Gene Expression Profile of 3D Spheroids in Comparison with 2D Cell Cultures and Tissue Strains of Diffuse High-Grade Gliomas.

The use of relevant, accessible, and easily reproducible preclinical models of diffuse gliomas is a prerequisite for the development of successful therapeutic approaches to their treatment. Here we studied the gene expression profile of 3D spheroids in a comparison with 2D cell cultures and tissue strains of diffuse high-grade gliomas. Using real time PCR, we evaluated the expression of Gfap, Cd44, Pten, S100b, Vegfa, Hif1a, Sox2, Melk, Gdnf, and Mgmt genes playing an important role in the progression of gliomas and regulating tumor cell proliferation, adhesion, invasion, plasticity, apoptosis, DNA repair, and recruitment of tumor-associated cells. Gene expression analysis showed that 3D spheroids are more similar to tumor tissue strains by the expression levels of Gfap, Cd44, and Pten, while the expression levels of Hif1a and Sox2 in 3D spheroids did not differ from those of 2D cell cultures, the expression levels S100b and Vegfa in 3D spheroids was higher than in other models, and the expression levels of Melk, Gdnf, and Mgmt genes changed diversely. Thus, 3D spheroid model more closely mimics the tumor tissue than 2D cell culture, but still is not the most relevant, probably due to too small size of spheroids, which does not allow reproducing hypoxia and apoptotic and necrotic processes in the tumor tissue.

[Advantages and disadvantages of bone graft materials activated by BMP-2 and constructs carrying its gene]. / Preimushchestva i nedostatki kostno-plasticheskikh materialov, aktivirovannykh BMP-2 i nesushchimi ego gen konstruktsiyami.

In the review gene constructs and proteins used to impart osteoinductive properties to bone graft materials are compared. On the basis of clinical and experimental data the experience and prospects of their application in maxillofacial surgery and dentistry are described. Information about complications associated with the use of bone morphogenetic protein-2 (BMP-2) and vectors carrying its gene is provided.

[Relative survival and cell adhesion of multipotent mesenchymal cells from primary teeth pulp on the surface membranes and sponges produced from collagen]. / Otnositel'naya vyzhivaemost' i adgeziya mul'tipotentnykh mezenkhimal'nykh kletok iz pul'py molochnykh zubov na poverkhnosti membran i gubok, proizvodimykh iz kollagena.

OBJECTIVE: To study the behavior of SHED cell culture on different types of materials for the regeneration of periodontal tissues with different porosity. MATERIAL AND METHODS: Porous collagen material Fibro-Gide (Geitstlich Pharma AG, Switzerland), designed to increase the volume of the gum and the barrier collagen membrane Bio-Gide (Geitstlich Pharma AG, Switzerland) were studied in vitro on SHED cultures. As a control sample, a Spongostan sponge made of gelatin (Johnson & Johnson Medical, UK) with the most pronounced porosity and wettability was used. Acute cytotoxicity was determined using a screening method for assessing the number of living cells in a sample (MTT test). SHED cells were sown on the materials to study the attachment of cells to materials and their migration inside the samples. Before seeding, the cells were stained with vital fluorescent dye PKH26 (red fluorescent cell linker kit, Sigma, Germany) for further visualization. RESULTS AND DISCUSSION: Using the MTT test it was shown that they do not have cytotoxic effects. At the same time by the 8th day of the experiment in the presence of Fibro-Gide and Bio-Gide the cells showed an increase in proliferative activity by 19% and 12%, respectively compared with the control group. The cells attached and spread out on the surface of the materials and migrated into the thickness of porous Fibro-Gide and Spongostan. CONCLUSION: The in vitro study showed that the most favorable material for SHED cell culture is the collagen material Fibro-Gide with sufficient porosity, elasticity and hydrophilicity. SHED cells attach to the collagen matrix and easily penetrate into the sample, filling the entire internal space, while the proliferative capacity of the cell culture increases.

Development prospects of curable osteoplastic materials in dentistry and maxillofacial surgery.

