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Creation of scaffold-based tissue-engineered constructs (SB TECs) is costly and requires coordinated qualified efforts. Cryopreservation enables longer shelf-life for SB TECs while enormously enhancing their availability as medical products. Regenerative treatment with cryopreserved SB TECs prepared in advance (possibly pret-a-porter) can be started straight away on demand. Animal studies and clinical trials indicate similar levels of safety for cryopreserved and freshly prepared SB TECs. Although cryopreservation of such constructs is more difficult than that of cell suspensions or tissues, years of research have proved the principal possibility of using ready-to-transplant SB TECs after prolonged cryostorage. Cryopreservation efficiency depends not only on the sheer viability of adherent cells on scaffolds after thawing, but largely on the retention of proliferative and functional properties by the cells, as well as physical and mechanical properties by the scaffolds. Cryopreservation protocols require careful optimization, as their efficiency depends on multiple parameters including cryosensitivity of cells, chemistry and architecture of scaffolds, conditions of cell culture before freezing, cryoprotectant formulations, etc. In this review we discuss recent achievements in SB TEC cryopreservation as a major boost for the field of tissue engineering and biobanking.

We studied the influence of sucrose applied in combination with different concentrations of penetrating cryoprotectants (DMSO, ethylene glycol, and glycerol) on the efficiency of cryopreservation of umbilical cord-derived multipotent stromal cells (MSC). The results indicate that these cells can be cryopreserved with the use of 5-10% DMSO or ethylene glycol with equal efficiency; addition of 0.2 M sucrose does not affect cell survival after thawing. The efficiency of glycerol as a cryoprotectant increases with increasing its concentration from 5 to 10%, but remains significantly lower than the efficiency of DMSO or ethylene glycol. Addition of sucrose to a final concentration of 0.2 M increases the efficiency of glycerol. The efficiency of combination of 10% glycerol and sucrose was comparable with that of combinations of DMSO and ethylene glycol with sucrose. The mechanism of the observed enhancement is apparently related to the influence of sucrose on the dynamic properties of the lipid membranes and facilitation of glycerol diffusion into the cells.

This paper investigates changes in the surface morphology, internal structure, and the molecular mass distribution of amorphous poly-DL-lactides during their hydrolytic degradation in the presence of extra-germinal mesenchymal stem cells (MSCs) (Wharton’s jelly of umbilical cord) of the rat and their metabolic products. It is shown that the degradation of initially monolithic polymer samples in culture and conditioned media is almost identical. However, in a culture medium containing MSCs, this process is much more intense. This effect can be interpreted in terms of the influence of enzymes secreted by living cells, which diffuse from the surface deep into the polymer sample and accelerate its hydrolysis, participating in a catalytic reaction with the ester bonds of polylactide molecules. We developed and verified a mathematical model that takes into account both noncatalytic and catalytic channels of hydrolysis, changes in the porosity of the polymer sample, and diffusion of short-length oligomers; this model is used to adequately interpret the experimental results.

The pancreas became one of the first objects of regenerative medicine, since other possibilities of dealing with the pancreatic endocrine insufficiency were clearly exhausted. The number of people living with diabetes mellitus is currently approaching half a billion, hence the crucial relevance of new methods to stimulate regeneration of the insulin-secreting β-cells of the islets of Langerhans. Natural restrictions on the islet regeneration are very tight; nevertheless, the islets are capable of physiological regeneration via β-cell self-replication, direct differentiation of multipotent progenitor cells and spontaneous α- to β- or δ- to β-cell conversion (trans-differentiation). The existing preclinical models of β-cell dysfunction or ablation (induced surgically, chemically or genetically) have significantly expanded our understanding of reparative regeneration of the islets and possible ways of its stimulation. The ultimate goal, sufficient level of functional activity of β-cells or their substitutes can be achieved by two prospective broad strategies: β-cell replacement and β-cell regeneration. The “regeneration” strategy aims to maintain a preserved population of β-cells through in situ exposure to biologically active substances that improve β-cell survival, replication and insulin secretion, or to evoke the intrinsic adaptive mechanisms triggering the spontaneous non-β- to β-cell conversion. The “replacement” strategy implies transplantation of β-cells (as non-disintegrated pancreatic material or isolated donor islets) or β-like cells obtained ex vivo from progenitors or mature somatic cells (for example, hepatocytes or α-cells) under the action of small-molecule inducers or by genetic modification. We believe that the huge volume of experimental and clinical studies will finally allow a safe and effective solution to a seemingly simple goal-restoration of the functionally active β-cells, the innermost hope of millions of people globally.

