Buccal Mesenchymal Stromal Cells as a Source of Osseointegration of Titanium Implants.

We studied the interaction of human buccal mesenchymal stem cells (MSCs) and osteoblasts differentiated from them with the surface of titanium samples. MSCs were isolated by enzymatic method from buccal fat pads. The obtained cell culture was presented by MSCs, which was confirmed by flow cytometry and differentiation into adipocytes and osteoblasts. Culturing of buccal MSCs on titanium samples was accompanied by an increase in the number of cells for 15 days and the formation of a developed network of F-actin fibers in the cells. The viability of buccal MSCs decreased by 8 days, but was restored by 15 days. Culturing of osteoblasts obtained as a result of buccal MSC differentiation on the surface of titanium samples was accompanied by a decrease in their viability and proliferation. Thus, MSCs from buccal fat pads can be used to coat implants to improve osseointegration during bone reconstruction in craniofacial surgery and dentistry. To improve the integration of osteoblasts, modification of the surface of titanium samples is required.

Studying the Proliferative Activity of Human Corneal Stromal Cell Populations.

The proliferative activity of populations of stromal cells (fibroblasts) obtained from human corneal lenticles under conditions of their differentiation into keratocytes was studied. It was shown that during differentiation, the number of dividing fibroblasts and the frequency of divisions, and motor activity of these cells (speed of movement along the cell trajectory and the length of the trajectory) sharply decreased. These findings indicate a decrease in the proliferative activity of fibroblasts under conditions of their differentiation and transformation into keratocytes. A period of 17 days is sufficient for differentiation of corneal fibroblasts into keratocytes.

Biocompatibility of Titanium Oxynitride Coatings Deposited by Reactive Magnetron Sputtering.

Titanium oxynitride coatings deposited by reactive magnetron sputtering improve biocompatibility of vascular stents by increasing NO production, viability, and adhesion of EA.hy926 cells. Thus, the application of titanium oxynitride coatings is a promising strategy for increasing the biocompatibility of nitinol stents.

Effect of Erythropoietin on Mononuclear Cells of the Bone Marrow and Spleen.

We studied the effect of preconditioning of human bone marrow mononuclear cells with erythropoietin on the immunophenotype of immunocompetent cells and paracrine activity of mouse splenocytes. The expression of erythropoietin receptors on immunocompetent human bone marrow cells was shown to change after a short-term (60 min) exposure to erythropoietin. The number of T helpers carrying erythropoietin receptors decreased and the number of T suppressors, B lymphocytes, and monocytes carrying erythropoietin receptors increased. The presence of 30% conditioned medium from human bone marrow mononuclear cells or 33.4 U/ml of erythropoietin reduced apoptosis/necrosis, increased intracellular activity of NADPH-dependent oxidoreductases of splenocytes, and did not affect oxidative phosphorylation (did not enhance lactate production and glucose uptake by cells).

Effect of Conditioned Media of Limbal Epithelial Cells and Corneal Stromal Cells on Functional Activity of Limb Mesenchymal Stem Cells.

We studied the effect of conditioned media from limbal epithelial stem cells, fibroblasts, and corneal keratocytes on the functional activity of human limbal mesenchymal stem cells. It was shown that the conditioned media from limbal epithelial stem cells reduced proliferative activity and inhibited migration of limbal mesenchymal stem cells. In the conditioned media of limbal epithelial stem cells, increased concentrations of VEGF and TNFα and reduced concentration of BDNF, vimentin, and fibronectin were found. The conditioned medium from corneal stromal cells did not affect functional activity of mesenchymal stem cells in the limbus. These data contribute to the understanding of the interaction of cells in the limbal niche and with corneal cells essential for the maintenance of the cellular homeostasis in the cornea.

Morphofunctional Properties of Corneal Stromal Cells.

Human corneal stromal cells were isolated by enzymatic digestion from a new source, lenticules obtained during laser vision correction by the ReLEx SMILe method. The resulting culture was mainly presented by fibroblast-like cells with a phenotype CD90-/CD73+/CD105+/keratocan-/lumican-/ALDH1A1+ that differentiate into keratocytes in a specialized medium. The concentration of fetal calf serum-derived growth factors affects the rate of proliferation, production of erythropoietin and brain neurotrophic factor by corneal fibroblasts, and to a lesser extent, their migration activity and production of extracellular matrix components. Thus, the high functional potential of fibroblast-like cells isolated from stromal lenticles can be used to develop cell technologies in ophthalmology.

