Matrix Metalloproteinases in Primary Culture of Cardiomyocytes.

The highly organized contractile apparatus of cardiomyocytes in heart tissue allows for their continuous contractility, whereas extracellular matrix components are synthesized and spatially organized by fibroblasts and endothelial cells. However, reorganization of the cardiomyocyte contractile apparatus occurs upon their 2D cultivation, which is accompanied by transient loss of their contractility and acquired capability of extracellular matrix synthesis (Bildyug, N. B., and Pinaev, G. P. (2013) Tsitologiya, 55, 713-724). In this study, matrix metalloproteinases were investigated at different times of cardiomyocyte 2D cultivation and 3D cultivation in collagen gels. It was found that cardiomyocytes in 2D culture synthesize matrix metalloproteinases MMP-2 and MMP-9, wherein their amount varies with the cultivation time. The peak MMP-9 amount is at early cultivation time, when the reorganization of cardiomyocyte contractile apparatus occurs, and the MMP-2 peak precedes the recovery of the initial organization of their contractile apparatus. Upon cardiomyocyte cultivation in 3D collagen gels, in which case their contractile apparatus does not rearrange, a steady small amount of MMP-2 and MMP-9 is observed. These data indicate that the cardiomyocyte contractile apparatus reorganization in culture is associated with synthesis and spatial organization of their own extracellular matrix.

[Filling the 3D scaffolds by the stromal bone marrow cells depends on the methods of cell seeding and the scaffold surface modifications].

The distribution of bone marrow stromal cells (BMSC) is studied in a 3D poly(L,L-lactide) scaffolds. It is shown that the population of cells seeding into the scaffold with a peristaltic pump (dynamic method) allows the penetration of cells inside of the scaffold compared with the application of the cell suspension on the scaffold surface (static method). In contrast to cells seeding to scaffold by dynamic method the cells seeding by static method migrate from scaffolds in the first few days almost completely. It is found that BMSCs cultured in 3D polylactide scaffold modified by fibrin form colonies, while BMSCs cultured in 3D polylactide-scaffold modified by collagen type I distribute inside scaffold such single cells.

[Contractile apparatus organization of cardiomyocytes upon their cultivation in collagen gels].

Cardiomyocytes are known to change their morphology and reorganize the contractile apparatus upon their culturing in 2D culture systems. Therefore, the monolayer cardiomyocyte cultures appear to be a poor model for studying of the biologically important processes in the heart. Present study relates to the searching for the most optimal culture conditions allowing cardiomyocytes to maintein their typical morphology and the native state of contractile apparatus. In this study, we investigated organization of the contractile apparatus of cardiomyocytes when cultured in 3D collagen gels of different density. It has been shown that cardiomyocytes preserve the initial organization of their contractile apparatus during prolonged cultivation in collagen gels at a concentration of 1 mg/ml, whereas partial reorganization of their contractile apparatus is observed at a concentration of 0.5 and 1.5 mg/ml. Thus, cardiomyocytes placed in 3D collagen gels with a protein concentration of 1 mg/ml could be considered as a culture be model which approximates to the natural conditions.

[Actin cytoskeleton organization and spreading of bone marrow stromal cells and cartilage cells during their combined and independent cultivation on different extracellular matrix proteins].

To clarify the mutual influence of bone marrow stromal cells (BMSCs) and cartilage cells we studied the organization of their actin cytoskeleton and cell spreading on different extracellular matrix proteins--laminin 2/4, collagen type I or fibronectin. It has been shown that the most pronounced difference in morphological characteristics of the cells such as their form, size and actin cytoskeleton organization occur in the case of interaction with fibronectin. So, after separate brief incubation of both cell types on fibronectin, the average area of BMSCs spreading was about 4 times greater than the area of the cartilage cell spreading. However, in the co-culture of these cells in a ratio of 1:1, the average jointed spreading area on fibronctin was nearly 1.5 times less than the theoretically calculated. To determine the nature of exposure of the cells to each other we have studied spreading of these cells in the media conditioned by another cell type. We have found that the area of BMSC's spreading in the medium conditioned by cartilage cells is markedly smaller than the area of spreading of the same cells in the control medium. These data suggest that the cartilage cells secrete factors that reduce BMSC's spreading.