In squamous cell carcinoma of the larynx, the population of epithelial cells in the tumor tissue is initially heterogeneous and, in addition to tumor cells invading the organ mucosa, includes normal epithelial cells of protein-mucous glands and cells of the stratified epithelium covering the mucous membrane. A search for differential markers to separate these subpopulations was carried out. The surface marker CD44 and cytokeratins 5 and 17 that are often used to verify carcinoma cells, are common markers for all epithelial cells of the larynx. In highly differentiated carcinoma, subpopulations of normal and tumor epithelial cells can be separated by the level of expression of cytokeratins 10 and 18 and nuclear markers Ki-67 and p63. However, in moderately differentiated carcinoma, tumor cells and normal cells of the basal layer of the stratified epithelium covering the mucous membrane of the larynx have similar phenotypes, which should be taken into account when conducting experimental studies.

Fast neutron therapy, which previously has demonstrated effective results, but along with a large number of complications, can again be considered a promising treatment method in the treatment of cancer. One of the ways of analyzing the relative biological efficiency and accurate biological dose of fast neutrons in body tissues is to improve the algorithms of computational biology and mathematical modeling. A high-performance computing code was written which allows to estimate in real-time mode the biological dose of the proton component from the action of neutron radiation with an energy of 14.8 MeV. A comparative analysis of the computing performance on various video cards was also performed.

We compared phagocytic activity of macrophages of monocyte origin and Kupffer cells under the influence of M1 and M2 inducers and without activation. Cultures of monocyte-derived macrophages and Kupffer cells were characterized by intensive expression of CD68 that was not affected by activation factors. At the same time, these cultures demonstrated different dynamics of phagocytic activity. Monocyte-derived macrophages initially had more pronounced absorption capacity that gradually increased during the experiment. Kupffer cells were characterized by abrupt fluctuations of phagocytic activity: sharp growth and rapid saturation. Despite these differences, the endosomes produced by monocyte-derived macrophages and Kupffer cells had similar degrees of maturity.

In the central nervous system and in the liver, the macrophage populations are represented exclusively by descendants of the hematopoietic progenitor cells of the yolk sac. The reasons for such differential distribution of macrophages are not fully understood. We found that, as can be judged by corresponding changes in the expression of CD86 and CD163 markers, the transient macrophages of monocytic lineage are more sensitive to activating stimuli. The two macrophage populations have distinct patterns of gene expression, which is particularly noticeable for M1- and M2-associated genes. For instance, Kupffer cells more readily develop and longer maintain the elevated expression levels of Il4, Il10, and Il13 upon the activation; by contrast, the macrophages of monocytic lineage express Il1b, Il12a, and Tnfα upon the activation. The obtained results allow us to conclude that the in vitro activated Kupffer cells of the liver are committed to M2 phenotype, whereas the in vitro activated monocyte-derived macrophages show a typical M1 behavior. These observations are likely to reflect the situation in the in vivo microenvironments.

