Changes in Rat Bone Tissue at the Site of the Defect In Vivo under the Effect of a Cryogenically Structured Albumin Sponge Containing a Bioregulator.

We performed a morphological study of the bone tissue after implantation of a cryogenically structured albumin sponge containing a bioregulator isolated from blood serum into an extensive experimental defect of the femur. By day 90, no complete reparation of the bone tissue was achieved in the control group (without implantation of 3D carrier), a loose spongy bone is formed at the site of the defect. After implantation of the 3D carrier without serum bioregulator, the defect was closed, but the formed bone was loose and contained no inflammation foci. After the defect was filed with the albumin sponge with the bioregulator, the repair pattern corresponded to the processes of epimorphic tissue regeneration. The results suggest that cryogenically structured protein material in combination with a serum bioregulator ensured complete restoration of the bone tissue.

Induction of Osteogenesis in Rat Bone Tissue Using Cryogenically Structured Porous 3D Materials Containing a Bioregulator.

We studied osteogenesis induction after implantation of porous cryogenically structured 3D scaffolds based on chitosan, alginate, and serum albumin and containing a bioregulator from bovine serum into in vivo modeled experimental bone defect in rats. It was shown that such 3D-matrices are effective as osteoconductors due to their effect on osteoblast precursors. All types of bioregulator-loaded 3D materials promote active bone repair, which manifested in restoration of the dense bone tissue, formation of the bone marrow, and the recovery of osteons on day 14 after surgery. In animals receiving implantation of control 3D scaffolds without the bioregulator, the formation of dense fibrous tissue and spongy bone was observed by this term.

Effect of Bioactive Peptide Complex Isolated from Bovine Serum on Proliferation and Migration of Mesenchymal Stromal Cells In Vitro and Reparation of Bone Defects In Vivo.

We studied the effect of a bioactive peptide complex isolated from bovine serum on the proliferative potential and migration rate of mesenchymal stromal cells in vitro, as well as on the healing of modeled bone defects in rats. This bioregulatory preparation stimulated proliferation of mesenchymal stromal cells from deciduous tooth pulp in vitro, but did not affect the rate of their migration in two-dimensional cultures. In vivo experiments showed that application of this preparation in combination with hydroxyapatite and chitosan gel accelerated bone tissue regeneration, thus ensuring restoration of morphologically normal bone matrix. Thus, cattle blood serum is an available source for the production of bioregulatory preparations for medical purposes.

Effect of Bioregulators Isolated from Rat Liver and Blood Serum on the State of Murine Liver in Roller Organotypic Culture after CCl4-Induced Fibrosis.

We studied the protective effect of bioregulators isolated from the liver and blood serum of mammals under conditions of manifest fibrosis. Fibrosis was induced by CCl4 administration for 30 days and then, the liver was cultured in a roller organotypic culture for 30 days in the presence of bioregulators. Hepatoprotective effect of bioregulators was evaluated on histological sections of the liver at different terms of culturing. Experiments with roller organotypic culture of the liver isolated from animals with in vivo CCl4-induced fibrosis demonstrated the protective effect of bioregulator of the liver origin, while bioregulator isolated from the blood was ineffective.

Analysis of a bioregulator isolated from rat testes in roller organotypic culture of mouse testes in vitro.

A new bioregulator, a representative of membranotropic homeostatic tissue-specific bioregulators acting in ultralow doses, was isolated from rat testes. A model of roller organotypic culturing of mouse testes was developed and the protective effect of the bioregulator was analyzed.

Burn-healing effects of a composition containing chitosan gel and a blood serum bioregulator.

We studied the effect of combined preparation on the basis of chitosan containing a bioregulator isolated from cattle serum in a concentration of 10(-10) mg/ml on healing of II-IIIa degree skin burns in mammals in vivo.

Study of wound-healing activity of bioregulators isolated from eye tissues and bovine serum in the model of experimental corneal injury in rabbits in vivo.

