Introduction. Artificial materials used in regenerative medicine induce a balanced inflammatory response after implantation, which is an important step for effective regeneration of damaged bone tissue. The contact of the implant with tissues and biological fluids is accompanied by the deposition of blood proteins on its surface, which contributes to the activation of the complement system and initiates blood clotting, leading to the formation of a fibrin clot. On the surface of the implant, fibrin ensures the adhesion of stem cells and their maturation into fibroblasts that produce collagen and its derivatives. The formed extracellular matrix is the basis for the formation of a tissue structure (callus). To prevent the development of postoperative pathological conditions caused by hypercoagulatory syndrome, therapeutic strategies with anticoagulants such as heparin are used. However, their use limits the formation of a fibrin clot in vivo, which may slow down the migration of mesenchymal stromal cells (MSCs) and the subsequent formation of callus. Aim. To investigate of the effect of heparin at pharmacological concentrations on stemness and the ability of MSCs from human adipose tissue to undergo osteogenic differentiation under conditions of in vitro cultivation. Materials and methods. To assess the morphofunctional state of cells cultured in the presence of heparin, 2 experimental groups were formed: 1) MSCs in the presence of heparin at a therapeutic concentration (1.3 IU/ml); 2) MSCs in the presence of heparin at a toxic concentration (13 IU/ml). Results and discussion. Flow cytometry results showed that the addition of heparin at both concentrations used in the study to MSC culture leads to an increase in the number of cells expressing the surface markers CD73 and CD90, indicating the maintenance of their stem state. On the other hand, a stimulatory effect of heparin at both concentrations used on the transcription of mRNA of osteogenic genes (BMP2, BMP6, ALPL, RUNX2, BGLAP and SMURF1) in MSCs was also observed, which may indicate the osteogenic potential of heparin for the cell culture studied. Conclusion. The results of the study are useful for regenerative medicine related to the use of MSCs in clinical practice; they may serve as a prerequisite for the development of new therapeutic strategies for orthopedic and traumatologic patients at high risk of postoperative thrombosis after endoprosthetics surgery and osteosynthesis.

Correct choice of nutrient media for culturing different types of cells in various applications is one of the most important aspects of modern biotechnology, since chemical composition of the culture media largely contains the necessary metabolites to support certain cells’ growth lines outside the body. Jurkat line of human leukemic T-lymphoblast-like cells (hereinafter Jurkat T-cells) is actively used for in vitro modeling of intracellular signaling and activation of normal blood T-lymphocytes mediated by the T-cell receptor/CD3/ CD4 complex in toxicological studies of immune and secretory responses, to test medicinal substances and ions. Also, Jurkat T-cells are widely used for ex vivo testing in immunology, oncology, toxicology, orthopedics, and traumatology. The existing standards and numerous studies are mainly based on short-term in vitro cultivation of Jurkat T-cells in RPMI 1640 nutrient medium. Meanwhile, the issues of long-term maintenance of the growth of Jurkat T-cells culture are poorly presented in the research literature. This study aimed for studying the activity of Jurkat T-cells over 7 to 14 days of in vitro culture and comparing the relative value of RPMI 1640 and αMEM media for the behavior of immunocompetent tumor cells. Using flow cytometry, multiplex analysis, and phase contrast Cell-IQ microscopy, the proportions of living cells and those dying by apoptosis and necrosis, secretion of cytokines and chemokines, and the dynamics of cell biomass propagation were studied. It was found that the αMEM medium in the complete nutrient medium, as compared with RPMI 1640, is more appropriate to in vitro promotion of cell viability (increased proportion of viable cells by 13.5% at the day 14), their secretory ability for 23 из 27 tested biomolecules, shortened adaptation time (на 32%) in culture before growth initiation, 5-fold increase of the Jurkat Т-cell cellularity by the day 7. Potential significance of the chemical components of nutrient media and secreted biomolecules for these results is discussed. As based on the results obtained, we concluded on superior properties of αMEM medium for long-term in vitro cultures of Jurkat T-cells. Consequently, the in vitro testing of medical devices intended for long-term contact with the body, including those for cancer patients, using Jurkat T-cell leukemia line in RPMI 1640 medium, may lead to wrong predictions on their biocompatibility and potential antitumor activity.