We studied the influence of sucrose applied in combination with different concentrations of penetrating cryoprotectants (DMSO, ethylene glycol, and glycerol) on the efficiency of cryopreservation of umbilical cord-derived multipotent stromal cells (MSC). The results indicate that these cells can be cryopreserved with the use of 5-10% DMSO or ethylene glycol with equal efficiency; addition of 0.2 M sucrose does not affect cell survival after thawing. The efficiency of glycerol as a cryoprotectant increases with increasing its concentration from 5 to 10%, but remains significantly lower than the efficiency of DMSO or ethylene glycol. Addition of sucrose to a final concentration of 0.2 M increases the efficiency of glycerol. The efficiency of combination of 10% glycerol and sucrose was comparable with that of combinations of DMSO and ethylene glycol with sucrose. The mechanism of the observed enhancement is apparently related to the influence of sucrose on the dynamic properties of the lipid membranes and facilitation of glycerol diffusion into the cells.

Macrophage populations in most mammalian organs consist of cells of different origin. Resident macrophages originate from erythromyeloid precursors of the yolk sac wall, maintenance of the numbers of such macrophages in postnatal ontogenesis is practically independent of bone marrow haematopoiesis. The largest populations of the resident macrophages of embryonic origin are found in the central nervous system (microglia) and liver (Kupffer cells). In contrast, skin dermis and mucous membranes become predominantly colonized by bone marrow-derived monocytes that show pronounced functional and phenotypic plasticity. In the present study, we compared Kupffer cells and monocytes using the immunophenotype, gene expression profile, proteome, and pool of microRNA. The observed differences did not consider the resident liver macrophages as purely M2 macrophages or state that monocytes have purely M1 features. Monocytes show signs of high plasticity and sensitivity to pathogen-associated molecular patterns (e.g., high levels of transcription for Tlr 2, 4, 7, and 8). In contrast, the resident liver macrophages were clearly involved in the regulation of specific organ functions (nitrogen metabolism, complement system protein synthesis).

Functional and anatomical connection between the liver and the spleen is most clearly manifested in various pathological conditions of the liver (cirrhosis, hepatitis). The mechanisms of the interaction between the two organs are still poorly understood, as there have been practically no studies on the influence exerted by the spleen on the normal liver. Mature male Sprague-Dawley rats of 250-260 g body weight, 3 months old, were splenectomized. The highest numbers of Ki67+ hepatocytes in the liver of splenectomized rats were observed at 24 h after the surgery, simultaneously with the highest index of Ki67-positive hepatocytes. After surgical removal of the spleen, expression of certain genes in the liver tissues increased. A number of genes were upregulated in the liver at a single time point of 24 h, including Ccne1, Egf, Tnfa, Il6, Hgf, Met, Tgfb1r2 and Nos2. The expression of Ccnd1, Tgfb1, Tgfb1r1 and Il10 in the liver was upregulated over the course of 3 days after splenectomy. Monitoring of the liver macrophage populations in splenectomized animals revealed a statistically significant increase in the proportion of CD68-positive cells in the liver (as compared with sham-operated controls) detectable at 24 h and 48 h after the surgery. The difference in the liver content of CD68-positive cells between splenectomized and sham-operated animals evened out by day 3 after the surgery. No alterations in the liver content of CD163-positive cells were observed in the experiments. A decrease in the proportion of CD206-positive liver macrophages was observed at 48 h after splenectomy. The splenectomy-induced hepatocyte proliferation is described by us for the first time. Mechanistically, the effect is apparently induced by the removal of spleen as a major source of Tgfb1 (hepatocyte growth inhibitor) and subsequently supported by activation of proliferation factor-encoding genes in the liver.

In this review we present current evidence on the possibility of umbilical cord tissue cryopreservation for subsequent clinical use. Protocols for obtaining umbilical cord-derived vessels, Wharton’s jelly-based grafts, multipotent stromal cells, and other biomedical products from cryopreserved umbilical cords are highlighted, and their prospective clinical applications are discussed. Examination of recent literature indicates we should expect high demand for cryopreservation of umbilical cord tissues in the near future.