[Reorganization of actin cytoskeleton in the initial stage of transendothelial migration of bone marrow multipotent mesenchymal stromal cells].

The analysis of actin cytoskeleton reorganization in rat bone marrow multipotent mesenchymal stromal cells after one hour adhesion to a monolayer of endothelial cell line EA.hy 926 allowed us to identify three types of cells interacting with the endothelial cells. Approximately half of multipotent mesenchymal stromal cells retained a rounded shape, most of them contained large round actin aggregates, had irregular borders and contacted with the surface of the endothelial cells by microvilli or protrusions similar to small lamellae. Almost all other cells were surrounded by narrow lamellae along the entire perimeter. In addition, a small amount.of elongated flattened cells that contacting with endothelial cells by means of focal contacts was observed. Microenvironmental factors such as proinflammatory cytokine tumor necrosis factor α or plasma proteins affected the ratio of stromal cell types, with different types of organization of the actin cytoskeleton in multipotent mesenchymal stromal cells population.

[Efficacy of dermoplasty and the dermal equivalent in treatment of vast leg ulcers of mixed genesis].

The most frequent causes of leg ulcers (90-95%) are chronic venous insufficiency (45-60%), obliterating atherosclerosis of the lower extremity arteries (10-20%), diabetes mellitus (15-25%) and their combinations (10-15%). The leg ulcers, specified as pyoderma gangrenosum, are the rare and severe pathology, which is very often misdiagnosed. The case history of a 58-year old female patient with vast leg ulcers of the both shanks is analyzed. The leg ulcers were caused by pyoderma gangrenosum and chronic venous insufficiency due to the varicose disease. Complete epithelization of both ulcers was achieved by means of dermoplasty combined using the dermal equivalent against the background of system immunosuppressive therapy.

[Migration rate of rabbit bone marrow stromal cells and rabbit dermal fibroblasts in different gels and activity of their MMPs].

This paper presents the results of the study of rabbit bone marrow stromal cells (BMSC) and rabbit dermal fibroblasts (DF) migration rates to collagen type I and fibrin gels. It has been shown that DF exhibit greater migration activity in collagen gel, whereas BMSC show a higher migration activity in fibrin gels. By studying the activity of matrix metalloproteinases (MMPs) synthesized by cells during cultivation in gels, it has been found for both cell types that the activity of MMP-9 is increased in fibrin gels and activity of MMP-2 is increased in collagen gels. Different speed of the migration of cells may be due to the properties of the cells, the activity of MMP synthesized by the cells and the influence of the microenvironment (collagen or fibrin) on the process of synthesis.

[Extracellular matrix dependence of the cardiomyocyte contractile apparatus organization].

In the process of cardiomyocyte culturing, their contractile apparatus undergoes reorganization involving the convertation of typical myofibrils into non-muscle type structures and subsequent restoration of the initial organization. The causes and mechanisms of the rearrangements described are unknown. In this study, we have shown that cultivation of cardiomyocytes on the individual extracellular matrix proteins, as well as on the matrix produced either by cardiac fibroblasts or by similar cardiomyocyte, reduses the time when the contractile apparatus is in a rearranged state, whereas soluble factors of the conditioned culture do not affect obviously these rearrangements. Using method of extracellular matrix isolation adapted for cardiomyocytes, we have shown that cardiomyocytes in culture produce their own extracellular matrix, which differs from the extracellular matrix of cardiac fibroblasts and varies with the culturing time.

[Novel splicing isoform of actin-binding protein alpha-actinin 4 in epidermoid carcinoma cells A431].