Molecular genetic mechanisms, signaling pathways, cultural conditions, factors, and markers of osteogenic differentiation of mesenchymal stem cells (MSC) are actively studied despite numerous works in this area of cellular technologies. This is largely due to the accumulating contradictions in seemingly classical knowledge, as well as permanent updating of the results in the field. In this regard, we focused on the main classical concepts and some new factors and mechanisms that have a noticeable regulatory effect on the differentiation potential of postnatal MSCs. The present review considers the significance of MSC sources for their differentiation capacity, as well as the role of the cellular microenvironment. The issues of classification, terminology, and functional activity of MSCs from various sources are discussed. The paracrine potential of MSCs in tissue regeneration has been considered; sufficient importance of inflammation in osteogenesis is noted, in particular, the presence of inflammatory cytokines and chemokines in the lesion focus, produced not only by microenvironmental cells but also by blood cells, including mononuclear leukocytes, migrating to the affected site. An important role in this review is given to biomechanical signals and to influence of conformational changes in cell cytoskeleton (cell shape) upon MSC differentiation, since the morphological features of cells and the structure of cytoskeleton are modulated by interactions of the cell surface with environmental factors, including hydrostatic pressure, fluid flow, compression/stretching loads. The data are presented concerning elasticity of extracellular matrix being a determining factor of cell differentiation. We conclude that one should switch from point studies of individual gene effects to multiple measurements of the gene-regulatory profile and biomolecules responsible for multiple, still poorly studied osteogenic factors of endogenous and exogenous origin. Among cornerstones in future (epi)genetic studies will be to decide if osteomodulatory effects are realized through specific signaling pathways and/or via cross-signaling with known genes controlling osteogenic differentiation of MSCs.

Background: Molecular genetic mechanisms, signaling pathways, conditions, factors, and markers of the osteogenic differentiation of mesenchymal stem cells (MSCs) are being actively studied and are among the most studied areas in the field of cellular technology. This attention is largely due to the mounting contradictions in the seemingly classical knowledge and the constant updating of results in the analyzed areas. In this regard, we focus on the main classical concepts and some new factors and mechanisms that have a noticeable regulatory effect on the differentiation potential of postnatal MSCs. Results: This review considers the importance of the sources of MSCs for the realization of their differentiation potential, molecular genetic factors and signaling pathways of MSC differentiation, the role of inflammatory cytokines and chemokines in osteogenesis, biomechanical signals, and the effect of conformational changes in the cellular cytoskeleton on MSC differentiation. Conclusion: It is concluded that it is necessary to move from studies focused on the effects of local genes to those taking multiple measurements of the gene-regulatory profile and the biomolecules critical for the implementation of numerous, incompletely studied osteogenic factors of endogenous and exogenous origin. Among the cornerstones of future (epi)genetic studies, whether osteomodulatory effects are realized through specific signaling pathways and/or whether cross-signaling with known genes drives the osteogenic differentiation of MSCs remains to be determined.

Synthetic materials used in regenerative medicine, upon implantation, induce the development of an inflammatory reaction necessary for the effective regeneration of damaged bone tissue. Implant contact with tissues is accompanied by the deposition of blood proteins and interstitial fluid on its surface, contributing to the activation of the complement system, components of innate immunity, initiating coagulation hemostasis, leading to the formation of a fibrin clot. An extracellular matrix based on fibrin, collagen and elastin forms on the implant’s surface, which provides the basis for the formation of tissue structure through the adhesion of stem cells to the forming bone callus before the formation of bone regenerate. To prevent the development of postoperative pathological conditions caused by hypercoagulable syndrome, therapeutic strategies are used to use anticoagulants (heparin, warfarin). However, their use limits the normal formation of a fibrin clot in vivo. This can slow down the migration of mesenchymal stem cells (MSC) and disrupt the formation of callus, inhibiting the processes of osseointegration of the implant and bone healing. The study’s goal was to study the effect of heparin in a gradient of low and high concentrations on the migration activity and stem capacity of human MSCs under in vitro cultivation conditions. According to the results of flow cytometry, it was revealed that high concentrations of heparin (130, 260 IU/ml) in a 2D cultivation model contribute to an increase in the number of cells expressing surface markers CD73 and CD90, which indicates that MSCs retain high clonogenic potential. A 3D model of in vitro cultivation with the addition of heparin and osteosubstituting implants bearing a CF coating with a roughness index of Ra = 2.6-4.9 μm contributed to preserving the “stemness” character of MSCs through the expression of surface markers CD73 and CD90. According to the results obtained using the xCELLigence system, heparin at a later time (from 20-40 hours) increases the invasion of MSCs through micropores that simulate the state of the blood vessel walls. However, in the presence of HAP nanoparticles that mimic the remodeling processes of the mineral bone matrix and/or resorption of bone cement, the effect of heparin was less pronounced. The results can be used in the field of regenerative medicine associated with the introduction of MSCs. The data can serve as a prerequisite for developing new therapeutic strategies for surgical patients with a high risk of postoperative thrombosis after osteosynthesis.

