V. V. Shupletsova

Dose-dependent effects of dexamethasone on functional activity of T-lymphocytes with different grades of differentiation

Glucocorticoids are anti-inflammatory and immunosuppressive agents with pleiotropic effects on the growth, differentiation, and functional activity of T lymphocytes. The comprehensive assessment of the influence of dexamethasone on the functional activity of T cells with different grades of differentiation was performed under experimental in vitro conditions. We established that dexamethasone suppressed the functional activity of CD45RA+ and CD45RO+ T lymphocytes. In the population of naive (CD45RA+) T cells, the inhibitory effect of dexamethasone on the early activation stages (IL-2-dependent, associated with the CD25 expression and IL-2 production) was more significant, whereas in the culture of primed memory cells (CD45RO+), dexamethasone affects the later stages (IL-2-independent, associated with the expression of proliferation molecule CD71). Multidirectional effects of dexamethasone on the level of expression of mRNA of telomerase catalytic unit (hTERT) were associated with the degree of T cells differentiation. Most likely, the role of glucocorticoid hormones in immunogenesis is mainly the suppression of the excessive growth of T cells and the maintenance of the clonal balance in lymphoid tissue.

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.

Morphofunctional changes of Jurkat T lymphoblasts upon short-term contact with a relief calcium phosphate surface

Human leukemic T lymphoblastoid cells (hereinafter, Jurkat T cells) have been used to model the morphofunctional reaction of T lymphocytes to 24-h in vitro contact with relief (roughness index Ra = 2.2–2.7 μm) pure titanium substrates (12 × 12 × 1 mm3) covered by a calcium phosphate (CP) bilateral coating prepared by the microarc method. Jurkat T cells placed in plastic multiwall plates (2D control of culture growth), as well as cells in contact for 24 h with oxide (TiO2) microarc coating on pure titanium substrate (3D control), served as comparison tests. From 27 to 98% of immortalized cells in the 2D control culture had the CD3+CD4+CD71+CD45RA+ immunophenotype and secreted IL-2, IL-4, IL-8, IL-10, and TNFα, but not IL-1b and IL-6. Other markers of cell activation, differentiation, maturation, and death (CD8, CD16, CD56, CD25, and CD95) were found in 0–2.5% of cells. IL-8 release elevated by the microtextured CP surface by 183 and 160% relative to 2D- and 3D-control cultures, respectively. The CD4/CD8 ratio dropped to 9: 1 (13: 1 and 82: 1 in 2D and 3D controls, respectively) due to CD4+ cell depletion and an increase in CD8+ cell number. The total cell number (TCN) in Jurkat T cell culture after 24-h contact with CP coating was decreased to 88% compared to 2D control (p < 0.04), which indicated cell division suppression. A drop in total cell number was accompanied with accelerated IL-8 secretion (r = −0.97, p < 0.00009). Low concentrations of IL-8 (pg/mL) induced apoptosis (r = 0.94, p < 0.0001). These results show that CP, rather than TiO2, coating is a preferential surface material for endoprosthesis replacement and fracture osteosynthesis in patients suffering from hematological and bone malignancies.

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, β–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.

Porous composite materials ZrO2(MgO)-MgO for osteoimplantology

The pore structure and phase composition of ceramic composite material ZrO2(Mg)-MgO at different sintering temperatures were studied. The main mechanical characteristics of the material were determined and it was shown that they are close to the characteristics of natural bone tissues. It was shown that material structure has a positive effect on the pre-osteoblast cells proliferation. In-vitro studies of pre-osteoblast cells, cultivation on material surface showed a good cell adhesion, proliferation and differentiation of MMSC by osteogenic type.

Rough Titanium Oxide Coating Prepared by Micro-Arc Oxidation Causes Down-Regulation of hTERT Expression, Molecular Presentation, and Cytokine Secretion in Tumor Jurkat T Cells

The response of the human Jurkat T cell leukemia-derived cell line (Jurkat T cells) after 24 h of in vitro exposure to a titanium substrate (12 × 12 × 1 mm3) with a bilateral rough (Ra = 2.2–3.7 μm) titanium oxide coating (rTOC) applied using the micro-arc method in a 20% orthophosphoric acid solution was studied. A 1.5-fold down-regulation of hTERT mRNA expression and decreases in CD3, CD4, CD8, and CD95 presentation and IL-4 and TNFα secretion were observed. Jurkat T cell inactivation was not correlated with the generation of intracellular reactive oxygen species (ROS) and was not mediated by TiO2 nanoparticles with a diameter of 14 ± 8 nm at doses of 1 mg/L or 10 mg/L. The inhibitory effect of the rTOC (Ra = 2.2–3.7 μm) on the survival of Jurkat T cells (Spearman’s coefficient rs = −0.95; n = 9; p < 0.0001) was demonstrated by an increase in the necrotic cell count among the cell population. In turn, an elevation of the Ra index of the rTOC was accompanied by a linear increase (r = 0.6; p < 0.000001, n = 60) in the magnitude of the negative electrostatic potential of the titanium oxide surface. Thus, the roughness of the rTOC induces an electrostatic potential and decreases the viability of the immortalized Jurkat T cells through mechanisms unrelated to ROS generation. This may be useful for replacement surgery applications of rough TiO2 implants in cancer patients.