Alpha-actinin 4 (ACTN4) belongs to actin binding proteins of the spectrin superfamily. Structural organisation of actin fibres and focal contacts is considered to be its primary function in a cell. Besides that, nucleocytoplasmic shuffling of ACTN4 and its involvement in nuclear processes were demonstrated. Lately, additional isoforms of ACTN4 resulted from an alternative splicing has been described in various cell types and malignant tumours. In this study, we present investigation of a novel ACTN4 isoform of 80 kDa. The isoform was found in human epidermoid carcinoma cells A431, and it was not detected in human skin fibroblasts, normal human keratinocytes and transformed human embryonic cells HEK293T. Analysis of ACTN4 mRNA in A431 cells showed the presence of a splice variant that lacked the exons 2-8. The deleted exons code two calponin homology domains responsible for ACTN4 binding to F-actin. Intracellular distribution of the described ACTN4 isoform (ACTN4ISO) overexpressed in HEK293T cells differed from that of the full size protein. In the cytoplasm, ACTN4ISO was allocated diffusively with no colocalisation with actin cytoskeleton structures. Intranuclear distribution of ACTN4ISO also differed from that of the full size ACTN4. Nevertheless, immunochemical analysis demonstrated possibility of ACTN4ISO to form heterodimers with the full size protein. Additional investigations of novel isoform interactions with ACTN4 protein partners might clarify its functional features in A431 cells.

[Isolation of tropomyosin particles from the cytosol of cultured cells and their protein composition analysis].

The presence of actin-binding protein, tropomyosin, shaped as particles or protein complexes that have no bonds with actin structures were found while the analisys of structural rearrangements of actin cytoskeleton. However, their functioning is still unknown. To study the composition and properties of these protein complexes a novel method of their separation from the cells without destroying the structures of the cytoskeleton have been developed. The protein composition of isolated tropomyosin particles has been analised by gel filtration, electrophoresis and Western blotting. They appeared to be a multimolecular complexes of about 700 kDa. Beside the tropomyosin and actin these complexes also contain the Hsp70, Hsp90 and myosin-9 identified by mass spectrometry analisys. Also, under inhibition of deacetylases by trichostatin A, changes in the number of particles and redistribution of tropomyosin between cytosol and cytoskeleton take place along with actin cytoskeleton rearrangements. The results obtained give a reason to assume that these multimolecular complexes may participate in the process of reorganization of the actin microfilaments.

[Stability of bone marrow stromal cells to low temperatures during differentiation].

On the bases of earlier conducted research about the stability of heterogeneous population of keratinocytes to low temperatures according to their stages of differentiation this experiment' studies in vitro the stability to low temperatures of rat bone marrow stromal cells before and after their adipocyte and osteocyte differentiation. Results show that bone marrow stromal cells after their differentiation into either adipocytes or octeocytes became least stable to low temperatures. Findings may serve as foundation for further studies that may explain the changes of processes and mechanisms that play a major role in BMSC stability to low temperatures according to their stage of differentiation.

[The effect of low temperatures on the viability of human epidermal keratinocytes found at different stages of differentiation].

The aim of this study was a comparative analysis to the degree of stability of human epidermal cells found at different stages of differentiation to low temperatures. The effect of different subzero temperatures of liquid nitrogen vapor on keratinocytes found both in human skin fragments and as isolated cells extracted from skin fragments has been studied. The degree of stability of epidermal cells low temperatures was evaluated by their ability to form a multilayer stratum in culture; hence this phenomenon explains the survival of a sufficient amount of proliferative cells after exposure to subzero temperatures. Quantitative analysis of the ratio of epidermal stem, transitory and differentiated cells in a population of viable cells before and after exposure to low temperatures were determined using antibodies corresponding to their different stages of differentiation. The results of this research show that the stability of human epidermal cells to low temperature differs depending on their stage of differentiation both in situ and in vitro. Epidermal stem cells and transitory cells are more stable than differentiated cells.

[Effect of formaldehyde in low concentrations on the proliferation and organization of the cytoskeleton of cultured cells].

3-4% (1.07-1.42 M) formaldehyde is one of the most popular and well-known organs, tissues and cells fixer. In this manuscript we have shown that formaldehyde in concentrations of up to 60 microM (0.0002%) does not have any negative effect on the viability of cell lines A431, HEK293 and primary rat fibroblasts, but it is also increases the proliferative activity of A431. The influence on A431 cells might be explained by the activation of epidermal growth factor receptors as a result of their interaction with formaldehyde.