The objective of this study was to evaluate physical, mechanical, and biological properties of the polydioxanone (PDO) monofilament meshes and polyglycolide (PGA) polyfilament meshes in comparison with Permacol® implants. In rat experimental model, a 1.5 × 2.0 cm defect in abdominal wall was reconstructed by using the Permacol surgical implant or knitted meshes produced from either PDO monofilament, or PGA multifilament. The implant sites were assessed for the tensile strength and the extents of material resorption, host inflammatory response and host tissue replacement on days 3, 10, 30, or 60 after the surgery. The PDO and PGA meshes were rapidly pervaded by the host connective tissue with elements of skeletal muscle histogenesis. The degree of adhesions was significantly higher in the Permacol group. All of the prostheses underwent resorption, which correlated with gradual decreases in the overall tensile strength of the site and the Col1a1 gene expression level. Elevated expression of Fgf2 gene maintained longer in the PDO group, and the Mmp9 gene expression level in this group was higher than in the other groups. Gene expression levels of inflammatory cytokines were higher in the Permacol group. The foreign body giant cell numbers were lower in the PDO and Permacol groups than in the PGA group. Minimal macrophage infiltration with predominance of M2 cells was observed in the PDO group. Overall, the PDO prosthesis turned out to be significantly better than the PGA or Permacol prostheses by a number of indicators of biocompatibility and efficacy. © 2018 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000-000, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 652-663, 2019.

Macrophages of mammals are a heterogeneous population of cells. This applies both to the functional parameters of macrophages and to the sources of their development. The comparative characteristics of macrophages of embryonic origin on the example of Kupffer cells and macrophages of bone marrow origin on the example of macrophages of monocyte derivatives were carried out. Cultures of Kupffer cells and macrophages of monocytic origin were obtained. The phenotype, profile of gene expression of native macrophages activated in direction M1 and M2 was studied. The phenotype of isolated cultures is characterized by methods of immunocytochemistry, flow cytometry. Gene expression was studied by real-time polymerase chain reaction. Under the influence of inducing factors, the phenotype of two populations of macrophages changes in a similar way: under the influence of M1-factors, the synthesis of CD86 and iNOs is activated in cells, under the influence of M2 - CD163 and Arg1. In Kupffer cells, expression of anti-inflammatory cytokines - il4, il13, is more pronounced, and in macrophages of monocytic origin of pro-inflammatory cytokines - il1b, tnfa, il12a. The induction of the genes of proinflammatory cytokines in Kupffer cells is slower compared to macrophages of monocytic origin.

AIM To investigate the influence of the umbilical cord-derived multipotent stromal cells (MSCs) on recovery of the liver after the subtotal resection, that is, removal of 80% of the organ mass, a renowned model of the small-for-size liver remnant syndrome. METHODS The MSCs were obtained from the intervascular tissue of umbilical cords, dissected from rat fetuses, by the explant culture technique. The vital labeling of MSCs with РКН26 was carried out on the 3rd passage. The subtotal resection was performed on male Sprague-Dawley rats. The experimental group animals received a transplant 106 MSCs infused into the spleen. Hepatocyte proliferation was assessed by counting of either mitotic figures or Ki67-positive cells in microscopic images. MSC differentiation was assessed with antibodies to hepatocyte-specific marker cytokeratin 18 (CK18), cholangiocyte-specific protein CK19, smooth muscle cell-specific protein α-SMA, the endothelial cell marker CD31, or the active fibroblast marker FAPα. Total macrophages of the liver were selectively stained in cryosections incubated with anti-CD68 antibodies (1:100, Abcam), while the M2a and M2c macrophage populations were selectively stained with anti-CD206 antibodies. Expression of interleukin and growth factor genes was evaluated with PCR-RT. RESULTS Intrasplenic allogeneic transplantation of the umbilical cord-derived multipotent stromal cells stimulates reparative processes within the residual liver tissue after subtotal resection (removal of 80% of the organ mass), as indicated by increased rates of hepatocyte proliferation and accelerated organ mass recovery. These effects may result from paracrine influence of the transplanted cells on the resident macrophage population of the liver. The transplantation favors polarization of macrophages to M2 phenotype (the M2-polarized macrophages specifically express CD206; they are known to suppress inflammation and support tissue repair). No differentiation of the transplanted cells into any of the liver cell types have been observed in the study. CONCLUSION We found no direct evidence for the paracrine effect of MSCs on liver regeneration after the subtotal liver resection in rats. However, the paracrine mechanism of the therapeutic activity of transplanted MSC is indirectly indicated by a decrease in the total number of CD68 + macrophages and an increase in the proportion of M2 pro-repair macrophages in the regenerating liver as compared to animals in which the transplantation was only mimicked.

