[The in vitro effect of antiseptics on epithelial cells of human cornea and conjunctiva]. / Vozdeistvie antiseptikov na epitelial'nye kletki rogovitsy i kon"yunktivy cheloveka in vitro.

Effective and safe antiseptic eye preparations are necessary for prevention and treatment of infectious and inflammatory eye diseases. PURPOSE: in vitro evaluation of the effect of antiseptic eye drops on corneal and conjunctival epithelial cells. MATERIAL AND METHODS: Antiseptic eye drops «Bactavit¼, «Vitabact¼ and «Ocomistin¼ were the object of the study. Immortalized human corneal epithelial cell lines (HCE) and human conjunctiva (Chang Conjunctiva, Clone 1-5c-4) were used as the test systems. The viability of the cells was assessed by their metabolic activity and morphology using the MTT test and phase-contrast microscopy. RESULTS: Antiseptic eye drops belonging to different groups of chemical compounds induced cytotoxic effects on the cells of corneal epithelium (HCE) and human conjunctiva (Chang Conjunctiva, Clone 1-5c-4) of varying degrees, leading to morphological and functional changes in those cells. CONCLUSION: The study confirms the possibility of using cultured cells for the in vitro comparative assessment of the cytotoxic effect of antiseptic ophthalmic agents.

Study of the Osteoindictive Properties of Protein-Modified Polylactide Scaffolds.

Bone marrow mesenchymal stromal cells are multipotent and can differentiate into cells of various tissues, which determines their high importance for clinical application. We performed an in vitro study of the osteogenic potential of mesenchymal stromal cells cultured on intact polylactide scaffolds or scaffolds modified with collagen I or fibrin. Scanning electron microscopy showed that the cells formed osteogenic nodules or osteogenic nodules on both intact and fibrin-modified polylactide scaffolds. Spectrophotometric detection of alkaline phosphatase activity on days 7 and 11 showed that mesenchymal stromal cell grown on intact polylactide scaffolds and on scaffolds modified with collagen type I or fibrin more intensively synthesized alkaline phosphatase than in the control (culture plastic). This dependence increases in the presence of osteogenic differentiation factors in the medium. After long-term culturing (4 weeks), the presence of calcium deposits detected by alizarin red staining confirmed the osteoinductive properties of intact and protein-modified polylactide scaffolds. These findings suggest that polylactide scaffolds and collagen I increase the osteogenic differentiation potential of mesenchymal stromal cells.

[Culture of mussel Mytiuls edulis I. mantle cells].

To date, cell lines derived from marine invertebrates have not been available. Hence primary cell cultures serve as model systems for various experiments. In present study we established primary culture of mussel Mytilus edulis L. mantle cells. Cells were isolated by means of explant culture or enzymatic dissociation of mantle tissue. They maintained viability up to 22 months regardless of culture initiation method. In course of culturing, cells, which were transferred onto new plates, successfully attached to a new surface. Physiological activity of cultured cells was also confirmed by formation of crystals, which appeared after 4-6 months. After continuous time of culturing, mantle cells can be cryopreserved using 5 % DMSO with post-freezing survival up to 50%. These results demonstrate that M. edulis mantle cells can maintain viability and physiological activity for exceptionally long time and can be cryopreserved for further examination.

[Tissue engineering in urology, new approaches for urinary bladder reconstruction].

Tissue engineering as applied to urologic pathology is covered extremely poor in the literature despite recently gaining popularity of regenerative medicine. The review reflects the current problems associated with reconstructive surgery of the urinary bladder, experience of the researchers from the United States in implementing cellular technologies for bladder replacement, the problems and prospects of this direction in case of such a severe pathology, as fibrous transformated bladder.

[Study of common cytotoxicity of aminoglycoside and fluoroquinolone antibiotics in cell cultures].