Currently, there is an increasing demand for biocompatible materials that can be used for bone reconstruction. However, there is still no consensus regarding adequate bone replacement material. The materials traditionally used for reconstructive surgeries, and methods of making bone-replacing implants from them have various disadvantages. They do not fully satisfy the biological and biomechanical characteristics of living tissues. This leads to a clinical situation called "implant failure” and consists of a violation of its integrity, loosening, attachment of infectious agents, and inflammation development. There are severe socio-economic losses for the patient himself and the state. The problem of infectious complications after surgical operations with the use of bone replacement implants is quite acute. Periprosthetic infection is a modern professional challenge for surgeons and bioengineers. However, antibiotic therapy, which is the only treatment of choice for periprosthetic infection, is characterized by various side effects and becomes ineffective due to microbes' antibiotic resistance. In this regard, for the fight against periprosthetic infection, metal ions with antimicrobial potential (copper, zinc) are considered promising, which are not destroyed during sterilization of medical devices and have their own biological (regulatory) activity. The presented data indicate researchers' interest in studying the interaction of immunocompetent and mesenchymal stem cells with biomedical materials with antimicrobial potential.

The paper reports a study of the morphofunctional activity of T-lymphocytes in response to the in vitro contact with calcium phosphate (CP) coating in the presence of the particles bearing antibodies against CD2, CD3, and CD28 antigens. VT1-0 titanium plates (10 × 10 × 1 mm 3 ) with a double-sided microarc rough ( R a = 2–5 µm) CP coating were used as the model samples of the mineral matrix of the bone tissue. Magnetic particles (MACSiBead TM T-cell Activation/Expansion Kit human) bearing antibodies to CD2, CD3, and CD28 antigens were used as a T-cell activator (TCA) simulating the signals produced by antigen-presenting cells (APCs). Mononuclear cells (MNCs) isolated from human blood (98.8% of CD45CD3 + cells) were cultured in the presence of samples with a CP coating and/or TCA (2 × 10 6 particles in 1.5 mL of nutritive medium in the proportion of 2 : 1 to cells) for 2 and 14 days. The CP coating and TCA triggered MNC culture adaptation in a synergic way via the mechanisms of hyperactivation and subsequent death of T-lymphocytes. Immune selection occurred through the accumulation of the naive CD45RA + /RO + T-lymphocytes and memory T-cells with the simultaneous depletion of the CD4 + and CD8 + T-cell pool. The shift in the T-lymphocyte populations was observed together with the increase (after 48 h of culturing) in the cell secretion activity with its subsequent decrease by the 14th day of observation. CP coating sustained (compared with the cell culture grown on plastic) the secretion ability of Th1 (IL-12, TNFα, and IFNγ) and Th2 (IL-4, IL-6, IL-10, and IL-13) lymphocytes. At the same time, the prolonged TCA signal after the 48-h activation caused the depletion of T-cell secretion. The suggestion that the observed in vitro effects may play role in the switching of signaling between T-lymphocytes, APCs, and CP materials at the cell–foreign body interface, which may result in a change in the inflammation phase, development of immune tolerance, successful osseointegration of the implant, or bone tissue remodeling impairment, is discussed.

Osteoimmunolgy describes the interactions between blood immune cells and human multipotent mesenchymal stromal cells (hMMSCs) as a basis of successful wound and fracture healing. The aim was to investigate in vitro interaction of adipose-derived hMMSCs and human blood mononuclear cells (hBMNCs) modulated by calcium phosphate (CaP) coating in 3D culture. Titanium plates (10 × 10 × 1 mm3) with micro-arc bilateral multilevel CaP coatings were used. Cell-IQ phase-contrast microscopy and the real-time cell analyzer (RTCA) showed the CaP coating and hBMNCs synergistic negative effects on hMMSC motility that could be conditioned by enhanced osteogenic differentiation of stromal cells. Indeed, a 10-fold increase in the bone mineralization around the CaP-coated samples was detected in the mixed (hBMNCs + hMMSCs) 3D culture. Thus, cellular and molecular crosstalk between hBMNCs and hMMSCs modulated by multilevel micro-arc CaP coating with bone-like topography is an effective 3D model to study in vitro the novel pathways of osteoimmunology.