The article presents a new method for treating iatrogenic damage to membranes in fetuscopic interventions. Since surgical treatment during pregnancy is associated with the use of trocar and amniotic fetal puncture, the most frequent complication of this procedure is premature discharge of amniotic fluid and abortion (28-67%). The new method of treatment is based on the use of the technique of safe and effective hermetic sealing of fetal membranes in fetoscopy, which will reduce the frequency of iatrogenic complications. The Ministry of Health of the Russian Federation has developed a method and created the main components for hermetic sealing of membranes: a tissue sealant from the blood components of the mother - a plasma enriched with platelets, which can be injected into the area of the defect of the fetal bladder in the form of a gel and an amniotic catheter providing effective sealing of the trocar hole from the inner and outer surfaces of the membranes. We described the results of a laboratory experiment on animals, discussed the effectiveness of a new method of treatment and the possibility of its practical application.

The mechanisms of proangiogenic activity of multipotent stromal cells from human umbilical cord were analyzed in vitro. The absence of secreted forms of proangiogenic growth factor VEGF-A in the culture medium conditioned by umbilical cord-derived multipotent stromal cells was shown by ELISA. However, the possibility of paracrine stimulation of cell proliferation, mobility, and directed migration of endothelial EA.hy926 cells was demonstrated by using MTT test, Transwell system, and monolayer wound modeling. The capacity of multipotent stromal cells to acquire the phenotype of endothelium-like cells was analyzed using differentiation media of three types. It was found that VEGF-A is an essential but not sufficient inductor of differentiation of umbilical cord-derived multipotent stromal cells into CD31+ cells.

Background In many clinical cases of extensive liver resection (e.g. due to malignancy), the residual portion is too small to maintain the body homeostasis. The resulting acute liver failure is associated with the compensatory growth inhibition, which is a typical manifestation of the ‘small for size’ liver syndrome. The study investigates possible causes of the delayed onset of hepatocyte proliferation after subtotal hepatectomy (80% liver resection) in rats. Results The data indicate that the growth inhibition correlates with delayed upregulation of the Tnf gene expression and low content of the corresponding Tnfα protein within the residual hepatic tissue. Considering the involvement of Tnf/Tnfα, the observed growth inhibition may be related to particular properties of liver macrophages – the resident Kupffer cells with CD68+CX1CR3−CD11b− phenotype. Conclusions The delayed onset of hepatocyte proliferation correlates with low levels of Tnfα in the residual hepatic tissue. The observed growth inhibition possibly reflects specific composition of macrophage population of the liver. It is entirely composed of embryonically-derived Kupffer cells, which express the ‘proregeneratory’ M2 macrophage-specific marker CD206 in the course of regeneration. Electronic supplementary material The online version of this article (10.1186/s12865-018-0260-1) contains supplementary material, which is available to authorized users.

Human umbilical cord represents a source of multipotent stromal cells of a supreme therapeutic potential. The cells can be isolated from either fresh or cryopreserved umbilical cord tissues. DMSO is a cryoprotectant most commonly used for preservation of umbilical cord tissues; however, cyto- and genotoxicity of this compound is evident and well documented. In the present study we performed successful cryopreservation of the umbilical cord tissue using other cryoprotectants: propylene glycol, ethylene glycol, and glycerol. Of these, 1.5 M ethylene glycol and 20% glycerol turned out to be the best in terms of the preservation of living cells within the frozen tissue, early onset of migration of these cells out of the thawed explants, and overall efficacy of multipotent stromal cell isolation. Cryobanking of tissues can improve availability of multiple cell products for medical purposes and promote the development of personalized medicine.