Cytotoxicity of Xenogeneic Pericardium Preserved by Epoxy Cross-Linking Agents.

The aim of the study was to assess the cytotoxic effect of xenopericardial biomaterial treated with di- and pentaepoxides on the cell cultures in vitro. MATERIALS AND METHODS: Samples of bovine and porcine pericardium were used in the work. Three different modes were employed for preservation: 1) 0.625% solution of glutaraldehyde (GA) and a two-fold change on days 2 and 7; 2) 5% solution of ethylene glycol diglycidyl ether (EGDE) changed on day 2; 3) 5% EGDE solution for 10 days, then 2% pentaepoxide solution also for 10 days. The cytotoxicity of the biomaterial was assessed by the extraction method. To determine the cytotoxicity of the biomaterial, EA.hy926 cells, multipotent mesenchymal stem cells (MMSCs), and fibroblasts were used. Cell viability was determined by the MTT test. The level of apoptosis and necrosis in the cell cultures was assessed by staining with acridine orange and ethidium bromide after cultivation with xenopericardial extracts employing different modes of preservation. RESULTS: Extracts of bovine and porcine pericardium preserved with GA have been found to have the greatest toxic effect on the cell cultures showing 20-33% reduction of cell viability. Extracts from bovine and porcine pericardium preserved with di- and pentaepoxy compounds do not have a toxic effect on endothelial cells, MMSCs, and fibroblasts since cell viability reduction is by no more than 15%. The lowest level of apoptosis and necrosis is observed in the cell cultures under the influence of extracts from the pericardium, preserved with diepoxide and pentaepoxide compounds. CONCLUSION: According to the MTT test for cytotoxicity and determination of the level of apoptosis and necrosis in cell cultures, bovine and porcine pericardia treated with di- and pentaepoxides have been established to have no cytotoxic effect on the culture of endothelial EA.hy926 cells, MMSCs, fibroblasts in vitro, whereas GA, in comparison with di- and pentaepoxides, has a toxic impact on the cells.

Effect of Erythropoietin on Morphofunctional Properties of Mesenchymal Stem Cells.

We studied the effect of erythropoietin on the morphofunctional status of bone marrow mesenchymal stem cells in patients with coronary heart disease. It was shown that the duration of cell exposure with erythropoietin had different effects on the expression levels of adhesion molecules, erythropoietin receptors, and co-expression of the erythropoietin receptor and common ß-chain of cytokines, apoptosis/necrosis, and the cell cycle. In most cases, erythropoietin increased proliferation, migration, and NO production by "aged" mesenchymal stem cells (passage 8) and passage 4 mesenchymal stem cells grown during the previous 3 passages in the presence of 33.4 U/ml erythropoietin. Erythropoietin increased the expression of the autophagy marker LC3B in mesenchymal stem cells grown in the presence of erythropoietin in the culture medium. Thus, long-term culturing of mesenchymal stem cells in the presence of erythropoietin in the culture medium increased their resistance to adverse microenvironment factors - oxidative stress and hyperglycemia.

[Therapeutic potential of a biomedical cellular product in rats with lower limb ischaemia]. / Terapevticheskii potentsial biomeditsinskogo kletochnogo produkta pri ishemii nizhnikh konechnostei u krys.

Critical ischaemia of lower limbs is a cause of death and invalidity in the whole world. Stem cells and products of their secretion find wide application in treatment of vascular diseases, including critical ischaemia of the lower limbs. Erythropoietin promotes an increase in the angiogenic potential of stem cells. The authors examined the therapeutic potential of a biomedical cellular product (mesenchymal stem cells and products of their secretion) and mesenchymal stem cells with erythropoietin on the processes of restoration of vessels in the hind legs of Wistar male rats following induction of lower limb critical ischaemia. Mesenchymal stem cells were derived from the bone marrow of male Wistar rats. Critical ischaemia of hind legs was modulated by transaction of the femoral artery. The parameters of microcirculation in the foot were assessed with the help of laser Doppler flowmetry. In the blood serum and crural muscles by means of solid-phase enzyme immunoassay we examined the levels of cytokines, growth factors, and persistent metabolites of nitrogen oxide - nitrites. Muscles morphology and the number of blood vessels were assessed by the findings of histological examination. It was shown that the biomedical cellular product alone and in combination with erythropoietin stimulated angiogenesis. The results of Doppler flowmetry revealed restoration of the parameters of microcirculation in the lower limb by 35-75% of the baseline values. Besides, we observed a decrease of muscle necrosis, connective tissue proliferation, and an increase in the number of the vessels supplying the muscles in the experimental groups. It was also determined that the biomedical cellular product influenced the levels of cytokines in blood serum and crural muscles. Hence, the obtained findings proved the therapeutic potential of the biomedical cellular product in critical ischaemia of lower limbs.