Introduction: Currently, traumatology actively uses ceramic materials with calcium phosphate coating (CP). Such materials have a stimulating effect on multipotent mesenchymal stromal cells (MMSCs) and immunocompetent cells. So, their use involves an assessment of the impact of these materials on the development of tumor cells. Methodology: The Jurkat 5332 cell line and human adipose-derived MMSCs (AMMSCs) were examined. 3D culture was simulated by adding to the cell culture the substrates from commercially pure titanium with rough (Ra=2-5 µm) CP microarc coating. The cultures were used: 2D with Jurkat T cells (JTCs) on plastic surface; 2D co-culture of JTCs and AMMSCs on plastics; 3D with JTCs and CP matrix; 3D with JTCs, AMMSCs, and CP matrix. Findings & Conclusion: Both 2D and 3D JTC cultures showed an increase in the CD45RO receptor expression that led to increasing number of CD45RO+CD45RA+ cells. Probably, JTCs restored the partial maturation and differentiation. Vice versa, the expression of CD45RO receptor and the number of CD45RO+CD45RA+ cells decreased in case of JTCs and AMMSCs co-cultivation. A similar reaction of the cells was revealed in the 3D culture of JTCs and AMMSCs. Thus, JTCs with AMMSCs co-cultivation may

The manufacture of biomaterial surfaces with desired physical and chemical properties that can directly induce osteogenic differentiation without the need for biochemical additives is an excellent strategy for controlling the behavior of mesenchymal stem cells (MSCs) in vivo. We studied the cellular and molecular reactions of MSCs to samples with a double-sided calcium phosphate (CaP) coating and an average roughness index (Ra) of 2.4–4.6 µm. The study aimed to evaluate the effect of a three-dimensional matrix on the relative mRNA expression levels of genes associated with the differentiation and maturation of MSCs toward osteogenesis (RUNX2, BMP2, BMP6, BGLAP, and ALPL) under conditions of distant interaction in vitro. Correlations were revealed between the mRNA expression of some osteogenic and cytokine/chemokine genes and the secretion of cytokines and chemokines that may potentiate the differentiation of cells into osteoblasts, which indicates the formation of humoral components of the extracellular matrix and the creation of conditions supporting the establishment of hematopoietic niches.

In evolutionary processes, human bone marrow has formed as an organ depot of various types of cells that arise from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Vital HSC activity is controlled through molecular interactions with the niche microenvironment. The review describes current views on the formation of key molecular and cellular components of the HSC niche, which ensure maintenance of home ostasis in stem cell niches, obtained from studies of their role in regulating the proliferation and differentiation of HSCs, including the physiological, reparative and pathological remodeling of bone tissue. Due to rapid developments in biotechnology, tissue bioengineering, and regenerative medicine, information can be useful for developing biomimetic and bioinspired materials and implants that provide an effective bone/bone marrow recovery process after injuries and, to a greater extent, diseases of various etiologies.

Maternally inherited Wolbachia symbionts infect D. melanogaster populations worldwide. Infection rates vary greatly. Genetic diversity of Wolbachia in D. melanogaster can be subdivided into several closely related genotypes coinherited with certain mtDNA lineages. mtDNA haplotypes have the following global distribution pattern: mtDNA clade I is mostly found in North America, II and IV in Africa, III in Europe and Africa, V in Eurasia, VI is global but very rare, and VIII is found in Asia. The wMel Wolbachia genotype is predominant in D. melanogaster populations. However, according to the hypothesis of global Wolbachia replacement, the wMelCS genotype was predominant before the XX century when it was replaced by the wMel genotype. Here we analyse over 1500 fly isolates from the Palearctic region to evaluate the prevalence, genetic diversity and distribution pattrern of the Wolbachia symbiont, occurrence of mtDNA variants, and finally to discuss the Wolbachia genotype global replacement hypothesis. All studied Palearctic populations of D. melanogaster were infected with Wolbachia at a rate of 33–100%. We did not observe any significant correlation between infection rate and longitude or latitude. Five previously reported Wolbachia genotypes were found in Palearctic populations with a predominance of the wMel variant. The mtDNA haplotypes of the I_II_III clade and V clade were prevalent in Palearctic populations. To test the recent Wolbachia genotype replacement hypothesis, we examined three genomic regions of CS-like genotypes. Low genetic diversity was observed, only two haplotypes of the CS genotypes with a ‘CCG’ variant predominance were found. The results of our survey of Wolbachia infection prevalence and genotype diversity in Palearctic D. melanogaster populations confirm previous studies. Wolbachia is ubiquitous in the Palearctic region. The wMel genotype is dominant with local occurrence of rare genotypes. Together with variants of the V mtDNA clade, the variants of the ‘III+’ clade are dominant in both infected and uninfected flies of Palearctic populations. Based on our data on Wolbachia and mtDNA in different years in some Palearctic localities, we can conclude that flies that survive the winter make the predominant symbiont contribution to the subsequent generation. A comprehensive overview of mtDNA and Wolbachia infection of D. melanogaster populations worldwide does not support the recent global Wolbachia genotype replacement hypothesis. However, we cannot exclude wMelCS genotype rate fluctuations in the past.

