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.

[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.

Imbalance of morphofunctional responses of Jurkat T lymphoblasts at short-term culturing with relief zinc- or copper-containing calcium phosphate coating on titanium.

Morphofunctional response of Jurkat T cells that were cultured for 24 h on substrates prepared from commercially pure titanium with relief microarc bilateral calcium phosphate coating containing copper or zinc was studied. Changes in the concentration of essential trace elements contained in this coating can cause significant imbalance of molecular processes of differentiation, secretion, apoptosis, and necrosis and reduce tumor cell survival.

Comparative investigations of structure and properties of micro-arc wollastonite-calcium phosphate coatings on titanium and zirconium-niobium alloy.

Investigation results of micro-arc wollastonite-calcium phosphate (W-CaP) biocoatings on the pure titanium (Ti) and Zr-1wt.%Nb (Zr-1Nb) alloy were presented. The voltages of 150-300 V generate the micro-arc oxidation (MAO) process with the initial amplitude current of 150-550 A and 100-350 A for Ti and Zr-1Nb substrates, respectively. The identical dependencies of changes of the coating thickness, surface roughness and adhesion strength on the process voltage were revealed for the both substrates. The W-CaP coatings with the thickness of 10-11 µm were formed on Ti and Zr-1Nb under the low process voltage of 130-150 V. Elongated wollastonite particles with the size in the range of 40-100 µm were observed in such coatings. The structure of the coatings on Ti was presented by the X-ray amorphous and crystalline phases. The X-ray reflexes relating to the crystalline phases of Ti and wollastonite were observed only in XRD patterns of the coatings deposited under 130-200 V on Ti. While, the crystalline structure with phases of CaZr4(PO4)6, ß-ZrP2O7, ZrO2, and Zr was detected in the coatings on Zr-1Nb. FT-IRS, XRD, SEM, and TEM data confirmed that the increase of the process voltage to 300 V leads to the dissociation of the wollastonite. No toxic effect of specimens on a viability, morphology and motility of human adipose-derived multipotent mesenchymal stem cells was revealed in vitro.

MORPHOFUNCTIONAL CHANGES OF JURKAT LINE T-LYMPHOBLASTS AT SHORT-TERM CONTACT WITH RELIEF CALCIUM PHOSPHATE SURFACE.

Human leukemic T-lymphoblastoid cells (hereinafter Jurkat T-cells) were used to model T-lymphocytes morphofunctional reaction to 24-h in vitro contact with relief (roughness index Ra = 2.2--2.7 mm) pure titanium substrates (12_12_1 mm3) covered by calcium phosphate (CP) bilateral coating that was prepared by micro-arc method. Jurkat T-cell culture on plastic surface of well plate (2D control of culture growth), as well as the cells that contacted for 24 h with oxide (TiO2) micro-arc coating on pure titanium substrate (3D control) served as comparison tests. 27--98 % of immortalized cells in 2D control culture had CD3+CD4+CD71+CD45RA+ immunophenotype and secreted IL-2, IL-4, IL-8, IL-10 and TNFa, but not IL-1b and IL-6. Other markers of cell activation, differentiation, maturation and death (CD8, CD16, CD56, CD25, CD95) were found at 0­2.5% of the cell population. Microtextured CP surface elevated statistically IL-8 outcome to 183 and 160 % of corresponding control values in 2D- and 3D-cultures of Jurkat T-cells. CD4/CD8 ratio fell to 9 : 1 (at 13 : 1 and 82 : 1 in 2D- and 3D-controls, respectively) both by CD4+ depletion and by CD8+ cell percent raise. Total amount of cells (TAC) in Jurkat T-cell culture after 24-h contact with CP coating was decreased to 88 % 2D control level (P < 0.04) that could favor to a suppression of cell division. TAC reduction in Jurkat T-cell culture was accompanied by accelerated IL-8 spontaneous secretion (r = ­0.97, P < 0.00009). For all this, IL-8 induced apoptosis (r = 0.94, P < 0.0001) in low concentrations (pg/ml). The obtained result is the feature of Jurkat T-cells reaction on CP but not TiO2 coatings, and it may be used in case of materials selection for endoprosthesis replacement and fracture osteosynthesis in patients suffering by hematological and bone malignancies.

[PHENOTYPIC CHARACTERIZATION AND FUNCTIONAL FEATURES OF MEMORY T- AND B-CELLS].

In this review, we systematized the data that characterize phenotypic properties and functional features of T- and B-lymphocytes of immune memory. We examin the organization of T-cells of immune memory in a population, and their selective distribution in the organism according to their effector potential.

[Dose-dependent effects of dexamethasone on functional activity of T-lymphocytes different grade of differentiation].

Glucocorticoids are anti-inflammatory and immunosuppressive agents which have pleiotropic effects on growth, differentiation and functional activity of T-lymphocytes. Under experimental conditions in vitro carried out a comprehensive assessment of the dexamethasone influence on the functional activity of T-cells with different differentiation degrees. It was established that the influence of dexamethasone on the functional activity of CD45RA+ and CD45RO+ T-lymphocytes, in general, has depressing character. It was revealed that in the population of naive (CD45RA+) T-cells dexamethasone exerts a more pronounced inhibitory effect on early (IL-2-dependent, associated with the CD25 expression and IL-2 production) activation stages, whereas in the culture primed memory cells (CD45RO+)--for later (IL-2-independent, associated with the expression of proliferation molecule CD71). Multidirectional effects of dexamethasone on the expression level of telomerase catalytic unit (hTERT) mRNA are associated with the degree of T cells differentiation. It isproposed, that the role of glucocorticoid hormones in immunogenesis is primarily aimed at suppression of excessive T cells growth and on the maintainance of the clonal balance in lymphoid tissue.