The article presents classification of the thermosetting materials for bone augmentation. The physical, mechanical, biological, and clinical properties of such materials are reviewed. There are two main types of curable osteoplastic materials: bone cements and hydrogels. Compared to hydrogels, bone cements have high strength features, but their biological properties are not ideal and must be improved. Hydrogels are biocompatible and closely mimic the extracellular matrix. They can be used as cytocompatible scaffolds for tissue engineering, as can protein- and nucleic acid-activated structures. Hydrogels may be impregnated with osteoinductors such as proteins and genetic vectors without conformational changes. However, the mechanical properties of hydrogels limit their use for load-bearing bone defects. Thus, improving the strength properties of hydrogels is one of the possible strategies to achieve the basis for an ideal osteoplastic material.

Comparative Analysis of the Paracrine Action of Neuronal and Glial Progenitor Cells Derived from Induced Human Pluripotent Stem Cells.

We performed comparative analysis of paracrine activity of neuronal and glial progenitors derived from induced pluripotent stem cells under conditions of hypoxia modeled by addition of cobalt dichloride. Neuronal and glial progenitors produced neuroprotective and neurotrophic effects on SHSY-5Y neuroblastoma cells in co-culture during the post-hypoxic recovery and reduced the number of apoptotic and necrotic cells. Moreover, they produced a neurotrophic effect and promote the formation and growth of neurites in neuroblastoma cells. The paracrine effect of glial progenitors was more pronounced, which can be explained by more intensive expression and secretion of neurotrophic factors in these cells.

Analysis of the Expression of Regulator Genes in Kupffer Cells and Monocytes.

Differences in the gene expression profiles in resident macrophages (in particular, Kupffer cells) and monocytes were revealed. However, these differences in gene expression profiles do not allow considering resident liver macrophages as purely M2 macrophages and monocytes as purely M1 macrophages. At the same time, a significant number of the genes upregulated in Kupffer cells are associated with normal regulation of liver functions (Arg 1, Flt, iNOs, and Kng). In monocytes, the expression of genes Alox15, Alox12, Tlr2, Tlr4, Tlr7, and Tlr8 (typical functional genes of macrophages) was also upregulated in comparison with Kupffer cells.

[Safety and efficacy of BMP-2 and BMP-7 use in dentistry]. / Bezopasnost' i éffektivnost' primeneniia morfogeneticheskikh belkov kosti 2 i 7 v stomatologii.

The article deals with bone morphogenetic proteins BMP-2 and BMP-7 with high osteoinductive potential. The materials containing these proteins are considered. Their safety and efficacy for regeneration of maxillofacial bone defects are evaluated. The prospects of bone tissue regeneration technologies development based on the use of bone morphogenetic proteins are described.

[Chitosan hydrogels biocompatibility improvement with the perspective of use as a base for osteoplastic materials in dentistry]. / Povyshenie biosovmestimosti khitozanovykh gidrogelei s perspektivoi ikh ispol'zovaniya v kachestve osnovy dlya kostno-plasticheskikh materialov v stomatologii.

Using chitosan as the basis for osteoplastic material, we were dealt with its low biocompatibility. The critical assessment of it is poorly presented in the literature and does not have systematic approaches to solving. The aim of the study was to determine the effect of factors affecting chitosan charge and its free amino groups number on the biocompatibility of hydrogels. Biocompatibility of chitosan compositions were studied in male Wistar rats (n=90). The subcutaneous implantation of chitosan discs and hydrogel caused abundant leukocyte infiltration. The addition of ß-glycerophosphate followed by dialysis slightly reduced the inflammatory response. Treatment with a solution of alkali NaOH and NaHCO3 buffer, on the contrary, intensified the inflammatory response. It is confirmed the effect of charged amino groups of chitosan on leukocyte taxis A decrease in the deacetylation degree (DD) of chitosan to 39.0% led to a statistically significant decrease in leukocyte infiltration. Saturation of chitosan hydrogels with PLA granules reduced by 16% the level of leukocyte infiltration, which was supposedly associated with a decrease in the volume of the hydrogel and an increase in the area of its interaction with blood plasma proteins, which reduce the positive charge of chitosan. The most significant reduction in leukocyte infiltration was achieved with a combination of deacetylated to 39.0% chitosan hydrogel with the addition of 16% by weight highly porous PLA granules.