Cytotoxicity of Aluminum-Silica Matrices Modified with Carbon Nanotubes.

We studied the effect of aluminum-silicon matrices modified with carbon nanotubes on proliferation and production of nitric oxide by splenocytes, thymocytes, and bone marrow mononuclear cells of female db/db mice. Synthesized matrices decreased cell proliferation and suppressed NO production by the studied cells.

Evaluation of the Effect of Plasma from Patients with Trophic Ulcers on the Function of Dermal Fibroblasts, Mesenchymal Stem Cells, and Endothelial Cells.

We studied the effect of platelet lysate and platelet-poor plasma from patients with trophic ulcers with and without type 2 diabetes mellitus on proliferation, migration, and apoptosis of human dermal fibroblast, mesenchymal stem cells, and endothelial cells. It is shown that plasma obtained from patients with type 2 diabetes mellitus produced inhibitory effects.

Cytokines of the Lymph and Blood Serum as Markers of Oncogenesis and Efficiency of Therapy in Chemically Induced Breast Cancer in Wistar Rats.

We studied the levels of serum and lymph cytokines involved in the pathogenesis of BC. BC was induced by injection of N-methyl-N-nitrosourea to Wistar rats. The animals underwent surgery, or received polychemotherapy (cyclophosphamide, methotrexate, and 5-fluorouracil), or surgical treatment was combined with polychemotherapy; in a special group, Panagen (fragmented DNA) was added to polychemotherapy. Cytokine concentration in the lymph was measured using Bio-Plex Pro Rat Cytokoness 24-Plex Assay test system (Bio-Rad). In rats with BC, the content of most studied cytokines (IL-1ß, IL-2, IL-4, IL-6, IL-7, IL-12, IL-13, MIP-1α, MIP-3α, RANTES, TNFα, and MCP-1) in the lymph and blood was significantly higher, while the content of IL-10 and GRO/KC was lower than in intact animals. Surgical resection of the tumor led to a significant decrease in the content of both pro- and anti-inflammatory cytokines in the lymph. Polychemotherapy led to a significant decrease in the content of IL-1ß, IL-4, IL-6, IL-7, MIP-1α, MIP-3α, and RANTES in the serum and lymph. Comparison of the cytokine content in the serum and lymph of operated animals after polychemotherapy with and without Panagen showed that the content of most cytokines (IL-5, IL-6, IL-7, IL-10, IL-13, IL-17A , IL-18, GRO/KC, IFNγ, and MIP-3α) was higher after Panagen administration.

Correlation of the Levels of microRNA with Cell Composition of Axillary Lymph Node in Wistar Rats with Breast Cancer.

We studied the levels microRNA (miR-21, miR-221, miR-222, and miR-429) in blood serum, thoracic duct lymph, and breast cancer tissue, as well as the cell composition of the axillary lymph node in Wistar female rats with chemically induced breast cancer. The levels of miR-221 and miR-429 in the tumor tissue and in the lymph correlated with the decrease in lymphocyte number in the medullary cords of the axillary lymph nodes.

Correlation Analysis of Morphological Changes of Structural and Functional Areas of Mesenteric Lymph Nodes with Cytokines of Lymph of Thoracic Lymphatic Duct in Experimental Breast Cancer.

We performed a correlation analysis of the morphometric parameters of mesenteric lymph nodes and cytokine content in the lymph of thoracic duct in rats with chemically induced breast cancer. The study showed that activity of the local immune response in the lymph nodes in breast cancer is aimed at antitumor protection. In breast cancer, the area of the paracortical zone remained at the level of the intact group, while the area of lymphoid nodules with germinative centers and the area of medullary substance increased; the number of macrophages in the thymus-dependent zone and zone responsible for humoral immunity also increased. The following positive correlations were revealed: in germinative centers and medullary substance, number of mitotic cells correlated with cytokine IL-5 content and the number of medium lymphocytes correlated with the content of chemokine MIP-1α; in the germinative centers, the number of immunoblasts correlated with the level of cytokine GRO/KC, in the paracortical zone, the number of macrophages correlated with the level of chemokine MCP-1, the number of reticular cells correlated with IL-6 and M-CSF content; in medullary substance, the number of small lymphocytes and mature cells plasma cells (their content was reduced) correlated with the level of chemokine GRO/KC, which can be caused by their migration from the lymph node.

