[Development of osteoplastic gene-activated 3D matrices based on adenoviral vectors]. / Razrabotka osteoplasticheskikh gen-aktivirovannykh 3D-matriksov na osnove adenovirusnykh vektorov.

Gene therapy is one of the most promising approaches in regenerative medicine for the restoration of extensive bone defects in dentistry and maxillofacial surgery. Matrices obtained using three-dimensional printing from bioresorbable polymers, impregnated with adenoviral constructs with genes for osteoinductive factors, can ensure safe and effective formation of bone tissue. OBJECTIVE: To study the properties of three-dimensional matrices based on polylactic-co-glycolic acid and adenoviral constructs with the GFP gene in vitro. MATERIALS AND METHODS: The matrices were obtained by antisolvent three-dimensional printing. Transduction efficiency was assessed by fluorescence microscopy and flow cytometry. The cytocompatibility of the matrices was assessed by the MTT test and by staining cells with fluorescent dyes. RESULTS: Matrices based on polylactic-co-glycolic acid have high cytocompatibility on adipose tissue-derived mesenchymal stem cells. Impregnation of adenoviral vectors with the green fluorescent protein gene in 3D matrices ensures the release of viral particles within a week, maintaining their high transducing ability. CONCLUSION: The developed method for obtaining gene-activated matrices can serve as the basis for the creation of effective osteoplastic materials for bone regeneration.

[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.

Methods of Generation of Induced Pluripotent Stem Cells and Their Application for the Therapy of Central Nervous System Diseases.

The use of induced pluripotent stem cells (IPSC) is a promising approach to the therapy of CNS diseases. The undeniable advantage of IPSC technology is the possibility of obtaining practically all types of somatic cells for autologous transplantation bypassing bioethical problems. The review presents integrative and non-integrative methods for obtaining IPSC and the ways of their in vitro and in vivo application for the study and treatment of neurological diseases.

Comparative Analysis of the Effects of Intravenous Administration of Placental Mesenchymal Stromal Cells and Neural Progenitor Cells Derived from Induced Pluripotent Cells on the Course of Acute Ischemic Stroke in Rats.

We compared the effects of placental mesenchymal stromal cells and neural progenitor cells derived from induced human pluripotent cells after their intravenous administration to rats in 24 h after transitory occlusion of the middle cerebral artery. The therapeutic effects were evaluated by the dynamics of animal survival, body weight, neurological deficit, and the volume of infarction focus in 7, 14, 30, and 60 days after surgery. Intravenous injection of neural progenitor cells produced a therapeutic effect on the course of experimental ischemic stroke by increasing animal survival in the most acute period and accelerating compensation of neurological deficit and body weight recovery. Neural progenitor cells were more effective than mesenchymal stromal cells from human placenta. The effectiveness of intravenous transplantation of neural progenitor cells in the model of occlusion of the middle cerebral artery is shown by us for the first time, although the therapeutic effect of their direct transplantation into the brain has already been described.

[Biocompatibility and osteoinductive properties of collagen and fibronectin hydrogel impregnated with rhBMP-2]. / Biosovmestimost' i osteogennye svoistva kollagen-fibronektinovogo gidrogelya, impregnirovannogo BMP-2.

The study aimed to demonstrate the biocompatibility and osteoinductive properties of a hydrogel based on highly purified collagen and fibronectin impregnated with rhBMP-2. In vitro and in vivo experiments have shown that the minimum effective dosage of rhBMP-2 is 10 µg/ml. The cytocompatibility of the collagen-fibronectin gel was determined using MTT test and staining with PKH-26. There was no inflammation reaction when the material was subcutaneously implanted in rats (n=30) in vivo. The collagen-fibronectin hydrogel containing 10 µg/ml rhBMP-2 showed high osteogenic properties. By the end of 28 days 8±4% of its volume was replaced by newly formed bone tissue in case of subcutaneous implantation, 17±10% in intramuscular implantation and 26±11% in intraosseous implantation in the calvarial critical-size. The optimal combination of biocompatible and osteogenic properties of collagen-fibronectin hydrogel impregnated with BMP-2 allows us to consider it as a promising basis for creating the new generation of osteoplastic materials for dentistry.

