Interaction of Graphene Oxide Nanoparticles with Human Mononuclear Cells in the Cell-IQ System.

The interaction of graphene oxide nanoparticles with human peripheral blood mononuclear cells was studied using the Cell-IQ continuous monitoring system for living cells. We used graphene oxide nanoparticles of various sizes coated with linear or branched polyethylene glycol (PEG) in concentrations of 5 and 25 µg/ml. After 24-h incubation with graphene oxide nanoparticles, the increase in the number of peripheral blood mononuclear cells at visualization points decreased; nanoparticles coated with branched PEG more markedly suppressed cell growth in culture. In the presence of graphene oxide nanoparticles, peripheral blood mononuclear cells retained high viability in culture after daily monitoring in the Cell-IQ system. The studied nanoparticles were engulfed by monocytes and the type of PEGylation had no effect on this process. Thus, graphene oxide nanoparticles reduced the increase in peripheral blood mononuclear cell mass during dynamic observation in the Cell-IQ system without reducing their viability.

Human Mesenchymal Stem Cells as a Carrier for a Cell-Mediated Drug Delivery.

A number of preclinical and clinical studies have demonstrated the efficiency of mesenchymal stromal cells to serve as an excellent base for a cell-mediated drug delivery system. Cell-based targeted drug delivery has received much attention as a system to facilitate the uptake a nd transfer of active substances to specific organs and tissues with high efficiency. Human mesenchymal stem cells (MSCs) are attracting increased interest as a promising tool for cell-based therapy due to their high proliferative capacity, multi-potency, and anti-inflammatory and immunomodulatory properties. In particular, these cells are potentially suitable for use as encapsulated drug transporters to sites of inflammation. Here, we studied the in vitro effects of incorporating synthetic polymer microcapsules at various microcapsule-to-cell ratios on the morphology, ultrastructure, cytokine profile, and migration ability of human adipose-derived MSCs at various time points post-phagocytosis. The data show that under appropriate conditions, human MSCs can be efficiently loaded with synthesized microcapsules without damaging the cell's structural integrity with unexpressed cytokine secretion, retained motility, and ability to migrate through 8 µm pores. Thus, the strategy of using human MSCs as a delivery vehicle for transferring microcapsules, containing bioactive material, across the tissue-blood or tumor-blood barriers to facilitate the treatment of stroke, cancer, or inflammatory diseases may open a new therapeutic perspective.

Osteogenic and Angiogenic Properties of Heparin as a System for Delivery of Biomolecules for Bone Bioengineering: a Brief Critical Review.

The review considers complex, controversial, and individual effects of heparin and its derivatives on the bone and circulatory systems in dependence of the dose, the state of the cells and tissues of the recipient. General data on the anticoagulant activity of heparin and its derivatives are presented; special attention is paid to the effect of heparin on mesenchymal cells and tissues and its role in angiogenesis. We also discuss the ability of heparin to bind osteogenic and angiogenic biomolecules in the context of the development of systems for their delivery and sustained controlled release and propose a schematic representation of the positive and side effects of heparin as a delivery system for biomolecules in tissue engineering.

Surface characterization and biological assessment of corrosion-resistant a-C:H:SiOx PACVD coating for Ti-6Al-4V alloy.

The paper focuses on the SiOx-doped amorphous hydrocarbon (a-C:H:SiOx) coating on the titanium (Ti-6Al-4V) alloy substrate obtained by plasma-assisted chemical vapor deposition (PACVD) in a mixture of argon gas and polyphenylmethylsiloxane vapor using a bipolar substrate bias. It is shown that the a-C:H:SiOx coating deposition results in the formation of a negative surface potential important for application of this coating for medical implants. The a-C:H:SiOx coatings improve the corrosion resistance of Ti alloy to 0.5 M NaCl solution and phosphate-buffered saline. In particular, the corrosion current density of the a-C:H:SiOx-coated sample in a 0.5 M NaCl solution at 22 °C decreases from 1∙10-8 to 1.7∙10-10 A/cm2, that reduces the corrosion rate from 9∙10-5 to 15∙10-7 mm/year. The a-C:H:SiOx coating facilitates the surface endothelization of an implant located in the thoracic aorta of a mini pig, and reduces the risk of thrombosis and implant failure. This effect can be explained by the ability of the a-C:H:SiOx coating ability to reduce in vitro a 24-hour secretion of pro-inflammatory interleukins (IL-6, IL-12(p70), IL-15, and IL-17) and cytokines (IFN-g and TNF-a) by blood mononuclear cells (MNCs) and elevates the concentration of anti-inflammatory interleukin IL-1Ra. In vitro analysis shows no cytotoxicity of the a-C:H:SiOx coating for the human blood MNCs, suggesting a promising PACVD on Ti alloys for cardiovascular implants, including pumps for mechanical heart support systems.