UNLABELLED: The aim of the study was to identify the overall cytotoxicity of aminoglycosides netilmicin and tobramycin, and fluoroquinolone ciprofloxacin. MATERIAL AND METHODS: We used three types of the cells: constantly transformed cell line CHO-K1, normal human skin fibroblasts and cells of normal human conjunctiva Clone 1-5C-4. Antibiotics activity was detected by their influence on cell viability. Quantitative and qualitative methods of evaluation have used to determine viability of the cells (quantitative assessment: a method of cloning cells and colorimetric method for assessing cell proliferation; qualitative assessment: lifetime visual observation under an inverted microscope for morphological status of cells in culture with photofixing). RESULTS: The most toxic effect for all types of the cells was shown in tobramycin. The least degree of toxicity for all types of the cells was determined in netilmicin. CONCLUSION: Studied antibiotics exert a cytotoxic effect in vitro and differ in their cytotoxic potential.

[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.

[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.

[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.

[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.

[Analysis of extracellular matrix proteins produced by cultured cells].

Extracellular matrix (ECM) is a highly organized multimolecular structure essential for vital function of any organism. Although a lot of data on the extracellular matrix components has been accumulated, an isolation of the entire set of these proteins still remains to be a complex procedure since ECM contains fibrillar proteins and proteoglycans, which form multidomain net-like structures. In the presented study, we developed a method for isolation of ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membrane. Following treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibrils significantly improved fractioning of ECM proteins. Extraction of the remained proteins from culture plate surface was preformed using a buffer developed on the basis of Laemmli probe buffer. With this method, we isolated ECM proteins synthesized by culturing cells and suitable for a future analysis by SDS PAGE and two-dimentional electrophoresis as well as for identification of individual proteins by mass-spectrometry. This study may allow comparing protein contents of ECMs isolated from different sources, and elucidate influences of various proteins on the protein and the properties of extracellular matrix of investigated cells.

[Characteristics of cytoskeleton organization of human normal postnatal, scar and embryonic skin fibroblasts spreading on different proteins of extracellular matrix].

Human dermal fibroblasts were obtained from various anatomical sites expressing different sets of genes. Distinction of their synthetic can be determined by the microenvironment and composition of receptors on the surface of the cells. Earlier, we have shown that the cytoskeleton of cultured cells rapidly reacts to any external modification, and characteristics of their spatial organization may reflect similarities and differences between cells if various origins under the same conditions. The extracellular matrix proteins influence on the character of structures forming by actin cytoskeleton. In this work, we analyzed cytoskeleton organization of the fibroblasts obtained from normal postnatal, scar an embryonic human skin, which were spreading on the basic proteins of extracellular matrix such as collagen Type I and IV, laminin 2/4, and fibronectin. The results of confocal microscopy showed that fibroblasts of various origins had considerable differences in organization of actin structures and distribution of focal contacts visualized by anti-vinculin antibodies. Furthermore, various fibroblasts spread on the same protein of the extracellular matrix revealed essential modifications of actin structures and focal contacts with the similarity of spatial organization of the cytoskeleton. Discovered variations of actin microfilaments organization are evidence of different extent of cell interactions with various proteins. These variations may depend on combination of integrins on the surface of a cell membrane. The data obtained give grounds to suppose considerable differences in morphogenic function of fibroblasts of various origins.

[Allocation and identification of fibrocytes from human peripheral blood].

Fibrocytes are the cells circulating in peripheral blood that synthesize a big number of various factors and take part in the start of reclaiming processes. The wound healing is a result of activity of fibrocytes. It is known that they participate in formation of hypertrophic and kelloid scars. The purpose of the present work was to research specific properties of fibrocytes in vitro. The data obtained testify that these cells really have hematopoietic origin and are undifferentiated. In this connection, while cultivating fibrocytes it is necessary to keep to some specific conditions: the use of the medium specific for stem cells and very high density of cultivation. In 10 days of culturing, the fibrocytes differentiate into fibroblasts. From the general pool of peripheral blood mononuclear cells, only fibrocytes are capable of DNA synthesis but in spite of it proliferative potential of these cells is very low.

[Application of dermal fibroblasts in complex treatment of patients with trophic ulcers of venous etiology].

The authors have analyzed the results of an experimental-clinical investigation devoted to studying the efficiency of local treatment of trophic ulcers at the stage of healing using transplantation of dermal allogenic fibroblasts. The method was proved to accelerate epithelization and to shorten the time of preparing to operations for correction of the venous blood flow.

[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.