[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.

[Differences in the cytocompatibility of bone-plastic materials from xenogeneic hydroxyapatite with stem cells from human exfoliated deciduous teeth and adipose tissue-derived mesenchymal stem cells]. / Razlichiia tsitosovmestimosti kostno-plasticheskikh materialov iz ksenogennogo gidroksiapatita s mul'tipotentnymi mezenkhimal'nymi stromal'nymi kletkami, poluchennymi iz pul'py vypavshikh molochnykh zubov i podkozhnogo lipoaspirata.

AIM: To compare the cytocompatibility of osteoplastic materials used in dentistry with stem cells from human exfoliated deciduous teeth (SHED) and adipose tissue-derived mesenchymal stem cells (AD-MSC). MATERIAL AND METHODS: Materials of the brands 'Bio-Oss', 'Indost', 'Bioplast', 'Viscoll' and 'Trikafor' were selected for study purposes. Cultures of SHED and AD-MSC were used for testing. The cytotoxic effect of the materials was determined using MTT test and vital staining with trypan blue. Cell adhesion was assessed by the vital staining of PKH-26. RESULTS: Water extracts of bone-plastic materials from xenogeneic hydroxyapatite of the brands 'Bio-Oss', 'Indost' and 'Bioplast' exert a cytotoxic effect on SHED and do not cause the death of AD-MSC. Materials based on collagen and ß-tricalcium phosphate possess high cytocompatibility with all cell cultures under study. CONCLUSION: From the point of cytocompatibility all the examined bone-plastic materials may be considered safe for the restoration of bone defects. It should be noted that SHED transplantation on the surface of materials containing xenogeneic hydroxypatite is unacceptable.

[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.

Adenoviral transduction of multipotent mesenchymal stromal cells from human adipose tissue with bone morphogenetic protein BMP-2 gene.

We determined conditions for effective transduction of multipotent mesenchymal stromal cells from human adipose tissue with adenoviral constructs carrying the gene of human bone morphogenetic protein BMP-2. The peak of transgene transcription and BMP-2 protein secretion in the transduced cultures was observed on day 6 after infection. The maximum transcription of BMP-2 gene and genes of osteogenic markers (bone sialoprotein, osteopontin, and osteocalcin) was observed in the medium containing sodium ß-glycerophosphate and ascorbic acid. Addition of D 3 vitamin did not enhance the expression of BMP-2 gene in transduced cells. The obtained cell cultures with high osteogenic potential can be used in bone tissue engineering.

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.

Improvement of cardiac contractile function in rats with postinfarction cardiosclerosis after transplantation of mononuclear and multipotent stroma bone marrow cells.

We compared the efficiency of autologous mononuclear cells and multipotent stromal cells of the bone marrow after their non-selective intracoronary transplantation on day 30 after acute coronary infarction in rats. Improvement of hemodynamic parameters of myocardial contractility (rates of left ventricular pressure rise and drop) in comparison with the initial values and deceleration of postinfarction prolongation of QRS and QT intervals were observed in rats of the experimental group in contrast to controls in 4 weeks after transplantation. These functional changes were more intensive after transplantation of multipotent stromal cells and were accompanied by more pronounced morphological signs of reverse myocardial remodeling: thickening of the scarred left ventricular wall, shrinkage of the scar, and decrease in left ventricular dilatation index.

Tissue engineering construct on the basis of multipotent stromal adipose tissue cells and Osteomatrix for regeneration of the bone tissue.

We developed a new method of creation of tissue engineering constructs for regeneration of the bone tissue based on the principle of free distribution of cells in a fibrin clot within a scaffold. The tissue engineering construct includes multipotent stromal adipose tissue cells committed in osteogenic lineage, platelet-rich plasma, and resorbed material on the basis of xenogeneic bone collagen. The culture of bone progenitor cells was characterized by the main markers of osteoblastic differon. The material meets all requirements for materials intended for tissue engineering. An innovative high-technological tissue engineering product for clinical application is prepared.