[Osteogenic and angiogenic properties of heparin as a system of biomolecule delivery for bone bioengineering: a brief critical review]. / Osteogennye i angiogennye svoistva geparina kak sistemy dostavki biomolekul pri bioinzhenerii kosti: kratkii kriticheskii obzor.

The review discusses the complex, ambiguous and individual effects of heparin and its derivatives on the bone and circulatory systems, in dependence of the dosage, the state of the cells and tissues of recipients. General data on the anticoagulant activity of heparin and its derivatives are presented; aspects of the effect of heparin on mesenchymal cells and tissues and its role in angiogenesis are considered in details. Particular attention is paid to the ability of heparin to bind osteogenic and angiogenic biomolecules: thus us especially important for the development of systems for their delivery and sustained controlled release. A schematic representation of the positive and side effects of heparin as a delivery system for biomolecules in tissue engineering is proposed.

Granulocyte colony-stimulating factor downregulates interferon-gamma receptor expression and stimulates interleukin-6 production in activated human macrophages.

We studied direct effects of human granulocyte colony-stimulating factor (G-CSF) on phenotypical properties of human macrophage cells in vitro. CD14+ monocyte/macrophages (Mc/Mphs) were isolated from blood of healthy donors by positive magnetic separation. G-CSF (0.01-1.0 ng/mL), when added to Mc/Mphs along with lipopolysaccharide (LPS, 1.0 µg/mL), was able to noticeably reduce proportions of CD119 (interferon-γ receptor 1)-positive cells, with no stable effects on CD16 (FcγRIII)+ and СD124 (IL-4 receptor subunit alpha)-positive cells. In addition, G-CSF markedly upregulated IL-6 production by LPS-activated Mph cells, without significantly affecting IL-1ß, IL-10 and tumor necrosis factor-α (TNF-α) secretion. Our data suggests that G-CSF could restrain Mph polarization to pro-inflammatory (M1) phenotype, thus potentially supporting pro-regenerative Mph activity with implications for immunotherapeutic interventions.

[Secretion of niche signal molecules in conditions of osteogenic differentiation of multipotent mesenchymal stromal cells induced by textured calcium phosphate coating]. / Sekretsiia signal'nykh molekul krovetvornykh nish v usloviiakh osteogennoi differentsirovki mul'tipotentnykh mezenkhimnykh stromal'nykh kletok, indutsirovannoi rel'efnym kal'tsii-fosfatnym pokrytiem.