We compared wound-healing activity of bioregulators isolated from cattle cornea, serum, and retinal pigment epithelium on in vivo model of experimental corneal injury in rabbits. Bioregulators were instilled into the eye as solutions at a concentration corresponding to 10(-12) mg protein/ml. The animals were sacrificed on day 21 after injury and the corneas were examined histologically. The best wound-healing effect was produced by bioregulators isolated from the cornea and serum and instilled successively into rabbit eyes with an interval of 15-20 min twice a day: multicellular epithelium was observed in the wound, and slight inflammation, in the stroma.

Effect of a composition containing chitosan gel and a bioregulator from blood serum on healing of purulent wounds in mice.

We studied the effect of combined preparation on the basis of chitosan containing a bioregulator isolated from cattle serum in a concentration of 10(-10) mg/ml on healing of purulent wounds in mammals in vivo.

[MALDI-TOF mass spectrometric identification of novel intercellular space peptides].

We performed the matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF) analysis of the peptides entering into the composition of not yet explored bioregulators derived from the extracellular matrix of the tissues of the various organs of the mammals, and also plants and fungi. The study included 15 different mammalian tissues, 13 species of plants, and 2 species of fungi. Exploring the bioregulators derived from eye tissues, we demonstrated that their composition includes peptide components with the same values of the molecular weight. The composition of the bioregulators derived from the tissues of various organs of mammals or different species of plants and fungi includes the peptides with different values of molecular weight. Obtained data indicate the growing evidence of the assumptions about the major function of the bioregulators of this group--their involvement in the regulation of tissue-organ homeostasis in the biological systems.

[Study of a new group of bioregulators isolated from the greater plantain (Plantago major L.)].

Proteins with physicochemical properties and biological activity similar to those of membrano-tropic homeostatic tissue-specific bioregulators that had been found earlier in various animal tissues were discovered in leaves of the common plantain (Plantago major L.). To study the specific activity of these plant proteins, we developed an experimental model for organotypic roller cultivation of newt (Pleurodeles waltl) skin tissue in vitro. We showed that the plant proteins of interest exert the wound-healing effect, which is characteristic of this plant, on the skin of vertebrates both in vitro and in vivo.

Effect of bioregulators isolated from the liver, blood serum, and bile of mammals on the state of newt liver tissue in organotypic culture.

We developed models of in vitro organotypic culturing of newt liver tissue with and without adhesion to the substrate. The effects of bioregulators isolated from mammalian liver, blood serum, and bile were studied on the developed models and their specificity was demonstrated. The state of the liver was evaluated by the area of clusters of pigmented cells and by the number of mitoses in the connective tissue cells of the cortical layer. These bioregulators exhibited their biological effects only under conditions of roller organotypic culturing of newt liver tissue.

On mechanisms underlying regeneration and reparation processes in tissues.

Studies of the probability of regulating the regenerative and reparative processes in pathologically modified tissues are reviewed. A ready cell system, providing the realization of reparative and regenerative processes in all organs (cell sources of regeneration), exists in all organisms. The authors suggest that active bioregulators, presented in this paper, are involved in the mechanisms of tissue regeneration by modulating the cell sources of regeneration.

[In vitro organotypic cultivation of adult newt and rat retinas].

Adult rat and newt retinas were studied during long organotypic 3D cultivation. A high proliferation level was discovered in the region of growth by applying DNA synthesis markers and in vitro mitosis registration in newt retina. Aggregates were formed in the retina spheroid cavity because dedifferentiated cells migrated into this region. Small cell populations in nuclear layers also had dividing and migration capacity. Rosette formation has been shown in newt retina. It is a characteristic of fetal retinal development under pathological conditions. The antiG FAP antibody dye demonstrated an increase in the parent M@uller cell population and generation of a small cell pool with short GFAP-extensions de novo. Recoverin expression studies detected its translocation from photoreceptor extensions to the cell bodies. Moreover, protein was presented in some cells inside the spheroid. It has been shown for the first time that cell proliferation occurred in the developing adult rat retinal spheroid in vitro; BrdU-positive cells and multiple mitoses were revealed in this zone. However, the source of proliferation was not in the peripheral retina, and stable macrophages and glial cells located among neurons of the inner nuclear layer had the ability to divide. The antiGFAP antibody showed an increase in GFAP fibers in the rat retina as well as in the newt retina. Recoverin translocated into photoreceptor perikaryons and the outer plexiform layer in cultivated rat retina. Interestingly, some cells with probably de novo expression of recoverin were discovered in rat and newt retinas.

