[Effect of type I collagen and fibronectin on cell morphology of human MSCs in vitro].

Limited knowledge about behaviour of stem cells in culture seems to be one of the reasons for problems in their successful introduction to applied medicine. To address this issue we have studied in vitro interaction of human mesenchymal stromal cells (MSCs) with various substrates (plastic, type I collagen, fibronectin, and mixtures of these proteins at various ratios) during the 16-18 h after cell plating. Several cell morphology features such as Area, Perimeter, spreading coefficient, polarization coefficient were determined. It has been shown that MSCs respond specifically to the substrate and can be classified into several groups according to the parameters studied. Collagen preferably fibronectin have opposite effects on polarization and spreading of the cells. Collagen preferably enhances polarization of the cells, whereas fibronectin stimulates proportional spreading of cells. Effect of collagen-fibronectin mixture on the cells cannot be considered as a simple additive effect. We assume that variation in the ratio of these proteins in the extracellular matrix might be one of the possible ways to influence the morphology of stem cells when they are induced to differentiate.

[Two spreading states of mesenchymal cells of the human embryo in vitro].

Spreading mesenchymal cells of human embryo on plastic and type I collagens (from rat, sheep and bull) was studied. Spreading of the cells on collagens was stronger than that in the control but no differences between the different collagens were revealed. The cell perimeter, the spreading coefficient and the cell projection area on the substrate were used as morphometric parametres. The spreading of cells was monitored for 0.5-2 h after plating. During the spreading both on plastic and on collagen, the groups of small cells were revealed as separate subpopulations. As a whole, such cells comprehend 9 % of the cell population in the control and 2% in experiment. We assume that this cell type is associated with a special independent functional state of the cells that precedes cell spreading.

[Method of preparation of tissue engineering and cell cultivation collagen by acid extraction of calf skin].

Seven methods of preparation of intact native collagen with telopeptides by acid extraction of calf skin have been compared; the hide was first dehaired by original mild enzymatical method using Bacillus licheniformis protease. The noncollagenous proteins and proteoglycans were previously removed by different ways and collagen was extracted by acid solvents and purified by salt precipitation. The dynamic of noncollagen impurities removing was followed by noncollagen proteins and hexuronic acids analysis in extracts. The purity of the resulting collagen was determined by polyacrilamide gel electrophoresis. The gel-forming capacity of the collagen was determined and the suitability of the product for tissue engineering was estimated by cultivation of fibroblasts and cardyomyocytes of new-born rats on collagen gells. All collagens obtained were not cytotoxic and had good gel-forming capacity. The preparation method with noncollagen impurities removing with 0.02 M K2HPO4 and collagen solution with 0.5 M acetic acid and 5 mM EDTA proved to be the best by final yield of the product and cell reaction to it. Hence, the optimal variant of collagen preparation method has been developed. The Russian Federation patent on this method was taken out.

[The hydrothermal contraction of collagen fibers as a method of its structure investigation].

The hydrothermal contraction of collagen fibers, that is sharp decrease of the fiber length in the narrow temperature range during their heating in water, is a typical example of phase transition which is analogous to melting. General thermodynamic consideration of the melting of oriented polymer fibers was carried out by Gee (1947) and Flory (1956). Flory derived and equilibrium dependence of force on temperature considering the melted polymer as an ideal rubber. We proposed an experimental method for quantitative investigation of this process including estimation of two critical parameters, which are the critical tension and the critical temperature. The necessary condition for the critical parameters estimation is the prior cross-linking of the fiber. We studied theoretically and by experiment the influence of different factors on these critical parameters. We demonstrated the critical tension of hydrothermal collagen contraction to be an important characteristic making possible the estimation of native collagen structure retaining and molecular orientation's degree. The critical tension value was used do advantage for the collagen structure characteristic in some mammoth fossils skin, in bovine skin in the process of leather manufacture and in artificial collagen fibers. The initial temperature of hydrothermal collagen contraction, what is known as shrinkage temperature using widely for the collagen tannage estimation, was shown to be dependent on the occurrence of non-collagenous sheath on native collagen fibers.

[Cultured skin cells interaction with polylactide surface coated by different collagen structures].

The purpose of this work was an optimization of polylactide film surfaces designed for human keratinocytes cultivation. The polylactide films were coated by collagen 1. The experiments showed that uniform covering of polymer surface by collagen, and formation of different collagen structures depend on the mode of the protein application. The differences in collagen distribution on the polymer surface influened the keratinocytes growth in culture. Analysis of keratinocytes alignment, as well as cytoskeleton organization demonstrated that fibrillar collagen promoted more even keratinocytes distribution in comparison with the distribution on molecular collagen.

[Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins].

Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading cells usually interacted with collagen and fibronectin rather that with laminin-2/4 or matrigel. The majority of these cells being represented by basal keratinocytes. Our data demonstrate that the applied method of keratinocyte selection may be directed for precise isolation of skin stem from a common cell population.

[Elaboration of biodegradable polymer substrate for cultivation of human dermal fibroblasts].

The influence of polylactic acid (PLA) surface films on the pattern of cell behavior was studied. The human dermal fibroblasts were cultivated on PLA covered glasses. The hydrophobic nature of PLA films depends on the availability of polymer solvent in the film preparation. PLA films obtained from a more polar solvent--aceton--appeared to be more hydrophilic than those obtained from methylene chloride. More hydrophilic polymer films also appeared to be more preferable for cell cultivation, and human dermal fibroblasts demonstrated a better adhesion and proliferation on hydrophilic rather than on hydrophobic PLA films.