Treatment of Intervertebral Disc Degeneration in Wistar Rats with Mesenchymal Stem Cells.

We studied the effect of erythropoietin on functional properties of mesenchymal stem cells under conditions of oxidative stress and their therapeutic potential in the treatment of intervertebral disc degeneration in Wistar rats. It was shown that erythropoietin stimulates proliferation under conditions of oxidative stress. Injection of bone marrow mesenchymal stem cells into the damaged intervertebral disc was followed by an increase in the height of the intervertebral disc and activation of repair processes in the nucleus pulposus. The combination of mesenchymal stem cells with erythropoietin provides the best effect of cell therapy in case of intervertebral disc damage.

Cytokine Profile in Experimental Models of Critical Limb Ischemia in Rats.

We studied the effect of intramuscular administration of a cellular product (mesenchymal stem cells, conditioned media, and erythropoietin) on cytokine levels in blood serum, conditioned media of bone marrow mononuclears, and calf muscles in Wistar rats with hind limb ischemia. It is shown that the cellular product reduces the proinflammatory background at the early stages of the experiment and increases the content proangiogenic factors.

[Effect of polyethylene terephthalate (Dacron®) on functional potential of human bone-marrow/progenitor stem cells]. / Vliianie poliétilentereftalata (Dakron®) na funktsional'nyi potentsial kostnomozgovykh stvolovykh/progenitornykh kletok cheloveka.

Vascular grafts made of polytetrafluoroethylene and polyethylene terephthalate have widely been used in cardiovascular surgery. The causes of delayed colonization of such grafts by endotheliocytes and mesenchymal stem cells have not been adequately investigated. The authors examined the effect of polyethylene terephthalate on the functional activity of human bone marrow mesenchymal stem cells and endothelial progenitor cells in vitro. Proliferation (MTT assay, real-time cellular impedance), migration (Boyden chamber, real-time cellular impedance), and nitric oxide production (spectrophotometciacally) by progenitor endothelial cells and mesenchymal stem cells were assessed with and without the presence of polyethylene terephthalate. The functional activity of the cells was shown to depend on the presence of polyethylene terephthalate in a well with cells. Thus, polyethylene terephthalate turned out to exhibit a toxic effect on progenitor endothelial and mesenchymal stem cells. Treatment of grafts with gelatine or fibronectin improved colonization of grafts with cells.

Effect of Polyethylene Terephthalate on Functional Properties of Endothelial and Mesenchymal Cells.

We studied the influence of vascular prostheses made of polytetrafluoroethylene and polyethylene terephthalate on the proliferation, migration, and NO production by bone marrow mesenchymal stem cells, human endothelial progenitor cells, and EA.hy926 endothelial cells, colonization of the prosthesis surface by endothelial and mesenchymal cells was also analyzed. Synthetic prostheses have a negative effect on cell proliferation and migration, while surface treatment with proteins (fibronectin or gelatin) promotes colonization of the prostheses with cells.

Therapeutic Potential of Biomedical Cell Product in DSS-Induced Inflammation in the Small Intestine of C57Bl/6J Mice.

We studied the effect of intravenous administration of biomedical cell product (bone marrow mesenchymal stem cells and media conditioned by these cells) on histological picture of the small intestine in mice with DSS-induced inflammation. It was shown that biomedical cell product promoted recovery of the intestinal mucosa in DSS-induced inflammation.

Effect of Intramuscular Administration of Mesenchymal Stem Cells and Erythropoietin on Angiogenesis in Critical Limb Ischemia.

We studied the effect of intramuscular injections of bone marrow mesenchymal stromal cells on microcirculation parameters in an ischemic limb of Wistar rats. It was shown that transplantation of mesenchymal stem cells with or without erythropoietin stimulates angiogenesis in the ischemic zone. An essential role in angiogenesis stimulation is played by cytokines and growth factors.