Short-term cell culturing on basement membrane matrix is a common and very convenient in vitro model of angiogenesis. We studied the possibility of interaction of multipotent stromal cells from the umbilical cord and Ea.hy926 endothelial cells on this model at the early and late periods of the experiment. Multipotent stromal cells alone and in combination with endothelial cells formed an unstable tubular network. Clusters formed after its disassembling later became the sprouting centers in co-culture of the two cell types, but not in pure culture of multipotent stromal cells. Multipotent stromal cells with CD31+ phenotype constitute the structural basis of newly formed stable 3D capillary-like network. Prolongation of the time of culturing and combination of the two in vitro models of angiogenesis (tubulogenesis and sprouting) allowed more complete assessment of the angiogenic potential of multipotent stromal cells.

The paper presents current evidence on the properties of human umbilical cord-derived mesenchymal stem cells, including origin, proliferative potential, plasticity, stability of karyotype and phenotype, transcriptome, secretome, and immunomodulatory activity. A review of preclinical studies and clinical trials using this cell type is performed. Prospects for the use of mesenchymal stem cells, derived from the umbilical cord, in cell transplantation are associated with the need for specialized biobanking and transplant standardization criteria.

Intrasplenic allogeneic transplantation of multipotent stromal cells from the umbilical cord stimulates hepatocyte proliferation and promotes recovery of liver weight in rats after subtotal resection (80% organ weight). It can be hypothesized that this effect of multipotent stromal cells is due to more rapid recovery of the number of mitochondria and normalization of mitochondrial function of liver hepatocytes.

Cell therapy is increasingly recognized as a beneficial practice in various cardiac conditions, but its fundamentals remain largely unclear. The fates of transplanted multipotent stromal cells in postinfarction cardiac microenvironments are particularly understudied. To address this issue, labeled multipotent stromal cells were infused into rat myocardium at day 30 after myocardial infarction, against the background of postinfarction cardiosclerosis. Therapeutic effects of the transplantation were assessed by an exercise tolerance test. Histological examination at 14 or 30 days after the transplantation was conducted by means of immunostaining and quantitative image analysis. An improvement in the functional status of the cardiovascular system was observed after both the autologous and the allogeneic transplantations. Location of the label-positive cells within the heart was restricted to the affected part of myocardium. The transplanted cells could give rise to fibroblasts or myofibroblasts but not to cardiac myocytes or blood vessel cells. Both types of transplantation positively influenced scarring processes, and no expansion of fibrosis to border myocardium was observed. Left ventricular wall thickening associated with reduced dilatation index was promoted by transplantation of the autologous cells. According to the results, multipotent stromal cell transplantation prevents adverse remodeling and stimulates left ventricular reverse remodeling.

The role of the lungs and kidneys in liver regeneration after subtotal hepatectomy was studied on a rat model. It was found that production of hepatocyte growth factor (HGF) in the lungs and kidneys and expression of cytokine genes Il1b, Il6, Il10, and tnfa significantly increased. Analysis of the dynamics of lung macrophage population showed that accumulation of HGF and the increase in the expression of cytokine genes in the lungs were accompanied by simultaneous increase in the number of CD68+ cells, which attested to the leading role of macrophages in activation of HGF synthesis in the lungs. Macrophage content in the kidneys after subtotal hepatectomy did not increase.