[Reconstruction of the connective tissue as a result of transplantation of fibrins dermal equivalent to the wounds of experimental animals].

We analyzed the effectiveness of wound healing in rats after application of the dermal equivalent (DE) based on fibrin with dermal fibroblasts. Histological studies of newly formed dermis biopsy samples selected during its recovery in the model wound in laboratory animals have shown a positive effect of DE on wound healing. It was found a significant increase in the area of collagen fibers, in the number of prekapillaries, capillaries and postcapillaries in the granulation tissue after application of DE compared with the control group, suggesting a more intense repair.

[Cells of coelomic liquid and cells of different tissues of sea star Asterias rubens L. isolated from intact and post-traumatic animals: behaviour and proliferation under cultivation in vitro].

Proposed sources of coelomocytes in Asteroidea after traumatic injures are coelomic epithelium, axial organ or Tidemann's bodies. To study the involvement of cell division in the process, proliferation of cells from different tissues of starfish Asterias rubens L. has been studied after bromdeoxyuridine incorporation in vivo. To study the differentiation of coelomocytes in vitro a method for isolation and cultivation of different tissue cells has been worked out and cell behaviour and proliferation in culture has been analyzed. The reliable BrdU incorporation has been found in coelomic epithelium cells in vivo. Coelomocytes and coelomic epithelium cells behaviour in culture dependent on the post-trauma period after which the cells were loaded into the culture whereas no difference was revealed for axial organ and Tidemann's bodies cells. Two-month cultivation of coelomic epithelium cells resulted in formation of colony-like accumulations of the cells with high nuclear-cytoplasm ratio which of colony-like accumulation of the cells with high nuclear-cytoplasm ratio which incorporated BrdU. Thus, coelomic epithelium cells seem to be more promising object for the study of A. rubens cell differentiation in vitro.

[Is "fibroblast" a specialized cell or a functional condition of mesenchymal cells derivatives?].

The debatable article is devoted to the analysis of consecutive changes of the notion about the origin, migration, morphofunctional heterogeneity, differentiation and proliferative potential of the basic cells of a connective tissue--fibroblasts. Despite of a plenty of an actual material on this section of cellular biology, till now there is no uniform concept about fibroblasts to a full degree defining their cytogenesis, features of phenotypic answers, and position in differon organization. In this article, the data available in literature are systematized and generalized. The modern outline of fibroblastic differon is offered for the subsequent determination of role and place of its various parts in normal physiological and pathological reactions of connective tissue.

[Cultivation of cells on a surface covered by microspheres with coupled histones].

The use of histones for modification of the surface intended for cultivation of cells was studied. The work was carried out on the cell line 293 of human embryonic kidney transformed by adenovirus (Ad5) and on the cell line BALB/3T3 clone A31 of mouse spontaneous transformed embryonic fibroblasts. We analyzed interaction of cells with histones of different types put on a hydrophobic surface or on dextran microspheres with diameters of 1.0 microm. It was shown, that all histones studied possessed adhesive ability, but their complexes consisting of total and core histones rendered the best influence on adhesion, morphology and growth of the cells in culture. Thus, cross-linked conjugates of histones immobilized on microspheres promoted in a greater degree formation of a network of cellular structures due to formation of intracellular contacts and simultaneous interaction of cells with several microspheres. Comparing with BALB/3T3 clone A31 the cell line 293 showed significant increase in proliferative activity in 11 days of cultivation on microspheres covered with cross-linked conjugates of histones. Our investigations have shown that the microspheres covered with cross-linked conjugates of histones can be used in the further at creation of the three-dimensional porous matrices intended for in vitro formation of tissue-like cellular structures in them.

[The cultivation of cells on the porous titanium implants with the different structure].

