds made from different silk proteins, fibroin of Bombyx mori and a recombinant analog of Nephila clavipes spidroin 1 known as rS1/9, and their biocompatibility and degradation behavior in vitro and in vivo. The vascularization and intergrowth of the connective tissue, which was penetrated with nerve fibers, at 8 weeks after subcutaneous implantation in Balb/c mice was more profound using the rS1/9 scaffolds. Implantation of both scaffolds into bone defects in Wistar rats accelerated repair compared to controls with no implanted scaffold at 4 weeks. Based on the number of macrophages and multinuclear giant cells in the subcutaneous area and the number of osteoclasts in the bone, regeneration was determined to be more effective after the rS1/9 scaffolds were implanted. Microscopic examination of the morphology of the matrices revealed differences in their internal microstructures. In contrast to fibroin-based scaffolds, the walls of the rS1/9 scaffolds were visibly thicker and contained specific micropores. We suggest that the porous inner structure of the rS1/9 scaffolds provided a better micro-environment for the regenerating tissue, which makes the matrices derived from the recombinant rS1/9 protein favorable candidates for future in vivo applications.

In vitro and in vivo biocompatibility studies of a recombinant analogue of spidroin 1 scaffolds

The goal of this study was to generate porous scaffolds from the genetically engineered protein, an analogue of Nephila clavipes spidroin 1 (rS1/9) and to assess the properties of new rS1/9 scaffolds essential for bioengineering. The salt leaching technique was used to make the rS1/9 scaffolds of interconnected macroporous structure with spontaneously formed micropores. The tensile strength of scaffolds was 18 ± 5 N/cm(2). Scaffolds were relatively stable in a phosphate buffer but degraded in oxidizing environment after 11 weeks of incubation. Applicability of the recombinant spidroin 1 as a substrate for cell culture was demonstrated by successful 3T3 cells growth on the surface of rS1/9 films (270 ± 20 cells/mm(2) vs. 97 ± 8 cells/mm(2) on the glass surface, p < 0.01). The 3T3 fibroblasts readily proliferated within the rS1/9 scaffold (from initially plated 19 ± 2 cells/mm(3) to 3800 ± 304 cells/mm(3) after 2 weeks). By this time, cells were uniformly distributed between the surface and deeper layers (27% ± 8% and 33% ± 4%, respectively; p > 0.05), whereas the initial distribution was 58% ± 7% and 11% ± 8%, respectively; p < 0.05). The rS1/9 scaffolds implanted subcutaneously into Balb/c mice were well tolerated. Over a 2-month period, the scaffolds promoted an ingrowth of de novo formed vascularized connective tissue elements and nerve fibers. Thus, scaffolds made of the novel recombinant spidroin 1 analogue are potentially applicable in tissue engineering.

Bacterial synthesis of silver sulfide nanoparticles

The metal-reducing bacterium Shewanella oneidensis MR-1 has been employed to obtain Ag2S nanoparticles from an aqueous solution of AgNO3 and Na2S2O3 at an ordinary temperature and pressure. The nanoparticles vary in size within 2–16 nm, and the fraction 6 to 12 nm in size constitutes about 70%. The maximum yield of nanoparticles in silver equivalent is 53%. Being visualized by transmission electron microscopy, the particles look like spheres with average diameters varying from 7 ± 2 nm to 9 ± 2 nm. The elemental composition of synthesized nanoparticles has been analyzed by energy-dispersive X-ray spectroscopy, and the estimated silver to sulfur atomic ratio is 2: 1. The presence of living bacterial cells is mandatory for the formation of Ag2S nanoparticles in the aqueous salt solution. Changes in the reaction conditions (reagent concentrations, temperature, and cell-incubation time in the reaction mixture) influence the yield of nanoparticles dramatically, but have little influence on their size.

Novel 3D-microcarriers from recombinant spidroin for regenerative medicine

Microcarriers generated from recombinant spidroin 1F9 are suitable for use as an injection material. The microcarriers were a heterogeneous mixture of microgel particles ranging from 50 to 300 µm in size with the predominance of particles of 50–150 µm. The surface of these microparticles had a complex topography and ensured efficient cultivation of primary and immortalized fibroblasts. Intradermal injections of microgel suspensions into the area of full-thickness skin wounds did not lead to the development of acute inflammation in mice; instead, they accelerated the recovery of skin tissue and stimulated neurogenesis and angiogenesis.