Reparation of the myocardium after transplantation of mononuclear bone marrow cells.

Safety and efficiency of intracoronary transventricular transplantation of autologous mononuclear bone marrow cells in rats with postinfarction cardiosclerosis were studied. The cells migrated to the damaged area and were detected only in the cicatricial tissue; they have fibroblast-like phenotype and some of them were stained with Fapα (marker of reactive fibroblasts). More active proliferation of non-muscular cells and formation and maturation of collagen fibers in the cicatricial tissue were observed after transplantation of mononuclear cells. This led to thickening of the cicatricial wall, but the size of the scar and index of dilatation of the left ventricle remained unchanged. The number and volume density of newly formed blood vessels in the damaged area increased after transplantation, but no labeled cells were seen in the vascular walls. It can be hypothesized that stimulation of neoangiogenesis is mediated by paracrine mechanisms, which also explains improvement of global contractility of the left ventricle (increased contractility index in functional tests). Thus, transplantation of mononuclear bone marrow cells leads to thickening and strengthening of the cicatricial wall, stimulates angiogenesis, and improves global myocardial contractility. However, no morphological signs of reverse remodeling of the left-ventricular myocardium were revealed.

Biocompatibility of tissue engineering constructions from porous polylactide carriers obtained by the method of selective laser sintering and bone marrow-derived multipotent stromal cells.

We studied the biocompatibility of porous polylactide carrier matrices obtained by means of surface selective laser sintering. Carrier matrices had no cytotoxic activity, but maintained adhesion and proliferation of cells. Subcutaneous transplantation of tissue engineering constructions from these carriers and bone marrow-derived multipotent stromal cells did not cause the inflammatory response and pathological changes in rats. The conditions for organotypic regeneration were provided at the site of transplantation (high degree of blood supply and considerable amount of immature precursor cells).

Regeneration of skull bones in adult rabbits after implantation of commercial osteoinductive materials and transplantation of a tissue-engineering construct.

We performed a comparative study of reparative osteogenesis in rabbits with experimental critical defects of the parietal bones after implantation of commercial osteoinductive materials "Biomatrix", "Osteomatrix", "BioOss" in combination with platelet-rich plasma and transplantation of a tissue-engineering construct on the basis of autogenic multipotent stromal cells from the adipose tissue predifferentiated in osteogenic direction. It was found that experimental reparative osteogenesis is insufficiently stimulated by implantation materials and full-thickness trepanation holes were not completely closed. After transplantation of the studied tissue-engineering construct, the defect was filled with full-length bone regenerate (in the center of the regenerate and from the maternal bone) in contrast to control and reference groups, where the bone tissue was formed only on the side of the maternal bone. On day 120 after transplantation of the tissue-engineering construct, the percent of newly-formed bone tissue in the regenerate was 24% (the total percent of bone tissue in the regenerate was 39%), which attested to active incomplete regenerative process in contrast to control and reference groups. Thus, the study demonstrated effective regeneration of the critical defects of the parietal bones in rabbits 120 days after transplantation of the tissue-engineering construct in contrast to commercial osteoplastic materials for directed bone regeneration.

Tissue engineering construction from 3D porous ceramic carriers and multipotent stromal cells for the repair of bone tissue defects.

The tissue engineering construction was developed from human bone marrow multipotent stromal cells and 3D porous foamed-ceramic carriers of a zirconium oxide-aluminum oxide system. The carriers had no cytotoxic activity and were potent in maintaining the cell adhesion and proliferation. We developed the method for inoculation and cultivation of bone marrow multipotent stromal cells on these carriers. The optimal time of incubation to obtain a tissue engineering construction was estimated. Bone marrow multipotent stromal cells could be cultured at a depth of 9 mm from the edge of the matrix. The tissue engineering construction holds promise for the repair of extensive defects in bone tissue.