[The prospects of collagen as a basis for curable and activated osteoplastic materials]. / Perspektivy ispol'zovaniia kollagenovogo gidrogelia v kachestve osnovy dlia otverzhdaemykh i aktivirovannykh kostno-plasticheskikh materialov.

In the review, the structure and biological properties of collagen, variants of its production from natural sources and purification are considered. Methods for modifying the physico-mechanical properties of collagen to create a curable, highly purified collagen hydrogel are described. The advantages of a cured highly purified collagen hydrogel as a basis for osteoplastic material and a means of delivery of growth factors are indicated. The registered osteoplastic materials based on the curable highly purified collagen hydrogel are described, and their comparative analysis is carried out. On the basis of the obtained data, a conclusion was made about the prospects of using collagen as a basis for curable and activated osteoplastic materials.

[Regeneration of dental pulp tissue using pulpal autologous mesenchymal stem cells and platelet-rich plasma]. / Regeneratsiia pul'py zuba s ispol'zovaniem autologichnykh mezenkhimal'nykh stvolovykh kletok pul'py i obogashchennoi trombotsitami plazmy.

Regeneration of pulp and dentin could be important in operative dentistry as a method to save teeth. Currently cell population from dental pulp of deciduous and permanent teeth of humans and laboratory animals are isolated and characterized. The paper presents a study on pulp regeneration using autologous mesenchymal stromal cells from pulp of molars in combination with fibrin clot, transplanted in pulp chamber of miniature pigs after pulp removal. The results proved that transplantation of autologous multipotent stromal cells of dental pulp in combination with autologous platelet-rich plasma in pulp chamber of miniature pigs after pulp removal leads to pulp restoration and reparative dentinogenesis with dentinal bridge formation on the 30th day. However, the completion of regeneration also results in a decrease in the pulp chamber volume due to the neodentin bedding. Tissue regeneration of dental pulp by direct pulp capping in the absence of inflammatory processes is a promising direction of the use of cellular technologies.

[The prospects of hydrogels usage as a basis for curable osteoplastic materials]. / Perspektivy ispol'zovaniia gidrogelei v kachestve osnovy dlia otverzhdaemykh kostno-plasticheskikh materialov.

The article deals with the main types of the polymers used in hydrogel preparation. Their biological, physical and chemical properties was compared. Ways of polymers hardening and prospects of medical application were considered. The prospect of use of chitosan hydrogels activated by osteoinductors as a material for bone augmentation were concluded.

Effect of Endothelial Cells on Angiogenic Properties of Multipotent Stromal Cells from the Umbilical Cord during Angiogenesis Modeling in the Basement Membrane Matrix.

Short-term cell culturing on basement membrane matrix is a common and very convenient in vitro model of angiogenesis. We studied the possibility of interaction of multipotent stromal cells from the umbilical cord and Ea.hy926 endothelial cells on this model at the early and late periods of the experiment. Multipotent stromal cells alone and in combination with endothelial cells formed an unstable tubular network. Clusters formed after its disassembling later became the sprouting centers in co-culture of the two cell types, but not in pure culture of multipotent stromal cells. Multipotent stromal cells with CD31+ phenotype constitute the structural basis of newly formed stable 3D capillary-like network. Prolongation of the time of culturing and combination of the two in vitro models of angiogenesis (tubulogenesis and sprouting) allowed more complete assessment of the angiogenic potential of multipotent stromal cells.

Mechanisms of therapeutic activity of multipotent cells in heart diseases.

Analysis of our findings and published reports on possible mechanisms of therapeutic activity of stem/progenitor cell transplantation in cardiovascular pathologies is presented.

Induction of osteogenic differentiation of multipotent mesenchymal stromal cells from human adipose tissue.