Intensification of bioelectricity generation in microbial fuel cells using Shewanella oneidensis MR-1 mutants with increased reducing activity

Electrogenicity of Shewanella oneidensis MR-1 mutants FRS1 and FRB1 with reducing activity 30–40% higher than in the original strain was studied in various microbial fuel cells (MFC) developed in the course of the work. The voltage and current density developed by the mutants were 1.7 times higher than in the case of S. oneidensis MR-1. A correlation was found between reducing activity of the cells and the voltage and current density developed in MFC. The possibility for enhanced bioelectricity production in MFC by genetic modification of S. oneidensis MR-1 was demonstrated.

Effect of NAD + -dependent formate dehydrogenase on anaerobic respiration of Shewanella oneidensis MR-1

An expression plasmid was constructed in order to carry out heterologous expression of the gene of the NAD+-dependent formate dehydrogenase (FDH) from methylotrophic bacterium Moraxella sp. in the cells of Shewanella oneidensis MR-1 under aerobic and anaerobic conditions. In both modes of cell cultivation, recombinant FDH activity was revealed in the cell lysate of the transformants. In the medium with lać tate as a carbon source, the rate of anaerobic respiration determined as the rate of conversion of fumarate (the electron acceptor) to succinate was higher in the transformant with recombinant FDH. Anaerobic cultivation of the FDH-containing transformant of S. oneidensis MR-1 in a microbial fuel cell (MFC) revealed increased current density.

Biodegradable matrices from regenerated silk of Bombix mori

The structure and biological properties of devices obtained from regenerated fibroin of Bombix mori silk in the form of films, threedimensional matrices, and tubes intended for regenerative medicine were studied. It is shown that regenerated fibroin forms a substrate that maintains adhesion and proliferation of eukary� otic cells by forming a structure that enables homoge� neous distribution of proliferating cells both on the cell surface and in deep matrix layers. The obtained devices are characterized by a high strength and elas� ticity. Matrices implanted into experimental animals undergo diodegradation and neovascularization with time. The properties of devices obtained from regener� ated fibroin allow them to be considered as a base in designing artificial biological analogues of tissue struc� tures. The key problem of modern medicine is the devel�

Study of some aspects of the mechanism of bacterial synthesis of silver sulfide nanoparticles by metal-reducing bacteria Shewanella oneidensis MR-1

In the present work it was shown that biosynthesis of silver sulfide nanoparticles from silver nitrate and sodium thiosulfate solutions of millimolar concentration occurs efficiently by living Shewanella oneidensis MR-1 cells, as well as by ultrasonically-disrupted cells and by the membrane fraction of the cells. The size of nanoparticles synthesized in the presence of living cells was 7.8 ± 1.5 nm, while in the presence of ultrasonically-disrupted cells — it was 6.5 ± 2 nm. The shape of nanoparticles in both cases was close to spherical. It was also shown, that synthesis of nanoparticles occurs in a cell-free solution of sodium thiosulfate that has been incubated with cells previously and to which then a silver nitrate solution was added. In this case the nanoparticles were of elongated shape and their size was (11 ± 4) × (24 ± 6) nm. In the control experiment, when only silver nitrate and sodium thiosulfate solutions not incubated with cells were used, the nanoparticles were not detected. It was shown that biosynthesis of nanoparticles occurs both in aerobic and anaerobic conditions. Nanoparticles are not formed by using thermally inactivated cells as it was shown by us previously. The results show the important role of the native structures of cells for the nanoparticles formation.

Characterization of biodegradable cell micro and macro carriers based on recombinant spidroin

Gels, microgels, and matrices were prepared based on a previously developed recombinant spidroin, 1F9, produced by Saccharomyces cerevisiae yeast strain; their physical, chemical, and biological properties were investigated. It was shown that microgels obtained from 2.5% hydrogel are sized in the range of 50–300 μm, with a predominance of particles of 50–150 μm in diameter. The microgel particles were stable in neutral, acidic, and alkaline media; the destruction of 50% (wt.) of the particles occurred in corrosive media (Fenton’s reagent) within three weeks. The microgel surface, which was studied by laser scanning confocal microscopy and scanning electron microscopy, has a complex relief in which there are both nano-(250–400 nm) and microstructures (3–7 μm). It was shown that particles of both microgel and matrices are capable of adhesion and support the proliferation of 3T3 murine fibroblasts in vitro.

Mutants of an electrogenic bacterium Shewanella oneidensis MR-1 with increased reducing activity

The mutants of Shewanella oneidensis MR-1 resistant to fosfomycin, a toxic analogue of phosphoenolpyruvate, were obtained. The mutants exhibited increased reducing activity and higher rates of lactate utilization. A correlation was shown between the rates of metabolism of oxidized substrates and the rate of reduction of methylene blue, a mediator of electron transport. The mutants of S. oneidensis MR-1 may be used in microbial fuel cells for intensification of energy production from organic compounds.