The data on human dermal fibroblasts and rabbit mesenchymal stromal cells cultivation on porous titanium implants are presented in given paper. Two types of implants were used: type 1--with irregular pores formed by pressed titanium particles and type 2--with regular pores formed by coalescence of one-size titanium particles into implant. The goal of this study was to choose the type of titanium implant porosity which ensures the tightest interaction of titanium implant with surrounding tissue cells after implantation in the body. Cells were cultivated on implants for 7 days and in both cases they formed confluent monolayer on the implants surfaces. That indicated adhesion, migration and proliferation of cells on such implants. Condition of cells cultured on titanium implants was controlled by scanning electron microscopy. The character of fibroblasts interaction with given implants was different depending on porosity type of implants. On implants with irregular pores, the cells were more spread and overlapped the pores spreading over neighbored particles. On implants with regular pores that formed by one-size particles into implant, the fibroblasts covered these particles not overlapping the pores and seldom interacted with neighbored particles by small outgrowths. There was no tight interaction of particles into implant. In implants formed by pressed particles, the cells grew not only on the surface but also in the depth of implant. Thereby, we suppose that more tight interaction of cells with titanium implant and, supposedly, tissues with implant in an organism will take place in the case of implant structure formed by pressed titanium particles.

Cell Cultivation on Porous Titanium Implants with Various Structures.

The paper presents data on the cultivation of human dermal fibroblasts and rabbit mesenchymal stromal cells on two types of porous titanium implants, i.e., those with irregular pores formed by pressed titanium particles and those with regular pores formed by the cohesion of one-size titanium particles inside the implant. The goal of this study was to determine what type of titanium implant porosity ensured its strongest interaction with cells. Cells were cultivated on implants for 7 days. During this period, they formed a confluent monolayer on the implant surface. Cells grown on titanium implants were monitored by scanning electron microscopy. Fibroblasts interaction with implants depended on the implant porosity structure. On implants with irregular pores cells were more spread. On implants with regular pores fibroblasts enveloped particles and were only occasionally bound with neighboring particles by small outgrowths. There was no tight interaction of particles inside the implant. In implants formed by pressed particles, cells grow not only on surface, but also in the depth of the implant. Thus, we suppose that a tighter interaction of cells with the titanium implant and, supposedly, tissues with the implant in the organism will take place in the variant when the implant structure is formed by pressed titanium particles, i.e., cellular interaction was observed inside the implant. In implants with irregular pores, cells grew both on the surface and in the depth. Thus, cells exhibited more adequate interactions with irregular pore titanium implants in vitro and hopefully the same interaction will be true in tissues after the implantation of the prosthesis into the organism.

[Ultrastructure of coelomic epithelium and coelomocytes of intact and wounded starfish Asterias rubens L].

Samples of coelomic epithelium and coelomocytes suspension of intact and wounded starfishes Asterias rubens L. were analyzed by electron microcopy. It has been demonstrated that coelomic epithelium is composed of three types of cells: flagellar (approx. 60%), secretory (approx. 3%) and myoepithelial (approx. 37%). Flagellar and secretory cells form the apical surface of coelomic epithelium. Secretory cells are represented by two subtypes: granular and mucous secretory cells. Myoepithelial cells are located in the basal zone of the epithelium. Adjacent flagellar cells are separated by intercellular gaps of various size in 4-5% of cases. These gaps are apparently the lacunae left by the flagellar cells after their departure to the coelomic cavity. The morphological pattern of transition of coelomic epithelium flagellar cells to the coelomocytes has been characterized. No significant structural alterations in organization of the coelomic epithelium were revealed after moderate wounding used in the present study. Small round-shaped young coelomycytes (approx. 3%) and bigger mature coelomocytes (approx. 97%) were found in coelomocytes suspension. A flagellum was revealed on the surface of one of the young coelomocytes. Surface of the mature coelomocytes forms the processes of various size and structure; their cytoplasm contains lysosomes and fagocytic vacuoles of different size. After wounding, activation of coelomocytes was noted finding expression in the sharp rise in the number and the length of their surface filopodia, and in the multicellular aggregates formation. By the sum of the ultrastructural data, histogenesis of coelomocytes from the flagellar cells of the coelomic epithelium is supposed to be a process of cellular transdifferentiation.