Experimental models for culturing of eye tissues of Pleurodeles waltl for evaluation of specific effects of sclera bioregulator in ultra-low doses.

Experiments on stationary culture of posterior eye and roll-bottle culture of the whole eye from adult water lizards Pleurodeles waltl showed that sclera bioregulator produces a stabilizing effects on adhesion interactions between the sclera, choroid, and pigment epithelium and on the maintenance of viability of sclera fibroblasts and pigment epithelium cells.

Expression of Ca-binding protein recoverin in normal, surviving, and regenerating retina of Pleurodeles waltl adult triton.

Immunohistochemical study of the expression of recoverin (photoreceptor protein) in the retina of Pleurodeles waltl adult triton was carried out in health, during regeneration after removal, and under conditions of long-lasting detachment. Studies with polyclonal (monospecific) antibodies to recoverin showed that normally it is present in the internal segment, connective cilium, in distal portions of the external segments of cones and rods, and in Landolt clubs of displaced bipolar cells. Detachment of the retina is associated with translocation of recoverin from the photoreceptor processes to perikaryons, and the content of recoverin-positive displaced bipolar cells increases. During regeneration of the retina after its excision via conversion of the pigmented epithelial cells, recoverin is synthesized in the prospective photoreceptor perikaryons and then accumulates in the forming inner segments. Hence, recoverin can serve as a reliable marker in studies of photoreceptor differentiation and functioning during regeneration or survival of the retina.

Structural-functional characteristics of a new bioregulator isolated from bovine pigmented epithelium tissue.

A new bioregulator operating in ultralow doses corresponding to 10(-17) mg/ml has been isolated from tissue of pigmented epithelium of bovine eyes. It has been established that the functional basis of this bioregulator is a complex of a low molecular weight regulatory peptide (4372 Da) and a modulator consisting of a mixture of proteins with molecular weights of 14.980-66.283 kDa. It has been shown that the regulatory peptide is responsible for membranotropic activity of the bioregulator, and the modulator proteins are responsible for biological action in ultralow doses. The data demonstrate an interrelation between nanocondition of the bioregulator and its ability to show activity in ultralow doses.

[Effect of adhesion on the status of corneal tissue during in vitro culturing].

Newt and rat cornea with and without the corneal limbus was cultured under conditions of adhesion to a substrate (stationary culturing) and in the absence of adhesion (roller culturing). It was found that the contact of the tissue with the substrate play the key role in the transduction of the regulatory signal that ensures the maintenance of cornea viability and affects proliferation and migration of cornea cells.

[Modulators of the regulatory protein activity acting at microdoses].

New, previously not studied bioregulators active in the ultra low doses corresponding of 10(-8) - 10(-17) mg/ml have been isolated from vitreoretinal tissue of eye. It has been shown that these bioregulators comprise some regulatory peptides-modulators represented by proteins with molecular weights 15-70 KDa one of which is bovine serum albumin. Correlation between the nanosize of bioregulators and their ability to show activity in ultra low doses is established.

[Role of a regulatory protein from the bovine cornea in activation of cell sources of corneal regeneration in vitro].

A highly purified regulatory protein isolated from the bovine cornea (RBC) was tested for the effect on the rat and newt corneas in vitro under different culture conditions. In the newt cornea, RBC stimulated limbus epithelial cells in roller cultures and cells in the basal layer of corneal epithelium in both roller and stationary cultures. In roller cultures of the rat cornea, RBC had no effect on weakly differentiated limbus cells but stimulated progenitor cells of the basal cornea layer to differentiation inbto definitive epithelial cells. In stationary cultures, RBC did not activate the involvement of cells into regeneration of the rat corneal tissue.