Nanoscale Electrical Potential and Roughness of a Calcium Phosphate Surface Promotes the Osteogenic Phenotype of Stromal Cells

Mesenchymal stem cells (MSCs) and osteoblasts respond to the surface electrical charge and topography of biomaterials. This work focuses on the connection between the roughness of calcium phosphate (CP) surfaces and their electrical potential (EP) at the micro- and nanoscales and the possible role of these parameters in jointly affecting human MSC osteogenic differentiation and maturation in vitro. A microarc CP coating was deposited on titanium substrates and characterized at the micro- and nanoscale. Human adult adipose-derived MSCs (hAMSCs) or prenatal stromal cells from the human lung (HLPSCs) were cultured on the CP surface to estimate MSC behavior. The roughness, nonuniform charge polarity, and EP of CP microarc coatings on a titanium substrate were shown to affect the osteogenic differentiation and maturation of hAMSCs and HLPSCs in vitro. The surface EP induced by the negative charge increased with increasing surface roughness at the microscale. The surface relief at the nanoscale had an impact on the sign of the EP. Negative electrical charges were mainly located within the micro- and nanosockets of the coating surface, whereas positive charges were detected predominantly at the nanorelief peaks. HLPSCs located in the sockets of the CP surface expressed the osteoblastic markers osteocalcin and alkaline phosphatase. The CP multilevel topography induced charge polarity and an EP and overall promoted the osteoblast phenotype of HLPSCs. The negative sign of the EP and its magnitude at the micro- and nanosockets might be sensitive factors that can trigger osteoblastic differentiation and maturation of human stromal cells.

Behavioral Changes of Multipotent Mesenchymal Stromal Cells in Contact with Synthetic Calcium Phosphates in vitro

Migration, proliferation, and osteogenic differentiation of human adipose-derived (AD) multipotent mesenchymal stromal cells (MMSCs) during in vitro modeling of indirect contact with calcium phosphate (CP) or nanoparticles of synthetic hydroxyapatite (HA) have been studied. The results were registered with electrode (real-time cell analysis, RTCA) or visual (Cell-IQ) systems of long-term observation of cell cultures. Bulk specimens were use in a Cell-IQ® v2 MLF device as pure titanium substrates (10 × 10 × 1 mm3) covered by a CP relief (roughness index Ra = 2.4–4.4 μm) bilateral coating that was prepared by the micr-arc method from an aqueous solution of orthophosphoric acid (20 wt %), calcium carbonate (9 wt %), and synthetic HA (6 wt %). HA crystallites (1 mg/mL) were fabricated by mechanochemical synthesis and served as an irritant in RTCA investigation. The Cell-IQ system identified a 3.5- to 10-fold decrease in cell number at the interface with CP coatings with differing roughness during 14-day cell culturing. After 21 days, it was accompanied by a weak reduction of MMSC antigen expression (CD73, CD90, and CD105) as opposed to an increase in MMSC osteogenic differentiation and intercellular-matrix mineralization. In turn, HA nanodispersion reduced the speed of MMSC migration by 1.5 times (P < 0.001) during 25-h RTCA recording, which simulated cell invasion through the microporous membrane (8-μm diameter). Inhibition of migration and cell division with increased osteogenic differentiation of MMSCs has been suggested to be a possible effect of biodegradation products of synthetic CP materials.

Modeling of the mesenchymal stem cell microenvironment as a prospective approach to tissue bioengineering and regenerative medicine (a short review)

One of the promising areas is the design and modification of materials for control over the fate of multipotent mesenchymal stromal cells (MMSCs) that will allow stroma of various human and animal organs and tissues to be constructed. However, the discussion about the existence and functioning of microenvironment for the MMSCs is just beginning to develop. The design of artificial materials that are able to reproduce biomimetically the cellular and tissue microenvironment and based on ideas and main elements borrowed from wildlife is current direction in a development of medical materials technology and tissue bioengineering. Scaffold technology is a promising experimental approach to simulate the properties of natural microenvironment of stem cells. Our aim is a short review of key elements of MMSC microterritories, its advanced investigations and the attempts of modeling in application to tissue bioengineering and regenerative medicine.

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.