Modern biomaterial biocompatibility research focuses the biomaterial hierarchic effects on multipotent mesenchymal stromal cell (MMSC) behavior (that occur at the nano-, micro- and macroscales) because MMSCs are the fundamental units that produce/regenerate bone tissue. Leukemia initiation and progression are connected with a disfunction of health cell microenvironment and MMSCs. Continuous monitoring of MMSC and tumor cell interaction is a promising tool for oncology, cellular biology, biotechnology and environmental research. The aim was to investigate a modulation of in vitro interaction of human MMSCs and leukemic T lymphoblast-like cells (Jurkat T cells) caused by micro-arc multilevel calcium phosphate (CP) coating with the help of Cell-IQ and RTCA advanced tools for continuous monitoring. An average velocity of cell division (AVCD) of human adipose-derived MMSCs (hAMMSCs) contacted in vitro with allogenic Jurkat line of human leukemic T lymphoblasts (Jurkat T cells) was studied by means of Cell-IQ v2 MLF integrated phase-contrast microscopic platform for real-time surveillance imaging of living cells. Both 50 mu L suspensions (5x10(4) viable karyocytes) of the CD73CD90CD105(+) adherent cells and Jurkat T cells were applied into the center of the well of 12-well plastic plates for 7 days at 100% humidity in a 5% CO2 atmosphere at 37 degrees C until a monolayer formation. A nutrient medium was once replaced. To determine cell invasion (chemotactic motility) through 8 mu m pores the real-time cell analysis (RTCA DP Analyzer) with the CIM-plate was used. AVCD of fibroblast-like adherent hAMMSCs was 0.27-0.63 divisions/h. CP coating diminished significantly the percent of dividing hAMMSCs contacted with leukemic Jurkat T cells. RTCA system showed significant hAMMSC invasion towards tumor cells and not vice versa. For all this, cellular interaction led to increasing viability of Jurkat T cells and decreasing hAMMSC viability. Thus, tumor Jurkat T cells could control a fate of health hAMMSCs and promote stromal microenvironment for survivability of tumor clones by means of secretable molecular products. Multilevel micro-arc CP coating forms bone-like inorganic structure that is capable to modulate in vitro interaction of human MMSCs and leukemic T lymphoblasts. The results obtained may be useful for replacement surgery applications of orthopedic implants in cancer patients.

Using a multiplex kit the secretion of a number of cytokines, chemokines, and growth factors has been investigated in vitro in a culture of human adipose-derived multipotent mesenchymal stromal cells (hAMMSCs) under conditions of their osteogenic differentiation caused by 14-day contact with a calcium phosphate (CP) surface of different roughness. Bilateral X-ray amorphous CP coatings were prepared on the samples of commercially pure titanium in the anodal regime using a microarc method. The electrolyte consisted of aqueous orthophosphoric acid (20 wt %), calcium carbonate (9 wt %), and synthetic hydroxyapatite nanopowder (6 wt %, particle diameter of 10–30 nm with single agglomerates up to 100 nm). hAMMSCs isolated from lipoaspirate were co-cultured after 4 passages with the CP-coated samples at a final concentration of 1.5 × 105 viable karyocytes per 1.5 mL of standard nutrition medium (without osteogenic stimulators) for 14 days (determination of the [CD45,34,14,20], CD73, CD90, and CD105 cell immunophenotype; analysis of secretory activity) and 21 days (alizarin red S cell culture staining) 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 a 2−3-fold increase in the proportion 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: leukemia inhibitory factor (LIF) and stem cell factor (SCF) cytokines in the case of the mean index of CP roughness Ra = 2.4–2.6 µm or stromal derived factor-1 (SDF-1α, CXCL12 chemokine) in the case of Ra = 3.1–4.4 µm.

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.

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.