Secretion of 21 cytokines, chemokines and growth factors (LIF, SCF, SDF-1a, SCGF-b, M-CSF, MCP-3, MIF, MIG, TRAIL, GRO-a; IL-1a, IL-2ra, IL-3, IL-12(p40), IL-16, IL-18, HGF, TNF-b, b-NGF, IFN-a2, CTACK) has been studied in vitro in the culture of human adipose-derived multipotent mesenchymal stromal cells (hAMMSCs) in conditions of its osteogenic differentiation caused by 14-day contact with calcium phosphate (CP) surface with different roughness. Bilateral X-ray amorphous CP coatings were prepared on the samples of commercially pure titanium in the anodal regime using a micro-arc method. An aqueous solution prepared from 20 wt% phosphoric acid, 6 wt% dissolved hydrohyapatite nanopowder (particle diameter 10-30 nm with single agglomerates up to 100 nm), and 9 wt% dissolved calcium carbonate was used to obtain CP coating. hAMMSCs isolated from lipoaspirate were co-cultured after 4 passages with the CP-coated samples at final concentration of 1.5´105 viable karyocytes per 1.5 mL of standard nutrition medium (without osteogenic stimulators) for 14 days (a determination of [CD45,34,14,20], CD73, CD90 и CD105 cell immunophenotype; an analysis of secretory activity) and 21 days (alizarin red S staining of culture) with medium replacement every 3-4 days. Under conditions of in vitro contact with rough CP coating hAMMSCs differentiated into osteoblasts synthesizing the mineralized bone matrix; this was accompanied by 2-3-fold increasing ratio of [CD45,34,14,20]+ hemopoietic cells. The following humoral factors of hemopoietic niches acted as the signal molecules escalating in vitro the hemopoietic base in 14 days of differentiating three-dimensional culture of hAMMSCs: either leukemia inhibitory factor (LIF) and stem cell factor (SCF) cytokines under mean index of CP roughness Ra=2.4-2.6 mm or stromal derived factor-1 (SDF-1a, CXCL12 chemokine) under Ra=3.1-4.4 mm.

Cell-IQ visualization of motility, cell mass, and osteogenic differentiation of multipotent mesenchymal stromal cells cultured with relief calcium phosphate coating.

The Cell-IQ continuous surveillance system allowed us to establish the following changes in a 14- day culture in vitro: a twofold suppression of the directional migration of multipotent mesenchymal stromal cells of human adipose tissue (MMSC-AT) towards the samples with a microarc calcium phosphate (CP) coating from synthetic hydroxyapatite; a tenfold decrease in the cell mass on the interphase with the samples, which was accompanied by a slight reduction in the expression of membrane determinants of stromal stem cells; and an enhancement of their osteogenic differentiation (osteocalcin secretion and mineralized matrix formation) on the 21st day of the study. Calcium phosphate particles, but not the calcium and phosphorus ions, may trigger the phenotypic transformation of the MMSC-AT behavior in vitro.

Erythropoietin exerts direct immunomodulatory effects on the cytokine production by activated human T-lymphocytes.

The effect of erythropoietin-ß (Epo-ß) on the functional profile of activated human T-lymphocytes remains largely unknown, which hinders clinical application of Epo as an immunomodulatory agent. We studied the direct impact of Epo on the activation status of human T lymphocytes following activation by particles loaded with antibodies (Abs) against human CD2, CD3, and CD28. T cell activation was assessed by the surface expression of CD38 activation marker. Epo did not significantly affect activation status of both CD4(+) and CD4(-) T cells, as well as of naive (CD45RA(+)CD197(+)), central memory (CD45RA(-)CD197(+)), effector memory (CD45RA(-)CD197(-)), and terminally-differentiated (CD45RA(+)CD197(-)) T cells. However, Epo markedly augmented production of IL-2, IL-4 and IL10 by activated T cells with concomitant reduction in IFN-γ secretion. Taken together, our data showed that Epo could directly down-regulate pro-inflammatory T cell responses without affecting T cell activation status.

[DIRECT EFFECT OF IL-7 ON THE GROWTH AND FUNCTIONAL ACTIVITY OF T-LYMPHOCYTES IN THE ABSENCE OF ANTIGENIC STIMULUS].

It is shown that in the absence of antigenic stimulus interleukin-7 (IL-7) is capable of increasing the content of cells expressing the CD25 molecule among both CD4-positive and CD4-negative effector memory T-cells (CD45RA-CD197-), as well as terminally differentiated T cells (CD45RA+CD197-) without causing a significant impact on the status of the activation of naive T cells (CD45RA+CD197+) and central memory T cells (CD45RA-CD197+). IL-7 was also able to enhance T-cell production of IL-2, interferon-γ (IFN-γ) and IL-10, but not IL-4. These data indicate the involvement of IL-7 into a direct upregulation of growth and functional activity of human T cells in the absence of antigenic stimulus and the relative scarcity of costimulatory effects.