Background Mesenchymal stromal/stem cells derived from human umbilical cord (UC-MSCs) uniquely combine properties of embryonic and postnatal MSCs and may be the most acceptable, safe, and effective source for allogeneic cell therapy e.g. for therapeutic angiogenesis. In this report we describe pro-angiogenic properties of UC-MSCs as manifested in vitro. Methods UC-MSCs were isolated from human Wharton’s jelly by enzymatic digestion. Presence of soluble forms of VEGF-A in UC-MSC-conditioned media was measured by ELISA. Effects of the conditioned media on human umbilical vein-derived endothelial EA.hy926 cells proliferation were measured by MTT-assay; changes in cell motility and directed migration were assessed by scratch wound healing and transwell chamber migration assays. Angiogenesis was modeled in vitro as tube formation on basement membrane matrix. Progressive differentiation of MSCs to endothelioid progeny was assessed by CD31 immunostaining. Results Although no detectable quantities of soluble VEGF-A were produced by UC-MSCs, the culture medium, conditioned by the UC-MSCs, effectively stimulated proliferation, motility, and directed migration of EA.hy926 cells. In 2D culture, UC-MSCs were able to acquire CD31+ endothelial cell-like phenotype when stimulated by EA.hy926-conditioned media supplemented with VEGF-A165. UC-MSCs were capable of forming unstable 2D tubular networks either by themselves or in combinations with EA.hy926 cells. Active spontaneous sprouting from cell clusters, resulting from disassembling of such networks, was observed only in the mixed cultures, not in pure UC-MSC cultures. In 3D mode of sprouting experimentation, structural support of newly formed capillary-like structures was provided by UC-MSCs that acquired the CD31+ phenotype in the absence of exogenous VEGF-A. Conclusion These data suggest that a VEGF-A-independent paracrine mechanism and at least partially VEGF-A-independent differentiation mechanism are involved in the pro-angiogenic activity of UC-MSCs.

We developed a new method of creation of tissue engineering constructs for regeneration of the bone tissue based on the principle of free distribution of cells in a fibrin clot within a scaffold. The tissue engineering construct includes multipotent stromal adipose tissue cells committed in osteogenic lineage, platelet-rich plasma, and resorbed material on the basis of xenogeneic bone collagen. The culture of bone progenitor cells was characterized by the main markers of osteoblastic differon. The material meets all requirements for materials intended for tissue engineering. An innovative high-technological tissue engineering product for clinical application is prepared.

The proliferation and death of hemopoietic cells in the regenerating liver of 17-day outbred albino rat fetuses were studied during 2 days after resection of 20% of the organ. The mitotic index of hemopoietic cells in the resected liver increased signifi cantly 24 h after the operation in comparison with the control fetuses. No increase in the counts of apoptotic hemopoietic cells was detected in the regenerating liver. Hence, resection of 20% of the liver in rat fetuses stimulated the proliferation of hemopoietic cells and did not stimulate their apoptosis.

Neoangiogenesis after transplantation of auto- and allogenic mononuclears and multipotent stromal cells from the bone marrow was studied on the model of inflammatory angiogenesis. Transplanted auto- and allogenic cells stimulate the formation of new blood vessels in the granulation tissue, this manifesting in an increase in the quantity and volume density of blood vessels. The most pronounced angiogenesis was observed after transplantation of allogenic mononuclears and multipotent stromal cells. It was associated with intense inflammatory infiltration, with less numerous and mature collagen fibers in the granulation tissue. Injection of allogenic cells led to stimulation and chronization of inflammation, infiltration with inflammatory and poorly differentiated cells, and more pronounced and lasting angiogenesis. However, neither auto-, nor allogenic transplanted labeled cells were detected in the walls of new blood vessels. Hence, it seems that bone marrow mononuclears and multipotent stromal cells stimulated angiogenesis mainly at the expense of production of angiogenic factors, and after transplantation of allogenic cells also by stimulating the inflammation.

Directions of migration of mononuclear bone marrow cells after intracoronary transventricular injection procedure developed by us were experimentally studied. After nonselective injection of cells into the right and left coronary arteries in rats, the labeled cells were detected only in the damaged zone of the myocardium. Localization of transplanted mononuclears in the scar attests to their homing into the damaged zone. Numerous cells were found in the red pulp of the spleen and solitary cells were detected in the liver and lungs. In the heart, the labeled transplanted cells were detected only in the scar zone at all terms of the study; they were not incorporated into the vascular walls, but were surrounded by thick bundles of collagen fibers and probably underwent differentiation into fibroblasts. No data on possible differentiation of the transplanted cells into vascular cells or cardiomyocytes were obtained.