[Endothelial vascular grafts (Experimental research)]. / Endotelizirovannye sosudistye protezy. (Eksperimental'noe issledovanie).

The authors present herein their findings obtained in bench-test and experimental studies, which made it possible to work out an original technology of creating an endothelial covering of the inner surface of vascular grafts made of polytetrafluorethylene. The new technology includes the definite sequential processes which are as follows: 1) creation of vascular endotheliocytes; 2) stimulation of growth and reproduction of endotheliocytes; 3) preparation of the graft, including creation of stable positive potential on its inner surface in order to create optimal conditions for endothelization; 4) graft endothelization itself. In order to assess efficacy of endothelial vascular grafts, we carried out a total of 105 experiments on dogs. The experimental conditions made it possible to comparatively study the standard and endothelialized grafts using them in the position of the aortic abdominal portion, carotid and femoral arteries. The new grafts turned out to possess satisfactory performance properties, which precluded formation of thromboses and hyperplasia of the noeintima, simultaneously providing good implantability.

[Features of formation of phases of dual-phase polymeric system in the presence of collagen I, laminin I, and their mixtures]. / Osobennosti formirovaniia faz dvukhfaznoi polimernoi sistemy v prisutstvii kollagena I, lamnina I i ikh smesi.

A study was made of the phase formation kinetics of a two-phase aqueous polymer system, consisting of 7.8% (w/w) dextran-500 and 4.6% (w/w) polyethylene glycol-6000, in the presence of various concentrations of collagen (0.07-0.20 mg/ml), laminin I (0.01-0.03 mg/ml) and their mixture. The phase formation was evaluated by registration of its optical density on a spectrophotometer. The obtained two-phase polymer system optical density curves and also the partitioning coefficients for the studied objects depend on surface properties of these objects. It has been shown that the surface properties of collagen I and laminin I differ according to differences in their molecules conformations, and that the phase formation kinetics points to their interaction during a mutual partitioning of these proteins in the system. The authors made a conclusion that collagen I and laminin I in the two-phase polymer system conditions could make complexes.

Matrigel increases the rate of split wound healing and promotes keratinocyte ;take' in deep wounds in rats.

The influence of matrigel, a mixture of the components of thebasement membrane, on the wound healing was studied in a modelof experimental wounds in rats. Matrigel was found to increasethe rate of epithelization of split-thickness wounds. The modelof deep wound was developed in which the host animal could notprovide enough migrating and proliferating keratinocytes tocover the wound area. The model is relevant to severe burns andinjuries in humans. When rat keratinocyte suspension wastransplanted into deep wounds, cell retention in the wound bedwas only observed if matrigel was added together with the cells.Increasing matrigel concentration in the wound was seen toenhance the rate of wound area coverage by the cells. Althoughthe process of healing seemed macroscopically normal, afterhistological screening of the biopsies cell in the wouldappeared as amorphous aggregates and tubules rather thenstratified epidermis.

[Effects of extracellular matrix elements on pseudopodial activity of rat keratinocytes]. / Vliianie élementov vnekletochnogo matriksa na psevdopodial'nuiu aktivnost' keratinotsitov krys.

Effects of extracellular matrix elements on the migration activity of keratinocytes have been studied in the primary culture obtained from newborn rats. Collagen of type I, matrigel, its fractions and the matrix produced by fibroblasts were used as substrata for cultivation. A method of migration activity estimation using latex spheres 0.8 mkm in diameter was first used. We have revealed that keratinocytes from the primary culture do not migrate on matrigel and fibroblast matrix, though displaying some pseudopodial activity. This activity dramatically increases on type I collagen, and a weak migration ability appears correlating with a particular structure of the actin cytoskeleton, i.e. with the appearance of special lamellopodia-connected filopodia.

Role of feeder cells in spreading and cytoskeleton organization of newborn rat keratinocytes.

We studied the effect of feeder cells (fibroblasts) and a mixture of the extracellular matrix components, Matrigel, on spreading and cytoskeleton organization of newborn rat keratinocytes (REK). REK formed lamellipodia on being plated together with feeder cells and on the Matrigel as a substrate whereas the same REK plated alone on a plastic surface formed filopodia. REK lamellipodia formation in co-cultures depended on the fibroblast addition time. Although conditioned medium from fibroblast cultures was not enough to induce lamellipodia, the extracellular matrix left after fibroblast removal was as effective as Matrigel. Our results indicate that lamellipodia formation seems to depend on the factor(s) secreted by fibroblasts and associated with the extracellular matrix.

[The stripping of cultures of rat keratinocytes]. / Stripping kul'tur keratinotsitov krys.

To develop a stripping model for newborn rat keratinocytes we chose appropriate culture conditions. As it has been shown for human keratinocytes, this model allows to separate the suprabasal cells from the basal cell layer, to obtain a pure population of basal-like cells. One of possible applications of this method was demonstrated in the study on the influence of extracellular matrix components on keratinocyte proliferation. A fraction of basal membrane gel, "the matrigel", was shown to decrease the uptake of 3H-thymidine by keratinocytes in the stripping model.

[The cultivation of hybridoma cells in agarose granules]. / Kul'tivirovanie kletok gibridomy v granulakh iz agarozy.

The results of cultivation of agarose gel immobilized hybridoma cells producing monoclonal antibodies to human alpha 2-interferon are presented. The immobilized cultured cells retained the higher viability for 10-28 days without changing the growth medium, in comparison with cultured cells in control suspension. The cells immobilized in agarose gel demonstrated a tendency to polyploidization as was revealed by the technique of DNA cytofluorimetry with Hoechst 33258.