We studied of osteogenic differentiation of multipotent mesenchymal stromal cells from human adipose tissue. Experiments showed that 1α,25-dihydroxycalciferol is a more effective inductor of osteogenesis than dexamethasone. Comparative analysis revealed activation of gene expression for the major osteogenic markers on day 7 of culturing in a medium containing 1α,25-dihydroxycalciferol. It was found that transcription of genes encoding type 1 collagen proteins, osteopontin, osteocalcin, and bone sialoprotein peaked on day 14 in culture, while the expression of alkaline phosphatase and bone morphogenetic protein-2 genes increased over 21 days. Intensive mineralization of the extracellular matrix was observed starting from day 14 in culture. On the basis of the analysis of these data, optimal terms for osteogenic induction (day 14) and an optimal inductor (1α,25-dihydroxycalciferol) were chosen and the protocol of effective osteogenic differentiation of multipotent mesenchymal stromal cells from human adipose tissue was developed for creation of tissue-engineered bone equivalents.

Effect of transplantation of bone marrow monomuclears on angiogenesis in rats.

We studied the effect of non-selective intracoronary transplantation of bone marrow mononuclears on day 30 after acute coronary infarction on angiogenesis in rats. On days 14 and 30 after transplantation of mononuclear cells, stable formation of new vessels was observed. The number of venules considerably increased after transplantation of mononuclear cells, which was seen from increased volume density of blood vessels and their caliber. Stable vascularization after transplantation of mononuclear cells improves blood supply, which is essential for reparation of the myocardium.

Pathways of bone marrow mononuclear cell differentiation after transplantation into postinfarction heart.

We studied homing and differentiation fate of transplanted bone marrow mononuclears after non-selective intracoronary injection on day 30 after acute myocardial infarction in rats. Mononuclear cells migrated to the cicatrix zone where they differentiated into fibroblasts and myofibroblasts. Mononuclear cells did not differentiate into cardiomyocytes, endothelial cells, or smooth muscle cells of vascular media. Stimulation of angiogenesis and reparation of the myocardium was observed under these conditions.

Isolation of insulin-producing cells from different populations of multipotent stromal cells of the umbilical cord and human adipose tissue.

Stromal cells of adipose tissue and human umbilical cord were isolated by the original method from general populations of multipotent subpopulation of multipotent stromal cells exhibiting perivascular phenotype (CD146+, CD31-). Effective directed differentiation of these cells into insulin-producing cells by transient transfection of the gene Pdx1 was demonstrated. Transfection multipotent stromal cells CD146-, CD31- derived from adipose tissue and umbilical cord and isolated by the standard method, did not result in activation of insulin gene transcription. It was shown that the expression of nestin was not necessary for effective pancreatic cell differentiation.

Preparation and characterization of culture of CD146+ cells from human adipose tissue.

A method for isolation of homogenous culture of cells expressing CD146 marker from adipose tissue lipoaspirate was developed. The resultant clonogenic cultures retained high proliferative activity, immunophenotype, and morphology after numerous passages. The presence of insulin in the medium served as the selective factor for maintenance of the population phenotype. These cultures effectively differentiate into CD31(+)endothelial cells and can be used in regenerative medicine.

Study of the safety of transplantation of cultured autologous human chondroblasts.

Study of tumorigenic activity of cultured human chondroblasts intended for autologous transplantations showed that subcutaneous injection of human chondroblast suspension in a dose of 10(7)cells/0.2 ml did not cause the development of tumors in C.B-17 SCID immunodeficient mice over 12 weeks. Subcutaneous formations of compact cartilaginous consistency persisted at the site of chondroblast transplantation in all animals; these formations gradually shrank. Subcutaneous injection of tumor cell suspension (A204 human embryonic sarcoma) to C.B-17 SCID mice under the same conditions caused 100% development of tumors (poorly differentiated sarcomas) by day 14.

Analysis of changes in cell volume of a mouse early embryo exposed to osmotic shock.

The impact of the osmotic component of the incubation medium for the volume of mouse early embryonic cell was studied by laser scanning microscopy. Common Dulbecco's medium caused a prolonged hyperosmotic effect. Adaptive phase of regulatory compensation for the osmotic shock was observed under hypotonic conditions. From these data, water permeability of the blastomer membrane is evaluated as 0.4 micro/(minxatm).