We studied morphological changes in the paraurethral area of Wistar rats after introduction of tissue engineering constructs on the basis of multipotent mesenchymal stem cells and gelatin sponge. The tissue engineering construct containing autologous culture of the stromal fraction of the adipose tissue was most effective. After introduction of this construct we observed more rapid degradation of the construct matrix and more intensive formation of collagen fibers.

Effect of dexamethasone on differentiation of multipotent stromal cells from human adipose tissue was evaluated. Addition of dexamethasone to growth medium resulted in active adipogenesis. Addition of dexamethasone to the osteogenic medium (containing active vitamin D3 form as the main inductor) led to simultaneous realization of the adipogenic and osteogenic potencies of multipotent stromal cells of the adipose tissue. Hence, the quality of the transplant on the basis of predifferentiated multipotent stromal cells from the adipose tissue for bone tissue repair can be deteriorated by dexamethasone directing some cells to adipogenic development.

The use of synthetic osteoplastic materials not always provides the required amount of the bone tissue. Transplantation of tissue-engineering constructs containing osteogenic precursor cells can be an alternative high-technology implantation method. Here we present the results of a pilot clinical study demonstrating safety of this method, accelerated healing of the operation wound, formation of young bone tissue after transplantation, and the possibility of mounting implants after 3 months in case of sufficient amount of the bone for primary fixation.

Expression of il1b, il6, il10, tnfa, hgf, tgfb, vegf, and fgf2 genes in the lungs and kidneys was examined on rat model of liver regeneration after subtotal hepatectomy. Enhanced expression of il6, il10, tnfa, hgf, and fgf2 genes was detected at the early terms after 80% liver resection.

In the liver of rats subjected to subtotal liver resection (80% organ weight), the expression of sox9 gene and SOX9 protein content increased and cells with hepatocyte morphology expressing SOX9 appeared; the proportion of cells expressing cytokeratin-19 also increased. Based on these data, we cannot completely exclude the involvement of resident progenitor cells and hepatocyte reprogramming in liver regeneration after subtotal resection, however, the contribution of these processes seems to be insignificant. The leading mechanism of liver mass recovery after subtotal resection is proliferation of hepatocytes.

Proliferation of hepatocytes is known to be the main process in the hepatectomy-induced liver regrowth; however, in cases of extensive loss it may be insufficient for complete recovery unless supported by some additional sources e.g. mobilization of undifferentiated progenitors. The study was conducted on rat model of 80% subtotal hepatectomy; the objective was to evaluate contributions of hepatocytes and resident progenitor cells to the hepatic tissue recovery via monitoring specific mRNA and/or protein expression levels for a panel of genes implicated in growth, cell differentiation, angiogenesis, and inflammation. Some of the genes showed distinctive temporal expression patterns, which were loosely associated with two waves of hepatocyte proliferation observed at 2 and 7 days after the surgery. Focusing on genes implicated in regulation of the progenitor cell activity, we came across slight increases in expression levels for Sox9 and two genes encoding tumor necrosis factor-like cytokine TWEAK (Tnfsf12) and its receptor Fn14 (Tnfrsf12a). At the same time, no increase in numbers of cytokeratin 19-positive (CK19+) cells was observed in periportal areas, and no CK19+ cells were found in hepatic plates. Since CK19 is thought to be a specific marker of both cholangiocytes and the hepatic progenitor cells, the data indicate a lack of activation of the resident progenitor cells during recovery of hepatic tissue after 80% subtotal hepatectomy. Thus, proliferation of hepatocytes invariably makes the major contribution to the hepatic tissue recovery, although in the cases of subtotal loss this contribution is distinctively modulated. In particular, induction of Sox9 and TWEAK/Fn14 regulatory pathways, conventionally attributed to progenitor cell activation, may incidentally stimulate mitotic activity of hepatocytes.